ABSTRACT
BARNWELL, CALLIE VIRGINIA. Early Embryonic Survival and Conceptus Elongation of
Bovine Embryos Produced In Vivo or In Vitro. (Under the direction of Charlotte E. Farin).
Early embryonic mortality is a major factor contributing to decreased reproductive
efficiency in domestic animals and in cattle most often occurs prior to maternal recognition
of pregnancy. Perturbations in embryo or conceptus development, as well as inadequate
uterine receptivity, contribute to the high incidence of embryonic mortality. Therefore, it is
critical to enhance our understanding of the mechanisms involved in optimal conceptusmaternal interactions.
The objective of the first study was to examine the relationship between recipient
serum progesterone levels, both at the time of embryo transfer and at conceptus recovery, on
conceptus development from in vivo (IVO) or in vitro (IVP) produced embryos. Single
Grade 1 blastocysts were transferred into heifers at Day 7 of development. Conceptuses were
recovered at Day 17 of gestation and classified as complete, degenerated, or no conceptus.
Compared with the IVO group, IVP embryos had more degenerated conceptuses.
Progesterone concentrations in recipients receiving IVO embryos were higher at Day 7
compared to those receiving IVP embryos. Heifers in the IVP groups had lower progesterone
concentrations at Day 7 when no conceptus was recovered at Day 17 compared with the IVO
group. There was no difference in progesterone concentration between treatment groups for
heifers with shorter conceptuses. However, when longer conceptuses were recovered, heifers
with IVP embryos had lower progesterone levels at Day 7 compared to those with IVO
embryos. In summary, serum progesterone concentration in recipients at the time of transfer
of IVO or IVP embryos was associated with conceptus development at Day 17 of gestation.
The objective of the second study was to characterize differential patterns of mRNA
expression between short and long bovine conceptuses recovered on Day 15 of gestation
from IVO or IVP embryos. In Experiment 1 and Experiment 2, embryos were produced in
vivo from superovulated Holstein donor cows and groups of Grade 1 and Grade 3 compact
morulas were transferred into recipient heifers at Day 6.5 of the cycle. For Experiment 3,
Grade 1 Holstein IVO and IVP blastocysts were transferred into recipient heifers at Day 7 of
the cycle. All conceptuses were then recovered at Day 15 of gestation, and measured to
assess overall length and area. Total RNA was extracted from conceptuses and analyzed on
individual GeneChip Bovine Genome Arrays. In Experiment 1, there were no differences in
gene expression profiles of conceptuses derived from Grade 1 and Grade 3 embryos. In
Experiment 2, a total of 348 genes were differentially expressed between short and long IVO
conceptuses. Of these, 221 genes were up-regulated and 127 were down-regulated in long
conceptuses compared to short conceptuses. Differentially expressed genes were largely
involved in metabolic and biosynthetic processes and immune response. Short and long IVO
conceptuses displayed differential regulation of genes previously identified as associated
with developmental competency to term and enhanced elongation. In contrast, in Experiment
3, there were no differentially expressed genes between short and long IVO conceptuses.
However, there was 1 gene differentially expressed between short and long IVP conceptuses.
In summary, differences in gene expression were identified between conceptuses recovered
on Day 15 of gestation that were classified based on length. We propose that conceptuses
which are at an earlier morphological stage than expected based on the day of recovery may
be of inferior developmental competency.
© Copyright 2014 by Callie Virginia Barnwell
All Rights Reserved
Early Embryonic Survival and Conceptus Elongation of Bovine Embryos Produced In Vivo
or In Vitro
by
Callie Virginia Barnwell
A dissertation submitted to the Graduate Faculty of
North Carolina State University
in partial fulfillment of the
requirements for the degree of
Doctor of Philosophy
Physiology
Raleigh, North Carolina
2014
APPROVED BY:
_______________________________
Charlotte E. Farin
Committee Chair
______________________________
C. Scott Whisnant
________________________________
William L. Flowers
________________________________
Christopher M. Ashwell
Minor Representative
ii
DEDICATION
In memory of Dr. Peter W. Farin.
iii
BIOGRAPHY
Education
PhD
Physiology, 2014, North Carolina State University, Raleigh, NC
BS
Biological Sciences, 2007, North Carolina State University, Raleigh, NC
Honors and Awards
Larry Ewing Memorial Trainee Travel Fund, Society for the Study of Reproduction, 2012
Graduate Assistantships in Areas of National Need (GAANN) Fellowship, Molecular
Biotechnology, 2011
Graduate Assistantships in Areas of National Need (GAANN) Fellowship, Molecular
Biotechnology, 2010
Provost's Fellowship, North Carolina State University, 2009
Professional Affiliations
International Embryo Transfer Society
Society for the Study of Reproduction
iv
ACKNOWLEDGMENTS
I would like to thank my advisor and mentor, Dr. Charlotte Farin, for all of her
expertise and guidance throughout my graduate program. I am grateful for your generosity
and support under difficult circumstances, and for always being present and involved. I
would like to thank my committee members, Drs. Scott Whisnant, Chris Ashwell, and
William Flowers, for their advice, guidance, and assistance. I would also like to express my
gratitude to Dr. Paul Mozdziak, for his support, which allowed me to continue with my
graduate program.
I would like to thank my colleague, Will Farmer, for his invaluable assistance, advice,
and partnership. I would also like to acknowledge the efforts of Ginger Hobgood, and all of
the others who volunteered their time to help with my projects. I am also indebted to Dr. Sam
Galphin, for offering his veterinary services in embryo transfer and recovery. A special
thanks goes to the employees of the NCSU Dairy Education Unit, namely Anthony Chesnutt,
John Zerrer, and Drew Gibson, who made the majority of my dissertation projects possible.
Thank you for being so accommodating and always willing to offer assistance where needed.
I would also like to thank my family and friends for all their support. Special thanks
are given to my parents, Tim and Kathryn, for their unwavering support. I would also like to
thank my husband, Greg Gibson, for sticking with me through it all.
Finally, I would like to thank the late Dr. Peter W. Farin, for his knowledge, support, and
guidance. I will be forever grateful.
v
TABLE OF CONTENTS
LIST OF TABLES ............................................................................................................. ix
LIST OF FIGURES ........................................................................................................... xi
CHAPTER 1: LITERATURE REVIEW .............................................................................1
IN VITRO PRODUCTION OF EMBRYOS .............................................................2
ASSESSING EMBRYO VIABILITY .......................................................................3
Methods of Embryo Evaluation .......................................................................4
Embryo Quality and Pregnancy Rate...............................................................6
Evaluation of In Vitro Produced Embryos.......................................................7
Gene Expression in Bovine Embryos ..............................................................9
PREGNANCY RECOGNITION AND EARLY CONCEPTUS
DEVELOPMENT ....................................................................................................12
Maternal Recognition of Pregnancy ..............................................................12
Normal Conceptus Development ...................................................................14
PROGESTERONE ..................................................................................................17
Patterns of Progesterone Concentration and Early Conceptus
Development ..................................................................................................17
Mechanisms of Progesterone Effects .............................................................21
ABNORMALITIES ASSOCIATED WITH IN VITRO EMBRYO
TECHNOLOGIES ...................................................................................................23
vi
Pre-implantation Development ......................................................................24
Peri-implantation Development .....................................................................25
Fetal Development .........................................................................................30
Placental Development ..................................................................................37
Underlying Mechanisms of AOS ...................................................................40
STATEMENT OF THE PROBLEM .......................................................................44
REFERENCES ........................................................................................................46
CHAPTER 2: MATERNAL SERUM PROGESTERONE CONCENTRATION AND
EARLY CONCEPTUS DEVELOPMENT OF BOVINE EMBRYOS PRODUCED IN VIVO
OR IN VITRO....................................................................................................................71
ABSTRACT .............................................................................................................71
INTRODUCTION ...................................................................................................72
METHODS ..............................................................................................................76
Embryo Production ........................................................................................76
Embryo Transfer and Conceptus Recovery ...................................................77
Radioimmunoassay ........................................................................................77
Statistical Analysis .........................................................................................78
RESULTS ................................................................................................................79
Conceptus Recovery ......................................................................................79
Progesterone Concentration at Time of Embryo Transfer .............................79
Progesterone Concentration at Time of Conceptus Recovery .......................80
vii
DISCUSSION ..........................................................................................................80
REFERENCES ........................................................................................................86
CHAPTER 3: DIFFERENCES IN THE TRANSCRIPTOME OF LONG AND SHORT
BOVINE CONCEPTUSES ON DAY 15 OF GESTATION PRODUCED IN VIVO OR IN
VITRO .............................................................................................................................112
ABSTRACT ...........................................................................................................112
INTRODUCTION .................................................................................................113
METHODS ............................................................................................................118
In Vivo Embryo Production .........................................................................118
In Vitro Embryo Production ........................................................................118
Embryo Transfer and Conceptus Recovery .................................................119
Total DNA Isolation and PCR-Based Sex Determination Assay ................120
Total RNA Isolation.....................................................................................121
Affymetrix Array Hybridization, Washing, Staining, and Scanning ...........121
Microarray Data Analysis ............................................................................122
cDNA Synthesis ...........................................................................................123
Quantitative Real-Time PCR .......................................................................123
Statistical Analysis .......................................................................................125
RESULTS ..............................................................................................................125
Experiments 1 & 2 .......................................................................................125
Day 15 Conceptus Recovery Summary ..................................................125
viii
Experiment 1 ................................................................................................126
Conceptuses Selected for Microarray Analysis Based on Grade............126
Experiment 2 ................................................................................................126
Conceptuses Selected for Microarray Analysis Based on Length ..........126
Conceptuses Selected for Microarray Analysis Based on Gender..........127
Differential Gene Expression between Long and Short Conceptuses ....127
Differential Gene Expression between Male and Female Conceptuses .129
Experiment 3 ................................................................................................130
Day 15 Conceptus Recovery Summary ..................................................130
Conceptuses Selected for Microarray Analysis Based on Length ..........130
Conceptuses Selected for Microarray Analysis Based on Production
System .....................................................................................................131
Gene Expression .....................................................................................131
Quantitative Real-Time PCR .......................................................................132
DISCUSSION ........................................................................................................133
REFERENCES ......................................................................................................144
CHAPTER 4: GENERAL CONCLUSIONS & FUTURE WORK.................................168
REFERENCES ......................................................................................................177
APPENDICES .................................................................................................................180
APPENDIX I. Differentially expressed genes between long and short
Day 15 conceptuses derived from Grade 1 IVO embryos. ......................................18
ix
LIST OF TABLES
Table 1.1
Criteria for assigning quality grades to bovine embryos as recommended
by the International Embryo Transfer Society ...............................................66
Table 1.2
Pregnancy rates of recipients following transfer of fresh bovine embryos
of different stages of development and quality grades produced either in
vivo or in vitro ...............................................................................................67
Table 1.3
Potential genes for assessing development and function of bovine
embryos ..........................................................................................................68
Table 2.1
Conceptus recovery at Day 17. Presented previously in modified form
[1] .................................................................................................................106
Table 2.2
Summary of average intact conceptus lengths (mean ± SEM) and ranges
for each treatment group ..............................................................................106
Table 2.3
Progesterone concentration (ng/mL; mean ± SEM) in recipient heifers at
Day 17 of gestation ......................................................................................111
Table 3.1
List of target genes, primer sequences, and product sizes of transcripts
validated by qPCR .......................................................................................155
Table 3.2
Summary of all measured Day 15 conceptuses recovered for
Experiments 1 and 2 (mean ± SEM) ............................................................155
Table 3.3
Summary of Day 15 conceptuses selected for microarray analysis in
Experiments 1 and 2 (mean ± SEM) ............................................................155
Table 3.4
The top 25 up-regulated and the top 25 down-regulated differentially
expressed genes in long conceptuses compared to short conceptuses
derived from Grade 1IVO embryos. A positive value indicates upregulation in long conceptuses while a negative value indicates upregulation in short conceptuses ....................................................................157
Table 3.5
The top biological processes overrepresented in genes solely upregulated in long conceptuses (top) and solely up-regulated in short
conceptuses (bottom) derived from IVO embryos.......................................160
x
Table 3.6
Molecular functions and cellular components overrepresented in genes
up-regulated in long conceptuses (top) and up-regulated in short
conceptuses (bottom) derived from IVO embryos.......................................161
Table 3.7
Top GeneGO pathway categories and their corresponding pathways
differentially expressed in long and short length conceptuses. The genes
that were relatively up-regulated in long conceptuses (underlined) and
up-regulated in short conceptuses (plain type) involved in the pathway
categories are listed. Only pathways significant at an adjusted P-value
≤ 0.05 are included.......................................................................................163
Table 3.8
Differentially expressed genes (n=12) between female and male Day 15
conceptuses derived from IVO embryos in Experiments 1 and 2. A
positive value indicates up-regulation in female conceptuses while a
negative value indicates up-regulation in male conceptuses .......................165
Table 3.9
Differentially expressed genes (n=10) between female and male Day 15
conceptuses derived from IVO embryos in Experiment 2. A positive
value indicates up-regulation in female conceptuses while a negative
value indicates up-regulation in male conceptuses ......................................165
Table 3.10 Summary of all measured Day 15 conceptuses recovered for Experiment
3 (mean ± SEM) ...........................................................................................166
Table 3.11 Summary of Day 15 conceptuses selected for microarray analysis in
Experiment 3 (mean ± SEM) .......................................................................166
Table A1
Differentially expressed genes (n=348) between long and short Day 15
conceptuses derived from Grade 1 IVO embryos. A positive value
indicates up-regulation in long conceptuses while a negative value
indicates up-regulation in short conceptuses. Adjusted P-value <0.05 .......182
xi
LIST OF FIGURES
Figure 1.1
Identification of abnormalities commonly associated with abnormal
offspring syndrome (AOS). In individual cases, each abnormality listed
may or may not be present .............................................................................69
Figure 1.2
Relative timing of pregnancy losses following the transfer of bovine
embryos produced in vivo (IVO), in vitro (IVP) or by somatic cell nuclear
transfer (SCNT) .............................................................................................70
Figure 2.1
Progesterone concentration (mean ± SEM) at Day 7 and treatment group.
Different letters denote significant differences between treatment groups
(P<0.05) .......................................................................................................107
a,b
Figure 2.2
Progesterone concentration (mean ± SEM) at Day 7 and conceptus state at
recovery at Day 17. a,b Different letters denote significant differences
between treatment groups within conceptus state (P<0.05).........................108
Figure 2.3
Progesterone concentration (mean ± SEM) at Day 7 and conceptus length
at Day 17. a,b Different letters denote significant differences between
treatment groups within conceptus length classification (P=0.05) ..............109
Figure 2.4
Progesterone concentration (mean ± SEM) at Day 7 and conceptus sex at
Day 17 ..........................................................................................................110
Figure 3.1
Examples of one short (left, 4.0 mm) and one long (right, 25.6 mm) Day
15 conceptus derived from Grade 1 IVO embryos ......................................156
Figure 3.2
The top GO biological processes overrepresented in the full list of DEGs
between long and short conceptuses derived from IVO embryos. DEGs upregulated (underlined) or down-regulated (plain type) in long conceptuses
compared to short are included for select processes ....................................159
Figure 3.3
The top 20 most significant GeneGo Pathway Maps containing DEGs
between long and short length conceptuses. P-value <0.05.........................162
Figure 3.4
Top 10 most significant GeneGo Process Networks containing genes (A)
up-regulated in long length conceptuses (B) and up-regulated in short length
conceptuses ..................................................................................................164
xii
Figure 3.5
Comparison of the fold changes of qPCR and microarray data for selected
genes identified as differentially expressed. The data shown for IL6, TP53I3,
TAGLN, AQP3, and JAM2 are from Experiment 2. The data shown for
RCAN2 are from Experiment 3. A positive value indicates up-regulation in
long conceptuses and a negative value indicates down-regulation in long
conceptuses compared to short conceptuses. Statistical significance of the
relative expression ratio is indicated (*P-value<0.05; **P-value <0.02) ....167
1
CHAPTER 1: LITERATURE REVIEW
Infertility and subfertility are major problems in domestic animals and particularly
affect production and economic efficiency in the cattle industry. Embryonic mortality is the
largest factor affecting reproductive efficiency; it is estimated that embryonic mortality rate
ranges from 40% to 56% in cattle [2-4]. Failure to establish pregnancy is due to both
embryonic and maternal factors, such as genetics, embryo source and quality, and the rate
and timing of the post-ovulatory rise in maternal progesterone concentration. The majority of
this loss (70% to 80%) occurs during the first three weeks of pregnancy, particular between
Day 8 and Day 16 post-insemination [4, 5]. This corresponds to the period leading up to
maternal recognition of pregnancy, during which the developing conceptus is undergoing a
radical increase in size and must produce adequate levels of interferon-tau to prevent
luteolysis.
In 2012 approximately 506,000 in vivo produced bovine embryos were transferred
commercially worldwide [6]. The mean survival rate to calving following the transfer of in
vivo produced embryos ranges from 31% to 60% [7]. In that same year 385,000 in vitro
produced bovine embryos were transferred commercially [6]. Mean survival rate to calving
after the transfer of in vitro produced embryos is lower, ranging from 30% to 40% [7]. In
vitro embryo production offers several advantages, including the production of embryos from
diseased or deceased animals, the generation of multiple calves per year from economically
2
important females, and serves as the platform for more advanced assisted reproductive
technologies.
However, the transfer of in vitro produced embryos is associated with lower
pregnancy rates in addition to the occurrence of abnormalities in fetuses, placentas, and
calves. Identification of methods to improve survival rates of both in vivo and in vitro
produced embryos could be a major benefit to the livestock industry.
IN VITRO PRODUCTION OF EMBRYOS
In vitro embryo production involves retrieval of oocytes from the ovary of the donor
cow using transvaginal ultrasound-guided ovum pick up or retrieval from ovaries collected at
an abattoir. Meoitically competent bovine oocytes can be harvested or aspirated from small
(3-5mm) and medium sized (6-10mm) antral follicles [8, 9]. Recovered oocytes are then
subjected to the sequential process of in vitro maturation (IVM), in vitro fertilization (IVF)
and in vitro culture (IVC).
Oocyte maturation involves synchronization of nuclear and cytoplasmic events to
allow successful fertilization. Nuclear maturation refers to the events associated with
germinal vesicle breakdown and the resumption of meiosis and ending with arrest at
metaphase II. Cytoplasmic maturation involves processes to prepare for oocyte activation
[10, 11]. These processes include the coordination and movement of cytoskeletal filaments
and redistribution of cytoplasmic organelles, as well as molecular maturation involving
accumulation, transcription, and processing of preformed mRNAs [12]. A few of the main
3
transcripts produced during this period include glutathione, ATP, and cell cycle regulators
maturation promoting factor (MPF), c-mos pro-oncogene (MOS), and mitogen-activated
protein kinase (MAPK). To support in vitro maturation of bovine oocytes, most laboratories
use TCM199 medium supplemented with 10% estrus cow serum or fetal calf serum,
estradiol, follicle stimulating hormone, and luteinizing hormone. Maturing oocytes are
typically incubated at 5% CO 2 at 38.5°C for 20-24 hours [13].
Prior to IVF, thawed frozen spermatozoa selected by using either a Percoll density
gradient with centrifugation, or a swim-up procedure [10]. Capacitation is accomplished by
inclusion of hyaluronic acid or heparain in the fertilization medium [10, 14]. Sperm are
coincubated with matured cumulus-oocyte complexes for 18 to 20 hours [15]. Following
fertilization co-culture, the cumulus cells are removed from the presumptive zygotes.
Presumptive zygotes are cultured until the blastocyst stage, approximately 168-192
hours post-insemination [16]. Media used for post-fertilization culture contain nutrients and
growth factors, and can range from undefined medium such as TCM199 supplemented with
serum, to semi-defined medium such as synthetic oviductal fluid (SOF) supplemented with
bovine serum albumin (BSA), to fully defined medium such as KSOM or SOF, in which all
components comprising the medium are known [10, 17]. Following culture, selected morula
or blastocyst stage embryos may be either transferred nonsurgically into recipients or frozen.
4
ASSESSING EMBRYO VIABILITY
The establishment of pregnancy and delivery of a live calf are the ultimate goals of an
embryo transfer program. A critical step in embryo transfer is the evaluation and selection of
suitable embryos either recovered from donor cows or harvested from in vitro embryo
production. Embryo selection is an important factor that contributes to pregnancy success
following transfer of embryos produced in vivo or in vitro.
Methods of Embryo Evaluation
Currently, there are several methods that may be used to estimate an embryo’s
viability. These include morphology, biopsy, differential staining, and assays of embryo
metabolism or respiration. The ideal assay of embryo viability should satisfy the following
criteria: 1) objective measurement, 2) non-invasive, 3) ability to distinguish individual or
multiple embryos at a time, 4) rapid, 5) technically simple and user-friendly, 6) affordable, 7)
highly predictable and 8) repeatable in all culture conditions [18, 19].
Visual assessment of embryo morphology is a commonly used method for evaluation
of bovine embryos prior to transfer. However, depending on the intended use of the
embryos, additional procedures such as embryo biopsy may be warranted. Embryo biopsy
and analysis of biopsied cells are technologies that allow for determination of genetic sex as
well as identification of genes associated with embryonic development, metabolism, and
other economically important traits.
5
Morphological evaluation of embryos using a stereomicroscope remains the most
widely used criterion for predicting embryo viability in cattle. The assessment of embryo
morphology involves assigning a stage of development and a quality grade to individual
embryos based on several observable characteristics. This method has the advantages of
being quick, non-invasive, and technically simple. In addition, it does not require extensive
equipment. However, visual evaluation of embryos is rather subjective and does require
training and experience in order to accurately assign the appropriate developmental stage and
grade.
When evaluating embryos, perhaps the most important morphological characteristic
to be considered is whether or not the embryo is at the appropriate stage of development
relative to the time post-fertilization. Systems used for evaluating embryos produced in vivo
or in vitro are based on deviations in morphology relative to embryo age and stage of
development. With these systems, age is based on days from estrus for in vivo derived
embryos or days from insemination for in vitro derived embryos.
Frequently used parameters for assessing embryo morphology include shape, color,
number of cells, compactness of the inner cell mass, number of extruded or degenerated
cells, number and size of vesicles, and the quality of the zona pellucida [20]. These
morphological parameters have been incorporated into embryo grading systems. During the
past three decades, several different ordinal scales have been used for grading bovine
embryos [20-24]. A widely used system for embryo evaluation is that recommended by the
International Embryo Transfer Society (IETS) [24]. The IETS system is based on formulated
6
guidelines for assigning each embryo a numerical code based on morphological integrity.
This system includes stage of development codes of 1 (unfertilized oocyte or 1-cell embryo)
to 8 (expanding hatched blastocyst), and quality grade codes of 1 (excellent or good) to 4
(dead or degenerating). The grading system is summarized in Table 1.1 [25]. It is
recommended that only excellent/good quality (Grade 1 or Code 1; zona pellucida intact)
embryos be used in international commerce [24].
Deviations in embryo morphology have been associated with lower pregnancy rates.
The proportion of recipients that become pregnant after transfer of fresh and frozen-thawed
embryos has generally ranged from 31-83% for in vivo produced embryos [21, 22, 26-28]
and 18-59% [26, 29-31] for in vitro produced (IVP) embryos; the wide range in values
reflects differences in embryo production method or culture, embryo quality and stage at
transfer, type of recipient, and farm or practitioner [32]. Higher pregnancy rates are usually
obtained when blastocysts are transferred before they begin to hatch from the zona pellucida
[33]. Wright [27] reported that higher pregnancy rates were obtained from the transfer of
blastocysts (64 to 66%) compared to the transfer of morulae (44 to 53%). However, other
investigators found no difference between pregnancy rates for groups of animals receiving
embryos of different stages of development (Table 1.2).
Embryo Quality and Pregnancy Rate
Multiple studies have demonstrated that there are significant differences in pregnancy
rates across different morphological grades [20-22, 27-31, 34]. In a retrospective analysis of
7
data from three commercial embryo transfer operations, embryo grade was identified as an
important factor in determining pregnancy rate [28]. Embryos of the highest grade were
associated with significantly higher pregnancy rates than embryos of lower grades. Similar
results, summarized in Table 1.2, have been reported by other investigators [22, 26-31].
Morphological evaluation is by no means a perfect predictor of pregnancy success.
Lindner and Wright (1983) observed that some embryos with a “poor” grade produced a
pregnancy, while other embryos of “good” quality failed to result in pregnancy [20].
Furthermore, visual evaluation of embryo quality is subjective and, thus, may be subject to
bias by the evaluator. A study of agreement on embryo stage of development and
morphological grade among experienced evaluators concluded that good agreement existed
when predicting embryo stage or the extremes of embryo grade [35]. However, evaluators
were in agreement less when selecting embryos of intermediate morphological quality.
Differences between experienced evaluators in selection of individual embryos for transfer
into recipients resulted in considerable variation in expected pregnancy rates [36].
Evaluation of In Vitro Produced Embryos
Compared to embryos produced in vivo, bovine embryos produced in vitro can be
more challenging to evaluate for embryo quality. Difficulties encountered when evaluating
in vitro produced embryos are likely due to their unique developmental and morphological
features. For example, Grisart et al. (1994) observed a delay in development rate of
presumptive zygotes cultured in vitro [37]. In general, the types of morphological defects
8
seen with in vitro produced embryos are similar to those of embryos produced in vivo.
However, compared to embryos produced in vivo, embryos produced in vitro have subtle
differences in gross and ultrastructural morphology [26, 38]. For example, morulae produced
in vitro have less pronounced compaction, variable amounts of coalescence of individual
blastomeres, a grainy cell mass, and less perivitelline space. Some embryologists have
reported that it is more difficult to discern extruded cells in the early blastocyst stages due to
a reduced perivitelline space [26, 33]. While in vivo produced embryos tend to be light
brown to brown in color and translucent at the blastocyst stage, in vitro produced embryos
may appear darker [20, 39]. The number of cells per embryo harvested from in vitro
production systems, however, has been similar to that of embryos produced in vivo [40]. In
vitro produced blastocysts of higher quality grades have more cells than blastocysts of lower
grades, and embryos that take longer to reach the blastocyst stage are of poorer quality [41].
At the ultrastructural level, embryos from in vitro co-culture systems have smaller and fewer
junctional complexes among the blastomeres [38]. In addition, blastomeres of in vitro
produced embryos may contain more cytoplasmic vacuoles [38].
When examining the ultrastructural morphometry of blastocysts, those produced in
vitro demonstrated deviations in volume densities in cellular structures involved in
metabolism as well as altered embryonic differentiation compared to those produced in vivo.
Blastocysts produced in vitro displayed decreased volume density of mitochondria and
nuclei, increased volume density of lipid, and increased proportional volume of vacuoles
[42]. Similarly, compact morula produced in vitro had increased volume density of lipid and
9
vacuoles, decreased proportional volume of total mitochondria, and increased cytoplasmicto-nuclear ratio compared to compact morula produced in vivo [43]. Differences also exist in
the extent of abnormal ploidy (chromosome number) amongst blastomeres of embryos
produced in vivo compared to their in vitro counterparts [44]. Whereas only 20-25% of
blastocysts from in vivo production systems demonstrate mixoploidy, approximately 45-70%
of in vitro produced blastocysts were determined to be mixoploid [45, 46]. It is important to
note that the incidence of mixoploidy detected was dependent on the method used to detect
chromosome number. Although embryos carrying increased proportions of mixoploid cells
can successfully establish and maintain pregnancy [44], severe increases in the number of
mixoploid blastomeres are not conducive to embryo survival [47].
Gene Expression in Bovine Embryos
The development of quantitative real-time PCR has allowed assessment of not only
qualitative aspects of gene expression but also quantitative changes in gene expression in
response to altered physiological states [48]. More recently, microarray technology has
emerged as a method to scan an entire genome for variations in mRNA levels associated with
a phenotype [49]. El-Sayed et al. [50] used microarray analysis to identify 52 genes that were
differentially expressed between IVP embryos that resulted in calf delivery following embryo
transfer compared to those that failed to produce a pregnancy. Biopsies from embryos that
resulted in calf delivery after embryo transfer had increased expression of mRNAs involved
in implantation (COX2, CDX2), carbohydrate metabolism (ALOX15), cell signaling (BMP15)
10
and signal transduction (PLAU). In contrast, embryos that did not result in pregnancy had
high levels of mRNA transcripts involved in inflammation (TNF), implantation inhibition
(CD9) and glucose metabolism (AKR1B1). In addition, they had higher levels of expression
for transcription factors involved in programmed cell death or tumor formation (MXS1,
PTTG1) [50].
A follow-up study investigated the gene expression profile of biopsies from in vivo
produced embryos based on pregnancy outcome following transfer to recipients [51]. There
was a high degree of similarity between transcript signatures of embryo biopsies resulting in
no pregnancy and pregnancy loss. Biopsies from embryos that resulted in no pregnancy were
enriched with transcripts related to tumor necrosis factor receptor binding (TNF), protein
binding (HSPD1), RNA binding (PAG2G4), calcium ion binding (S100A10 and S100A14),
and oxidoreductase activity (ND1, FL405, and AKR1B1). Highly expressed transcripts in the
pregnancy loss group also included those involved in oxidoreductase activity (FL405 and
FL396) and calcium ion binding (S1000A10), as well as structural constituent of ribosome
(RPS15A and RPL3), ATPase inhibitor activity (ATPIF1), protein binding (CD9 and
HSPD1), and nucleotide binding (EFF1A1). They reported 41 differentially expressed genes
(DEGs) between biopsies resulting in no pregnancy and those resulting in calf delivery. For
pregnancies resulting in calf delivery, transcripts involved in protein binding (KRT8 and
ELOVL1), DNA binding (H3F3B and H2FA), structural constituent of ribosome (RPLP0),
placenta specific (PLAC8), growth factor activity (BMP15), and signal transducer activity
(RGS2) were upregulated in embryo biopsies. There were 43 DEGs between embryo biopsies
11
resulting in pregnancy loss between Day 28 and Day 42 compared to calf delivery.
Furthermore, the authors compared the results of this study [51] with their previous study
[50] in which biopsies from IVP embryos were assessed. There were 21 DEGs between
biopsies resulting in no calf delivery versus calf delivery for both in vivo and in vitro derived
embryos [51]. Of those, 18 showed the same trend of gene expression for both embryo
production methods, and are proposed to be associated with developmental competence
independent of environmental origin of the blastocysts. These results contradict other studies
that report major differences in the global gene expression profile between in vivo and in
vitro produced embryos; however, these biopsy studies differentiated between groups of
embryos with different developmental outcomes. The high signature similarities between in
vivo and in vitro derived embryos resulting in calf delivery suggest that differences in
expression profiles between embryos of different developmental competencies are more
consequential than differences arising as a result of embryo source.
Held et al. [52] sought to correlate mRNA expression patterns of isolated blastomeres
from 2-cell in vitro produced bovine embryos with the developmental competence of their
sister blastomere. Using microarray analysis, 77 genes were identified as differentially
regulated between developmentally competent (developed to the blastocyst stage) and
incompetent blastomeres. Competent sister blastomeres expressed elevated levels of mRNAs
associated with oxidative stress responses, antioxidant activity and oxidative phosphorylation
[52]. Several other studies, summarized in Table 1.3, have reported candidate genes whose
12
transcripts or products may be involved in embryo survival or that are differentially regulated
between in vivo and in vitro produced embryos [53-63].
The transcriptome of embryos at various stages of development has been associated
with both pre- and post-transfer developmental outcome [50, 52, 64]. In the future, specific
mRNA signatures for embryos destined to exhibit specific abnormal offspring syndrome
(AOS) subtypes following embryo transfer may also be identifiable. This type of analysis
will support the development of pre-transfer embryo screening procedures. Furthermore, in
vitro embryo production methods could be altered to eliminate the occurrence of AOS. For
example, inclusion of specific supplements to medium used for production of bovine
embryos in vitro, including IGF1, CSF2 and hyaluronan, were demonstrated to improve
pregnancy rate post-transfer [65].
PREGNANCY RECOGNITION AND EARLY CONCEPTUS DEVELOPMENT
Maternal Recognition of Pregnancy
It is estimated that moderate-producing dairy cows experience about a 40%
embryonic and fetal mortality rate; about 70-80% of this loss is sustained between Days 8
and 16 of gestation [2, 3]. Maternal recognition of pregnancy in cattle occurs around Day 16
of gestation and requires interactions between the uterus and the conceptus which must
prevent the luteolytic cascade and prolong the lifespan of the corpus luteum (CL). Pregnancy
recognition signals from the trophoblast of the preimplantation conceptus act in a paracrine
manner to prevent the pulsatile release of prostaglandin F2α (PGF) from the endometrium
13
which would otherwise cause the regression of the CL [66, 67]. The agent of maternal
recognition of pregnancy in ruminants is interferon-tau (IFN-τ); its expression is
developmentally regulated up to the period of pregnancy recognition.
Tau interferons are a subclass of the omega interferons and members of the Type I
IFN family; IFN-τ genes have anti-viral and anti-proliferative activity [68]. IFN-τ is
produced by mononuclear cells of the embryonic trophectoderm and exerts an antiluteolytic
effect on the endometrium. IFN-τ acts by directly preventing the transcription of the estrogen
receptor gene and indirectly blocking transcription of the oxytocin receptor gene in the
luminal epithelium and superficial glandular epithelium of the endometrium. As the release
of PGF occurs in response to oxytocin binding its receptor on the endometrium, the absence
of the oxytocin receptor results in the prolonged lifespan of the CL and continued production
of progesterone [69-71].
Bovine IFN-τ secretion begins as early as Day 7 of pregnancy; secretion peaks around
Day 17 (around the time of maternal recognition of pregnancy), and can be detected as late as
Day 25 [72, 73]. At peak expression, IFN-τ mRNA is the most highly concentrated mRNA
present in the developing conceptus [68]. IFN-τ production by the conceptus is positively
associated with maternal plasma progesterone concentration [74]. Initiation of IFN-τ
transcription occurs independently of the maternal uterine environment, as IFN-τ is
expressed in blastocysts following in vitro fertilization and maturation [68]. Transcription
factors such as Ets-2, AP-1, ras/MAPK, Cdx2, and the growth factor GM-CSF have been
implicated in activating IFN-τ gene expression [68, 75, 76]. IFN-τ expression is terminated
14
following implantation, as evidenced by the fact that ovine IFN-τ expression stops in regions
where the trophoblast connects to the uterine epithelium [71].
In addition to its antiluteolytic effects, IFN-τ can influence uterine receptivity and
conceptus elongation through a paracrine manner by stimulating the expression of several
IFN-τ-stimulated genes (ISGs) in the endometrium. IFN-τ binds to the Type I IFN receptor
on the endometrium and activates the JAK-STAT-IRF signalling pathway used by other
interferons in the Type I family [66]. Expression of ISGs generally declines as IFN-τ
expression declines. A study by Forde et al. [77] used RNAseq to identify 402 differentially
expressed genes in the endometrium between pregnant and cyclic heifers, several of which
are regulated by IFN-τ expression in vivo. Their results suggested that Type 1 IFN signaling
(IFN-τ secretion) was primarily responsible for the changes in the transcriptome between the
pregnant and nonpregnant animals. They hypothesized that as IFN-τ mRNA can be detected
at the blastocyst stage, the embryo may have effects on the endometrium that have yet to be
discovered.
Normal Conceptus Development
Elongation of the conceptus is associated with increased secretion of IFN-τ; therefore
conceptuses must reach adequate size to produce sufficient quantities of IFN-τ to prevent
luteolysis [78]. Elongation in bovine pre-implantation embryos begins as early as the
spherical blastocyst on Day 7 of gestation [79]. The conceptus transitions through
intermediate ovoid (Day 12-13) and tubular (Day 14-15) forms to reach the filamentous (Day
15
16-17) conceptus form that will begin implantation around Day 19-20 of gestation [79].
Several studies have reported on conceptus length at these stages [79-85]. Although
individual size can vary greatly, spherical embryos measure approximately 150µM, ovoid
embryos range from 1-10mm in length whereas tubular and filamentous embryos range from
30-60mm and 100-300mm in length, respectively. Elongation is initiated in trophoblast cells
around Day 12 and these cells begin to elongate at Day 13 [78]. The entire process is
completed by Day 24 [78]. During the period of elongation, the conceptus increases in size
more than 1000-fold [82, 83]. Protein content also increases with increased conceptus surface
area [82, 86]. A large increase in IFN-τ gene expression occurs on Day 15 and is due to the
transition of the conceptus to an elongated, tubular form [73].
Upon hatching from the zona pellucida around Day 8-9, developing embryos have a
trophectoderm comprised of cuboidal cells connected by tight junctions and desmosomes, as
well as a well-defined inner cell mass (ICM) that is encased by a thin layer of trophoblast
cells, called the Rauber's layer [87]. This layer is disrupted by adjacent underlying ICM cells
which form tight junctions around Day 11; the hypoblast forms a thin cell layer lining the
ICM and trophoblast. Around Day 12 the epiblast (originally the ICM) displaces the Rauber's
layer and establishes the embryonic disc, though the embryonic disc remains relatively
constant in size [80, 86, 87]. By Day 14, the embryos are ovoid to tubular and tight junctions
and desmosomes are present between the adjacent epiblast cells, as well as between epiblast
and trophoblast cells [87].
16
The embryo is relatively autonomous up to the blastocyst stage and does not require
contact with the maternal system. Blastocysts can be developed in vitro and are routinely
transferred to synchronized recipients. However, the process of elongation is maternally
driven, as evidenced by the fact that elongation does not occur in vitro or in vivo in the
absence of uterine glands [88, 89]. Elongation is dependent upon endometrial secretions,
termed histotroph, present in the uterine lumen. In addition to secreting proteins for maternal
recognition of pregnancy, elongating conceptuses produce proteins that stimulate spatiotemporal alterations in gene expression in the endometrium necessary for conceptus survival
and implantation. This includes suppressing the immunological rejection of the conceptus by
the uterus around the time of implantation [90]. It is possible that the absence, or temporal
delay, of certain components of histotroph could be responsible for pregnancy loss during
this peri-implantation period.
Understanding the molecular signals associated with endometrial receptivity and
embryo implantation is critical to improving pregnancy success. Many studies have focused
on gene expression of the Day 7 blastocyst, or gene expression in the maternal endometrium.
Relatively few studies have examined gene expression in the elongating conceptus,
particularly on a global scale [59, 78, 84, 91-94]. Machado et al. [84] compared Day 7 and
Day 14 bovine embryos produced either in vivo or in vitro. They detected higher expression
levels of three genes out of eight studied in Day 7 IVP embryos compared to Day 7 in vivo
produced embryos. Interestingly, these differences were not observed at Day 14, potentially
suggesting that differences originating from production methods were minimized for these
17
transcripts following exposure to the maternal environment. Ushizawa et al. [94] utilized a
custom utero-placental cDNA microarray to examine gene expression between Day 7, Day
14, and Day 21 bovine embryos. They reported that 680 genes were upregulated and 26 were
downregulated between Day 7 and Day 14; 452 were upregulated while 2 were
downregulated between Day 14 and Day 21. Genes affected included transcriptional
regulators, oncogenes, those involved in tumor suppression, cell cycle control and apoptosis.
Mamo et al. [93] utilized RNA sequencing to characterize the changes in the transcriptome of
Day 7, 10, 13, 16, and 19 bovine embryos. They reported nine gene clusters with stagespecific expression profiles, including those upregulated at Day 16 and Day 19 likely to be
involved in maternal recognition of pregnancy and implantation. Hue et al. [80] compared
molecular datasets for differentially expressed genes during conceptus elongation from
Ushizawa et al. [94] and Mamo et al. [93], as well as from their own laboratory, and found
that there were four differentially expressed genes common to all three studies. These were
IGFBP7, PAG2, PAG11, and TKDP3. They also reported that the Ingenuity Pathways
Analysis top functions of the genes from these combined datasets examining the pre- and
peri-implantation periods included "lipid metabolism", "connective tissue development and
cellular assembly," "cellular growth and differentiation," and "inflammatory responses." It is
also of note that one study reported a major X-chromosome inactivation between blastocyststage embryos and Day 14 conceptuses; this reduction in the transcriptional sexual
dimorphism of several autosomal genes indicates this early elongating period is also
susceptible to sex-specific embryo loss [95].
18
PROGESTERONE
Patterns of Progesterone Concentration and Early Conceptus Development
Progesterone is a steroid hormone secreted by the corpus luteum in cattle. Continued
secretion of luteal progesterone is critical for the establishment of pregnancy. Maternal
recognition of pregnancy relies on sufficient production of IFN-τ by the embryo to override
luteolysis; embryos more advanced in development or size are able to produce greater
amounts of IFN-τ thereby ensuring continued production of progesterone needed to establish
and maintain pregnancy. Early studies focused on the relationship between circulating
progesterone concentration early in the luteal phase and pregnancy rate. Niemann et al. [96]
studied the relationship between recipient plasma progesterone levels and pregnancy rates in
cattle following embryo transfer. They found that the average progesterone concentration at
Day 7 following estrus across all recipients was 3.21 ± 0.19 ng/mL. They also discovered
that pregnancy rates tended to be higher when plasma progesterone was between 2 and 5
ng/mL, with the highest pregnancy rates (63.2%) achieved with progesterone concentrations
between 2 and 3 ng/mL. Interestingly, that research group reported that pregnancy rates were
lower when progesterone levels fell below 2 ng/mL (35.3%) or above 5 ng/ml (28.6%).
Similarly, cows with a progesterone concentration <5ng/mL on Day 14 were more likely to
lose the pregnancy between Days 28 and 42 [97]. These results suggested that there may be
an optimal range of progesterone concentration at Day 7 that is conducive to pregnancy.
Stronge et al. [98] compared the concentration of milk progesterone on Days 5, 6, and 7 and
19
embryo survival. Optimal embryo survival rates occurred when milk progesterone
concentration was 7.4, 13.2, and 16.8 ng/mL on Days 5, 6, and 7, respectively.
The rate and timing of the post-ovulatory rise in progesterone concentration is also
important in influencing fertility and embryo development. Mann and Lamming [99]
investigated the pattern of circulating progesterone concentration in cows daily from Day 1
after insemination until slaughter at Day 16. They reported that cows without an embryo at
Day 16 underwent a significantly delayed increase in progesterone concentration as
compared to those with an embryo. The overall concentration of progesterone during this
time was also lower (>1 ng/mL difference) in cows without an embryo as compared to those
with an embryo at Day 16. This study further related the pattern of progesterone
concentration to embryo development assessed based on IFN-τ secretion. Cows with poorly
developed embryos, that is, having little to no IFN-τ production, also displayed a delayed
increase in progesterone concentration and an overall lower concentration of progesterone
compared to cows with well developed embryos. They concluded that maternal recognition
of pregnancy relies, at least in part, on an appropriate pattern of progesterone concentration
from ovulation to Day 16. A similar study in sheep found that lower concentrations of
progesterone in the early luteal phase resulted in delayed conceptus development and less
secretion of ovine IFN-τ [100]. Kenyon et al. [97] reported that the fold change in
progesterone concentration from Day 0 to 7 and from Day 7 to 14 was associated with
pregnancy status on Day 63. In fact, among cows pregnant at Day 63, the minimum fold
change in progesterone concentration from Day 0 to 7 was 2.71; the minimum fold change
20
from Day 7 to 14 was 1.48. These results further confirm that a fast, early rise in
progesterone concentration is associated with pregnancy success following embryo transfer.
Mann et al. [101] further elucidated the effects of the timing of the post-ovulatory rise
in progesterone on early embryo development by supplementing cows either early (Days 5 to
9) or late (Days 12 to 16) and assessing embryo length and uterine IFN-τ concentration.
These authors demonstrated that early progesterone supplementation (Days 5 to 9) resulted in
a four-fold increase in embryo length and a six-fold increase in IFN-τ concentration
compared to supplementation later in the luteal phase (Days 12 to 16) or to nonsupplemented controls. In beef heifers, progesterone supplementation between Days 3 to 6.5
was also correlated with increased embryo survival [102]. Stronge et al. [98] also compared
the relationship between the rate of increase in milk progesterone from Days 4 to 7 and
embryo survival. The optimum rate of increase in milk progesterone during this period that
was conducive to embryo survival was 4.7 ng/mL per day.
Other studies have examined whether recipient serum progesterone concentration
influences embryo survival and size of in vivo or in vitro produced embryos. Lonergan et al.
[103] studied the effects of embryo source (in vivo vs. in vitro) and progesterone level of the
recipient (high vs. normal) on Day 7 on bovine embryo development at Day 13. Average
progesterone concentration differed between superovulated females, representing a high
progesterone environment, and non-superovulated (control) females from Day 3 to Day 13.
They reported that progesterone environment had a significant effect on embryo size at Day
13. Embryos from high progesterone recipients had a significantly larger mean area (3.86 ±
21
0.45 mm2) than those recovered from the control progesterone recipients (1.66 ± 0.38 mm2).
Furthermore, the progesterone environment an embryo was exposed to prior to Day 7
influenced embryo size at collection, as in vivo produced embryos derived from
superovulated donors had a larger average area compared to in vitro produced embryos that
were not exposed to progesterone prior to transfer. However, progesterone environment from
Day 7 to Day 13 also appeared to influence embryo size at Day 13. When in vivo produced
embryos derived from superovulated donors were transferred into high progesterone
recipients they attained a greater average area compared to those transferred into controllevel progesterone recipients. Carter et al. [104] investigated the effect of supplemented
progesterone (using a PRID) starting from Day 3 on bovine embryo development at Days 5,
7, 13, and 16. In that study, progesterone supplementation did not influence embryonic
survival or development to the blastocyst stage. However, the proportion of viable embryos
recovered in the high progesterone group was numerically greater compared to controls on
Day 13 and on Day 16. Furthermore, progesterone supplementation was associated with
increased embryo length on Day 13 and Day 16, coincident with the period during which the
conceptus is elongating. Similarly, enhanced conceptus size following progesterone
supplementation has also been reported for Days 14 [105] and 16 [101].
Mechanism of Progesterone Effects
Progesterone exerts its effect on embryo development indirectly through
progesterone-regulated changes in endometrial gene expression. Clemente et al. [106]
22
established that progesterone was acting indirectly via the endometrium and was not acting
directly on the embryo itself. Although progesterone receptor mRNA was detected in the
embryo following the morula stage, addition of progesterone to the culture medium of in
vitro produced embryos did not affect blastocyst development in vitro nor following transfer
to recipients [106]. However, progesterone supplementation of recipients prior to transfer of
in vitro produced embryos at Day 7 significantly increased conceptus length and area at Day
14 compared to those recovered from control recipients [106]. Therefore, the embryo does
not need to be present in the uterus during the time of early progesterone exposure to receive
the benefits.
Forde et al. [107] investigated the effects of elevated progesterone on endometrial
gene expression in early pregnant heifers as well as the temporal changes in this expression
using Affymetrix Bovine GeneChips. Elevated progesterone was associated with 36
differentially expressed genes (DEGs) on Day 5 and 124 DEGs on Day 7. In contrast, there
were only a few DEGs on Days 13 and 16. Based on results comparing gene expression
between treatment groups at different stages of development, they concluded that elevated
progesterone advanced endometrial gene expression. In another study by the same research
group, a low progesterone model was created by administering PGF 2α on Days 3, 3.5, and 4,
and was used to study the effects of low progesterone concentration on endometrial gene
expression and conceptus development [108]. After characterizing changes in gene
expression that occurred during the luteal phase, they examined the effects of low
progesterone on expression of selected transcripts. Forde et al. [108] reported that the
23
induction of low progesterone delayed the normal temporal changes that occurred in this
time. Conceptus length and area at Day 14 was significantly decreased in low progesterone
recipients compared to controls. Based on microarray analysis of endometrial gene
expression, low progesterone affected the expression of 498 DEGs on Day 7 and 351 DEGs
on Day 13 [109]. Furthermore, based on comparison to related studies, it was found that the
expression of genes likely to be involved in conceptus elongation were downregulated in
recipients with low progesterone on Day 13 compared to controls. Taken together, the result
of these two studies found a significant number of endometrial genes affected by altered
progesterone concentration, whether progesterone is artificially elevated (4157 DEGs) [108]
or is artificially lowered (809 DEGs) [109], as compared to the temporal changes that occur
during a normal estrous cycle.
A candidate gene approach has been used to identify and characterize genes that are
up-regulated in the uterine endometrium at the initiation of elongation (Day 13) and that
encode for secreted proteins present in the histotroph [110]. Both circulating progesterone
concentration and the presence of a conceptus altered the expression of 5 genes in the
endometrium, with several of the corresponding proteins identified as present in the
histotroph. Those genes were PLIN2, TINAGL1, NPNT, LCAT, NMN and APOA1. It is
likely that they may play a role in conceptus elongation.
Collectively, information on the role of progesterone in conceptus survival and
elongation support the concept that an optimal uterine environment must exist for survival
and elongation of the conceptus. However, the developing conceptus also plays a role in
24
influencing the spatio-temporal changes in endometrial function. Evidence exists that the
endometrium can sense and respond differently to distinct types or qualities of embryos
present. This supports the suggestion that signals from different embryos may vary and can,
in turn, elicit different responses from the endometrial transcriptome [111, 112].
Understanding the dialogue at the embryonic and maternal interface is of upmost importance
in understanding endometrial receptivity and conceptus survival.
ABNORMALITIES ASSOCIATED WITH IN VITRO EMBRYO TECHNOLOGIES
The transfer of in vitro produced embryos or somatic cell nuclear transfer (cloned)
embryos may result in abnormalities of fetuses, placentas and calves. In cattle and sheep,
these abnormalities were initially recognized as oversize offspring and were referred to as
Large Offspring Syndrome. In addition to large phenotypes, other problems have been
reported including increased rates of early embryonic death and abortion, extended gestation
lengths, abnormal placentas as well as increased rates of hydrallantois, congenital
deformities, and perinatal death [26, 34, 113-116]. Fetuses and placentas from in vitro
produced embryos may range from normal development to subtle abnormalities such as
abnormal development of fetal skeletal muscle or placental blood vessels, to more obvious
problems such as increased body weight. However, because all of these pregnancies do not
result in fetal or placental overgrowth, the term Abnormal Offspring Syndrome (AOS) was
proposed to more accurately denote the range of characteristics seen with this syndrome
[117].
25
In cattle, the expression of anomalies associated with AOS can range from changes in
gene expression in preimplantation-stage embryos [118] to moderate alterations with little
apparent phenotypic compromise [119] to severe alteration in phenotype accompanied by
abortion or neonatal death (Figure 1.1) [117, 120, 121]. Expression of AOS is influenced by
culture conditions [122-124], species [125], extent of embryo manipulation [126, 127] and
even levels of maternal nutrients [128]. Characteristics associated with AOS may be grouped
into four types [117]. Briefly, Type I AOS represents early embryonic or conceptus death
prior to completion of organogenesis at approximately Day 42 of gestation. Type II AOS
represents abnormal development of the placental membranes and fetus between completion
of organ differentiation and full term (Day 42 to approximately Day 280 of gestation) as well
as fetal death during this period. Type III AOS represents a full-term fetus or placenta with
severe developmental abnormalities and no evidence of compensatory response by the fetus
or placenta. Newborn calves are often severely compromised with altered clinical,
hematological, or biochemical parameters, with death occurring at parturition or during the
neonatal period. In Type IV AOS a full term fetus or placenta with moderate abnormalities is
observed; however, the feto-placental unit has compensated and adapted to the compromising
genetic or physiological insults and the fetus survives. These calves may be normal or they
may have clinical or biochemical abnormalities.
26
Pre-implantation Development
Compared to AI or the transfer of in vivo-produced embryos, in vitro produced
embryos can experience even greater rates of early embryonic death; however, this can be
influenced by the quality grade of the embryo transferred. For example, survival rates for
bovine embryos of quality grade 1 were the same whether derived from in vivo or in vitro
production systems; however, for embryos of quality grade 2, survival rates were lower if
from in vitro production systems [34]. As described previously, there are morphological
differences between in vivo and in vitro produced preimplantation embryos. Pregnancy rates
and calving rates following the transfer of IVP embryos vary both across and within
laboratories, although they can be lower than those seen following artificial insemination
(AI) or the transfer of in vivo-produced embryos [26, 36, 129-132]. Other factors such as
embryo evaluator [35] and recipient characteristics [131] can also influence the successful
establishment and maintenance of pregnancy following transfer of non-frozen in vivo and in
vitro produced bovine embryos. Early comparisons of gene expression for bovine
preimplantation embryos produced both in vivo and in vitro demonstrated that expression of
both imprinted and nonimprinted genes can be affected by in vitro embryo production [55,
57, 59, 60, 91, 133-137].
Peri-implantation Development
It is estimated that moderate-producing dairy cows experience about a 40%
embryonic and fetal mortality rate; about 70-80% of this loss is sustained between Days 8
27
and 16 of gestation [2]. Maternal recognition of pregnancy occurs around Day 15-16 of
gestation in cattle, and requires adequate expression of interferon-tau (IFN-τ) from the
developing conceptus to prevent the luteolytic cascade [73, 138]. Higher rates of pregnancy
loss occurred for conceptuses around the peri-implantation period following the transfer of in
vitro produced (IVP) embryos [1, 91, 139, 140]. Although a portion of this loss could be
attributed to difficulties in recovering these conceptuses, significant embryonic loss has been
reported in the two-week period following the transfer of IVP and somatic cell nuclear
transfer (SCNT; cloned) embryos. For example, recovery rates of 51.2% were reported at
Day 12 of gestation but only 33% to 38% of the IVP embryos transferred were recovered at
Day 14 [91, 141]. Another study reported that at Day 14, 75% of IVP embryos transferred
were not recovered (from 120 embryos transferred), compared to 85% to 93% for SCNT
embryos (from 102 and 195 embryos transferred, respectively) [140]. At Day 25 of
gestation, only 19% of conceptuses derived from IVP and 15% of conceptuses from cloned
embryos were recovered compared to 67% of conceptuses produced by AI [142].
In addition to lower recovery rates, conceptuses derived from IVP embryos also
exhibit some morphological differences compared to those derived in vivo. IVP conceptuses
were longer than in vivo produced conceptuses both at Day 12 [91] and Day 17 [139] of
gestation. Sawai et al [85] failed to detect a significant difference in trophoblastic length
between in vivo and SCNT conceptuses at Day 15. Betsha et al. [143] reported comparable
recovery rates at Day 16 following the transfer of in vivo produced, in vitro produced, and
SCNT embryos. However, in that study SCNT embryos were considerably shorter than their
28
in vivo or in vitro produced counterparts. In contrast, Machado et al. [84] did not detect any
significant differences in conceptus length at Day 14 between those conceptuses derived
from IVP or in vivo produced embryos. Furthermore, other investigators found no statistical
difference in the recovery rate of conceptuses on Day 14 [84] or of intact conceptuses on Day
16 [59] between IVP and in vivo groups. Bertolini et al. [59] did report an increase in total
structures or degenerated non-hatched embryos recovered on Day 16 in their IVP group
compared with in vivo controls. In that study, conceptuses derived from IVP embryos had
smaller embryonic discs and tended to have shorter trophoblastic lengths compared to in vivo
produced controls. Differences in conceptus lengths between studies could be attributed to
differences between in vitro conditions used to produce embryos, the number of embryos
transferred per recipient, as well as to the timing of recovery relative to maternal recognition
of pregnancy. Contrasting results indicate more research is needed to determine whether
embryo source is a significant factor in conceptus growth.
The presence of an embryonic disc is another important feature to consider, as
recovery rate is generally higher when an embryonic disc is detected with no signs of
conceptus degeneration [141]. One study detected an embryonic disc in roughly 25% of
conceptuses recovered at Day 14, with no significant differences in the proportion of discs
detected between IVP and in vivo produced groups [84]. Compared to conceptuses derived
from the transfer of IVP embryos, 20% fewer conceptuses from SCNT embryos had an
embryonic disc at recovery on Day 17 [92]. Other studies have observed similar percentages
of peri-implantation conceptuses with embryonic discs [59, 141]. Culture conditions can also
29
influence the presence of an embryonic disc in developing conceptuses; one study reported a
higher percentage of embryonic discs present when KSOM medium (72%) was utilized as
compared to SOF medium (46%) [141].
Peri-implantation conceptuses derived from IVP or SCNT embryos may have
different developmental and ultrastructural properties [140]. SCNT conceptuses were
developmentally delayed at Day 14 and Day 21 when compared to the expected
developmental timeline described for in vivo conceptuses [140]. When comparing
conceptuses from IVP- and SCNT-derived embryos, SCNT conceptuses at both Days 14 and
21 of gestation were shorter in length and displayed more ultrastructural deficiencies at both
time points compared to IVP conceptuses [140]. For example, SCNT conceptuses exhibited
a greater occurrence of apoptosis, more vacuoles, fewer mitochondria in the hypoblast,
disintegration of the basement membrane between the hypoblast and epiblast, less defined
tight junctions between trophectoderm cells, fewer desmosomes and fewer polyribosomes.
Differences in gene expression have been observed between conceptuses derived
from the transfer of IVP, SCNT, or in vivo produced embryos [59]. For example, in vivo
control embryos showed a temporal reduction in transcriptional activity from Day 7 to Day
16 that was not seen during this period in IVP embryos. Furthermore, on Day 16 of gestation
conceptuses from IVP derived embryos had greater expression of IGF1R mRNA transcript
compared to in vivo controls [59]. While Day 7 IVP blastocysts had a lower relative level of
IFN-τ transcript compared to in vivo produced controls, developmentally delayed Day 16
IVP conceptuses had greater content of IFN-τ mRNA [59]. Global transcriptome analysis
30
found 365 DEGs between Day 16 conceptuses derived from embryos produced in vitro
compared to artificial insemination [143]. Genes involved in metabolic pathways were
upregulated in IVP conceptuses, whereas those involved in tight junctions, agrin interaction
at neuromuscular junction, and TNRF-1 signally were significantly downregulated compared
to AI controls.
Betsha et al. [143] also reported 477 DEGs between Day 16 conceptuses derived from
SCNT and AI embryos. Of the 274 genes commonly altered in conceptuses from IVP and
SCNT embryos, 204 transcripts had a lower expression compared to AI controls. Genes
affected included those involved in tight junction signaling pathways, small molecules
biochemistry, carbohydrate and lipid metabolism, as well as DNA replication,
recombination, and repair. These results highlight the influence of pre-elongation culture
conditions on embryo development. Compared to IVP conceptuses, conceptuses derived
from the transfer of SCNT embryos lacked expression of NANOG and FGF4 and
overexpressed IFN-τ mRNA at Day 17 of gestation [92]. Using a custom microarray, 47
genes were identified as differentially expressed at Day 17 between conceptuses from SCNT
and IVP embryos. Furthermore, expression of genes involved in trophoblast proliferation
(Mash2), differentiation (Hand1) and trophoblast function (PAG-9) were altered in SCNT
conceptuses at Day 17 compared to conceptuses from either IVP or AI [144]. Conceptuses
derived from SCNT embryos had significantly lower expression of IGFBP-3 mRNA at Day
15 compared to in vivo controls [85]. At Day 25 of gestation, conceptuses derived from IVP
or SCNT embryos had a greater expression of IGF-I mRNA compared to AI controls. In
31
addition, conceptuses derived from SCNT embryos had greater expression of IGF-II mRNA
than IVP-derived conceptuses [142]. Interestingly, gene expression profiles from uterine
endometria of pregnancies derived from SCNT and IVP embryos differ [111]. These
observations support the suggestion that during the peri-implantation period, abnormal
embryo-maternal communication could be responsible for the altered phenotype in cloned
animals. In effect, altered expression of genes vital to normal trophoblast differentiation and
subsequent placental development associated with IVP and SCNT may be factors
contributing to increased pregnancy losses and other abnormalities observed later in
development.
Fetal Development
Development of fetuses following the transfer of IVP or SCNT embryos is associated
with increased fetal mortality and a multitude of fetal abnormalities. Following the period of
embryonic loss in cattle, fetuses derived from IVP or SCNT embryos experience higher rates
of gestational loss compared to those produced in vivo (Figure 1.2). A large scale study
analyzing results from over 30 data sets on IVP and SCNT reported that fetal losses were
higher for both IVP and SCNT embryos as compared to AI or ET pregnancies, even after
adjusting for effects such as season, parity and sex of calf [129]. Furthermore, IVP and
SCNT calves experienced longer gestation lengths, higher birth weights and increased
incidences of dystocia and perinatal losses compared to AI or ET calves.
32
Higher rates of pregnancy loss following the transfer of IVP embryos are seen during
the late embryonic period (Day 30) and early fetal period (up to Day 90) of gestation
compared to in vivo controls [117]. While the rate of pregnancy loss from approximately 2
months of gestation to term is less than 10% in recipients carrying in vivo-produced embryos
[145-147], this loss can be two to three times greater in recipients carrying IVP embryos [26,
148-150]. In addition to greater early embryonic losses, IVP pregnancies experience fetal
losses, particularly between Days 30 and Day 65 of gestation, with one study reporting the
majority of the losses occurring between Days 30 and 44 [150]. By comparison, pregnancies
derived from embryos produced in vivo had lower rates of loss that were distributed more
evenly between Days 30 and 90 [150]. Furthermore, pregnancy rate at Day 60 of gestation
was 42% lower in recipients with Grade 1 IVP embryos as compared to those with Grade 1
in vivo produced embryos [34]. Pregnancy rate at Day 50-60 of gestation following the
transfer of Grade 1 fresh IVP embryos (59%) was significantly lower compared to that of
Grade 1 fresh in vivo produced embryos (76%) [26]. Based on data from a large scale study,
pregnancy rate between Days 90 and 150 of gestation was similar for both fresh and frozen
IVP embryos (43.5%) [151].
The occurrence of early fetal mortality is even greater following the transfer of cloned
embryos. Losses of 50% to 100% of pregnancies from cloned embryos can occur between
days 30 and 60 of gestation, compared to a 2% to 10% loss of pregnancies from natural
service that normally occurs during this period [120, 126, 152, 153]. A second period of high
pregnancy loss occurs during the second trimester of pregnancy for cloned pregnancies [120,
33
126, 154]. In many of these cases, while few gross abnormalities may be noted in the
aborted fetuses, the placentas are often grossly abnormal [120, 126, 154]. Cloned
pregnancies may also be subjected to greater losses during the third trimester, often
characterized by placental abnormalities including hydrallantois, reduced placentome number
and edema in the placental membranes [120, 126]. Based on a nationwide survey in Japan on
the survival of embryos and calves derived from SCNT from 1998 to 2007, the survival rate
of SCNT embryos and calves had not improved during that 10 year period [155]. Pregnancy
rates at Day 30 following the transfer of SCNT embryos ranged from 21.6-32.9% and were
lower compared to pregnancy rates following the transfer of in vivo produced (50-52%) and
IVP embryos (37-46%) [155].
In addition to an increased incidence of fetal mortality, fetuses derived from IVP and
SCNT embryos exhibit a variety of morphological abnormalities during early gestation.
Compared to AI controls, SCNT and IVP fetuses at Day 80 of gestation had increased body
weight, liver weight and thorax circumference [156]. Furthermore, these investigators found
that the ratio of crown-rump length to thorax circumference was a defining morphological
characteristic for the phenotype of disproportionate fetal overgrowth. In cloned fetuses,
decreased crown rump length and/or fetal heart beat were predictive ultrasound markers for
fetal death that occurred in about 27% of clones that died prior to Day 90 of gestation [157].
These investigators noted, based on ultrasound assessments after Day 100 of gestation, that
risk factors for loss of SCNT-derived fetuses included placental edema, hydrops and
increased abdominal circumference.
34
The frequency of congenital malformations, which include hydroallantois and
abnormal limbs, is increased in calves from IVP embryos (3.2%) compared to AI controls
(0.7%) [151]. At Day 90 of gestation, IVP pregnancies were associated with increased fetal
crown-rump lengths and organ weights compared to in vivo controls [158]. At Day 180 of
gestation, IVP pregnancies were associated with greater uterine, placental and fetal weights
compared to in vivo controls. Fetal weight, fetal liver, fetal heart, and placentome weight
were all correlated and were significantly greater in IVP pregnancies [158]. At Day 222 of
gestation, fetuses from IVP embryos were 17% larger than in vivo-produced controls and
exhibited significant increases in fetal body weight, heart girth, heart weight and long-bone
length [34]. Furthermore, fetuses from IVP embryos exhibited no difference in gross visceral
organ weight but had smaller skeletal muscle measurements per kilogram body weight
compared to fetuses from the IVO group, suggesting that IVP fetuses develop
disproportionately to their body weight. Histologically, IVP fetuses at Day 222 of gestation,
exhibited a ratio of secondary-to-primary skeletal muscle fiber number that was significantly
greater than that for in vivo-derived controls. Similarly, the proportional volume of tissue
present between myofibrils was also greater in muscle of IVP fetuses compared to controls
[159]. Expression of myostatin mRNA was decreased in the skeletal muscle of fetuses from
the IVP group, while expression of GAPDH tended to be increased as compared to the IVO
group. These observations are consistent with the observed changes in muscle fiber ratios
since myostatin is an inhibitor of muscle fiber development and GAPDH is a key enzyme in
glycolysis - a pathway that is heavily used by secondary muscle fibers [159].
35
Perinatal mortality rates of 8.6% to 15.6% from IVP calves at birth or shortly after
birth were reported in recipient heifers and were as high as 17.9% for recipient cows [26,
149, 151]. In contrast, perinatal mortality rates in AI calves was approximately 6.2% to 9.0%
for in vivo and ET pregnancies [151, 160]. Some studies have reported an increased gestation
length for IVP fetuses [151] whereas others have not [161]. The incidence of stillbirth and
perinatal mortality within the first 48 hours post partum was increased in IVP calves when
compared to in vivo derived controls but was not due to altered gestation length [162].
Stillbirth and postnatal mortality were also increased in SCNT pregnancies. Only about 70%
of cloned cattle survive the perinatal period and these losses are generally sustained within
the first week of postnatal life (reviewed in [163]). Based on the 2012 national survey by the
Japanese Ministry of Agriculture, of 594 cloned calves born in Japan between 1998 and
2007, 14.8% were stillborn and 95% were dead within 24 hours after parturition [164]. The
calving rate for SCNT pregnancies (5.1%-12.6%) was also lower than for in vivo/ET
(23.9%-34.8%) and for IVP (20.2%-22.3%) pregnancies [155]. Based on data from 3,374
SCNT embryos transferred over 5 years of commercial cloning practice in the United States,
Argentina and Brazil, 41% of the embryos were alive at Day 30 of gestation but only 9% of
the total embryos transferred resulted in live births [157]. Of these, 388 live SCNT calves, or
about 42%, died between parturition and 150 days of life [157]. The efficiency of cell lines
for successful production of viable SCNT pregnancies ranged from 0 to 45%, with about
24% of cell lines failing to produce live calves [157].
36
The most conspicuous abnormalities seen in the perinatal period following the
transfer of IVP embryos is the occurrence of high birth weight calves in association with a
broader distribution of birth weight compared to in vivo controls. High rates of perinatal
pregnancy loss associated with IVP pregnancies can be largely attributed to dystocia
occurring as a result of large sized fetuses [117, 161]. Dystocia can have long-term effects
on recipient animal health, production and future reproductive performance [117]. High birth
weight in association with IVP of transferred embryos has been reported for several Bos
taurus breeds including Holstein, Angus, Simmental and Japanese black [129, 151, 162, 165,
166]. Transfer of IVP derived embryos in Bos indicus Gyr cattle also resulted in higher birth
weights than AI controls, with particular effect observed for male calves [167]. Interestingly,
IVP calves demonstrated not only higher birth weights but also elevated plasma fructose
concentrations after birth when compared to in vivo controls [158]. These factors appeared
to be associated with metabolic distress in the IVP calves [158].
Common morphological abnormalities reported following the transfer of SCNT
embryos included enlarged umbilical cord, respiratory problems, prolonged recumbency, and
contracted flexor tendons [157]. In addition, cloned calves demonstrated other abnormalities
at birth including hypothermia, hypoglycemia, severe metabolic acidosis and hypoxia,
although these parameters were not significantly correlated with birth weight. Because
plasma insulin concentrations were significantly increased and plasma glucagon
concentrations correlated with birth weight, aberrant energy availability and energy
utilization in utero were proposed to induce conditions similar to that seen with human
37
gestational diabetes [168]. At birth, cloned calves often exhibit respiratory distress
syndrome [169] and, in some cases, exhibited left-sided cardiopathy with ventricular dilation
and pulmonary hypertension [169]. In addition, calves that appeared abnormal at birth also
exhibited an edematous placenta [169]. Other studies have reported enlarged umbilical
vessels and varying degrees of edema as well as illnesses such as ruminitis, abomasomitis
and coccidiosis at birth [121, 126, 152, 170, 171]. Mean placentome number was also lower
and mean placentome weight higher in placentas of cloned calves compared to IVP controls
[171].
Fetuses and calves from IVP or SCNT embryos exhibit a right-skewed weight
distribution compared to those produced from AI or in vivo produced embryo transfer (ET)
[26, 129, 171, 172]. Cloned calves were 20% larger at birth compared to ET or AI calves
and exhibited a greater variation in birth weight [173]. Interestingly, cloned calves derived
from oocytes matured in vitro had a significantly higher birth weight than those cloned calves
derived from oocytes matured in vivo. In fact, the mean birth weight of the female in vitro
derived calves was significantly higher than that of the in vivo derived male calves [174].
The proportion of male offspring resulting from the transfer of IVP embryos was reported to
be increased from the expected 1:1 ratio compared to AI controls where a normal sex ratio
was maintained [151, 167] [59]. In contrast, other studies have not reported a difference in
the proportion of males born following the transfer of IVP embryos [26, 34, 175].
Fetuses derived from IVP embryos may also differ in gene and protein expression
compared to those produced in vivo. At Day 90 of gestation, IVP pregnancies were
38
associated with lower fetal D-glucose concentrations than in vivo controls [150]. Expression
of IGF-II mRNA was 2-fold greater in Day 63 liver tissue but decreased in skeletal muscle
tissue of fetuses derived from IVP embryos compared to that of in vivo derived controls
[176]. Furthermore, skeletal muscle tissue from fetuses at Day 222 of gestation derived from
the transfer of IVP embryos had lower expression of myostatin mRNA and tended to have
increased expression of GAPDH mRNA compared to in vivo controls [177]. In contrast,
despite differences in fetal and placental morphology at Day 90 or Day 180 of gestation
between pregnancies derived from IVP or in vivo produced embryos, no significant
difference in levels of mRNA expression were detected in fetal liver and placental tissues
from these pregnancies when a 13k oligonucleotide microarray was used for analysis [178].
Because these results contrasted with data from earlier studies, Jiang et al. [178] proposed
that abnormal gene expression could be stage and tissue specific. Alternatively, the limited
number of observations used within each treatment and stage subgroup, coupled with a
limited size expression array, may have precluded identification of differences in mRNA
expression.
Placental Development
In addition to fetal abnormalities, the production of calves following the transfer of
IVP or SCNT embryos is associated with defects in placental development and vasculature.
Placentas of IVP or SCNT pregnancies can exhibit differences in morphology, function, gene
expression, hormone production or protein production compared to placentas from in vivo
39
derived embryos or AI pregnancies pregnancies [117, 120, 126, 171, 179, 180].
Perturbations in placentation and placental function could affect growth and development of
those fetuses produced from in vitro techniques. Abnormalities in placental development are
even more pronounced in pregnancies from SCNT embryos; in fact, the majority of
pregnancy loss in SCNT pregnancies is attributed to placental deficiencies [120, 169]. In
contrast, for in vivo derived pregnancies, the majority of losses are due to fetal abnormalities
[26].
Loss of IVP pregnancies during early gestation can be attributed to abnormalities and
failures in placental development. Common morphological abnormalities reported in IVP
placentas include enlarged placentas (placentomegaly), increased placental fluid volume
(hydrallantois) and reduced placentome surface area (reviewed in [117]). In an analysis of
placentas at Day 70 of gestation from embryos produced in vitro using either a semi-defined
culture medium (mSOF) or an undefined, serum-supplemented medium (M199) and
compared to in vivo produced embryo transfer controls [179], placentas from embryos
produced in mSOF medium were heavier, had decreased placental efficiency, fewer
placentomes and decreased placental fluid compared to those from the M199 or in vivo
control groups. In addition, placentas from the mSOF group had decreased blood vessel
density and levels of VEGF mRNA in the cotyledons, indicative of impaired vascular
development. Furthermore, an increased density of fetal pyknotic cells in placentomes from
the mSOF group was observed compared to the M199 and in vivo control groups, indicative
of increased cell death in the fetal villi [179]. Other placental abnormalities that were
40
observed in IVP pregnancies during early gestation include differences in placentome size
distribution, weight and surface area at Days 90 and 180 of gestation [150, 158].
Furthermore, placentomes directly surrounding the fetus were observed to be longer and
thinner during the first trimester of IVP pregnancies compared to controls. At Day 180 of
gestation, increased placentome surface area and weight was associated with increased
glucose and fructose accumulation in the fetal plasma and fluids [158]. While IVP calves
had placentas with significantly fewer cotyledons than placentas from in vivo controls,
placentas from IVP pregnancies had a larger total cotyledonary surface area. It was
hypothesized that increased substrate utilization in the larger IVP fetuses could be
responsible for the higher fetal weights observed in that treatment group [150, 158].
First trimester placentas of SCNT pregnancies can lack proper vascularization and
exhibit abnormalities including hypoplasia and reduced cotyledonary development [120, 181184]. Similarly, pregnancy loss following transfer of SCNT embryos later in gestation was
attributed to a number of factors including reduced cotyledonary or placentome number,
hydrallantois, placental and fetal edema [126]. Improper allantoic development is often the
leading cause of embryo mortality after Day 30 of pregnancy, as the allantois is necessary for
the establishment of the chorioallantoic placenta. Placentomes are formed starting around
Day 30 [185]. SCNT embryos can have impaired cotyledonary formation or other problems
that prevent the proper development of placentome structure and number [181]. Placentome
number is more variable in SCNT pregnancies compared to AI controls [120, 181] and there
41
is a high likelihood of pregnancy loss by Day 90 of gestation when placentome numbers are
very low [117, 120, 181].
Later in gestation, placentas from either IVP or SCNT pregnancies can exhibit other
anomalies in morphology or function. At Day 222 of gestation, placentas recovered from IVP
pregnancies had an increased proportional volume of blood vessels in the maternal caruncles
when compared to in vivo controls [186]. Furthermore, placentas from the IVP group had an
increased ratio of the tissue volume occupied by blood vessels to placentome surface area,
indicative of potentially increased vascularization compared to controls. In contrast, no
differences were identified in the volume density of fetal villi at Day 70 between IVP and in
vivo groups, suggesting perturbations in villous formation likely occur later in gestation
[179]. Similarly, there was no difference in placental weight at Day 70 of gestation whereas
at Day 222 of gestation, placentas from IVP pregnancies were heavier than in vivo controls
[186]. These results also support the suggestion that deviations in placental growth
associated with IVP pregnancies occurs later in gestation [179]. Fetal overgrowth associated
with in vitro production systems likely leads to an increased need for nutrients and gas
exchange and placentas from IVP pregnancies may develop compensatory mechanisms
within the vascular system to address the decreased surface area available for fetal and
maternal contact [186]. The incidence of hydrallantois in IVP pregnancies is about 1 in 200
pregnancies and is significantly greater than the 1 in 7,500 frequency reported for in vivo
derived pregnancies [26].
42
During the second half of gestation, SCNT placentas may be exhibit placentomegaly
and hydrallantois with edema of the fetal and placental tissues as the leading cause of fetal
mortality in these pregnancies [181]. Furthermore, umbilical vessel diameter is generally
larger and the umbilical cords have a larger luminal space due to the lack of luminal folds in
SCNT pregnancies compared to AI control pregnancies [181, 182]. Placental overgrowth in
SCNT pregnancies could be due to inherent defects in the placenta rather than due to fetal
overgrowth and can occur in up to 50% of late gestation SCNT pregnancies [182]. Placental
overgrowth occurred prior to fetal overgrowth and the ratio of fetal to placentome weight was
lower in SCNT pregnancies as compared to AI and IVF controls [182].
Underlying Mechanisms of AOS
Abnormal Offspring Syndrome has not been consistently linked to single genes or a
specific pathophysiology. The occurrence of AOS is influenced by in vitro embryo culture
conditions [187] and levels of maternal nutrients [128]. The abnormalities seen in bovine
embryos, fetuses, placentas, and offpsring with AOS are consistent with disruption of
epigenetic reprogramming and the expression of imprinted genes as well as altered regulation
of nonimprinted genes [73]. Genomic imprinting is a form of gene regulation where genes
are expressed in a monoallelic, parent-of-origin specific manner due to epigenetic
modifications [73, 188]. Epigenetic modifications are chemical alterations to the DNA or to
histone proteins that alter chromatin structure without altering the nucleotide sequence [189,
190].
43
In mammals the major epigenetic mark is DNA methylation, however expression of
imprinted genes is also regulated by other epigenetic modifications such as covalent
modification of histones leading to RNA silencing [190, 191]. Normally, demethylation of
paternal DNA occurs after fertilization and demethylation of maternal DNA occurs during
early cleavage divisions [192-194]. DNA methyltransferases are responsible for the erasure,
acquisition, and maintenance of methylation imprints in gametes as well as for establishing
and maintaining DNA methylation patterns during epigenetic reprogramming of the embryo
[195, 196]. Embryonic methylation patterns are re-established during development to the
blastocyst stage in cattle via DNA methyltransferase 1 (DNMT lo) [194, 197]. De novo
remethylation is governed by the actions of DNMT 3a and DNMT 3b [193]. Altered mRNA
expression of DNMT1 and DNMT3a have been observed in IVP and SCNT preimplantation
embryos, suggesting aberrations in DNMT activity in the in vitro produced preimplantation
embryo likely contribute to the physiological changes associated with AOS [118]. Evidence
suggests that imprinted gene expression in preimplantation embryo is sensitive to altered
epigenetic modifications that may result from exposure to the in vitro culture environment
during this period [118, 134, 176, 198-200].
Acquisition of maternal epigenetic modifications regulating imprinted gene
expression occurs during late follicular development; therefore, aspiration and in vitro
maturation of bovine oocytes may also contribute to epigenetic dysregulation [201]. In
bovine oocytes there are substantial developmental and physiological differences between in
vitro matured COCs compared to those that are matured in vivo. Several studies reported
44
that 50 to 80% of in vivo matured oocytes develop to the blastocyst stage, whereas only 15 to
40% of in vitro matured oocytes reach the blastocyst stage [202-205]. Furthermore, a fetal
overgrowth phenotype was also observed in transgenic calves derived from in vitro matured
oocytes when compared to those derived from in vivo matured oocytes [174].
Many imprinted genes regulate the expression of non-imprinted genes, and abnormal
phenotypes may arise from aberrant expression of non-imprinted genes as a downstream
effect of epigenetic dysregulation on imprinted genes [117, 206, 207]. Alternatively, in vitro
culture environments and SCNT protocols may have adverse effects on the DNA
modifications regulating the expression of non-imprinted genes independent of genomic
imprinting. IVP and SCNT protocols also negatively impact the expression of genes that
regulate the acquisition and maintenance of epigenetic modifications necessary for the
appropriate expression of imprinted genes.
The in vitro production and transfer of bovine embryos is not only important in the
commercial bovine embryo transfer industry, but also serves as the basis of other embryo
biotechnologies in cattle. Both IVP and SCNT embryos have a variety of uses both in
research and in commercial fields. For example, IVP embryos can be used to improve
pregnancy rates in dairy cows suffering from summer heat stress [149], while SCNT embryos
have the potential to rescue endangered or extinct species [152, 208]. However, the transfer
of these embryos is associated with lower pregnancy and calving rates, as well as the
occurrence of AOS in a proportion of resulting conceptuses, fetuses, or offspring. As the
utilization of these embryo technologies will likely continue to increase, it is important to
45
develop methods to either prevent these abnormal phenotypes or screen potential candidates
for these abnormalities. This will require further elucidation of the mechanisms responsible
for the discrepancies between individuals produced in vivo, in vitro, or by SCNT.
46
STATEMENT OF THE PROBLEM
Early embryonic mortality is a major factor contributing to decreased reproductive
efficiency in domestic animals and in cattle most often occurs prior to maternal recognition
of pregnancy. Successful recognition and subsequent maintenance of pregnancy depends on
optimal conceptus-maternal communication. Perturbations in embryo or conceptus
development, as well as inadequate uterine receptivity, contribute to the high incidence of
embryonic mortality. Therefore, it is critical to enhance our understanding of the mechanisms
involved in optimal conceptus-maternal interactions and understand how factors such as the
maternal progesterone environment, embryo production method, and embryo morphology
and gene expression affect this dialogue.
Maintaining adequate levels of circulating progesterone is critical for establishing an
optimal uterine environment conducive to pregnancy success. Through its regulation of gene
expression in the endometrium, progesterone enhances conceptus elongation and is
associated with increased fertility in cattle. The size of the elongating conceptus is linked to
increased production of IFN-τ, the agent of maternal recognition of pregnancy.
Another factor that contributes to pregnancy success is the quality of the embryo,
which often relies on proper selection of in vivo produced embryos prior to transfer. While
morphological evaluation is the most widely used assessment technique, it requires training
and may be best paired with a more objective measure, such as an assessment of gene
expression. In vitro embryo production is a widely-used technique both in research and in the
commercial livestock industry, however it is associated with lower pregnancy rates and the
47
occurrence of some developmental abnormalities. Therefore, it is important to identify
methods to both improve embryo selection and enhance the survival of both in vitro and in
vivo produced embryos. This could be accomplished through a better understanding of
interactions that occur between the progesterone environment of the recipient female and the
embryo source.
There has been significant progress made in the past several years to describe and
characterize gene expression in the uterine endometrium. However, to date there is very little
information on the genetic mechanisms involved in proper conceptus development and
elongation during the peri-implantation period. Furthermore, most studies that have
investigated gene expression patterns in the developing bovine embryo have focused on preimplantation stage blastocysts. Currently, there is inadequate information on the genes and
pathways governing the growth and development of the peri-implantation conceptus,
particularly when considering that the majority of embryonic loss occurs during this time
frame. In addition, very little information exists comparing conceptus elongation and the
corresponding transcriptomes between conceptuses derived from in vitro and in vivo
produced embryos. Therefore, the objectives of these studies were to:
1. investigate the interactions of progesterone environment at the time of transfer and
embryo production method on pregnancy success and early conceptus development;
2. identify and characterize differences in gene expression between Day 15 conceptuses
derived from embryos of different morphological grades; and
48
3. identify and characterize differences in gene expression between long and short
conceptuses at Day 15 of gestation that were derived from either in vivo or in vitro
produced embryos.
49
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Table 1.1: Criteria for assigning quality grades to bovine embryos as recommended by the
International Embryo Transfer Society [25].
Code
1
2
3
4
Grade
Excellent or Good
Description
Symmetrical and spherical embryo mass with individual
blastomeres (cells) that are uniform in size, color, and
density. This embryo is consistent with its expected
stage of development. Irregularities should be relatively
minor, and at least 85% of the cellular material should
be intact, viable embryonic mass. This judgment should
be based on the percentage of embryonic cells
represented by the extruded material in the perivitelline
space. The zona pellucida should be smooth and have no
concave or flat surfaces that might cause the embryo to
adhere to a Petri dish or a straw.
Fair
Moderate irregularities in shape of the embryonic mass
or in size, color and density of individual cells. At least
50% of the cellular material should be an intact, viable
embryonic mass.
Poor
Major irregularities in shape of the embryonic mass or
in size, color and density of individual cells. At least
25% of the cellular material should be an intact, viable
embryonic mass.
Dead or Degenerating Degenerating embryos, oocytes or 1-cell embryos:
nonviable.
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Table 1.2: Pregnancy rates of recipients following transfer of fresh bovine embryos of
different stages of development and quality grades produced either in vivo or in vitro.
Embryo
source
Stage of
development
Percent pregnant (no. of transfers) by
embryo grade
Grade 1
Grade 2
Grade 3
Reference
In vivo
Late morula + early
blastocyst
71% (7)
56% (672)
44% (130)
Shea, 1981
Morula to expanded
blastocyst
64% (1748)
45% (438)
33% (100)
Wright, 1981
Morula to expanded
blastocyst
76%a
65%a
54%a
Hasler et al., 1995
Morula to expanded
blastocyst
73.2% (4163)
68.3% (3156)
56.3% (1641)
Hasler, 2001
Late morula +
blastocyst
54% (61)
51% (41)
26% (27)
Reichenbach et al.,
1992
Blastocyst
46.9%b
35.8%b
19.2%b
Looney et al., 1994
Morula
56% (90)
43% (23)
Early to midblastocyst
56-58% (969)
41-46%
(283)
Expanded to hatched
blastocyst
50-62% (483)
56% (36)
Morula + blastocyst
18.5% (81)c
In vitro
Hasler et al., 1995
Block et al., 2009
28.6% (70)d
Expanded blastocyst
38.3% (81)c
29.5% (78)d
a
320 total transfers among all 3 embryo grades; b813 total transfers among all 3 embryo grades; cControl group,
and dHyaluronan treated group.
72
Table 1.3: Potential genes for assessing development and function of bovine embryos.
Gene Function
Gene Name
Gene Abbreviation
References
Bos taurus bone morphogenetic protein 15
BMP15
El-Sayed et al., 2006
Bos taurus urokinase-type plasminogen activator
PLAU
El-Sayed et al., 2006
Leukemia inhibitory factor, beta receptor
LIF, LR-β
Eckert and Niemann, 1998
Insulin-like growth factor 1 receptor
IGF1R
Bertolini et al., 2002
Growth factor & cell signaling
Insulin-like growth factor 2 & receptor
IGF2, IGF2R
Bertolini et al., 2002
Fibroblast growth factor 2 (basic)
FGF2
Lazzari et al., 2002
Transcription factors
Bos taurus msh homeobox 1
MSX1
El-Sayed et al., 2006
Homo sapiens pitutary tumor-transforming 1
PTTG1
El-Sayed et al., 2006
Caudal type homeobox transcription factor 2
CDX2
El-Sayed et al., 2006
Bos taurus arachidonate 15-lipoxygenase
ALOX15
El-Sayed et al., 2006
Bovine aldose reductase family 1, member B1
AKR1B1
Metabolism
Glucose transporter 1, 3, 4
GLUT1,GLUT3, GLUT4
El-Sayed et al., 2006
Wrenzycki et al., 2001; Bertolini et al., 2002;
Lazzari et al., 2002
Glucose-6-phosphate dehydrogenase
G6PD
Wrenzycki et al., 2002
Phosphoglycerate kinase 1
PGK1
Wrenzycki et al., 2002; El-Sayed et al., 2006
Bos taurus elongation factor 1 alpha 1
EEF1A1
El-Sayed et al., 2006
Maternal recognition & implantation
Interferon tau
IFNT
Wrenzycki et al., 2001
Bos taurus prostaglandin G/H synthase-2
COX2/PTGS2
El-Sayed et al., 2006
Bos taurus CD9 antigen
CD9
El-Sayed et al., 2006
Connexin 31, 43
CX31, CX43
Rizos et al., 2002; Wrenzycki et al., 1996
Cadherin 1, type 1 (E-cadherin)
CDH1
Wrenzycki et al., 2001
Desmocollin 1, 2
DSC1, DSC2
Wrenzycki et al., 2001
Bos taurus thioredoxin mRNA
TXN
El-Sayed et al., 2006
Copper/Zinc superoxide dismutase
Cu/Zn-SOD
Rizos et al., 2002
Heat shock protein
HSP
Wrenzycki et al., 2001
DNA methyltransferase 1, 3 alpha
DNMT1, DNMT3A
Wrenzycki et al., 2001; Wrenzycki and
Niemann, 2003
Bos taurus tumor necrosis fator
TNF
El-Sayed et al., 2006
Mammalian achaete-scute homologue 2
MASH2
Wrenzycki et al., 2001
X-inactive specific transcript
XIST
Wrenzycki et al., 2002
Embryo compaction/cavitation
Stress adaptation
Other
73
Figure 1.1: Identification of abnormalities commonly associated with abnormal offspring
syndrome (AOS). In individual cases, each abnormality listed may or may not be present.
74
Figure 1.2: Relative timing of pregnancy losses following the transfer of bovine embryos
produced in vivo (IVO), in vitro (IVP) or by somatic cell nuclear transfer (SCNT).
75
CHAPTER 2: MATERNAL SERUM PROGESTERONE CONCENTRATION AND
EARLY CONCEPTUS DEVELOPMENT OF BOVINE EMBRYOS PRODUCED IN
VIVO OR IN VITRO
ABSTRACT
The hormone progesterone is essential for proper embryonic development. The
objective of this study was to examine the relationship between recipient serum progesterone
levels, both at the time of embryo transfer and at conceptus recovery, on conceptus
development from in vivo- or in vitro-produced embryos. Embryos were produced in vivo by
superovulation of Holstein cows (IVO; n = 17) or in vitro with either serum-containing
(IVPS; n = 27) or serum-restricted medium (IVPSR; n = 34). Single Grade 1 blastocysts from
each embryo production system were transferred into heifers at Day 7 of development.
Conceptuses were recovered at Day 17 of gestation and classified as complete, degenerated,
or no conceptus. Compared with the IVO group, in vitro-produced embryos had more
(P = 0.055) degenerated conceptuses (IVO, 0%; IVPS, 18.5%; IVPSR, 20.6%). There were
no differences in progesterone concentrations at the time of transfer when recipients received
either male or female embryos (P>0.05). Progesterone concentrations in recipients receiving
in vivo-produced embryos were higher (P<0.05; 3.74 ± 0.4 ng/mL; LSM ± SEM) at Day 7
compared to those receiving in vitro-produced embryos (IVPS 2.4 ± 0.2, IVPSR 2.58 ± 0.3
ng/mL). Heifers in the in vitro treatment groups had lower (P<0.01) progesterone
concentrations at Day 7 when no conceptus was recovered at Day 17 (IVPS 2.1 ± 0.4, IVPSR
76
2.7 ± 0.3 ng/mL) compared with the IVO group (4.5 ± 0.5 ng/mL). There was no difference
in progesterone concentration between treatment groups for heifers with shorter conceptuses
(≤194 mm). However, when longer (>194 mm) conceptuses were recovered, heifers with in
vitro produced embryos had lower (P=0.05) progesterone levels at Day 7 compared to those
with in vivo produced embryos (IVPS 2.2 ± 0.5, IVPSR 2.3 ± 0.5, IVO 3.9 ± 0.5 ng/mL). In
summary, serum progesterone concentration in recipients at the time of transfer of in vivo- or
in vitro-produced embryos was associated with conceptus development at Day 17 of
gestation.
INTRODUCTION
Progesterone is important for establishing and maintaining pregnancy, as well
ensuring proper embryo development. An elevated concentration of maternal serum
progesterone early in pregnancy has been associated with higher pregnancy rates and
embryonic growth rates in cattle [66, 96, 103]. Normal plasma progesterone concentrations
on Day 7 after estrus range from 1-6 ng/mL with an average concentration of 3.21 ng/mL
[96]. Pregnancy rates were lower when progesterone levels at the time of transfer on Day 7
fell below 2 ng/mL or rose above 5 ng/mL [96]. Furthermore, cows with serum progesterone
concentrations less than 5 ng/mL on Day 14 were more likely to lose their pregnancies
between Days 28 and 42 [97]. These results suggest there is an optimal range of progesterone
concentration at Day 7 of gestation that is conducive to pregnancy.
77
There is a high degree of pregnancy loss in cattle between fertilization and term,
ranging from 40% to 56% [2-4]. The majority of this loss (70% to 80%) occurs in the first
three weeks of pregnancy, particularly during the period leading up to maternal recognition
of pregnancy (Day 8 to Day 16 post-insemination) [4, 5]. There is mounting evidence that the
early maternal environment is critical in determining the quality of the blastocyst and,
consequently, its ability to produce adequate amounts of IFN-τ necessary for pregnancy
establishment. Understanding how embryo development is controlled is critical for
determining ways to reduce the high rates of early embryonic mortality [99, 103].
There appears to be a relationship between early conceptus development and both the
rate and timing of the progesterone increase following conception [97, 99, 104]. Cows
without an embryo at Day 16 post-insemination had a delayed increase in progesterone
concentration from Day 1 to Day 16 compared to those with an embryo present [99].
Furthermore, when considering cows that had an embryo, there was a delayed increase in
progesterone in animals who also had undetectable concentrations of IFN-τ. Kenyon et al
[97] suggested that a fast, early rise in progesterone concentration was associated with
pregnancy success following embryo transfer. They reported that the fold change in
progesterone concentration from Day 0 to 7 and from Day 7 to 14 was associated with
pregnancy status on Day 63. The minimum fold change required for pregnancy establishment
was 2.71 and 1.48 for the Day 0 to 7 and Day 7 to 14 intervals, respectively [97].
The period of greatest embryonic loss in cattle occurs between blastocyst formation
and the initiation of elongation (Days 7 to 16 post-fertilization) [2]. During this period the
78
embryo undergoes a dramatic increase in size and protein content [82, 83]. High
concentrations of circulating progesterone, both prior to and during this period, are strongly
associated with an increase in conceptus length. Mann et al [101] reported that the timing of
progesterone supplementation is important because supplementation between Days 5 to 9
during the postovulatory rise resulted in a several-fold increase in trophoblast length and
IFN-τ production compared to supplementation between Days 12 to 16. Similarly,
administration of progesterone in the first several days of pregnancy stimulated conceptus
growth and development to Day 14 for in vivo [105] and in vitro produced embryos [106].
Furthermore, mean embryo area was significantly increased at Day 13 in recipients that were
superovulated compared to controls [103]. Embryonic length was also significantly increased
on Day 13 and Day 16 of pregnancy in animals that received progesterone supplementation
compared to controls [104]. In contrast, conceptus length and area were significantly
decreased at Day 14 in recipients with an induced low progesterone environment compared
to those with a normal progesterone environment [108]. Interestingly, early progesterone
supplementation may not accelerate embryonic development until after hatching of the
blastocyst from the zona pellucida. Both Carter et al [104] and Satterfield et al [209] failed to
find a significant relationship between progesterone supplementation and development to the
blastocyst stage in cattle or sheep, respectively.
Studies have shown lower pregnancy rates following the transfer of in vitro produced
embryos compared to in vivo produced embryos [26, 129-132, 139, 142] as well as higher
rates of early embryonic death during the peri-implantation period based on pregnancy or
79
recovery rate [1, 91, 103, 140, 141]. In vitro produced embryos are also associated with a
range of developmental abnormalities of the fetus, placenta, and calf that have been termed
Abnormal Offspring Syndrome [1, 26, 117]. At the blastocyst stage, in vitro produced
embryos exhibit some morphological differences from those produced in vivo, such as a
darker color [20, 39], less pronounced compaction [26], fewer junctional complexes among
blastomeres [38], and a reduced perivitelline space [26, 210]. Elongating conceptuses derived
from in vitro produced embryos exhibit some morphological differences compared to those
derived in vivo. One study reported that conceptuses derived from in vitro produced embryos
were longer at Day 12 [91], while another reported the in vitro derived conceptuses were
shorter at Day 13 [103] than their in vivo counterparts. Others failed to detect differences in
conceptus length during this period [59, 84]. The contrasting results between studies could be
attributed to differences between in vitro conditions used to produce embryos, the number of
embryos transferred per recipient, or the timing of recovery relative to the onset of maternal
recognition of pregnancy. The transfer of in vitro produced embryos may also be associated
with an increase in the recovery of degenerated, non-hatched embryos during the periimplantation period [59].
While previous studies have largely focused on the relationship between the progesterone
environment and conceptus development, there are few data incorporating the effects of
embryo source and progesterone environment on subsequent conceptus development. The
objective of this study was to examine the relationship between maternal serum progesterone
concentrations at the time of embryo transfer (Day 7) and conceptus recovery (Day 17) on
80
pregnancy rate and early conceptus development from embryos produced in vivo or in vitro.
Endogenous progesterone concentration was analyzed for its relationship with conceptus
status upon recovery, conceptus length, and conceptus sex.
METHODS
Embryo Production
The following procedures were approved by the Institutional Animal Care and Use
Committee at North Carolina State University. Embryos produced in vivo (IVO) were
recovered from superovulated Holstein cows [179]. Donor cows were pre-synchronized by
administering two i.m. injections of 25mg PGF2α given 14 days apart (Lutalyse; Pfizer
Animal Health, Groton, CT). The cows were subsequently superovulated with 400mg FSH
given in decreasing doses by i.m. injection over a 4 day period (Folltropin-V; Bioniche
Animal Health, USA). Cows were given two i.m. injections of PGF2α on the morning and
evening of the third day of the FSH treatment to lyse the CL and induce estrus. Donor cows
were artificially inseminated at 12 and 24 hours after estrus (Day 0 = estrus) using thawed
frozen semen from a single Holstein bull. Embryos were recovered on Day 7 by non-surgical
uterine lavage.
Embryos produced in vitro were generated as previously described [34, 179]. Briefly,
ovaries were collected from Holstein cows at a local abattoir and transported in saline
supplemented with 0.75µg/mL penicillin. Oocytes were aspirated from 2-10 mm antral
follicles, recovered, and matured in TCM-199 (Gibco BRL, Grand Island, NY) supplemented
81
with 10 µg/mL LH, 5 µg/mL FSH, 1 µg/mL estradiol, 200 µM pyruvate, 50 µg/mL
gentamicin, and 10% estrous cow serum for 20 h at 39°C in an atmosphere of 5% CO2 in air
and 100% humidity. Oocytes were then fertilized in vitro for 18 to 20 h with thawed frozen
semen from the same bull as used for the IVO group [179]. At 20 h post-insemination,
zygotes were separated into one of two treatment groups (IVPS: in vitro produced with
serum-containing medium throughout culture; IVPSR: in vitro produced with serumrestricted medium). Embryos were cultured in TCM-199 with 10% estrous cow serum
(IVPS) or in TCM-199 with 1% BSA for 72 h followed by TCM-199 with 10% estrous cow
serum for the next 168 h (IVPSR) [139]. All cultures were conducted at 39°C in an
atmosphere of 5% CO2 in air.
Embryo Transfer and Conceptus Recovery
Angus heifers were administered 25 mg PGF2α by i.m. injection given 12 days apart
to synchronize estrous. Single Grade 1 [25] blastocysts were transferred non-surgically into
recipients at Day 7 of the estrus cycle (IVO n=17, IVPS n=27, IVPSR n=34). Conceptuses
were recovered following slaughter 10 days after transfer (Day 17 of gestation), snap-frozen
in liquid nitrogen and stored at -80°C. Sex of the conceptus was determined by polymerase
chain reaction using a Y-chromosome specific probe [211]. Recovered conceptuses were
classified as intact, degenerated, or absent. Conceptus length was recorded and conceptuses
were grouped based on the average length of the in vivo produced conceptuses (194 mm).
82
Conceptuses with lengths ≤194 mm were classified as "short", whereas those with lengths
>194 mm were classified as "long."
Radioimmunoassay
Blood samples were collected from recipient heifers at the time of embryo transfer
(Day 7) and at the time of conceptus recovery (Day 17) from the caudal vein. Samples were
incubated at 4°C for 12-24 h and centrifuged at 2200 rpm for 20 min. Serum was collected
and stored at -20C. Progesterone concentration in serum was determined by RIA (Siemens
Healthcare Diagnostics, Duluth, GA). Samples from Day 7 and Day 17 were run in duplicate
in separate assays. Intra-assay coefficient of variation (CV) for the Day 7 assay was 7.02%
and for the Day 17 intra-assay CV, 4.96%. The inter-assay CV was 7.34% between these two
assays.
Statistical Analysis
All statistical analyses were performed using SAS (SAS Institute; Cary, NC). Data on
pregnancy rate and conceptus status were analyzed using Fisher’s Exact Test. Conceptus
length was analyzed using ANOVA. Because no degenerated conceptuses were recovered in
the IVO group, a two-way ANOVA could not be run. Therefore, a one-way ANOVA within
each level of conceptus state was used to determine the effect of treatment on progesterone
concentration. The final model for progesterone concentration at Day 7 included the main
83
effect of treatment group (IVO, IVPS, IVPSR), conceptus length (short, long), conceptus sex,
and their interactions.
The final model for progesterone concentration at Day 17 included the main effect of
treatment group, conceptus length, conceptus sex, and their interactions. The significance
level was set at P≤0.05 and Duncan’s Multiple Range Test was used to separate means when
a significant difference was detected. Data are presented as least-squares means ± SEM.
RESULTS
Conceptus recovery
A total of 78 blastocysts were transferred to recipients on Day 7 of the estrous cycle
amongst the three treatment groups. There was no difference (P>0.05) in pregnancy rate at
Day 17 of gestation between the IVO group (11/17, 64.7%) and the in vitro groups (IVPS
16/27, 59.3%; IVPSR 18/34, 52.9%). However, the two in vitro treatment groups tended to
have a higher percentage (P=0.055) of degenerated conceptuses recovered (IVPS 18.5%,
IVPSR 20.6%) compared to the IVO group (0%; Table 2.1). There was no significant
difference (P>0.05) in mean or median conceptus lengths between treatment groups (Table
2.2).
Progesterone concentration at time of embryo transfer
At the time of transfer, progesterone concentration was higher (P<0.05) in recipients
receiving IVO embryos (3.74 ± 0.4 ng/mL) compared to recipients in the IVP groups (IVPS
84
2.4 ± 0.2, IVPSR 2.58 ± 0.3 ng/mL; Figure 2.1). Interestingly, for embryos produced in vitro,
progesterone concentration in the recipient heifers was lower (P<0.01) when no conceptus
was recovered (IVPS 2.1 ± 0.4, IVPSR 2.7 ± 0.3 ng/mL) compared to in vivo controls when
no conceptus was recovered (4.5 ± 0.5 ng/mL). There were no differences (P>0.05) in
progesterone concentrations between treatment groups when intact (IVO 3.3 ± 0.4, IVPS 2.6
± 0.4, IVPSR 2.6 ± 0.4 ng/mL) or degenerating conceptuses (IVPS 2.6 ± 0.6, IVPSR 2.3 ±0.5
ng/mL) were recovered (Figure 2.2).
There was no difference (P>0.05) in progesterone concentration at the time of transfer
between treatment groups for heifers with shorter conceptuses (≤194 mm; IVO 2.6 ± 0.8,
IVPS 3.1 ± 0.8, IVPSR 3.2 ± 0.9 ng/mL). However, when longer (>194 mm) conceptuses
were recovered, heifers with in vitro produced embryos had lower (P=0.05) progesterone
levels at Day 7 (IVPS 2.2 ± 0.5, IVPSR 2.3 ± 0.5 ng/mL) compared to those with in vivo
produced embryos (3.9 ± 0.5 ng/mL; Figure 2.3).
There was no effect (P>0.05) of treatment on progesterone levels in recipients with
either male (IVO 3.9 ±0.6, IVPS 3.2 ± 0.6, IVPSR 2.4 ±0.5 ng/mL) or female (IVO 2.6 ±0.7,
IVPS 2.1 ± 0.5, IVPSR 3.1 ± 0.7 ng/mL) conceptuses at the time of transfer (Figure 2.4).
Progesterone concentration at time of conceptus recovery
No significant differences in progesterone concentrations at Day 17 of gestation were
found between treatments (Table 2.3). There were also no differences between treatment
85
groups when the change in progesterone concentration from Day 7 to Day 17 was analyzed
(data not shown).
DISCUSSION
Within the first few weeks of gestation, bovine embryos must secrete appropriate
amounts of interferon-tau (IFN-τ) to inhibit the luteolytic mechanism and maintain secretion
of progesterone required for further development. Associations between low maternal
progesterone concentrations and pregnancy failure have been reported [96, 97, 99, 103] while
early progesterone supplementation improved pregnancy rates and advanced embryo
development [104, 105, 107]. Progesterone exerts its effect on the embryo indirectly through
actions on the endometrium [106]. Addition of progesterone to cultures of in vitro produced
embryos did not affect blastocyst development [106]. In contrast, progesterone
supplementation of recipients prior to transfer of in vitro produced embryos at Day 7 resulted
in conceptuses with increased length and area at Day 14 compared to conceptuses recovered
from non-supplemented recipients [106].
In the current study, there was a tendency to recover more degenerated conceptuses at
Day 17 from the two IVP groups compared to the IVO group. This is consistent with the
results of Bertolini et al [59] who recovered more total structures (including intact and
broken conceptus pieces or degenerated nonhatched embryos) at Day 16 from an IVP group
compared with in vivo controls. In our study there were no degenerated conceptuses
recovered from heifers receiving in vivo produced embryos. While the progesterone
86
environment of the recipient was unknown at the time of transfer, those recipients receiving
IVO embryos did have a higher progesterone concentration at Day 7 compared to those
receiving IVP embryos. The lower progesterone environment in recipients from the two IVP
groups is likely responsible for the disparity in the number of degenerated conceptuses
recovered between treatment groups. However, because the transfer of in vitro produced
embryos has been associated with lower pregnancy rates and/or lower recovery rates during
early gestation, it is also possible that the IVP embryos in our study were compromised in
their ability to survive and signal their presence to the maternal system. It may be that a
relatively high progesterone environment around Day 7 may be required to "rescue" IVP
embryos that are already compromised in their developmental competency due to exposure to
in vitro culture conditions. Furthermore, IVO embryos may be able to tolerate a relatively
wide range in progesterone concentration, given that there may be an upper limit of
progesterone concentration conducive to pregnancy success [96].
Interestingly, despite exposure to a relatively high progesterone environment at Day
7, conceptuses derived from IVO embryos were not significantly longer than the IVP-derived
conceptuses. Taken together, these results suggest that a single assessment of the
progesterone environment at Day 7 is not necessarily predictive of conceptus length at Day
17. As reported in other studies [97, 99, 101, 105, 106], measuring the increase in
progesterone concentration leading up to, and around Day 7, would better predict conceptus
elongation.
87
Embryos produced in vitro had significantly lower progesterone concentrations in
recipient heifers at Day 7 when no conceptus was recovered at Day 17 compared to in vivo
controls. This observation also supports the hypothesis that bovine embryos produced in vitro
may require a higher level of progesterone in their environment prior to and around Day 7 in
order to survive. This requirement may arise from altered embryo-maternal communication
during the peri-implantation period as a consequence of in vitro embryo production methods.
The endometrial transcriptome can be influenced by several factors, including circulating
progesterone concentration. Early progesterone supplementation advanced endometrial gene
expression by altering the timing or duration of expression pattern for genes that contribute to
the composition of histotroph [107]. Furthermore, inducing a delay in the post-ovulatory rise
of progesterone altered normal temporal changes in endometrial gene expression and led to a
reduction in the ability of the uterus to support conceptus development following embryo
transfer [108, 109]. Evidence also exists that endometrium can sense and respond differently
to embryos with different developmental potentials or embryos originating from different
production methods [111, 112]. In addition, gene expression at the blastocyst stage is
associated with embryo competency and pregnancy success following transfer to recipients
[51, 212]. Altered gene expression as seen in IVP embryos [55, 59, 91, 134] may
compromise embryo competency, which can be further compounded by altered endometrial
gene expression due to inadequate circulating progesterone.
When longer (>194mm) conceptuses were recovered at Day 17, heifers that received
in vitro produced embryos had significantly lower progesterone concentrations at Day 7
88
compared to those which received in vivo produced embryos. Thus, while progesterone
concentration may be important in the establishment and maintenance of pregnancy of in
vitro produced embryos, it may not have as great an influence on conceptus elongation as it
does for in vivo produced embryos. Furthermore, while there was no statistical difference
between mean or median conceptus lengths between treatments, the two IVP groups had the
greatest range in conceptus lengths and contained the longest conceptuses. Furthermore, 4
out of 23 (17.4%) in vitro-derived conceptuses were longer than the longest in vivo-derived
conceptus. It appears that the in vitro produced embryos were elongating more than their in
vivo counterparts despite the relatively lower progesterone environment. These results
suggest that there is a difference in autonomy between in vitro and in vivo produced
embryos. In vitro produced embryos may be more dependent upon intrinsic factors for
development and growth with the effects of the embryo production method superseding the
effects of the maternal environment on conceptus elongation.
Progesterone-induced changes in gene expression of uterine tissues during early
blastocyst stages may be responsible for the longer conceptus lengths observed in the present
and previous studies [101, 103-106]. Lonergan et al [103] studied the effects of embryo
source (in vivo vs in vitro) and progesterone level of the recipient (high, superovulated vs
normal, non-superovulated) on Day 7 on bovine embryo development at Day 13.
Interestingly, there was circumstantial evidence that the difference in mean area at Day 13
between in vitro produced embryos transferred into high progesterone recipients compared to
normal progesterone recipients was not as great as the difference in mean area between in
89
vivo produced embryos recovered from superovulated donors and transferred into either high
or normal progesterone recipients. These results suggest that the progesterone environment at
the time of transfer may not influence elongation of in vitro produced embryos to as great an
extent as it influences elongation of the in vivo produced embryos. These observations are
consistent with our observations that the long in vitro-derived conceptuses experienced a
lower progesterone environment at the time of transfer compared to long in vivo-derived
conceptuses.
In summary, serum progesterone concentrations in recipient heifers at the time of
transfer of in vivo or in vitro produced embryos were related to conceptus survival. The
transfer of in vitro produced embryos was associated with a lower recovery rate and higher
proportion of degenerated conceptuses recovered at Day 17 as compared to in vivo controls.
There was no difference in progesterone concentration between treatment groups for heifers
with shorter conceptuses. However, when longer conceptuses were recovered, heifers with in
vitro produced embryos had lower progesterone levels at Day 7 compared to those with in
vivo produced embryos. In conclusion, this study provides evidence to suggest that there may
be a difference in autonomy between in vitro and in vivo produced embryos during the
period of conceptus elongation, with in vitro produced embryos relying more on intrinsic
factors to influence elongation. Hence, the maternal environment at the time of embryo
transfer may have a greater influence on conceptus elongation of in vivo produced embryos
compared to in vitro produced embryos.
90
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Table 2.1: Conceptus recovery at Day 17. Presented previously
in modified form [1].
IVO
IVPS
IVPSR
No. of embryos transferred
17
27
34
Pregnant (%)
64.7
59.3
52.9
Degenerated (%)
0a
18.5b
20.6b
Pregnant, non-degenerated (%)
100
68.8
61.1
a.b
P=0.055
Table 2.2: Summary of average intact conceptus lengths (mean ± SEM)
and ranges for each treatment group.
Treatment
Group
IVO
IVPS
IVPSR
N
Average
Length (mm)
Median
Length (mm)
Range Low-High
(mm)
11
11
12
194.3 ± 40.6
231.1 ± 38.2
300.8 ± 48.0
222.0
200.0
311.5
18-373
90-502
68-701
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Figure 2.1: Progesterone concentration (mean ±
SEM) at Day 7 and treatment group. IVO n=17,
IVPS n=27, IVPSR n=34. a,bDifferent letters
denote significant differences between
treatment groups (P<0.05).
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Figure 2.2: Progesterone concentration (mean ± SEM) at Day 7 and conceptus state at recovery at Day 17. Complete conceptus:
IVO n=11, IVPS n=11, IVPSR n=11. Degenerated or fragmented conceptus: IVO n=0, IVPS n=5, IVPSR n=7. No conceptus: IVO
n=6, IVPS n=11, IVPSR n=16. a,b Different letters denote significant differences between treatment groups within conceptus state
(P<0.05).
115
Figure 2.3: Progesterone concentration (mean ± SEM) at Day 7 and conceptus length at Day 17.
Short: IVO n=5, IVPS n=5, IVPSR n=4. Long: IVO n=6, IVPS n=6, IVPSR n=8. a,b Different
letters denote significant differences between treatment groups within conceptus length
classification (P=0.05).
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Figure 2.4: Progesterone concentration (mean ± SEM) at
Day 7 and conceptus sex at Day 17. Male: IVO n=6, IVPS n=6,
IVPSR n=8. Female: IVO n=5, IVPS n=9, IVPSR n=5.
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Table 2.3: Progesterone concentration (ng/mL; mean ± SEM) in recipient heifers at Day 17 of gestation.
Treatment
IVO
IVPS
IVPSR
Complete
conceptus
Degenerated
or fragmented
conceptus
6.1 ± 0.9
7.6 ± 0.9
7.4 ± 0.9
5.4 ± 1.4
4.3 ± 1.3
No conceptus
Small
(≤ 194 mm)
Large
(>194 mm)
Male
Female
5.8 ± 1.4
5.7 ± 1.1
6.0 ± 0.8
5.1 ± 1.1
7.8 ± 1.1
7.5 ± 1.3
7.0 ± 1.4
7.4 ± 1.4
7.4 ± 1.2
6.3 ± 1.2
8.4 ± 1.2
8.0 ± 1.0
6.0 ± 1.3
6.5 ± 1.0
6.1 ± 1.3
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CHAPTER 3: DIFFERENCES IN THE TRANSCRIPTOMES OF SHORT AND
LONG BOVINE CONCEPTUSES ON DAY 15 OF GESTATION FROM EMBRYOS
PRODUCED IN VIVO OR IN VITRO
ABSTRACT
The majority of pregnancy loss in cattle occurs between days 8 and 16 of gestation
coincident with the initiation of conceptus elongation and the onset of maternal recognition
of pregnancy. Conceptus elongation is associated with an increase in interferon-tau synthesis
and is driven by conceptus-endometrial interactions. The objective of this experiment was to
characterize differential patterns of mRNA expression between short and long bovine
conceptuses recovered on Day 15 of gestation from embryos produced in vivo or in vitro. In
Experiment 1 and Experiment 2, embryos were produced in vivo (IVO) from superovulated
Holstein donor cows and groups of Grade 1 and Grade 3 compact morulas were transferred
into recipient heifers at Day 6.5 of the cycle. For Experiment 3, Grade 1 Holstein IVO and in
vitro produced (IVP) blastocysts were transferred into recipient heifers at Day 7 of the cycle.
Conceptuses were then recovered at Day 15 of gestation, and measured to assess overall
length and area. Total RNA was extracted from conceptuses and analyzed on individual
GeneChip Bovine Genome Arrays (Affymetrix, Inc.). Experiment 1 compared gene
expression between conceptuses derived from Grade 1 and Grade 3 IVO embryos.
Experiment 2 compared gene expression between IVO conceptuses classified as either short
(mean ± SEM length of 4.2 ± 0.1 mm) or long (24.7 ± 1.9 mm). Experiment 3 compared gene
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expression between IVO and IVP conceptuses classified as either short (IVO = 25.0 ± 4.4
mm; IVP = 16.5 ± 2.9 mm) or long (IVO = 41.3 ± 1.3mm; IVP = 45.2 ± 7.2 mm). In
Experiment 1, there were no differences in gene expression profiles of Day 15 conceptuses
derived from Grade 1 and Grade 3 embryos. In Experiment 2, a total of 348 genes were
differentially expressed between short and long IVO conceptuses. Of these, 221 genes were
up-regulated and 127 down-regulated in long conceptuses compared to short conceptuses. In
Experiment 3, there were no differentially expressed genes between short and long IVO
conceptuses, however there was 1 gene differentially expressed between short and long IVP
conceptuses. In summary, differences in gene expression were identified between
conceptuses recovered on Day 15 of gestation that were classified based on length. These
data can be used to identify genes and cellular pathways involved in enhanced conceptus
elongation that may have the potential to serve as markers for pregnancy success.
INTRODUCTION
Cattle experience a high degree of pregnancy loss between fertilization and term,
ranging from roughly 40 to 56% [3, 4, 7]. This loss is even greater following the transfer of
in vitro produced embryos, as mean survival rate to calving is only 30% to 40% [7], thus
representing pregnancy losses of 60-70% throughout gestation. In 2012, approximately
506,000 in vivo produced and 385,000 in vitro produced embryos were transferred
commercially worldwide [6]. Embryonic mortality is therefore a major factor contributing to
economic loss in commercial practices. Communication between the developing conceptus
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and the maternal endometrium is vital for the successful establishment and maintenance of
pregnancy. A better understanding of the mechanisms involved in the development of the
elongating conceptus could help identify methods to improve survival rates of both in vivo
and in vitro produced embryos.
Embryo selection is an important factor that contributes to pregnancy success
following transfer of embryos produced in vivo or in vitro. Morphological evaluation of
embryos for stage of development and quality grade is the most widely used method for
predicting embryo viability in cattle. Multiple studies have demonstrated that there are
significant differences in pregnancy rates across different morphological grades [20-22, 2731, 34]. Wright [27] reported a pregnancy rate of 65% after the transfer of IVO Grade 1
embryos compared to 33% following the transfer of Grade 3 embryos. A large retrospective
analysis of embryo transfer data from three commercial operations identified embryo grade
as an important factor in determining pregnancy rate [28]. In that study, rates were
significantly higher following the transfer of IVO Grade 1 embryos (73%) as compared to
Grade 3 embryos (56%). Similar results have been reported following the transfer of IVP
Grade 1 (54%) and Grade 3 (26%) embryos [29]. However, morphological evaluation is not
always the best predictor of pregnancy success, as there are "poor" grade embryos that will
produced a pregnancy while "good" quality embryos may fail [20]. It is therefore of interest
to develop an objective and highly predictive method of pre-transfer embryo screening. A
few studies have associated the transcriptome of bovine embryos with both pre- and posttransfer developmental outcome [50-52, 64]. However, comparisons in the post-transfer
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studies have utilized only Grade 1 embryos. A total of 41 genes were differentially expressed
between biopsies of Grade 1 IVO embryos resulting in no pregnancy and those resulting in
calf delivery [51], while 52 differentially expressed genes (DEGs) were identified from the
same comparison between biopsies of Grade 1 IVP embryos [50]. Between these two studies,
18 genes were commonly differentially expressed with the same trend of expression for both
IVO and IVP blastocysts resulting in calf delivery [51].
Roughly 70% to 80% of pregnancy loss in cattle is sustained during the first two to
three weeks of pregnancy, particularly during the period between Days 8 and 16 of gestation
preceding maternal recognition of pregnancy [2, 213]. Embryonic loss during the periimplantation period is even higher following the transfer of in vitro produced embryos [1, 91,
103, 140, 141]. As bovine embryos are generally assessed at Day 7 prior to transfer, the
majority of research has focused on the gene expression or developmental potential of
blastocysts at this time. Furthermore, the vast majority of studies focusing on the period prior
to and during maternal recognition of pregnancy concentrate on gene expression in the
maternal endometrium. To date, only a few studies have focused on the elongating conceptus
with particular emphasis on global gene expression [59, 78, 84, 91-94].
The agent of maternal recognition of pregnancy is interferon-tau (IFNT), which is
produced solely by the conceptus trophectoderm. IFNT prevents luteolysis by inhibiting
production of PGF2α by the endometrium. Conceptuses must therefore reach adequate size in
order to produce sufficient quantities of IFNT to prevent luteolysis. Conceptus elongation
involves transitions from a spherical blastocyst on Day 7 of gestation, through intermediate
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ovoid (Day 12 to 13) and tubular (Day 14 to 15) forms, finally reaching a filamentous
conceptus around Day 16 to 17 [79]. Elongation is initiated in trophoblast cells around Day
12 and is completed by Day 24. During this time, the conceptus increases in size more than
1000-fold [82, 214] concurrent with an increase in protein content [82, 86]. Trophoblast
length doubles daily between Days 9 and 16, with a particularly sharp increase from Day 13
to 14 [215]. Elongation is maternally driven, as it does not occur in vitro [88] or in vivo in
the absence of uterine glands [89]. Progesterone from the corpus luteum acts indirectly via
the endometrium to stimulate embryonic growth and conceptus elongation [106].
Endometrial secretions, known as histotroph, stimulate cell proliferation and migration as
well as mediate attachment and adhesion of the conceptus to the endometrium [216]. While
the importance of endometrial receptivity and gene expression during the pre- and periimplantation period is well established [67, 107, 108, 217-219], there is evidence that the
developing embryo can alter the endometrial transcriptome [77, 112, 220]. Thus, proper
communication between the conceptus and endometrium is vital for pregnancy
establishment.
Numerous studies have reported a wide variation in length between conceptuses
recovered on the same day, whether due to experimental conditions such as embryo source
[84, 91, 103, 143] or enhanced by an environment containing elevated maternal serum
progesterone environment [101, 103-106]. However, due to superovulation this variation in
trophoblast length still exists within a flush from a single animal [59, 106, 215] and within
flushes from multiple recipients in the same treatment group [59, 84, 87, 106, 214, 221, 222]
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for both in vivo and in vitro-derived conceptuses. Betteridge et al [214] reported a wide
variation in conceptus length within day recovered from superovulated donors as early as
Day 10. At Day 15, conceptus length ranged from 0.255-135 mm [214]. Machado et al [84]
found that while there was no significant difference in length at Day 14 between IVO and in
IVP-derived conceptuses in their study, both groups exhibited a wide range in length, varying
from 0.60-90.00 mm and 0.57-57.10 mm, respectively. Conceptus elongation may also be
affected by the type of embryo transfer recipient, as conceptuses recovered from heifers at
Day 11 to 14 were twice as long as those recovered from cows [215]. Furthermore,
conceptuses recovered from heifers exhibited greater variability in length compared to those
recovered from cows when examining a cohort from a single flush.
Variation in length among conceptuses recovered on the same day is likely due to
different developmental competencies, as enhanced conceptus length is associated with
greater IFNT production necessary to prevent the luteolytic cascade [78, 223]. Berg et al
[215] attributed disproportionally short conceptus lengths to a problem in trophoblast
development, subsequently ending in a failure in pregnancy recognition. A recent study
reported that blastocyst cell number at Day 7 was associated with conceptus length at Day 14
[224]. If conceptuses with shorter trophoblast lengths are developmentally compromised,
then a comparison of gene expression with those longer, more viable conceptuses may be of
great value. Specifically, developmentally competent conceptuses may be more
transcriptionally active, and may turn on genes that can mediate successful communication
with the maternal system.
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The objectives of the current study were to investigate the global gene expression of
Day 15 conceptuses based on three main comparisons. First, to compare conceptuses that are
derived from the transfer of IVO Grade 1 or Grade 3 embryos to identify potential markers of
embryo competency. Second, to further explore the processes involved in conceptus
elongation, particularly as they relate to developmental competency, by comparing Day 15
conceptuses that are short or long in length and derived from IVO embryos. Third, to
compare short and long length conceptuses derived from IVO and IVP blastocysts.
METHODS
In vivo embryo production
All procedures involving animals were approved by the Institutional Animal Care and
Use Committee at North Carolina State University. Embryos produced in vivo (IVO) were
recovered from superovulated Holstein cows. Donor cows were synchronized by application
of an Eazi-Breed CIDR (Zoetis, Florham Park, New Jersey) for seven days. One day after
insertion of the CIDR, animals received 100 µg of the GnRH-analog, Cystorelin (Merial
Limited, Duluth, GA). Two days later, cows were subsequently superovulated with 400 mg
of FSH (Folltropin-V; Bioniche Animal Health, USA) given by i.m. injection in decreasing
doses over a 4 day period. Cows were given two i.m. injections of PGF2α 12 hours apart (35
mg then 25 mg) on the fourth day of the FSH treatment. Donor cows were artificially
inseminated at 12 and 24 hours after estrus (Day 0 = estrus) using thawed frozen semen from
a single Holstein bull. Embryos were recovered by non-surgical uterine lavage on Day 6.5
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for Experiments 1 and 2 and on Day 7 for Experiment 3. Embryos were evaluated for stage
of development and morphological grade [25].
In vitro embryo production
In vitro produced blastocysts were generated using two culture systems as previously
described [179]. Briefly, ovaries of Holstein cows were obtained from a local abattoir, and
cumulus-oocyte complexes (COCs) were aspirated from follicles and washed five times in
modified Tyrode medium (TL-Hepes). COCs were matured for approximately 22 h in TCM199 supplemented with 10% heat-inactivated estrus cow serum (ECS), 5 µg/ml FSH, 250 µM
sodium pyruvate, and 50 µg/ml gentamicin. All cultures were incubated at 39°C, 5% CO 2 in
air with 100% humidity. Following maturation, COCs were washed and placed in
fertilization medium that consisted of heparin-supplemented Tyrode lactate pyruvate medium
with 6 mg/ml fatty acid-free BSA. Thawed frozen semen from the same Holstein bull used
for in vivo embryo production was used for in vitro fertilization. Motile spermatozoa were
collected using the swim-up procedure and used for fertilization in 0.75 ml of fertilization
medium at a concentration of 1.0x106 cells per ml. Spermatozoa and COCs were coincubated
for 18-20 h. Presumptive zygotes were washed six times with TL-Hepes and placed into one
of two culture systems for the remainder of the embryo culture period.
Two post-fertilization culture systems were used. The first was TCM-199
supplemented with 10% ECS and the second was a modified synthetic oviductal fluid (SOF)
containing 0.6% BSA. Embryos in the TCM-199 group were cultured with their cumulus
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investments in 1 ml of medium at 39°C, 5% CO 2 in air with 100% humidity. TCM-199
culture medium was changed at 48-h intervals throughout a 168-h culture period. Embryos in
the SOF group had their cumulus investments removed following fertilization by moderate
vortexing. These embryos were cultured, undisturbed, in 1 ml of medium at 39°C in an
atmosphere of 90% N2, 5% O2, and 5% CO2 throughout a 168-h culture period.
Embryo transfer and conceptus recovery
Recipient Holstein heifers were synchronized by administering 25mg of PGF 2α given
by i.m. injection 10 days apart (Lutalyse; Pfizer Animal Health, Groton, CT). For
Experiments 1 and 2, Grade 1 (n=31) and Grade 3 (n=22) IVO embryos were transferred in
groups of 2-6 embryos per recipient. For Experiment 3, Grade 1 IVO embryos (n=8) and
Grade 1 IVP embryos (TCM-199 n=13; cSOF n=24) were transferred in groups of 1-5
embryos per recipient. Conceptuses were recovered by non-surgical uterine lavage at Day 15
of gestation using a silicone two-way catheter and flush medium (Complete Flush medium,
BioLife, Agtech, Inc.; Manhattan, KS) modified to enlarge the cannulae to allow passage of
post-hatching embryos in an intact state. Flush medium was collected from the uterus in 1L
sterile bottles. Following recovery, conceptuses were located under a stereomicroscope and
measured for length and width. When conceptuses were at least 4 mm long, a small 1 mm
section of the trophoblast was removed by scalpel, snap-frozen in liquid nitrogen, and stored
individually at -80°C for DNA isolation. The remainder of the conceptus tissue was also
snap-frozen, and stored individually at -80°C for RNA isolation.
127
Total DNA isolation and PCR-based sex determination assay
Total DNA was purified from a small 1 mm section of individual conceptus tissue
(when allowed) using the DNeasy Blood & Tissue Kit as per manufacturer's instructions
(Qiagen; Redwood City, CA). The sex of the conceptus was determined using a set of Ychromosome specific primers [225] (BOV97M, basepair position 12 to 142 [226]) as
previously described [198]. Amplification products were visualized by agarose gel
electrophoresis and sex was determined based on the presence or absence of the Y-specific
amplification product.
Total RNA isolation
Total RNA was purified from each individual conceptus for both IVO and IVP
groups according to the manufacturer's protocol (RNeasy Mini Kit, Qiagen; USA). For all
experiments, RNA quantity was measured by NanoDrop (Thermo Fisher Scientific; USA).
For Experiments 1 and 2, RNA quality and integrity was assessed by NanoDrop ratio of
absorbance values at 260 and 280 nm (260/280 = 2.11 ± 0.01) and by visualization of 28S
and 18S rRNA bands in 1% agarose gels. For Experiment 3, quality and integrity (260/280 =
2.14 ± 0.003; rRNA 28S/18S = 1.6 ± 0.1) of total extracted and purified RNA were
determined by the Agilent 2100 Bioanalyzer (Santa Clara, CA).
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Affymetrix array hybridization, washing, staining, and scanning
For each conceptus sample, 250 ng of total RNA was processed for microarray
hybridization to Affymetrix Bovine GeneChips using the GeneChip 3’ IVT Express Kit as
per manufacturer instructions (Affymetrix; Santa Clara, CA). There was one conceptus per
array. Briefly, the RNA was reverse transcribed into cDNA using T7 Oligo(dT) primer.
Following the first and second strand cDNA synthesis, in vitro transcription was carried out
at 40°C for 16 hours to generate biotin-labeled aRNA. The aRNA was fragmented at 94°C
for 35 min. The aRNA was prepared for hybridization according to the GeneChip 3’ IVT
Express Kit User Manual. Briefly, the hybridization cocktail was hybridized to the arrays at
45°C for 16 hours. Following hybridization the arrays were washed with the Affymetrix
Fluidics Station 450 wash stations. The arrays were scanned with the Affymetrix GeneChip
Scanner 3000 7G Plus with Autoloader. GeneChip Command Console Software was used for
control of all of the instrumentation.
Microarray data analysis
Quality assessment as well as microarray data normalization, background correction,
and summarization (using the Robust Multi-array Average algorithm) were performed using
Affymetrix Expression Console software. An ANOVA was performed to determine
differentially expressed genes using JMP Genomics 5.0 (SAS; Cary, NC). P-values were
adjusted using the Benjamini-Hochberg procedure to control the false discovery rate (FDR)
129
[227]. Differentially expressed genes were defined using the criteria of an adjusted P-value
<0.05, and fold-change ≥1.5.
The list of DEGs corresponding to an Entrez Gene identifier were further classified to
their gene ontology (GO) of biological processes, molecular functions, and cellular
components using the bioinformatics tool DAVID (http://david.abcc.ncifcrf.gov/) [228]. GO
enrichment analysis groups genes according to processes that occur more often in the gene
list than could be predicted by chance based on the transcripts present on the array. DAVID
uses a modified Fisher Exact P-value to measure this gene enrichment in the annotation
terms. GO analysis does not take into account gene expression levels.
For pathway analysis, the list of DEGs was organized using Entrez Gene identifiers,
folds changes, and adjusted P-values as observations. Pathway analysis was performed using
MetaCore by GeneGo (Thomson Reuters) to identify significant pathways and networks
affected by the DEGs. MetaCore uses a variation of the Fisher Exact test for multiple sample
testing using the Benjamini-Hochberg FDR adjustment.
cDNA synthesis
Reactions were carried out in a final volume of 50 µL to allow addition of 2 µg of
total RNA from the smallest conceptuses. To start, 2 µg of total RNA from each conceptus
sample was DNase treated by incubation of the RNA with 3 µl of DNase and 4 µl of DNase
buffer at 37°C for 20 min. DNase was inactivated at 75°C for 10 min. Following the
manufacturer’s instructions, 2 µg of DNase-treated RNA was incubated with 1.25 µL of
130
random primers (Promega; Madison, WI), 2.5 µL of 10 mM dNTP mix (PCR Nucleotide
Mix, Roche; Mannheim, Germany) and distilled water at 65°C for 15 min, followed by
incubation on ice for 1 min. To these tubes, 10 µL of 5X First Strand Buffer, 2.5 µL of 0.1 M
DTT and 2.5 µL of 200U/µl reverse transcriptase (Superscript III, Invitrogen; Carlsbad, CA)
were added. Samples were incubated at 25°C for 5 min, 50°C for 60 min, and then the
enzyme was inactivated by heating to 70°C for 15 min. The synthesized cDNA was purified
using the QIAquick Purification Kit (Qiagen; USA) according to the manufacturer’s
instructions. The purified cDNA was eluted into a 100 µL volume of elution buffer, yielding
a final cDNA concentration of 20 ng/µl.
Quantitative real-time PCR
For validation of microarray results from Experiments 2 and 3, six DEGs and one
housekeeping gene (H2AZ) were selected for quantitative real-time polymerase chain
reaction (qPCR). Primers were designed using NCBI Primer-Blast (Table 3.1;
www.ncbi.nlm.nih.gov/tools/primer-blast/). Primer specificity was verified by sequencing of
a PCR amplified product using the LifeTech 3730xl sequencing platform (Life Technologies;
Grand Island, NY).
For Experiment 2, three short length and three long length Grade 1IVO-derived
conceptuses were used for qPCR analysis of five transcripts. These transcripts were IL6,
TP53I3, TAGLN, AQP3, and JAM2. For Experiment 3, three short length and three long
131
length IVP-derived conceptuses were used for qPCR analysis of five transcripts. These
transcripts included RCAN2, TP53I3, TAGLN, AQP3, and JAM2.
Each reaction was carried out in duplicate and consisted of 100 ng cDNA, 2.5 µL
each forward and reverse primers (10 µM/µL), 12.5 µL of SYBR Green Master Mix (BioRad Laboratories; Hercules, CA), and 2.5 µL of RNase- and DNase-free water. qPCR was
performed on an iQ5 Real-Time PCR Detection System (Bio-Rad). The cycling conditions
were 2 min at 50°C, 3 minutes at 95°C, and 40 cycles of melting at 95°C for 20 seconds
followed by annealing and extension at 60°C for 20 seconds. Samples were subjected to a
melt curve analysis following amplification to verify the absence of nonspecific products.
The expression level of each transcript was normalized to H2AZ transcript levels (target
transcript C t - H2AZ C t ). The relative expression fold change ratios in the analyzed
conceptuses were obtained using the 2-ΔΔCt method [229].
Statistical analysis
All statistical analysis was performed using SAS (SAS Institute, Inc., Cary, NC).
Differences in recovery rates and gender ratios were analyzed by Fisher's Exact test or Chisquare. Data on length and area was analyzed by ANOVA using the PROC GLM procedure
in SAS, with treatment (as it pertained to embryo grade, length, gender, or production system
in each comparison) as the fixed variable. Statistical analysis of qPCR results was performed
by comparing normalized ΔC t values by the two-tailed Student's t-test.
132
RESULTS
Experiments 1 & 2
Day 15 conceptus recovery summary
The recovery rate (number of conceptuses recovered/number of embryos transferred)
at Day 15 was 61.3% (19/31) for Grade 1 embryos. The recovery rate for Grade 3 embryos
was 18.2% (4/22), which was significantly lower (P-value<0.03) than the recovery rate of the
Grade 1 embryos. Length and area of all recovered conceptuses is summarized in Table 3.2.
Note that two conceptuses derived from Grade 1 embryos were too degenerated to measure
length and area, and were not included in the final data set. Conceptus lengths ranged from
ovoid with a length of 1.0 mm to tubular with a length of 29.8 mm. There was no significant
difference (P>0.05) in average length or area at Day 15 between conceptuses derived from
the two different embryo grades.
Experiment 1
Conceptuses selected for microarray analysis based on grade
Four conceptuses derived from the Grade 1 embryo group and three conceptuses
derived from Grade 3 embryos were selected for microarray analysis (Table 3.3). There were
no differences in the male:female ratio between and within groups (P-value>0.05).
There were no differentially expressed genes (DEGs) detected between Day 15 IVO
conceptuses derived from Grade 1 or Grade 3 embryos.
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Experiment 2
Conceptuses selected for microarray analysis based on length
All of the conceptuses were selected based on length and were derived from Grade 1
embryos. Five conceptuses were selected and classified as short and five conceptuses were
selected and classified as long (Table 3.3). Conceptuses were designated as ovoid, early
tubular, or tubular based on shape and size as defined by Degrelle et al. [79, 230] and Grealy
et al. [82]. Conceptuses were considered ovoid in morphology when the length was more
than 1.5 times the width, but the length was <10 mm. Conceptuses designated as early
tubular were 10-30 mm long, and tubular conceptuses were <100 mm long. In the present
study, conceptuses classified as short were considered ovoid in shape, while conceptuses
classified as long were early tubular in morphology (Figure 3.1). There were no differences
in the male:female ratio between and within groups (P-value>0.05).
Conceptuses selected for microarray analysis based on gender
All conceptuses utilized in Experiments 1 and 2 were used to compare gene
expression between Day 15 conceptuses based on gender. Gene expression was also
compared between male and female conceptuses derived only from Grade 1 embryos in
Experiment 2. There were no differences in the male:female ratio between and within groups
for either comparison (P-value>0.05). There was no difference in average length or area
between male and female conceptuses for either comparison (P-value>0.05).
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Differential gene expression between long and short conceptuses
There were 348 differentially expressed genes (DEGs) detected between Day 15 IVO
long length conceptuses compared to short length conceptuses. Of these, 221 and 127 DEGs
were up-regulated and down-regulated in long conceptuses compared to short conceptuses,
respectively. The top 25 up-regulated and the top 25 down-regulated genes in long
conceptuses compared to short conceptuses are summarized in Table 3.4. The full list of
DEGs is found in Table A1. The complete list of DEGs was submitted for further analysis to
determine the functional classifications as assigned by gene ontology (GO) terms for
overrepresentation by DAVID. DEGs were classified according to their GO terms for
biological process, molecular function, and cellular component. There were 41 biological
processes overrepresented in DEGs related to conceptus elongation at Day 15 (P-value
≤0.05; Figure 3.2). The two top biological processes enriched with DEGs were generally
related to metabolic and biosynthetic processes including lipid biosynthetic process,
coenzyme and cofactor metabolism, cellular aldehyde and cholesterol metabolism or immune
response which included acute inflammatory response, regulation of phatocytosis, regulation
of immune effector process, response to wounding, regulation of adaptive immune response,
antigen processing and presentation of peptide antigen via MHC class I.
To further elucidate the GO terms enriched with transcripts more highly expressed at
each conceptus length, GO enrichment analysis was performed on a list containing only
genes that were up-regulated in long conceptuses compared to short conceptuses. Biological
processes overrepresented in long conceptuses are summarized in Table 3.5. Biological
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processes enriched with transcripts with higher expression in long conceptuses included
those related to metabolism, inflammatory response, and tissue development. Molecular
functions and cellular components overrepresented in genes up-regulated in long conceptuses
are summarized in Table 3.6. DEGs were primarily localized to the microsome, vesicular
fraction, and external side of the plasma membrane.
GO enrichment for genes specifically up-regulated in short conceptuses were also
examined (Table 3.5). Biological processes overrepresented in genes with higher expression
in short conceptuses were also related to metabolism, but also included ribosome and
ribonucleoprotein complex biogenesis. Molecular functions and cellular components
overrepresented in genes up-regulated in short conceptuses are summarized in Table 3.6.
DEGs were primarily localized in the vacuole.
The list of DEGs was also submitted for pathway analysis in MetaCore by GeneGo.
The GeneGo Pathway Maps are a series of sequential, multi-step, well-defined actions. The
top 20 most significant GeneGo Pathway Maps containing DEGs involved in conceptus
elongation are summarized in Figure 3.3. DEGs between long and short length conceptuses
were primarily involved in the immune response and lipid metabolism pathway categories.
Individual pathways significant at P-value ≤ 0.05 within these two major pathway categories,
as well as the genes involved in these pathways, are summarized in Table 3.7. The majority
of the pathways and DEGs involved in lipid metabolism were up-regulated in long
conceptuses compared to short conceptuses. Other pathways of interest included those
involved in carbohydrate and steroid metabolism, and development.
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To attain a broader, more dynamic view of biological functions and pathway
interactions, the list of DEGs solely up-regulated in long conceptuses and solely up-regulated
in short conceptuses was submitted for network analysis. The top scoring GeneGo Process
Networks containing genes up-regulated in long and up-regulated in short conceptuses
compared to their counterparts are summarized in Figure 3.4. Networks containing DEGs upregulated in long conceptuses were involved in cellular reorganization and development,
proliferation, and transport. Networks containing DEGs up-regulated in short conceptuses
included those involved in immune response, inflammation, and apoptosis.
Differential gene expression between female and male conceptuses
When comparing all conceptuses from Experiments 1 and 2, there were 12 genes
differentially expressed between conceptuses of different gender (7 up-regulated and 5 downregulated in female conceptuses compared to male conceptuses; Table 3.8). Expression of X
(inactive)-specific transcript (XIST) was 37-fold greater in female conceptuses compared to
male conceptuses. In addition there appeared to be a number of other genes that were
differentially expressed between male and female conceptuses. Three of the genes more
highly expressed in male conceptuses were Y-linked genes including ubiquitously
transcribed tetratricopeptide repeat gene, Y-linked (UTY), oral-facial-digital syndrome 1 Ylinked (OFD1Y), and zinc finger, RNA-binding motif and serine/arginine rich 2 (ZRSR2Y).
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When comparing only the conceptuses utilized in Experiment 2, there were 10 genes
differentially expressed between conceptuses of different gender (7 up-regulated and 3 downregulated in female conceptuses compared to male conceptuses; Table 3.9).
Experiment 3
Day 15 conceptus recovery summary
The recovery rate (number of conceptuses recovered/number of embryos transferred)
was 50.0% (4/8) for the IVO group, 38.5% (5/13) for the TCM-199 group, and 29.2% (7/24)
for the cSOF group. The two IVP groups had a combined recovery rate of 32.4% (12/37).
There was not a significant difference in recovery rate between IVO, TCM-199, and cSOF
grops nor between IVP combined and IVO groups (P-value>0.05). Conceptus length ranged
from ovoid (2.5 mm) to late tubular (99.8 mm). There was no significant difference (P>0.05)
in average length or area between treatment groups (Table 3.10).
Conceptuses selected for microarray analysis based on length
Average lengths and areas of the selected Day 15 conceptuses for each comparison
are summarized in Table 3.11. For the length comparison of the IVP treatment group, five
conceptuses were selected and classified as short while four conceptuses were selected and
classified as long. Conceptuses in the short group were tubular, except for one ovoid
conceptus. Conceptuses classified as long were all tubular. There were no differences in the
male:female ratio between and within groups (P-value>0.05).
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For the length comparison of the IVO treatment group, there were two short and two
long conceptuses. The two conceptuses in the short group were early tubular and the two
long conceptuses were tubular in shape. There were no differences in the male:female ratio
between and within groups (P-value>0.05).
Conceptuses selected for microarray analysis based on production system
For the first production comparison, all conceptuses from the IVO (n=4) and all
conceptuses from IVP groups (n=9) were compared. Furthermore, a subsequent production
comparison was controlled for average length. In this comparison, four conceptuses were
selected from the IVP group which combined had similar average length to the four IVO
conceptuses.
Gene expression
There was no difference (P-value>0.05) in gene expression profiles at Day 15 in
conceptuses derived from Grade 1 blastocysts from the TCM-199 (n=6) compared to the
SOF (n=3) IVP culture systems. Therefore, conceptuses derived from both culture systems
were combined into a single IVP treatment group. No difference in gene expression profiles
was found between Day 15 conceptuses regardless of whether they were derived from Grade
1 blastocysts from either IVO or IVP embryo production systems. This was true for both the
comparison of all IVO and IVP conceptuses, as well as the comparison between four IVO
and four IVP conceptuses of similar average lengths. In addition, there was no difference (P-
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value>0.05) in gene expression profiles between male and female conceptuses in Experiment
3 (4 males, 9 females).
In contrast to our results in Experiment 2 within the IVO conceptuses (n=5 per
group), there was no difference in gene expression between short and long IVO conceptuses
derived from Grade 1 blastocysts (n=2 per group).
There was only 1 DEG detected between long and short conceptuses on Day 15
derived from IVP embryos. This gene was regulator of calcineurin 2 (RCAN2) which was
up-regulated 4.09-fold (P-value<0.05) in long IVP-derived conceptuses compared to short
IVP-derived conceptuses.
Quantitative Real-Time PCR
The microarray results were verified for selected differentially expressed genes by
qPCR (Figure 3.5). For Experiment 2, all five genes tested were significantly differentially
expressed with a fold change ratio >1.5 between long and short conceptuses. The direction of
change was in agreement with the microarray results.
For Experiment 3, the relative expression fold change of RCAN2 was >1.5 between
long and short conceptuses and was statistically significant. The direction of change was in
agreement with the microarray results. Also in agreement with the microarray results, the
relative expression differences for the other transcripts studied were low and not statistically
significant (data not shown).
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DISCUSSION
There were no significant differences in gene expression between conceptuses
derived from Grade 1 or Grade 3 embryos. There are two possible explanations that may
account for this result. First, it is possible that the differences in morphology seen at Day 7
are subsequently minimized following exposure to the uterine environment. Consistent with
this idea, differences in gene expression between Day 7 IVP and IVO embryos were also not
observed in Day 14 embryos [84]. An alternative explanation may be that embryonic loss of
Grade 3 embryos may occur prior to Day 15 of gestation. Thus, the conceptuses studied in
Experiment 1 may represent those with greater developmental competency, contributing to a
lack of difference in gene expression in Day 15 conceptuses derived from Grade 1 versus
Grade 3 embryos identified on Day 7.
Elongation of the ruminant and porcine conceptus is required for the establishment
and maintenance of pregnancy, which also coincides with the period of greatest embryonic
loss. It is possible, therefore, that early embryonic death may be a result of inadequate
elongation. A wide range of bovine conceptus lengths is commonly observed when harvested
during the peri-implantation period [59, 84, 87, 106, 214, 215, 221, 222]. This raises several
questions regarding the viability of the elongating embryos showing impeded growth as
compared to their counterparts. Conceptuses that demonstrate impeded growth may be less
developmentally competent, due to inherent defects in the individual that would impair their
survival even in the most receptive environment. Alternatively, shorter conceptuses may be
developmentally delayed, causing an asynchrony with the uterine environment, but may
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survive if transferred into a more synchronous recipient. Therefore, understanding the nature
of the differences in gene expression between shorter conceptuses with impeded growth
compared to longer conceptuses that are recovered on the same day of gestation may provide
insights that will clarify how competent short conceptuses really are.
Based on a meta-analysis of gene expression in the elongating conceptus, genes
commonly identified between three studies were involved in functions such as lipid
metabolism, connective tissue development and cellular assembly, cellular growth and
differentiation, and inflammatory response [80]. Four genes were common to the three
studies in the meta-analysis and included insulin-like growth factor binding protein 7
(IGFBP7), pregnancy-associated glycoproteins 2 (PAG2) and 11 (PAG11), and trophoblast
Kunitz domain protein 3 (TKDP3). Results of Experiment 2 in our study are in general
agreement with these findings. In our study, IGFBP7 and a different member of the TKDP
family, TKDP2, were more highly expressed in long conceptuses compared to short
conceptuses. Interestingly, in Experiment 2, PAG11 was one of the top three most highly
expressed transcripts in both long and short conceptuses, however expression of PAG12 was
8.9-fold higher in long conceptuses compared to short conceptuses. PAGs are a family of
aspartic proteinases expressed exclusively by trophoblast cells [231], and have been used to
monitor embryonic and fetal viability in cattle [232].
Lipid metabolism is frequently identified as a being a top process differentially
expressed between somatic cell nuclear transfer (SCNT), IVP, and IVO elongating
conceptuses [80]. Lipid metabolism, as well as other forms of cellular metabolism, were also
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part of the top molecular functions containing DEGs between normal and retarded buffalo
embryos during the period of highest embryonic mortality [233]. Lipids and fatty acids are
used for energy production, and are precursors for prostaglandin and steroid biosynthesis. In
particular, the gene apiolipoprotein A-1 (APOA1), has been found to be expressed in cattle
during conceptus elongation [93, 234, 235]. APOA1 was one of the twenty most highly
expressed genes at five studied time points from Day 7 to Day 19 of gestation [93, 234]. In
our study, APOA1 was up-regulated in long conceptuses compared to short conceptuses.
Prostaglandin-endoperoxidase synthase 2 or prostaglandin G/H synthase and
cyclooxygenase (PTGS2), also known as COX-2, is expressed in the endometrium as well as
in the trophectoderm of the elongating conceptus in ruminants [236]. PTGS2 is up-regulated
by IFNT [237], and its expression in Day 7 bovine embryos has been identified as a predictor
of developmental competency to term [50, 51]. Furthermore, PTGS2 has been linked to
conceptus elongation in pigs [238] and in sheep, as administration of a prostaglandin
synthase inhibitor during early pregnancy in ewes retarded conceptus elongation at Day 14
[239]. PTGS2-derived prostaglandin from the conceptus likely has paracrine and autocrine
effects on development and may mediate the effects of progesterone and IFNT in the
endometrium [67, 239]. In our study, PTGS2 was up-regulated in long conceptuses compared
to short conceptuses. This may suggest that shorter conceptuses have reduced developmental
competency as compared to their longer counterparts, as it is down-regulated in biopsies
from both in vivo and in vitro produced embryos resulting in no pregnancy or no calf
delivery [50, 51, 64].
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Conceptus elongation is required for continued survival, as increased surface area
enables maternal-conceptus cross-talk and nutrient exchange. Inadequate elongation could be
a major cause of the high incidence of early embryonic death seen during the periimplantation period. In the current study, several genes known to be related to the elongation
process were more highly expressed in the long length conceptuses. For example, prolinerich 15 (PRR15) is required for normal conceptus development in sheep [240]. Interestingly,
PRR15 mRNA was not detected in ovine conceptuses that were spherical in shape at Day 11
of gestation, but PRR15 expression increased quadratically from Day 13 to peak at Day 16,
coincident with the period in sheep when conceptus elongation stops [240]. A few genes
more highly expressed in the long conceptuses were also up-regulated in porcine conceptuses
of tubular shape compared to ovoid shape [238]. These included genes involved in
steroidogenesis (aromatase, CYP19A1), oxidative stress response (microsomal glutathione Stransferase 1, MGST1), and prostaglandin synthesis (COX-2/PTGS2). Interestingly,
expression of midkine (MDK) was highest in tubular porcine conceptuses compared to ovoid
or filamentous, however MDK was more highly expressed in short conceptuses in the present
study [238]. This may be indicative of differences between the two species, or MDK may
have multiple roles in elongation.
The top network containing DEGs with higher expression in long conceptuses was
related to actin filaments of the cytoskeleton. DEGs involved in this network included
TAGLN, TPM2, TPM4, SPTBN2, MYO1B, PAK1, and MTSS1.The actin cytoskeleton is
essential for conceptus elongation, as it generates force for controlling the shape and
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structure of cells [241]. Interestingly, IL6 was up-regulated in long conceptuses and has been
shown to induce an increase in endothelial permeability in vitro by rearranging actin
filaments and changing the shape of bovine endothelial cells [242], which could be important
for mediating conceptus elongation. Another DEG related to conceptus elongation was
junctional adhesion molecule 2 (JAM2). Betsha et al [143] reported that JAM2 was more
highly expressed in disproportionally short Day 16 conceptuses derived from IVP and SCNT
embryos. This is consistent with our results, as JAM2 was up-regulated in short conceptuses
compared to long conceptuses in Experiment 2. They hypothesized that down-regulation of
JAM2 is required for enhanced trophoblast elongation.
Although IFNT secretion is correlated to conceptus length [59, 243, 244], there was
not a significant difference in the level of expression of IFNT between long and short
conceptuses. In fact, IFNT was the most highly expressed transcript in both groups. Our
observations are consistent with those of Robinson et al. [244] who reported that the increase
in IFNT secretion into the uterus around the time of maternal recognition of pregnancy was
due to an increase in embryo size, reflecting an increase in the mass of trophoblast, not an
up-regulation of mRNA expression. In addition to IFNT, the elongating conceptus secretes
cytokines that can modulate the maternal immune system. Expression of interleukin-6 (IL6)
during the peri-implantation period is reported to be important for the establishment and
maintenance of pregnancy in sheep, pigs, and cows [245, 246]. IL6 was up-regulated in long
conceptuses compared to short conceptuses in the current study. This observation may
suggest that short conceptuses are less competent than their longer counterparts.
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Immune responses play a critical role in the establishment and maintenance of
pregnancy in ruminants. Trophoblast cells must come into direct contact with the uterine
epithelium and protect the developing fetus (embryonic disc) from immune rejection by the
maternal system. Therefore, the maternal immune system is generally suppressed in order to
protect the allogenic conceptus from maternal immune attack [247]. Immunorejection may be
an important contributing factor in early pregnancy loss in cattle, since expression of
immune-system related genes are suppressed in receptive compared to nonreceptive
endometrium [64]. Results of our study indicate that long and short conceptuses display
differential regulation of immune-related genes, as several of the top pathways and networks
affected include those related to the complement system, inflammation, and phagocytosis. It
is unclear whether shorter conceptuses are being targeted for attack by the maternal immune
system, or whether shorter conceptuses are simply expressing delayed immune-related gene
expression because they are behind in development. In humans, pregnancy is associated with
a shift from a cell-mediated immune response to an antibody-based response, though there
exists a delicate balance between the two [248]. Antibody-based immune responses are
associated with increased levels of certain interleukins [245], including IL6. IL6 has been
implicated as a modulator of local inflammation and autoimmunity in humans [249]. In our
study, IL6 was up-regulated in long conceptuses, perhaps suggesting the long conceptuses
have or are shifting to antibody-based immune responses.
There are two classes of major histocompatibility complex class I (MHC-I)
molecules, classical and nonclassical. Classical MHC are highly polymorphic and are thus
146
capable of presenting many different types of antigens, including paternal antigens found on
invasive trophoblast cells in cattle and horses [250, 251]. Consequently, trophoblast cells
may fail to express classical MHC-I molecules containing paternal antigens. Nonclassical
MHC-I proteins express a "zero" antigen to evade uterine Natural Killer cells while being
recognized as self antigen by leukocytes [252]. MHC-I molecules are generally not expressed
from cells of non-invasive regions of the human placenta nor in non-invasive trophoblast
cells of the placenta in ungulates [253-255]. MHC-I expression is down-regulated in sheep
and cattle trophoblast cells during the first and second trimesters of pregnancy, the period
when intimate contact between the maternal and fetal tissues is initiated [256, 257].
Futhermore, inappropriate first trimester MHC-I expression was reported during clone
pregnancies in cows [257]. This is suspected to be involved in pregnancy loss due to attack
by activated cytotoxic T lymphocytes (CTLs). In Experiment 2, expression of the transcript
BOLA-N/JSP.1, which shares homology with classical human MHC-I molecules, was downregulated in long conceptuses compared to short conceptuses. Furthermore, expression of the
cell surface glycoprotein CD8A, which is found on CTLs and interacts with MHC-I
molecules, showed the same pattern of down-regulation in long conceptuses compared to
their shorter counterparts. Expression of CD8A, along with other immune-related genes, was
down-regulated in the endometrium of heifers with successful pregnancy and calf delivery
[258]. This would seem to suggest that shorter Day 15 conceptuses, like those of the cloned
pregnancies, may not be adequately suppressing expression of classical MHC-I molecules
and therefore may be subject to immune attack from the maternal system.
147
Interestingly, down-regulated expression of β-2-microglobulin (B2M), the light chain
of the MHC-I molecule, is also associated with reduced developmental competency in both
in vivo and in vitro produced embryos [50, 51]. Furthermore, B2M is proposed to be an
important intermediate mitogen for cytokines such as IFNT and IL6 to mediate conceptus
and endometrial growth [259]. In our study, IL6 and B2M were down-regulated in short
conceptuses relative to long conceptuses, further supporting the notion of the reduced
viability of short conceptuses. B2M is an interferon-stimulated gene which is expressed in
the developing placenta in mice [260] and in the conceptus trophectoderm in sheep [261]. β2-microglobulin is a component of all MHC-I molecules, including the nonclassical MHC-I.
β-2-microglobulin is also a component of another MHC-I family protein, the neonatal IgG
transporter, FcRn [262]. FcRn is involved in regulating IgG transport and catabolism [263].
Interestingly, the gene for the high affinity receptor for the Fc portion of IgG (FCGRA1) was
also comparatively up-regulated in long conceptuses in our study. Studies in mice and
humans have shown FcγR1 can bind monomeric IgG and may be pro-inflammatory [264,
265]. Other immune-related DEGs in the current study included the comparative upregulation of T cell receptor alpha (TCRA) in long conceptuses, and the higher expression of
Ig kapp chain (IGK) and the TNF receptor-associated factors TRAF3 and TRAF4 in short
conceptuses.
Genes involved in activation of the complement system, part of the innate immune
response, were differentially regulated in long versus short conceptuses. Expression of the
classical complement pathway genes, C1R and C1S, were more highly expressed in long
148
conceptuses compared to short conceptuses. In the classical complement pathway, C1 is
primarily activated by antigen-bound antibody molecules, specifically the Fc portion of
bound IgG. C1R and C1S are up-regulated in the endometrium of Day 18 pregnant versus
cyclic cows [220], and similar patterns of regulation are seen in humans [266]. In addition to
their role in the complement system, C1S and C1R have been shown to cleave the heavy
chain of MHC class I antigens, and C1S has proteolytic activity against the light chain B2M
[267, 268]. Cleavage of MHC-I molecules releases the foreign antigen-binding site and the
T-cell receptor recognition regions, indicating a role of C1S complement cleavage in the
immune response [268]. In contrast, C3, which is involved in all three complement pathways,
was more highly expressed in short conceptuses. These results highlight the complexity and
the importance of the immune system during conceptus elongation, and may provide further
evidence that improper, or delayed, regulation of immune-related genes may contribute to
early embryonic loss.
Differences in gene expression between male and female conceptuses in Experiment
2 revealed a large up-regulation of the transcript XIST. XIST is involved in initiating X
chromosome inactivation and maintaining normal development in female embryos [269].
Interestingly, XIST expression in bovine embryos is initiated around the 2-cell stage, with an
increase in expression during blastocyst elongation [270]. XIST was detectable in all female
bovine conceptuses by Day 14 of development [270]. Several of the transcripts differentially
expressed between male and female conceptuses were autosomal genes. This is consistent
with results from Bermejo-Alvarez et al. [271], and suggests that the sex chromosomes can
149
indirectly influence sexual dimorphism. Consequently, the same research group proposed
that the period prior to maternal recognition of pregnancy may be associated with sexspecific embryo loss [95].
In contrast to the numerous differences between long and short IVO conceptuses in
Experiment 2, there were no significant differences in the transcriptome of short and long
IVO conceptuses in Experiment 3. Furthermore, there was only 1 differentially expressed
gene identified between long and short conceptuses derived from IVP embryos in
Experiment 3. There are several potential factors that could have contributed to this apparent
disparity in results. First, in Experiment 2 the IVO conceptuses were derived from Grade 1
embryos assessed and transferred at the compact morula stage, whereas the IVO and IVP
embryos were assessed and transferred at the blastocyst stage in Experiment 3. Therefore, it
is possible that assessing embryo morphology at the morula stage may be too early to predict
embryo viability. As a result, there may have been more embryos with reduced
developmental competence transferred in Experiment 2 compared to Experiment 3. Second,
it is also important to note that Experiment 2 compared five short and five long IVO
conceptuses, while the comparison of IVO conceptuses in Experiment 3 only utilized two
short and two long conceptuses. Finally, a third explanation for the disparity in our results
could be related to the differences in morphology and length between conceptuses studied in
Experiment 2 and Experiment 3. Conceptuses classified as short in Experiment 2 were an
average of 4 mm long and ovoid in shape, while conceptuses classified as long were tubular
in shape, with an average length around 25 mm. In contrast, all but one conceptus selected
150
for analysis in Experiment 3 was tubular in shape, and the average lengths for both length
groups were higher for the IVP-derived conceptuses. The transcriptome of tubular
conceptuses of differing lengths may not differ as much as the transcriptomes of ovoid and
tubular conceptuses. In addition, the relative lack of DEGs between long and short IVPderived conceptuses may indicate that differences in morphology of IVP-derived conceptuses
is not as directly related to gene expression as seen in the IVO-derived conceptuses.
In summary, we report differences in gene expression between IVO-derived Day 15
bovine conceptuses of differing trophoblast lengths. Differentially expressed genes were
largely involved in metabolic and biosynthetic processes and immune response. Long and
short IVO conceptuses from Experiment 2 displayed differential regulation of genes
previously identified as associated with developmental competency to term and enhanced
elongation. Therefore, in conclusion, we propose that conceptuses with impeded growth may
be of inferior developmental competency. The relative lack of differentially expressed genes
identified in Day 15 short and long conceptuses derived from IVO and IVP embryos in
Experiment 3 may be due to differences in experimental factors between Experiments 2 and
3. Specifically, we propose that conceptuses at an earlier morphological stage of
development than expected based on the day of gestation are likely developmentally
compromised. Those conceptuses that are at the expected morphological stage, but perhaps
vary in length within that morphological stage, likely are of similar and superior
developmental competency. Using our study as an example, we would expect conceptuses
collected at Day 15 to be at the tubular stage of elongation. Therefore, ovoid conceptuses
151
collected at Day 15 may have reduced viability. In contrast, conceptuses identified as tubular,
regardless of length, likely do not differ in developmental competency and represent
conceptuses of superior viability. In conclusion, differences in gene expression between
conceptuses collected at the same day of development may be more heavily influenced by
conceptus morphology than differing conceptus lengths within a morphological stage.
152
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Table 3.1: List of target genes, primer sequences, and product sizes of transcripts validated by qPCR.
Target
Accession
Sequence
Forward Primer
Reverse Primer
Product
Size (bp)
AQP3
NM_001079794.1
CGGTGGTTTCCTCACCATCA
GGCCCAGATCGCATCGTAAT
229
TP53I3
NM_001075378.1
GCGGACTTACTCCAGAGACAA
ACATACTGAGCCTGACCCCC
173
IL6
NM_173923.2
TCCAGAACGAGTATGAGG
CATCCGAATAGCTCTCAG
236
TAGLN
NM_001046149.1
CAACATGGCCAACAAGGGTC
CAGCTTGCTCAGAATCACGC
199
JAM2
NM_001083736.1
CGCGGTGTTTCCTTTGTCTA
TGCCGGAGCCACTAATACTTC
204
H2AFZ
NM_174809.2
AGGACGACTAGCCATGGACGTGTG
GTTCCGATGTTAACGACCACCACC
209
RCAN2
NM_001015632.1
GCTGCCCAGCTCTTTCTTGT
AGGCCCTCAAATTTTTCCTTAACC
140
Table 3.2: Summary of all measured Day 15 conceptuses recovered for Experiments 1 and 2 (mean ± SEM).
Embryo
Grade
N
Average
Length (mm)
Median
Length (mm)
Length Range
Low-High (mm)
Average
Area (mm2)
Median Area
(mm2)
1
3
17
4
10.5 ± 2.4
11.6 ± 3.9
4.7
12.9
1.7-29.8
1.0-19.7
14.0 ± 3.3
14.2 ± 5.0
6.5
15.9
Table 3.3: Summary of Day 15 conceptuses selected for microarray analysis in Experiments 1 and 2 (mean ±
SEM).
Comparison
Treatment
N
Gender Ratio
(male:female)
Average
Length (mm)
Median
Length (mm)
Average
Area (mm2)
Median
Area (mm2)
Grade
Grade 1
4
2:2
19.9 ± 2.6
20.5
30.3 ± 4.8
31.1
Grade 3
3
2:1
15.2 ± 2.4
14
18.8 ± 2.9
16.2
Short
5
2:3
4.2 ± 0.1
4.1
5.6 ± 0.6
5.9
Long
5
1:4
24.7 ± 1.9
25.6
32.3 ± 3.3
34.0
Length
165
Figure 3.1: Examples of one short (left, 4.0 mm) and one
long (right, 25.6 mm) Day 15 conceptus derived from
Grade 1 IVO embryos.
166
Table 3.4: The top 25 up-regulated and the top 25 down-regulated differentially expressed genes in long
conceptuses compared to short conceptuses derived from Grade 1 IVO embryos. A positive value indicates upregulation in long conceptuses while a negative value indicates up-regulation in short conceptuses.
Gene Symbol1
Gene Title
C15H11orf96
PAG12
TP53I3
IL6
TAGLN
AHSG
PRUNE2
BMP2
Bt.23770.1.A1_at
S100A11
MSRB3
SRPX
PRR15
SSBP2
ELOVL5
CYP19A1
IGFBP7
MBOAT1
GATM
Bt.16188.1.S1_at
IDH1
PTER
PAQR8
Bt.29415.1.A1_at
NDRG1
IFITM3
PRSS35
BOLA-N /// JSP.1
TMPRSS2
C3
EGFLAM
LOC514189
CTNNAL1
CERS4
Bt.13920.1.S1_at
NAAA
NDN
XPNPEP2
NRP1
GSDMC
JAM2
DNASE1L3
SYBU
chromosome 15 open reading frame, human C11orf96
pregnancy-associated glycoprotein 2-like /// pregnancy-associated glycoprotein 12
tumor protein p53 inducible protein 3
interleukin 6 (interferon, beta 2)
transgelin
alpha-2-HS-glycoprotein
prune homolog 2 (Drosophila)
bone morphogenetic protein 2
--S100 calcium binding protein A11
methionine sulfoxide reductase B3
sushi-repeat containing protein, X-linked
proline rich 15
single-stranded DNA binding protein 2
ELOVL fatty acid elongase 5
cytochrome P450, family 19, subfamily A, polypeptide 1
insulin-like growth factor binding protein 7
membrane bound O-acyltransferase domain containing 1
glycine amidinotransferase (L-arginine:glycine amidinotransferase)
--isocitrate dehydrogenase 1 (NADP+), soluble
phosphotriesterase related
progestin and adipoQ receptor family member VIII
--N-myc downstream regulated 1
interferon induced transmembrane protein 3
protease, serine, 35
MHC class I antigen /// MHC Class I JSP.1
transmembrane protease, serine 2
complement component 3
EGF-like, fibronectin type III and laminin G domains /// pikachurin-like
uncharacterized LOC514189
catenin (cadherin-associated protein), alpha-like 1
ceramide synthase 4
--N-acylethanolamine acid amidase
necdin homolog (mouse)
X-prolyl aminopeptidase (aminopeptidase P) 2, membrane-bound
neuropilin 1
gasdermin C
junctional adhesion molecule 2
deoxyribonuclease I-like 3
syntabulin (syntaxin-interacting)
Fold
Change
11.14
8.90
8.77
5.42
4.95
4.50
4.45
4.36
4.35
4.13
4.02
3.93
3.92
3.79
3.70
3.63
3.50
3.46
3.35
3.22
3.21
3.14
3.07
2.97
2.86
-2.04
-2.04
-2.05
-2.11
-2.11
-2.13
-2.22
-2.24
-2.26
-2.31
-2.41
-2.56
-2.59
-2.62
-2.66
-2.66
-2.71
-2.88
Adjusted
P-value
0.015
0.021
0.012
0.013
0.012
0.031
0.014
0.014
0.016
0.042
0.018
0.015
0.014
0.031
0.015
0.028
0.013
0.039
0.014
0.031
0.030
0.030
0.031
0.012
0.020
0.012
0.017
0.025
0.021
0.025
0.038
0.031
0.013
0.043
0.017
0.039
0.014
0.013
0.023
0.023
0.013
0.027
0.022
167
Table 3.4 Continued
1
FOLR1
AQP3
SLC5A5
PDGFRA
ATP6V0D2
folate receptor 1 (adult)
aquaporin 3 (Gill blood group)
solute carrier family 5 (sodium iodide symporter), member 5
platelet-derived growth factor receptor, alpha polypeptide
ATPase, H+ transporting, lysosomal 38kDa, V0 subunit d2
-3.10
-3.16
-3.27
-3.47
-3.72
0.031
0.012
0.012
0.012
0.020
LOC100848243
ZNF503
uncharacterized LOC100848243
zinc finger protein 503
-3.79
-6.76
0.015
0.032
Transcripts without annotations were identified by probe set ID
168
Figure 3.2: The top GO biological processes overrepresented in the full list of DEGs between long and short
conceptuses derived from IVO embryos. DEGs up-regulated (underlined) or down-regulated (plain type) in long
conceptuses compared to short are included for select processes.
169
Table 3.5: The top biological processes overrepresented in genes solely up-regulated in long conceptuses (top)
and solely up-regulated in short conceptuses (bottom) derived from IVO embryos.
Biological Process
Up-regulated in long conceptuses
lipid biosynthetic process
cellular aldehyde metabolic process
acute inflammatory response
cellular amino acid derivative metabolic process
phospholipid biosynthetic process
response to wounding
muscle organ development
cholesterol metabolic process
regulation of immune effector process
steroid metabolic process
cholesterol biosynthetic process
sterol metabolic process
sterol biosynthetic process
heart trabecula formation
defense response
carboxylic acid biosynthetic process
organic acid biosynthetic process
inflammatory response
glycerophospholipid biosynthetic process
phosphatidic acid metabolic process
response to interferon-gamma
phosphatidic acid biosynthetic process
striated muscle tissue development
muscle tissue development
regulation of adaptive immune response
regulation of adaptive immune response based on somatic recombination of
immune receptors built from immunoglobulin superfamily domains
Up-regulated in short conceptuses
ribosome biogenesis
ribonucleoprotein complex biogenesis
coenzyme metabolic process
coenzyme biosynthetic process
L-serine metabolic process
cofactor metabolic process
folic acid and derivative biosynthetic process
one-carbon metabolic process
cofactor biosynthetic process
No. of DEGs
P-value
9
4
5
6
4
7
5
4
4
5
3
4
3
2
7
5
5
5
3
2
2
2
4
4
3
0.001
0.001
0.002
0.003
0.014
0.015
0.015
0.019
0.019
0.021
0.022
0.024
0.027
0.027
0.028
0.029
0.029
0.033
0.039
0.040
0.040
0.040
0.042
0.046
0.048
3
0.048
4
4
4
3
2
4
2
3
3
0.015
0.025
0.034
0.046
0.057
0.067
0.073
0.077
0.085
170
Table 3.6: Molecular functions and cellular components overrepresented in genes up-regulated in long
conceptuses (top) and up-regulated in short conceptuses (bottom) derived from IVO embryos.
Term
No. of DEGs
P-value
Up-regulated in long conceptuses
Molecular function
calcium-dependent phospholipid binding
phospholipid binding
lipid binding
calcium ion binding
transferase activity, transferring nitrogenous groups
aromatase activity
heme binding
electron carrier activity
tetrapyrrole binding
3
5
7
11
3
2
4
5
4
0.008
0.011
0.037
0.043
0.051
0.060
0.082
0.089
0.097
Cellular Component
microsome
vesicular fraction
external side of plasma membrane
cell surface
endoplasmic reticulum
5
5
4
5
10
0.012
0.013
0.063
0.070
0.091
Molecular function
vitamin binding
vitamin B6 binding
pyridoxal phosphate binding
4
3
3
0.031
0.040
0.040
Cellular Component
vacuole
4
0.065
Up-regulated in short conceptuses
171
Figure 3.3: The top 20 most significant GeneGo Pathway Maps
containing DEGs between long and short length conceptuses.
P-value <0.05.
172
Table 3.7: Top GeneGO pathway categories and their corresponding pathways differentially expressed in long
and short length conceptuses. The genes that were relatively up-regulated in long conceptuses (underlined) and
up-regulated in short conceptuses (plain type) involved in the pathway categories are listed. Only pathways
significant at an adjusted P-value ≤ 0.05 are included.
Top Pathway
Map Categories
Pathway Maps
Genes
Immune
Response
Classical complement pathway; Alternative complement
pathway; Lectin induced complement pathway; iC3b-induced
phagocytosis via alpha-M/beta-2 integrin; IL-33 signaling
pathway
C1S, C1R, C3, BOLAN/JSP.1, B2M, CD8A,
ITPR1, PAK1, PRKCI,
H2B, IL33, IL6
Lipid
Metabolism
Unsaturated fatty acid biosynthesis; n-3 Polyunsaturated fatty
acid biosynthesis; n-6 Polyunsaturated fatty acid biosynthesis;
Phospholipid metabolism p.1; Triacylglycerol metabolism
p.1; N-Acylethanolamines, HSRL5-transacylation pathway;
G-alpha(q) regulation of lipid metabolism; PDGF activation
of prostacyclin synthesis; Fatty Acid Omega Oxidation;
Leukotriene 4 biosynthesis and metabolism
ACSL5, ELOVL4,
ELOVL5, FADS1,
LPCAT3, AGPAT1,
MBOAT1, ALDH3A2,
ALDH9A1, PTGS2,
ITPR1, PRKCI, DPEP1
173
Figure 3.4: Top 10 most significant GeneGo Process Networks containing genes (A) up-regulated in long length conceptuses (B) and up-regulated
in short length conceptuses.
174
Table 3.8: Differentially expressed genes (n=12) between female and male Day 15 conceptuses derived from
IVO embryos in Experiments 1 and 2. A positive value indicates up-regulation in female conceptuses while a
negative value indicates up-regulation in male conceptuses.
Gene Symbol1
Gene Title
XIST
ECH1
IDH3G
X (inactive)-specific transcript
enoyl CoA hydratase 1, peroxisomal
isocitrate dehydrogenase 3 (NAD+) gamma
inhibitor of kappa light polypeptide gene enhancer in B-cells,
IKBKG
kinase gamma
Bt.13308.1.S1_at --LOC100849028
uncharacterized LOC100849028
ASB11
ankyrin repeat and SOCS box containing 11
KDM6A ///
lysine (K)-specific demethylase 6A /// histone demethylase UTYLOC100851307
like /// ubiquitously transcribed tetratricopeptide repeat gene, Y/// UTY
linked
OFD1Y
oral-facial-digital syndrome 1 Y-linked
NUDT10
nudix (nucleoside diphosphate linked moiety X)-type motif 10
Bt.28573.1.A1_at --zinc finger (CCCH type), RNA-binding motif and serine/arginine
ZRSR2Y
rich 2
1
Transcripts without annotations were identified by probe set ID.
Fold
Change
Adjusted
P-value
37.11
2.34
1.68
0.000
0.018
0.000
1.54
1.53
1.53
1.50
0.000
0.014
0.020
0.008
-1.70
-2.14
-2.67
-4.91
0.002
0.001
0.044
0.000
-6.21
0.000
Table 3.9: Differentially expressed genes (n=10) between female and male Day 15 conceptuses derived from
IVO embryos in Experiment 2. A positive value indicates up-regulation in female conceptuses while a negative
value indicates up-regulation in male conceptuses.
Gene Symbol1
XIST
ECH1
AUTS2
IDH3G
MTCP1NB
LOC100852254
/// UPF3B
BRCC3
OFD1Y
Bt.28573.1.A1_at
Gene Title
X (inactive)-specific transcript
enoyl CoA hydratase 1, peroxisomal
autism susceptibility candidate 2
isocitrate dehydrogenase 3 (NAD+) gamma
mature T-cell proliferation 1 neighbor
regulator of nonsense transcripts 3B-like /// UPF3 regulator of
nonsense transcripts homolog B (yeast)
BRCA1/BRCA2-containing complex, subunit 3
oral-facial-digital syndrome 1 Y-linked
--zinc finger (CCCH type), RNA-binding motif and serine/arginine
ZRSR2Y
rich 2
1
Transcripts without annotations were identified by probe set ID.
Fold
Change
Adjusted
P-value
47.26
2.46
1.74
1.67
1.60
0.000
0.048
0.014
0.032
0.016
1.53
1.50
-2.19
-5.45
0.014
0.009
0.035
0.000
-6.36
0.000
175
Table 3.10: Summary of all measured Day 15 conceptuses recovered for Experiment 3 (mean ± SEM).
Treatment
N
Average
Length (mm)
Median
Length (mm)
Length Range
Low-High (mm)
Average
Area (mm2)
Median
Area (mm2)
IVO
IVP TCM-199
IVP cSOF
4
7
5
33.2 ± 5.1
30.8 ± 8.1
39.8 ± 16.4
34.7
21.4
32.7
20.6-42.6
6.4-66.2
2.5-99.8
39.7 ± 14.1
35.8 ± 11.8
44.9 ± 16.6
29.6
20.1
43.4
Table 3.11: Summary of Day 15 conceptuses selected for microarray analysis in Experiment 3 (mean ± SEM).
Comparison
Treatment
N
Gender Ratio
(male:female)
Average
Length (mm)
Median
Length (mm)
Average
Area (mm2)
Median
Area (mm2)
Production
IVO
IVP*
IVP**
4
4
9
2:2
4:0
7:2
33.2 ± 5.1
34.3 ± 4.7
29.3 ± 6.1
34.7
36.5
21.7
39.7 ± 14.1
39.9 ± 6.9
35.2 ± 9.2
29.6
41.1
22.1
IVP Length
IVP Short
IVP Long
5
4
3:2
4:0
16.5 ± 2.9
45.4 ± 7.2
19.1
41.4
16.6 ± 3.0
58.4 ± 13.0
18.2
49.4
IVO Short
2
1:1
IVO Long
2
1:1
*
IVP conceptuses selected based on length
**
All IVP conceptuse
25.0 ± 4.4
41.3 ± 1.3
25.0
41.3
25.4 ± 6.9
54.1 ± 27.2
25.3
54.1
IVO Length
176
30
**
qPCR
Microarray
25
20
Fold Change
15
10
**
**
*
5
0
-5
*
-10
-15
**
-20
IL6
TP53I3
TAGLN
AQP3
JAM2
RCAN2
Figure 3.5: Comparison of the fold changes of qPCR and microarray data for selected genes identified as
differentially expressed. The data shown for IL6, TP53I3, TAGLN, AQP3, and JAM2 are from Experiment 2.
The data shown for RCAN2 are from Experiment 3. A positive value indicates up-regulation in long
conceptuses and a negative value indicates down-regulation in long conceptuses compared to short conceptuses.
Statistical significance of the relative expression ratio is indicated (*P-value<0.05; **P-value <0.02).
177
CHAPTER 4: GENERAL CONCLUSIONS & FUTURE WORK
The high incidence of early embryonic loss seen in cattle occurs primarily during the
peri-implantation period. A greater knowledge of the complex mechanisms involved in
conceptus-endometrial interactions governing the establishment of pregnancy and conceptus
elongation is needed to understand the causes of this reduced reproductive efficiency and to
elucidate strategies to improve embryo survival. The research described in this dissertation
addresses the influence of maternal progesterone concentration at the time of transfer on
survival and development of in vivo and in vitro produced bovine embryos during the periimplantation period. Furthermore, this research addresses the effects of embryo morphology,
embryo production method, and conceptus elongation on the survival and gene expression of
conceptuses at Day 15 of development. The goal of this research was to gain a better
understanding of the mechanisms involved in early pregnancy success and conceptus
elongation in cattle.
A successful pregnancy requires a receptive endometrium, a competent embryo, and
proper communication between the two. Uterine receptivity develops as a response to
circulating progesterone and can be influenced by signals from the developing conceptus,
such as IFNT and prostaglandins. Progesterone alters the endometrial transcriptome, and thus
can influence histotroph secretion into the uterine lumen. Progesterone exerts its effects on
the endometrium by modulating the timing of the normal changes that occur in the
endometrial transcriptome [106-108, 272]. Inadequate progesterone during the first seven
days after estrus is associated with a delay in the expression of genes in the endometrium, a
178
delay in the down-regulation of the progesterone receptor, a reduction or delay in conceptus
elongation, and greater embryonic loss [96, 102, 108].
Embryo competency is associated with morphology at the time of transfer and gene
expression at the blastocyst stage. Foremost, the elongating conceptus must reach an
adequate size in order to produce sufficient quantities of IFNT to signal its presence to the
maternal system. In the absence of this signal, genes responsible for uterine receptivity are
turned off and luteolysis occurs. IFNT is also responsible for stimulating the expression of
other genes in both the maternal endometrium and developing conceptus that are associated
with uterine receptivity and conceptus elongation, respectively. Conceptus-endometrial
interactions are complex and require coordinated alterations in gene expression.
In my first study, there was no difference in pregnancy rate at Day 17 of gestation
between the IVO, IVPS, and IVPSR groups. However, the two IVP groups tended to have a
higher percentage of degenerated conceptuses recovered compared to the IVO group. It is
likely that the increase in degenerated conceptuses recovered from heifers receiving IVP
embryos is a result of a lower progesterone environment at Day 7 in these heifers as
compared to those receiving IVO embryos. This is consistent with results from other studies
showing the early progesterone environment is associated with pregnancy rate [4, 66, 96,
103]. However, it is also possible that the IVP embryos were developmentally compromised
as a result of exposure to in vitro culture conditions. In vitro embryo production is associated
with lower pregnancy rates and altered gene expression at the blastocyst stage compared to
those produced in vivo. Furthermore, the progesterone concentration in heifers receiving IVP
179
embryos was also lower when no conceptus was recovered compared to IVO controls. These
results suggest that a higher progesterone environment prior to and around Day 7 may be
required for survival of in vitro produced embryos. The altered gene expression patterns
reported in blastocyst-stage IVP embryos may be compromising their ability to communicate
properly with the maternal endometrium, which has also been compromised by inadequate
circulating progesterone. In effect, this research suggests that the progesterone environment
at the time of transfer is critical for the survival of IVP embryos, perhaps even more so than
for the survival of IVO embryos.
There was no significant difference in mean conceptus length at Day 17 between IVO
and IVP-derived conceptuses, even though the IVO embryos were transferred into recipients
with a higher progesterone environment at Day 7. Heifers receiving IVP embryos also had
lower progesterone levels on Day 7 at the time of transfer compared to those receiving IVO
embryos when longer conceptuses were recovered at Day 17. These results suggest that the
progesterone environment at the time of transfer does not influence elongation in IVPderived conceptuses as much as it does for IVO-derived conceptuses. These observations
may also indicate that in vivo and in vitro produced embryos respond differently to the early
progesterone environment. IVP embryos appear to require a higher progesterone
concentration around Day 7 of gestation compared to IVO embryos. However, progesterone
is not as influential on the elongation of IVP embryos, which likely rely on more intrinsic
factors to guide this process.
180
Despite differences in embryo morphology and recovery rate following transfer, there
were no differences in gene expression between Day 15 conceptuses derived from Grade 1
and Grade 3 embryos. We hypothesize that either the loss of Grade 3 embryos occurs prior to
Day 15, or that differences that exist at the morula stage are minimized by exposure to the
uterine environment. The latter hypothesis is supported by results from another study that
observed differences in the transcriptome of Day 7 embryos but not Day 14 embryos [84].
Future work could involve recovering conceptuses derived from Grade 1 and Grade 3
embryos earlier in development (perhaps on each day from Day 10 to 14), to elucidate when
and why Grade 3 embryos are associated with increased loss. If fewer Grade 3 embryos are
recovered compared to Grade 1 embryos, but those that are recovered have similar gene
expression profiles, then it is likely that the majority of Grade 3 embryos are lost prior to a
certain day of development. Thus, only the most competent embryos continue in
development. Alternatively, if differences in gene expression exist between Grade 1 and
Grade 3 embryos until a certain day of development, then it is likely that exposure to the
maternal environment is enabling some of the Grade 3 embryos to "catch up" in gene
expression.
Because conceptus elongation is required for the establishment and maintenance of
pregnancy, we hypothesized that the extent of conceptus elongation may be related to
developmental competency. Conceptuses displaying impeded growth during the periimplantation period may be developmentally compromised, whether due to an inherent defect
of the embryo or a developed asynchrony with the maternal environment. Following the
181
transfer of Grade 1 embryos in the second study, Day 15 conceptuses of short and long
length displayed differential expression of 348 genes. There were 221 differentially
expressed genes (DEGs) comparatively up-regulated in long conceptuses and 127 DEGs
comparatively up-regulated in short conceptuses. DEGs were generally related to metabolic
and biosynthetic biological processes, namely lipid metabolism, as well as immune response.
In our research, the majority of pathways and DEGs involved in lipid metabolism were upregulated in long conceptuses. Interestingly, other studies have indicated lipid metabolism as
a top process differentially expressed by elongating conceptuses of different production
methods [80] and by buffalo embryos of normal and retarded growth during the period of
their highest embryonic mortality [233]. Failure to express, or up-regulate, the expression of
genes related to the production of lipids and fatty acids by Day 15 of development may be
one piece of evidence pointing to the reduced developmental competency of shorter
conceptuses.
Studies have also reported changes in the expression of immune system-related genes
in the maternal endometrium during the peri-implantation period [64, 247, 258]. The
maternal immune system is generally suppressed during pregnancy to prevent rejection of the
conceptus, which presents as an allogeneic tissue. In Experiment 2 of our second study, short
and long conceptuses displayed differential regulation of many immune-system related
genes. Interestingly, genes associated with classical MHC-I molecules were down-regulated
in long conceptuses when compared to short conceptuses. A failure to suppress expression of
182
these genes may indicate that the short conceptuses were being subjected to immune attack
from the maternal system.
Short and long conceptuses also displayed differential regulation of genes known to
be associated with developmental competency to term. Genes such as PTGS2, B2M, and IL6
were up-regulated in long conceptuses, and have been linked to developmental competency
to term [50, 51, 246]. Other genes known to be associated with pregnancy establishment and
normal conceptus elongation, such as IGFBP7, TKDP2, PAG12, PRR15, APOA1 [80, 93,
234, 235, 240], were up-regulated in long conceptuses. These results are consistent with our
hypothesis that conceptuses which display impeded growth have reduced developmental
competency.
Several questions are raised as a result of this research. First, why are shorter
conceptuses associated with decreased viability? While the evidence does suggest shorter
conceptuses may have reduced developmental competency, the source of this defect is
unclear. Shorter conceptuses may have an inherent defect and, thus, are destined to fail
regardless of the uterine environment. Alternatively, shorter conceptuses may be
developmentally delayed compared to their longer counterparts, resulting in an asynchrony
with the maternal environment. Of course these two hypotheses could be interrelated, as
improper gene expression due to a defect in the embryo could result in impeded growth and
developmental asynchrony. Future studies could address this question. For example, gene
expression of short Day 15 conceptuses could be compared to gene expression of Day 14
conceptuses of a similar average length. It would be of interest to know if the transcriptome
183
of short Day 15 conceptuses is more similar to that of these Day 14 conceptuses, indicating a
developmental delay. Conversely, if the short Day 15 conceptuses still show differences in
the expression of genes related to developmental competency as compared to "normal"
length Day 14 conceptuses, it may indicate that short conceptuses are inherently inferior. The
second question that is raised is: can the genes related to developmental competency and
enhanced conceptus elongation identified in the current study, as well as other studies as
described in this dissertation, be used to improve embryo survival? Future work could
identify whether the products of these genes are secreted by the embryo, and could be
detected and/or measured in a culture dish prior to transfer.
Short and long Day 15 conceptuses displayed an interesting differential regulation of
many immune-system related genes. We could speculate as to why this might occur. The
shorter conceptuses may fail to regulate, namely suppress, genes involved in immunological
tolerance between the maternal and fetal systems, such as genes associated with MHC-I
molecules or antigens that were found to be differentially expressed in our research.
Alternatively, the maternal immune system could become immunologically activated by
improper expression of chemokines and cytokines produced by the conceptus. Elongating
conceptuses in ruminants express IL6 [246] which, interestingly, was comparatively downregulated in short conceptuses. Secretion of IFNT increases as conceptus elongation proceeds
[59, 243, 244] and, in turn, influences the expression of other genes in the endometrium
and/or conceptus [66, 70] and can alter certain immune responses (reviewed in [90]). Thus, it
184
may be that the maternal endometrium responds by attacking the short conceptuses, which
are, instead, treated as foreign tissue or pathogens.
Our study, and many other studies, reported a wide variation in conceptus lengths
from a cohort of conceptuses recovered from a single recipient. However, it is important to
note that in these studies multiple embryos were transferred into each recipient. It is,
therefore, interesting to speculate that these conceptuses may also be competing with each
other within the uterus. Short conceptuses may not only face immunological attack from the
maternal system but also may face attack from other longer conceptuses within the same
recipient uterus. In cattle, a single conceptus normally develops within the uterus. The
singleton conceptus would normally be primarily concerned with establishing
communication with the maternal endometrium. However, when multiple conceptuses are
present within the uterus, some may interpret signals from other less developed conceptuses
as being produced by infectious agents.
Interestingly, progesterone acts as an immunosuppressant. Studies in rats and sheep
report that progesterone can prevent or delay rejection of tissue allografts or xenografts in the
uterus and can reduce responses to bacterial pathogens (reviewed in [247]). Results from our
first study suggest that the progesterone environment at the time of transfer is more
influential on the elongation process of in vivo produced embryos. Results from our second
study found differential gene expression between short and long conceptuses derived from
IVO embryos. Some of these differentially expressed genes included those related to immune
185
processes. Thus, it may be that progesterone's immunosuppressive actions may also be
involved in regulating conceptus elongation.
In Experiment 2 we identified 348 genes that were differentially expressed between
short and long Day 15 conceptuses derived from IVO embryos. In contrast, in Experiment 3
we found no DEGs between short and long IVO-derived conceptuses, and only one DEG was
identified between short and long conceptuses derived from IVP embryos. A possible
explanation for these discrepant results may be that differences in conceptus morphology
existed between the short and long conceptuses that were used for each experiment. In
Experiment 2, ovoid and tubular conceptuses were compared while in Experiment 3
conceptuses that were primarily mid tubular were evaluated. Future work should limit
comparisons to conceptuses with differing morphology (for example, ovoid vs tubular or
tubular vs filamentous). A second explanation for our discrepant results may be that in
Experiment 2 embryos were assessed and transferred at the morula stage whereas in
Experiment 3, embryos were at the blastocyst stage. It is possible that assessing embryo
morphology at the morula stage may be too early to predict embryo viability, and thus more
embryos with reduced developmental competence were transferred in Experiment 2
compared to Experiment 3.
In conclusion, the results of this dissertation indicate that embryos from different
production systems may respond differently to the progesterone concentration in recipients
prior to and around Day 7 of the cycle. Furthermore, IVO-derived conceptuses with impeded
186
growth at Day 15 display differential regulation of gene expression compared to their longer
counterparts which may indicate a reduced developmental competence to term.
187
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215
APPENDICES
216
APPENDIX I
DIFFERENTIALLY EXPRESSED GENES BETWEEN LONG AND SHORT DAY 15 CONCEPTUSES
DERIVED FROM GRADE 1 IVO EMBRYOS.
217
Table A1. Differentially expressed genes (n=348) between long and short Day 15 conceptuses derived from Grade 1 IVO embryos. A positive value
indicates up-regulation in long conceptuses while a negative value indicates up-regulation in short conceptuses. Adjusted P-value <0.05.
Entrez Gene ID
Gene Symbol
Gene Title
100270684
C15H11orf96
chromosome 15 open reading frame, human C11orf96
337906 /// 782451
PAG12
pregnancy-associated glycoprotein 2-like /// pregnancy-associated glycoprotein 12
Fold
Change
Adjusted
P-value
11.14
0.015
8.90
0.021
508875
TP53I3
tumor protein p53 inducible protein 3
8.77
0.012
280826
IL6
interleukin 6 (interferon, beta 2)
5.42
0.013
513463
TAGLN
transgelin
4.95
0.012
280988
AHSG
alpha-2-HS-glycoprotein
4.50
0.031
518308
PRUNE2
prune homolog 2 (Drosophila)
4.45
0.014
615037
BMP2
bone morphogenetic protein 2
4.36
0.014
---
Bt.23770.1.A1_at
---
4.35
0.016
337886
S100A11
S100 calcium binding protein A11
4.13
0.042
617849
MSRB3
methionine sulfoxide reductase B3
4.02
0.018
337918
SRPX
sushi-repeat containing protein, X-linked
3.93
0.015
538952
PRR15
proline rich 15
3.92
0.014
616564
SSBP2
single-stranded DNA binding protein 2
3.79
0.031
617293
ELOVL5
ELOVL fatty acid elongase 5
3.70
0.015
281740
CYP19A1
cytochrome P450, family 19, subfamily A, polypeptide 1
3.63
0.028
616368
IGFBP7
insulin-like growth factor binding protein 7
3.50
0.013
541284
MBOAT1
membrane bound O-acyltransferase domain containing 1
3.46
0.039
414732
GATM
glycine amidinotransferase (L-arginine:glycine amidinotransferase)
3.35
0.014
---
Bt.16188.1.S1_at
---
3.22
0.031
218
Table A1 continued
281235
IDH1
isocitrate dehydrogenase 1 (NADP+), soluble
3.21
0.030
782020
PTER
phosphotriesterase related
3.14
0.030
531018
PAQR8
progestin and adipoQ receptor family member VIII
3.07
0.031
---
Bt.29415.1.A1_at
---
2.97
0.012
504499
NDRG1
N-myc downstream regulated 1
2.86
0.020
---
Bt.27089.1.S1_at
---
2.83
0.014
536203
ABCG2
ATP-binding cassette, sub-family G (WHITE), member 2
2.81
0.048
281614
ALCAM
activated leukocyte cell adhesion molecule
2.71
0.020
---
Bt.21877.1.S1_at
---
2.71
0.013
536187
CAPRIN2
caprin family member 2
2.70
0.043
497015
TPM2
tropomyosin 2 (beta)
2.67
0.012
100139952
GFI1
growth factor independent 1 transcription repressor
2.66
0.014
505622
WBSCR17
Williams-Beuren syndrome chromosome region 17
2.66
0.015
507526
CDH17
cadherin 17, LI cadherin (liver-intestine)
2.62
0.017
---
Bt.14368.1.A1_at
---
2.60
0.028
280968
TPPP
tubulin polymerization promoting protein
2.58
0.045
---
Bt.16356.1.S1_at
---
2.56
0.026
518050
ANXA3
annexin A3
2.55
0.019
513004 /// 782311
TPST1
tyrosylprotein sulfotransferase 1-like /// tyrosylprotein sulfotransferase 1
2.54
0.012
286852
PPP3CA
protein phosphatase 3, catalytic subunit, alpha isozyme
2.53
0.018
517063
AK4
adenylate kinase 4
2.53
0.037
100336454
VSTM4
V-set and transmembrane domain containing 4
2.53
0.027
219
Table A1 continued
616676
GALM
galactose mutarotase (aldose 1-epimerase)
2.50
0.029
768230
CYYR1
cysteine/tyrosine-rich 1
2.49
0.028
523223
PSTPIP2
proline-serine-threonine phosphatase interacting protein 2
2.47
0.013
281444
RBP4
retinol binding protein 4, plasma
2.46
0.023
515687
GUSB
glucuronidase, beta
2.45
0.015
---
Bt.14368.2.S1_at
---
2.44
0.030
282023
PTGS2
prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase)
2.42
0.046
533729
PAK1
p21 protein (Cdc42/Rac)-activated kinase 1
2.40
0.031
---
Bt.18428.1.A1_at
---
2.36
0.014
613349
DYNLT3
dynein, light chain, Tctex-type 3
2.35
0.017
---
Bt.19495.1.A1_at
---
2.33
0.029
510305
TMEM45B
transmembrane protein 45B
2.29
0.020
281869
INPP1
inositol polyphosphate-1-phosphatase
2.29
0.014
---
Bt.8419.1.S1_at
---
2.28
0.039
---
Bt.11909.1.A1_at
---
2.27
0.048
317659
DNAJC6
DnaJ (Hsp40) homolog, subfamily C, member 6
2.26
0.017
511716 /// 785508
EPHX2
epoxide hydrolase 2, cytoplasmic /// epoxide hydrolase 2, cytoplasmic-like
2.25
0.014
---
Bt.16100.2.S1_at
---
2.24
0.027
614509
MXI1
MAX interactor 1
2.23
0.015
---
Bt.9158.1.A1_at
---
2.23
0.013
---
Bt.23992.1.A1_at
---
2.21
0.043
615135
IPMK
inositol polyphosphate multikinase
2.18
0.023
220
Table A1 continued
536335
KIF27
kinesin family member 27
2.18
0.048
511671
CXCL16
chemokine (C-X-C motif) ligand 16
2.17
0.017
505060
CYP51A1
cytochrome P450, family 51, subfamily A, polypeptide 1
2.15
0.013
510393
BLNK
B-cell linker
2.14
0.023
785489
MBOAT2
membrane bound O-acyltransferase domain containing 2
2.13
0.041
541012
TCP11L2
t-complex 11 (mouse)-like 2
2.12
0.014
533107
FADS1
fatty acid desaturase 1
2.12
0.048
---
Bt.8879.2.S1_at
---
2.12
0.033
---
Bt.13994.1.S1_at
---
2.12
0.023
513967
ALDH3A2
aldehyde dehydrogenase 3 family, member A2
2.11
0.014
---
Bt.8879.1.A1_at
---
2.10
0.031
514345
MCOLN3
mucolipin 3
2.09
0.036
282466
S100A10
S100 calcium binding protein A10
2.08
0.032
282227
FCGR1A
Fc fragment of IgG, high affinity Ia, receptor (CD64)
2.07
0.043
539290
PLSCR4
phospholipid scramblase 4
2.06
0.015
532015
ELOVL4
ELOVL fatty acid elongase 4
2.06
0.050
---
Bt.11704.1.S1_at
---
2.06
0.026
100139988
RHOQ
ras homolog gene family, member Q
2.05
0.032
---
Bt.17155.1.A1_at
---
2.04
0.026
100337074
LOC100337074
Leukocyte elastase inhibitor-like
2.03
0.032
---
Bt.16100.1.S1_at
---
2.02
0.031
281662
CAPN2
calpain 2, (m/II) large subunit
2.02
0.015
221
Table A1 continued
511690
ST6GALNAC2
ST6 (alpha-N-acetyl-neuraminyl-2,3-beta-galactosyl-1,3)-N-acetylgalactosaminide alpha-2,6sialyltransferase 2
2.01
0.031
506968
NADKD1
NAD kinase domain containing 1
2.00
0.031
---
Bt.1220.1.S1_at
---
2.00
0.033
281203
GNG2
guanine nucleotide binding protein (G protein), gamma 2
1.99
0.013
282303
PECAM1
platelet/endothelial cell adhesion molecule
1.99
0.013
513597
MYOF
myoferlin
1.98
0.020
---
Bt.6261.1.S1_at
---
1.97
0.048
---
Bt.25216.1.A1_at
---
1.96
0.027
535227
CYP2U1
cytochrome P450, family 2, subfamily U, polypeptide 1
1.95
0.047
504847
CCNI
cyclin I
1.94
0.015
503553
TKDP2
trophoblast Kunitz domain protein 2
1.94
0.043
282358
SLC2A3
solute carrier family 2 (facilitated glucose transporter), member 3
1.94
0.032
---
Bt.21042.1.S1_at
---
1.93
0.050
---
Bt.21620.1.A1_at
---
1.93
0.024
520865
AASS
aminoadipate-semialdehyde synthase
1.92
0.035
508488
GALNT12
UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 12 (GalNAc-T12)
1.92
0.030
---
Bt.22349.1.A1_at
---
1.92
0.012
---
Bt.8678.1.A1_at
---
1.92
0.046
534389
FAM105A
family with sequence similarity 105, member A
1.91
0.031
100336865
SPTBN2
spectrin, beta, non-erythrocytic 2
1.88
0.030
280832
KIT
v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog
1.87
0.049
---
Bt.14566.1.A1_at
---
1.86
0.019
222
Table A1 continued
523498 /// 788499
MTSS1
MTSS1 protein-like /// metastasis suppressor 1
1.85
0.034
538226
SESN1
sestrin 1
1.83
0.019
---
Bt.22349.2.S1_at
---
1.83
0.013
785621
TCRA
T cell receptor, alpha
1.82
0.013
514159 /// 100851804
ACSL5
acyl-CoA synthetase long-chain family member 5 /// long-chain-fatty-acid--CoA ligase 5-like
1.82
0.024
---
Bt.27336.1.A1_at
1.82
0.017
534290
ANO10
anoctamin 10
1.81
0.014
615359
PARP4
poly (ADP-ribose) polymerase family, member 4
1.81
0.042
511583
FAM114A1
family with sequence similarity 114, member A1
1.80
0.031
535588
ABHD5
abhydrolase domain containing 5
1.79
0.024
---
Bt.18952.1.A1_at
---
1.78
0.032
---
Bt.28431.1.S1_at
---
1.77
0.019
532283
KLHDC8A
kelch domain containing 8A
1.77
0.025
535277
TPM4
tropomyosin 4
1.77
0.049
767827
C1S
complement component 1, s subcomponent
1.77
0.022
281643
BCAT2
branched chain amino-acid transaminase 2, mitochondrial
1.76
0.014
505355
ECI2
enoyl-CoA delta isomerase 2
1.75
0.023
---
Bt.11757.1.A1_at
---
1.75
0.015
---
Bt.10166.1.A1_at
---
1.74
0.045
515361
LPCAT3
lysophosphatidylcholine acyltransferase 3
1.74
0.045
534463
MFSD6
major facilitator superfamily domain containing 6
1.73
0.031
223
Table A1 continued
530341
LOC530341
cortactin-binding protein 2-like
1.72
0.050
---
Bt.14566.2.S1_at
---
1.72
0.027
510329
ATP2C2
ATPase, Ca++ transporting, type 2C, member 2
1.72
0.048
512171
ADAMTS1
ADAM metallopeptidase with thrombospondin type 1 motif, 1
1.71
0.048
506569
TTC3
tetratricopeptide repeat domain 3
1.71
0.014
616166
RALGPS1
Ral GEF with PH domain and SH3 binding motif 1
1.70
0.022
---
Bt.19303.1.A1_at
---
1.70
0.034
281779
GFPT1
glutamine--fructose-6-phosphate transaminase 1
1.70
0.041
---
Bt.20994.1.S1_at
---
1.69
0.048
---
Bt.18532.1.A1_at
---
1.69
0.020
100848079
LOC100848079
trimethyllysine dioxygenase, mitochondrial-like
1.69
0.035
510651
C5H12orf35
chromosome 5 open reading frame, human C12orf35
1.69
0.032
537539
ALDH9A1
aldehyde dehydrogenase 9 family, member A1
1.68
0.049
---
Bt.28282.1.S1_at
---
1.68
0.013
---
Bt.24224.1.S1_at
---
1.68
0.033
---
Bt.16114.1.S1_at
---
1.68
0.022
---
Bt.723.1.S1_at
---
1.68
0.015
---
Bt.20683.1.S1_at
---
1.67
0.024
---
Bt.1756.2.S1_at
---
1.67
0.037
511899
INSIG1
insulin induced gene 1
1.66
0.038
100847108
LOC100847108
uncharacterized LOC100847108
1.65
0.041
505569
CLCN2
chloride channel 2
1.65
0.049
224
Table A1 continued
281945
NR2F2
nuclear receptor subfamily 2, group F, member 2
1.64
0.038
---
Bt.22644.2.A1_at
---
1.64
0.028
511591
SORT1
sortilin 1
1.63
0.014
613665
LEPROT
leptin receptor overlapping transcript
1.63
0.031
539347
C5H12orf23
chromosome 5 open reading frame, human C12orf23
1.62
0.013
616979
RAB31
RAB31, member RAS oncogene family
1.62
0.015
526609
SLC31A2
solute carrier family 31 (copper transporters), member 2
1.62
0.017
100297914
LOC100297914
uncharacterized LOC100297914
1.61
0.023
540614
SEPT8
septin 8
1.61
0.042
540076
KIF21A
kinesin family member 21A
1.61
0.014
281039
CAST
calpastatin
1.61
0.013
---
Bt.21083.1.S1_at
---
1.60
0.034
286772
NPC1
Niemann-Pick disease, type C1
1.60
0.015
---
Bt.2838.1.A1_at
---
1.60
0.024
281781
B4GALT1
UDP-Gal:betaGlcNAc beta 1,4- galactosyltransferase, polypeptide 1
1.60
0.040
---
Bt.28126.1.S1_at
---
1.60
0.014
407767
HMGCS1
HMGCS1 protein-like
1.59
0.013
---
Bt.23648.1.A1_at
---
1.59
0.014
538841
ZSWIM6
zinc finger, SWIM-type containing 6
1.59
0.026
---
Bt.6642.1.S1_a_at
---
1.59
0.038
---
Bt.13765.1.S1_at
---
1.59
0.025
100848443
LOC100848443
uncharacterized LOC100848443
1.58
0.023
225
Table A1 continued
---
Bt.6121.2.S1_at
---
1.58
0.022
538516
PDLIM3
PDZ and LIM domain 3
1.58
0.034
---
Bt.8674.1.A1_at
---
1.58
0.016
535378
PARD3B
par-3 partitioning defective 3 homolog B (C. elegans)
1.58
0.041
514291
EMILIN2
elastin microfibril interfacer 2
1.57
0.030
505238
RALB
v-ral simian leukemia viral oncogene homolog B (ras related; GTP binding protein)
1.57
0.013
510749
PANK3
pantothenate kinase 3
1.57
0.043
511080
CCDC111
coiled-coil domain containing 111
1.57
0.037
617975
H1F0
H1 histone family, member 0
1.56
0.045
281626
ANXA5
annexin A5
1.56
0.026
514951
RBFOX2
RNA binding protein, fox-1 homolog (C. elegans) 2
1.56
0.035
788673
HECA
headcase homolog (Drosophila)
1.55
0.028
509422
FKBP7
FK506 binding protein 7
1.55
0.037
526395
SERF1A
small EDRK-rich factor 1A (telomeric)
1.55
0.021
505325
LRCH1
leucine-rich repeats and calponin homology (CH) domain containing 1
1.55
0.044
---
Bt.3072.2.S1_at
---
1.55
0.043
511007
FBXL20
F-box and leucine-rich repeat protein 20
1.55
0.022
614824
HKDC1
hexokinase domain containing 1
1.55
0.032
512299
GNPNAT1
glucosamine-phosphate N-acetyltransferase 1
1.54
0.039
281767
FDFT1
farnesyl-diphosphate farnesyltransferase 1
1.54
0.021
280729
B2M
beta-2-microglobulin
1.54
0.025
510634
TEN1
TEN1 telomerase capping complex subunit homolog (S. cerevisiae)
1.54
0.022
226
Table A1 continued
510489
SH3KBP1
SH3-domain kinase binding protein 1
1.53
0.016
614672
SH3BGRL3
SH3 domain binding glutamic acid-rich protein like 3
1.53
0.048
---
Bt.26930.1.S1_at
---
1.53
0.019
528478
PRKCI
protein kinase C, iota
1.52
0.028
286815
GCH1
GTP cyclohydrolase 1
1.51
0.024
616222
COPZ2
coatomer protein complex, subunit zeta 2
1.51
0.043
514013
APBB2
amyloid beta (A4) precursor protein-binding, family B, member 2
1.51
0.013
512656
VPS13B
vacuolar protein sorting 13 h omolog B (yeast)
1.51
0.027
510334
NEURL
neuralized homolog (Drosophila)
1.51
0.016
281631
APOA1
apolipoprotein A-I
1.51
0.014
---
Bt.13650.2.A1_at
---
1.51
0.022
---
Bt.14682.1.A1_at
---
1.51
0.025
515125 /// 516010
SMAD2 /// SMAD3
SMAD family member 2 /// SMAD family member 3
1.51
0.014
---
Bt.24418.1.A1_at
---
1.50
0.040
617543
REEP5
receptor accessory protein 5
1.50
0.017
---
Bt.19260.1.A1_at
---
1.50
0.028
785086
CREB3L2
cAMP responsive element binding protein 3-like 2
1.50
0.031
515067
PGAM2
phosphoglycerate mutase 2 (muscle)
1.50
0.029
281625
ANXA4
annexin A4
1.50
0.032
282137
AGPAT1
1-acylglycerol-3-phosphate O-acyltransferase 1 (lysophosphatidic acid acyltransferase, alpha)
1.50
0.032
767968
BEX2
brain expressed X-linked 2
1.50
0.037
511581
C1R
complement component 1, r subcomponent
1.50
0.039
227
Table A1 continued
537282
MYO1B
myosin IB
1.50
0.041
---
Bt.27841.1.A1_at
---
1.50
0.038
282191
COL4A1
collagen, type IV, alpha 1
1.50
0.037
493719
MGST1
microsomal glutathione S-transferase 1
1.50
0.050
532399
NT5C
5', 3'-nucleotidase, cytosolic
-1.50
0.049
510820
TSR1
TSR1, 20S rRNA accumulation, homolog (S. cerevisiae)
-1.50
0.031
506182
TRAF3
TNF receptor-associated factor 3
-1.50
0.014
497030
GTDC2
glycosyltransferase-like domain containing 2
-1.50
0.017
510085
SLC16A3
solute carrier family 16, member 3 (monocarboxylic acid transporter 4)
-1.50
0.019
513666 /// 541108 ///
788724 /// 100297758 ///
100851089
HIST2H2AA4 ///
HIST2H2AC
histone cluster 2, H2aa4 /// histone cluster 2, H2ac
-1.50
0.027
281296
MAPT
microtubule-associated protein tau
-1.50
0.037
506914
CEP41
centrosomal protein 41kDa
-1.50
0.024
513045
MYBBP1A
MYB binding protein (P160) 1a
-1.51
0.017
534683
COMMD1
copper metabolism (Murr1) domain containing 1
-1.51
0.014
617403
TMEM52
transmembrane protein 52
-1.51
0.015
515001
NAT10
N-acetyltransferase 10 (GCN5-related)
-1.51
0.014
530342
FAM173A
family with sequence similarity 173, member A
-1.51
0.014
100850521
LOC100850521
uncharacterized LOC100850521
-1.52
0.034
781568 /// 783305
TRAF4
TNF receptor-associated factor 4-like /// TNF receptor-associated factor 4
-1.52
0.030
---
Bt.21137.1.S1_at
---
-1.53
0.022
532995
KIAA0391
KIAA0391 ortholog
-1.53
0.013
228
Table A1 continued
540720
MRPL46
mitochondrial ribosomal protein L46
-1.54
0.015
100848808
LOC100848808
uncharacterized LOC100848808
-1.54
0.013
281912
MFAP2
microfibrillar-associated protein 2
-1.54
0.048
537692
MBLAC2
metallo-beta-lactamase domain containing 2
-1.54
0.031
---
Bt.2385.1.S1_at
---
-1.54
0.015
534995
SYNGR1
synaptogyrin 1
-1.55
0.012
613430
GRAMD4
GRAM domain containing 4
-1.55
0.035
515057
GAR1
GAR1 ribonucleoprotein homolog (yeast)
-1.55
0.032
286872
MSX1
msh homeobox 1
-1.55
0.049
514971
CYP2S1
cytochrome P450, family 2, subfamily S, polypeptide 1
-1.55
0.036
506890
IGK
Ig kappa chain
-1.56
0.027
513433
MMACHC
methylmalonic aciduria (cobalamin deficiency) cblC type, with homocystinuria
-1.56
0.018
510315
CIRH1A
cirrhosis, autosomal recessive 1A (cirhin)
-1.56
0.012
505554
NEU1
sialidase 1 (lysosomal sialidase)
-1.56
0.026
100848535
LOC100848535
zinc finger protein 239-like
-1.57
0.041
317697
ITPR1
inositol 1,4,5-trisphosphate receptor, type 1
-1.58
0.032
281888 /// 100138974
KRT10
keratin 10 /// keratin 13-like
-1.58
0.044
532842
MRPL32
mitochondrial ribosomal protein L32
-1.58
0.013
506950
MAP7D3
MAP7 domain containing 3
-1.58
0.025
511442
KNOP1
chromosome 25 open reading frame, human C16orf88
-1.59
0.012
541070
MYLIP
myosin regulatory light chain interacting protein
-1.59
0.038
511427
KRI1
KRI1 homolog (S. cerevisiae)
-1.59
0.019
229
Table A1 continued
784070
HDGFRP3
hepatoma-derived growth factor, related protein 3
-1.60
0.023
327664
PYGM
phosphorylase, glycogen, muscle
-1.60
0.014
513309
GFM1
G elongation factor, mitochondrial 1
-1.60
0.013
533613
RABEPK
Rab9 effector protein with kelch motifs
-1.61
0.013
511236
TAF1B
TATA box binding protein (TBP)-associated factor, RNA polymerase I, B, 63kDa
-1.61
0.040
514685
DPEP1
dipeptidase 1 (renal)
-1.61
0.016
505878
SOX15
SRY (sex determining region Y)-box 15
-1.62
0.015
509264
PHF17
PHD finger protein 17
-1.62
0.034
613376
RPIA
ribose 5-phosphate isomerase A
-1.62
0.015
507054
IL33
interleukin 33
-1.62
0.031
522370
PPAN
peter pan homolog (Drosophila)
-1.63
0.012
---
Bt.26026.1.A1_at
---
-1.63
0.031
511610
SURF6
surfeit 6
-1.63
0.019
---
Bt.23918.1.A1_at
---
-1.63
0.048
613639
TSPAN12
tetraspanin 12
-1.64
0.023
534904
GALNT11
UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 11 (GalNAc-T11)
-1.64
0.013
---
-1.64
0.025
--781648
LAP3
leucine aminopeptidase 3
-1.64
0.016
784844
TCEB3
transcription elongation factor B (SIII), polypeptide 3 (110kDa, elongin A)
-1.64
0.013
532663
ENPP2
ectonucleotide pyrophosphatase/phosphodiesterase 2
-1.65
0.012
280852 /// 618377
MDK
midkine-like /// midkine (neurite growth-promoting factor 2)
-1.65
0.045
230
Table A1 continued
614890
PTPMT1
protein tyrosine phosphatase, mitochondrial 1
-1.65
0.017
768068
505183 /// 506306 ///
521580 /// 527244 ///
537985 /// 615043 ///
616776 /// 616868 ///
618012 /// 781410 ///
782971 /// 787269 ///
787465 /// 787581 ///
100295221
KLHL12
kelch-like 12 (Drosophila)
-1.65
0.014
H2B /// HIST1H2BL ///
HIST2H2BE
histone H2B-like /// histone H2B /// histone cluster 1, H2bl /// histone cluster 2, H2be
-1.65
0.038
---
Bt.17497.1.S1_at
---
-1.67
0.013
767910
PLAC8
placenta-specific 8
-1.67
0.035
540231
FAM83B
family with sequence similarity 83, member B
-1.67
0.014
534718
STXBP6
syntaxin binding protein 6 (amisyn)
-1.68
0.014
282604 /// 788912
FDXR
ferredoxin reductase /// NADPH:adrenodoxin oxidoreductase, mitochondrial-like
-1.68
0.019
504950
NPM3
nucleophosmin/nucleoplasmin 3
-1.68
0.032
534832
KIF13A
kinesin family member 13A
-1.68
0.015
280994
ALPL
alkaline phosphatase, liver/bone/kidney
-1.68
0.013
507197
SHMT2
serine hydroxymethyltransferase 2 (mitochondrial)
-1.69
0.012
538579
RIMKLB
ribosomal modification protein rimK-like family member B
-1.69
0.043
786013
AATF
apoptosis antagonizing transcription factor
-1.69
0.013
---
Bt.24511.1.A1_at
---
-1.69
0.015
281917
MOCS1
molybdenum cofactor synthesis 1
-1.69
0.015
---
Bt.5961.1.S1_at
---
-1.69
0.019
505043
CD320
CD320 molecule
-1.69
0.013
538826
SSFA2
sperm specific antigen 2
-1.71
0.025
231
Table A1 continued
281678
CEBPD
CCAAT/enhancer binding protein (C/EBP), delta
-1.71
0.024
510524
CTSH
cathepsin H
-1.72
0.019
533156
DAPP1
dual adaptor of phosphotyrosine and 3-phosphoinositides
-1.72
0.016
613696
PLEKHO2
pleckstrin homology domain containing, family O member 2
-1.73
0.026
515742
TMEM150A
transmembrane protein 150A
-1.74
0.039
100174924
LOC100174924
uncharacterized LOC100174924
-1.75
0.013
613483
C18H16orf74
chromosome 18 open reading frame, human C16orf74
-1.76
0.048
534296
MTHFD1L
methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1-like
-1.78
0.013
281060
CD8A
CD8a molecule
-1.79
0.040
100848739
LOC100848739
uncharacterized LOC100848739
-1.79
0.038
519439
SOX18
SRY (sex determining region Y)-box 18
-1.79
0.020
---
Bt.21137.2.S1_at
---
-1.80
0.014
533166
SORL1
sortilin-related receptor, L(DLR class) A repeats containing
-1.81
0.049
777787
FABP7
fatty acid binding protein 7, brain
-1.83
0.027
525380
IER2
immediate early response 2
-1.83
0.008
532421
ZNF567
zinc finger protein 567
-1.88
0.017
526046
SURF2
surfeit 2
-1.88
0.014
414346
PPIF
peptidylprolyl isomerase F
-1.89
0.015
533044
PSAT1
phosphoserine aminotransferase 1
-1.92
0.022
508387
CCL26
chemokine (C-C motif) ligand 26
-1.94
0.015
517539
MTHFD2
methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 2, methenyltetrahydrofolate cyclohydrolase
-1.95
0.015
232
Table A1 continued
280715
AK1
adenylate kinase 1
-1.98
0.014
338077
FUT5
fucosyltransferase 5 (alpha (1,3) fucosyltransferase)
-2.04
0.044
282255 /// 777594 ///
786073
IFITM3
interferon induced transmembrane protein 3 /// interferon induced transmembrane protein 3 (1-8U) ///
interferon-induced transmembrane protein 3 (1-8U)-like
-2.04
0.012
614940
PRSS35
protease, serine, 35
-2.04
0.017
407173 /// 100125916 ///
100126048
BOLA-N /// JSP.1 ///
LOC100125916
MHC class I antigen /// MHC Class I JSP.1 /// uncharacterized protein 100125016
-2.05
0.025
511037
TMPRSS2
transmembrane protease, serine 2
-2.11
0.021
280677
C3
complement component 3
-2.11
0.025
534427 /// 100847583
EGFLAM
EGF-like, fibronectin type III and laminin G domains /// pikachurin-like
-2.13
0.038
514189
LOC514189
uncharacterized LOC514189
-2.22
0.031
515749
CTNNAL1
catenin (cadherin-associated protein), alpha-like 1
-2.24
0.013
505233
CERS4
ceramide synthase 4
-2.26
0.043
---
Bt.13920.1.S1_at
---
-2.31
0.017
515375
NAAA
N-acylethanolamine acid amidase
-2.41
0.039
386664
NDN
necdin homolog (mouse)
-2.56
0.014
504822
XPNPEP2
X-prolyl aminopeptidase (aminopeptidase P) 2, membrane-bound
-2.59
0.013
539369
NRP1
neuropilin 1
-2.62
0.023
508492
GSDMC
gasdermin C
-2.66
0.023
538846
JAM2
junctional adhesion molecule 2
-2.66
0.013
512512
DNASE1L3
deoxyribonuclease I-like 3
-2.71
0.027
618577
SYBU
syntabulin (syntaxin-interacting)
-2.88
0.022
233
Table A1 continued
516067 /// 539750
FOLR1
folate receptor 1 (adult)
-3.10
0.031
780866
AQP3
aquaporin 3 (Gill blood group)
-3.16
0.012
505310
SLC5A5
solute carrier family 5 (sodium iodide symporter), member 5
-3.27
0.012
282301
PDGFRA
platelet-derived growth factor receptor, alpha polypeptide
-3.47
0.012
511839
ATP6V0D2
ATPase, H+ transporting, lysosomal 38kDa, V0 subunit d2
-3.72
0.020
100848243
LOC100848243
uncharacterized LOC100848243
-3.79
0.015
789846
ZNF503
zinc finger protein 503
-6.76
0.032