Centrosome phosphorylation and the developmental expression of meiotic competence in mouse oocytes.

Previous studies suggested that the transition from an incompetent to a competent meiotic state during the course of oogenesis in the mouse involved a G2/M-like cell cycle transition (Wickramasinghe et al, 1991. Dev. Biol. 143, 162). The present studies tested the hypothesis that centrosome phosphorylation, an event normally induced by MPF, is required for this developmental transition and the expression of meiotic competence in cultured growing mouse oocytes. Multiple fluorescence labeling techniques were used to evaluate centrosome number, phosphorylation status, and microtubule nucleating capacity in competent and incompetent oocytes. Experimental conditions were established for reversibly altering the phosphorylation status of the centrosomes and the effects of these treatments on meiotic resumption were examined. Phosphorylated centrosomes nucleating short microtubules were observed in competent oocytes, whereas nonphosphorylated centrosomes and interphase microtubule arrays were found in incompetent oocytes. Upon recovery from nocodazole-induced microtubule depolymerization, short microtubules formed from centrosomes in competent oocytes, whereas long microtubules reappear in the cytoplasm of incompetent oocytes. Perturbation of the phosphorylation state of oocytes with activators of protein kinase A or protein kinase C resulted in the formation of long interphase microtubules in competent oocytes while centrosome phosphorylation was maintained. Treatment of competent oocytes with the phosphorylation inhibitor 6-dimethylaminopurine also led to formation of long microtubules, although under these conditions centrosomes were dephosphorylated. When competent oocytes were treated simultaneously with puromycin and the phosphodiesterase inhibitor isobutyl methylxanthine (IBMX) for 6 hr, centrosomes became dephosphorylated; centrosomes were rephosphorylated when competent oocytes were further cultured in IBMX without puromycin. Conditions that induced centrosome dephosphorylation in competent oocytes resulted in the loss of the ability to express meiotic competence in culture, whereas maintenance of centrosome phosphorylation in these oocytes was correlated with the ability to resume meiosis. These results suggest that the G2/M transition that occurs when mouse oocytes progress from an incompetent to a competent state in vivo involves the phosphorylation of centrosomes and that the maintenance of centrosome phosphorylation is required for the in vitro expression of meiotic competence.     

Cyclin B1 turnover and the mechanism causing insensitivity of fully grown mouse oocytes to cycloheximide inhibition of meiotic resumption

Cyclin B1 turnover and the insensitivity of fully-grown mouse oocytes to cycloheximide (CHX) inhibition of germinal vesicle breakdown (GVBD) were examined by assaying GVBD and cyclin B1 levels after treatment of oocytes with various combinations of eCG and CHX. Whereas over 95% of oocytes underwent GVBD after culture for 24 h with CHX alone, only 10% did so after culture with CHX + eCG (P < 0.05). In addition, preculture with eCG alone had no effect, but preculture with eCG + CHX prevented GVBD during a second culture with CHX alone. Therefore, we inferred that eCG delayed GVBD long enough for CHX inhibition of protein synthesis to allow cyclin B1 to decrease below a threshold where GVBD became dependent upon its de novo synthesis. However, western blot revealed no cyclin B1 synthesis, but cyclin B1 degradation, as long as GVs were maintained intact with eCG. Regarding the function of CHX in preculture without protein synthesis to block subsequent GVBD, whereas eCG delayed GVBD for only 3 h, CHX had an ongoing effect that further postponed GVBD, thus allowing cyclin B1 to decrease below the threshold. When oocytes precultured with eCG + CHX were further cultured without eCG and CHX, cyclin B1 first decreased but then, because of the ongoing effects of CHX, increased to a level sufficient to induce GVBD. The content of P34Cdc2 was not altered under any of the culture conditions (P > 0.05). We concluded that insensitivity of mouse germinal vesicle (GV) oocytes to CHX was due to the presence of sufficient cyclin B1, and that cyclin B1 level in such oocytes was maintained by an equilibrium between synthesis and degradation.     

Inhibition of nuclear maturation in fully grown porcine and mouse oocytes after their fusion with growing porcine oocytes

Porcine ovarian oocytes, isolated from follicles of 5 mm in diameter (large oocytes), were fused either together or with oocytes isolated from follicles of 0.5 mm in diameter (small oocytes). In giant cells composed of two large oocytes (control) germinal vesicle breakdown (GVBD) occurred and two metaphase I chromosome sets (M I) were observed 24 to 30 h after fusion. By contrast, in giant cells composed of one large and one small porcine oocyte, both germinal vesicles (GVs) remained well conserved after 24-30 h of culture. An identical situation was observed after fusion and cultivation of small porcine and large mouse oocytes isolated from preovulatory follicles. The results demonstrate the presence of inhibiting activity in the ooplasm of small porcine oocytes that prevents nuclear maturation of large porcine and mouse oocytes fused to them. This maturation inhibiting activity can be overcome by preincubating large porcine oocytes for more than 14 h before fusion with small oocytes. During preincubation the ooplasm produces sufficient amount of maturation promoting factor (MPF) to overcome the inhibiting activity present in small porcine oocytes thus inducing GVBD and chromatin condensation both in small and large oocytes.     

Comparison of embryonic developmental competence of mouse oocytes grown with and without serum.

The first objective of this study was to determine whether oocyte growth in serum-free medium affects the solubility of the zona pellucida to alpha-chymotrypsin digestion, which is an index of zona pellucida "hardening" and reflects the potential penetrability of the zona pellucida by sperm. Oocyte-granulosa cell complexes were isolated from the preantral follicles of 12-day-old mice and cultured for 10 days in medium containing 5% fetal bovine serum (FBS) or in serum-free medium. The zonae pellucidae of oocytes grown in serum-free medium were four times as hard as freshly isolated germinal vesicle (GV)-stage oocytes grown in vivo or oocytes grown in vitro in FBS-containing medium. The hardening of the zonae pellucidae of oocytes grown in serum-free medium was prevented by addition of fetuin. The second objective was to compare the competence to undergo embryogenesis of oocytes that grew in serum-free vs. FBS-containing medium. Approximately 70% of the oocytes underwent maturation regardless of whether the medium was serum-free or contained FBS. Of the mature ova grown in medium containing FBS, 53% cleaved to the two-cell stage after insemination compared with only 6% of the ova grown in serum-free medium. Addition of fetuin to the serum-free medium used for oocyte growth increased the frequency of cleavage to the two-cell stage. Of the embryos derived from oocytes that grew in FBS-containing medium, 70% completed the two-cell stage to blastocyst transition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nitric oxide and meiotic competence of porcine oocytes

Reproductive biotechnology such as in vitro fertilization, the creation of transgenic animals or cloning by nuclear transfer depends on the use of fully grown, meiotically competent oocytes capable of completing meiotic maturation by reaching the stage of metaphase II. However, there exists only a limited quantity of these oocytes in the ovaries of females. In view of their limited number, growing oocytes without meiotic competence represent a possible source. The mechanisms controlling the acquisition of meiotic competence, however, are still not completely clear. A gas with a short half-life, nitric oxide (NO), produced by NO-synthase (NOS) enzyme can fulfill a regulatory role in this period. The objective of this study was to ascertain the role of NO in the growth phase of pig oocytes and its influence on the acquisition of meiotic competence with the help of NOS inhibitors, NO donors and their combinations. We demonstrated that the selective competitive iNOS inhibitor aminoguanidine and also the non-selective NOS inhibitor l-NAME block meiotic maturation of oocytes with partial or even full meiotic competence at the very beginning. NOS inhibitors influence even competent oocytes in the first stage of meiotic metaphase. However, blockage is less effective than at the beginning of meiotic maturation. The number of parthenogenetically activated competent oocytes greatly increased in a pure medium after inhibitor reversion. A large quantity of NO externally added to the in vitro cultivation environment disrupts the viability of oocytes. The effectiveness of the inhibitor can be reversed in oocytes by an NO donor in a very low concentration. However, the donor is not capable of pushing the oocytes farther than beyond the first stage of meiotic metaphase. The experiments confirmed the connection of NO with the growth period and the acquisition of meiotic competence. However, it is evident from the experiments that NO is not the only stimulus controlling the growth period.     

MPF components and meiotic competence in growing pig oocytes

Growing pig oocytes (≤90 μm in diameter) are unable to resume meiosis in vitro. The objective of our present experiments has been to identify the reasons for meiotic competence in these cells. By comparing histone H1 kinase activity in growing and fully grown oocytes we demonstrate that incompetence is associated with an inability to activate H1 kinase in growing oocytes. Immunoblotting was used to determine whether this kinase inactivity resulted from a lack of either p34^cdc2 protein or B-type cyclin. The results established that each of these cell cycle molecules are present in comparable amounts in both growing and fully grown oocytes. In the third series of studies experiments were carried out in an attempt to induce p34^cdc2 activation during growth. Treatment with okadaic acid, an inhibitor of phosphatase 1 and 2A known to stimulate and accelerate the transition into M-phase of the meiotic cycle in a number of different species, was able to induce p34^cdc2 kinase activity and facilitated the G₂- to M-phase in growing oocytes. We conclude that although growing oocytes in pigs have sufficient key cell cycle components for the G₂ to M transition, they remain incapable of converting these components to active maturation-promoting factor (MPF) until growth is virtually completed. Inhibition of phosphatase 1 or 2A induces the formation of active MPF during growth by an as yet unidentified pathway. © 1994 Wiley-Liss, Inc.     

Meiotic competence of in vitro grown goat oocytes

The objective of the present study was to grow meiotically incompetent goat oocytes from early antral follicles in vitro and to render them competent to undergo germinal vesicle breakdown. Cumulus-oocyte complexes with pieces of parietal granulosa cells were isolated from follicles 0.35-0.45 mm in diameter using both mechanical and enzymatic methods. The cumulus-oocyte complexes were divided into two groups according to oocyte diameter (group A: < 95 microm; group B: > 95 microm) and cultured for 8 or 9 days on granulosa cell monolayers. Within 8 days of culture, the mean oocyte diameter increased from 86 +/- 0.4 microm to 95 +/- 0.7 microm in group Aand from 106 +/- 0.2 microm to 109 +/- 0.5 microm in group B. After 9 days of culture, the mean diameter of oocytes from groups A and B were 99 +/- 0.5 microm and 112 +/- 0.4 microm, respectively. The meiotic competence of oocytes grown in vitro was evaluated by in vitro maturation. Within 8 days of culture, only 3% of oocytes from group A and 6% of oocytes from group B acquired the ability to undergo germinal vesicle breakdown. After 9 days of culture, 7% of group A oocytes and 42% of group B oocytes were competent to resume meiosis. The expression of p34(cdc2) in oocytes grown in vitro was analysed by the western blot technique. During 9 days of culture, p34(cdc2) accumulated in both groups of growing oocytes, but its concentration was lower than in fully grown oocytes used as controls. The results showed for the first time that goat oocytes from early antral follicles can grow, accumulate p34(cdc2) and acquire the ability to resume meiosis, when cultured for 9 days on granulosa cell monolayers.     

Meiotic competence and acetylation pattern of UV light classified mouse antral oocytes after meiotic arrest with isobutylmethylxanthine

Chromatin transformation from a diffused or NSN configuration to a compacted or SN shape that forms a ring around the nucleolus is regarded as one of the modifications necessary for successful embryonic development. But the process of the transformation is poorly understood. In this study we cultured mouse antral oocytes under meiotic arrest with IBMX for periods between 3 and 24 hr. We observed the chromatin status of the oocytes before and after culture under UV illumination. We reported here that the NSN configured oocytes transformed temporally through an intermediate form into the SN configuration while under meiotic arrest in vitro. Meiotic rate was improved in the NSN oocytes after the meiotic arrest but decreased in the SN oocytes. We also reported that chromatin of both the NSN and SN oocytes was acetylated and the two groups underwent the same pattern of H4/K5 deacetylation during meiotic maturation. We hypothesized that the transformation of mouse oocyte from the NSN to SN type may be time rather than oocyte size specific and the abrupt deacetylation of NSN oocyte during spontaneous maturation may explain its poor meiotic and developmental competence.     

APCcdh1 activity in mouse oocytes prevents entry into the first meiotic division

Fully grown mammalian oocytes maintain a prophase I germinal-vesicle stage arrest in the ovary for extended periods before a luteinizing hormone surge induces entry into the first meiotic division. Cdh1 is an activator of the anaphase-promoting complex (APC) and APCcdh1 is normally restricted to late M to early G1 phases of the cell cycle. Here, we find that APCcdh1 is active in mouse oocytes and is necessary to maintain prophase arrest.     

Involvement of thiol-disulfide groups in the sensitivity of fully grown mouse oocytes to calcium-free medium

The lethality caused by calcium-free medium (CFM) to fully grown mouse oocytes significantly decreases if a disulfide reducing agent (dithiothreitol, reduced glutathione, or L-cysteine) is added to the medium. In this condition, most of the surviving oocytes do not spontaneously resume meiosis. We also show that the sulfhydryl content of fully grown oocytes, estimated by monobromobimane labeling, rapidly decreases during culture in CFM. The hypothesis is discussed that lethality of oocytes cultured in CFM may be a consequence of an alteration of thiol-disulfide balance.     

Double-strand breaks induce short-scale DNA replication and damage amplification in the fully grown mouse oocytes

Break-induced replication (BIR) is essential for the repair of DNA double-strand breaks (DSBs) with single ends. DSBs-induced microhomology-mediated BIR (mmBIR) and template-switching can increase the risk of complex genome rearrangement. In addition, DSBs can also induce the multi-invasion-mediated DSB amplification. The mmBIR-induced genomic rearrangement has been identified in cancer cells and patients with rare diseases. However, when and how mmBIR is initiated have not been fully and deeply studied. Furthermore, it is not well understood about the conditions for initiation of multi-invasion-mediated DSB amplification. In the G2 phase oocyte of mouse, we identified a type of short-scale BIR (ssBIR) using the DNA replication indicator 5-ethynyl-2’-deoxyuridine (EdU). These ssBIRs could only be induced in the fully grown oocytes but not the growing oocytes. If the DSB oocytes were treated with Rad51 or Chek1/2 inhibitors, both EdU signals and DSB marker γH2A.X foci would decrease. In addition, the DNA polymerase inhibitor Aphidicolin could inhibit the ssBIR and another inhibitor ddATP could reduce the number of γH2A.X foci in the DSB oocytes. In conclusion, our results showed that DNA DSBs in the fully grown oocytes can initiate ssBIR and be amplified by Rad51 or DNA replication.     

Meiotic competence acquisition is associated with the appearance of M-phase characteristics in growing mouse oocytes

To determine whether the acquisition of meiotic competence during the growth phase of oogenesis is associated with the appearance of M-phase characteristics, oocytes obtained from 13- to 30-day-old mice were evaluated by fluorescence microscopy with respect to chromatin and microtubule organization , in vitro maturation ability, and the distribution of M-phase phosphoproteins. Meiotically incompetent oocytes were distinguished from their competent counterparts in displaying elaborate interphase-like arrays of cytoplasmic microtubules and dispersed germinal vesicle chromatin. Meiotically competent oocytes were larger in size, exhibited condensation of chromatin around the nucleolus, and displayed a progressive diminution of cytoplasmic microtubules in conjunction with the appearance of multiple microtubule organizing centers. After 24 hr in culture, medium- to large-sized oocytes exhibiting perinucleolar chromatin condensation resume meiosis whereas smaller meiotically incompetent oocytes retain GVs with diffuse chromatin. Moreover, indirect immunofluorescence studies using the M-phase phosphoprotein specific monoclonal antibody MPM-2 indicate that the appearance of reactive cytoplasmic foci is directly correlated with nuclear changes characteristic of meiotically competent oocytes. Thus, the earliest transition to a meiotically competent state during oocyte growth in the immature mouse ovary is characterized by stage-specific and coordinated modifications of nuclear and cytoplasmic components.     

Bovine immature oocytes acquire developmental competence during meiotic arrest in vitro

To test the hypothesis that oocytes require time to acquire developmental competence during meiotic arrest, we investigated the effects of butyrolactone I (BL I), a potent and specific inhibitor of cyclin-dependent kinase, on the developmental competence of bovine oocytes after in vitro fertilization (IVF) following release from meiotic arrest. In the present study, 4 culture conditions were used: addition of BSA or fetal bovine serum (FBS) under 2 oxygen tensions (5% vs. 20%) during meiotic arrest with 100-microM BL I. The developmental competence to the blastocyst stage was higher (P < 0.01) in oocytes that were arrested in FBS-supplemented medium under 5% O2 (37%) than in oocytes that were arrested under other conditions (5%-24%) or that matured directly following follicle aspiration (23%). The time course of nuclear maturation of BL I-treated oocytes was also examined. The results demonstrated that oocytes treated with BL I start germinal vesicle (GV) breakdown and reach the metaphase II stage 5.5-6.0 h earlier than nonarrested oocytes. The developmental rates to the blastocyst stage of BL I-treated oocytes matured for 15.5 and 21 h were higher (P < 0.05) than those of nontreated oocytes matured for 21 and 26.5 h, respectively. These results demonstrate that bovine immature oocytes, which were arrested at the GV stage with BL I in FBS-supplemented medium under low oxygen tension, acquire higher developmental competence during meiotic arrest.     

Fatty acid oxidation and meiotic resumption in mouse oocytes

We have examined the potential role of fatty acid oxidation (FAO) in AMP‐activated protein kinase (AMPK)‐induced meiotic maturation. Etomoxir and malonyl CoA, two inhibitors of carnitine palmitoyl transferase‐1 (CPT1), and thus FAO, blocked meiotic induction in dbcAMP‐arrested cumulus cell‐enclosed oocytes (CEO) and denuded oocytes (DO) by the AMPK activator, AICAR. C75, an activator of CPT1 and FAO, stimulated meiotic resumption in CEO and DO. This effect was insensitive to the AMPK inhibitor, compound C, indicating an action downstream of AMPK. Palmitic acid or carnitine also promoted meiotic resumption in DO in the presence of AICAR. Since C75 also suppresses the activity of fatty acid synthase (FAS), we tested another FAS inhibitor, cerulenin. Cerulenin stimulated maturation in arrested oocytes, but to a lesser extent, exhibited significantly slower kinetics and was effective in CEO but not DO. Moreover, etomoxir completely blocked C75‐induced maturation but was ineffective in cerulenin‐treated oocytes, suggesting that the meiosis‐inducing action of C75 is through activation of FAO within the oocyte, while that of cerulenin is independent of FAO and acts within the cumulus cells. Finally, we determined that long chain, but not short chain, fatty acyl carnitine derivatives were stimulatory to oocyte maturation. Palmitoyl carnitine stimulated maturation in both CEO and DO, with rapid kinetics in DO; this effect was blocked by mercaptoacetate, a downstream inhibitor of FAO. These results indicate that activation of AMPK stimulates meiotic resumption in mouse oocytes by eliminating a block to FAO. Mol. Reprod. Dev. 76: 844–853, 2009. © 2009 Wiley‐Liss, Inc.