BIOLOGY OF REPRODUCTION 58, 952-962 (1998)
Developmental Consequences of Karyokinesis Without Cytokinesis during the First
Mitotic Cell Cycle of Bovine Parthenotes'
Rabindranath De La Fuente and W. Allan King2
Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph,
Guelph, Ontario, Canada N1G 2W1
ABSTRACT
Bovine parthenogenetic embryos and bovine embryos produced by in vitro fertilization were compared for chromosomal
complement and developmental potential. Oocytes (n = 1885)
were matured in vitro, fertilized (n = 1151) or activated (n =
734) by exposure to 5 RiM ionomycin for 4 min, and then treated
with 1.9 mM 6-dimethylaminopurine for 5 h to inhibit protein
kinase functions and promote mitosis. Mean cleavage rates at
4.2% for
4.7% for fertilization and 60.1
48 h were 76.3
activation (p < 0.05). A similar percentage of embryos had
reached the blastocyst stage on Day 8 post fertilization/postactivation (16.4 ± 3.3%) and (15.8 ± 1.0%), respectively. Blastocysts (n = 53) produced by in vitro fertilization had higher
total cell numbers (116.9 ± 5.5) than parthenotes (n = 71, 67.2
+ 3.5 cells, p < 0.05). Differential staining indicated a significant reduction in the number of blastomeres allocated to both
the inner cell mass and trophectodermal lineages in parthenotes
(p < 0.05). All parthenotes (n = 65) were polyploid or mixoploid, with observed karyotypes of 4n (61.53%), 2n/4n
(30.76%), 2n/8n (4.61%), and 3n (3.07%). In contrast, only 9
control blastocysts (n = 53) revealed abnormal metaphases
(16.9%). At 6 h postactivation (hpa), 70.7% of parthenotes (n
= 65) demonstrated a fully formed pronucleus; and at 10 hpa
(n = 86), 89% had completed pronuclear formation. Pronuclear
DNA replication was observed by 6 hpa and resulted in the formation of a second pronucleus in 76.9% of activated oocytes
(n = 104) by 24 hpa. These pronuclear kinetics lead to a high
number of embryos with binucleate blastomeres upon cleavage.
Thus, alterations in the DNA content (ploidy) of bovine parthenogenetic blastocysts reflect ongoing karyokinesis without cytokinesis during the first mitotic cell cycle after exposure to a
protein kinase inhibitor.
INTRODUCTION
Analysis of the zygotic transition to the first mitosis in
the bovine parthenote is essential to improve oocyte activation and nuclear transfer procedures in domestic species.
Moreover, production of parthenotes is also relevant for the
analysis of mechanisms regulating embryonic development
and growth in uniparental embryos. Murine parthenotes
have been valuable models for investigating the roles of the
maternal and paternal genomes on embryonic gene expression and development [1-4]. Differences in protein synthesis requirements during completion of the second meiotic
division between bovine and murine oocytes [5], as well as
different patterns of centrosomal inheritance [6, 7], make
the bovine oocyte an interesting model for parthenogenesis.
Unlike the situation with the mouse, where maternal inheritance of centrosomal components may facilitate the proAccepted November 19, 1997.
Received August 5, 1997.
'This research was supported by grants from the Natural Sciences and
Engineering Research Council of Canada, Cattle Breeding Research Council, and the Ontario's Ministry of Agriculture Food and Rural Affairs. R.D.
was the recipient of a Government of Canada Award.
2Correspondence. FAX (519) 767-1450; e-mail: [email protected]
duction of diploid parthenotes upon artificial activation [6],
bovine zygotes depend on both paternal and maternal contributions for centrosome formation [7].
In mammalian oocytes, meiosis is controlled by a complex cascade of protein phosphorylation/dephosphorylation
induced by protein kinases [8, 9]. Activation of regulatory
proteins by protein kinases coordinates the optimal interaction between nuclear and cytoplasmic components in the
oocyte, resulting in a single round of DNA replication followed by two successive rounds of chromosome segregation [10]. Reductional divisions to ensure a haploid DNA
content in the female gamete are thus accomplished through
the functional integration of chromosomes and microtubular components at the meiotic spindle [11]. After fertilization, resumption of the second meiotic division results in
the extrusion of a second polar body. Furthermore, the biochemical environment established within the ooplasm after
sperm penetration allows the optimal progression of paternal chromatin decondensation and remodeling before pronuclear formation [12].
The induction of parthenogenesis in mammalian oocytes
requires a calcium-releasing stimulus to complete the second meiotic division. In turn, resumption of a mitotic cell
cycle requires subsequent exposure to protein synthesis inhibitors [13] or broad-spectrum protein kinase inhibitors
[14]. Parthenogenesis has been induced in bovine oocytes
by exposure to a Ca2 + ionophore followed by cycloheximide [15] or 6-dimethylaminopurine (6-DMAP), resulting
in the resumption of embryonic cell cycles and a high percentage of blastocyst formation [16]. The effects of 6DMAP on chromatin and microtubule configurations during
meiosis are mediated by the inhibition of protein kinases
and, consequently, protein phosphorylation [14, 17]. Dephosphorylations induced by 6-DMAP inactivate c-mos and
mitogen-activated protein (MAP) kinase [18, 19], as well
as a range of downstream unidentified kinases leading to
the rapid formation of a nuclear envelope after oocyte activation, but with consequences for the normal progression
of postfertilization events [14]. Treatment with 6-DMAP
after bovine oocyte activation induces pronuclear formation
and drives the parthenote into interphase of the first mitotic
cell cycle, presumably as a uniform diploid [16]. However,
examination of chromosomal complements in bovine parthenotes occurring spontaneously [20] or produced by activation protocols [21] suggests the presence of heteroploidy in a high number of embryos. Analysis of the first embryonic cell cycle is thus essential to determine the origin
of abnormal karyotypes in the bovine parthenote.
Murine parthenotes are characterized by poor development of trophectoderm derivatives [1]. Defective cell allocation to both trophectoderm and inner cell mass (ICM)
lineages has been observed as early as the blastocyst stage
[22, 23]. However, no information is available on the mechanisms regulating the allocation of blastomeres to the ICM
or trophectoderm in the bovine parthenote.
The objective of the present study was to examine the
952
IN VITRO DEVELOPMENT OF BOVINE PARTHENOTES
developmental potential and chromosomal complement of
uniparental or biparental embryos produced, respectively,
by activation with ionomycin and 6-DMAP treatment or by
in vitro fertilization. Our results suggest that after a rapid
pronuclear formation in parthenotes, karyokinesis occurs
without cytokinesis during the first mitotic cell cycle. Analysis of pronuclear formation indicates that a rapidly formed
pronucleus engages in DNA synthesis by 6 h postactivation
(hpa), resulting in the formation of a second nuclear envelope by 24 hpa. Such pronuclear kinetics induces a variety of cell genotypes including binucleate blastomeres and
polyploid chromosome complements.
953
for 4 min at room temperature. After activation, oocytes
were transferred for 5 min to a Hepes-TALP solution containing 30 mg/ml BSA and rinsed twice in Hepes-TALP
with 1 mg/ml BSA. Activated oocytes were rinsed twice in
3 ml of IVC medium containing 1.9 mM 6-DMAP (Sigma)
and transferred (in groups of 30) to a 50-pl droplet of the
same solution under silicone oil. After exposure to 6DMAP for 5 h in a humidified atmosphere of 5% CO 2 in
air at 39C, the oocytes were washed four times with fresh
IVC medium and cocultured with BOEC as described
above for control embryos.
Analysis of Pronuclear Formation
MATERIALS AND METHODS
In Vitro Bovine Embryo Production
Embryos were produced by in vitro oocyte maturation,
fertilization, and culture as previously described [24]. Briefly, oocytes were obtained by follicular aspiration and collected into Hepes-buffered Ham's F-10 (Canadian Life
Technologies, Burlington, ON, Canada). Cumulus-oocyte
complexes (COCs) with uniform cytoplasm and three to
five layers of cumulus cells were selected for in vitro maturation in Hepes-buffered tissue culture medium (TCM)199 (Canadian Life Technologies) supplemented with 0.2
M L-glutamine (Sigma Chemical Co., St. Louis, MO), 0.2
M sodium pyruvate (Sigma), 0.6% penicillin-streptomycin
(10 000 U/ml penicillin G, 10 000 mg/ml streptomycin; Canadian Life Technologies), and 10% steer serum (SS) under
silicone oil (Fisher Scientific, Nepean, ON, Canada). The
COCs were cultured for 22-24 h at 39°C in a humidified
atmosphere of 5% CO 2 in air and subsequently denuded
from the cumulus cells by vigorous pipetting in 3 ml Hepes-buffered Tyrode's albumin-lactate-pyruvate medium
(TALP) [25]. Denuded oocytes were then rinsed in TALP
supplemented with 20 ,ug heparin (Sigma)/ml (IVF-TALP).
Twenty oocytes were transferred into a 95-,l droplet of
IVF-TALP containing 5 jl of a bovine oviductal epithelial
cell (BOEC) suspension under silicone oil.
Frozen-thawed sperm were subjected to a swim-up procedure for 1 h in TALP to recover the motile fraction. The
supernatant was centrifuged at 300 x g for 5 min, and a 5l aliquot of sperm pellet (approximately 1 X 106 cells/ml)
was added to the 95-,ul droplets of IVF-TALP containing the
oocytes. The time (h) after initial sperm and COC coincubation from this point onward is referred to as hours postinsemination (hpi). At 18 hpi, presumptive zygotes were washed
twice in 1 ml of in vitro culture (IVC) medium consisting of
TCM-199 (Canadian Life Technologies), supplemented with
10% SS, 0.2 M sodium pyruvate, 0.6% penicillin-streptomycin, and 0.35% BSA (Sigma). Groups of 30 zygotes were
cocultured with BOEC (5-jl suspension) in 50 1 of IVC
medium for 8 days in a humidified atmosphere of 5% CO 2
in air at 39°C. On Days 3 and 5 postinsemination, 25 x1l of
IVC medium was added to the culture droplets.
Activated oocytes were examined for pronuclear status
at 6, 10, and 24 h after exposure to 5 p.M ionomycin (Sigma). Presumptive parthenotes were fixed overnight in methanol:acetic acid (3:1) and whole mounted on poly-L-lysine
(Sigma)-coated slides as previously described [16]. A 22 X
22-mm microscope coverglass (Fisher) with glass beads
(90- to 120-jim diameter) on the corners was placed over
the parthenotes. Activated oocytes were then stained with
1% aceto-orcein in 40% acetic acid before the coverglass
was sealed with rubber cement for analysis under phase
contrast with a Leitz-Aristoplan microscope (Wild Leitz
GmbH, Wetzlar, Germany). Nuclear configurations were
classified as metaphase II by the presence of a single polar
body and discernible chromosomes close to the site of first
polar body extrusion. Activated oocytes with one polar
body and highly condensed nuclei were classified as condensed chromatin. Pronuclear formation was determined by
the presence of a single polar body and a complete nuclear
envelope surrounding highly decondensed chromatin.
Analysis of DNA Synthesis
Pronuclear DNA synthesis was analyzed after
[3 H]thymidine incorporation and autoradiography according to Plante et al. [26]. Parthenotes and control embryos
were incubated in IVC medium supplemented with 2 .Ci/ml [3 H]thymidine (Amersham, Oakville, ON, Canada)
for a period of 2 h at 6, 8, 10, and 20 hpa/hpi, respectively,
washed twice in freshly prepared culture medium, and
transferred to a hypotonic solution (1.0% sodium citrate)
for 7-10 min before being spread on glass slides. Embryos
were spread with methanol:acetic acid (1:1) and subsequently fixed overnight in methanol:acetic acid (3:1). Radiolabeled slides were dipped into NTB2 Kodak emulsion
(Kodak Tetrachem, Rexdale, ON, Canada), maintained in a
water bath at 40°C, and air dried in complete darkness for
3-4 h. The slides were stored in light-proof boxes at 4°C
for 6 days and subsequently developed in D-19 (Kodak
Tetrachem) for 3 min and counterstained with a 4.0% buffered Giemsa solution. Qualitative analysis of silver grain
deposition was performed under a x40 objective on a Leitz
Aristoplan light microscope.
ParthenogeneticActivation
Laser Scanning Confocal Microscopy
Oocyte activation and parthenogenesis were induced as
described by Susko-Parrish et al. [16]. After 24 h of in vitro
maturation, COCs were treated with 1 mg/ml hyaluronidase
(Sigma) in a Hepes-buffered TALP solution for 5 min to
remove the cumulus cells. Denuded oocytes were then
rinsed twice in Hepes-TALP with 1 mg/ml BSA (Sigma).
Oocytes were activated by exposure to 5 puM ionomycin
(Sigma) in Hepes-TALP supplemented with 1 mg/ml BSA
Nuclear configurations in parthenotes and control embryos
were analyzed, respectively, at 6, 10, and 24 hpa or after in
vitro fertilization, and at the 4-cell (48 h), morula (Day 5),
and blastocyst (Day 8) stages, by confocal microscopy with
serial reconstructions. All zonae pellucidae were removed by
exposure to 0.1% pronase in Ham's F-10 at 390C for 3-7 min
before blastomere fixation with 3% paraformaldehyde in PBS
for 30 min. Membrane permeation was accomplished with
954
DE LA FUENTE AND KING
PBT buffer consisting of 0.25% Tween 20 (Bio-Rad Laboratories, Hercules, CA) and 0.4% BSA (Sigma) in PBS for
30 min. Embryos were then stained for 30 min with 100
,ig/ml fluorescein isothiocyanate (FITC)-labeled wheat germ
lectin (WGA; Sigma) in PBS. DNA was counterstained with
5 Ig/ml propidium iodide (Sigma) in PBS for 5 min. Analysis
of nuclear configurations and enumeration of blastomeres
were performed with a Bio-Rad 600 MRD confocal microscope with a 560-nm excitation wavelength (rhodamine filter)
and a 488-nm excitation wavelength (FITC filter). For the
localization of pronuclear structures, a single 7-xm section
was recorded in each channel and superimposed for the observation of decondensed DNA. Cleavage-stage embryos and
compact morulae (Day 5) were analyzed by scanning 7-pm
sections on the z-axis on each channel and further serial reconstruction to estimate the number of nuclei per blastomere.
A second group of cleavage-stage parthenotes and control embryos were evaluated under fluorescence microcopy for nuclei
and blastomere numbers at 48 h postactivation or fertilization.
Differential Staining of ICM and Trophectoderm
For the differential staining of trophectoderm and ICM,
zona-free blastocysts (Day 8) were initially exposed to 100
,ug/ml FITC-labeled WGA in PBS for 20 min at 39°C, followed by fixation with 3% paraformaldehyde in PBS for
30 min and permeation in PBT buffer as described above.
Embryos were counterstained with 5 pg/ml propidium iodide in PBS for 5-10 min. After staining, embryos were
transferred to poly-L-lysine (Sigma)-coated slides and
whole mounted with an antifading medium, DABCO (1,4diazobicyclo[2,2,2] octane; Aldrich Chemical Co., Milwaukee, WI), in glycerol and 0.2 M Tris-HCl (pH = 8.6), under
a coverslide that was then sealed. Exposure of zona-free
blastocysts to FITC-labeled WGA allows the exclusive labeling of the trophectoderm cell surface. Phagocytic activity in trophectoderm cells also contributes to the incorporation of FITC labeling on the trophectoderm epithelium.
Counterstaining with propidium iodide after fixing and permeation of embryos stained both trophectoderm and ICM
cell nuclei. Blastomeres allocated to the ICM were readily
distinguishable, as they were exclusively labeled by propidium iodide.
Quantification of blastomeres allocated to the ICM and
trophectoderm was performed with fluorescence microscopy. Zona-free blastocysts were exposed to 100 g/ml
FITC-labeled WGA in PBS for 20 min at 39°C. Blastocysts
were then fixed in 100% ethanol for 10-20 min, followed
by permeation with PBT buffer supplemented with 5 ig/ml
propidium iodide for 10 min. A final rinse of 20-30 sec in
FITC solution was undertaken to remove excess BSA before overnight fixation in 100% ethanol. Embryos were
then mounted in antifading medium as described above and
compressed under a coverslide. The number of nuclei allocated to the ICM was determined with a Leitz-Aristoplan
fluorescence microscope equipped with a dual rhodamineFITC filter of 560-nm wavelength. Trophectoderm nuclei
from the same embryo were identified under a rhodamine
filter with a 546-nm wavelength.
Blastocyst Cell Number and Chromosomal Analysis
Parthenotes and control embryos at the blastocyst stage
on Day 8 of development were exposed to 0.05 $jg/mlcolcemid (Sigma) for 4 h in IVC medium to arrest metaphase
cells. After exposure to this microtubule inhibitor, embryos
were transferred to a hypotonic solution of 1% sodium ci-
TABLE 1. Nuclear status in bovine oocytes following activation with 5
jIM ionomycin and 1.9 mM 6-DMAP.
Nuclear configurations
Time
postactivationa
6 h
10 h
24 h
MII
1PN/1 Pb
2PN/1 Pb
nb
(%)
(%)
(%)
65
86
104
13 (20)
2 (2.3)
10 (9.6)
46 (70.7)
77 (89)
11 (10.5)
1 (1.5)
2 (2.3)
80 (76.9)
Cond.
chrom.
(%)
5 (7.6)
3 (3.4)
1 (0.9)
a Time postactivation corresponds to hours after exposure to ionomycin.
b Pooled data from 4 independent experiments.
MII, metaphase II arrest; PN, pronucleus; 1Pb, first polar body; Cond.
chrom., condensed chromatin.
trate (Fisher) for 4-7 min and fixed for analysis of total
cell number and chromosome complement. Individual blastocysts were then spread onto glass slides with methanol:
acetic acid (1:1) and fixed overnight with methanol:acetic
acid (3:1) before air drying and staining with 4% Giemsa
in PBS for 4 min [27].
Apoptosis Detection
Detection of apoptotic nuclei was accomplished by in
situ nuclear labeling with terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) reaction
(Boehringer Mannheim, Laval, PQ, Canada). Zona-free embryos at the blastocyst stage (Day 8) were fixed in 3%
paraformaldehyde solution in PBS for 30 min, rinsed twice
in PBS, and permeated with 0.1% Triton X (Bio-Rad) in
0.1% sodium citrate for 2 min on ice according to manufacturer's specifications. Parthenotes and control embryos
were resuspended in 50 [l TUNEL reaction mixture or in
50 pl TUNEL label alone as a negative control. Embryos
were incubated for 60 min at 37°C in a humidified atmosphere. Positive controls were incubated with 50 U/ml RQ1
RNase-free DNase (Promega, Madison, WI) for 20 min at
37°C before exposure to the TUNEL reaction mixture. Embryos were rinsed twice in PBS and counterstained with 5
jig/ml propidium iodide in PBT buffer. Blastocysts were
then mounted in antifading medium and analyzed by confocal microscopy as described above. Analysis of nuclear
morphology and labeling was performed on 7-pLm sections
also as described.
Statistical Analysis
Cleavage rate and percentage of blastocysts in parthenogenetic and control embryos were compared using chisquare analysis. The percentage of embryos showing binucleate blastomeres at 48 h postactivation/in vitro fertilization
in parthenotes and control embryos, respectively, was compared by chi-square analysis and Fisher's exact test. Total
cell number and mitotic index were compared by a onetailed t-test. Allocation of blastomeres to ICM or trophectoderm lineages was compared by t-test using the Statistical
Analysis Systems (Version 6.10; SAS, Cary, NC).
RESULTS
Pronuclear Formation following Parthenogenetic Activation
Nuclear configurations of bovine embryos observed by
aceto-orcein staining after parthenogenetic activation are
summarized in Table 1. By 6 hpa, most parthenotes (>
70%) had a fully formed pronucleus and one polar body.
The remaining putative parthenotes demonstrated a range
IN VITRO DEVELOPMENT OF BOVINE PARTHENOTES
TABLE 2.
955
Mean ( SEM) cleavage rate and development to blastocyst in bovine parthenotes.
No. (%) of oocytes
Embryos
No. of oocytes
Cleaved at 48 h
Control
1151
879 (76.3 + 4.7)a
186 (16.4
3.3)c
20.9 ± 3.2c
734
431 (60.1 - 4.2)b
114 (15.8 ± 1.0)c
26.8 _ 2.2c
Parthenotes
Blastocyst Day 8
±
Percentage from cleaved
- Different superscripts within columns indicate significant differences (p < 0.05); data from 5 independent experiments.
of nuclear configurations, with some oocytes remaining at
metaphase II arrest or showing clusters of condensed chromatin. In addition, a small percentage (< 2%) of parthenotes in this group contained two pronuclei. By 10 hpa, the
percentage of parthenotes with a single pronucleus and one
polar body had significantly increased (p < 0.05). A small
percentage of metaphase II-arrested oocytes and parthenotes with condensed chromatin remained. The proportion
of parthenogenetic embryos with two pronuclei remained
low (< 2.5%). By 24 hpa, a second pronucleus was observed in more than 76% of parthenotes. Only 10% of parthenotes with a single pronucleus remained, and less than
1% showed condensed chromatin.
Pronuclear DNA Synthesis after Exposure to 6-DMAP
The onset of pronuclear DNA synthesis among parthenotes is illustrated in Figures 1 and 2. [3 H]Thymidine incorporation and autoradiographic analysis demonstrated
that 25 of 34 (73.5%) parthenogenetic embryos (pooled
data from two experiments) were actively engaged in DNA
synthesis as early as 6 hpa as indicated by silver grain deposition over decondensed chromatin in a large pronucleus
(Fig. 1, A and B, and Fig. 2). In contrast, DNA synthesis
was not observed in any control zygote (n = 22) at 6 hpi.
However, two clusters of condensed chromatin, corresponding to the male and female genomes, were found in opposing regions of the oocyte upon serial reconstruction with
confocal microscopy (Fig. 1C). At 8 hpa, 25 of 42 (60%)
parthenotes had a large, centrally located pronucleus that
demonstrated DNA synthesis (Fig. 2). DNA synthesis was
minimal in corresponding control embryos, with only 4 of
27 (15%) of these presenting clusters of decondensing chromatin and slight silver grain deposition. Serial reconstruction with confocal microscopy of control embryos revealed
two eccentrically located nuclei with decondensing chromatin at the initial stages of pronuclear formation. By 10
hpa, DNA synthesis was observed in 99 of 139 (71%) parthenotes, and the intensity of labeling was increased in
comparison to that observed in parthenotes at 6 hpa. At this
stage, most pronuclei had migrated to a central position
within the oocyte. By 10 hpi, 32 of 66 (48.4%) control
zygotes showed evidence of pronuclear formation with
DNA synthesis in at least one pronucleus. The remaining
embryos presented chromatin clusters at very early stages
of decondensation with no silver grain deposition above
background. By 20 hpa, parthenotes were engaged in a second round of DNA synthesis, with 30 of 51 (58.8%) presenting evidence of DNA replication in two pronuclei. By
TABLE 3.
Embryos
Control
Parthenotes
24 hpa, two closely apposed large pronuclei had migrated
to a central location within the oocyte (Fig. 1, D and E).
Evidence for asynchronous DNA synthesis was present in
30 of 45 (66.6%) control embryos, as [3 H]thymidine incorporation was observed in only one or in both pronuclei
in various embryos. In contrast with parthenotes, control
zygotes at 24 hpi demonstrated chromosome alignment at
the mitotic spindle in preparation for cytokinesis (Fig. F).
Binucleate Blastomeres in Bovine Parthenotes
Upon cleavage at 48 hpa, binucleate blastomeres were
commonly observed in parthenogenetic embryos (Fig. 1G).
Confocal and fluorescence microscopy revealed the presence of one or more large binucleate blastomeres in 20 of
37 (54.1%) cleaved parthenotes (pooled data from two separate experiments). In contrast, the majority of control embryos at a corresponding stage of development presented a
single nucleus per blastomere (Fig. 1H). Only 7 of 43
(16.3%) control embryos presented binucleate blastomeres.
Anucleate blastomeres were also observed in 7 of 37
(18.9%) and 3 of 43 (6.9%) parthenotes and control embryos, respectively. The mean cell number (t SEM) in
parthenotes at this stage, 3.16 + 0.2, was significantly lower than that of control embryos (n = 43; 5.07 + 0.4, p <
0.05). Confocal microscopy revealed that binucleate blastomeres in parthenotes seemed to be associated with a
smaller number of cells at each developmental stage analyzed and with large cells that had been extruded into the
blastocoelic cavity. In a few control embryos, blastomeres
with fragmented nuclei had also been extruded into the
blastocoelic cavity.
In Vitro Development in Bovine Parthenotes
Mean cleavage rate ( SEM) at 48 hpi (control) or activation in 5 independent experiments is illustrated in Table
2. At 48 hpi, a higher percentage (p < 0.05) of control
embryos had cleaved to the 2- to 4-cell stage. The percentage of cleaved oocytes in the fertilization group ranged
from 62% to 91%. Two- and four-cell embryos were predominantly observed at this time. However, a subset of fastdeveloping embryos with 4-8 blastomeres was evident.
The percentage of activated oocytes that had cleaved by 48
hpa ranged from 44% to 68%. At this stage, parthenotes
consisted mainly of 2-4 blastomeres, with only a small
number of 4- to 6-cell embryos observed. The proportion
of parthenotes that reached the blastocyst stage by Day 8
of development did not differ from that for control embryos
Total cell number and chromosome complement in bovine parthenotes.
No. of
blastocysts
Total cell number
(± SEM)
% 2n (n)
% 2n/4n (n)
53
71
116.9 ± 5.5a
67.2 + 3. 5 b
83.02 (44)
0 (0)
9.4 (5)
30.8 (19)
Ploidy
% 3n (n)
% 4n (n)
% 2n/8n (n)
3.7 (2)
3.1 (3)
3.7 (2)
61.5 (40)
0 (0)
4.6 (4)
Different superscripts within a column indicate significant differences (p < 0.05); pooled data from 5 independent experiments.
956
DE LA FUENTE AND KING
FIG. 1. Pronuclear formation and DNA synthesis in parthenotes after exposure to 6-DMAP. Control embryos are shown on the right. Cytoplasmic
staining with FITC-labeled WGA (green). A) A large pronucleus with decondensed chromatin counterstained with propidium iodide (red) was observed
at 6 hpa (X60). B) Pronuclear DNA replication as determined by [Hthymidine incorporation and autoradiography in parthenotes at 6 hpa (x100). C)
Control zygotes at 6 hpi (x60) revealed condensed chromatin configurations (stained with propidium iodide) corresponding to the paternal and maternal
genomes in a single 7-1.m scan obtained with confocal microscopy. D) Formation of a second pronucleus in parthenotes at 24 hpa. E) Both pronuclei
957
IN VITRO DEVELOPMENT OF BOVINE PARTHENOTES
0 80-
200
.
M
60.
'U
1
-100
40-
* Control
O] Parthenotes
U
20-
I
I
0
6
8h
Bh
10h
20h
Time post Activation
FIG. 2.
Analysis of DNA synthesis in parthenotes (n = 266) at various
stages hpa. Embryos were labeled with 2 p.Ci/ml [3Hthymidine in TCM199 medium for 2 h at times indicated and were spread on slides for
autoradiography. Data are pooled from 2 independent experiments.
(Table 2). Morphological evaluation of blastocysts using
both light and confocal microscopy revealed a variety of
phenotypes, with some parthenotes being indistinguishable
from control embryos and presenting an ICM comparable
to that observed in control blastocysts. However, a high
proportion of parthenotes were characterized by a small
ICM and contained extruded blastomeres that appeared to
be located within the blastocoelic cavity.
Table 3 summarizes the mean (+ SEM) total cell number
on Day 8 of development in parthenotes and control embryos.
Parthenogenetic embryos had lower total cell numbers (range
25-184) at the blastocyst stage compared to controls (p <
0.05). Irregularly sized micronuclei and blastomere nuclei
were observed in some parthenotes, particularly in those with
the lowest cell numbers. After a 4-h exposure to colcemid,
the mitotic index (number of metaphase nuclei/total number
of nuclei) was significantly reduced in parthenotes (10%)
compared to controls (12.2%; p < 0.05). Informative metaphases were obtained from 92.9% (66 of 71) of parthenotes
(Table 3). The remaining 5 embryos showed highly condensed chromosomes or overlapping metaphases that precluded reliable interpretation. No parthenotes had diploid metaphases (i.e., 2N = 60). Tetraploidy (i.e., 4N = 120) was the
most common type of abnormality, followed by mixoploidy
with diploid and tetraploid metaphaies found in the same embryo. A small percentage of parthenotes displayed triploid and
octaploid metaphases. In contrast, all control embryos gave
interpretable metaphase spreads. The majority revealed a diploid chromosome complement. Mixoploid chromosome complements were observed in a small percentage (< 10%) of
control blastocysts. However, most of these were accounted
for by diploid-tetraploid chromosome configurations, with <
4% of embryos showing only triploid or tetraploid configurations.
demonstrated a second round of DNA replication by 20 hpa (x100). F)
In control zygotes, the paternal and maternal genomes were observed
aligned at the mitotic spindle by 24 hpi before cytokinesis. Upon cleavage
at 48 hpa, binucleate blastomeres were observed in a high percentage of
parthenotes. G) Serial reconstruction obtained with confocal microscopy
demonstrating a 4-cell parthenote with a binucleate blastomere (x60).
DNA was counterstained with propidium iodide. H) Control embryo at
48 hpi. Blastomeres in an 8-cell embryo presented a single nucleus (H).
0
Total Cell Number
ICM
Trophectoderm
FIG. 3. Total cell number ( SEM) and allocation of blastomeres to ICM
and trophectoderm in bovine parthenotes (n = 16) and control (n = 16)
expanded blastocysts on Day 8 of development. Embryos were stained as
described in Materials and Methods and analyzed with fluorescence microscopy. * Significant differences (p < 0.05). Data are pooled from 2
independent experiments.
Blastomere Allocation to the ICM and Trophectoderm
Cell Line
The differential staining technique described in the present study was adapted for both confocal and fluorescence
microscopy. Substitution of paraformaldehyde with 100%
ethanol for the fixing step allowed counting of individual
nuclei under fluorescence microscopy. The allocation of
blastomeres to the ICM and trophectoderm lineages in parthenotes and control blastocysts cultured in vitro is summarized in Figure 3. Parthenotes (n = 16; two separate
experiments) had fewer total cells (82.8
4.8) than controls (n = 16; 140.7 + 5.2, p < 0.05). Fewer cells were
scored in both ICM (24 + 2.0) and trophectoderm (58.9 ±
3.6) of parthenotes on Day 8 of development than in controls (55.2 + 2.0 ICM and 85.4 ± 4.5 trophectoderm; p <
0.05). The ratio of ICM over total cell number in parthenotes was 0.24 and was significantly different from that for
controls (0.39; p < 0.05). In both parthenotes and control
blastocysts, micronuclei were observed mainly in the ICM.
On the other hand, large nuclei and binucleate cells were
present in the trophectoderm. Figure 4 shows representative
serial reconstructions of 25 scans (7 ptm each) on the zaxis obtained with confocal microscopy from differentially
stained control (A) and parthenote (B) blastocysts. A prominent ICM was observed in control blastocysts, whereas
parthenotes displayed a small cluster of ICM. A few blastomeres with irregular shaped nuclei were found extruded
into the blastocoelic cavity in parthenotes (Fig. 4B). Extruded blastomeres were also found attached to external trophectoderm cells. Retrospective analysis of morula-stage
parthenotes (Fig. 4D) revealed the presence of disorganized
nuclei and a smaller cluster of inner cells as compared with
controls at a corresponding stage. Extruded blastomeres
with fragmented nuclei, however, were also observed in the
nascent blastocoelic cavity in control compact morulae
(Fig. 4C).
Apoptosis in the ICM and Trophectoderm Cell Lineages
Cell death scored by nuclear fragmentation in blastomeres from parthenotes and control embryos was confirmed
to be mediated by apoptosis (Fig. 4E). In the ICM, apoptotic nuclei were found either at the blastocoelic surface or
internally, surrounded by morphologically viable cells. On
958
DE LA FUENTE AND KING
FIG. 4. Morphological assessment of early development in control (A, C) and parthenotes (B, D) at different stages of development in vitro. Serial
reconstructions obtained with confocal microscopy (x25). Differential staining of trophectoderm (green) and ICM (red) in control (A) and parthenotes
(B) at the blastocyst stage on Day 8 postfertilization/activation, respectively. Notice the presence of extruded blastomeres into the blastocoelic cavity of
parthenotes (B, arrow). C) Control early cavitating blastocyst on Day 5 of development, with extruded blastomeres into the blastocoelic cavity (arrow)
and perivitelline space (arrowhead). D) Corresponding parthenote at the compact morula stage showing a smaller cluster of inner cells and disorganized
IN VITRO DEVELOPMENT OF BOVINE PARTHENOTES
the other hand, in the trophectodermal epithelium, apoptotic
nuclei showed a less demarcated pattern of nuclear fragmentation. Blastomeres extruded into the blastocoelic cavity had apoptotic nuclei in both controls and parthenotes.
However, binucleate cells extruded into the blastocoelic
cavity of parthenotes did not show signs of nuclear fragmentation or apoptosis as detected by TUNEL (Fig. 4F).
DISCUSSION
The establishment of efficient oocyte activation strategies for nuclear transfer procedures, and the production of
parthenotes with high potential for development to the blastocyst stage, require a better understanding of the mechanisms ensuring a diploid chromosome complement during
the transition to the first embryonic cell cycle. In the present study, nuclear and chromosomal configurations were
analyzed during early development of bovine parthenotes.
Our results have shown that after parthenogenetic activation
and during the first mitosis, karyokinesis takes place without cytokinesis in the majority of embryos. Analysis of nuclear configurations revealed that a fully formed pronucleus
was actively involved in DNA synthesis as early as 6 hpa
in most parthenotes. Premature DNA synthesis resulted in
the formation of a second pronucleus by 24 hpa. Upon
cleavage, a variety of cell genotypes were identified in
which binucleate blastomeres were observed among otherwise mononucleated blastomeres from the same embryo.
Chromosomal analysis of these developing embryos revealed a high incidence of tetraploid metaphases. Parthenotes at the blastocyst stage had fewer cells in both ICM
and trophectoderm and a smaller proportion of cells allocated to the ICM. Apoptosis was present in both ICM and
trophectoderm of parthenotes and control embryos. Binucleate cells extruded into the blastocoelic cavity of parthenotes, however, did not show evidence of nuclear fragmentation or apoptosis, indicating that different mechanisms may regulate cell numbers in different cell lineages
as early as the blastocyst stage in the bovine embryo. Comparable blastocyst rates can be achieved in control and parthenogenetic embryos, suggesting that embryonic mortality
due to chromosomal abnormalities takes place after blastulation in the bovine embryo.
Pronuclear formation in the mouse zygote is at least partially regulated by MAP kinase activity [28-30]. Both cmos and MAP kinase can be detected at the meiotic spindle,
where they seem to be involved in the maintenance of chromosome condensation and in preventing the formation of a
nuclear envelope [18, 19, 30-33]. Injection of a constitutively expressed MAP kinase into mouse oocytes or activation of MAP kinase with okadaic acid precludes the formation of a nuclear membrane [34, 35]. Furthermore, both
kinases can be detected at high levels for at least 5-7 h
postfertilization, after which decreasing levels of c-mos and
MAP kinase activity coincide with protein dephosphorylation, signaling the transition from a meiotic to a mitotic
cell cycle and coordinating the progression of postfertilization events before male and female pronuclear structures
appear [12, 35, 36].
nuclei. E) Localization of FITC-labeled apoptotic nuclei (arrow) at the ICM
of parthenotes at the blastocyst stage on Day 8 of development. Nuclear
counterstaining with propidium iodide (red). F) Note the absence of nuclear fragmentation or TUNEL labeling in binucleate cells (arrow) extruded into the blastocoelic cavity of parthenotes.
959
In the present study, a fully formed pronucleus was observed in parthenotes as early as 6 hpa. Pronuclear formation after parthenogenetic activation has been shown to
occur shortly after exposure to 6-DMAP in mouse [14] and
bovine oocytes [16]. This suggests that inhibition of protein
phosphorylation by exposure to 6-DMAP [14, 17] resulted
in premature pronuclear formation after c-mos and MAP
kinase inactivation. In mouse oocytes, MAP kinase activity
is maintained by a process requiring protein phosphorylation, and its activity is directly inhibited by exposure to 6DMAP [18, 19]. In addition, oocytes from c-mos-deficient
mice fail to activate MAP kinase during meiosis and consequently present a high rate of spontaneous parthenogenetic activation with several chromosomal abnormalities
[32, 33]. Analysis of bovine parthenotes with confocal microscopy in the present study revealed a centrally located
large pronucleus with highly decondensed chromatin in the
majority of embryos after exposure to 6-DMAP. This configuration resembles some of the pronuclear structures observed in c-mos-deficient mice [37, 38].
Pronuclear formation in parthenotes after exposure to 6DMAP was also associated with premature DNA synthesis.
In addition to [3H]thymidine incorporation, the increased
number of chromosomes at first mitosis confirmed the continuous and replicative nature of the observed DNA synthesis. During meiotic maturation in Xenopus oocytes, cmos coordinates the reduction of genetic material during
the transition from metaphase I to metaphase II without
DNA replication [39]. Thus, the DNA synthesis observed
in the majority of successfully activated bovine oocytes is
likely to be due to the chemical inactivation of the c-mos/
MAP kinase pathway after exposure to 6-DMAP. Treatment
with 6-DMAP has also been shown to induce DNA replication in invertebrate oocytes at various stages of meiosis.
In this case, however, the effects of 6-DMAP appear to be
mediated by different protein kinases or phosphatases, as a
c-mos homologue has yet to be described in invertebrate
oocytes [40]. Premature DNA synthesis in a rapidly formed
(diploid) pronucleus led to completion of S phase and chromosome condensation into a metaphase plate by 15-17 hpa
(data not shown). Furthermore, subsequent karyokinesis before the first cleavage resulted in the reassembly of a second, centrally located pronucleus in close apposition with
the first nuclear envelope by 24 hpa in most parthenotes.
Moreover, both pronuclei were found to be engaged in a
second round of DNA replication by 20 hpa. Impaired cytokinesis has previously been reported [32] in oocytes from
c-mos-deficient mice in which 8% of spontaneously activated oocytes presented two pronuclei 24 h after germinal
vesicle breakdown.
The cleavage rate observed at 48 hpa was lower than in
control embryos. Previous studies, however, report similar
cleavage rates for bovine parthenotes [16, 21]. A reduced
cleavage rate might be the result of activation failure, as
indicated by the presence of a small percentage of metaphase
II-arrested oocytes, and/or of incomplete activation in those
oocytes showing condensed chromatin as previously observed in metaphase III-arrested mouse oocytes [41]. After
exposure to 6-DMAP, bovine parthenotes were able to form
anastral bipolar spindles and cleave at the time expected for
fertilized controls [7]. However, in the present study, analysis
of nuclear configurations upon cleavage revealed a significant increase in the frequency of parthenotes with binucleate
blastomeres at 48 hpa as compared to controls. Blastomeres
with a single nucleus were also present among parthenotes
with one or two binucleate blastomeres, and although direct
960
DE LA FUENTE AND KING
assessment of their DNA content was not performed, the
presence of diploid metaphases at the blastocyst stage indicates that some diploid blastomeres do arise upon cleavage.
Binucleate and multinucleated blastomeres have been previously described in 17-30% of human embryos produced
by in vitro fertilization, and the presence of a tetraploid DNA
content suggested acytokinesis as the mechanism of formation [42-44]. Binucleate cells have also been described in
Swiss 3T3 fibroblasts overexpressing c-mos. In 30% of transformed cells, meiotic-like spindle attachment to the plasma
membrane interfered with cytokinesis but not with karyokinesis, resulting in the formation of binucleate cells with a
tetraploid DNA content. It was suggested that similar mechanisms may be responsible for the genetic instability observed in cancer cells [45].
Tetraploid metaphases were found in > 60% of parthenotes at the blastocyst stage, corresponding with a similar
percentage of binucleate parthenotes observed at 48 hpa.
Similarly, 50-70% of mouse embryonic fibroblasts deficient in the tumor suppressor protein p53 presented tetraploid and octaploid metaphases after exposure to microtubule inhibitors or serial passages in vitro. In this case, development of tetraploidy has been associated with the loss
of p53 function as a spindle assembly checkpoint [46]. Several cell cycle checkpoints in somatic cells coordinate the
progression of a single round of DNA replication followed
by chromosome segregation before cell division [47]. However, it has been suggested that during early embryonic development, cells lack such checkpoints and hence the progression of rapid cell cycles is allowed [48]. The lack of
checkpoints during early mammalian development may explain the high incidence of nuclear abnormalities such as
binucleate blastomeres with abnormal karyotypes [49].
The presence of octaploid metaphases in a small percentage of blastocysts suggests that several rounds of DNA
replication take place in some blastomeres. Multinucleated
blastomeres were not observed at 48 hpa, indicating that
such cells appear later during embryo development, possibly by acytokinesis of an already binucleate cell. The proliferative capacity of such blastomeres is not clear. Tesarik
et al. [50] have demonstrated the presence of DNA synthesis in blastomeres with multiple nuclei. However, an intrinsic repair mechanism has been proposed in which a mosaic
embryo could regain a euploid status by avoiding multinucleated blastomere proliferation and eliminating abnormal cells [50]. Confocal microscopy revealed the presence
of extruded blastomeres into the blastocoelic cavity of parthenotes or in the perivitelline space upon removal of the
zona pellucida in a few controls, suggesting the presence
of an active mechanism for blastomere elimination in bovine blastocysts. Selective elimination of cells with abnormal mitosis has also been described in the Drosophila melanogaster embryo, in which affected nuclei are removed
from the dividing population of cells, suggesting the presence of an embryonic mechanism to extrude cells with abnormal karyotypes. It has been suggested that such a mechanism compensates for the lack of cell cycle checkpoints
during early development [51].
Morphological evaluation at 48 hpa demonstrated that a
reduced cell number was already present in parthenotes,
which consisted mainly of 2-4 blastomeres. An increased
amount of DNA may interfere with cytokinesis, and the
presence of a binucleate blastomere may also delay further
cleavage. In spite of the wide prevalence of chromosomal
abnormalities in parthenotes, the rate of development to the
blastocyst stage was similar to that of controls. High rates
of blastocyst formation have been previously obtained in
bovine parthenotes produced under similar conditions
[16, 21]. However, a lower total cell number was observed
than in control embryos. Morphological evaluation of parthenotes at the blastocyst stage revealed a variety of phenotypes, with some embryos showing a patent ICM and
some embryos more closely resembling trophoblastic vesicles. Such phenotypic variability was also reflected in a
wide range in total cell number. Differential staining suggests that a lower cell number was mainly due to a decrease
in the proportion of blastomeres allocated to the ICM in
parthenotes. However, an adverse effect was evident in both
ICM and trophectoderm cell lineages. A decrease in total
cell number has been observed in mouse diploid parthenotes, in which numbers of both trophectoderm and ICM
cells declined in comparison to those in control fertilization-derived embryos at the expanded blastocyst stage [23].
It is possible that the rates of cell proliferation are affected
by the presence of polyploidy in parthenotes, with binucleate cells taking longer at S phase or metaphase stage of the
cell cycle. In addition, blastomeres extruded into the blastocoelic cavity of parthenotes could be contributing further
to the reduction in cell number. On the other hand, the
possibility of an adverse effect of uniparental genomes on
cell proliferation cannot be overlooked. It has been suggested that genomic imprinting has an effect on cell proliferation as early as the blastocyst stage [22, 23]. The effects on cell proliferation and differentiation during parthenogenesis might be a consequence of altered expression
patterns for imprinted genes [22]. Moreover, a twofold reduction in the expression levels of several genes associated
with cell cycle progression has been observed in mouse
parthenotes compared to fertilized controls [3]. Adverse effects of parthenogenesis may also be due to overexpression
of X-linked genes [2], as dosage compensation takes place
at a later stage in mouse parthenotes [52, 53].
Programmed cell death (PCD) regulates cell numbers
and eliminates abnormal cells at various developmental
stages [54]. At the blastocyst stage, PCD regulates the number of ICM cells by eliminating redundant blastomeres with
potential for trophectoderm differentiation [55, 56]. However, the role, if any, of PCD in the trophectoderm is not
clear. It is possible that different mechanisms operate in
different cell lineages. The absence of apoptosis in binucleate cells in parthenotes would support this hypothesis.
PCD can be activated by several pathways [57], including
p53-mediated cell cycle checkpoints. Insights into the
mechanisms operating during early development will be instrumental to determine whether binucleate blastomeres
have a delayed (developmentally regulated) onset of PCD,
or whether a permissive state exists in different cell lines,
especially those of the extraembryonic tissues or their precursors. Cell proliferation was severely reduced in embryos
homozygous for a null mutation of the tumor suppressor
genes Brca 1 and Apc [58, 59], where deficient development of ICM and extraembryonic tissues resulted in embryonic mortality. However, polyploid trophoblast giant
cells were not affected, suggesting that different growthregulatory mechanisms may be operating in different cell
lineages [58, 59].
Although at a reduced rate, chromosomally abnormal
human embryos have been shown to undergo implantation,
although with a considerable increase in subsequent embryo loss and spontaneous abortion [42]. Similar blastocyst
rates obtained in bovine parthenotes also seem to indicate
their ability to overcome some of the early milestones of
IN VITRO DEVELOPMENT OF BOVINE PARTHENOTES
embryonic development, namely the transition from maternal to zygotic gene expression and blastocyst formation, at
the same rate as in vitro-fertilized embryos. In this regard,
transcription has been previously described in spontaneously activated parthenotes at the 2- and 4-cell stage [60].
The presence of tetraploid cells in control blastocysts
was lower than previously reported for in vitro-produced
bovine embryos [61-63]. On Days 7-9 of development,
2n/4n chromosome complements were observed in 32.4%
of blastocysts corresponding to binucleate cells at the trophectoderm cell layer [61]. Polyploid cells have also been
found in mouse morulae [64] and in the trophoblast of
sheep [65], pig [66], and bovine elongating blastocysts [67],
where they form trophoblast binucleate cells. In contrast,
> 90% of parthenogenetic embryos presented polyploid
metaphases, with a tetraploid chromosome complement as
the most common type of abnormality. Development to the
blastocyst stage has also been described for triploid mouse
embryos. Although with reduced developmental potential
as compared to that of control diploid embryos, fertilization
of metaphase I-arrested oocytes resulted in formation of
blastocysts, 75% of which were triploid [68]. This suggests
that embryonic mortality resulting from chromosome abnormalities might occur after the blastocyst stage. Mouse
embryos lacking cyclin A, an important regulator of the
G1/S and G2/M transition in mammalian cell cycles, were
able to develop to the blastocyst stage, presumably due to
maternal mRNA reserves that escape degradation until the
postimplantation period, when the embryo dies [69]. It is
possible that bovine parthenotes develop to the blastocyst
stage relying on maternal translational products and that the
adverse effects of parthenogenesis manifest at later stages
of development. The low incidence of chromosome abnormalities observed after blastocyst elongation in ruminants
[65, 67] might suggest that embryonic mortality due to
chromosomal effects takes place during the initial phases
of the elongation period [65]. Our data narrow the potential
time point of initiation of embryo demise due to chromosome aberrations to a stage after blastocyst formation in the
bovine embryo.
ACKNOWLEDGMENTS
The authors are grateful to Dr. B.A. Croy and Dr. M. Viveiros for
critical review of the manuscript, Dr. J. Lamarre for useful discussions,
Dr. M. Farquhar for assistance with confocal microscopy, A. Valiant for
assistance with statistical analysis, and S. Kawarsky for useful discussions
and assistance with figure preparation. We are also thankful to Dr. A.
Reyes (Centro Medico Nacional Siglo XXI, M6xico) for his encouragement and invaluable advice.
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