Regulation of spontaneous and induced resumption of meiosis in mouse oocytes by different intracellular pathways

The mitogen-activated protein kinase-dependent and the cAMP-protein kinase A-dependent signal transduction pathways were studied in cultured mouse oocytes during induced and spontaneous meiotic maturation. The role of the mitogen-activated protein kinase pathway was assessed using PD98059, which specifically inhibits mitogen-activated protein kinase 1 and 2 (that is, MEK1 and MEK2), which activates mitogen-activated protein kinase. The cAMP-dependent protein kinase was studied by treating oocytes with the protein kinase A inhibitor rp-cAMP. Inhibition of the mitogen-activated protein kinase pathway by PD98059 (25 micromol l(-1)) selectively inhibited the stimulatory effect on meiotic maturation by FSH and meiosis-activating sterol (that is, 4,4-dimethyl-5alpha-cholest-8,14, 24-triene-3beta-ol) in the presence of 4 mmol hypoxanthine l(-1), whereas spontaneous maturation in the absence of hypoxanthine was unaffected. This finding indicates that different signal transduction mechanisms are involved in induced and spontaneous maturation. The protein kinase A inhibitor rp-cAMP induced meiotic maturation in the presence of 4 mmol hypoxanthine l(-1), an effect that was additive to the maturation-promoting effect of FSH and meiosis-activating sterol, indicating that induced maturation also uses the cAMP-protein kinase A-dependent signal transduction pathway. In conclusion, induced and spontaneous maturation of mouse oocytes appear to use different signal transduction pathways.     

Effect of inhibition of sterol delta 14-reductase on accumulation of meiosis-activating sterol and meiotic resumption in cumulus-enclosed mouse oocytes in vitro

Two sterols of the cholesterol biosynthetic pathway induce resumption of meiosis in mouse oocytes in vitro. The sterols, termed meiosis-activating sterols (MAS), have been isolated from human follicular fluid (FF-MAS, 4,4-dimethyl-5 alpha-cholest-8,14,24-triene-3 beta-ol) and from bull testicular tissue (T-MAS, 4,4-dimethyl-5 alpha-cholest-8,24-diene-3 beta-ol). FF-MAS is the first intermediate in the cholesterol biosynthesis from lanosterol and is converted to T-MAS by sterol delta 14-reductase. An inhibitor of delta 7-reductase and delta 14 reductase, AY9944-A-7, causes cells with a constitutive cholesterol biosynthesis to accumulate FF-MAS and possibly other intermediates between lanosterol and cholesterol. The aim of the present study was to evaluate whether AY9944-A-7 added to cultures of cumulus-oocyte complexes (COC) from mice resulted in accumulation of MAS and meiotic maturation. AY9944-A-7 stimulated dose dependently (5-25 mumol l-1) COC to resume meiosis when cultured for 22 h in alpha minimal essential medium (alpha-MEM) containing 4 mmol hypoxanthine l-1, a natural inhibitor of meiotic maturation. In contrast, naked oocytes were not induced to resume meiosis by AY9944-A-7. When cumulus cells were separated from their oocytes and co-cultured, AY9944-A-7 did not affect resumption of meiosis, indicating that intact oocyte-cumulus cell connections are important for AY9944-A-7 to exert its effect on meiosis. Cultures of COC with 10 mumol AY9944-A-7 l-1 in the presence of [3H]mevalonic acid, a natural precursor for steroid synthesis, resulted in accumulation of labelled FF-MAS, which had an 11-fold greater amount of radioactivity incorporated per COC compared with the control culture without AY9944-A-7. In contrast, incorporation of radioactivity into the cholesterol fraction was reduced 30-fold in extracts from the same oocytes. The present findings demonstrate for the first time that COC can synthesize cholesterol from mevalonate and accumulate FF-MAS in the presence of AY9944-A-7. Furthermore, AY9944-A-7 stimulated meiotic maturation dose dependently, indicating that FF-MAS, and possibly other sterol intermediates of the cholesterol synthesis pathway, play a central role in stimulating mouse oocytes to resume meiosis. The results also indicate that oocytes may not synthesize steroids from mevalonate.     

Nuclear and cytoplasmic maturation of mouse oocytes after treatment with synthetic meiosis-activating sterol in vitro.

Synthetically produced meiosis-activating sterol, a sterol originally derived from follicular fluid (FF-MAS), induces meiotic maturation of mouse oocytes in vitro. We therefore compared FF-MAS-induced maturation of naked mouse oocytes arrested in prophase I by either hypoxanthine (Hx) or forskolin (Fo) with spontaneous maturation of naked oocytes. FF-MAS-treated oocytes overcame the meiotic block by Hx or Fo, although germinal vesicle breakdown was delayed by 11 h and 7 h, respectively. We also investigated the influence of FF-MAS on chromosome, microtubule, and ultrastructural dynamics in Hx-cultured oocytes by immunocytochemistry and electron microscopy. Similarly to spontaneously matured oocytes, chromosomes became aligned, a barrel-shaped spindle formed, and overall organelle distribution was normal in FF-MAS-matured oocytes. The number of small cytoplasmic asters was elevated in FF-MAS-treated oocytes. Although the number of cortical granules (CGs) was similar to that in spontaneously matured oocytes, the overall distance between CGs and oolemma was increased in the FF-MAS group. These observations suggest that the initiation of meiotic maturation in FF-MAS-treated oocytes in the presence of high cAMP levels leads to a delayed but otherwise normal nuclear maturation. FF-MAS appears to improve oocyte quality by supporting microtubule assembly and by delaying CG release, which is known to contribute to reduced fertilization.     

Cytoplasmic reorganization during the resumption of meiosis in cultured preovulatory rat oocytes

Changes in organelle topography and microtubule configuration have been studied during the resumption and progression of meiosis in cultured preovulatory rat oocytes. Germinal vesicle breakdown (GVBD) was reversibly inhibited by dibutyryl cAMP (DcAMP) or nocodazole, a microtubule-disrupting agent. The microtubule stabilizing agent taxol did not inhibit GVBD, but did impair further maturation. The migration of acidic organelles and chromatin in living oocytes was analyzed using the vital stains acridine orange and Hoechst 33258, respectively. Germinal vesicle stage oocytes undergo perinuclear aggregation of acidic organelles during GVBD and these organelles subsequently disperse into the cell cortex as the first meiotic spindle migrates to the oocyte periphery. DcAMP and nocodazole block the perinuclear aggregation of acidic organelles, whereas, in taxol-treated oocytes, organelle aggregation and GVBD occur but the dispersion of acidic organelles was arrested. Dose-response studies on the effects of nocodazole showed that GVBD was generally retarded and that a 50% inhibition of GVBD was achieved at concentrations in excess of 1.0 microM. Concentrations of taxol at 10 microM or above effectively inhibited both chromatin condensation and meiotic spindle formation. Indirect immunofluorescence microscopy with anti-tubulin antibodies revealed dissolution of microtubules with 1.0 microM nocodazole. Taxol had little effect on microtubule organization in germinal vesicle or chromatin condensation stage oocytes; however, when oocytes that had formed first meiotic spindles were treated with taxol, numerous microtubule asters appeared which were preferentially associated with the oocyte cortex. The changes in organelle topography, microtubule configuration, and drug sensitivity are discussed with respect to the regulation of cytoplasmic reorganization during the meiotic maturation of rat preovulatory oocytes.     

Immunopurified anti-müllerian hormone does not inhibit spontaneous resumption of meiosis in vitro of rat oocytes

The ability of immunopurified, biologically potent anti-Müllerian hormone (AMH) to inhibit the spontaneous resumption of meiosis of rat oocytes was tested in vitro. Two different batches of AMH in the range of 0.75-9.0 micrograms/protein did not suppress the spontaneous resumption of meiosis. Neither did AMH (6 micrograms/ml) induce meiotic resumption of follicle-enclosed oocytes in culture. It is concluded, therefore, that AMH has no oocyte meiosis-inhibiting activity.     

PKB/AKT is involved in resumption of meiosis in mouse oocytes

BACKGROUND INFORMATION: In fully grown mouse oocytes, a decrease in cAMP concentration precedes and is linked to CDK1 (cyclin-dependent kinase 1) activation. The molecular mechanism for this coupling, however, is not defined. PKB (protein kinase B, also called AKT) is implicated in CDK1 activation in lower species. During resumption of meiosis in starfish oocytes, MYT1, a negative regulator of CDK1, is phosphorylated by PKB in an inhibitory manner. It can imply that PKB is also involved in CDK1 activation in mammalian oocytes. RESULTS: We monitored activation of PKB and CDK1 during maturation of mouse oocytes. PKB phosphorylation and activation preceded GVBD (germinal vesicle breakdown) in oocytes maturing either in vitro or in vivo. Activation was transient and PKB activity was markedly reduced when virtually all of the oocytes had undergone GVBD. PKB activation was independent of CDK1 activity, because although butyrolactone I prevented CDK1 activation and GVBD, PKB was nevertheless transiently phosphorylated and activated. LY-294002, an inhibitor of phosphoinositide 3-kinase-PKB signalling, suppressed activation of PKB and CDK1 as well as resumption of meiosis. OA (okadaic acid)-sensitive phosphatases are involved in PKB-activity regulation, because OA induced PKB hyperphosphorylation. During resumption of meiosis, PKB phosphorylated on Ser(473) is associated with nuclear membrane and centrosome, whereas PKB phosphorylated on Thr(308) is localized on centrosome only. CONCLUSIONS: The results of the present paper indicate that PKB is involved in CDK1 activation and resumption of meiosis in mouse oocytes. The presence of phosphorylated PKB on centrosome at the time of GVBD suggests its important role for an initial CDK1 activation.     

Reducing CYP51 inhibits follicle-stimulating hormone induced resumption of mouse oocyte meiosis in vitro

Meiosis activating sterol, produced directly by lanosterol 14-α-demethylase (CYP51) during cholesterol biosynthesis, has been shown to promote the initiation of oocyte meiosis. However, the physiological significance of CYP51 action on oocyte meiosis in response to gonadotrophins’ induction remained to be further explored. Herein, we analyzed the role of CYP51 in gonadotrophin-induced in vitro oocyte maturation via RNA interference (RNAi). We showed that although both luteinizing hormone (LH) and follicle-stimulating hormone (FSH) significantly induced meiotic resumption in follicle-enclosed oocytes (FEOs), the effect of LH on oocyte meiosis resumption in FEOs was weaker than FSH. Moreover, both FSH and LH were able to upregulate CYP51 expression in cultured follicular granulosa cells when examined at 8 h or 12 h posttreatments, respectively. Interestingly, whereas knockdown of CYP51 expression via small interference RNA (siRNA) moderately blocked (23% reduction at 24 h) FSH-induced oocyte maturation [43% germinal vesicle breakdown (GVBD) rate in RNAi vs. 66% in control, P < 0.05] in FEOs, similar treatments showed no apparent effects on LH-induced FEO meiotic maturation (58% GVBD rate in RNAi vs. 63% in control, P > 0.05). Moreover, the results in a cumulus-enclosed oocytes (CEOs) model showed that approximately 30% of FSH-induced CEOs’ meiotic resumption was blocked upon CYP51 knockdown by siRNAs. These findings suggest that FSH, partially at least, employs CYP51, and therefore the MAS pathway, to initiate oocyte meiosis.     

Granulosa cells inhibit the resumption of meiosis in bovine oocytes in vitro

The oocytes of cattle are not as sensitive as those of laboratory animals to purines, cAMP, or follicular extracts. To study the resumption of meiosis, a method is needed that is capable of inhibiting meiosis completely for a minimum of 24 h. This study was designed to evaluate interrelationships in granulosa-oocyte-cumulus complexes using fresh granulosa cells aspirated from small follicles (1-5 mm) in which the cumulus is normally firmly attached. Selected oocyte-cumulus complexes obtained from a slaughterhouse (n = 2,236) were co-incubated with one of the following: various concentrations of fresh granulosa cells in tissue cultures medium (TCM) 199 or bovine follicular fluid (BFF) either without or after one washing and/or freezing; resuspended granulosa cells previously cultured for 7 days; blood cells; or medium alone. Additionally, oocyte-cumulus complexes were embedded in agar cylinders before incubation with or without cells. The rate of maintenance of intact germinal vesicles (GV) in oocytes after 24 h ranged from 40-77% when 5-100 x 10(6) unwashed cells/ml BFF were used, compared to only 16% in oocytes cultured in BFF alone. The pattern was the same when washed cells were used (30-77%, using 5-100 x 10(6) cells/ml BFF), but they were not as effective as unwashed cells. With TCM-199 and the same five concentrations of cells (5, 10, 25, 50, and 100 x 10(6)/ml), a similar inhibition was obtained with greater than or equal to 25 but not with 5 (3%) or 10 (5%) x 10(6)/ml.(ABSTRACT TRUNCATED AT 250 WORDS)     

Temporary inhibition of meiosis resumption in vitro by adenylate cyclase stimulation in immature bovine oocytes

This experiment was designed to analyze the effect of adenylate cyclase stimulation on cumulus-enclosed immature oocytes. More than 1400 selected (complete and unexpanded cumulus) oocytes from follicles 1 to 5 mm in diameter were recovered from ovaries obtained at slaughter and cultured for 24 h in TCM-199+10% fetal calf serum (FCS), with or without the adenylate cyclase stimulator, and in the presence or absence of bovine follicular fluid (BFF, 50%), or in complete BFF. In a second set of experiments, oocytes treated for 24 h were further cultured for a second 24 h with TCM-FCS alone. Oocytes were classified as germinal vesicle (G); intermediate (I; up to Metaphase I); matured (M; Anaphase I to Metaphase II); or degenerated (D), and cumulus expansion was evaluated. Products used were sodium fluoride (NaF), isobutylmethylxanthine (IBMX), adenosine (ADE) and forskolin (FK), all known to stimulate accumulation of cAMP in cells without the involvement of a hormone receptor except for adenosine, which acts as a substrate or as an agonist. The results indicate that NaF (0.01 M), IBMX (0.2 mM), FK (0.1 mM) and complete BFF can significantly reduce the proportion of oocytes reaching the mature state. Combination of NaF or FK to BFF (50%) are also effective at the significant level. Cumulus expansion was always limited when meiotic progress was affected or when adenosine was present in the culture media. When oocytes were cultured for a second 24 h in the control media, only NaF had a significant residual effect, but many oocytes were showing degenerative changes after the second incubation period. This method provides a new means to block oocyte nuclear maturation.     

RINGO efficiently triggers meiosis resumption in mouse oocytes and induces cell cycle arrest in embryos.

RINGO was identified as a Cdc2-binding and activating protein which is necessary and sufficient to trigger G2/M progression in Xenopus oocytes. We have investigated whether the function of RINGO is conserved in mouse oocytes. We show that RINGO induces Germinal Vesicle BreakDown (GBVD) in mouse oocytes. Mos is known to induce GVBD in mouse oocytes, and is also involved in the metaphase II arrest, which is due to the CSF (CytoStatic Factor) activity. We found that RINGO also has CSF activity and induces cleavage arrest after injection into one blastomere of a late two-cell mouse embryo, like Mos. However, RINGO also inhibits polar body extrusion of wild type mouse oocytes. The same effect of RINGO on first and second polar body extrusion was observed in Mos -/- mouse oocytes. The injection of RINGO mimics Mos effects: GVBD induction and efficient cleavage arrest. However, our results in mouse oocytes suggest that RINGO may have additional functions in meiosis regulation.     

Involvement of MEK-mitogen-activated protein kinase pathway in follicle-stimulating hormone-induced but not spontaneous meiotic resumption of mouse oocytes

Mitogen-activated protein (MAP) kinase has been reported to be activated during oocyte meiotic maturation in a variety of mammalian species. However, the mechanism(s) responsible for MAP kinase activation and the consequence of its premature activation during gonadotropin-induced oocyte meiotic resumption have not been examined. The present experiments were conducted to investigate the possible role of MAP kinase in FSH-induced and spontaneous oocyte meiotic resumption in the mouse. MAP kinase kinase (MAPKK, MEK) inhibitor, PD98059 or U0126, produced a dose-dependent inhibitory effect on both FSH-induced oocyte meiotic resumption and MAP kinase activation in the oocytes. However, the same inhibitor did not block spontaneous meiotic resumption of either denuded or cumulus cell-enclosed mouse oocytes, despite the activity of MAP kinase being totally inhibited. Immunoblotting the oocytes and the cumulus cells with the anti-active MAP kinase antibody showed that MAP kinase activity in the oocytes was detected at 8 h of FSH treatment, prior to germinal vesicle breakdown and increased as maturation progressed in the following culture period. In the cumulus cells, MAP kinase was activated even faster, its activity was detected at 1 h of FSH stimulation and increased gradually until 8 h of FSH treatment, then decreased and diminished after 12 h of FSH action. These data demonstrated that the MEK-MAP kinase pathway is implicated in FSH-induced but not spontaneous oocyte meiotic resumption.     

Effect of cumulus and granulosa cells on meiosis resumption in murine oocytes in vitro

Effects of different follicular cell types on resumption of meiosis were studied. Cumulus enclosed oocytes (CEO), denuded oocytes (DO), cumulus cells (CCs) and mural granulosa cells (GCs) were used. Oocytes were obtained from mature gonadotrophin-stimulated and unstimulated mice. The resumption of meiosis was assessed by the germinal vesicle breakdown (GVBD) at the end of cultivation. It has been shown that GCs produced a meiosis activating substance due to gonadotrophin stimulation; for meiosis resumption connections between CCs and the oocyte were not necessary, but the very production of the meiosis activating substance, was, however, dependent on the initial connection between CCs and the oocyte. The presence of oocyte was necessary for stimulating CCs to produce a diffusible heat stable meiosis activating substance; gonadotrophins induced CCs to produce a diffusible thermostable meiosis activating substance. This substance induced, in a paracrine fashion, resumption of meiosis directly. It is proposed that the heat stable meiosis activating component of the used media from gonadotrophins-stimulated CEO may belong to a kind of meiosis activating sterols, previously isolated from human follicular fluid and from adult bull testes.     

Involvement of mitogen-activated protein kinase (MAPK) pathway in LH- and meiosis-activating sterol (MAS)-induced maturation in rat and mouse oocytes

Gonadotropic stimulation of meiotic resumption in mice is dependent upon mitogen-activated protein kinase (MAPK) activation in the somatic compartment of the follicle. By contrast, spontaneous resumption of meiosis is independent of MAPK activation. In view of the suggested role of meiosis-activating sterol (MAS) in oocyte maturation we have (i) compared MAPK activation in rat preovulatory follicles stimulated by LH or by accumulation of endogenous MAS by using an inhibitor of MAS conversion, AY9944; (ii) examined whether stimulation of meiosis by MAS is dependent upon MAPK activation using denuded oocytes (DO) of Mos- null mice (hereafter Mos(-/-)) with oocytes unable to activate MAPK. Rat preovulatory follicles responded to LH or AY9944 stimulation by MAPK activation. Inhibition of MAPK phosphorylation blocked both LH- and AY9944 triggered resumption of meiosis. In mouse cumulus-enclosed oocytes (CEOs) and DOs AY9944 stimulated GVB in wild-type and Mos(-/-) mouse CEOs cultured with hypoxanthine (Hx). Addition of MAS or AY9944 to mouse DOs cultured with Hx induced resumption of meiosis only in wild-type and Mos(+/-) oocytes, but they were ineffective in Mos(-/-) oocytes. The observed sluggish activation of MAPK induced by AY9944 in rat follicle-enclosed oocytes (FEO) may cause the delay in meiotic resumption in response to MAS and AY9944 stimulation. Further, it is incompatible with the suggested role of MAS as an obligatory mediator of LH in the induction of meiotic maturation. MAPK/MOS activation, whether in the somatic compartment or in denuded oocytes, is required for MAS- like LH-, FSH-, or EGF-induced resumption of meiosis.     

Role of divalent cations in the resumption of meiosis of rat oocytes.

Resumption of meiotic maturation was induced in follicle-enclosed rat-oocytes by treatment with the divalent cationophore A23187 (10(-5)M). However, the same effect was attained by incubation in Ca++-deficient medium, even in the presence of EDTA or EGTA (1mM). The stability of the first polar body was increased under Ca++-deficient conditions. Neither the ionophore nor Ca++-deficient medium interfered with the spontaneous maturation of isolated oocytes of the rat. The experiments with cultured follicles suggest that redistribution of divalent cations may participate in the physiological control of meiosis in mammalian oocytes.     

Lead cations affect the control of both meiosis arrest and meiosis resumption of the mouse oocyte in vitro at least via the PKC pathway

The aim of this study was to determine in vitro whether lead has a direct cytotoxic effect on the female gamete or through its surrounding somatic cells. We had previously demonstrated that it partly accumulates in the mouse ovary and induces follicle and oocyte apoptosis. The data reported here demonstrate for the first time that low levels of Pb(NO3)2 (相似文献   

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