Preimplantation embryonic development of spontaneous mouse parthenotes after oocyte meiotic maturation in vitro

Of eggs ovulated in LT/Sv mice, 10–20% undergo spontaneous parthenogenetic activation, and 40–50% of the parthenotes develop to blastocysts when cultured in simple defined medium from the one-cell stage. Similar percentages of oocytes isolated from Graafian follicles undergo parthenogenetic activation after spontaneous maturation in simple defined medium, but embryonic development proceeds no further than the two-cell stage. The simple defined medium that supported preimplantation development of ovulated eggs and spontaneous maturation of extrafollicular oocytes contained no serum, free amino acids, or vitamins. The present experiments were conducted to determine what conditions during spontaneous maturation of extrafollicular oocytes could promote the ability of oocytes to develop to blastocysts after parthenogenetic activation and mimic the environment of preovulatory follicles. Cumulus-enclosed oocytes that were matured in simple medium supplemented with fetal bovine serum (FBS) developed to blastocysts after spontaneous parthenogenetic activation. Furthermore, minimum essential medium (MEM), a complex medium containing free amino acids and vitamins, could substitute completely for FBS for maturing oocytes from (C57BL/6J × LT/Sv)F₁ mice, and to a lesser extent for maturing LT/Sv oocytes. Therefore, even though germinal vesicle breakdown in mouse oocytes and preimplantation development of mouse eggs can occur in the absence of an exogenous supply of free amino acids and vitamins, a complete, or normal, mouse oocyte maturation cannot. These results also demonstrated that gonadotropins are not necessary during oocyte meiotic maturation for parthenogenetically activated eggs to develop through the preimplantation stages. Luteinizing hormone or 17β-estradiol in MEM during oocyte maturation had no effect on the subsequent development of parthenotes. In contrast, follicle stimulating hormone (FSH) and progesterone in the maturation medium decreased the number of ova that subsequently cleaved, and FSH decreased the number of cleaved eggs that developed to blastocysts.     

Cytogenetic analysis of bovine parthenotes after spontaneous activation in vitro

We conducted a cytogenetic study of bovine parthenotes derived from oocytes matured and cultured in vitro. In vitro maturation was carried out by culturing follicular oocytes for 24 h in TCM199 supplemented with estrous cow serum (ECS) and hormones at 39 degrees C in 5% CO2. Matured oocytes were incubated for 20 h in sperm TALP without the addition of spermatozoa, after which they were cultured in maturation droplets for 48 to 72 h. Spontaneous activation occurred in 9.5% of the matured oocytes. Cytogenetic analysis of 24 parthenotes revealed that 62.5% exhibited a normal, diploid chromosome complement. The remaining 37.5% had various ploidy anomalies: haploidy (25%), triploidy (4.2%) and tetraploidy (8.3%). Parthenotes exhibited different developmental stages. The number of blastomeres ranged from 2 to 8 within a parthenote. Only 1 parthenote was comprised 9 to 16 cells. The results showed that spontaneous parthenogenetic activation which occurs in an IVM/IVF system may interfere with embryo production efficiency.     

Developmental capacity of mouse oocytes that undergo maturation in vitro: Effect of the hormonal state of the oocyte donor

In a previous study, it was shown that cumulus cell-enclosed germinal vesicle (GV)-stage oocytes, isolated from pregnant mares' serum gonadotropin (PMSG)-primed immature (22–24 day old) mice and that underwent spontaneous maturation in vitro, exhibited frequencies of embryonic development similar to oocytes stimulated to mature and ovulate in vivo by administration of gonadotropins [Schroeder AC, Eppig JJ, (1984) Dev Biol 102:493–497]. In the present study, the effect of the hormonal state of the oocyte donor on the capacity of in vitro matured oocytes to be fertilized and undergo pre- and post-implantation development was explored further. Oocytes were isolated at the GV-stage from the following groups of mice: 1) unprimed immature mice; 2) adult cycling mice; 3) unprimed Snell dwarf (dw) mice that have undetectable levels of growth hormone (GH), prolactin, and thyroid-stimulating hormone (TSH); and 4) primed and unprimed hypogonadal (hpg) mice that have undetectable levels of circulating gonadotropins. Oocytes maturing in vitro after isolation from normal unprimed immature or adult mice at all stages of the estrous cycle acquired full developmental capacity. GV-stage oocytes isolated from dwarf mice showed embryonic development equivalent to normal ( + /?) littermate controls. Therefore, GH, TSH, or prolactin are not required during oogencsis in vivo to promote the acquisition of competence to complete embryogenesis after maturation in vitro. Oocytes from hypogonadal mice had a much reduced capacity for preimplantation development when compared with normal littermates. Administration of PMSG to the hypogonadal mice significantly increased the developmental capacity of oocytes that underwent maturation in vitro. Gonadotropins, therefore, have a beneficial effect on the oocytc's capacity for embryonic development.     

The relationship between parthenogenetic embryonic development and cumulus cell-oocyte intercellular coupling during oocyte meiotic maturation

Intercellular coupling between cumulus cells and oocytes persists after oocyte meiotic maturation has been initiated. The experiments described here focus on the relationship between oocyte-cumulus cell intercellular coupling during maturation and the subsequent embryonic development of spontaneous mouse parthenotes. Several lines of evidence suggest that this coupling during oocyte maturation is required for the acquisition of the capacity for spontaneous mouse parthenotes to develop embryologically. First, the period of time that LT/Sv oocytes remained coupled to cumulus cells during oocyte maturation in vivo corresponded to that required for the acquisition of the capacity for parthenogenetic embryonic development. Second, the longer that cumulus cells were present during Fpontaneous oocyte maturation in vitro, the higher was the percentageofova undergoing subsequent parthenogenetic development. Third, cumulus cell-free oocytes cocultured with cumulus cell-enclosed oocytes during the maturation period in vitro did not develop embryologically. Fourth, intercellular coupling between cumulus cells and oocytes persisted throughout the oocyte maturation period in vitro. Fifth, incubation of oocyte-cumulus cell complexes in medium containing follicle-stimulating hormone (FSH) promoted uncoupling and decreased the percentage of ova undergoing parthenogenetic development. Thus, cell-to-cell communication, mediated via the intercellular coupling pathway between cumulus cells and oocytes, plays an important role during oocyte maturation and relates to subsequent preimplantation development.     

Effects of meiosis-inhibiting agents and equine chorionic gonadotropin on nuclear maturation of canine oocytes

Experiments were conducted to determine the effects of meiosis-inhibiting-agents and gonadotropins on nuclear maturation of canine oocytes. The culture medium was TCM199 + 10 ng/ml epidermal growth factor supplemented with 25 microM beta-mercaptoethanol, 0.25 mM pyruvate, and 1.0 mM L-glutamine (Basal TCM). Initially, oocytes were cultured in Basal TCM alone or in Basal TCM + dibutylryl cyclic adenosine monophosphate (0.5, 1, 5, or 10 mM dbcAMP) for 24 hr. Dibutylryl cAMP inhibited resumption of meiosis in a dose-dependent manner; 60% of oocytes remained at the germinal vesicle (GV) stage after being cultured for 24 hr in 5 mM dbcAMP. The meiosis-inhibitory effect of dbcAMP appeared to be reversible, as the oocytes resumed meiosis and completed nuclear maturation after being cultured for an additional 48 hr in its absence. Oocytes were then cultured in Basal TCM alone or in Basal TCM + roscovitine (12.5, 25, or 50 microM) for 24 hr. Although approximately 60% of oocytes cultured in 25 microM roscovitine remained at the GV stage, this percentage was not significantly different from the 48% that also remained at the GV stage when cultured in its absence. Oocytes were cultured in Basal TCM + 25 microM roscovitine for 17 hr, exposed briefly to equine chorionic gonadotropin (eCG), and then cultured in Basal TCM for 48 hr. Short exposure of oocytes to eCG was beneficial, as it significantly increased the proportion of oocytes developing beyond germinal vesicle breakdown (P < 0.05) with approximately 20-30% of these were metaphase I (MI) oocytes. Study of the kinetics of nuclear maturation demonstrated that large numbers of oocytes remained at MI even after being cultured for 52 hr following brief exposure to eCG. This study showed that in vitro maturation of canine oocytes can be somewhat improved by short exposure of oocytes to eCG. However, further studies are still required to derive effective methods to mature canine oocytes in vitro.     

Developmental capacity of mouse oocytes matured in vitro: effects of gonadotrophic stimulation, follicular origin and oocyte size.

Development of mammalian oocytes is usually correlated with ovarian follicular development. This correlation was tested by determining whether gonadotrophic stimulation of follicular development in immature mice resulted in a coordinated increase in the embryonic developmental capacity of the oocytes. Oocyte cumulus cell complexes were isolated at the germinal vesicle stage from small, medium and large antral follicles of 26-day-old mice and matured and fertilized in vitro. The frequency with which embryos from oocytes from small follicles completed the two-cell to blastocyst transition was lower than for embryos from oocytes from large follicles (33% and 79%, respectively). Germinal-vesicle stage oocyte-cumulus cell complexes were isolated from 22-26-day-old mice that were unprimed or primed by injection of equine chorionic gonadotrophin 48 h before isolation. Oocytes were matured in control medium, or in medium containing 1 microgram follicle-stimulating hormone (FSH) ml-1, and then fertilized in vitro. Priming did not increase the number of embryos completing the two-cell stage to blastocyst transition in the 22-day-old group nor did FSH treatment of maturing oocytes when the oocytes were isolated from unprimed 22-day-old mice. In contrast, priming increased the percentage of embryos completing the two-cell stage to blastocyst transition in the 26-day-old group by 20%. FSH treatment of maturing oocytes from the unprimed, 26-day-old group increased the number of embryos completing the transition to the same level as those in the primed 26-day-old group, but FSH did not increase the frequency of transition in the primed 26-day-old group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction of mouse oocyte maturation in vivo by perturbants of purine metabolism

Previous studies have suggested a critical role for purines in the maintenance of mouse oocytes in meiotic arrest. If so, then disrupting specific purine metabolic pathways in vivo might induce the resumption of oocyte maturation. To test this hypothesis, immature mice were primed with pregnant mare's serum gonadotropin and 24 h later received, by i.p. injection, one of several drugs that inhibit specific enzymes in the purine metabolic pathways. Cumulus cell-enclosed oocytes were isolated from the ovaries at varying times after drug treatment and assessed for germinal vesicle breakdown (GVBD). The inosine monophosphate dehydrogenase inhibitors, mycophenolic acid (MA) and bredinin (Br), each induced GVBD in a dose-dependent fashion, Br being the more effective agent. When the kinetics of oocyte maturation were examined, 71% of the oocytes from MA-treated mice had undergone GVBD 21-22 h after drug administration. Moreover, 100% GVBD was observed in oocytes from Br-treated mice after 6 h. The action of these drugs appeared to be a direct one and not mediated through stimulation of pituitary gonadotropin release or atresia. Azaserine, an inhibitor of de novo purine synthesis, also induced GVBD in a dose-dependent manner. However, sodium hadacidin and dl-alanosine, inhibitors of adenylosuccinate synthetase, failed to elicit a maturational response in oocytes in vivo. These data support an essential role for guanyl and/or xanthyl derivatives in the maintenance of meiotic arrest in vivo.     

Developmental Potential of Prepubertal Mouse Oocytes Is Compromised Due Mainly to Their Impaired Synthesis of Glutathione

Although oocytes from prepubertal animals are found less competent than oocytes from adults, the underlying mechanisms are poorly understood. Using the mouse oocyte model, this paper has tested the hypothesis that the developmental potential of prepubertal oocytes is compromised due mainly to their impaired potential for glutathione synthesis. Oocytes from prepubertal and adult mice, primed with or without eCG, were matured in vitro and assessed for glutathione synthesis potential, oxidative stress, Ca²⁺ reserves, fertilization and in vitro development potential. In unprimed mice, abilities for glutathione synthesis, activation, male pronuclear formation, blastocyst formation, cortical granule migration and polyspermic block were all compromised significantly in prepubertal compared to adult oocytes. Cysteamine and cystine supplementation to maturation medium significantly promoted oocyte glutathione synthesis and blastocyst development but difference due to maternal age remained. Whereas reactive oxygen species (ROS) levels increased, Ca²⁺ storage decreased significantly in prepubertal oocytes. Levels of both catalytic and modifier subunits of the γ-glutamylcysteine ligase were significantly lower in prepubertal than in adult oocytes. Maternal eCG priming improved all the parameters and eliminated the age difference. Together, the results have confirmed our hypothesis by showing that prepubertal oocytes have a decreased ability to synthesize glutathione leading to an impaired potential to reduce ROS and to form male pronuclei and blastocysts. The resulting oxidative stress decreases the intracellular Ca²⁺ store resulting in impaired activation at fertilization, and damages the microfilament network, which affects cortical granule redistribution leading to polyspermy.     

Reduced oxygen concentration improves the developmental competence of mouse oocytes following in vitro maturation

Reduced atmospheric oxygen concentration is beneficial to embryo development; however, optimal oxygen concentration for oocyte maturation remains undetermined. Likewise, there is no consensus of appropriate medium supplementation during maturation. The objective of this study was to determine whether oxygen tension (20% or 5% O2) and epidermal growth factor (EGF) affect oocyte metabolism and subsequent embryo development. Cumulus-oocyte complexes (COCs) were collected from 28-day-old equine chorionic gonadotropin (eCG) primed or unprimed F1 (C57BL/6xCBA) mice. COCs were matured in defined medium in one of four groups: 20% O2, 20% O2 + EGF, 5% O2, 5% O2 + EGF. In vivo matured COCs were also collected for analysis. COCs from unprimed mice, matured in 5% O2 +/- EGF or 20% O2 + EGF had higher metabolic rates than COCs matured in 20% O2 (P < 0.05). COCs from primed mice had higher metabolic rates when matured in the presence of EGF, regardless of oxygen tension (P < 0.01). Oxygen uptake and mitochondrial membrane potential were higher for in vivo matured oocytes and oocytes matured under 5% O2 compared to oocytes matured under 20% O2 (P < 0.05). Blastocyst formation was not different between maturation groups (primed or unprimed); however, embryo cell numbers were 20-45% significantly higher when COCs were matured at 5% O2 (P < 0.05). Results suggest that oocytes matured in physiological concentrations of oxygen have improved development and metabolic activity, more closely resembling in vivo maturation. These findings have implications for oocyte maturation in both clinical and research laboratories.     

Mouse oocyte mitogenic activity is developmentally coordinated throughout folliculogenesis and meiotic maturation.

Oocytes secrete soluble factors that regulate the growth and differentiation of follicular cells, including maintenance of the distinctive cumulus cell phenotype. This study determines whether the mitogenic activity of oocytes is developmentally regulated and examines the responsiveness of follicular cells to oocytes at different stages of follicular development. Prepubertal SV129 mice of varying ages were primed with 5 IU equine chorionic gonadotropin (eCG) and oocytes/zygotes collected either 46 h post-eCG (immature oocytes), 12 h after administration of 5 IU human CG (hCG; ovulated ova), or 12 h post-hCG and mating (zygotes). Mural granulosa cells (MGC) from antral follicles and GC from preantral follicles were cultured +/- denuded oocytes (DO) for 18 h, followed by a 6-h pulse of [(3)H]thymidine as an indicator of cellular DNA synthesis. Coculturing MGC with meiotically maturing oocytes led to a dose-dependent increase in [(3)H]thymidine incorporation (20-fold above control levels at 0.5 DO/microl). However, [(3)H] counts remained unchanged from control levels when cultured with meiotically incompetent DO from 11- to 15-day-old mice (3% germinal vesicle breakdown; GVB), irrespective of dose of DO or developmental status of GC (MGC or preantral GC). In some treatments, spontaneous meiotic resumption of competent oocytes was prevented by culturing with 5 microM milrinone, a selective inhibitor of oocyte-specific cyclic nucleotide phosphodiesterase. The mitogenic capacity of oocytes was found to decline during and after oocyte maturation. [(3)H]Thymidine incorporation in MGC was highest (11-fold above controls) when cultured with meiotically inhibited (milrinone-treated) GV DO, stimulated 5.5-fold by culture with maturing oocytes, 3-fold with ovulated ova, and unstimulated by zygotes. [(3)H]Thymidine incorporation in MGC was not altered by the dose of milrinone, either in the presence or absence of DO. Metaphase I marked the beginning of the decline in the capacity of oocytes to promote MGC DNA synthesis. These results demonstrate that the capacity of oocytes to promote proliferation of granulosa cells follows a developmental program, closely linked to oocyte meiotic status, increasing with the acquisition of meiotic competence and declining during and after oocyte maturation.     

In vitro ageing of pig oocytes: effects of the histone deacetylase inhibitor trichostatin A.

After in vitro maturation, the unfertilized pig oocytes underwent the process called ageing. This process involves typical events such as fragmentation, spontaneous parthenogenetic activation or lysis. Inhibition of histone deacetylase, using its specific inhibitor trichostatin A (TSA), significantly delayed the maturation of pig oocytes cultured in vitro. The ageing of oocytes matured under the effect of TSA is the same as the ageing in oocytes matured without TSA. The inhibition of histone deacetylase during oocyte ageing significantly reduced the percentage of fragmented oocytes (from 30% in untreated oocytes to 9% in oocytes aged under the effect of 100 nM of TSA). Oocytes matured in vitro and subsequently aged for 1 day under the effects of TSA retained their developmental capacity. After parthenogenetic activation, a significantly higher portion (27% vs. 15%) of oocytes developed to the blastocyst stage after 24 h ageing under 100 nM TSA when compared with oocytes activated after 24 h ageing in a TSA-free medium. The parthenogenetic development in oocytes aged under TSA treatment is similar to the development of fresh oocytes (29% of blastocyst) artificially activated immediately after in vitro maturation.     

Haploidy but not parthenogenetic activation leads to increased incidence of apoptosis in mouse embryos.

Aneuploidy underlies failed development and possibly apoptosis of some preimplantation embryos. We employed a haploid model in the mouse to study the effects of aneuploidy on apoptosis in preimplantation embryos. Mouse metaphase II oocytes that were activated with strontium formed haploid parthenogenetic embryos with 1 pronucleus, whereas activation of oocytes with strontium plus cytochalasin D produced diploid parthenogenetic embryo controls with 2 pronuclei. Strontium induced calcium transients that mimic sperm-induced calcium oscillations, and ploidy was confirmed by chromosomal analysis. Rates of development and apoptosis were compared between haploid and diploid parthenogenetic embryos (parthenotes) and control embryos derived from in vitro fertilization (IVF). Haploid mouse parthenotes cleaved at a slower rate, and most arrested before the blastocyst stage, in contrast to diploid parthenotes or IVF embryos. Developmentally retarded haploid parthenotes exhibited apoptosis at a significantly higher frequency than did diploid parthenotes or IVF embryos. However, diploid parthenotes exhibited rates of preimplantation development and apoptosis similar to those of IVF embryos, indicating that parthenogenetic activation itself does not initiate apoptosis during preimplantation development. These results suggest that haploidy can lead to an increased incidence of apoptosis. Moreover, the initiation of apoptosis during preimplantation development does not require the paternal genome.     

Effect of low-temperature bovine ovary storage on the maturation rate and developmental potential of follicular oocytes after in vitro fertilization, parthenogenetic activation, or somatic cell nucleus transfer

The present study examined the competence of oocytes from bovine ovaries stored at low temperatures for at least 1 day, which is the necessary time period to complete inspection for bovine spongiform encephalopathy. Storage of ovaries at 10 degrees C for 24 h did not affect oocyte maturation (68% versus 68%) or the potential of oocytes to develop into day 8 blastocysts after in vitro fertilization (25% versus 27%), parthenogenetic activation (19% versus 25%), or somatic cell nucleus transfer (27% versus 32%) compared with controls. In vitro-fertilized and parthenogenetic oocytes from ovaries stored at 10 degrees C for 48 h had a significantly decreased maturation rate and developmental potential, but nucleus-transferred oocytes that received cultured cumulus cells did not (27% versus 32%). Thus, bovine ovaries can be stored at 10 degrees C for at least 24 h without decreasing oocyte maturation competence or the developmental potential of in vitro-fertilized, parthenogenetically activated, and somatic cell nucleus-transferred oocytes, at least to the blastocyst stage. The present study provides valuable information with regard to removing bovine ovaries from abattoirs after testing for bovine spongiform encephalopathy.     

Development of parthenogenetic rat embryos

In an effort to establish cloning technology for the rat, we tested several methods (electric stimulation, treatment with ethanol or strontium) for the parthenogenetic activation of rat oocytes. We observed marked individual differences among rats of the outbred Wistar strain in their ability to yield activatable oocytes. These differences were independent of the activation protocol and may be due to a genetic predisposition that is crucial for the parthenogenetic activation of oocytes. The activation of oocytes was dependent upon the time between superovulation of the donor animal and the collection of the embryos. Aged oocytes (derived about 24 h after superovulation) were more prone to activation by each method than were younger oocytes, and some even underwent spontaneous activation without treatment and exhibited pronuclear formation and blastocyst development. All activation methods were effective in generating parthenogenetic rat embryos, and rat parthenotes developed until implantation. However, in general, short-term (15 min) and long-term (2 h) strontium treatment was superior to stimulation by ethanol or electric pulse for parthenogenetic activation. These results will be helpful in achieving successful cloning in the rat.     

Conditions for in vitro maturation and artificial activation of ferret oocytes

The ferret represents an attractive species for animal modeling of lung diseases because of the similarity between ferret and human lung biology and its relatively small size and short gestation time. In an effort to establish experimental protocols necessary for cloning ferrets, optimized conditions for in vitro maturation and artificial activation of ferret oocytes were examined. Cumulus-oocyte complexes were harvested from ovaries of superovulated ferrets, and in vitro maturation was evaluated in three different culture media: medium 1 (TCM-199 + 10% FBS), medium 2 (TCM-199 + 10% FBS with eCG [10 IU/ml] and hCG [5 IU/ml]), or medium 3 (TCM-199 + 10% FBS with eCG, hCG, and 17beta-estradiol [2 microg/ml]). After 24 h of maturation in vitro, the maturation rate of oocytes cultured in medium 2 (70%, n = 79) was significantly greater (P < 0.01) than those of oocytes cultured in the other two media (27%-36%, n = 67-73). At 48 h, similar maturation rates (56%-69%, n = 76-87) were observed for all three types of media. For activation experiments, oocytes cultured in medium 2 were stimulated with electrical and chemical stimuli either individually or in combination. Treatment with cycloheximide and 6-dimethylaminopurine (6-DMAP) following electrical stimulation resulted in 43% (n = 58) of the oocytes developing to the blastocyst stage. Such an activation rate represented a significant improvement over those obtainable under other tested conditions, including individual treatment with electrical pulses (10%, n = 41), cycloheximide (3%, n = 58), or 6-DMAP (5%, n = 59). Blastocysts derived from in vitro activation appeared to be normal morphologically and were composed of an appropriate number of both inner cell mass (mean +/- SEM, 10.3 +/- 1.1; n = 11) and trophectoderm (60.8 +/- 2.9, n = 11) cells. These results have begun to elucidate parameters important for animal modeling and cloning with ferrets.     

Lanosterol 14alpha-demethylase expression in the mouse ovary and its participation in cumulus-enclosed oocyte spontaneous meiotic maturation in vitro

The expression of lanosterol 14alpha-demethylase (LDM) in the mouse ovary after gonadotrophin administration was examined and the action of follicle fluid meiosis activating sterol (FF-MAS), derived from lanosterol by the action of LDM, on oocyte spontaneous maturation was also evaluated in cumulus cell enclosed oocytes (CEOs). Expression of LDM was primarily in oocytes in primordial and secondary follicles prior to administration of gonadotrophins, but obvious LDM expression was apparent in ovarian somatic cells 48 h after administration of equine chorionic gonadotrophin (eCG), especially in luteal and cumulus cells 54 h after eCG or 48 h after eCG plus 6 h after human chorionic gonadotrophin (hCG). The LDM expression in oocytes was only slightly elevated in larger growing follicles after eCG treatment. On the contrary, 48 h after hCG treatment, the elevated expression of LDM was only detected in interstitial cells. Therefore, eCG may be the primary gonadotrophin for LDM expression, and furthermore for production of FF-MAS in mouse cumulus cells (which are indispensable for oocyte maturation in vivo). Conversely, inhibitors of LDM, either 40 microM azalanstat or 50 microM RS-21745, significantly inhibited oocyte germinal vesicle breakdown (GVB) after 4h of in vitro culture; GVB rates decreased to 14 or 20%, compared to 90% in spontaneous maturation, respectively. There was no significant increase in GVB in CEOs following specific inhibitor of sterol Delta14-reductase and Delta7-reductase, AY9944-A-7 (5-100 microM), until marked oocytes degeneration appeared (50 microM). The phenomena may be ascribed to slow, passive accumulation of FF-MAS by AY9944-A-7, which cannot be associated with fast spontaneous progression. Furthermore, in spontaneous-matured CEOs, LDM was expressed preferentially in cumulus cells instead of oocytes. Therefore, FF-MAS may have a positive role in the spontaneous maturation of CEOs. In conclusion, there was an eCG-dependent dual LDM expression pattern on both oocytes and somatic cells in growing follicles in vivo, which may increase LDM expression and FF-MAS production in cumulus cells for oocyte maturation. For the first time, the inhibitory effect of LDM inhibitors on spontaneous maturation, together with the strong LDM expression in spontaneous matured CEOs, indicated that FF-MAS produced by cumulus cells might participate in spontaneous maturation of mouse CEOs.     

Maintenance of meiotic arrest and developmental potential of porcine oocytes after parthenogenetic activation and somatic cell nuclear transfer

Several studies report that meiotic maturation of porcine oocytes can be reversibly preserved. The present study examined how long meiotic maturation can be suppressed. The first experiment determined the preservation medium suitable for reversibly suppressing meiotic maturation of porcine oocytes. The second experiment examined the in vitro developmental potential of oocytes maintained in meiotic arrest after parthenogenetic activation and nuclear transfer of somatic cells. Preservation of cumulus-oocyte complexes with NCSU-37 medium containing 10% follicular fluid, 1 mM dibutyryl cyclic AMP, and follicular shell pieces for 24-96 h at 39 degrees C did not affect oocyte maturation compared with controls (94-98% vs. 98%). The potential of parthenogenetically activated and nuclear-transferred oocytes maintained in meiotic arrest for 24-48 h to develop into blastocysts was not significantly different from that of controls (20-25% vs. 18% and 8-11% vs. 9%, respectively). The present study demonstrated that meiotic maturation of porcine oocytes can be suppressed after preservation for 48 h at 39 degrees C without decreasing oocyte maturation competence or the ability of oocytes to develop to at least the blastocyst stage.     

Possible involvement of Class III phosphatidylinositol-3-kinase in meiotic progression of porcine oocytes beyond germinal vesicle stage

Phosphatidylinositol-3-kinases (PI3Ks) play pivotal roles in meiotic progression of oocytes from metaphase I to metaphase II stage. Using a Class III-specific inhibitor of PI3K, 3-methyladenine (3MA), this study shows that Class III PI3K may be essential for meiotic progression of porcine oocytes beyond germinal vesicle (GV) stage. Treatment of immature porcine oocytes with 3MA for 22-42 h arrested them at the GV stage, irrespective of the presence or absence of cumulus cells. Furthermore, a significantly high proportion (60.9 ± 13.8%) of 3MA-treated oocytes acquired a nucleolus completely surrounded by a rim of highly condensed chromatin (GV-II stage). The GV-arresting effect of 3MA was, however, completely reversible upon their further culture in the absence of 3MA for 22 h. When cumulus-oophorus-complexes (COCs), arrested at the GV stage for 22 h by 3MA, were further cultured for 22 h in the absence of 3MA, 96.1 ± 1.5% of oocytes reached the MII stage at 42 h of IVM and did not differ from non-treated control oocytes with respect to their ability to fertilize, cleave and form blastocyst (P > 0.05) upon in vitro fertilization (IVF) or parthenogenetic activation (PA). These data suggest that 3MA efficiently blocks and synchronizes the meiotic progression of porcine oocytes at the GV stage without affecting their ooplasmic maturation in terms of post-fertilization/activation in vitro embryonic development. Our data also provide indirect evidence for the likely participation of Class III PI3K in meiotic maturation of porcine oocyte beyond the GV stage.     

Freeze-fracture electron microscopy of membrane changes in progesterone-induced maturing oocytes and eggs of Xenopus laevis

Using freeze-fracture electron microscopy, compositional changes were analysed in the surface membrane of Xenopus oocytes during maturation after in vitro progesterone treatment, as well as in eggs before and after fertilization. Investigated stages were as follows: (1) defolliculated full-grown oocytes; (2) defolliculated oocytes after 5 min exposure to 5 micrograms/ml progesterone; (3) ditto at germinal vesicle breakdown (GVBD) after 5 h progesterone treatment; (4) unfertilized eggs at oviposition and (5) zygotes 30 min post-fertilization. Comparing the patterns of intramembranous particle (IMP) density and IMP size during these stages the following changes were found: a transient decrease in IMP density was found after 5 min progesterone treatment; a 48% increase during maturation; a further 17% increase after fertilization. In defolliculated oocytes tight-junction-like structures were found, but no gap junctions. These results are discussed with reference to progesterone action, membrane remodelling, protein synthesis and membrane lipid organization.