BIOLOGY OF REPRODUCTION 71, 1991–2002 (2004)
Published online before print 11 August 2004.
DOI 10.1095/biolreprod.104.031831
Large-Scale Generation and Analysis of Expressed Sequence Tags from
Porcine Ovary1
Honglin Jiang,3,4 Kristin M. Whitworth,4 Nathan J. Bivens,4 James E. Ries,4 Rami J. Woods,4
Lawrence J. Forrester,4 Gordon K. Springer,4 Nagappan Mathialagan,5 Cansu Agca,4 Randall S. Prather,4
and Matthew C. Lucy2,4
Department of Animal Science,4 University of Missouri, Columbia, Missouri 65211
Monsanto Company,5 St. Louis, Missouri 63198
ABSTRACT
One method to identify the factors that control ovarian function is to characterize the genes that are expressed in ovary. In
the present study, cDNA libraries from fetal, neonatal, and prepubertal porcine ovaries, pubertal ovaries on different days of
the estrous cycle (Days 0 [follicle], 5, and 12 [follicle and corpus
luteum]), and follicles isolated from weaned sows (diameter, 2,
4, 6, and 8 mm) were constructed and sequenced. A total of
22 176 cDNAs were sequenced, of which 15 613 were of sufficient quality for clustering. Clustering of cDNAs resulted in
8507 contigs, 6294 (74%) of which were comprised of a single
sequence. Sixty-eight percent of the contigs had consensus sequences that were homologous to existing Tentative Consensus
(TC) sequences or mature transcripts (ET) in The Institute for
Genomic Research Porcine Gene Index. The consensus sequences were classified according to the Gene Ontology Index. Most
cDNA-encoded proteins were components of the nucleus, ribosome, or mitochondrion. The proteins primarily functioned in
binding, catalysis, and transport. Nearly 75% of the proteins
were involved in metabolism and cell growth and/or maintenance. Analysis of the cDNA frequency across different libraries
demonstrated differential gene expression within different-size
follicles, between follicles and corpora lutea, and across developmental time-points. The expression of selected genes (analyzed by ribonuclease protection assay and Northern blotting)
was consistent with the frequency of their respective cDNA in
the individual libraries. This porcine ovary unigene set will be
useful for identifying factors and mechanisms controlling ovarian follicular development in a variety of species.
corpus luteum, follicle, gene regulation, ovary
INTRODUCTION
The ovarian follicle, consisting of an oocyte and surrounding layers of granulosa and theca cells, fulfills the
essential ovarian functions of steroidogenesis and gamete
production. During reproductive life, ovarian follicles undergo dynamic changes in morphology and function as they
progress through the primordial, primary, secondary, antral,
Supported in part by a grant from the Monsanto Company (St. Louis,
MO) and the University of Missouri (Food for the 21st Century Program).
2
Correspondence: Matthew C. Lucy, 164 Animal Science Research Center,
University of Missouri, Columbia, MO 65211. FAX: 573 882 6827;
e-mail: [email protected]
3
Current address: Department of Animal and Poultry Sciences, Virginia
Tech, Blacksburg, VA.
1
Received: 10 May 2004.
First decision: 16 June 2004.
Accepted: 23 July 2004.
Q 2004 by the Society for the Study of Reproduction, Inc.
ISSN: 0006-3363. http://www.biolreprod.org
and preovulatory follicular stages [1]. An even greater morphological and functional change occurs when follicular
cells differentiate into the corpus luteum following the LH
surge and ovulation [2]. Few follicles actually progress
through all of the successive stages, because a high rate of
follicular atresia leads to follicular death before ovulation
[3, 4].
The mechanisms underlying ovarian follicular development have been intensely studied. It is generally accepted
that antral and preovulatory follicular development is primarily controlled by pituitary gonadotropins [1]. Early
stages of follicular development (preantral and small antral
stages) are controlled by a variety of locally produced hormones and growth factors [5, 6]. Despite continuing progress in the area of ovarian biology, the factors controlling
some of the key events in follicular development, including
the initiation of primordial follicle growth, antrum formation, follicular dominance, and follicular atresia, remain to
be identified.
Large-scale sequencing of cDNA libraries is a rapid and
efficient means of discovering new genes and providing
both quantitative and qualitative information regarding
gene expression within specific tissues or cell types [7–12].
We carried out large-scale sequencing of cDNA from the
pig ovary to identify novel intraovarian factors and mechanisms involved in ovarian development. Here, we report
a catalog of genes expressed in the porcine ovary (expressed sequence tags [ESTs]) as well as a preliminary view
of their expression patterns throughout ovarian development, follicular growth, and ovulation.
MATERIALS AND METHODS
Library Construction and Sequencing
Ovarian tissue (whole ovary, dissected follicles, or corpora lutea) (Table 1) was collected from cross-bred pigs (Sus scrofa domestica), frozen
in liquid nitrogen shortly after collection, and stored at 2808C until RNA
extraction. The tissues from several individual pigs were pooled for RNA
extraction. The procedures used for tissue collection were approved by the
Animal Care and Use Committee of the University of Missouri-Columbia.
The procedures for constructing the Day 0 follicle (pd0fol), Day 5
ovary (pd5ov), Day 12 follicle (pd12fol), and Day 12 corpus luteum
(pd12CL) cDNA libraries were reported previously [12]. The methods
described herein were used for production of the 2-mm follicle (p2mm),
4-mm follicle (p4mm), 6-mm follicle (p6mm), 8-mm follicle (p8mm), fetal
ovary (pfeto), neonatal ovary (pnatal), and prepubertal ovary (pputal) libraries. Total cellular RNA was extracted using the Trizol reagent (Life
Technologies, Gaithersburg, MD) according to the manufacturer’s instructions. Poly(A) RNA was isolated from total cellular RNA using the Oligotex mRNA kit (Qiagen, Chatsworth, CA). The integrity and purity of
extracted RNA were verified by measuring the ratio of absorbance at 260
and 280 nm and by electrophoresis of an RNA aliquot on formaldehydeagarose gels.
1991
1992
JIANG ET AL.
TABLE 1. Sources of tissues used to construct cDNA libraries.
Library
Library code
Animal type
Tissue type
2-mm follicle
4-mm follicle
6-mm follicle
8-mm follicle
Day 0 follicle
Day 5 ovary
p2mm
p4mm
p6mm
p8mm
pd0fol
pd5ov
Sow
Sow
Sow
Sow
Gilt
Gilt
Follicle
Follicle
Follicle
Follicle
Follicle
Whole ovary
Day 12 corpus luteum
pd12cl
Gilt
Corpus luteum
Day 12 follicle
pd12fol
Gilt
Follicle
Fetal ovary
pfeto
Fetus
Whole ovary
Neonatal ovary
pnatal
Gilt
Whole ovary
Prepubertal ovary
pputal
Gilt
Whole ovary
The cDNA libraries were constructed by using the SuperScript Plasmid
System for cDNA Synthesis and Plasmid Cloning (Life Technologies) according to the manufacturer’s instructions with a modified reverse transcription reaction [13]. The reverse transcription was performed by annealing 1 mg of poly(A) RNA with 10 mg of an oligo(dT) primer
(GCTGCTCGCGGCCGC-tag-T18, where tag 5 library-specific five-base
sequence identifier) at 428C or by annealing 1 mg of poly(A) RNA with
4 mg of an anchored oligo(dT) primer (GCTGCTCGCGGCCGC-tagT18VN, where V 5 A, G, or C and N 5 A, G, C, or T) at 508C. An
excessive amount of oligo(dT) primer or an anchored oligo(dT) primer
was used to reduce the number of clones with long poly(A) tails in the
cDNA libraries [13]. The cDNAs were ligated into either the pT7T3 vector
(pd0fol, pd5ov, pd12fol, and pd12CL cDNA libraries) or the pSport1 vector (p2mm, p4mm, p6mm, p8mm, pfeto, pnatal, and pputal libraries; Life
Technologies) at the NotI and SalI sites. Ligated vector-cDNAs were transformed into DH10B Escherichia coli-competent cells (Life Technologies)
by electroporation. The complexity of the library was estimated according
to the manufacturer’s instructions (Life Technologies).
The quality of each cDNA library was assessed before large-scale
cDNA sequencing was performed. Bacteria clones from each library were
picked and individually grown overnight at 378C in deep-well, 96-well
plates containing 1.4 ml of Terrific Broth with ampicillin [14]. Plasmid
extractions from bacterial cultures were performed by using the QIAprep
96 Turbo Miniprep kit (Qiagen) according to the manufacturer’s instructions. The average cDNA insert size and the percentage of clones without
an insert were determined by EcoRI and HindIII restriction-enzyme digestion of plasmid isolated from 96 randomly picked clones. Libraries with
an average insert size of greater than 1 kilobase (kb) and more than 95%
of clones having a cDNA insert were subjected to preliminary cDNA
sequencing. Then, 288 randomly picked clones were sequenced. The ABI
Prism Big Dye Terminator cycle sequencing chemistry (Applied Biosystems, Foster City, CA) with a T3 (cDNA ligated into the pT7T3 vector)
or Sp6 (cDNA ligated into the pSport1 vector) sequencing primer was
used for the sequencing reactions. The sequencing reaction products (39
cDNA sequence) were analyzed on an ABI 377 Automated DNA sequencer (Applied Biosystems). The cDNA sequence data from the preliminary
sequencing were evaluated. A cDNA library was subjected to large-scale
sequencing if it met the following criteria: 1) More than 95% of the clones
contained inserts, 2) the average insert size was greater than 1 kb, 3) less
than 5% of the clones contained ribosomal RNA, 4) less than 10% of the
clones contained long poly(A) tails (.40 adenines), and 5) the complexity
of the library was at least 106 colony-forming units. The large-scale sequencing was performed as described for the preliminary sequence analysis above.
Sequence Clustering and Annotation
The cDNA sequences from the 11 libraries were clustered into contigs
using the SeqMan II software system (DNASTAR, Inc., Madison, WI).
The original trace files from the automated DNA sequencing were loaded
into the software system. The trace files were evaluated for quality using
the Trace Quality Evaluation system within SeqMan II. Poor-quality sequence, vector sequence, and poly(A) tails were trimmed from the sequence. The trimmed cDNA sequences were assembled into contigs using
Description
Dissected from sow ovary on Days 1–5 after weaning.
Dissected from sow ovary on Days 1–5 after weaning.
Dissected from sow ovary on Days 1–5 after weaning.
Dissected from sow ovary on Days 1–5 after weaning.
Dissected from gilt ovary on the day of estrus.
Ovary collected on day 5 of the estrous cycle. Includes
small antral follicles and corpora lutea.
Dissected from gilt ovary on Day 12 of the estrous cycle.
Dissected from gilt ovary on Day 12 of the estrous cycle (3–5-mm antral follicles).
Ovary collected on approximately Day 60 of gestation.
Contains primordial germ cells.
Ovary collected on approximately Day 22 of age. Contains primordial and primary follicles.
Ovary collected on approximately Day 155 of age. Primordial through antral-stage follicles.
the assembly process within SeqMan II. A 90% minimum match percentage was used. The actual average match percentage for entry into the
contig was 97.1%. A consensus sequence for each contig was exported
from SeqMan II.
The consensus cDNA sequence for each cluster was compared with
The Institute for Genomic Research (TIGR) Porcine Gene Index nucleotide sequence databases (Porcine Index, release 8.0; TIGR, Rockville, MD;
http://www.tigr.org/tdb/tgi) using the BLAST program [15]. The contig
consensus sequence was considered to be homologous to a TIGR Tentative
Consensus (TC) sequence or mature transcript (ET) sequence when the
BLAST score was greater than 200. Contigs that were homologous to the
TIGR TC sequence were annotated with a gene name by using the TIGR
EST Annotator.
Functional classifications from Gene Ontology (GO) Consortium [16]
were assigned to each contig using the homologous TIGR TC number and
the functional classifications assigned by TIGR. A set of higher-level parent classifications (GO Slim) [17] were used for the GO assignments. The
GO Slim assignments were performed manually by visually inspecting the
Tree view and identifying the appropriate GO Slim category. Genes whose
component, process, or function fell into multiple GO Slim categories were
given equal weighting across categories.
Electronic Northern Blot Analyses
Contigs were comprised of cDNA clones arising from the original
cDNA libraries. An ‘‘electronic Northern’’ was performed by analyzing
the frequency of library clones within each contig. Three collective libraries were selected for analyses. A collective follicle library (combined
p2mm, p4mm, p6mm, and p8mm libraries) was compiled and analyzed,
because the individual follicle libraries represented follicles at different
stages of preovulatory development [18]. The pd12fol and pd12CL libraries were constructed from follicles and corpora lutea collected from
the same ovaries on the same day of the estrous cycle. Therefore, a collective Day 12 ovary library (pd12fol and pd12CL) also was complied
and analyzed for cDNAs that were differentially expressed in follicles and
corpora lutea. Finally, a collective library representing developmental stages (fetal, neonatal, and prepubertal) was analyzed to identify cDNAs
whose presence was specific to developmental stages of the ovary. Independence was examined using the chi-square test. The observed value was
the number of clones arising from each library. Each library should contribute a proportional number of clones to the contig if the frequency of
clones was independent of library. The expected value (assuming independence) therefore was the total number of clones in the contig multiplied
by the proportion of accepted sequences in the subject library (relative to
the total number of sequence in the collective libraries).
Comparison of mRNA Amount in Tissues with cDNA
Frequency in Libraries
Ribonuclease protection (RP) and Northern blot assays were used to
determine the mRNA expression levels of selected genes in ovaries, follicles, and corpora lutea. The expression levels of seven mRNAs were
measured. These mRNAs were selected because their cDNAs appeared to
be differentially represented within closely related libraries. The expres-
1993
EXPRESSED SEQUENCE TAGS FROM PORCINE OVARY
TABLE 2. Characteristics of individual cDNA sequences evaluated for quality and clustered by using the SeqMan II program.a
Library
2 mm follicle (p2mm)
4 mm follicle (p4mm)
6 mm follicle (p6mm)
8 mm follicle (p8mm)
Day 0 follicle (pd0fol)
Day 5 ovary (pd5ov)
Day 12 corpus luteum (pd12cl)
Day 12 follicle (pd12fol)
Fetal ovary (pfeto)
Neonatal ovary (pnatal)
Prepubertal ovary (pputal)
Total
a
b
Sequence
attempts (n)
2592
2592
2592
2592
1152
384
2016
1632
2016
2304
2304
22 176
Accepted
sequences (%)
1805
1721
1983
2047
725
89
1219
945
1505
1851
1723
15 613
(70)
(66)
(77)
(79)
(63)
(23)
(60)
(58)
(75)
(80)
(75)
(70)
Contigs (n)
1358
1350
1404
1469
614
79
901
781
1153
1529
1333
8507
Singleton contigs
(n [%])
1160
1160
1171
1266
540
72
798
683
1034
1375
1151
6294
(85)
(86)
(83)
(86)
(88)
(91)
(89)
(87)
(90)
(90)
(86)
(74)
Contigs in TIGR
(n [%])b
1096
1017
1179
1094
484
63
666
602
826
1046
1070
5797
(81)
(75)
(84)
(74)
(79)
(80)
(74)
(77)
(72)
(68)
(80)
(68)
The consensus cDNA sequence for each cluster was compared against with the TIGR Porcine Gene Index.
Blast score greater than or equal to 200 when blasted against the TIGR Porcine Gene Index 8.0 (January 6, 2004; http://www.tigr.org/tdb/tgi).
sions of three mRNAs were compared for fetal, neonatal, and prepubertal
tissues, and the expression of three mRNAs were compared for 2-, 4-, 6-,
and 8-mm follicle tissues. The expression of one mRNA was compared
between follicle and corpus luteum. Total cellular RNA was extracted from
tissues as described above. Radiolabeled probes were synthesized from
selected cDNA plasmids. The RP and Northern blot assays were performed as single experiments using previously described procedures [19,
20]. The mRNA expression level was subjectively compared to the number
of cDNA isolated from each library.
for individual libraries ranged from 23% (Day 5 ovary library) to 80% (neonatal ovary library). The average
trimmed sequence length across all libraries was 348 6 1
(mean 6 SEM) base pairs (bp; range, 99 to 659 bp) (Fig.
1). Clustering of the cDNA sequences resulted in 8507 contigs (Table 2). Single-sequence contigs comprised 74% of
the total, and 99% of the contigs had 20 or fewer members
(Fig. 2).
Data and cDNA Clones Access
Annotation of cDNA and Contigs
The sequence for each cDNA library clone reported herein has been
deposited in GenBank. A list of cDNA library clones within each contig,
an annotated list of the contigs (contig number, gene name, frequency of
clones from each library, etc.), and the consensus sequence for each contig
are available at http://www.swine.rnet.missouri.edu.
Sixty-eight percent of the contigs (n 5 5797) were defined as homologous (BLAST score, $200) to existing TC
or ET in the TIGR Porcine Gene Index (Fig. 3). The average BLAST score for homologous contigs was 601 6 3.
Forty-eight contigs has 20 or more members (Table 3).
Nearly half of the largest contigs had consensus sequences
that were homologous to genes involved in protein synthesis (initiation factors, elongation factors, and ribosomal proteins). Other large contigs had consensus sequences for enzymes involved in intracellular energy production (e.g.,
components of the electron-transport chain), for structural
components of the cell, for proteins involved in protein
turnover, or for proteins involved in transport.
The consensus sequences for contigs were homologous
to a number of genes that function in the endocrinology of
ovarian cells (Table 4). The sequenced cDNA encoded insulin-like growth factor (IGF) system genes (including IGFbinding proteins), inhibin/activin system genes, relaxin and
RESULTS
cDNA Sequencing and Clustering
A total of 22 176 cDNA were sequenced and subjected
to the Trace Quality Evaluation system within SeqMan II.
Seventy percent of the cDNA sequences (n 5 15 613) contained sufficient cDNA sequences of acceptable quality for
clustering (Table 2). The percentage of accepted sequences
FIG. 1. The length of trimmed cDNA sequence (cDNA length after removal of poor-quality sequence, vector sequence, and poly[A] tail) for
cDNA clones submitted for clustering. The number of cDNA clones (frequency) within different categories of trimmed sequence length is presented. A trimmed sequence length of 100 represents clones with 99–
100 bp of trimmed sequence. Trimmed sequence lengths of 150, 200,
and 250 bp (etc.) on the abscissa represent ranges of trimmed sequence
lengths (101–150, 151–200, 201–250 bp, etc., respectively).
FIG. 2. The number of contigs with 20 or fewer members. The frequency
of contigs is represented on the ordinate (logarithmic scale) and the size
of the contig (1, 2, 3, or 4 cDNA members, etc.) is represented on the
abscissa.
1994
JIANG ET AL.
FIG. 3. The number of contigs with different BLAST scores from a nucleotide homology search of the TIGR Porcine Gene Index. A BLAST score
of 50, 100, 150, etc. on the abscissa represents a range of BLAST scores
(,50, 51–100, 101–150, etc., respectively).
relaxin-like protein genes, and transforming growth factor
b family genes. The inhibin a chain (contig 756) represented 0.29% of the sequenced cDNA. The cDNAs for
three separate prostaglandin synthesis enzymes and four
separate steroidogenic enzymes were sequenced. Prostaglandin F synthase 1 (contig 273) was one of the most
abundant cDNAs and represented 0.30% of cDNA sequences. Steroid acute regulatory protein (StAR; contig 3043)
represented 0.12% of the sequenced cDNAs. The cDNAs
for hormone receptors and components of hormone secondmessenger systems (cAMP, inositol triphosphate, phosphatidylinositol 3-kinase, mitogen-associated protein kinase,
Janus kinase, and sma/mad) were also sequenced.
The cDNAs were classified according to the GO Index
(cellular component, molecular function, and biological
process). We found that 797 contigs (3242 individual
cDNA clones) had GO cellular component annotations in
the TIGR Porcine Gene Index. Each contig contained one
or more cDNA clones. Based on the number of cDNA
clones (an indirect indicator of mRNA species within the
cell), the majority of cellular mRNA encoded components
of the ribosome, mitochondrion, and nucleus (Fig. 4). We
found that 996 contigs (3370 individual cDNA clones) had
a GO function annotation. The consensus sequences for
most contigs were homologous to genes whose products
were involved in binding or catalytic activity (enzymes).
We found 949 contigs (2948 individual cDNA clones) with
a GO process annotation. Nearly three-quarters of the
cDNAs sequenced encoded genes involved in cellular metabolism and cell growth and/or maintenance.
Frequency of cDNAs in Different Libraries (Electronic
Northern Blot Analysis)
The frequency of library cDNA clones within the largest
individual contigs is shown for follicle libraries (p2mm,
p4mm, p6mm, and p8mm) (Table 5), Day 12 ovary libraries
(p12fol and p12CL) (Table 6), and whole-ovary libraries
(pfeto, pnatal, and pputal) (Table 7). Independence of
cDNA distribution within the libraries was tested by chisquare (statistical significance inferred at minimum threshold of P , 0.10). A lack of independence (statistically significant result) indicated that within a contig, specific
cDNAs were found in a higher percentage in some libraries
relative to others.
We found 18 large contigs ($10 members) whose cDNA
clones were not equally distributed across follicle libraries
(Table 5). Nine contigs (contig 2 [elongation factor 1a],
contig 1 [cytochrome c oxidase], contig 160 [cytochrome
b], contig 18 [identity unknown], contig 222 [actin a], contig 1711 [translation elongation factor eEF-2], contig 5141
[retinoic acid receptor-responder protein 1], contig 163
[ADP ATP carrier protein isoform T2], and contig 5152
[glutathione S-transferase]) had cDNA clones that were predominantly found in the collective p6mm and p8mm libraries. Three contigs (contig 288 [ferritin, heavy polypeptide], contig 273 [prostaglandin F synthase 1], and contig
756 [inhibin a]) had cDNA predominately found in the
p6mm library. Four contigs (contigs 36 and 37 [glutathione
S-transferase], contig 651 [cathepsin L], and contig 1448
[ATP synthase g]) had cDNA clones predominately found
in the p8mm library. Two contigs (contig 161 [cytochrome
c oxidase III] and contig 370 [thymosin b4]) had cDNA
clones primarily found in p2mm and p4mm libraries. The
remaining contigs with more than 10 members had cDNA
equally distributed across follicle libraries.
Eight contigs had cDNA clones that were not equally
distributed within the collective p12fol and p12cl libraries
(contigs with four or more members) (Table 6). Eleven contigs had cDNAs that were found primarily in pd12CL, and
seven contigs had cDNAs that were found primarily in
pd12fol. The greatest difference in cDNA distribution toward pd12CL was found for contigs 1 (cytochrome c oxidase), 12 (prostate-secreted seminal plasma protein [PSP94 protein]), 33 (ATP synthase A chain), 3043 (StAR),
5146 (tissue metalloproteinase inhibitor [TIMP]-1), and
5207 (CYP1B1). The cDNAs for a variety of proteins and
enzymes involved in progesterone synthesis were found in
higher proportions within pd12CL (contigs 3043 and 5325
[StAR], contig 2308 [CYPXIA1; side chain-cleavage enzyme], and contig 282 [3-b-hydroxysteroid dehydrogenase]). The greatest difference in cDNA distribution toward
pd12fol was found for contigs 278 (nexin-1), 18 (unknown), 368 (translationally controlled tumor protein
[TCTP]), 7 (collagen a2[I] chain), 255 (unknown), and
2326 (alanine glyoxylate aminotransferase). The cDNAs
within contigs 7, 18, and 368 (found in high abundance in
pd12fol) were also abundant in the individual follicle libraries (Table 5).
Eleven contigs had cDNA clones that were not equally
distributed within the collective pfeto, pnatal, and pputal
libraries (contigs with six or more members) (Table 7).
Three contigs (contig 33 [ATP synthase A chain], contig
161 [cytochrome c oxidase III], and contig 2285 [guanine
nucleotide-binding protein b subunit-like protein]) contained cDNAs that were predominately found in the pfeto
library. Three contigs contained cDNAs that were predominately found in the pnatal library (contig 412 [collagen,
type III, a1], contig 533 [eukaryotic translation initiation
factor 4A], and contig 5141 [retinoic acid receptor-responder protein 1]). Five contigs contained cDNAs that were predominately found in the pputal library (contig 288 [ferritin,
heavy polypeptide], contig 430 [monooxygenase], contig
64 [selenoprotein P-like protein], contig 756 [inhibin a
chain], and contig 34 [vimentin]). The five contigs whose
cDNAs were predominately found in the pputal library
(created from tissue-containing antral follicles) were among
the largest contigs for the individual follicle libraries (Table
5).
Comparison of mRNA Amount in Tissues with cDNA
Frequency in Libraries
Seven mRNAs were independently examined for expression level within ovarian tissues using RP assay (Fig.
1995
EXPRESSED SEQUENCE TAGS FROM PORCINE OVARY
TABLE 3. Contig number, TIGR TC number, gene name, total number of isolated clones, and percentage of all sequences for contigs with 20 or more
members (all libraries).a
Contig
number
1
2
33
160
161
434
36
274
288
1308
7
368
273
1788
756
12
34
67
412
1309
64
2036
414
435
104
651
1133
13
1712
18
162
430
5122
164
167
413
2515
3770
2281
748
1313
1787
5130
5123
533
741
1732
5125
a
TIGR TC
TC116362
TC103875
TC116169
TC116172
TC103937
TC116175
TC116463
TC103916
TC103870
TC103959
TC116269
TC116297
TC104571
TC116311
TC104824
TC117722
TC116272
TC116295
TC103911
TC103988
TC104140
TC103960
TC116176
TC103503
TC104062
TC116298
TC115698
TC103942
TC104193
TC107052
TC116338
TC103694
TC103919
TC116277
TC103994
TC103287
TC103878
TC116268
TC116335
TC116555
TC103159
TC116317
TC116409
TC116192
TC116418
TC116332
TC116301
TC103354
Gene name
Cytochrome c oxidase (EC 1.9.3.1) chain I—pig mitochondrion
Elongation factor 1-a 1
ATP synthase A chain (EC 3.6.1.34) (Protein 6)
Cytochrome b
Cytochrome c oxidase III
60S acidic ribosomal protein PO (L10E)
Glutathione S-transferase
40S ribosomal protein S2
Ferritin, heavy polypeptide
Ribosomal protein S3a
Collagen a2(I)
Translationally controlled tumor protein (TCTP) (p23)
Prostaglandin-F synthase 1 (EC 1.1.1.188)
Ribosomal protein L7a
Inhibin a
Prostate secreted seminal plasma protein (PSP-94 protein)
Vimentim
40S ribosomal protein SA (P40)
Collagen, type III, a1
40S ribosomal protein S7 (S8)
Selenoprotein P-like protein
60S ribosomal protein L6
Ribosomal protein L10
60S Ribosomal protein L4
Ribosomal protein S8
Cathepsin L (EC 3.4.22.15)
Tumor-specific transplantation antigen P198 homolog p23
Polyubiquitin 3
Bone proteoglycan II (PG-S2) (Decorin)
Unknown
60S ribosomal protein L9
Monooxygenase, nonspecific
Heat shock cognate 71-kDa protein
Ribosomal protein L11
60S ribosomal protein L17 (L23)
Nonhistone chromosomal protein HMG-17
Ferritin, light polypeptide
Ribosomal protein S9
Ribosomal protein L5
Cytochrome b5
40S ribosomal protein S6 (Phosphoprotein NP33)
Integral membrane protein 2B (Transmembrane protein BR1)
Eukaryotic translation elongation factor 1 gamma
Glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12)
Eukaryotic translation initiation factor 4A
Diamine acetyltranferase (EC 23.1.57)
40S ribosomal protein S3
60S ribosomal protein L8
Total clones (n) % of sequences
222
215
114
89
86
71
63
63
63
56
53
49
47
47
46
44
44
42
42
39
37
37
34
33
32
32
32
31
31
30
29
29
28
27
27
27
25
24
23
22
22
22
22
21
20
20
20
20
1.42
1.38
0.73
0.57
0.55
0.45
0.40
0.40
0.40
0.36
0.34
0.31
0.30
0.30
0.29
0.28
0.28
0.27
0.27
0.25
0.24
0.24
0.22
0.21
0.20
0.20
0.20
0.20
0.20
0.19
0.19
0.19
0.18
0.17
0.17
0.17
0.16
0.15
0.15
0.14
0.14
0.14
0.14
0.13
0.13
0.13
0.13
0.13
A complete list of contigs can be found at http://www.swine.rnet.missouri.edu.
5, A and B) or Northern blot analysis (Fig. 5C). In most
cases, the level of mRNA expression within a tissue reflected the relative number of cDNA clones isolated from the
respective libraries. For example, the RP assay did not detect the inhibin a subunit mRNA in fetal or neonatal ovary,
but a radiographic signal was readily detected for inhibin
a subunit in prepubertal ovary (Fig. 5A). The corresponding clone frequency within contig 756 (inhibin a) was one,
zero, and seven for pfeto, pnatal, and pputal, respectively.
Likewise, the mRNA expression level within fetal, neonatal, and prepubertal tissues for selenoprotein P (contig 64)
and an unknown gene (contig 2542) (Fig. 5A) as well as
the mRNA expression level for 2-, 4-, 6-, or 8-mm follicles
for inhibin b (contig 3428) and LH-receptor mRNA (contig
1894) (Fig. 5B) were visually correlated with the number
of clones isolated from the respective libraries. The 17ahydroxylase mRNA was highest in 6-mm follicles (Fig.
5B), but the corresponding number of cDNA clones for
p2mm, p4mm, p6mm, and p8mm was one, zero, four, and
five, respectively. The apparent decrease in 17a-hydroxylase mRNA in 8-mm follicles was not reflected in the
cDNA frequency across libraries. The PSP-94 cDNA was
found almost exclusively in the corpus luteum (one cDNA
from pd12fol compared to 41 clones from pd12CL), and
Northern blot analyses demonstrated expression of PSP-94
(;600 bp) in corpus luteum but not in the follicle or nonluteal ovary (Fig. 5C).
DISCUSSION
Large-scale cDNA sequencing projects have generated
millions of ESTs from numerous species and a variety of
tissues (including porcine ovary) [7–12]. The present EST
project is unique from others, because whole ovary repre-
1996
JIANG ET AL.
TABLE 4. Contig number, TIGR TC number, gene name, total number of isolated clones, and percentage of all sequences for representative contigs
whose cDNAs encode genes involved in the endocrine regulation of ovarian cells.
Contig
number
TIGR TC
Gene name
Total clones (n)
% of sequences
756
3775
2120
156
1887
6872
4489
5090
5255
2738
4322
5955
TC104824
TC117675
TC104817
TC117324
TC104032
TC117989
TC119778
TC118084
TC120386
TC103983
TC117225
TC117725
Hormones and hormone-binding proteins
Inhibin a chain
Follistatin (activin-binding protein)
Relaxin
Relaxin-like factor (RLF/INSL3)
Monocyte chemotactic protein 1 (MCP-1)
Interleukin-18
Transforming growth factor b-1
Transforming growth factor b-3
Insulin-like growth factor-I
Insulin-like growth factor-II
Insulin-like growth factor binding protein-2
Insulin-like growth factor binding protein-5
46
1
4
2
2
1
1
1
1
3
2
2
0.29
0.01
0.03
0.01
0.01
0.01
0.01
0.01
0.01
0.02
0.01
0.01
273
1714
6549
TC104571
TC104909
TC105381
Prostaglandin synthesis
Prostaglandin-F synthase 1
Prostaglandin-E synthase
Prostaglandin-D synthase
47
2
1
0.30
0.01
0.01
1953
3043
2308
281
5149
1576
TC116550
TC116845
TC104489
TC117802
TC104115
TC108675
Steroidogenesis
Sterol carrier protein-2
Steroidogenic acute regulatory protein
Cytochrome P450 side-chain cleavage
3-b-Hydroxysteroid dehydrogenase
Cytochrome P450 steroid 17 a-hydroxylase/17 20 lyase
Cytrochrome P450 Aromatase
1
19
5
8
11
1
0.01
0.12
0.03
0.05
0.07
0.01
5888
1894
452
3323
5800
6335
TC106004
NP276344
TC104214
TC108406
TC105022
TC119247
Hormone receptors
Atrial natriuretic peptide receptor B (guanylate cyclase)
Luteinizing hormone receptor
Progesterone membrane-binding protein
GnRH receptor (type II)
Interleukin-11-receptor a chain
Steroidogenic factor 1
2
5
4
1
1
1
0.01
0.03
0.03
0.01
0.01
0.01
5542
5568
6096
2751
5808
3144
2038
3430
7751
6123
353
5536
1502
2599
2801
1244
TC104369
TC124885
TC105650
TC104116
TC119549
TC104440
TC106516
TC117154
TC107962
TC104637
TC105997
TC116679
TC105469
TC116574
TC117952
TC105621
Second-messenger systems
cAMP-dependent protein kinase type I-a regulatory chain
cAMP-dependent protein kinase type II-b regulatory chain
Phospholipase C b-2
Calmodulin
Phosphatidylinositol 3-kinase regulatory subunit
RhoC
Growth factor receptor-bound protein 14
Mitogen-activated protein kinase 1a
Mitogen-activated protein kinase kinase 1
Mitogen-activated protein kinase 3 (ERK-1)
Mitogen-activated protein kinase phosphatase 6
Janus kinase 1
SMAD 4
Mitogen-activated protein/microtubule affinity-regulating kinase 3
Serine/threonine protein phosphatase 2A regulatory subunit A
Serine/threonine protein phosphatase 2A regulatory subunit B
3
3
2
11
1
4
3
1
1
2
1
3
2
6
1
1
0.02
0.02
0.01
0.07
0.01
0.03
0.02
0.01
0.01
0.01
0.01
0.02
0.01
0.04
0.01
0.01
senting specific stages of development (fetal, neonatal, and
prepubertal) and preovulatory ovarian follicles sampled at
different diameters were used to generate individual libraries for cDNA sequencing. Each clone was sequenced from
its original library so that the frequency of cDNA within
each tissue could be determined. Oligonucleotide tags were
used during library construction to confirm the origin of
each cDNA clone. The 15 613 sequences clustered into
8507 contigs whose sequences were blasted against the
TIGR Porcine Gene Index. Sixty-eight percent of the contigs were homologous to existing sequences within the
TIGR Porcine Gene Index. The genes were associated with
common cell functions, such as energy metabolism, protein
synthesis, signal transduction, cell communication, transport, development, and cell-cycle regulation (Fig. 4). Genes
associated with ovary-specific functions, such as steroido-
genesis, were also identified (Table 4). The remaining 30%
of the consensus sequences were not homologous to porcine sequences at TIGR and may represent novel mRNA
species. Given the large number of unique sequences in the
TIGR Porcine Gene Index, our observed frequency of novel
consensus sequences (32%) suggests a high rate of new
gene discovery in porcine ovary. The original cDNA sequences and their respective library clusters represent a scientific resource for ovarian biologists who study ovarian
gene expression (http://www.swine.rnet.missouri.edu).
As revealed by the cDNA frequency, cytochrome c oxidase cDNA was the most abundant cDNA, representing
1.42% of the 15 613 clones (Table 3). Cytochrome c oxidase is an enzyme in the mitochondrial electron-transport
chain (intracellular energy production). Other highly expressed genes in porcine ovary were also involved in intra-
EXPRESSED SEQUENCE TAGS FROM PORCINE OVARY
FIG. 4. The percentage of cDNA within different categories of the GO
Index. The cDNAs were classified according to GO Slim categories for
cellular component (top), molecular function (middle), and biological
process (bottom).
cellular energy production (ATP synthase and cytochrome
c oxidase III). Elongation factor 1a had the second-highest
cDNA frequency (1.38% of the cDNA clones), and other
genes involved in protein synthesis (including ribosomal
proteins) were also highly expressed. More than 50% of the
cDNA sequences encoded proteins involved in metabolism
(GO Slim process) (Fig. 4), and nearly 50% of the genes
encoded components of the ribosome or mitochondria (GO
Slim component) (Fig. 4). Thus, the bulk of the mRNA
within an ovarian cell encodes genes that are involved in
the intracellular activities that are not unique to ovarian
cells (metabolism, protein synthesis, etc.), and only a small
1997
FIG. 5. RP and Northern blot analyses for selected mRNA representing
cDNA sequenced in the project. The analyses were performed on mRNA
from A) whole porcine ovary (fetal, neonatal, or prepubertal; ribonuclease
protection assay); B) ovarian follicles collected at different diameters after
weaning (2, 4, 6, or 8 mm; ribonuclease protection assay); or C) porcine
ovary (whole nonluteal ovary [Ovary], corpus luteum [CL], or follicles
[Fol]; Northern blot analyses for PSP-94). In A and B, the contig number
and the annotated gene name are presented to the right of the blot and
the number of cDNA isolated from each library is shown below the individual lanes. For PSP-94 (C), 41 cDNAs were isolated from pd12cl, and
one cDNA was isolated from pd12fol. In general, the number of library
cDNAs within the respective contig reflected the level of mRNA expression within the tissue.
percentage of the major cDNA species appeared to be specific to reproductive tissues. Reproductive genes with a
high expression level (high frequency of sequenced cDNA)
included inhibin a and PSP-94. The abundance of cDNA
for these two reproductive genes in ovary is unusual, because most other genes previously associated with ovarian
function were found in relatively low numbers. For exam-
1998
JIANG ET AL.
TABLE 5. The number of sequenced cDNAs within individual follicle libraries (p2mm, p4mm, p6mm, and p8mm).a
Contig
2
1
160
36
288
273
756
161
34
651
33
7
368
18
1133
430
2515
5122
372
13
413
221
412
1041
5130
5123
370
222
37
1311
64
1712
1711
476
5141
163
997
1448
5152
416
5149
a
b
TIGR TC
TC103875
TC116362
TC116172
TC116463
TC103870
TC104571
TC104824
TC103937
TC116272
TC116298
TC116169
TC116269
TC116297
TC107052
TC115698
TC103694
TC103878
TC103919
TC118187
TC103942
TC103287
TC116199
TC103911
TC104001
TC116409
TC116192
TC116291
TC116201
TC116465
TC104054
TC104140
TC104193
TC116425
TC116368
TC116449
TC116313
TC116411
TC104248
TC117178
TC116566
TC104115
Name
Elongation factor 1-a 1
Cytochrome c oxidase (EC 1.9.3.1) chain 1
Cytochrome b
Glutathione S-transferase
Ferritin, heavy polypeptide
Prostaglandin-F synthase 1 (EC 1.1.1.188)
Inhibin a chain
Cytochrome c oxidase III
Vimentin
Cathepsin L (EC 3.4.22.15)
ATP synthase A chain (EC 3.6.I.34) (Protein 6)
Collagen a 2(I)
Translationally controlled tumor protein (TCTP) (p23)
Unknown
Tumor-specific transplantation antigen P198 homolog p23
Monooxygenase, nonspecific
Ferritin, light polypeptide
Heat shock cognate 71-kDa protein
Smfn protein
Polyubiquitin 3—human
Nonhistone chromosomal protein HMG-17
Actin, g
Collagen, type III, a1
Apolipoprotein E
Eukaryotic translation elongation factor 1 g
Glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12)
Thymosin b 4
Actin a
Glutathione S-transferase
Apolipoprotein AI
Selenoprotein P-like protein
Bone proteoglycan II precursor (PG-S2)
Translation elongation factor eEF-2
Polyadenylate-binding protein 1
Retinoic acid receptor-responder protein 1
ADP ATP carrier protein isoform T2
Omithine decarboxylase antizyme
ATP synthase g chain mitochondrial precursor
Glutathione S-transferase
Mitochondrial SP-22 protein
Steroid 17-alpha-hydroxylase/17 20 lyase
p2mm
p4mm
p6mm
p8mm
P,
22
16
9
2
6
4
3
16
12
6
10
6
8
2
6
8
6
2
1
6
3
6
2
2
2
2
8
1
0
2
3
2
1
3
0
1
1
2
0
2
1
15
4
2
3
5
4
1
7
6
1
8
5
7
0
6
4
2
7
3
1
5
0
1
3
3
0
4
0
2
0
2
1
0
1
1
0
3
0
1
1
0
40
21
17
10
29
25
27
7
6
9
3
7
6
7
5
4
5
4
5
4
4
5
6
3
4
6
1
4
2
4
4
3
6
3
3
6
3
1
6
2
4
50
34
28
37
8
7
7
6
10
14
7
7
4
15
3
3
5
3
7
4
3
4
5
6
4
5
0
7
8
6
2
5
3
3
6
3
3
7
3
5
5
0.001
0.001
0.001
0.001
0.001
0.001
0.001
0.050
NSb
0.050
NS
NS
NS
0.001
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
0.010
0.050
0.050
NS
NS
NS
0.100
NS
0.100
0.100
NS
0.050
0.100
NS
NS
The largest contigs ($ 10 total members) are shown. Ribosomal subunit genes are not shown.
NS, not significant.
ple, only a single copy of aromatase (contig 1576), FSH
receptor (contig 7993), zona pellucida protein 3 (contig
7528), and IGF-I (contig 5255) were sequenced. Fewer than
five cDNAs were sequenced for several other genes that
play physiologically important roles within the ovary (IGFII, IGF-binding protein-2, LH receptor) (Table 4). Tissuespecific ovarian genes therefore may be found only in contigs with a small number of members.
The cDNA libraries sequenced in the present study were
not subtracted or normalized. The frequency of cDNA sequences therefore may approximate the expression level of
the mRNA in the tissue from which the cDNA library was
made [21–24]. For this reason, the frequency of each cDNA
from each library was counted. Analysis of the cDNA frequency across different libraries suggested differential patterns of expression within different-size follicles (diameter,
2–8 mm) (Table 5), between follicles and corpora lutea (Table 6), and across developmental time-points (fetal, neonatal, and prepubertal ovary) (Table 7). The expression of
selected genes was consistent with the frequencies of their
respective cDNA in the individual libraries (analyzed by
RP assay or Northern blot) (Fig. 5).
Distinct biochemical processes may underlie the morphological and physiological changes in follicles as they
grow from 2 to 8 mm in diameter. Follicles of 2 to 4 mm
in diameter either may continue growth or may undergo
atresia [25]. Follicles of 6 mm in diameter are highly estrogenic, and in our previous analyses, follicles of 6 mm in
diameter had more aromatase mRNA than those of 2, 4, or
8 mm in diameter [18]. Follicles of 8 mm in diameter had
less aromatase, because they had been exposed to the LH
surge and had undergone luteinization before ovulation.
Eight contigs had cDNA clone frequency that was highly
disproportionate (P ,0.001) across follicle libraries (Table
5). Five contigs had cDNA clone frequencies that increased
in the collective p6mm and p8mm libraries (contig 2 [elongation factor 1a], contig 1 [cytochrome c oxidase], contig
160 [cytochrome b], contig 36 [glutathione S-transferase],
and contig 18 [unknown]). The physiological basis for the
observed changes in gene frequency is open to further scientific investigation. The increase in elongation factor 1a
mRNA and cytochrome c oxidase mRNA may reflect an
increase in protein synthesis and energy metabolism, respectively, in large follicles. The increase in glutathione Stransferase that we observed in the p8mm library is supported by previous research showing an increase in glutathione S-transferase in large follicles [26]. The increase in
glutathione S-transferase is believed to support progester-
1999
EXPRESSED SEQUENCE TAGS FROM PORCINE OVARY
TABLE 6. The number of sequenced cDNAs within individual day 12 libraries (pd12fol and pd12CL).a
Contig
1
12
33
3043
161
748
64
5139
36
1531
2943
2
741
5146
278
2331
18
368
5207
412
2252
7
255
2326
160
282
581
2308
5325
1914
2453
245
273
1998
2120
5158
1031
1496
a
b
TC
TC116362
TC117722
TC116169
TC116845
TC103937
TC116555
TC104140
TC117113
TC116463
TC105082
TC117132
TC103875
TC116332
TC116432
TC117595
TC104136
TC107052
TC116297
TC117961
TC103911
TC104215
TC116269
TC120237
TC117678
TC116172
TC117803
TC106109
TC104489
TC116845
TC110813
TC103991
TC117806
TC104571
TC119205
TC104817
TC103355
TC104176
TC108301
Name
Cytochrome c oxidase (EC 1.9.3.1) chain I
Prostate secreted seminal plasma protein precursor (PSP-94 protein)
ATP synthase A chain (EC 3.6.1.34) (Protein 6)
Steroidogenic acute regulatory protein
Cytochrome c oxidase III
Cytochrome b5
Selenoprotein P-like protein
NADH-ubiquinone oxidoreductase chain 6 (EC 1.6.5.3)
Glutathione S-transferase
NADH-ubiquinone oxidoreductase chain 2 (EC 1.6.5.3)
3-Hydroxy-3-methylglutaryl-coenzyme A synthase
Elongation factor 1 a 1
Diamine acetyltransferase (EC2.3.1.57)
TIMP-1
Nexin-I
PTH-responsive osteosarcoma D1 protein
Unknown
TCTP (23)
CYP1B1 cytochrome P450 enzyme
Collagen, type III, a1
Cytochrome oxidase subunit II
Collagen a 2(I)
Unknown
Alanine glyoxylate aminotransferase
Cytochrome b
3-b-Hydroxysteroid dehydrogenase
Nuclear receptor NR5A2
P450scc cytochrome P450 11A1 (EC 1.14.15.6) (CYPXIA1)
Steroidogenic acute regulatory protein mitochondrial
IQ motif containing GTPase-activating protein 2
Prosaposin
UDP glucose pyrophosphorylase
Prostaglindin F synthase 1 (EC 1.1.1.188)
Solute carrier family 35, member F5
Relaxin
Glutathione peroxidase
Heat shock protein 84
Karyopherin (importin) b
pd12fol
pc12cl
P,
21
1
7
1
4
3
7
4
6
3
1
5
2
0
5
1
5
5
0
1
3
4
4
4
0
0
0
0
0
3
3
1
1
1
1
1
2
2
67
41
34
15
10
9
4
7
2
5
6
2
5
6
1
5
0
0
5
4
2
0
0
0
4
4
4
4
4
1
1
3
3
3
3
3
2
2
0.001
0.001
0.001
0.010
NSb
NS
NS
NS
0.100
NS
NS
NS
NS
0.050
0.050
NS
0.010
0.010
0.050
NS
NS
0.050
0.050
0.050
0.100
0.100
0.100
0.100
0.100
NS
NS
NS
NS
NS
NS
NS
NS
NS
The largest contigs ($ 4 total members) are shown. Ribosomal subunit genes are not shown.
NS, Not significant.
one synthesis in preovulatory follicles that have been exposed to the LH surge and are undergoing luteinization
[27]. The cytochrome b cDNA was increased in large follicles as well. Cytochrome b is a component of the oxidase
system of neutrophils, and neutrophils are attracted to the
follicle during ovulation [28]. The increase in cytochrome
b therefore may reflect an infiltration of neutrophils into the
preovulatory follicle. Contig 18 represents a cDNA that encodes an unknown protein. The cDNA sequence is found
within the TIGR database but, nonetheless, cannot be tied
to any known gene. The identity and function of this unknown protein that is increased in large porcine follicles is
a potential subject for future investigations.
Contig 288 (ferritin, heavy polypeptide), contig 273
(prostaglandin F synthase 1), and contig 756 (inhibin a)
were specifically increased in the p6mm library at P ,
0.001. The high expression of inhibin a is consistent with
the role of inhibin as an endocrine factor secreted from
large follicles [29]. Others have reported an increase in inhibin a mRNA in midstage preovulatory follicles, with a
subsequent decrease in inhibin a mRNA is late-stage preovulatory follicles [30]. Ovarian follicles synthesize prostaglandins E, D, and F, as evidenced by our sequencing of
synthase enzymes for the respective enzymes (Table 4) and
previously published literature [31]. Prostaglandin synthesis
increases in preovulatory follicles, particularly around the
time of the LH surge [32], and an increase in prostaglandin
synthesis is required for ovulation in pigs [33]. A clear
increase in ferritin cDNA was noted within the p6mm library, and to our knowledge, changes in ferritin mRNA
have not been previously reported in preovulatory follicles.
Iron is required for coordinating the heme within P450 enzymes, which is a class of enzymes that includes those
involved in steroidogenesis [34]. Ferritin sequesters and
stores intracellular iron in a nontoxic form [35]. The increase in ferritin in preovulatory follicles may play an important role in iron homeostasis during the preovulatory
period.
Twelve contigs had cDNA frequencies that differed at P
, 0.05 across the pd12fol and pd12CL libraries (Table 6).
Six of the contigs (contig 1 [cytochrome c oxidase], contig
12 [PSP-94], contig 33 [ATP synthase], contig 3043
[StAR], contig 5146 [TIMP-1], and contig 5207 [CYP1B1])
contained cDNAs that were more abundant in the pd12CL
library. An increase in cytochrome c oxidase and ATP synthase activity in corpora lutea relative to follicles has been
demonstrated in previous studies [36, 37]. Cytochrome c
oxidase and ATP synthase are mitochondrial enzymes, and
the tissue differences may simply reflect the greater density
of mitochondria in corpus luteum relative to follicle. Greater StAR mRNA within corpora lutea relative to follicles
has also been found in porcine ovary [38]. The difference
in cDNA frequency may reflect the greater mitochondrial
density as well as greater steroidogenic capacity of corpus
2000
JIANG ET AL.
TABLE 7. The number of sequenced cDNA within individual ovary libraries (pfeto, pnatal, and pputal).a
Contig
2
1
33
161
160
412
7
368
1712
13
288
69
430
64
1133
5130
756
34
1787
1433
1893
413
5122
2546
2285
533
1764
552
2515
1892
5141
5123
760
222
109
1711
383
a
b
TC
TC103875
TC116362
TC116169
TC103937
TC116172
TC103911
TC116269
TC116297
TC104193
TC103942
TC103870
TC116177
TC103694
TC104140
TC115698
TC116409
TC104824
TC116272
TC116317
TC103884
TC104425
TC103287
TC103919
TC116429
TC104004
TC116418
TC104011
TC104069
TC103878
TC103911
TC116449
TC116192
TC105289
TC116201
TC116634
TC116425
TC118023
Name
Elongation factor 1-a I
Cytochrome c oxidase (EC 1.9.3.1) chain I
ATP synthase A chain (EC 3.6.1.34)
Cytochrome c oxidase III
Cytochrome b
Collagen, type III, aI
Collagen a 2(I)
TCTP (p23)
Bone proteoglycan II precursor (PG-S2) (Decorin)
Polyubiquitin 3
Ferritin, heavy polypeptude
Tubulin a-1 chain
Monooxygenase, nonspecific
Selenoprotein P-like protein
Tumor-specific transplantation antigen P198 homolog p23
Eukaryotic translation elongation factor 1 g
Inhibin a chain
Vimentin
Integral membrane protein 2B (Transmembrane protein BRI)
Chain B of porcine hemoglobin
Unknown
Nonhistone chromosomal protein HMG-17
Heat shock cognate 71-kDa protein
p97
Guanine nucleotide-binding protein b subunit-like protein
Eukaryotic initiation factor 4A-II (eIF-4A-II)
Smooth muscle protein SM22
Heterogeneous nuclear ribonucleoprotein H (hnRNP H)
Ferritin, light polypeptide
Collagen, type III, aI
Retinoic acid receptor responder protein 1
Glyceraldehyde 3-phosphate dehydrogenase
Elongation factor 1 d
Actin a
Translation elongation factor eEF-I b chain
Translation elongation factor eEF-2
Unknown
pfeto
pnatal
pputal
P,
17
13
26
20
9
2
3
7
1
4
2
5
1
0
3
2
1
2
0
2
4
1
1
3
5
2
1
0
2
2
0
2
0
0
3
3
1
34
18
6
3
7
17
8
7
8
3
2
4
0
1
2
4
0
0
3
1
2
3
3
3
2
5
1
4
4
2
5
0
2
4
1
1
3
28
21
10
9
13
3
8
4
7
7
9
3
9
9
5
3
7
6
5
5
2
4
4
2
0
0
5
3
1
3
1
4
4
2
2
2
2
NSb
NS
0.001
0.001
NS
0.001
NS
NS
NS
NS
0.050
NS
0.001
0.001
NS
NS
0.010
0.050
NS
NS
NS
NS
NS
NS
0.050
0.100
NS
NS
NS
NS
0.050
NS
NS
NS
NS
NS
NS
The largest contigs ($ 6 total numbers) are shown. Ribosomal subunit genes are not shown.
NS, Not significant.
luteum relative to follicle. Tissue inhibitors of metalloproteinases play a role in remodeling of the extracellular matrix [39]. The expression of TIMP-1 mRNA increases during late preovulatory development and increases further
within the corpus luteum [40]. To our knowledge, specific
expression of CYP1B1 (a P450 enzyme involved in steroid
metabolism) [41] in corpus luteum has not been previously
reported.
The abundance of prostate-secreted plasma protein (PSP94 or b-microseminoprotein) in corpus luteum relative to
follicle was unexpected (Table 6). The PSP-94 protein is a
major component of seminal fluid from a variety of species.
Although PSP-94 has been used as a marker of prostatic
development in males [42], a specific function for PSP-94
in prostate physiology has not been identified. The PSP-94
protein will bind immunoglobulin and may influence the
activity of immune cells [43]. A recent study found an inhibitory effect of PSP-94 in prostate tumor growth [44].
Thus, PSP-94 may control cell growth and development
either through an indirect effect on immune cells or through
a direct effect on luteal cells themselves. We examined
PSP-94 in cow and pig corpus luteum and found that the
mRNA was only expressed in pig corpus luteum (unpublished observations). Furthermore, the mRNA was maximally expressed in midcycle corpora lutea. Thus, the porcine corpus luteum may be unique from the corpus luteum
of other species in that it specifically expresses PSP-94
(Fig. 5). The highly specific expression of PSP-94 mRNA
suggests a unique role for this protein in porcine corpus
luteum.
Six contigs had cDNAs that were more abundant in
pd12fol compared to pd12cl (P , 0.05; contig 278 [nexin1], contig 18 [unknown], contig 368 [TCTP], contig 7 [collagen a2 (I) chain], contig 255 [unknown], and contig 2326
[alanine glyoxylate aminotransferase]) (Table 6). The
cDNAs within contigs 7, 18, and 368 were also abundant
in the individual follicle libraries (Table 5). The relative
abundance of nexin-1 (a serine protease inhibitor) in follicles compared to corpus luteum confirms a recent report
showing a high level of nexin-1 expression in preovulatory
follicle [45]. Nexin-1 mRNA decreases after the LH surge.
Thus, the decrease in a specific protease inhibitor may facilitate ovulation. Translationally controlled tumor protein
was recently implicated as a protein that binds elongation
factor eEF1A during the elongation step of protein synthesis [46]. The abundance of TCTP cDNA in follicle libraries
(Tables 5 and 6) highlights the importance of protein synthesis during follicular development. The presence of collagen a2 (I) in pd12fol may reflect the relative abundance
of specific collagen subunits in follicles versus corpora lutea [47]. The two contigs classified as unknown should be
investigated further as genes controlling unique aspects of
follicular development.
Several genes were differentially expressed when the
pfetal, pnatal, and pputal libraries were examined. Ovarian
follicular development in pigs is different from that in hu-
EXPRESSED SEQUENCE TAGS FROM PORCINE OVARY
mans and other farm animals, because the neonatal ovary
(sampled herein) is compact, with a dense population of
primordial and primary follicles. Oocytes may still reside
in egg nests at birth, and a period of 1–2 wk after birth
may be required before follicular populations are fully established and primordial follicles enter the growth phase
[48]. The ovary then undergoes a protracted period of follicular growth before puberty. Three genes had contigs that
were highly represented in either fetal or neonatal ovary (P
, 0.001; contig 33 [ATP synthase A chain], contig 161
[cytochrome c oxidase III], and contig 412 [collagen, type
III, a1]) (Table 7). The fetal ovary is densely packed with
oocytes, and oocytes have a large number of mitochondria.
Thus, the high frequency of ATP synthase and cytochrome
c oxidase in the pfeto libraries may reflect the preponderance of mitochondria in developing oocytes. Ovarian follicles change the collagen composition of their basal lamina
when they commence growth [49]. The shift in expression
toward collagen type III a1 within the pnatal library may
be indicative of an active phase of follicular growth in the
neonatal ovary. Additional cDNAs had a frequency that
appeared to be greater in either the pfeto (contig 2285 [guanine nucleotide-binding protein b subunit-like protein]) or
pnatal (contig 5141 [retinoic acid-responder protein 1] and
contig 533 [eukaryotic translation elongation factor 1g]) libraries. Five contigs (contig 288 [ferritin, heavy polypeptide], contig 430 [monooxygenase], contig 64 [selenoprotein P-like protein], contig 756 [inhibin a chain], and contig
34 [vimentin]) had cDNAs that were predominately derived
from prepubertal ovaries (pputal library). The prepubertal
ovaries used to construct the pputal library contained antral
follicles. Each of the five contigs was among the largest
contigs in the collective follicle libraries (Table 5). The observed frequencies of cDNA in the pputal library therefore
supported the sequencing data from the follicle libraries
(Table 5).
In summary, the present study has provided a catalog of
8507 contigs derived from 15 613 cDNA sequences obtained in porcine ovarian tissue. The list of ovarian cDNAs
and their frequencies in each ovarian library will be a useful
index of ovarian gene expression. The observed differences
in cDNA frequency across libraries appeared to be logical
given existing knowledge of ovarian biology. For most contigs, the frequency of the sequenced genes was too few to
reliably study gene expression across tissues. Greater sensitivity for gene expression analysis will be achieved using
solid-phase microarrays constructed from the cDNAs generated herein. This second phase of the current project is
underway.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
ACKNOWLEDGMENTS
The authors thank B. Blaue, C. K. Boyd, and P. M. Roozen for technical assistance.
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