Meiotic Maturation Of Bovine Oocytes Research Articles

H1foo is essential forin vitromeiotic maturation of bovine oocytes

Oocyte-specific linker histone, H1foo, is localized on the oocyte chromosomes during the process of meiotic maturation, and is essential for mouse oocyte maturation. Bovine H1foo has been identified, and its expression profile throughout oocyte maturation and early embryo development has been established. However, it has not been confirmed if H1foo is indispensable during bovine oocyte maturation. Effective siRNAs against H1foo were screened in HeLa cells, and then siRNA was microinjected into bovine oocytes to down-regulate H1foo expression. H1foo overexpression was achieved via mRNA injection. Reverse transcription polymerase chain reaction (RT-PCR) results indicated that H1foo was up-regulated by 200% and down-regulated by 70%. Based on the first polar body extrusion (PB1E) rate, H1foo overexpression apparently promoted meiotic progression. The knockdown of H1foo significantly impaired bovine oocyte maturation compared with H1foo overexpression and control groups (H1foo overexpression = 88.7%, H1foo siRNA = 41.2%, control = 71.2%; P < 0.05). This decrease can be rescued by co-injection of a modified H1foo mRNA that has escaped from the siRNA target. However, the H1e (somatic linker histone) overexpression had no effect on PB1E rate when compared with the control group. Therefore we concluded that H1foo is essential for bovine oocyte maturation and its overexpression stimulates the process.

Effect of Roscovitine Pretreatment on the Meiotic Maturation of Bovine Oocytes and their Subsequent Development after Somatic Cell Nuclear Transfer

Effect of Roscovitine Pretreatment on the Meiotic Maturation of Bovine Oocytes and their Subsequent Development after Somatic Cell Nuclear Transfer

Enhancement of Bovine oocyte maturation by leptin is accompanied by an upregulation in mRNA expression of leptin receptor isoforms in cumulus cells

In this study, the mechanisms of supposed leptin action on oocyte maturation were examined. Expression of leptin mRNA, as determined with RT-PCR, was present in oocytes but not in cumulus cells. The long isoform of the leptin receptor (ObR-L) was expressed exclusively in cumulus cells after 7 and 23 hr of maturation. In oocytes the expression of the short receptor isoform (ObR-S) and all the receptor isoforms combined (ObR-T) did not change during maturation, as determined by quantitative RT-PCR, but in cumulus cells there was a significant increase in ObR-S transcripts after 7 hr of maturation. To determine if leptin plays a role in resumption of meiosis, oocytes meiotically arrested by the connection of the cumulus to a piece of granulosa layer were exposed to leptin. After 23 hr of culture, the proportion of oocytes that had resumed meiosis did not differ from the control. Exposure of COCs to leptin (1,000 ng/ml) resulted after 17 hr of maturation in a smaller proportion of oocytes that was still in metaphase-I stage (M-I) compared to the control group. Fertilization of oocytes after maturation in the presence of leptin resulted in a larger proportion of embryos that had developed to the 8-cell stage on Day 5 compared to the control group and in more blastocysts on Day 8 of culture. It is concluded that leptin enhances meiotic maturation of bovine oocytes, and that this effect is cumulus cell-mediated.

Activation of Akt (protein kinase B) stimulates metaphase I to metaphase II transition in bovine oocytes

In somatic cells, the serine/threonine kinase Akt (or protein kinase B) was shown to contribute to processes linked to cellular growth, cell survival and cell cycle regulation. In contrast to these findings, the function of Akt during the meiosis of mammalian oocytes remains to be investigated. We analysed the phosphorylation pattern and the activity of Akt during meiotic maturation (transition from prophase I to metaphase II) of bovine oocytes. The oocytes were matured in vitro (IVM) for 0, 10 and 24 h to reach the germinal vesicle (GV), metaphase I (M I) and metaphase II (M II) stages respectively. The abundance and phosphorylation pattern of Akt was revealed by Western blotting using total Akt or phosphoso-Akt-specific antibodies. The activity of this particular kinase was determined by an in vitro kinase assay. Furthermore, functional properties were analysed by cultivating oocytes in the presence of the Akt inhibitor SH6. The results showed that the overall abundance of Akt did not change significantly during IVM. On the other hand, Akt became phosphorylated at Thr 308 and Ser 473, reaching its maximum at the M I phase. In the GV and M II stages, only low basal phosphorylation levels were observed on both sides. This phosphorylation profile corresponded strictly to the activity of the kinase. The cultivation of oocytes in the presence of the phosphatidylinositol analogue SH6 for 24 h showed that, with higher concentrations, up to 65% of the oocytes were arrested in the M I stage. This result indicated that Akt is involved in the M I/M II transition during the meiotic maturation of bovine oocytes. The physiological aspects of the Akt function will be discussed.

Regulation of translation during in vitro maturation of bovine oocytes: the role of MAP kinase, eIF4E (cap binding protein) phosphorylation, and eIF4E-BP1.

Meiotic maturation of mammalian oocytes (transition from prophase I to metaphase II) is accompanied by complex changes in the protein phosphorylation pattern. At least two major protein kinases are involved in these events; namely, cdc2 kinase and mitogen-activated protein (MAP) kinase, because the inhibition of these kinases arrest mammalian oocytes in the germinal vesicle (GV) stage. We show that during meiotic maturation of bovine oocytes, the translation initiation factor, eIF4E (the cap binding protein), gradually becomes phosphorylated. This substantial phosphorylation begins at the time of germinal vesicle breakdown (GVBD) and continues to the metaphase II stage. The onset of eIF4E phosphorylation occurs in parallel with a significant increase in overall protein synthesis. However, although eIF4E is nearly fully phosphorylated in metaphase II oocytes, protein synthesis reaches only basal levels at this stage, similar to that of prophase I oocytes, in which the factor remains unphosphorylated. We present evidence that a specific repressor of eIF4E, the binding protein 4E-BP1, is present and could be involved in preventing eIF4E function in metaphase II stage oocytes. Recently, two protein kinases, called Mnk1 and Mnk2, have been identified in somatic cells as eIF4E kinases, both of which are substrates of MAP kinase in vivo. In bovine oocytes, a specific inhibitor of cdk kinases, butyrolactone I, arrests oocytes in GV stage and prevents activation of both cdc2 and MAP kinase. Under these conditions, the phosphorylation of eIF4E is also blocked, and its function in initiation of translation is impaired. In contrast, PD 098059, a specific inhibitor of the MAP kinase activation pathway, which inhibits the MAP kinase kinase, called MEK function, leads only to a postponed GVBD, and a delay in MAP kinase and eIF4E phosphorylation. These results indicate that in bovine oocytes, 1) MAP kinase activation is only partially dependent on MEK kinase, 2) MAP kinase is involved in eIF4E phosphorylation, and 3) the abundance of fully phosphorylated eIF4E does not necessarily directly stimulate protein synthesis. A possible MEK kinase-independent pathway of MAP kinase phosphorylation and the role of 4E-BP1 in repressing translation in metaphase II oocytes are discussed.

Involvement of connexin 43 in meiotic maturation of bovine oocytes

In ovarian follicles, cumulus cells provide the oocyte with small molecules that permit growth and control maturation. These nutrients reach the germinal cell through gap junction channels, which are present between the cumulus cells and the oocyte, and between the cumulus cells. In this study the involvement of intercellular communication mediated by gap junction channels on oocyte maturation of in vitro cultured bovine cumulus-oocyte complexes (COCs) was investigated. The stages of oocyte maturation were determined by Hoechst 33342 staining, which showed that 90% of COCs placed in the maturation medium for 24 h progress to the metaphase II stage. Bovine COC gap junction communication was disrupted initially using n-alkanols, which inhibit any passage through gap junctions. In the presence of 1-heptanol (3 mmol l(-1)) or octanol (3.0 mmol l(-1) and 0.3 mmol l(-1)), only 29% of the COCs reached metaphase II. Removal of the uncoupling agent was associated with restoration of oocyte maturation, indicating that treatment with n-alkanols was neither cytotoxic nor irreversible. Concentrations of connexin 43 (Cx43), the major gap junction protein expressed in the COCs, were decreased specifically using a recombinant adenovirus expressing the antisense Cx43 cDNA (Ad-asCx43). The efficacy of adenoviral infection was > 95% in cumulus cells evaluated after infection with recombinant adenoviruses expressing the green fluorescence protein. RT-PCR performed on total RNA isolated from Ad-asCx43-infected COCs showed that the rat Cx43 cDNA was transcribed. Western blot analysis revealed a three-fold decrease in Cx43 expression in COCs expressing the antisense RNA for Cx43. Injection of cumulus cells with Lucifer yellow demonstrated further that the resulting lower amount of Cx43 in infected COCs is associated with a two-fold decrease in the extent of coupling between cumulus cells. In addition, oocyte maturation was decreased by 50% in the infected COC cultures. These results indicate that Cx43-mediated communication between cumulus cells plays a crucial role in maturation of bovine oocytes.

Involvement of connexin 43 in meiotic maturation of bovine oocytes

Involvement of connexin 43 in meiotic maturation of bovine oocytes

Butyrolactone I reversibly inhibits meiotic maturation of bovine oocytes,Without influencing chromosome condensation activity.

In this study, butyrolactone I (BL I), a potent and specific inhibitor of cyclin-dependent kinases, was shown to block germinal vesicle (GV) breakdown (GVBD) in bovine oocytes in a concentration-dependent manner; GVBD was almost totally inhibited over the course of 24-48 h of culture when 100 microM BL I was included in tissue culture medium 199 containing either polyvinyl alcohol or BSA. Correlated with this inhibition was the failure of either p34(cdc2) kinase or mitogen-activated protein (MAP) kinase to become activated, and it was unlikely that BL I directly inhibited MAP kinase, since 100 microM BL I did not inhibit MAP kinase activity present in extracts obtained from metaphase II-arrested bovine eggs that possess high levels of MAP kinase activity. Nevertheless, the formation of highly condensed bivalents was observed in 78% of the BL I-treated GV-intact oocytes. This result suggests that chromosome condensation during first meiosis in bovine oocytes does not require the activity of either p34(cdc2) kinase or MAP kinase. Treatment of BL I-arrested oocytes with okadaic acid (OA) did not result in either the activation of p34(cdc2) kinase or MAP kinase, or inducement of GVBD. The BL I-induced block of GVBD for 24 h was reversible, and a subsequent 24-h culture resulted in 90% of oocytes reaching metaphase II with emission of the first polar body. Correlated with the progression to and arrest at metaphase II was the full activation of both p34(cdc2) and MAP kinases. The reversibility after 48 h of culture in BL I was partially decreased when compared to that achieved after an initial 24-h culture. Fertilization in vitro of these eggs resulted in a high incidence of both sperm penetration and pronucleus formation (88% and 70%, respectively).

Meiotic Maturation of Bovine Oocytes Cultured In Vitro with EGF and Wortmannin.

This study was designed to examine the effect of EGF on meiotic maturation of bovine oocytes and to evaluate the ability of wortmannin, a specific PI 3-kinase inhibitor, to modulate EGF-induced oocyte maturation. EGF significantly improved meiotic maturation of both cumulus-intact and denuded oocytes. Wortmannin effectively and irreversibly blocked oocytes from progressing to the MII stage. When oocytes were transferred to wortmannin-treated medium after being cultured for 2 h in wortmannin-free medium, 36% of them progressed to the MII stage. By delaying the time of transfer to 4 h, 64% of the oocytes developed to MII, and by further delaying to 6 h, the rate of MII formation was almost similar to that of oocytes cultured without wortmannin supplementation. Culturing of the oocytes with EGF for 2 h did not improve their meiotic maturation. Increasing the incubation period with EGF for 4 or 6 h resulted in a maturation rate similar to that of those cultured with EGF for the total period of incubation. Data suggest that EGF is capable of stimulating bovine oocyte meiotic maturation regardless of the presence of cumulus cells, and that PI 3-kinase is involved in this process. Results also show that inhibition of PI 3-kinase from 6 h after the onset of culture does not block meiotic progression, and that EGF induces its positive maturation signal during the first 4 h of culture period.

Effect of bovine seminal ribonuclease and bovine pancreatic ribonuclease A on bovine oocyte maturation

Bovine seminal ribonuclease (BS-RNase) contains the MxM (noncovalent dimer) and M=M (free monomer) in constant ratio. The aim of this work was to evaluate the effect of BS-RNase, its monomer and dimer forms, and also various mutants of this enzyme on meiotic completion in cattle oocytes. It was found that BS-RNase has irreversible effects on the meiotic maturation of bovine oocytes in vitro, particularly on the completion of meiosis. The effect of BS-RNase is dose-dependent. In medium supplemented with 1 microg/ml, the results were comparable with those of the control (70% MII oocytes after 24 hr of culture). Whereas 5 microg/ml reduced the number of MII oocytes to 50%, 10 and 25 microg/ml arrested this process completely. The MxM form and RNase A at 5 microg/ml inhibited the maturation rate by 71 and 48%, respectively, but a less significant effect was observed for the M=M form, or the carboxymethylated monomers MCM31 and MCM32 (21%, 16%, and 42% MII oocytes, respectively, in comparison with control). These data demonstrate that bovine ribonucleases can have variable detrimental effects on the maturation of bovine oocyte. J. Exp. Zool. 287:394-399, 2000.

Possible role for phosphatidylinositol 3-kinase in regulating meiotic maturation of bovine oocytes in vitro

In this study 2 phosphatidylinositol 3-kinase (PI 3-kinase)-specific inhibitors, wortmannin and 2-[4-Morpholinyl]-8-phenyl-4H-1-benzopyran-4-one (LY294002), were used to investigate whether PI 3-kinase is involved in the signal transduction that leads to bovine oocyte maturation. Bovine follicular oocytes were cultured in vitro for 24 h in a basic medium consisting of tissue culture medium-199 supplemented with LH, FSH, fetal cow serum, Na-pyruvate and gentamicin. The oocytes were then examined for the stage of meiotic progression and degree of cumulus expansion. In Experiment 1, in cumulus-oocyte complexes (COCs), wortmannin, at any level tested (10 −8 M, 10 −7M or 10 −6M), had no effect on resumption of meiosis as judged by germinal vesicle breakdown and progression to prometaphase I or metaphase I. However, wortmannin significantly (P< 0.01) decreased the proportion of oocytes developing to metaphase II in a dose-dependent manner. In Experiment 2, when denuded oocytes were cultured with wortmannin at 0, 10 −7M and 10 −6 M concentrations, the same pattern of response for COCs was observed, with no effect on meiotic resumption and a significant (P< 0.01) decrease in the proportion of oocytes reaching metaphase II. In Experiment 3, half of the recovered COCs were denuded and both denuded and intact COCs were cultured in the presence of 0, 2.5 × 10 −5 M, 5.0 × 10 −5 M and 7.5 × 10 −5 M LY 294002 before being examined for meiotic progression. Whereas LY294002, at any examined level, had no effect on the percentage of oocytes developing to metaphase I, it significantly (P<0.01) decreased the proportion of metaphase II oocytes when used at 5.0 × 10 −5 or 7.5 × 10 −5 M for both intact COCs and denuded oocytes. In Experiment 4, no significant difference in the degree of cumulus expansion was scored after the COCs were cultured in the presence of wortmannin or LY294002 or in the absence of either treatment. These results provide indirect evidence for a role of PI 3-kinase in the bovine oocyte itself in regulating meiotic progression beyond metaphase I.

The influence of different types of media supplement on the meiotic maturation of bovine oocytes in vitro

This work was designed to evaluate the influence of different types of media supplements in the maturation of preovulatory oocytes in cattle. We studied 4 different supplements: serum from estrous cattle (ECS), serum from fetal calves (FCS), amniotic fluid from cattle (bAF) and follicular fluid from cattle (bFF). Preovulatory bovine oocytes recovered from ovaries of mature cows after slaughter were cultured for 24 h in TCM-199 medium containing 20% of one of these protein supplements. The percentages of oocyte maturation were significantly higher (P < 0.001) in ECS (78%), FCS (79%) and bAF (77%) than in bFF (52%). These data indicate that supplementation with ECS, FCS or bFF is adequate for maturation of bovine oocytes without addition of other compounds.

Meiotic maturation of bovine oocytes in vitro: improvement of meiotic competence by dibutyryl cyclic adenosine 3′,5′-monophosphate.

The present study was undertaken to determine the precise stage of growth at which the ability to resume meiosis is acquired in bovine oocytes. Oocytes of various sizes were isolated from ovaries by mechanical dissection using an 18-gauge needle followed by a razor blade. This method yielded an average of 26.2 +/- 7.4 growing and fully grown oocytes from an ovary. Cumulus-enclosed oocytes were cultured in vitro in tissue culture medium 199 containing 10% fetal calf serum. Oocytes less than or equal to 90 microns in diameter did not resume meiosis. However, germinal vesicle breakdown was observed in oocytes whose diameters exceeded 91 microns. Polar body formation was observed in oocytes with diameters exceeding 101 microns. About 80% of the oocytes with diameters greater than or equal to 121 microns were able to extrude the polar body. The percentage of large oocytes (101 to 120 microns) with first polar body increased when incubated in medium containing dibutyryl cyclic adenosine 3',5'-monophosphate; however, oocytes 90 to 101 microns did not extrude the first polar body even when cultured in a medium containing dibutyryl cyclic adenosine 3',5'-monophosphate. These observations indicate that the capability to resume meiosis is acquired gradually during development of oocytes and that dibutyryl cyclic adenosine 3',5'-monophosphate can improve the meiotic competence of bovine oocytes in culture.

Follicular fluid obtained after the LH surge-stimulates meiotic maturation of bovine oocytes

Follicular fluid obtained after the LH surge-stimulates meiotic maturation of bovine oocytes

Chromosome configurations and time sequence of the first meiotic division in bovine oocytes matured in vitro.

Bovine oocytes aspirated from small follicles were cultured for various time periods. Subsequently, the oocytes were fixed and stained with Giemsa and analyzed for their chromosome configuration. The appearance of the various chromosome configurations and their time sequence were documented. The influence of the presence or absence of follicle-stimulating hormone (FSH) during culture was studied. No difference was found in the proportion of oocytes completing meiotic maturation in the presence or absence of FSH. On average, 75% of the oocytes reached metaphase II after 20 h. FSH showed two main effects: 1) all the cumulus oocyte complexes incubated longer than 10 h in the presence of FSH showed cumulus expansion, and 2) the time period required for chromosome condensation was prolonged for 3 h in the presence of FSH. However, the time sequence in vitro in the presence as well as in the absence of FSH paralleled the time sequence found in vivo, where variations of several hours have been reported. The delay in chromosome condensation in the presence of FSH was assumed to be due to a transient increase in cyclic adenosine 3',5'-monophosphate in the cumulus oocyte complexes. As demonstrated for FSH, the described culture system allowed the study of individual factors for their influence on meiotic maturation of bovine oocytes.

Stage-Dependent Effects of Inhibiting Ribonucleic Acids and Protein Synthesis on Meiotic Maturation of Bovine Oocytes In Vitro

Ribonucleic acid synthesis continues at a low rate within 1h of germinal vesicle breakdown. To determine if this newly synthesized mRNA is required for the resumption of meiosis in cattle oocytes, cumulus-enclosed oocytes were removed from 2 to 4-mm antral follicles directly into Dulbecco's medium with or without the RNA inhibitor, α-amanitin, or the protein synthesis inhibitor, cycloheximide (10μg/ml). They were washed twice and matured for 28 or 48h in medium 199, with Earle's salts, 2.2g/L NaHCO3 and L-glutamine supplemented with 20% heated fetal calf serum to which were added gonadotropins (10μg/ml NIH-LH-S18; 10μg/ml NIH-FSH-S8; 20ng/ml NIH-P-S9), estradiol-17β (1.5μg/ml), solcoseryl (30 μl/ml), and Dibekacin sulfate (100μg/ml) with or without the inhibitors. After 28h of maturation, 95.8% of control oocytes had undergone germinal vesicle breakdown and formation of a metaphase plate compared with only 1.3% of those continuously exposed to cycloheximide and 38% of those continuously exposed to amanitin. Exposure to cycloheximide or amanitin for only the first 16h of culture followed by 12h of inhibitor free culture resulted in 96.6% germinal vesicle breakdown with cycloheximide but only 56.5% germinal vesicle breakdown with amanatin. We conclude newly synthesized mRNA and protein synthesis is required for both full cumulus cell expansion and germinal vesicle breakdown.