Assessment of active translation in cumulus-enclosed and denuded oocytes during standard in vitro maturation and early embryo development.

STUDY QUESTION: Which actively translated maternal transcripts are differentially regulated between clinically relevant in vitro and in vivo maturation (IVM) conditions in mouse oocytes and zygotes? SUMMARY ANSWER: Our findings uncovered significant differences in the global transcriptome as well as alterations in the translation of specific transcripts encoding components of energy production, cell cycle regulation, and protein synthesis in oocytes and RNA metabolism in zygotes. WHAT IS KNOWN ALREADY: Properly regulated translation of stored maternal transcripts is a crucial factor for successful development of oocytes and early embryos, particularly due to the transcriptionally silent phase of meiosis. STUDY DESIGN, SIZE, DURATION: This is a basic science study utilizing an ICR mouse model, best suited for studying in vivo maturation. In the treatment group, fully grown germinal vesicle oocytes from stimulated ovaries were in vitro matured to the metaphase II (MII) stage either as denuded without gonadotropins (IVM DO), or as cumulus-oocyte complexes (IVM COC) in the presence of 0.075 IU/ml recombinant FSH (rFSH) and 0.075 IU/ml recombinant hCG (rhCG). To account for changes in developmental competence, IVM COC from non-stimulated ovaries (IVM COC-) were included. In vivo matured MII oocytes (IVO) from stimulated ovaries were used as a control after ovulation triggering with rhCG. To simulate standard IVM conditions, we supplemented media with amino acids, vitamins, and bovine serum albumin. Accordingly, in vitro pronuclear zygotes (IMZ) were generated by IVF from IVM DO, and were compared to in vivo pronuclear zygotes (IVZ). All experiments were performed in quadruplicates with samples collected for both polyribosome fractionation and total transcriptome analysis. Samples were collected over three consecutive months. PARTICIPANTS/MATERIALS, SETTING, METHODS: All ICR mice were bred under legal permission for animal experimentation (no. MZE-24154/2021-18134) obtained from the Ministry of Agriculture of the Czech Republic. Actively translated (polyribosome occupied) maternal transcripts were detected in in vitro and in vivo matured mouse oocytes and zygotes by density gradient ultracentrifugation, followed by RNA isolation and high-throughput RNA sequencing. Bioinformatic analysis was performed and subsequent data validation was done by western blotting, radioactive isotope, and mitotracker dye labelling. MAIN RESULTS AND THE ROLE OF CHANCE: Gene expression analysis of acquired polysome-derived high-throughput RNA sequencing data revealed significant changes (RPKM ≥ 0.2; P ≤ 0.005) in translation between in vitro and in vivo matured oocytes and respectively produced pronuclear zygotes. Surprisingly, the comparison between IVM DO and IVM COC RNA-seq data of both fractionated and total transcriptome showed very few transcripts with more than a 2-fold difference. Data validation by radioactive isotope labelling revealed a decrease in global translation bof20% in IVM DO and COC samples in comparison to IVO samples. Moreover, IVM conditions compromised oocyte energy metabolism, which was demonstrated by both changes in polysome recruitment of each of 13 mt-protein-coding transcripts as well as by validation using mitotracker red staining. LARGE SCALE DATA: The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE241633 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE241633). LIMITATIONS, REASONS FOR CAUTION: It is extremely complicated to achieve in vivo consistency in animal model systems such as porcine or bovine. To achieve a high reproducibility of in vivo stimulations, the ICR mouse model was selected. However, careful interpretation of our findings with regard to assisted reproductive techniques has to be made by taking into consideration intra-species differences between the mouse model and humans. Also, the sole effect of the cumulus cells' contribution could not be adequately addressed by comparing IVM COC and IVM DO, because the IVM DO were matured without gonadotropin supplementation. WIDER IMPLICATIONS OF THE FINDINGS: Our findings confirmed the inferiority of standard IVM technology compared with the in vivo approach. It also pointed at compromised biological processes employed in the critical translational regulation of in vitro matured MII oocytes and pronuclear zygotes. By highlighting the importance of proper translational regulation during in vitro oocyte maturation, this study should prompt further clinical investigations in the context of translation. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Czech Grant Agency (22-27301S), Charles University Grant Agency (372621), Ministry of Education, Youth and Sports (EXCELLENCE CZ.02.1.01/0.0/0.0/15_003/0000460 OP RDE), and Institutional Research Concept RVO67985904. No competing interest is declared.

Delayed early embryonic lethality following disruption of the murine cyclin A2 gene.

In higher eukaryotes, cell cycle progression is controlled by cyclin dependent kinases (Cdks) complexed with cyclins. A-type cyclins are involved at both G1/S and G2/M transitions of the cell cycle. Cyclin A2 activates cdc2 (Cdk1) on passage into mitosis and Cdk2 at the G1/S transition. Antisense constructs, or antibodies directed against cyclin A2 block cultured mammalian cells at both of these transitions. In contrast, overexpression of cyclin A2 appears to advance S phase entry and confer anchorage-independent growth, and can lead to apoptosis. A second A-type cyclin, cyclin A1 has been described recently which, in the mouse, is expressed in germ cells but not somatic tissues. To address the possible redundancy between different cyclins in vivo and also the control of early embryonic cell cycles, we undertook the targeted deletion of the murine cyclin A2 gene. The homozygous null mutant is embryonically lethal, demonstrating that the cyclin A2 gene is essential. Surprisingly, homozygous null mutant embryos develop normally until post-implantation, around day 5.5 p.c. This observation may be explained by the persistence of a maternal pool of cyclin A2 protein until at least the blastocyst stage, or an unexpected role for cyclin A1 during early embryo development.

Storage of bovine isolated follicles: a new alternative way to improve the recovery rate of viable embryos from ovarian follicles of slaughtered cows.

The vitality of bovine oocytes stored in isolated follicles was examined. The aim of this work was to prolong the time of in vitro manipulation of oocytes before their maturation and develop a new alternative of oocyte "capacitation" to improve the quality of in vitro produced embryos. Follicles were dissected from the ovaries of slaughtered cows; subsequently, follicles were divided according to their diameter into three categories (2-3, 3-4 and 4-6 mm), and stored at 17-18 degrees C for 24 or 48 h in a modified tissue culture medium-199 (TCM-199) with reduced pH. After that time, the cumulus-oocyte complexes (COCs) were isolated, matured, fertilized, and embryos cultured in vitro for a total of 9 days. The percentage of total blastocysts, and hatched blastocysts developed from oocytes, initially kept ("capacitated") for 24h at 17-18 degrees C, within follicles of 3-6mm size categories, were significantly higher than that oocytes of the control [of control oocytes] (44.9 and 30.3% versus 36.2 and 20.4%, respectively). The oocytes of follicles stored for 48 h at 17-18 degrees C already had decreased developmental capacity. Interesting data were obtained when COCs of the 3-4 and 4-6 categories were additionally divided into two subgroups according to their presumed developmental history (originating from the supposed growing "fit" in contrast to the supposed regressing "unfit" follicles). The higher improvement in the rate of hatched blastocysts from 24h stored oocytes was observed only in the subgroup originated from "fit" COCs (15.3 versus 25.0%, and 20.0 versus 34.4%, in the 3-4 and 4-6mm categories, respectively). The transfer of 26 blastocysts (developed of follicles kept for 24h at 17-18 degrees C) to 26 recipient heifers resulted in 18 pregnancies. Storage of follicles at 17-18 degrees C in vitro resulted not only in recovery of higher numbers of blastocysts of better quality but also facilitated the safe transport of follicles for a long distance. The extended, time of follicle storage before the proper oocyte maturation allowed also the synchronization of an appropriate number of recipient animals according to the number of isolated follicles.

Association of p34cdc2 kinase and MAP kinase with microtubules during the meiotic maturation of Xenopus oocytes.

p34cdc2 protein is found in prophase, metaphase and activated Xenopus oocytes at a similar level whereas its kinase activity oscillates within meiosis. Using an anti-PSTAIRE antibody that recognizes Xenopus p34cdc2, it was demonstrated that the major part of p34cdc2 was associated with microtubules isolated in vitro from Xenopus oocytes. Conversely, tubulin was recovered in association with p34cdc2 in p13-Sepharose pellets. The abundance of the fraction of p34cdc2 which was associated with microtubules did not oscillate during the meiotic maturation and the activation process. By contrast, the histone H1 kinase activity of p34cdc2 estimated in microtubular oocyte pellets was much higher in metaphase than in prophase oocytes. Cyclin B, which is associated in vivo with p34cdc2 in prophase and metaphase oocytes, was also present in the microtubular fractions. However, cyclin was not necessary for the binding of p34cdc2 to microtubules since p34cdc2 from activated eggs, where cyclin was missing, still copurified with microtubules. Purified MAP2, but not tubulin, was able to bind to p34cdc2, demonstrating that the association between p34cdc2 and microtubules was mediated by microtubule-associated proteins. During the meiotic maturation of Xenopus oocytes, several protein kinases were activated, among them MAP kinase. MAP kinase also associated with microtubules. It was demonstrated that both p34cdc2 kinase and MAP kinase purified from Xenopus oocytes were able to phosphorylate in vitro rat brain MAP2. However both protein kinases phosphorylated different domains of MAP2, suggesting that they might regulate microtubules in different ways.

Accumulation of the proteolytic marker peptide ubiquitin in the trophoblast of mammalian blastocysts.

Ubiquitination is a universal protein degradation pathway in which the molecules of 8.5-kDa proteolytic peptide ubiquitin are covalently attached to the epsilon-amino group of the substrate's lysine residues. Little is known about the importance of this highly conserved mechanism for protein recycling in mammalian gametogenesis and fertilization. The data obtained by the students and faculty of the international training course Window to the Zygote 2000 demonstrate the accumulation of ubiquitin-cross-reactive structures in the trophoblast, but not in the inner cell mass of the expanding bovine and mouse blastocysts. This observation suggests that a major burst of ubiquitin-dependent proteolysis occurs in the trophoblast of mammalian peri-implantation embryos. This event may be important for the success of blastocyst hatching, differentiation of embryonic stem cells into soma and germ line, and/or implantation in both naturally conceived and reconstructed mammalian embryos.

Interplay between CDC2 kinase and MAP kinase pathway during maturation of mammalian oocytes.

Two principal kinases, p34cdc2 kinase and MAP kinase play a pivotal role in maturation of mammalian oocytes. In the porcine and bovine oocytes both kinases are activated around the time of germinal vesicle breakdown (GVBD). Butyrolactone I (BL I), a specific inhibitor of cdk kinases, prevents effectively and reversibly resumption of meiosis in the porcine and bovine oocytes. Neither p34cdc2 kinase nor MAP kinase are activated in oocytes inhibited in the GV stage. The bovine oocytes maintained for 48 h in the medium supplemented with BL I, progress subsequently to metaphase II in 91%, their cumuli expand optimally and after in vitro fertilization they possess two pronuclei. When the cdc2 kinase is blocked in the porcine oocytes by BL I, MAP kinase, activated by okadaic acid treatment, is able to substitute cdc2 kinase and induce GVBD. The histone H1 kinase activity sharply decreases in the metaphase II oocytes treated by BL I and one or two female pronuclei are formed. These data indicate that BL I is a useful tool either for the two step in vitro culture of mammalian oocytes or for their activation in nuclear transfer experiments.

Dynamics of meiosis and protein kinase activities in bovine oocytes correlated to prolactin treatment and follicle size.

Oocyte developmental competence depends on the size of the original follicle and is affected by compounds like prolactin. We wished to investigate nuclear and cytoplasmic maturation of bovine oocytes correlated to their origin and response to prolactin treatment, by monitoring at frequent intervals meiotic configuration of chromosomes and activity of histone H1 and MAP-kinase. Bovine ovaries were obtained from a slaughterhouse and oocytes were recovered by follicle isolation. Oocytes (n = 1,397) with a compact cumulus were selected from small (2 to 3 mm) and large (4 to 5 mm in diameter) follicles and cultured up to 28 h in TCM 199+20% bull serum with or without 50 ng/mL bovine prolactin. Four groups of oocytes were formed: originating from small or large follicles, and treated or not treated with prolactin. At the scheduled time intervals for in vitro maturation, cumulus oocyte complexes from the 4 groups were randomly selected and the oocytes were analyzed for histone H1 and MAP-kinase, and for chromatin configuration. The first meiotic division took longer to complete in oocytes from large follicles (P < 0.01). Under the influence of prolactin the meiosis was prolonged in oocytes both from small and large follicles (P < 0.05). Histone H1 and MAP-kinases started to be activated at approximately the same time, around 6 h after beginning maturation. But after this time, significantly lower levels of both kinase activities were found in oocytes treated with prolactin, especially those treated during Meiosis I (P < 0.05). Our results indicate a correlation of chromatin configuration and histone H1/MAP-kinase activities.

Cell-cycle aspects of growth and maturation of mammalian oocytes.

In this review, recent data concerning growth and maturation of nonmammalian and mammalian female germ cells are compiled with regard to the increased understanding of somatic cells mitotic cycles, from yeast to human tissues. These data allow us to conclude that growing oocytes of nonvertebrates, lower vertebrates, and mammals resemble somatic cells in the G1 phase of the mitotic cycle in their metabolic and cell cycle behavior. Transcriptional and translational activity of growing oocytes and G1 somatic cells is not compatible with the activation of maturation promoting factor (MPF), with chromatin condensation or with nuclear membrane disintegration. Growing oocytes, even when they are in the dictyate stage of the first meiotic division, promptly inactivate MPF introduced into their cytoplasm by fusion or microinjection, just as do somatic interphase cells. In mammals, the LH surge induces "de novo" RNA and protein synthesis in granulosa cells. This metabolic change in granulosa cells abolishes their inhibitory activity, and meiosis in fully grown oocytes in preovulatory follicles is then resumed. Resumption of meiosis requires an activation of pre MPF molecules within oocytes. This can be achieved either without (mouse, rat, and rabbit) or with (pig, sheep, and cow) an active protein synthesis by the oocytes. The species specificity is probably dependent on the presence or absence of cyclin-like and/or mos-like molecules in fully grown oocytes. Both major events during GVBD, chromatin condensation, and nuclear envelope disintegration require protein phosphorylation. Experimentally, these two phosphorylation activities can be separated one from another. The active MPF molecules are amplified autocatalytically in amphibian and starfish oocytes. However, an increase of MPF activity in mouse and pig oocytes, similarly as in Rana pipiens and sturgeon oocytes, requires an active protein synthesis.

Behaviour of mouse primary spermatocyte nuclei after fusion to enucleated metaphase II oocytes.

Primary spermatocytes originating from prepubertal mouse testes were electrofused to metaphase II (MII)-stage oocytes, enucleated either by the conventional micromanipulation method or by chemical treatment with etoposide and cycloheximide. These experiments were followed by assessment of morphological changes in transferred nuclei using light microscopy, by chromosomal analyses and by screening of hybrids for the presence or absence of DNA synthesis using anti-bromodeoxyuridine antibody and immunofluorescence staining of the hybrids. The results show differences between the two types of ooplasts in susceptibility to activation stimuli. However, when activated, both types of ooplasts gave rise to hybrids of similar morphology. From 35.3% to 63% of activated hybrids originating from chemically or microsurgically enucleated oocytes, respectively, contained one large pronucleus in cytoplasm, 62% or 31.6% hybrids from those two groups, respectively, possessed two smaller pronuclei and a few contained three or four pronuclei. No DNA synthesis was detected in any hybrid containing one or more pronuclei. The chromosome spreads of hybrids with premature chromosome condensation (PCC) morphology (before activation) show that most of the hybrids had a diploid (2n) number of chromosomes. The nature and regularity of the cell division cycle in the hybrids are discussed.

The effect of various capacitation active compounds and capacitation time on the in vitro fertility and protein tyrosine phosphorylation profiles of bovine sperm.

In this paper the effects of capacitation and fertilisation stimulating compounds (heparin, caffeine, glucose, D-penicillamine, bovine serum (BOS), bovine serum albumin (BSA), polyvinyl alcohol (PVA)) were analysed in several in vitro fertilisation protocols. Attention was paid to the rate of penetrated oocytes, kinetics of penetration and to polyspermic fertilisation. Cryopreserved bovine sperm and in vitro matured bovine oocytes were used throughout all the fertilisation experiments. As detected in the first 8 h fertilisation experiment with non-incubated sperm, the supplementation of medium with heparin, BOS and glucose supported the fertilisation rate most effectively (100%), including the kinetics of pronuclei formation (52.4%). The absence of BOS resulted in a decreased fertilisation rate (62.7%) as well as a delay in pronuclei formation (13.6%), similar to that after substitution of heparin with caffeine (73.0% and 25.4%, respectively). The penetration rate in the control medium with BOS (without heparin and caffeine) was surprisingly high, especially in medium without glucose (62.2%). The positive effect of glucose on sperm penetration was observed mainly in a chemically defined medium with PVA. High polyspermy rates were observed throughout all experiments in the media containing heparin or caffeine and BOS as the macromolecular component. D-Penicillamine was not shown to be a fertilisation-stimulating molecule. However, as detected in the second experiment in which oocytes were fertilised with 5 h incubated sperm, its positive effect on the prolongation of a fertile life span of cryopreserved spermatozoa was significant. The presence of either caffeine or heparin in the fertilisation medium (FM) with BOS during sperm incubation induced tyrosine phosphorylation of an approximately 90 kDa protein, detected after 5 h of sperm incubation. The absence of BOS reduced tyrosine phosphorylation of this protein in fertilisation medium with heparin. The percentage of motile spermatozoa and those with intact acrosomes were monitored throughout all experiments.

The behaviour of mitotic nuclei after transplantation to early meiotic ooplasts or mitotic cytoplasts.

This study evaluates the ability of the cytoplasm to determine the nature of the division cycle (meiotic or mitotic) in nuclei obtained from mitotically dividing cells. Using mouse oocytes in different stages of development two types of cytoplasm were prepared: firstly, early meiotic ooplasts were obtained by enucleation of non-matured, prophase-stage oocytes; secondly, mitotic cytoplasts were prepared by enucleation and activation of metaphase II (MII)-stage oocytes. These two types of cytoplasts were then used in fusion experiments, in which mouse primitive type A spermatogonia (prospermatogonia) or mouse fibroblasts were used as a source of donor nuclei. While the fusion of prospermatogonia with mitotic cytoplasts resulted, as expected, in normal premature chromosome condensation (PCC) and subsequent pronuclear formation (58.1%), the majority of hybrids obtained by fusion of prospermatogonia with early meiotic ooplasts (40.3%) displayed unique morphology consisting of two sets of chromosomes organised in two spindle centres connected by microtubules. Each set of chromosomes contained the haploid (1n) number of chromosomes as revealed by chromosome analyses. The same morphology was observed also in 44.2% of hybrids in which the differentiated nuclei of fibroblasts were used as a source of donor mitotic nuclei. In both cases the hybrids were blocked at this stage with high activity of maturation promoting factor (MPF), resistant to any kind of activation and not able to undergo further development. These results suggest that the early meiotic ooplasm was able to induce the initiation of a meiosis-like reducing division in mitotic nuclei originating both from the germline cells and from more differentiated somatic cells.

Inhibition of protein synthesis affects histone H1 kinase, but not chromosome condensation activity, during the first meiotic division of pig oocytes.

The influence of protein synthesis on the regulation of the first meiotic division was studied in pig oocytes. We show that histone H1 kinase activity gradually increases during in vitro culture of pig oocytes, reaching maximum in metaphase I stage after 24 hr of culture. However, in the presence of the protein synthesis inhibitor cycloheximide, histone H1 kinase is not activated during the whole culture period, and after 24 hr it is approximately at the same level as in prophase-stage oocytes. The gradual increase in phosphorylation of six proteins of molecular weights 39, 48, 53, 66, 96, and 120 kDa, observed during the first 24 hr of culture, was not detected when cycloheximide was added to the culture medium. Similarly, the decrease in phosphorylation of a 90-kDa protein was not seen in cycloheximide-treated oocytes. On the other hand, the levels of both MPF components, p34cdc2 and cyclin B, which were found to be nearly constant during the first meiotic division, were not influenced by cycloheximide treatment as revealed by Western blotting. The process of germinal vesicle breakdown (GVBD) was totally blocked by cycloheximide. The condensation of chromatin, however, was not influenced, suggesting that GVBD and chromosome condensation could be regulated independently. The different degrees of MPF activation involved in these processes, as well as the nature of the protein(s) which must be synthesized for triggering GVBD, are discussed.

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).

Chromatin condensation and histone H1 kinase activity during growth and maturation of rabbit oocytes.

Fully grown rabbit oocytes, isolated from preovulatory follicles, exhibit highly condensed bivalents within an intact germinal vesicle while a very low level of histone H1 kinase activity could be detected in their extracts. Chromatin condensation started in growing oocytes isolated from antral follicles presenting a diameter of 0.5 mm. This event was accompanied by a transient rise in histone H1 kinase activity which culminated in large antral follicles measuring 0.75 to 1 mm in diameter. However, the extent of histone H1 kinase activity observed in these growing oocytes remained far less important than that recorded in extracts prepared from in vitro cultured metaphase I and metaphase II oocytes. Moreover, this activity was insufficient to induce germinal vesicle breakdown which will only occur with an increasing efficiency, following in vitro culture of medium, large, and fully grown antral follicles.

Time sequence of germinal vesicle breakdown in pig oocytes after cycloheximide and P-aminobenzamidine block.

All porcine oocytes cultured 20 hr in medium with 10 micrograms/ml cycloheximide rested in the germinal vesicle (GV) stage but with the highly condensed bivalents in nucleoplasm. When these oocytes were washed and cultured in the control medium for 2, 4, and 6 hr, germinal vesicle breakdown (GVBD) was completed in 0, 86, and 100% of them, respectively. When similarly inhibited oocytes cultured successively only 2.5 hr in the control medium were given again in cycloheximide enriched medium (3.5 hr), nearly all of them reached late diakinesis stage again. It means that oocytes cultured for 20 hr and washed free of this inhibitor of protein synthesis completed GVBD rapidly (4 hr) and protein synthesis crucial for nuclear membrane disintegration occurred already during the first 2 hr after washing of inhibitor. All oocytes cultured for 20 hr in medium with 1 mM p-aminobenzamidine rested in GV with chromatin around the compact nucleolus. The successive culture in cycloheximide (20 hr) and p-aminobenzamidine (10 hr) prevented GVBD in all oocytes, too. In contrast, when the oocytes washed after cycloheximide block (20 hr) were cultured in p-aminobenzamidine enriched medium 2 and 3 hr and again for 6 hr in cycloheximide medium, the nuclear membrane dissolved in 62 and 68% of oocytes, respectively. These data suggest that inhibition of protein synthesis in pig oocytes does not prevent the high condensation of bivalents in GV. However, nuclear membrane breakdown requires the successive protein synthesis and proteolysis.

Okadaic acid accelerates germinal vesicle breakdown and overcomes cycloheximide- and 6-dimethylaminopurine block in cattle and pig oocytes.

Pig and cattle oocytes, when released from the follicle, spontaneously resume first meiotic division within 20 or 8 hr, respectively. In oocytes of both species, the activity of histone H1 kinase increases during maturation, exhibiting a maximum in metaphase I. Treatment of these oocytes with okadaic acid results in acceleration of germinal vesicle breakdown (GVBD) and of histone H1 kinase activation. This effect is more important in pig oocytes, in which the acceleration rises for 6 hr, as compared to 2 hr in cattle. Moreover, under these conditions, H1 kinase activity measured after 12 hr of culture appears higher than that observed in control metaphase I oocytes. When added to prophase oocytes, both cycloheximide and 6-DMAP (6-dimethylaminopurine) block GVBD and histone H1 kinase activation. Okadaic acid, at a concentration of 2.5 microM, is able to release the inhibitory effect exerted by cycloheximide on histone H1 kinase activity; however, GVBD occurred only in two-thirds of pig and one-quarter of cattle oocytes after 20 hr of culture. In addition, okadaic acid fully reverses the effect of 6-DMAP on H1 kinase activity and on GVBD in both species. The opposite effects of 6-DMAP and okadaic acid on MPF activation are discussed, as well as the nature of the protein, which has to be synthesized during the first meiotic division and may be involved in the MPF activation cascade.

RNA and protein synthesis requirements for the resumption of meiosis in rabbit oocytes: the role of cumulus cells.

In vitro maturation of rabbit cumulus-enclosed oocytes was fully inhibited in alpha-amanitin- (100 micrograms/ml) and cycloheximide- (5 micrograms/ml) supplemented media. The inhibition was reversible and substantially reduced by delaying the addition of alpha-amanitin (2h) or cycloheximide (3 h). In contrast, both drugs did not inhibit germinal vesicle breakdown in denuded oocytes. Co-culture of granulosa cells (1 x 10(6)/ml) with denuded oocytes did not substitute for an intact cumulus. The data presented here suggest that the resumption of meiosis in rabbit cumulus-enclosed oocytes is dependent upon early transcriptional and translational events which probably occur within the cumulus cells.

Activation of bovine oocytes by specific inhibition of cyclin-dependent kinases.

Activation of bovine oocytes by experimental procedures that closely mimic normal fertilization and allow to obtain haploid oocytes is essential both for intracytoplasmic sperm injection (ICSI) and for nuclear transfer. Therefore, with the goal of producing haploid activated oocytes, this study evaluated whether bohemine, either alone or in combination with ionomycin, is able to activate young matured bovine oocytes. Furthermore, the effect of bohemine on the patterns of DNA synthesis after pronuclear formation as well as changes in histone H1 kinase and MAP kinase activities during the process of activation were studied. Our results with bohemine show that the specific inhibition of CDKs in metaphase II bovine oocytes induces parthenogenetic activation in a dose-dependent manner (25, 50, and 100 microM, respectively), either alone (3%, 30%, and 50%) or in combination with ionomycin (30%, 70%, and 87.5%). A single pronucleus and extrusion of the second polar body was observed (97%) when Ca(2+) influx was stimulated in the presence of bohemine, although pronuclear formation without polar body extrusion was observed when bohemine was used alone. Bohemine-activated oocytes started to synthesize DNA in the first hour (37%) after their removal from bohemine-supplemented medium (6-7 hr post-activation; hpa). A high synchrony in the S-phase was registered with more than 85% of parthenotes actively synthesizing DNA 8 hpa. By contrast, DNA synthesis was absent in oocytes cultured for 4, 6, and 8 hpa in the presence of bohemine and a low rate was observed by those cultured for 18 hr (30%) in bohemine-supplemented medium. This confirms the ability of the inhibitor to arrest the cell cycle in the G1/S boundary for at least 8 hr. A drop in histone H1 kinase activity was observed in bohemine-activated oocytes. The activity of MBP kinase decreased later than histone H1 kinase and even 4 hr after inomycin-bohemine treatment at least half of this activity was still detectable. Then, the MBP kinase activity decreased and the lowest level could be seen 6-8 hpa. In summary, our study shows that in vitro matured bovine oocytes can be successfully activated by a synthetic inhibitor of CDKs. This effect can be improved by combination with ionomycin. The targeting of CDKs in the way to activate bovine oocytes can be an approach to improve the efficiency of mammalian oocyte activation.

Co-culture with pig membrana granulosa cells modulates the activity of cdc2 and MAP kinase in maturing cattle oocytes.

Bovine cumulus-enclosed oocytes, initially cultured up to diakinesis (8 h of initial culture) or metaphase I (12 h of initial culture), were subsequently co-cultured for 6 h in contact with pig membrana granulosa (PMG) cells and then assayed for histone H1 and MAP kinase activities. In addition, the phosphorylation state of ERK 1,2 proteins was determined by Western blotting. The alterations in nuclear envelope breakdown, meiotic spindle formation and the patterns of chromosome condensation were analysed by immunofluorescence and transmission electron microscopy. The diakinesis-stage oocytes (initially cultured for 8 h) already possessed high histone H1 kinase and MAP kinase activities that were correlated with condensed and partially individualised chromosomes. The ERK 1 and most ERK 2 proteins were partly phosphorylated. Following the 6 h co-culture of these oocytes with PMG a rapid decrease in MAP kinase activity and a slower decrease in histone H1 kinase occurred, as well as ERK 1 and ERK 2 dephosphorylation. Both kinase activities and ERK 1,2 phosphorylation were fully restored following the release of the oocytes from co-culture and a subsequent culture in the absence of PMG. Moreover, the clumped bivalents were reindividualised and 56% of these oocytes reached metaphase II after 20 h of culture without PMG. The metaphase I oocytes, initially cultured for 12 h, displayed a fusiform meiotic spindle and a metaphase array of chromosomal bivalents, accompanied by high levels of both histone H1 and MAP kinase activity. Co-culture of MI oocytes with PMG abolished the activity of both kinases and caused the dephosphorylation of ERK 1 and ERK 2. Furthermore, the spindle microtubules were depolymerised and the chromosomal bivalents clumped into a single mass. Neither of the protein kinase activities nor the meiotic spindle were restored following subsequent culture in the absence of PMG for up to 20 h. These observations indicate that under in vitro conditions membrana granulosa cells can cause a prompt decrease in histone H1 and MAP kinase activities, and metaphase I oocytes. While these events are fully reversible in late diakinesis oocytes, metaphase I oocytes did not complete maturation after release from co-culture.

A distinct cyclin A is expressed in germ cells in the mouse.

In this paper, the existence of two A-type cyclins in the mouse is demonstrated. In the adult mouse, the expression of cyclin A1, which has greatest sequence identity with Xenopus cyclin A1, is restricted to germ cells. In contrast cyclin A2, which has greatest sequence identity with human cyclin A and Xenopus cyclin A2, is expressed in all tissues analysed. In order to explore the function of cyclin A1 in germ cells, its expression during the meiotic cell cycle and its associated kinase subunits have been characterised in the testis. The levels of cyclin A1 mRNA rise dramatically in late pachytene spermatocytes and become undetectable soon after completion of the meiotic divisions; thus its expression is cell cycle regulated. In lysates of germ cells from adult testes, cyclin A1 is present in p13suc1 precipitates, and cyclin A1 immunoprecipitates possess histone H1 kinase activity. Three kinase partners of cyclin A1 were identified: p34cdc2, a polypeptide of 39 x 10(3) M(r) that is related to p33cdk2 and, in lesser quantities, p33cdk2. Cyclin A1 was also detected in oocytes; in metaphase I and metaphase II oocytes, a proportion of the cyclin A1 colocalises with the spindle, possibly suggestive of a functional interaction. These data indicate that mammalian germ cells contain cyclin A1-dependent kinases that either act as a substitute for, or in addition to, the cyclin A2-dependent kinases characterised in somatic tissues.