Incidence Of Germinal Vesicle Breakdown Research Articles

259 Cdk7 AND CYCLIN H, BUT NOT Mat1, ARE INVOLVED IN MEIOTIC RESUMPTION OF PORCINE IMMATURE OOCYTE

The complex kinase Cdk-activating kinase (CAK) consists of the catalytic subunit Cdk7, regulatory subunit Cyclin H, and assembly factor Mat1. The CAK is essential for maturation-promoting factor (MPF) activation by phosphorylating threonine 161 (T161) of Cdc2 in mitosis. Although it is known that meiotic resumption of oocytes is regulated by MPF activity, the role of CAK in meiosis is still unclear. In the present study, we attempted to confirm the involvement of CAK in meiotic resumption of porcine immature oocyte. Cumulus–oocyte complexes (COC) were collected from antral follicles of gilts and cultured up to 48 h in TYH medium containing 20% porcine follicular fluid, 3.2 mg/mL of BSA, and 1.0 IU mL–1 of pregnant mare serum gonadotropin. The T161 phosphorylation level of Cdc2 in cultured oocytes was analysed by Western blot analysis. The transcripts were collected from noncultured or cultured oocytes, and Cdk7, Cyclin H, and Mat1 expression were detected by RT-PCR. Overexpression of Cdc2 or inhibition of Cdk7, Cyclin H, and Mat1 during oocyte maturation was performed by microinjection of mRNA or antisense RNA into ooplasm of immature COC and verified by Western blot or semiquantitative RT-PCR. Maturation-promoting factor kinase activity was assayed by Histone H1 kinase activity assay. Statistical analyses in this study were carried out by Student’s t-test. The T161 phosphorylation of Cdc2 was found during the culture period from 18 h to 48h, which was after germinal vesicle breakdown (GVB). Overexpression of Cdc2 increased the incidence of GVB at 18 h, but overexpression of mutant Cdc2 (replaced T161 by alanine) had no influence on GVB. These results indicate that T161 phosphorylation of Cdc2 is important for meiotic resumption. Next, we attempted to confirm the CAK function during oocyte maturation. Transcripts of Cdk7, Cyclin H, and Mat1 were detectable throughout the culture period. Inhibition of Cdk7 and Cyclin H caused a decrease in T161 phosphorylation and MPF activity, and the incidence of GVB was significantly lower than in nontreated oocytes. In contrast, Mat1-inhibited oocytes resumed meiosis and developed to the metaphase II stage, and the incidence was not different between Mat1-inhibited oocytes and nontreated oocytes. These results suggest that Cdk7 and Cyclin H are working as CAK and activate Cdc2 by T161 phosphorylation, although Mat1 is dispensable during oocyte maturation.

Effect of the factor inhibiting germinal vesicle breakdown on the disruption of gap junctions and cumulus expansion of pig cumulus-oocyte complexes cultured in vitro

The present study was undertaken to explore the regulatory mechanisms for meiotic resumption of pig cumulus-oocyte complexes (COCs) by assessing the nuclear status of oocytes, the degree of gap junction cell-to-cell communication and cumulus expansion after culture of various numbers of COCs in 10 microl droplets of medium for 24 h. Gap junction communication was examined by confocal laser scanning microscopy after injection of a fluorescent dye, lucifer yellow, into the oocytes. When one, three or six COCs were cultured in a 10 microl droplet, germinal vesicle breakdown was observed in > 70% of oocytes; increasing the number of COCs in a droplet further actually decreased the proportion of oocytes undergoing germinal vesicle breakdown (10 COCs: 49%; 20 COCs: 21%; 40 COCs: 13%). When six COCs were cultured in a 10 microl droplet of conditioned medium (prepared previously by culturing 20 intact and oocytectomized COCs for 24 h), the proportion of oocytes undergoing germinal vesicle breakdown was significantly reduced compared with the proportion in fresh medium. An increase in the proportion of category 1 COCs (all gap junctions within cumulus cells and between cumulus cells and oocyte are functionally maintained) was achieved by increasing the number of COCs cultured in a 10 microl droplet. The addition of conditioned medium to the fresh medium at a concentration of 50% significantly inhibited cumulus expansion. From these results, it is concluded that the factors secreted by cumulus cells regulate the disruption of gap junctions and cumulus expansion, and concurrently control the incidence of germinal vesicle breakdown in pig COCs.

Effect of the factor inhibiting germinal vesicle breakdown on the disruption of gap junctions and cumulus expansion of pig cumulus-oocyte complexes cultured in vitro

The present study was undertaken to explore the regulatory mechanisms for meiotic resumption of pig cumulus-oocyte complexes (COCs) by assessing the nuclear status of oocytes, the degree of gap junction cell-to-cell communication and cumulus expansion after culture of various numbers of COCs in 10 microl droplets of medium for 24 h. Gap junction communication was examined by confocal laser scanning microscopy after injection of a fluorescent dye, lucifer yellow, into the oocytes. When one, three or six COCs were cultured in a 10 microl droplet, germinal vesicle breakdown was observed in > 70% of oocytes; increasing the number of COCs in a droplet further actually decreased the proportion of oocytes undergoing germinal vesicle breakdown (10 COCs: 49%; 20 COCs: 21%; 40 COCs: 13%). When six COCs were cultured in a 10 microl droplet of conditioned medium (prepared previously by culturing 20 intact and oocytectomized COCs for 24 h), the proportion of oocytes undergoing germinal vesicle breakdown was significantly reduced compared with the proportion in fresh medium. An increase in the proportion of category 1 COCs (all gap junctions within cumulus cells and between cumulus cells and oocyte are functionally maintained) was achieved by increasing the number of COCs cultured in a 10 microl droplet. The addition of conditioned medium to the fresh medium at a concentration of 50% significantly inhibited cumulus expansion. From these results, it is concluded that the factors secreted by cumulus cells regulate the disruption of gap junctions and cumulus expansion, and concurrently control the incidence of germinal vesicle breakdown in pig COCs.

Effects of hormones and osmolarity in the culture medium on germinal vesicle breakdown of porcine oocytes

The present study was conducted to examine effects of hormones and osmolarity on germinal vesicle breakdown (GVBD) and histone H1 kinase (HIK) activity in porcine oocytes cultured in vitro. The basic medium used for culture of oocytes was modified Tyrode's solution in which the osmolarity was adjusted to 134 to 495 mOsm by changing the concentration of sodium chloride (NaCl). When the hormones were present, osmolarity of medium that allows GVBD of oocytes was less than 400 mOsm. However, the range of osmolarity of medium that allows meiotic maturation of oocytes was 210 to 362 mOsm. On the other hand, without hormonal supplement, the incidence of GVBD in oocytes decreased as the osmolarity of the medium increased in the rage of 210 to 362 mOsm. By increasing the osmolarity of the medium from 210 to 362 mOsm by addition with sorbitol instead of NaCl, the incidence decreased from 89.1 % to 13.3 %. In oocytes cultured in medium of 210 mOsm without hormones, the percentage of oocytes that underwent GVBD and had increased H1K activity 20 h after culture was significantly higher (P<0.05) than those of oocytes cultured in the same medium supplemented with hormones or medium of 362 mOsm. These results indicate that in vitro induction of GVBD in porcine oocytes is strongly affected by osmolarity of the medium in the absence of hormones. The results also suggest that, under low osmolarity (210 mOsm), GVBD is accelerated with rapid increase of H1K activity.

Protein kinase C and intracellular calcium are involved in follicle-stimulating hormone-mediated meiotic resumption of cumulus cell-enclosed porcine oocytes in hypoxanthine-supplemented medium.

The present experiments were conducted to examine the hypothesis that follicle-stimulating hormone (FSH) can stimulate the hydrolysis of phosphoinositide, generating the intracellular second messengers to activate protein kinase C and mobilizing intracellular calcium, thus inducing oocyte meiotic resumption. Pig cumulus cell-enclosed oocytes (CEO) were cultured for 24 hr in 4 mM hypoxanthine (HX)-supplemented medium and treated with different agents in the following designs: (1) CEO were treated with neomycin (an inhibitor of phosphoinositide hydrolysis) in the presence of FSH or only treated with 7,12-dimethylbenzin(a) anthracene (DMBA, a tumor promoter which can cause phosphorylation of phospholipase C (PLC), formation of inositol triphophate, and mobilization of intracellular calcium) to mimic the direct activation of PLC; (2) CEO were challenged by FSH, together with sphingosine or staurosporine (two kinds of PKC inhibitors); or treated with phorbol myristate acetate (PMA, an activator of PKC) separately; (3) CEO were primed with BAPTA/AM (an intracellular calcium chelator) or BAPTA/AM +FSH for 60 min, and then transferred into a new culture medium supplemented with FSH but without BAPTA/AM; total culture time was 24 hr. At the end of the culture, the incidence of germinal vesicle breakdown (GVBD) was calculated. The results showed that: (1) FSH (100 U/liter) could stimulate pig CEO to override the arrest of HX and resume meiosis; DMBA (10(-8)-10(-5) M) itself also had such a kind of effect; whereas neomycin, at the level of 10-20 mM, could dramatically inhibit the stimulatory effect of FSH. (2) Staurosporine (10(-9)-10(-6) M) or sphingosine (10(-8)-10(-5) M) could also inhibit the effect of FSH in a dose-dependent manner on stimulating CEO to resume meiosis. However, PMA (10(-8)-10(-5) M) alone had a dual effect on the meiotic resumption of pig CEO. PMA, at the level of 10(-8)-10(-6) M, could stimulate CEO to resume meiosis, and at high concentration of 10(-5) M , it could even enhance the inhibitory effect of HX. (3) Priming CEO with BAPTA/AM only or BAPTA/AM +FSH for 60 min could significantly inhibit the effect of FSH in a dose-dependent manner. These results indicate that in the process of ligand-mediated meiotic resumption of pig CEO, FSH can stimulate the hydrolysis of phosphoinositide leading to the activation of PKC and mobilization of intracellular calcium; and suggest that multiple signaling pathways and signal interaction are involved in this process.

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 heteroclitus ovarian follicles leading to the inhibition of oocyte meiosis reinitiation (oocyte maturation) in vitro induced by the naturally occurring maturation-inducing steroid 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (17,20 beta P) 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 17,20 beta P in IBMX-free culture media in the presence or absence of 10(-4) M 5-HT. The exposure of follicles to 17,20 beta P 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 17,20 beta P, 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 17,20 beta P-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 17,20 beta P-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.

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.

Effects of Phosphatidylinositol 3-Kinase Inhibitors, Wortmannin and LY294002, on Germinal Vesicle Breakdown (GVBD) in Porcine Oocytes.

To determine whether phosphatidylinositol 3-kinase (PI 3-kinase) cascade plays any role in mammalian oocyte meiotic progression, porcine oocytes were matured in vitro with PI 3-kinase specific inhibitors, wortmannin and LY294002. In cumulus-oocyte complexes (COCs), the addition of 10-8, 10-7 or 10-6 M wortmannin or 0.5, 5.0 or 7.5 ×10-5 M LY294002 to the medium significantly reduced the proportion of oocytes exhibiting germinal vesicle breakdown (GVBD) compared with the inhibitor-free control. In denuded oocytes (DOs), no significant differences in the incidence of GVBD were found between 10-8 M wortmannin treatment and controls, and between 10-8 and 10-7 M treatments, respectively. LY294002 treatment at all concentrations tested did not significantly affect GVBD in DOs. When COCs were cultured in the presence of wortmannin or LY294002, the proportions of oocytes arrested at GVII were significantly higher than the control. The degree of cumulus cell expansion was significantly decreased with wortmannin or LY294002 treatment compared to the control. We concluded that activation of PI 3-kinase in cumulus cells is closely associated with meiotic resumption of the porcine oocytes and the cumulus cell expansion.

Preincubation of cumulus-oocyte complexes before exposure to gonadotropins improves the developmental competence of porcine embryos matured and fertilized in vitro

The objectives of the present study were to examine whether delayed exposure of porcine cumulus-oocyte complexes (COCs) to gonadotropins affects the diameter of oocytes, the nuclear morphology of the germinal vesicle, the rate of germinal vesicle breakdown (GVBD), and the embryonic developmental rate of inseminated oocytes following maturation and fertilization in vitro (IVM/IVF). After preincubation (experimental) or no preincubation (control) in BSA-free NCSU23 medium containing 1096 porcine follicular fluid for 12 h, COCs were cultured for maturation in the same medium supplemented with gonadotropins for 20 h and then without those gonadotropins for 20 h. During the preincubation period, the nuclear morphology of the germinal vesicles became more homogeneous. Incidence of GVBD after 20 h of maturation culture was not different between the control and experimental group. When cultured in NCSU23 medium for 7 d following IVF, the incidence of embryos that developed to the blastocyst stage (23.1 ± 3.1%) was higher in the experimental group than in the control group (8.7 ± 1.2%). Blastocysts in the experimental group had a larger number of cells than control blastocysts. Following embryo transfer into the oviduct of recipient gilts, IVM/IVF embryos had elongated by Day 12 of gestation. These results indicate that preincubation of porcine COCs, before exposure to gonadotropins to induce the resumption of meiosis, increases the rate of development of IVM/IVF embryos to the blastocyst stage.

Stimulatory effect of insulin-like growth factor I and epidermal growth factor on the maturation of rabbit oocytes in vitro.

The effects of different combinations of epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) on cumulus expansion and meiotic maturation were examined in rabbit oocytes. Selected rabbit follicular oocytes were matured in vitro and were classified as cumulus-oocyte complexes or denuded oocytes. They were cultured in TCM 199, and were treated with growth factors at different concentrations: EGF at 0, 1, 10, 50 and 100 ng ml-1, IGF-I at 0, 50, 100 and 200 ng ml-1 and EGF plus IGF-I at 10 + 50; 10 + 100; 50 + 50 and 50 + 100 ng ml-1, respectively. After 6 h of culture, the oocytes were assessed for nuclear maturation and after 16 h of culture, for cumulus expansion and maturation stage. After culture for 6 h, the incidence of germinal vesicle breakdown was higher (P < 0.05) in all of the growth factor treatments tested compared with controls. After culture for 16 h, EGF enhanced the incidence of cumulus expansion at all of the concentrations tested. Cumulus expansion was greatest with 50 mg EGF ml-1 plus 100 ng IGF-I ml-1 (72.0% versus 2.4% in controls). Treatment with IGF-I significantly increased (P < 0.05) the incidence of metaphase II stage, and maximum stimulation occurred at 100 ng IGF-I ml-1 (84.5% versus 31.1% in controls). However, IGF-I did not affect cumulus expansion. When denuded oocytes were used, no positive effects on nuclear maturation rates were observed for any treatment. These results suggest that: (1) EGF, either alone or with IGF-I, stimulates cumulus expansion; (2) the addition of IGF-I or EGF plus IGF-I significantly enhances nuclear maturation in immature rabbit oocytes; and (3) this effect is mediated by the presence of cumulus cells.

Effects of gonadotropins upon the incidence and kinetics of meiotic maturation of macaque oocytes in vitro

The specific aim of this study was to determine the effects of gonadotropins in vitro upon the incidence of and precise time interval to germinal vesicle breakdown (GVB) and extrusion of the first polar body (PB1) in oocytes from nonstimulated rhesus monkeys. Cumulus-enclod germinal vesicle (GV) stage oocytes from 10 normal, cycling rhesus monkeys in the follicular phase of the menstrual cycle were cultured with either: (1) 1.0 micrograms/ml human follicle-stimulating hormone (hFSH), (2) 10 micrograms/ml human luteinizing hormone (hLH), (3) 1.0 microgram/ml hFSH and 10 micrograms/ml hLH, or (4) no gonadotropins (controls). Oocytes (n = 234) were examined at 3-hr intervals from 0 to 21 hr and at 4-hr intervals from 24 to 52 hr for GVB and PB1. Neither the incidence of GVB (hFSH: 63.5%; hLH: 56.1%; both gonadotropins: 63.1%; no gonadotropins: 53.6%) nor extrusion of PB1 (hFSH: 41.3%; hLH: 36.4%; both gonadotropins: 36.9%; no gonadotropins; 31.9%) differed (P > 0.05) among treatments. The time to GVB was accelerated (P < 0.05) by gonadotropins (hFSH: 10.8 +/- 1.7 hr; hLH: 10.1 +/- 1.8 hr; both gonadotropins: 8.8 +/- 1.1 hr) when compared to controls (17.4 +/- 2.0 hr). However, the time interval to extrusion of PB1 did not differ (P > 0.05) among treatments (hFSH: 32.3 +/- 1.2 hr; hLH: 35.1 +/- 1.4 hr; both gonadotropins: 35.2 +/- 1.3 hr; no gonadotropins: 34.1 +/- 1.2 hr). The mean interval to extrusion of PB1 was 34.1 +/- 0.6 hr.(ABSTRACT TRUNCATED AT 250 WORDS)

Defining a role for calcium in the resumption and progression of meiosis in the pig oocyte

The role of calcium (Ca++) during spontaneous meiotic maturation of pig oocytes was examined. The hypothesis that elevations of endogenously derived intracellular Ca++ are prerequisite for germinal vesicle breakdown (GVBD) and progression through meiosis was tested. In addition, investigations were carried out to determine whether GVBD and meiotic progression were dependent upon extracellular Ca++ influx. Elevation of endogenously derived Ca++ was inhibited directly by loading cells with BAPTA, a specific Ca++-chelator, or indirectly with neomycin. Extracellular Ca++ influx was prevented by culturing oocytes in Ca(++)-deficient medium, with EGTA, or in the presence of the Ca++ channel blocker verapamil. Pretreatment with BAPTA/AM and subsequent culture in the absence of added exogenous Ca++ resulted in a similar inhibition of GVBD (1 microM BAPTA/AM vs. untreated, P < 0.01). After 4 h following follicular release, oocytes were no longer sensitive to BAPTA/AM treatment. Neomycin also significantly inhibited GVBD (0.5 mM neomycin vs. untreated, P < 0.05) as well as meiotic progression past metaphase I (0.25 mM neomycin vs. untreated, P < 0.05). The incidence of GVBD was not significantly affected when oocytes were simply cultured in Ca++ deficient medium or when cultured in the presence of EGTA or verapamil. However, progression of meiosis past GVBD to metaphase II was suppressed by reducing levels of Ca++ in the culture medium (0.68 mM Ca++ vs. 1.7 mM Ca++, P < 0.05) and by treatment with verapamil (0.25 mM verapamil vs. untreated, P < 0.05). Meiotic progression was completely blocked at metaphase I in the presence of 0.85 mM EGTA.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of Gonadotropin on Ovarian Intrafollicular Processes during the Development of Oocyte Maturational Competence in a Teleost, the Atlantic Croaker: Evidence for Two Distinct Stages of Gonadotropic Control of Final Oocyte Maturation1

Full-grown oocytes of Atlantic croaker are insensitive to maturation-inducing steroid (MIS) unless they are primed with gonadotropin (GtH). The objective of this study was to examine the mechanism of GtH-induced maturational competence in croaker oocytes. Specifically, we determined the in vitro secretion of steroids by intact ovarian follicles of unprimed or hCG-primed fish, the direct effects of steroids on maturational competence, and the effects of steroid (cyanoketone), protein (cycloheximide), and RNA (actinomycin D) synthesis inhibitors on hCG-induced maturational competence and steroidogenesis in vitro. The steroid content of the incubation medium after hCG treatment was measured by RIA. The effects of hCG or exogenous steroid treatment on maturational competence were determined by recording the incidence of germinal vesicle breakdown (GVBD) after MIS-induced GVBD in a standard bioassay. Our major findings were: (1) induction of maturational competence occurred after exposure of ovarian follicles to hCG either in vivo or in vitro; (2) MIS secretion was detected in follicles of hCG-primed fish but not unprimed fish, and no MIS secretion was observed during hCG induction of maturational competence in vitro; (3) treatment with cyanoketone blocked the hCG-dependent secretion of testosterone and estradiol but not the development of maturational competence; (4) treatment with MIS or various other exogenous steroids in the absence of hCG did not induce maturational competence; and (5) hCG-induced maturational competence was inhibited by cycloheximide and actinomycin D. Therefore, the mechanisms of GtH induction of oocyte maturation in Atlantic croaker can be described in two distinct stages: a delta-4 steroid-(including MIS) and estrogen-independent priming stage followed by a MIS-mediated GVBD stage. The priming stage may involve mechanisms requiring RNA as well as protein synthesis.

Differential effects of testosterone and dibutyryl cyclic AMP on the meiotic maturation of mouse oocytes in vitro

Fully grown, meiotically immature mouse oocytes were isolated and cultured under varying conditions with the aim of determining a) whether the inhibitory effects of testosterone on oocyte meiotic maturation require the synthesis of new oocyte proteins and b) if the meiosis-inhibiting effects of testosterone and dibutyryl cyclic AMP (dbcAMP) are distinct and can be differentiated. We found that the inclusion of puromycin in culture medium containing testosterone has no effect on the meiosis-inhibiting potency of testosterone or upon the reversibility of testosterone effects. We conclude that testosterone inhibits oocyte meiosis by a mechanism that is independent of protein synthesis. We also found that oocytes exposed to testosterone recover more rapidly, as evidenced by the timing of germinal vesicle breakdown (GVBD) following placement in a control medium, than do oocytes exposed to dbcAMP. Through further investigation of this phenomenon we have determined the sequence of testosterone and dbcAMP effects relative to the time course of GVBD. A testosterone-sensitive event occurs 20 min prior to GVBD, while the dbcAMP-sensitive event precedes GVBD by 41 min. The nature of this difference may involve the differential interaction of testosterone and dbcAMP with a set of puromycin-sensitive proteins that are required for GVBD. When oocytes were initially cultured in medium containing both puromycin and either testosterone or dbcAMP and then moved to medium containing puromycin alone the incidence of GVBD was reduced relative to oocytes never exposed to puromycin. This observation suggests that mouse oocytes contain proteins that are required for GVBD and that experience a high turnover rate. The degree of reduction in GVBD was a function of the length of puromycin exposure and was significantly greater in dbcAMP- than in testosterone-exposed oocytes. If oocytes were initially cultured in medium containing puromycin and dbcAMP, the rate of GVBD upon removal of dbcAMP was initially slow but increased with time. This observation is consistent with the hypothesis that dbcAMP inhibits oocytes at a point prior to the functioning of the puromycin-sensitive proteins. However, if oocytes were cultured in medium containing puromycin and testosterone the rate of GVBD following testosterone removal was not significantly reduced relative to oocytes that were not exposed to puromycin. This observation suggests that testosterone acts to inhibit meiosis at a site beyond the function of the puromycin-sensitive proteins or that testosterone causes a reduction in the turnover rate of these proteins.(ABSTRACT TRUNCATED AT 400 WORDS)

Cooperative inhibitory effect of follicular fluid and cAMP on hamster oocyte maturation.

Porcine or human follicular fluid inhibited the spontaneous maturation of isolated hamster oocytes in vitro during the first 1.5 h of culture. Moreover, the presence of 50% follicular fluid combined with 100 microM dbcAMP cooperatively reduced the incidence of germinal vesicle breakdown. The addition of FSH also inhibited the resumption of meiosis, and the presence of LH did not overcome the inhibitory effects of follicular fluid and tended to impede isolated hamster oocyte maturation in vitro.

Meiosis-inhibiting effects in vivo of antiserum to progesterone on follicular ova in immature rats treated with gonadotropins.

Effects of the neutralization of endogenous progesterone with rabbit antiserum to progesterone (anti-progesterone) on germinal vesicle breakdown of ova in follicles of small (less than 125 micrometers), intermediate (125-250 micrometers) and large (greater than 250 micrometers) diameter were examined by a quantitative histological technique. Immature rats were treated with 5 IU pregnant mare's serum gonadotropin (PMS) then with 10 IU human chorionic gonadotropin (hCG). Administration of anti-progesterone together with hCG 6 h later significantly decreased the incidence of germinal vesicle breakdown of ova in the large follicles, but not in the intermediate ones. This treatment did not affect the proportion of intermediate to large follicles in the population. Replacement with progesterone 1 h after the simultaneous injection of hCG and anti-progesterone partly reversed the reduced incidence of meiosis. An injection of rabbit antiserum to estrone, in addition to the replacement with progesterone 1 h after the simultaneous injections of hCG and anti-progesterone, restored the incidence of meiosis to a value comparable to the values found for control rats treated sequentially with PMS and hCG. We concluded that the hCG-induced preovulatory rise in progesterone has a limited but definite stimulatory effect on the resumption of meiosis in the ova of large follicles and that it mediates the meiosis-inducing action of hCG.

PROPIONATE AND CYCLOHEXIMIDE REVERSIBLY BLOCK PROGESTERONE INDUCED CALCIUM SURGE IN AMBYSTOMA MEXICANUM OOCYTES.

The protoprotein aequorin was used in order to monitor Ca2+ transients in conditions where progesterone induced maturation was reversibly inhibited. Propionate but not isethionate Cl-free medium impaired both meiosis reinitiation and the Ca2+ transient, unless oocytes were returned to normal Cl-containing medium. Similar results were obtained with the protein synthesis inhibitor cycloheximide. In both cases, the incidence of germinal vesicle breakdown (GVBD) and the time schedule relating it to the Ca2+ surge appeared not very different from that found from control oocytes. The evidence suggests that both treatments act on the initial step by which progesterone triggers the intracellular Ca2+ release needed for maturation promoting factor (MPF) elaboration. No definitive conclusion can be reached however from these experiments concerning the need for protein synthesis during meiosis reinitiation.

Progesterone induction of oocyte maturation in Rana pipiens: Reversibility of cycloheximide inhibition

AbstractThe reversibility of cycloheximide inhibition of progesteroneinduced oocyte maturation was investigated. Progesterone treated immature oocytes underwent nuclear (germinal vesicle breakdown) and cytoplasmic maturation unless inhibited by incubation in the continuous presence of cycloheximide. However, when previously treated oocytes were simply washed in culture media on the day following the initial treatment, they matured without additional exposure to progesterone. The incidence of germinal vesicle breakdown observed after washing and reincubation was similar to that seen in paired oocytes retreated with progesterone, cycloheximide‐pretreated control oocytes newly treated with progesterone or control oocytes treated only with progesterone on the first day. Oocytes exposed to low concentrations of progesterone matured the following day whereas paired oocytes inhibited by cycloheximide during the first day did not mature at similar low doses following washing on the second day. The time of germinal vesicle breakdown in oocytes previously inhibited with cycloheximide was either slightly delayed or unaltered as compared to that of control oocytes newly treated with progesterone. The results indicate that an initial step of progesterone induced maturation in Rana is reversibly blocked but not detrimentally altered by cycloheximide. The evidence suggests that the effects of cycloheximide may be mediated through effects on the formation and/or autocatalytic production of maturation promoting factor (MPF). The cellular and molecular significance of these data to the process of meiotic arrest and oocyte maturation are discussed.

Inhibition of maturation of isolated rat oocytes by porcine follicular fluid.

The inhibitory action of porcine follicular fluid (FF1) on the spontaneous maturation of isolated rat oocytes was studied. Both FF1 and a low-molecular-weight fraction thereof (PFF1) inhibited the maturation of oocytes in culture. When the oocytes were scrutinized for maturation after culturing for 6 h in a medium containing 50% (v/v) FF1, the incidence of germinal vesicle breakdown (GVB) was reduced from 75 to 53% (P &lt; 0·001) in oocytes collected 20 h after administration of pregnant mare serum gonadotrophin (PMSG), and from 94 to 53% (P &lt; 0·01) in oocytes collected from preovulatory follicles 44 h after treatment with PMSG. Prolongation of the culture period to 20 h resulted in an increase in the number of oocytes undergoing GVB to 58% (harvested 20 h after PMSG treatment; P &lt; 0·001) or 71% (44 h after treatment with PMSG; P &gt; 0·05). Inhibition of GVB by PFF1 occurred at concentrations ≥ 0·085 mg protein/ml; the extent of inhibition was related directly to the concentration of inhibitor and inversely to the duration of culture and the developmental stage of the follicles from which the oocytes were derived. The inhibition of the resumption of meiosis by FF1 or PFF1 was overcome by ovine LH (5 μg/ml). However, GVB was delayed even in the presence of LH, compared with controls cultured in the absence of the inhibitor. These results demonstrate that the maturation-inhibiting action of porcine follicular fluid is not species specific and that the sensitivity of the rat oocyte to the inhibitor changes during the course of follicular development.