Serotonin inhibition of steroid-induced meiotic maturation in the teleost Fundulus heteroclitus: Role of cyclic AMP and protein kinases

The transduction of the serotonin (5-HT) signal in Fundulus heteroclitusovarian follicles leading to the inhibition of oocyte meiosis reinitiation (oocyte maturation) in vitro induced by the naturally occurring maturation-inducing steroid 17α,20β-dihydroxy-4-pregnen-3-one (17,20βP) was investigated. Steroid-induced oocyte maturation was inhibited by 5-HT in a dose-dependent manner; maximum inhibition (90%) was observed with 10⁻⁴ M 5-HT. Groups of follicle-enclosed oocytes were cultured in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) and treated with increasing doses of 5-HT. Serotonin was found to slightly increase the levels of follicular 3′,5′-cyclic adenosine monophosphate (cAMP) in a dose-dependent manner; 10⁻⁴ M 5-HT induced approximately a 3-fold increase in cAMP with respect to the controls. The changes in cAMP were then evaluated in follicles treated with 17,20βP in IBMX-free culture media in the presence or absence of 10⁻⁴ M 5-HT. The exposure of follicles to 17,20βP alone produced a small and transient reduction in cAMP (40%) within 1–3 hr of steroid stimulation, and these early changes in cAMP appeared associated with a high incidence of germinal vesicle breakdown (80% GVBD) by 24 hr of incubation. Under these conditions, treatment of follicles with 5-HT also increased significantly the production of cAMP, and when 5-HT was combined with 17,20βP, the steroid-mediated reduction in cAMP was prevented and the levels of GVBD inhibited by 95%. Meiosis also was reinitiated with either the protein kinase A (PKA) inhibitor H8 or the protein kinase C (PKC) activator PMA, and the 5-HT inhibitory action on GVBD was found to be 100-fold reduced or completely ineffective, respectively. Preincubation of follicles with the PKC inhibitor GF109203x abolished PMA-induced GVBD in a dose-dependent manner, whereas this inhibitor had no effect on 17,20βP-triggered meiotic maturation, indicating that activation of PKC is apparently sufficient but not necessary to reinitiate meiosis. Taken together, these findings suggest that 5-HT may inhibit 17,20βP-induced meiotic reinitiation through the activation of a cAMP-PKA transduction pathway and that PKC possibly induces oocyte maturation by a different pathway than the steroid and thus is not affected by 5-HT. Mol. Reprod. Dev. 49:333–341, 1998. © 1998 Wiley-Liss, Inc.     

The effect of four C21-steroids on oocyte maturation in Siberian sturgeon (<Emphasis Type="Italic">Acipenser baeri</Emphasis> Brandt)

The effect of four C21-steroids, progesterone (P₄), 17,20β-dihydroxy-4-pregnen-3-one (17,20βP), 17,20β,21-trihydroxy-4-pregnen-3-one (20βS) and 11-desoxycortisol (S), on in vitro oocyte maturation in Siberian sturgeon (Acipenser baeri Brandt) females was demonstrated using short-term (5 and 30 min) exposures of ovarian follicles to steroid solutions followed by incubation in steroid-free medium. The study aimed to find out which of the four candidates for a maturationinducing steroid (P₄, 17,20βP, 20βS or S) induces a fastest germinal vesicle breakdown (GVBD) in oocytes of Siberian sturgeon. Dissolution of the oocyte nucleus or GVBD was taken as a criterion of oocyte maturation. Dose-response profiles of hormone activities as well as effects of the hormones under short-term exposures of follicles to their equal doses were compared. P₄ was found to be a most active GVBD inducer compared to other C21-steroids, S was the second in its activity, whereas 17,20βP and 20βS were less efficient. A comparison of the present and previously obtained data on the dynamics of C21-steroids in vivo and their effect on ovarian follicles in vitro indicates an important role of the above hormones, particularly P₄ and 20βS, in the regulation of the final stage of oocyte maturation in sturgeons.     

Pharmacological Characterisation of [35S]-GTPγS Binding to Chinese Hamster Ovary Cell Membranes Stably Expressing Cloned Human 5-HT1D Receptor Subtypes

Abstract

[³⁵S]-GTPγS binding has been used to study the function of cloned human 5-HT_1D receptor subtypes stably expressed in chinese hamster ovary (CHO) cells. 5-HT stimulated [³⁵S]-GTPγS binding to membranes from cells expressing 5-HT_1Dα or 5-HT_1Dβ receptors. In membranes containing 5-HT_1Dβ receptors, 5-CT and sumatriptan stimulated binding to a similar extent as 5-HT while yohimbine, metergoline and 8-OHDPAT were partial agonists. The order of potency for agonists was 5-CT > 5-HT > metergoline > sumatriptan > yohimbine > 8-OHDPAT. The stimulation of binding by 5-HT in membranes containing 5-HT_1Dβ receptors was potently antagonised by methiothepin (pA₂ 8.9 ± 0.1). The overall pharmacological profile for the human 5-HT_1Dβ receptor, defined using [³⁵S]-GTPγS binding, agreed well with that reported for inhibition of forskolin-stimulated adenylyl cyclase. In addition, methiothepin and ketanserin inhibited basal [³⁵S]-GTPγS binding to membranes containing 5-HT_1Dα or 5-HT_1Dβ receptors, suggesting that these compounds show negative efficacy at 5-HT_1D receptor subtypes. The data show that [³⁵S]-GTPγS binding is a suitable method for studying the interaction between cloned human 5-HT_1D receptors and G-proteins.     

Regulation of mouse oocyte meiotic maturation: implication of a decrease in oocyte cAMP and protein dephosphorylation in commitment to resume meiosis

Mouse oocytes are reversibly inhibited from resuming meiotic maturation in vitro by cAMP phosphodiesterase inhibitors such as 3-isobutyl-1-methyl xanthine (IBMX) and cAMP analogs such as dibutyryl cAMP (dbcAMP). Oocytes cultured in IBMX-containing medium were transferred to and cultured in IBMX-free medium for various periods of time prior to their return to either IBMX- or dbcAMP-containing medium. Results from these experiments defined a period of time in which oocytes became committed to resuming meiosis. Forskolin, which elevated the intracellular oocyte cAMP concentration, transiently inhibited oocytes from resuming meiosis. Levels of cAMP were determined in oocytes incubated in medium that allows resumption of meiosis. The level of oocyte cAMP decreased significantly during the time in which oocytes become committed to resuming meiosis. This decrease in oocyte cAMP was not observed in oocytes inhibited from resuming meiosis by IBMX. In addition, cAMP levels were determined in preovulatory antral follicles, cumulus cell-oocyte complexes, and oocytes during gonadotropin-induced resumption of meiosis in vivo. A decrease in oocyte cAMP preceded resumption of meiosis as manifested by germinal vesicle breakdown (GVBD). This decrease apparently occurred before or during a period of time in which follicle and cumulus cell cAMP were increasing. Associated with commitment to resume meiosis was a characteristic set of changes in oocyte phosphoprotein metabolism that preceded GVBD. These changes are, to date, some of the first reported biochemical changes that precede GVBD. Results from these experiments are discussed in terms of a possible role cAMP may play in regulation of resumption of meiosis in mammals.     

Cumulus cells secrete a meiosi-inducing substance by stimulation with forskolin and dibutyric cyclic adenosine monophosphate

The role of the cumulus cells in initiating the resumption of meiosis after exposure to forskolin and dbcAMP was studied in the mouse. The resumption of meiosis was monitored by the percentage of germinal vesicle breakdown (GVBD) and polar body formation (PB). The cumulus-enclosed oocytes (CEO) and denuded oocytes (DO) were cultured with and without hypoxanthine (HX) in the culture medium. Three types of experiments were performed: (1) Effect of forskolin on spontaneous resumption of meiosis, i.e. cultures without HX, and two experiments in which HX is present throughout the culture: (2) Effect of transient exposure to forskolin or dibutyric-cyclic adenosinemonophosphate (dbcAMP) on GVBD prior to continued culture without forskolin or dbcAMP (oocyte priming). (3) Priming of CEO with forskolin for 2 hr, separation of cumulus cells and oocytes, followed by coculture of rejoined cumulus cells and oocytes, or coculture of the cumulus cells and new, unprimed DO. (1) Forskolin inhibited a spontaneous resumption of meiosis in a dose-dependent manner during the first 5 hr of culturing. After 22 hr all controls and CEO resumed meiosis, whereas only half of the DO did. (2) At least 1 hr of priming the CEO with forskolin is needed to induce GVBD and PB formation, but forskolin inhibited the resumption of meiosis when present for 24 hr. Similar results were obtained with a high concentration of dbcAMP. (3) A separation and rejoining of oocytes and cumulus cells after priming induced the resumption of meiosis in a significantly greater number of oocytes than in the control oocytes which were not primed. The GVBD of unstimulated DO also increased significantly when cocultured with cumulus cells from primed CEO. The percentage of GVBD in unprimed DO and in DO isolated from primed CEO was the same. We suggest that within 1–2 hr, forskolin and cAMP stimulate cumulus cells to produce a diffusible meiosis-inducing substance which overcomes HX-inhibition and induces oocyte maturation, including both GVBD and PB formation. The CEO must be primed for more than 2 hr before the resumption of meiosis in DO isolated from such CEO is induced. Oocyte-cumulus connections are crucial as far as initiating the production of a meiosis-inducing substance is concerned. Oocyte-cumulus connections are not needed for transferring this substance to the oocyte. © 1994 Wiley-Liss, Inc.     

丝裂原活化蛋白激酶(MAPK)参与调节斑马鱼卵母细胞第一次减数分裂的恢复

旨在阐明MAPK的激活在斑马鱼(Danio rerio)卵母细胞第一次减数分裂恢复中的作用。用17α,20β双羟孕酮(17α,20β-DHP)单独作用及17α,20β-DHP与U0126(MAPK上游信号MEK阻断剂)共同作用两种方法,分别培养斑马鱼卵母细胞。采集不同培养时间的卵母细胞,经裂解变性后利用SDS-PAGE电泳和Western Blot蛋白质免疫印迹技术,检测MAPK磷酸化变化,并且观察生发泡破裂(GVBD)发生情况。结果表明,斑马鱼卵母细胞在17α,20β-DHP诱发下6h基本发生GVBD,MAPK活性在2h明显升高,一直到6h都保持在较高水平。而加入U0126后,斑马鱼卵母细胞MAPK活性受到明显抑制,同一时间GVBD发生率均低于对照组斑马鱼卵母细胞。以上结果表明,MAPK参与调节斑马鱼卵母细胞第一次减数分裂的恢复,是斑马鱼卵母细胞GVBD正常发生所必需的。     

抗真菌药物影响生殖细胞发育机理的研究

目的：探讨抗真菌药物对促性腺激素诱导的卵母细胞成熟的机理。方法：用小鼠卵母细胞体外培养模型,将小鼠卵母细胞培养在含有次黄嘌呤（ＨＸ,卵母细胞成熟抑制物）的培养注中,观察促卵泡生成素（ＦＳＨ）、两性毒素Ｂ、酮康唑对卵母细胞减数分裂恢复的影响。结果：①ＦＳＨ（１０￣２００ＩＵ／Ｌ）显著刺激卵丘－卵母细胞复合体（ＣＥＯ）克服ＨＸ的抑制而恢复减数分裂,该作用具有剂量依赖性（Ｐ〈０．０５）;②两性霉素Ｂ（０     

New insight into the role of phosphodiesterase 3A in porcine oocyte maturation

Background  

The ovulatory surge of gonadotropins triggers oocyte maturation and rupture of the ovarian follicle. The resumption of nuclear maturation in the oocyte from the prophase stage is characterized by germinal vesicle breakdown (GVBD). It has previously been shown that specific inhibition of cAMP degradation by PDE3 prevents the resumption of oocyte meiosis. However, no report has characterized the activity of PDE3 in the porcine oocyte, or the implication of the cAMP-PDE3 pathway in the entire nuclear maturation process. In this study, PDE3 activity in the oocyte was assessed during in vitro maturation (IVM) and the possible roles of the cAMP-PDE3 pathway in the resumption and progression of meiosis were investigated in terms of different models of oocyte maturation.     

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.     

Resumption of meiosis induced by meiosis-activating sterol has a different signal transduction pathway than spontaneous resumption of meiosis in denuded mouse oocytes cultured in vitro.

The sterol 4,4-dimethyl-5-cholesta-8,14,24-trien-3-ol (follicular fluid meiosis-activating sterol [FF-MAS]) isolated from human follicular fluid induces resumption of meiosis in mouse oocytes cultured in vitro. The purpose of this study was to examine the hypothesis that differential signal transduction mechanisms exist for FF-MAS-induced and spontaneous in vitro resumption of meiosis in mouse oocytes. Mouse oocytes were dissected from ovaries originating from mice primed with FSH 48 h before oocyte collection. Mechanically denuded germinal vesicle (GV) oocytes were in vitro matured in medium supplemented with hypoxanthine and FF-MAS or allowed to mature spontaneously; both groups were exposed to individual compounds known to inhibit specific targets in the cell. After 20-22 h of in vitro maturation, resumption of meiosis was assessed as the frequency of oocytes in GV breakdown (GVBD) stage. Pertussis toxin (2.5 microg/ml) did not influence resumption of meiosis in either group. Dibutyryl cyclic GMP (320 microM) inhibited FF-MAS-induced GVBD, but not spontaneous GVBD, whereas the subtype 5 phosphodiesterase-inhibitor zaprinast (50 microM) inhibited GVBD in both groups. Microinjection of the catalytic subunit of cAMP-dependent protein kinase into oocytes inhibited spontaneous GVBD, but not FF-MAS-induced GVBD. An inhibitor of cytoplasmic polyadenylation, cordycepin (80 microM), inhibited or retarded spontaneous GVBD to a further extent than it did FF-MAS-induced GVBD. Spontaneous GVBD was more sensitive to the histone H1 kinase-inhibitor olomoucine (250 microM) than was FF-MAS-induced GVBD. Addition of the mitogen-activated protein kinase (MAPK)-inhibitor PD 98059 (50 microM), phospholipase C-inhibitor U-73122 (10 microM), p21(ras)-inhibitor lovastatine (250 microM), and the src-like kinase inhibitor PP2 (20 microg/ml) inhibited FF-MAS-induced GVBD, but not spontaneous GVBD. Both MAPKs, extracellular regulated kinase (ERK) 1 and ERK2, were phosphorylated under FF-MAS-induced meiotic resumption, in contrast to spontaneous meiotic resumption, in which ERK1 and ERK2 phosphorylation occurred 2 h after GVBD. In the present study, we show that FF-MAS acts through an MAPK-dependent pathway, and we suggest that src-like kinase, p21(ras), and phosphoinositide signaling lie upstream of MAPK in the FF-MAS-activated signaling pathway. Clearly, striking pathway differences are present between spontaneous versus FF-MAS-induced meiotic resumption.     

Mitogen-activated protein kinase activity during goat oocyte maturation and the acquisition of meiotic competence

Changes in MPF and MAPK activities during meiotic maturation of goat oocytes were investigated. Detection of MPF activity occurred concomitantly with GVBD, increased at MI, decreased during anaphase-telophase I transition, and increased thereafter in MII oocytes. The appearance of MAPK activity was delayed compared to MPF activity. MAPK activity increased after GVBD and persisted during the MI-MII transition. Whether MAPK was implicated in goat oocyte meiotic competence was also investigated by using oocytes from different follicle size categories that arrest at specific stages of the maturation process (GV, GVBD, MI, and MII). Results indicate that the ability of goat oocytes to resume meiosis is not directly related to the presence of Erk2. The ability to phosphorylate MAPK is acquired by the oocyte during follicular growth after the ability to resume meiosis. GVBD-arrested oocytes exhibited a high level of MPF activity after 27 hr of culture. However, 28% of oocytes from this group contained inactive MAPK, and 72% exhibited high MAPK activity. In addition, 29% of GVBD-arrested oocytes contained a residual interphasic network without recruitment of microtubules around the condensed chromosomes; 71% of GVBD-arrested oocytes displayed recruitment of microtubules near the condensed chromosomes and contained asters of microtubules distributed throughout the cytoplasm. These results indicate that oocytes arrested at GVBD were not exactly at the same point in the meiotic cell cycle progression, and suggest that MAPK could be implicated in the regulation of microtubule organization. The data presented here suggest that in goat oocytes, MAPK is not implicated in the early events of meiosis resumption, but rather in post-GVBD events such as spindle formation and MII arrest. © 1996 Wiley-Liss Inc.     

Cumulus cells of oocyte-cumulus complexes secrete a meiosis-activating substance when stimulated with FSH

The effect of the different follicular cell types on resumption of meiosis was studied during stimulation with FSH. Cumulus enclosed oocytes (CEO), denuded oocytes (DO), and cumulus and mural granulosa cells were used. The resumption of meiosis and oocyte maturation were assessed by the determination of the germinal vesicle breakdown (GVBD) and polar body formation (PB) at the end of a 24 hr culture period in the presence of 4 mM hypoxanthine (HX). The effects of recombinant LH (r-LH) and hCG were also evaluated. Oocyte exposure to the gonadotrophins varied from 5 min to 24 hr (i.e., priming time). Oocytes were obtained from immature gonadotrophin-stimulated and -unstimulated mice. 1. FSH (1 IU/L-75 IU/L) provoked a dose-dependent increase in GVBD and PB in CEO, but not in DO, in stimulated and unstimulated mice. Eight IU/L was sufficient for inducing resumption of meiosis. In contrast, LH and hCG (both 1 IU/L-1500 IU/L) were without effect on GVBD and PB in CEO and DO of oocytes from stimulated and unstimulated mice. A combination of 8IU/L FSH and 4–8 IU/L hCG produced an additive effect, whereas combinations with LH and higher concentrations of hCG had no such effect. 2. A 2 hr priming with FSH (8 IU/L-75 IU/L) induced a dose-dependent oocyte maturation in CEO. Thirty minutes of priming with FSH (75 IU/L) was sufficient for induction of meiotic resumption in CEO. 3. Priming CEO with FSH for 2 hr followed by the separation and repooling of oocytes and cumulus cells induced oocyte maturation. GVBD of new, unprimed DO added to cumulus cells of primed CEO increased slightly but was significant, whereas GVBD in DO isolated from the primed CEO only increased marginally. DO cocultured with FSH-primed cumulus masses seem to be prevented from resuming meiosis. 4. Priming a coculture of granulosa cells and DO with FSH for 2 hr caused a significant increase in GVBD compared to the control, evaluated after 24 hr. In contrast, a 24 hr FSH-priming of a coculture of granulosa cells and DO was without effect on GVBD. 5. A spent medium in which unstimulated cumulus cells or mural granulosa cells had grown was without effect on GVBD in DO. However, a small fraction of the DO resumed meiosis after culture in a spent medium derived from a 2 hr priming of CEO and spent media from 24 hr priming of CEO induced a 2–3 times higher GVBD frequency in the DO compared to the controls. Heat treatment of spent media (70°C, 30 min) from a 24 hr FSH-priming of CEO still induced GVBD in naive DO. The results showed that FSH, in a concentration of as little as 8 IU/L, but not r-LH and hCG, induced within 30 minutes the cumulus cells to produce and after 2 hr to secrete a diffusible heat stable meiosis activating substance. This substance overcame, in a paracrine fashion, the inhibiting effect of HX and induced oocyte maturation directly in DO. The production of this substance, however, was dependent on the initial connection between the cumulus cells and the oocyte, indicating an important 2-way communication between these 2 cell types. The mural granulosa cells did not produce a meiosis inducing activity by stimulation with FSH, but significantly, more DO matured after coculture with the nonstimulated granulosa cells for 24 hr than for 2 hr. It is proposed that the heat stable meiosis activating component of the spent media from the FSH-stimulated CEO belongs to the meiosis activating sterols, MAS, previously isolated from human follicular fluid and from adult bull testes. Mol. Reprod. Dev. 46:296–305, 1997. © 1997 Wiley-Liss, Inc.     

Phosphorylation of mitogen-activated protein kinase is regulated by protein kinase C, cyclic 3',5'-adenosine monophosphate, and protein phosphatase modulators during meiosis resumption in rat oocytes

Mitogen-activated protein (MAP) kinase, protein kinase C (PKC), cAMP, and okadaic acid (OA)-sensitive protein phosphatases (PPs) have been suggested to be involved in oocyte meiotic resumption. However, whether these protein kinases and phosphatases act by independent pathways or interact with each other in regulating meiosis resumption is unknown. In the present study, we aimed to determine the regulation of meiosis resumption and MAP kinase phosphorylation by PKC, cAMP, and OA-sensitive PPs in rat oocytes using an in vitro oocyte maturation system and Western blot analysis. We found that ERK1 and ERK2 isoforms of MAP kinases existed in a dephosphorylated (inactive) form in germinal vesicle breakdown (GVBD)-incompetent and GVBD-competent germinal vesicle intact (GVI) oocytes as well as GVBD oocytes at equivalent levels. These results indicate that MAP kinases are not responsible for the initiation of normal meiotic resumption in rat oocytes. However, when GVBD-incompetent and GVBD-competent oocytes were incubated in vitro for 5 h, MAP kinases were phosphorylated (activated) in GVBD-competent oocytes, but not in meiotic-incompetent oocytes, suggesting that oocytes acquire the ability to phosphorylate MAP kinase during acquisition of meiotic competence. We also found that both meiosis resumption and MAP kinase phosphorylation were inhibited by PKC activation or cAMP elevation. Moreover, these inhibitory effects were overcome by OA, which inhibited PP1/PP2A activities. These results suggest that both cAMP elevation and PKC activation inhibit meiosis resumption and MAP kinase phosphorylation at a step prior to OA-sensitive protein phosphatases. In addition, inhibitory effects of cAMP elevation on meiotic resumption and MAP kinase phosphorylation were not reversed by calphostin C-induced PKC inactivation, indicating that cAMP inhibits both meiotic resumption and MAP kinase activation in a PKC-independent manner.     

Towards a new understanding on the regulation of mammalian oocyte meiosis resumption

Mammalian oocytes reach prophase of first meiosis around the time of birth, and remain at this stage for months or years, depending on the species. Only after puberty will the fully-grown oocytes begin to resume meiosis which is stimulated by gonadotropin surge. It has long been known that a high level of intra-oocyte cyclic adenosine 3',5'-monophosphate (cAMP) prevents oocyte meiosis resumption as indicated by germinal vesicle breakdown (GVBD). Recently, guanosine triphosphate-binding (G) protein-coupled receptors/G proteins/adenyl cyclase pathway endogenous to the oocyte as well as cAMP diffusion from the somatic compartment through gap junctions have been implicated in maintaining cAMP at levels that prevent oocytes from resuming meiosis. Another second messager molecule, guanosine 3',5'-cyclic monophosphate (cGMP), has also recently been found to play important roles in maintaining oocyte meiosis arrest. cGMP in the follicular somatic cells diffuses into the oocyte and causes an increase in oocyte cAMP, presumably by acting on phosphodiesterase 3 (PDE3). The cGMP level in the somatic compartment of the follicle decreases in response to luteinizing hormone (LH), and this change may be mediated through the epidermal growth factor (EGF)-like factors and specific cGMP-phosphodiesterase subtype activity. It is well known that gonadotropic stimulation of meiotic resumption depends on mitogen-activated protein kinase (MAPK) activation in the somatic compartment of the follicle; recent studies show that LH, through cAMP/protein kinase A (PKA) and protein kinase C (PKC) pathways, induces the synthesis of paracine factors such as EGF-like facors and meiosis activating sterol (MAS) to regulate oocyte GVBD via the MAPK pathway in follicle cells. A recent granulosa cell-specific knockout study has for the first time provided in vivo evidence for the important role of extracellular regulated kinase 1 and 2 (ERK1/2), two main forms of MAPK, and their downstream molecules in granulosa cells in oocyte meiosis resumption. Unresolved questions and future directions on research regarding signaling changes in follicle cells and oocytes as well their communication in response to the gonadotropin surge are addressed in this review.     

Multiple triggers of oocyte maturation in nemertean worms: the roles of calcium and serotonin

To analyze the process of oocyte maturation in nemertean worms, oocytes with a large nucleus (=germinal vesicle, or GV) were removed from gravid ovaries of Cerebratulus lacteus and Micrura alaskensis. Following transfer to natural seawater (NSW), fully grown oocytes spontaneously matured as indicated by their completion of germinal vesicle breakdown (GVBD), whereas GVBD was reversibly blocked if the oocytes were initially placed in calcium-free seawater (CaFSW). Similarly, calcium ionophore treatments triggered GVBD in calcium-containing artificial seawater (ASW) but not in CaFSW, suggesting that external calcium influx may facilitate maturation. However, compared to the overall levels of maturation elicited by ASW, significantly higher percentages of GVBD were achieved with NSW or with ASW that had been conditioned with marine sediment. Moreover, calcium channel blockers decreased GVBD rates in ASW but not in NSW, which is consistent with the view that substances other than external calcium ions can trigger maturation. Accordingly, oocytes underwent equally high levels of GVBD when treated with serotonin (=5-hydroxytryptamine, or 5-HT) in ASW or CaFSW. The 5-HT-induced maturation was blocked by inhibitors of 5-HT receptors but continued to occur in the presence of calcium channel blockers or the calcium chelator BAPTA. In addition, oocytes microinjected with fluorescent calcium indicators underwent GVBD in response to 5-HT without displaying marked calcium transients during confocal imaging runs. Collectively, such findings suggest that nemertean oocytes can mature via multiple pathways that may include external calcium influx or a 5-HT-induced signaling cascade that lacks prominent calcium fluctuations. J. Exp. Zool. 287:243-261, 2000.     

A serotonin receptor antagonist induces oocyte maturation in both frogs and mice: evidence that the same G protein-coupled receptor is responsible for maintaining meiosis arrest in both species

Accumulating evidence has indicated that vertebrate oocytes are arrested at late prophase (G2 arrest) by a G protein coupled receptor (GpCR) that activates adenylyl cyclases. However, the identity of this GpCR or its regulation in G2 oocytes is unknown. We demonstrated that ritanserin (RIT), a potent antagonist of serotonin receptors 5-HT2R and 5-HT7R, released G2 arrest in denuded frog oocytes, as well as in follicle-enclosed mouse oocytes. In contrast to RIT, several other serotonin receptor antagonists (mesulergine, methiothepine, and risperidone) had no effect on oocyte maturation. The unique ability of RIT, among serotonergic antagonists, to induce GVBD did not match the antagonist profile of any known serotonin receptors including Xenopus 5-HT7R, the only known G(s)-coupled serotonin receptor cloned so far in this species. Unexpectedly, injection of x5-HT7R mRNA in frog oocytes resulted in hormone-independent frog oocyte maturation. The addition of exogenous serotonin abolished x5-HT7R-induced oocyte maturation. Furthermore, the combination of x5-HT7R and exogenous serotonin potently inhibited progesterone-induced oocyte maturation. These results provide the first evidence that a G-protein coupled receptor related to 5-HT7R may play a pivotal role in maintaining G2 arrest in vertebrate oocytes.     

Effects of protein kinase C activators on germinal vesicle breakdown and polar body emission of mouse oocytes

Protein phosphorylation mediated by cAMP-dependent protein kinase is instrumental in maintaining meiotic arrest of mouse oocytes. To assess whether protein phosphorylation mediated by calcium/phospholipid-dependent protein kinase (protein kinase C) might also inhibit the resumption of meiosis, we treated oocytes with activators of this enzyme. The active phorbol esters 12-O-tetra-decanoyl phorbol-13-acetate (TPA) and 4 beta-phorbol 12,13-didecanoate (4 beta-PDD) inhibited germinal vesicle breakdown (GVBD), as did a more natural activator of protein kinase, C, sn-1,2-dioctanoylglycerol (diC8). An inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD), did not inhibit GVBD. We then examined whether protein kinase C activators inhibit a step in the cAMP-modulated pathway that regulates resumption of meiosis. TPA did not inhibit the maturation-associated decrease in oocyte cAMP. Microinjected heat-stable protein inhibitor of cAMP-dependent protein kinase failed to induce GVBD in the presence of TPA. Both TPA and diC8 partially inhibited specific changes in oocyte phosphoprotein metabolism that are tightly correlated with resumption of meiosis; these agents also induced the apparent phosphorylation of specific oocyte proteins. These results suggest that protein kinase C activators may inhibit resumption of meiosis by acting distal to a decrease in cAMP-dependent protein kinase activity, but prior to changes in oocyte phosphoprotein metabolism that are presumably required for resumption of meiosis. Finally, we compared the effects of db-cAMP and protein kinase C activators on polar body emission following GVBD. TPA, 4 beta-PDD or diC8, but not 4 alpha-PDD or db-cAMP, inhibited polar body emission in a dose-dependent manner. The morphology and cytology of oocytes in which polar body emission was inhibited by TPA or 4 beta-PDD differed from that of oocytes treated with diC8. Thirty to 60% of the former were round in shape and exhibited a clump of chromosomes but no spindle; the remainder were distended in shape and exhibited a metaphase I spindle. All oocytes treated with diC8, however, were round, had dispersed chromosomes, and no spindle. These results suggest that, in contrast to resumption of meiosis, polar body emission is inhibited by activation of protein kinase C but not cAMP-dependent protein kinase.     

小鼠年龄及卵丘—卵母细胞间联接的解离对卵母细胞体外成熟的影响

实验研究了小鼠卵母细胞体外过程中卵丘－卵母细胞间的相互作用。实验小鼠为雌性Ｂ６Ｄ２杂交一代。激素处理４８小时后分离出卵后天和卵母细胞复合体,并培养在含有次黄嘌呤的培养液中。２４小时后检查卵母细胞核成熟情况。     

Protein phosphorylation in meiotically competent and incompetent mouse oocytes

Specific changes in the two-dimensional gel electrophoretic pattern of mouse oocyte phosphoproteins precede germinal vesicle breakdown (GVBD). We report that changes in the relative abundance of phosphoamino acids occurred prior to GVBD. We also report data that further strengthen the close association of the changes in phosphoprotein patterns with resumption of meiosis. The calmodulin antagonist W7, which transiently inhibits GVBD, inhibited partially at least two of the maturation-associated phosphoprotein changes, the dephosphorylation of a 60,000 Mr phosphoprotein and the phosphorylation of a 36,000 Mr protein. In oocytes from juvenile mice that were incompetent to resume meiosis, neither these changes nor the phosphorylation of proteins of Mr 24,000 and 28,000 occurred; all these changes occurred, however, in oocytes from juvenile mice that were competent to resume meiosis. The microinjection of the heat-stable inhibitor of cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKI), which induces GVBD in fully grown oocytes, did not induce GVBD in meiotically incompetent oocytes. Microinjected PKI did not induce the increased protein phosphorylations associated with maturation, but it did induce the dephosphorylation of the 60,000 Mr phosphoprotein. These results provide molecular markers for commitment to resume meiosis in GV-intact oocytes and indicate a potential basis for meiotic incompetence.