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PubMed2000

Centrosome inheritance in sheep zygotes: centrioles are contributed by the sperm.

The inheritance and duplication of the sperm centriole in the sheep zygote was studied by transmission electron microscopy. We found two centrioles at one pole and a single centriole at the opposite pole of the first mitotic spindle, in monospermic eggs, 20-21 hours postinsemination. This indicated both duplication and relocation of centrioles to opposite spindle poles during fertilization. The absence of centrioles in mature sheep oocytes was confirmed. Following activation by the calcium ionophore A 23187, mature oocytes entered mitosis and formed a bipolar spindle 18 hours later. Centrioles were not detected in the mitotic spindle of parthenogenotes. Androgenetic eggs were obtained by excision of the anaphase II/telophase II meiotic spindle of fertilized eggs. They were capable of undergoing mitosis and formed one or two bipolar spindle(s) in monospermic and dispermic eggs, respectively, 20-24 hours postinsemination. In two monospermic androgenetic eggs, two centrioles were found at one pole and a single centriole at the opposite pole of the first mitotic spindle. Three centrioles were also observed in another androgenetic egg in prometaphase of the first mitotic division, in close vicinity to the sperm neck-piece. These data provide evidence that the sperm centriole do reproduce and occupy a pivotal position on opposite spindle poles at syngamy. Altogether, the present findings suggest that centrioles of sheep zygotes are paternally derived.

Animals↗
PubMed1999

Development of an efficient method to produce uniformly haploid parthenogenones.

Bovine ovaries (paired by cow) were obtained from a local abattoir and cumulus oocyte complexes were aspirated within six hours of slaughter. Two methods for activation [(1) calcium ionophore (ionomycin) alone (n = 191); and (2) ionomycin followed by the protein synthesis inhibitor cycloheximide (n = 207)] were evaluated for production of bovine parthenogenones. Activation with ionomycin alone resulted in a development rate of 33%, while activation with ionomycin and cycloheximide sequentially resulted in a development rate to two-cell stage of 49%. A procedure was developed to expedite accurate evaluation of activated oocytes for uniformly haploid development. Uniformly haploid parthenogenones that cleaved at least once in four days of in vitro culture were individually prepared for genetic analysis. Three techniques: (1) phosphate buffered saline; (2) TL-HEPES with 0.2% ovine serum albumin; and (3) TL-HEPES with 0.2% polyvinyl pyrrolidone were compared to harvest parthenogenones for genetic analysis. The only effective method that did not create spurious results during later genetic analysis was TL-HEPES with 0.2% polyvinyl pyrrolidone. Based on the results of this study, we estimate that an average of 5-7 uniformly haploid bovine parthenogenones can be realized from each donor (using pairs of ovaries). These parthenogenones, when maintained as family units, will be valuable for accomplishment of female-specific genetic linkage analysis.

Animals↗
PubMed1996

Mosaic methylation of Xist gene before chromosome inactivation in undifferentiated female mouse embryonic stem and embryonic germ cells.

Epigenetic modification is implicated in the choice of the X chromosome to be inactivated in the mouse. In order to gain more insight into the nature of such modification, we carried out a series of experiments using undifferentiated mouse cell lines as a model system. Not only the paternally derived X (XP) chromosome, but the maternally derived one (XM) was inactivated in the outer layer of the balloon-like cystic embryoid body probably corresponding to the yolk sac endoderm of the post-implantation embryo in which XP is preferentially inactivated. Hence, it is likely that the imprint responsible for the nonrandom XP inactivation in early mouse development has been erased or masked in female ES cells. CpG sites in the 5' region of the Xist gene were partially methylated in female ES and EG and parthenogenetic ES cell lines as in the female somatic cell in which the silent Xist allele on the active X is fully methylated, whereas the expressed allele on the inactive X is completely unmethylated. In the case of undifferentiated ES cells, however, methylation was not differential between two Xist alleles. This observation was supported by the demonstration that single-cell clones derived from female ES cell lines were not characterized by either allele specific Xist methylation or nonrandom X inactivation upon cell differentiation. Apparently these findings are at variance with the view that Xist expression and X inactivation are controlled by preemptive methylation in undifferentiated ES cells and probably in epiblast.

Alleles↗
PubMed1998

The mouse Gtl2 gene is differentially expressed during embryonic development, encodes multiple alternatively spliced transcripts, and may act as an RNA.

We have isolated a novel mouse gene (Gtl2) from the site of a gene trap integration (Gtl2lacZ) that gave rise to developmentally regulated lacZ expression, and a dominant parental-origin-dependent phenotype. Heterozygous Gtl2lacZ mice that inherited the transgene from the father showed a proportionate dwarfism phenotype, whereas the penetrance and expressivity of the phenotype was strongly reduced in Gtl2lacZ mice that inherited the transgene from the mother. Gtl2 expression is highly similar to the beta-galactosidase staining pattern, and is down-regulated but not abolished in mice carrying the Gtl2lacZ insertion. In early postimplantation embryos, Gtl2 is expressed in the visceral yolk sac and embryonic ectoderm. During subsequent development and organogenesis, Gtl2 transcripts are abundant in the paraxial mesoderm closely correlated with myogenic differentiation, in parts of the central nervous system, and in the epithelial ducts of developing excretory organs. The Gtl2 gene gives rise to various differentially spliced transcripts, which contain multiple small open reading frames (ORF). However, none of the ATG codons of these ORFs is in the context of a strong Kozak consensus sequence for initiation of translation, suggesting that Gtl2 might function as an RNA. Nuclear Gtl2 RNA was detected in a temporally and spatially regulated manner, and partially processed Gtl2 transcripts were readily detected in Northern blot hybridizations of polyadenylated RNA, suggesting that primary Gtl2 transcripts are differently processed in various cell types during development. Gtl2 transcript levels are present in parthenogenic embryos but may be reduced, consistent with the pattern of inheritance of the Gtl2lacZ phenotype.

Alternative Splicing↗
PubMed1997

Cell proliferation is reduced in parthenogenetic mouse embryos at the blastocyst stage: a quantitative study.

BACKGROUND: Parthenogenetic and androgenetic embryos fail to develop to term, possibly because of genomic imprinting, an epigenetic alteration of certain genes, depending on the parent of origin. The effect of this phenomenon has been studied mainly in mid-gestation embryos, without morphological abnormalities detected during the preim-plantation period. Nevertheless, parthenogenetic mouse embryos never develop to the blastocyst stage in the same numbers as do fertilized ones, and up to 50% fail to implant, suggesting that genomic imprinting may be responsible for this lack of viability. METHODS: We have made a quantitative and morphometric analysis of the cell proliferation capacity at the end of the preimplantation period in parthenogenetic mice to study if such parameter is affected by the monoparental constitution of the embryos. RESULTS AND CONCLUSIONS: We have found that, apart from the different morphology and cell number induced by culture conditions, parthenogenetic mouse blastocysts have a significantly smaller cell number than do fertilized control embryos. Our interpretation of these results is that the monoparental constitution of these embryos may be responsible for the lack of some factor required to sustain cell proliferation after the morula stage.

Animals↗
PubMed1996

Metabolism and cell allocation during parthenogenetic preimplantation mouse development.

Diploid parthenogenetic postimplantation mouse embryos, containing two maternal genomes, are characterized by poor development of extraembryonic membranes derived from the trophectoderm and primitive endoderm of the blastocyst. This is thought to be caused by a deficiency of expression of paternally derived imprinted genes. Here we have compared the inner cell mass, from which the primitive endoderm and fetal lineages are derived, and the trophectoderm, which forms a major component of the placenta, in parthenogenetic and fertilized preimplantation embryos. We have also studied the metabolism from the 1-cell to the blastocyst stage. Cell numbers were reduced in the ICM and TE of parthenogenetic blastocysts compared to fertilized blastocysts. This was thought to be due to the increased levels of cell death observed in these lineages. Pyruvate and glucose uptake by parthenogenetic embryos was similar to that by fertilized embryos throughout preimplantation development. However, at the expanded blastocyst stage glucose uptake by parthenogenetic embryos was significantly higher than by fertilized embryos. The implications of the actions of imprinted genes and of X-inactivation is discussed.

Animals↗
PubMed1996

The development of preimplantation mouse parthenogenones in vitro in absence of glucose: influence of the maternally inherited components.

Mouse preimplantation embryo development is characterized by a switch from a dependence on the tricarboxylic acid cycle pre-compaction to a metabolism based on glycolysis post-compaction. In-view of this, the role of glucose in embryo culture medium has come under increased analysis and has lead to improved development of outbred mouse embryos in glucose free medium. Another type of embryo that has proven difficult to culture is the parthenogenetic (PN) mouse embryo. With this in mind we have investigated the effect of glucose deprivation on PN embryo development in vitro. Haploid and diploid PN embryos were grown in medium M16 with or without glucose (M16-G) and development, glycolytic rate, and methionine incorporation rates assessed. Haploid PN and normal embryo development to the blastocyst stage did not differ in either M16 or M16-G. In contrast, although diploid PN embryos formed blastocysts in M16 (28.3%), they had difficulty in undergoing the morula/blastocyst transition in M16-G (7.6%). There was no significant difference in mean cell numbers of haploid PN, diploid PN and normal embryos cultured in M16 and M16-G at the morula and blastocyst stage. Transfer of diploid PN embryos from M16-G to M16 at the four- to eight-cell stage dramatically increased blastocyst development. At the morula stage diploid PN embryos grown in M16-G exhibited a higher glucose metabolism and protein synthesis compared to those grown in M16 and to haploid PN embryos. Difficulties of diploid PN embryos in undergoing the morula/blastocyst transition in absence of glucose infer the existence of a link between the maternally inherited components and the preimplantation embryos dependence on glucose.

Animals↗
PubMed1996

Morphological development, cell number, and allocation of cells to trophectoderm and inner cell mass of in vitro fertilized and parthenogenetically developed buffalo embryos: the effect of IGF-I.

The morphology and number of cells in the trophectoderm (TE) and inner cell mass (ICM) of buffalo blastocysts derived from in vitro fertilization and cultured in the presence or absence of insulin-like growth factor-I (IGF-I) were analyzed by differential fluorochrome staining technique. The total cell number (TCN), TE number, and ICM cell number were significantly higher in blastocysts developed in vitro in the presence of IGF-I as compared to blastocysts developed without IGF-I (P < 0.01). It was observed that the buffalo blastocyst took 5-9 days postfertilization to develop in vitro. In order to correlate the time required for blastocyst development and the allocation of cells to TE and ICM, blastocysts were designated as fast (developing on or before day 7) or slow (developing after day 7). The TCN, TE, and ICM cells of fast-developing blastocysts cultured in the presence of IGF-I were significantly higher than slow-developing blastocysts (P < 0.01). The blastocysts developed on day 6 had a mean total cell number 118.6 +/- 21.4, which significantly decreased to 85.6 +/- 17.4, 62.0 +/- 14.5, and 17.0 +/- 4.0 on days 7, 8, and 9, respectively (P < 0.05). Normal development of buffalo embryo showed that, on average, embryos reached compact morula stage at the earliest between days 4.5-5.5. Blastocysts developed, at the earliest, between days 5.0-6.0, and it took them, on average, 6.5 days to hatch from the zona pellucida. TCN, TE, and ICM increased three times from morula to blastocyst; however, the proportion of ICM to TCN remained the same, in both embryonic stages. TE approximately doubled in hatched blastocysts, as compared to unhatched blastocysts (P < 0.05). However, ICM cells were decreased. The time required for development of parthenogenetic blastocysts was observed to be greater as compared to in vitro fertilized (IVF) blastocysts. The total cell number of parthenogenetic blastocysts was 100.8 +/- 11.3, including 59.2 +/- 8.4 cells of TE and 42.1 +/- 6.9 cells of ICM.

Animals↗
PubMed1996

Intracellular pH change does not accompany egg activation in the mouse.

In the sea urchin, some other marine invertebrates, and the frog, Xenopus, egg activation at fertilization is accompanied by an increase in intracellular pH (pHi). We measured pHi in germinal vesicle (GV)-intact mouse oocytes, ovulated eggs, and in vivo fertilized zygotes using the pH indicator dye, SNARF-1. The mean pH, was 6.96 +/- 0.004 (+/- SEM) in GV-intact oocytes, 7.00 +/- 0.01 in ovulated, unfertilized eggs, and 7.02 +/- 0.01 in fertilized zygotes, indicating no sustained changes in pHi after germinal vesicle breakdown (GVBD) or fertilization. To examine whether transient changes in pHi occur shortly after egg activation, mouse eggs were parthenogenetically activated by 7% ethanol in phosphate buffered saline (PBS); no significant change in pHi followed ethanol activation. Since increased Na+/H+ antiporter activity is responsible for pHi increase in the sea urchin, pHi was measured in the absence of added bicarbonate or CO2 (a condition under which the antiporter would be the only major pHi regulatory mechanism able to operate, since the others were bicarbonate-dependent) in GV-intact oocytes, ovulated eggs, and in vivo fertilized zygotes to determine whether a Na+/H+ antiporter was activated. There was no physiologically significant difference in pHi after GVBD or fertilization, when pHi was measured in bicarbonate-free medium, nor any change upon parthenogenetic activation. Thus, a change in pHi is not a feature of egg activation in the mouse.

Animals↗
PubMed1997

Parthenogenetic activation of Chinese hamster oocytes by chemical stimuli and its cytogenetic evaluation.

This study was undertaken parthenogenetically to activate Chinese hamster oocytes in vitro by chemical stimuli. Oocytes were exposed to five different chemical agents, ethanol (EtOH), strontium chloride (SrCl2), cycloheximide (CHX), phorbol ester (PMA), and ionophore A23187 (IA23). No parthenogenetic activation was observed in the oocytes treated with 8% EtOH for 8-11 min, 1.7 mM and 5.0 mM SrCl2 for 1 hr, 100 microM and 400 microM CHX for 2 hr, and 81 nM and 162 nM PMA for 5 min. In contrast, 89.7% of oocytes parthenogenetically extruded the second polar body in treatment with 3 microM IA23 for 5 min, but only 22.6% of them formed a pronucleus and developed to 2-cell embryos. The remaining ova stopped their cell cycle immediately after completion of the second meiotic division. They had unichromatid chromosomes (monads), which are called MIII chromosomes. Treatment with 5 microM IA23 for 5 min was so deleterious that > 90% of oocytes were degenerated. However, oocyte activation was significantly improved when the treatment with 3 microM IA23 for 5 min was followed by treatment with 8% EtOH for 10 min, 100 microM CHX for 2 hr, 81 nM PMA for 5 min or 3 microM IA23 for 5 min: rates of pronuclear formation were 54.4%, 84.3%, 34.2%, and 54.6%, respectively. More than 80% of pronucleate ova successfully developed into 2-cell stage. Additive treatment with 5 mM SrCl2 for 1 hr had no positive effect on pronuclear formation. Incidences of aneuploidy (4.6%) and structural chromosome aberrations (1.0%) in parthenogenons produced by combined stimuli of IA23 and CHX were not significantly different from those (3.8% and 1.6%, respectively) in female pronuclei of ova fertilized in vitro, showing that combined treatments with IA23 and CHX cause neither nondisjunction at the second meiotic division nor structural aberrations in MII chromosomes. The present technique for parthenogenetic activation of Chinese hamster oocytes may be useful as an assessment system to detect aneugenic and clastogenic effects of mutagens on mammalian oocytes.

Animals↗
PubMed1997

Role of secreted proteins and gonadotrophins in promoting full maturation of porcine oocytes in vitro.

Experiments were designed to identify the extent to which follicle cells and hormones contribute to the developmental competence of porcine oocytes matured in vitro. Oocyte-cumulus complexes were collected from ovaries by dissection and cultured in 2 ml of TCM199-based medium in 5% CO2 in humidified air at 38.5 degrees C. This basic maturation system was supplemented, for either the first 24 hr only or for the 48-hr culture period, with 1) everted follicle shell alone, 2) gonadotrophic hormones alone, or 3) both follicle shells and hormones. The effect of these treatments was evaluated on 1) meiotic maturation rates, 2) the capacity of matured eggs to undergo activation and early cleavage, and 3) changes to the profile of proteins secreted into the culture medium. The results showed that 1) supplementation with either follicle shell or hormones alone increased the rates of meiotic maturation over the nonsupplemented control group, and 2) combined follicle shell and hormonal supplementation yielded the highest rates for maturation, activation, and cleavage but only when hormonal supplementation was removed after the first 24 hr of culture. Proteins of 30, 37, 45, and 46 kD, but of unknown function, were secreted during the first 24 hr into the culture medium in groups supplemented with follicle shells. The addition of hormones did not affect this pattern of secreted proteins. It is possible that some secreted proteins may act to facilitate full maturation of pig oocytes.

Animals↗
PubMed1997

Protein kinase C is required for the disappearance of MPF upon artificial activation in mouse eggs.

The aim of the present study was to investigate the implication of protein kinase C (PKC) in the mouse egg activation process. We used OAG (1-oleoyl-2-acetyl-sn-glycerol) as a PKC activator, calphostin C as a specific PKC inhibitor, and the calcium ionophore A23187 as a standard parthenogenetic agent. The exposure of zona-free eggs to 150 microM or 50 microM OAG for 10 min resulted in meiosis II completion in approximately 80% of instances. By contrast, at a lower concentration (25 microM), the PKC stimulator was ineffective as parthenogenetic agent. Shortly after the application of 150 microM OAG, the cytosolic Ca2+ concentration ([Ca2+]i) increased transiently in all the eggs examined, whereas after the addition of 50 microM OAG, [Ca2+]i remained unchanged for at least 20 min. During this period, the activity of M-phase promoting factor (MPF) dramatically decreased and most of the eggs entered anaphase except when the PKC was inhibited by calphostin C. Similarly, MPF inactivation and meiosis resumption were prevented in calphostin C-loaded eggs following treatment with A23187, even though the ionophore-induced Ca2+ signalling was not affected. Taken together, our results indicate that stimulation of PKC is a sufficient and necessary event to induce meiosis resumption in mouse eggs and strongly suggest that, in this species, the mechanism by which a transient calcium burst triggers MPF inactivation involves a PKC-dependent pathway.

Animals↗
PubMed1997

Haploid maternal genome derived from early diplotene oocytes can substitute for the female pronucleus in preimplantation mouse development.

We describe the preimplantation development of mouse embryos that have received the haploid maternal genome derived from early diplotene nuclei of primordial oocytes (PO). Two generations of recipient egg-cells were used. Induction of two meiotic divisions of the PO nucleus and the reduction of the number of chromosomes to the haploid level were achieved in preovulatory oocytes (primary recipients). The developmental potential of the obtained haploid genome was examined in zygotes (secondary recipients). The nuclei of PO obtained from newborn mice were transferred by cell electrofusion to in vitro maturing (IVM) and enucleated preovulatory mouse oocytes. The reconstructed oocytes which had completed maturation, i.e., reached metaphase II, were artificially activated (8% ethanol + CHX). Activated oocytes were used as donors of haploid pronuclei of PO origin which were transferred (by karyoplast fusion) to partially enucleated zygotes containing only the male pronucleus. Thus, reconstituted zygotes were transplanted to the ligated oviducts of the cycling mice and 27% of them developed to the blastocyst stage. Our experiments demonstrate that 1) the nucleus of PO can be induced to premature meiotic divisions in an IVM enucleated preovulatory oocyte; 2) in the presence of a normal male pronucleus, the haploid pronucleus of PO origin can substitute for a female pronucleus during preimplantation development.

Animals↗
PubMed1998

Parthenogenetic development and protein patterns of newly matured bovine oocytes after chemical activation.

Development of an effective activation protocol is of great importance for studying oocyte competence and embryo cloning. Experiments were designed to examine effects of intracellular calcium elevating agents such as calcium ionophore A23187 (CaA) and ethanol, or protein synthesis and phosphorylation inhibitors such as cycloheximide (CH) and 6-dimethylaminopurine (6-DMAP), or a sequential combination of these agents on both parthenogenetic development and protein patterns of newly matured bovine oocytes. Oocytes were matured for 24 hr in M-199 supplemented with follicle-stimulating hormone (FSH), luteinizing hormone (LH), and estradiol at 39 degrees C in humidified air. They were then activated by various treatments and cultured in KSOM. Protein patterns at 15 hr after treatment were determined on 8-15% gradient SDS-PAGE and silver stained. Results demonstrated that none of the chemical agents--CaA, ethanol, 6-DMAP, or cycloheximide--could effectively induce parthenogenetic development of young bovine oocytes. When compared with the single treatments, sequentially combined treatments of CaA with 6-DMAP or with cycloheximide plus cytochalasin D (CD) significantly increased the rates of cleavage (78-82% versus 3-13%) and blastocyst development (31-40% versus 0%), which were comparable with those of IVF group (80% and 35%, respectively; P > 0.05). Supplementation with CD to the combined CaA and CH treatment improved rates of cleavage and blastocyst development versus without CD supplementation (31% versus 7%; P < 0.05). Fluorescent microscopy revealed that 95% (n = 40) of oocytes treated with CaA plus 6-DMAP had one pronucleus (PN) and one polar body (PB), while 88% (n = 40) in the CaA plus cycloheximide-treated group had one PN and two PBs and 85% (n = 40) in CaA plus cycloheximide and CD group had two PNs and one PB. Treatment by CaA alone resulted in 73% of oocytes (n = 40) arrested at a metaphase stage with two PBs (named as metaphase III or MIII). Protein patterns were similar for chemically activated and in vitro-fertilized (IVF) oocytes in that the 138- and 133-kDa proteins, whose functions are not yet known, were present in the metaphase-stage (MII 24 hr, MII 40 hr, and MIII) oocytes but were absent in PN-stage oocytes regardless of treatment. Therefore, these proteins seem to be metaphase-associated proteins. Taken together, we conclude that optimal parthenogenetic development of newly matured bovine oocytes can be obtained by calcium ionophore treatment followed by incubation in either 6-DMAP or cycloheximide plus cytochalasin D and that the reduction of the 138- and 133-kDa proteins might be necessary for the full activation of bovine oocytes.

Animals↗
PubMed1998

In vitro and in vivo survival of frozen-thawed bovine oocytes after IVF, nuclear transfer, and parthenogenetic activation.

Cryopreservation of bovine oocytes would be beneficial both for nuclear transfer and for preservation efforts. The overall objective of this study was to evaluate the viability as well as the cryodamage to the nucleus vs. cytoplasm of bovine oocytes following freezing-thawing of oocytes at immature (GV) and matured (MII) stages using in vitro fertilization (IVF), parthenogenetic activation, or nuclear transfer assays. Oocytes were collected from slaughterhouse ovaries. Oocytes at the GV, MII, or MII but enucleated (MIIe) stages were cryopreserved in 5% (v/v) ethylene glycol; 6% (v/v) 1,2-propanediol; and 0.1-M sucrose in PBS supplemented with 20% (v/v) fetal bovine serum. Frozen-thawed oocytes were subjected to IVF, parthenogenetic activation, or nuclear transfer assays. Significantly fewer GV oocytes survived (i.e., remained morphologically intact during freezing-thawing) than did MII oocytes (47% vs. 84%). Subsequent development of the surviving frozen-thawed GV and MII oocytes was not different (58% and 60% cleavage development; 7% and 12% blastocyst development at Day 9, respectively, P > 0.05). Parthenogenetic activation of frozen-thawed oocytes resulted in significantly lower rates of blastocyst development for the GV than the MII oocyte groups (1% vs. 14%). Nuclear transfer with cytoplasts derived from frozen-thawed GV, MII, MIIe, and fresh-MII control oocytes resulted in 5%, 16%, 14%, and 17% blastocyst development, respectively. However, results of preliminary embryo transfer trials showed that fewer pregnancies were produced from cloned embryos derived from frozen oocytes or cytoplasts (9%, n = 11 embryos) than from fresh ones (19%, n = 21 embryos). Transfer of embryos derived by IVF from cryopreserved GV and MII oocytes also resulted in term development of calves. Our results showed that both GV and MII oocytes could survive freezing and were capable of developing into offspring following IVF or nuclear transfer. However, blastocyst development of frozen-thawed oocytes remains poorer than that of fresh oocytes, and our nuclear transfer assay suggests that this poorer development was likely caused by cryodamage to the oocyte cytoplasm as well as to the nucleus.

Animals↗
PubMed1999

Offspring born from chimeras reconstructed from parthenogenetic and in vitro fertilized bovine embryos.

Chimeric embryos were produced by aggregation of parthenogenetic (Japanese Red breed) and in vitro fertilized (Holstein breed) bovine embryos at the Yamaguchi Research Station in Japan and by aggregation of parthenogenetic (Red Angus breed) and in vitro fertilized (Holstein breed) embryos at the St. Gabriel Research Station in Louisiana. After embryo reconstruction, live offspring were produced at each station from transplanting these embryos. The objective of this joint study was to evaluate the developmental capacity of reconstructed parthenogenetic and in vitro fertilized bovine embryos. In experiment I, chimeric embryos were constructed: by aggregation of four 8-cell (demi-embryo) parthenogenetic and four 8-cell stage (demi-embryo) IVF-derived blastomeres (method 1) and by aggregation of a whole parthenogenetic embryo (8-cell stage) and a whole IVF-derived embryo (8-cell stage) (method 2). Similarly in experiment II, chimeric embryos were constructed by aggregating IVF-derived blastomeres with parthenogenetic blastomeres. In this experiment, three categories of chimeric embryos with different parthenogenetic IVF-derived blastomere ratios (2:6; 4:4, and 6:2) were constructed from 8-cell stage bovine embryos. In experiment III, chimeric embryos composed of four 8-cell parthenogenetic and two 4-cell IVF-derived blastomeres or eight 16-cell parthenogenetic and four 8-cell IVF-derived blastomeres were constructed. Parthenogenetic demi-embryos were aggregated with sexed (male) IVF demi-embryos to produce chimeric blastocysts (experiment IV). In the blastocyst stage, hatching and hatched embryos were karyotyped. In experiment V, chimeric embryos that developed to blastocysts (zona-free) were cryopreserved in ethylene glycol (EG) plus trehalose (T) with different concentrations of polyvinylpyrrolidone (PVP; 5%, 7.5%, and 10%). In experiment I, the aggregation rate of the reconstructed demi-embryos cultured in vitro without agar embedding was significantly lower than with agar embedding (53% for 0% agar, 93% for 1% agar, and 95% for 1.2% agar, respectively). The aggregation was also lower when the aggregation resulted from a whole parthenogenetic and IVF-derived embryos cultured without agar than when cultured with agar (70% for 0% agar, 94% for 1% agar, and 93% for 1.2% agar, respectively). The development rate to blastocysts, however, was not different among the treatments. In experiment II, the developmental rates to the morula and blastocyst stages were 81%, 89%, and 28% for the chimeric embryos with parthenogenetic:IVF blastomere ratios of 2:6, 4:4, and 6:2, respectively. In experiment III, the developmental rate to the morula and blastocyst stages was 60% and 65% for the two 4-cell and four 8-cell chimeric embryos compared with 10% for intact 8-cell parthenogenetic embryos and 15% for intact 16-cell parthenogenetic embryos. To verify participation of parthenogenetic and the cells derived from the male IVF embryos in blastocyst formation, 51 embryos (hatching and hatched) were karyotyped, resulting in 27 embryos having both XX and XY chromosome plates in the same sample, 14 embryos with XY and 10 embryos with XX. The viability and the percentage of zona-free chimeric embryos at 24 hr following cryopreservation in EG plus T with 10% PVP were significantly greater than those cryopreserved without PVP (89% vs. 56%). Pregnancies were diagnosed in both stations after the transfer of chimeric blastocysts. Twin male (stillbirths) and single chimeric calves were delivered at the Yamaguchi station, with each having both XX and XY chromosomes detected. Three pregnancies resulted from the transferred 40 chimeric embryos at the Louisiana station. Two pregnancies were lost prior to 4 months and one phenotypically-chimeric viable male calf was born. We conclude that the IVF-derived blastomeres were able to stimulate the development of bovine parthenogenetic blastomeres and that the chimeric parthenogenetic bovine embryos were developmentall

Animals↗
PubMed1999

Cell allocation and chromosomal complement of parthenogenetic and IVF bovine embryos.

Considerable concerns exist regarding the quality of parthenogenetically activated embryos in terms of sufficient numbers of cells comprising the inner cell mass (ICM) and trophectoderm (TE) and the ploidy. Therefore, these two parameters were used to assess the quality of embryos derived from parthenogenetic activation by using calcium ionophore A23187 (CaI) followed by either 6-dimethylaminopurine (6-DMAP, 3.5 hr or 6.5 hr) or cycloheximide (CHX) plus cytochalasin D (CD). The conventional in vitro (IVF) produced embryos served as a control. Double staining of the parthenogenetic blastocysts showed that the total cell number (TC) of embryos from the 6-DMAP 3.5 hr (87.0 +/- 5.3) and CHX+CD (79.0 +/- 6.1) groups was not different (P > 0.05), but was lower than that of control embryos (116.0 +/- 5.8, P < 0.001). The mean ratios of inner cell mass (ICM) and trophectoderm (TE) cells in the 6-DMAP 3.5 hr group (0.57 +/- 0.04) and the control IVF group (0.50 +/- 0. 02) did not differ significantly. Both were higher than those of the CHX+CD group (0.36 +/- 0.02; P < 0.05). Further analysis of chromosomal compositions of developing stage embryos at day four after IVF or parthenogenetic activation demonstrated that prolonged treatment with 6-DMAP for 6.5 hr resulted in a significantly lower percentage of diploid embryos and a significantly higher percentage of abnormal ploidy embryos compared to treatment with 6-DMAP for 3.5 hr or with CHX and IVF. In conclusion, parthenogenetic activation of bovine oocytes with CaI followed by 6-DMAP for 3.5 hr could produce better quality embryos in terms of total cell numbers, the number of cells allocated to the ICM, and the ploidy of embryos.

Adenine↗