Role of protein kinase C activation in oocyte maturation and steroidogenesis in ovarian follicles of Rana pipiens: Studies with phorbol 12-myristate 13-acetate

In ovarian follicles of Rana pipiens, frog pituitary homogenates (FPH) elevate intrafollicular progesterone levels which in turn is thought to induce meiotic resumption in the prophase I arrested oocytes. Calcium plays a role in FPH and steroid-provoked responses in the somatic and gametic components of the follicle, presumably via effects exerted at the plasma membrane of their respective target cells. Many membrane active hormones which utilize Ca²⁺ in their intracellular transduction also provoke membrane phosphoinositide hydrolysis yielding inositol triphosphate (IP₃) and diacyl glycerol (DAG), an activator of the CA²⁺-dependent protein kinase C (PKC). The actions of phorbol 12-myristate 13-acetate (TPA), a potent synthetic activator of PKC, on progesterone production and oocyte maturation was examined in in vitro cultured ovarian follicles. TPA induced germinal vesicle breakdown (GVBD) in intact follicles and in oocytes denuded of somatic components, while the inactive compound phorbol 13-monoacetate was ineffective. Further, TPA induction of GVBD exhibited similarities to progesterone-induced GVBD, being inhibited by treatments which elevate cAMP or inhibit protein synthesis. TPA alone did not elevate intrafollicular or medium progesterone levels, as occurred in FPH-treated follicles. TPA partially inhibited intrafollicular progesterone accumulation induced by FPH or treatments which elevate cAMP levels. These data suggest that activation of PKC plays a role in oocyte maturation independent of follicular progesterone production as occurs in response to FPH. Further, it appears that the somatic cells of the amphibian follicle also possess PKC which when activated, antagonizes cAMP generating pathway in these cells. Results indicate that protein kinase can influence oocyte maturation in Rana follicular oocytes by several mechanisms.     

Hormonal dissociation of ovulation and maturation of oocytes: Ovulation of immature amphibian oocytes by prostaglandin

Prostaglandin involvement in ovulation and maturation of amphibian (Rana pipiens) ovarian follicular oocytes was investigated using in vitro-cultured ovarian follicles. Exposure of follicles to PGF₂α during culture stimulated variable but generally low levels of ovulation without concomitant induction of maturation. Addition of PGF₂α to cultured follicles markedly enhanced the incidence of ovulation in follicles exposed to progesterone or frog pituitary homogenate (FPH). Onset of the ovulatory process was further accelerated following addition of PGF₂α to FPH-treated follicles. PGE, in contrast to PGF₂α, exhibited no stimulatory effects on ovulation and consistently inhibited ovulation induction by FPH and progesterone. Cytological analysis of follicles undergoing ovulation revealed that ovulation of immature oocytes induced by PGF₂α varied markedly from that seen following FPH or progesterone stimulation of follicles in vivo or in vitro. Immature oocytes in contrast to maturing oocytes were typically ovlulated with follicle cells still attached to the vitelline membrane. The observations indicate that PGF₂α effected follicle rupture and contraction of the follicular epithelium and theca without prior separation of the follicle cells from the oocyte. Selective inhibitors of steroid synthesis (cyanoketone) and protein synthesis (cycloheximide) inhibited FPH-induced ovulation and maturation. PGF₂α reversed the inhibitory effects of cyanoketone and cycloheximide on FPH-induced ovulation but not maturation of oocytes. Neither prostaglandins alone or in combination with progesterone or FPH induced ovulation of oocytes following removal of the follicular epithelium. Ovulatory effects of PGF₂α appear to be mediated through the follicular epithelium. Results indicate that ovulation and maturation of amphibian oocytes can be induced independently of each other by separate classes of hormones. Normal synchronization of ovulation and maturation of oocytes may require the combined action of prostaglandins and steroids acting within different follicular compartments.     

Evidence for Follicle Wall Involvement in Ovulation and Progesterone Production by Frog (Rana pipiens) Follicles in vitro

Involvement of different cellular investments of the amphibian ovarian follicle wall in the ovulatory process, progesterone production, and oocyte maturation was investigated. Following microdissection, to selectively remove one or more layers (surface epithelium, theca, follicle cells) of the follicle wall, dissected and undirected ovarian follicles were treated with frog pituitary homogenate (FPH) or progesterone. Intact follicles ovulated in response to pituitary homogenate and this was associated with contractions of the follicle wall. Ovulation and follicular contractions were not observed following removal of the surface epithelium without removing the thecal layer. Oocyte maturation occured in response to FPH following removal of the surface epithelium alone or together with the theca, but not in the absence of the follicle cells. Intact follicles were most responsive to FPH with respect to progesterone production, and removal of all somatic cells from oocytes obliterated FPH stimulated progesterone production. Oocytes, regardless of wether any or all follicular wall layers were removed, matured but did not ovulate following exposure to progesterone. The results suggest that the surface epithelium, but not the theca, is required for FPH-induced extrusion (ovulation) of the oocyte from ovarian follicle wall. Additionally, the somatic tissue rather than the oocyte appears to be the cells producing progesterone following FPH treatment. The results indicate that separate cellular layers (individually and/or as a result of interactions) of the follicle wall carry out different functions during follicular differentiation and mediation of ovulation. Data provide functional evidence for a role of the surface epithelium in controlling the process of ovulation and follicular contraction.     

Induction and inhibition of meiotic maturation of amphibian (Rana dybowskii) follicular oocytes by forskolin and cAMP in vitro

Previous studies have demonstrated that direct or indirect elevation of cAMP levels in cultured amphibian ovarian follicles simultaneously stimulated production of oocyte maturation-inducing steroid (progesterone) by the follicles and inhibited oocyte maturation induced by endogenous or exogenous hormone. The duration of cAMP stimulation influenced arrest and reinitiation of oocyte meiotic maturation in ovarian follicles of Rana dybowskii. Addition of forskolin (adenylate cyclase stimulator) to cultured follicles inhibited both progesterone- and frog pituitary homogenate (FPH)-induced oocyte maturation. Similar inhibitory results were obtained when hormone-treated follicles were cultured in the continual presence of cAMP. Oocyte maturation increasingly occurred in follicular oocytes when cAMP or forskolin addition was delayed following treatment with FPH or progesterone. Transient exposure (6-8 hr) of ovarian follicles to forskolin or cAMP markedly stimulated oocyte maturation as well as accumulation of progesterone as measured by radioimmunoassay within the ovarian follicles. Forskolin was more effective than cAMP, at the dose tested, in stimulating progesterone production and accumulation by the follicles. The data demonstrate that transient manipulation (elevation) of cAMP levels in cultured follicles, without added FPH or steroid, was sufficient to initiate oocyte maturation. Results suggest that, with transient exposure to forskolin or exogenous cAMP, there is a sequential increase and decrease in endogenous cAMP levels in the somatic cells and germ cell components of the ovarian follicle. These changes appear to mediate production of maturation-inducing steroid and secondarily allow its effects on the oocyte to be expressed.     

Involvement of protein kinase C in the regulation of oocyte maturation in amphibians (Rana dybowskii)

Ovarian oocytes of Rana dybowskii, isolated early in the hibernation period (late autumn), failed to mature, i.e., germinal vesicle breakdown (GVBD), in response to progesterone during in vitro follicle culture. Oocytes collected during the middle hibernation period matured in response to progesterone, whereas those collected late during the hibernation period (close to the breeding season) underwent spontaneous maturation without added hormone (Kwon et al., '89). The maturational response (GVBD) of oocytes, collected at the three stages of hibernation, to protein kinase C (PKC) activation was investigated and compared to that of progesterone stimulation. A phorbol ester, phorbol 12-myristate 13-acetate (TPA) was used for PKC activation. TPA addition to cultured follicles collected during the early or middle period of hibernation induced oocyte GVBD. The incidence of maturation (% GVBD) induced by TPA varied markedly between animals. TPA (10 microM) induced oocyte maturation in the presence or absence of follicle cells. The time course of the TPA-induced maturation was similar to that of progesterone-stimulated maturation (ED50, 7-9 h). TPA also accelerated the onset of maturation of the follicular oocytes exhibiting spontaneous in vitro maturation. Both TPA- and progesterone-stimulated maturation was blocked by treatment with cycloheximide (1 microgram/2 ml), forskolin (9 microM) (an adenylate cyclase stimulator), and verapamil (0.27 mM) (a calcium transport blocker). Treatment of oocytes with a calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) (100 microM) or a PKC inactivator 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine (H-7) (50 microM) likewise suppressed TPA- or progesterone-induced maturation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium effects on progesterone accumulation and oocyte maturation in cultured follicles of Rana pipiens

Pituitary homogenates (FPH) provoke a cascade of responses in the amphibian ovarian follicle, culminating in progesterone biosynthesis and oocyte maturation (GVBD). Calcium may play an important role as an intracellular second messenger in regulating these physiological responses. Experiments were carried out on cultured, isolated follicles of Rana pipiens to assess the effects of varying extracellular calcium on follicular progesterone accumulation and oocyte maturation. In hormonally unstimulated follicles, an increase in extracellular Ca2+ alone produced a significant increase in progesterone in methanol extracts of follicles after 4 hours of culture, and in some cases also provoked oocyte maturation assessed after 24 hours of culture. In no case did elevated Ca2+ alone stimulate maximal progesterone accumulation as compared with FPH-stimulated follicles, although the time-course of accumulation was similar. The calcium ionophore, A-23187, similarly increased progesterone accumulation in a dose-dependent manner when introduced in amphibian Ringer's (1.35 mM Ca2+), but inhibited progesterone elevation caused by increasing calcium concentrations in the culture media and FPH stimulation. Depleting free calcium from the culture medium with graded doses of the chelator EGTA decreased FPH-induced progesterone accumulation and inhibited FPH- and progesterone-induced GVBD. The calcium channel blocker, verapamil, also inhibited FPH-induced progesterone accumulation and GVDB in a dose-dependent manner, while having no effect on progesterone-induced meiotic resumption. These data strongly implicate intracellular calcium levels regulating progesterone production by ovarian follicle cells and subsequent oocyte maturation.     

Dichotomous effects of forskolin on somatic and germ cell components of the ovarian follicle: evidence of cAMP involvement in steroid production and action

The role of cyclic AMP (cAMP) in ovarian follicular functions in Rana pipiens was investigated with the use of the adenylate cyclase stimulator, forskolin, which is thought to elevate intracellular level of cAMP. Effects of forskolin on oocyte germinal vesicle breakdown (GVBD) and on progesterone production by the follicles were assessed during the course of in vitro culture. Addition of forskolin to culture medium suppressed both progesterone-and frog pituitary homogenate (FPH)-induced meiotic maturation of the oocytes. Inhibitory effects of forskolin were essentially reversible and forskolin completely inhibited GVBD when added during the first four hours of incubation following exposure to progesterone. Forskolin alone stimulated a low level progesterone production by isolated follicles, but markedly stimulated progesterone production when it was supplemented with a low dose of FPH (0.005 pituitary equivalent/ml). Thus, forskolin acts synergistically with FPH on follicle cells to stimulate progesterone production. A higher dose of FPH (0.05 pitui. eq./ml) produced no additional synergistic effect of forskolin. Therefore, forskolin appears to have two contradictory functions in ovarian follicles: it augments FPH induced follicle secretion of meiosis initiator, progesterone, and simultaneously suppresses the maturation of the oocytes triggered by exogenous progesterone or FPH. The data presented indicate that there are two independent adenylate cyclase systems in the ovarian follicles which have separate functions: one in the follicle cells and the other in the oocyte. The two enzyme systems are thus compartmentalized and regulate different biological functions using the same messenger, cAMP. The data provide evidence that in amphibians, as in mammals, pituitary hormones regulate steroid hormone production by follicle cells via a cyclic AMP system. Thus, control of oocyte maturation induction appears to be determined by the relative levels of cAMP present in the follicle cells and oocytes.     

Steroidogenesis in Fundulus heteroclitus. IV. Dichotomous effects of a phorbol ester on ovarian steroid production and oocyte maturation.

The possible role of protein kinase C (PKC) activation in mediating the stimulatory actions of a Fundulus pituitary extract (FPE) on ovarian steroidogenesis and oocyte maturation was investigated. The phorbol ester, phorbol 12-myristate 13-acetate (PMA), alone slightly increased basal 17 alpha-hydroxy,20 beta-dihydroprogesterone (DHP) and 17 beta-estradiol (E2) synthesis and significantly stimulated germinal vesicle breakdown (GVBD). Addition of FPE promoted synthesis of DHP, testosterone (T), and E2, and initiated GVBD. Phorbol ester inhibited FPE-induced steroidogenesis but increased the number of oocytes that underwent GVBD. Phorbol ester also markedly impeded induction of steroidogenesis by dibutyryl cAMP and differentially affected the conversion of 25-hydroxycholesterol, pregnenolone, or progesterone to DHP, T, and E2: DHP production was not affected; T production diminished; and E2 synthesis increased (T aromatization also increased). These results suggest an inhibitory role for the PKC pathway on FPE-induced ovarian steroid production, with PMA appearing to affect various steroidogenic steps. The stimulatory action of PMA on oocyte maturation seems to be independent of follicular steroid production since aminoglutethimide, an inhibitor of steroidogenesis, did not block PMA-induced GVBD. Moreover, PMA had a marked stimulatory effect on GVBD in denuded oocytes. Thus, in contrast to the inhibitory role found for the PKC pathway on ovarian follicular steroidogenesis, activation of PKC in the oocyte may serve as a signal-transducing mechanism leading to GVBD.     

Stimulatory and Inhibitory Effects of Human Follicular Fluid on Amphibian Oocyte Maturation and Ovulation hi vitro

Follicular fluid was collected from individual human ovarian follicles and its effects, alone or in combination with frog pituitary homogenate (FPH), on oocyte maturation and ovulation were assessed following incubation with amphibian ovarian follicles in vitro. Oocyte maturation, with little or no concomitant ovulation, was induced by variable amounts of follicular fluid. Some of the individual follicular fluid samples were very active in inducing oocyte maturation, whereas others were inactive. Frog pituitary homogenates exhibited biologic activity (induced oocyte maturation and ovulation) when incubated in the presence of most follicular fluid samples. However, follicular fluid samples from two individuals inhibited ovulation but not maturation in FPH-treated follicles. These results demonstrate that amphibian follicles remain viable and undergo a number of physiologic changes in the presence of unfractionated human follicular fluid. Under appropriate conditions both stimulatory and inhibitory effects of follicular fluid were observed. These data suggest that amphibian ovarian follicles may provide a simple and independent means for detecting and assaying a number of biologic activities present in follicular fluid obtained from single human and other mammalian ovarian follicles. Such results may provide the basis for dissociating endocrine and cellular interactions which occur during normal and abnormal follicular differentiation.     

Cholesterol mediation of progesterone production and oocyte maturation in cultured amphibian (Rana pipiens) ovarian follicles

Treatment of isolated amphibian ovarian follicles with frog pituitary homogenate (FPH) increases follicular progesterone levels, which, in turn, initiate oocyte maturation. Recent studies have demonstrated that follicular progesterone production requires concomitant protein synthesis at some stage preceding pregnenolone formation. Experiments were carried out to determine whether cholesterol metabolism plays a role in mediating these biochemical and physiological processes. Aminoglutethimide (AGI, and inhibitor of P450 side-chain cleavage enzyme) inhibited FPH-induced intrafollicular progesterone accumulation and oocyte maturation (or germinal vesicle breakdown, GVBD) in a dose-dependent manner. Follicular progesterone accumulation and GVBD were both stimulated, in the absence of FPH, after addition of 25-OH-cholesterol, but not cholesterol, to the culture medium. Higher levels of progesterone were present in defolliculated oocytes as compared to intact ovarian follicles after incubation with 25-OH-cholesterol. The results indicate that the surface epithelium and theca layer in the follicle wall retard 25-OH-cholesterol access to steroid-producing follicle cells. AGI blocked 25-OH-cholesterol-induced accumulation of progesterone and GVBD in defolliculated oocytes, suggesting that 25-OH-cholesterol does not directly induce GVBD and is metabolized by the follicle cells. The capacity of follicles to accumulate progesterone following preincubation with FPH or 25-OH-cholesterol along with AGI was compared. Intrafollicular levels of progesterone increased after AGI- and 25-OH-cholesterol-treated follicles were washed. In contrast, progesterone levels decreased in follicles pretreated with AGI and FPH after washing. The results indicate that considerable 25-OH-cholesterol, but not endogenous cholesterol (FPH stimulation), remains available for steroidogenesis after removal of AGI. A significant, but incomplete, inhibition of progesterone accumulation occurred when follicles were incubated in the presence of 25-OH-cholesterol and cycloheximide. This partial blockage produced by the protein synthesis inhibitor indicates that some basal protein synthesis is required for progesterone accumulation from exogenous 25-OH-cholesterol. We conclude that intracellular cholesterol stores in the follicle wall are utilized to mediate FPH induction of progesterone accumulation and oocyte maturation in amphibian follicles.     

In-vitro Production of Meiosis Inducing Substance (MIS) by Isolated Amphibian (Rana pipiens) Follicle Cells*

Previous studies indicated that pituitary hormone induced oocyte maturation in preovulatory amphibian ovarian follicles is mediated by somatic elements of the follicle. In this study procedures were developed for isolating and culturing follicle cells and their ability to produce meiosis inducing substance (MIS) was assessed. Defolliculated oocytes surrounded by a single layer of follicle cells but not denuded oocytes matured in response to frog pituitary hormone (FPH) stimulation. Cultured follicle cells secreted MIS following stimulation with FPH. The amount of MIS activity produced was related to the number of follicle cells cultured and the dose of FPH utilized. Radioimmunoassay (RIA) analysis of medium from follicle cell cultures demonstrated that FPH stimulated steroid (progesterone) secretion from these cells. Addition of cAMP to follicle cell cultures enhanced FPH stimulated steroid production. The results indicate that follicle cells retain FPH responsiveness when uncoupled from the immature oocyte and exhibit both MIS and steroid secretory functions.     

Studies on the Mechanism of Action of Estradiol in Regulating Follicular Progesterone Levels: Effects on cAMP Mediated Events and 3β-Hydroxysteroid Dehydrogenase

Previous studies demonstrated that estradiol interferes with pituitary-induced progesterone production and oocyte maturation in cultured amphibian ( Rana pipiens ) ovarian follicles. To elucidate the mode of action of estradiol in modulating follicular progesterone accumulation we have examined its effects on cAMP-induced progesterone production and enzymatic conversion of pregnenolone to progesterone by 3β-hydroxysteroid dehydrogenase (3β-HSD). Follicular cAMP levels were manipulated with forskolin (an adenylate cyclase activator), isobutyl methyl xanthine (IBMX-phosphodiesterase inhibitor) and exogenously added cAMP. Progesterone production induced by forskolin alone or forskolin in combination with frog pituitary homogenate (FPH) was inhibited by estrogen. Addition of estradiol to culture medium markedly inhibited follicular progesterone accumulation following treatment of follicles with cAMP and IBMX. In the presence of exogenous pregnenolone, non-FPH stimulated ovarian follicles effectively converted the 3β-HSD substrate to progesterone. Treatment of follicles with estradiol inhibited conversion of pregnenolone to progesterone. The results indicate that estradiol acts, following FPH stimulation, at one or more steps subsequent to elevation of cAMP levels to regulate intrafollicular progesterone accumulation and oocyte maturation. Estrogen appears to directly influence the enzymatic (3β-HSD) conversion of pregnenolone to progesterone.     

Protein synthesis involvement in regulating pituitary-induced progesterone levels in ovarian follicles of Rana pipiens

Involvement of protein synthesis in frog pituitary homogenate (FPH)-induced progesterone production and/or accumulation in ovarian follicles was investigated. In amphibians, cycloheximide (C), an inhibitor of protein synthesis, inhibits progesterone and FPH-induced germinal vesicle breakdown (GVBD). However, the site and mechanisms of action of cycloheximide within ovarian follicles have not been elucidated. Intrafollicular progesterone produced by FPH is considered to mediate oocyte maturation; thus, cycloheximide may interfere with production and/or action of progesterone. Simultaneous treatment of FPH-stimulated follicles with cycloheximide inhibited FPH-induced progesterone accumulation (measured by RIA) and the accompanying-GVBD in a dose-dependent fashion. Inhibitory effects of cycloheximide on either FPH-induced progesterone production or GVBD were not reversed when follicles were washed and returned to fresh medium devoid of FPH and cycloheximide. However, subsequent restimulation of washed follicles with FPH resulted in increased progesterone levels and oocyte maturation. The extent of reversibility, in terms of GVBD and progesterone production, after FPH restimulation varied between animals. Pretreatment of follicles with cycloheximide for 6 hours, without FPH, had little or no effect on progesterone production when follicles were washed and treated with FPH. Delayed addition of cycloheximide to follicles following FPH stimulation blocked further progesterone accumulation as indicated by measurement of intrafollicular progesterone at the time of cycloheximide addition and at the end of the incubation period. The results indicate that cycloheximide rapidly inhibits progesterone production and that continuous protein synthesis is required for progesterone accumulation. Furthermore, protein synthesis does not appear to be required for progesterone metabolism since intrafollicular progesterone declined with prolonged culture even in the presence of cycloheximide. The nature of protein(s) involved in follicular progesterone production remains to be elucidated. FPH mediation of oocyte maturation within ovarian follicles appears to depend upon protein synthesis in somatic follicle cells, which is required for progesterone production, and in the oocyte, to mediate the response to the steroid trigger.     

An essential role for putrescine biosynthesis during meiotic maturation of amphibian oocytes

The objective of this study was to investigate the role of polyamines during meiotic maturation of Xenopus oocytes. The results indicate a rapid and significant increase in the activity of ornithine decarboxylase (ODC), the rate-limiting enzyme in the polyamine biosynthetic pathway, during the meiotic maturation induced by either progesterone or human chorionic gonadotropin (HCG). This increase in the enzyme activity was followed by an accumulation of putrescine without any effect on the levels of spermidine or spermine. The inhibition of ODC activity and the accumulation of putrescine levels by α-difluoromethyl ornithine (DFMO), a catalytic irreversible inhibitor of ODC, also resulted in the inhibition of maturation mediated by progesterone in Xenopus oocytes. DFMO caused an inhibition of both maturation and ovulation induced by HCG in ovarian fragments. This inhibition was readily reversible by exogenous supply of putrescine to the medium. These observations suggest that putrescine plays an important role during the meiotic maturation of amphibian oocytes.     

Role of phosphatidylinositide metabolism in ras-induced Xenopus oocyte maturation.

Microinjection of Xenopus oocytes with ras protein (p21) was used to investigate the role of phospholipid metabolism in ras-induced meiotic maturation. Induction of meiosis by ras was compared with induction by progesterone, insulin, and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Neomycin, which specifically binds to phosphatidylinositides and inhibits their metabolism, blocked meiotic maturation induced by ras or insulin but not by progesterone or TPA. In addition, p21 and TPA, but not insulin or progesterone, stimulated the incorporation of 32Pi into oocyte lipids. ras protein specifically stimulated 32P incorporation into phosphatidylinositides, whereas both ras and TPA stimulated 32P incorporation into phosphatidylcholine and phosphatidylethanolamine. The stimulatory effect of p21 on phosphatidylinositide metabolism correlated with the dose response and kinetics of ras-induced meiotic maturation. In addition, the ras oncogene protein was more potent than the proto-oncogene protein both in inducing meiotic maturation and in stimulating phosphatidylinositide metabolism. These results indicate that phosphatidylinositide turnover is required for ras-induced meiosis and suggest that phosphatidylinositide-derived second messengers mediate the biological activity of ras in Xenopus oocytes.     

Induction and inhibition of amphibian (Rana pipiens) oocyte maturation by protease inhibitor (TPCK)

We report for the first time that oocyte nuclear and cytoplasmic maturation are triggered in vitro in non-hormone-treated amphibian (Rana pipiens) ovarian follicles following transient exposure to synthetic chymotrypsin inhibitor Nα-tosyl-L-phenylalanine-chloromethyl ketone (TPCK). The mechanism of action of TPCK in regulating oocyte maturation was investigated and compared to that induced by progesterone or pituitary hormone. Follicular oocytes failed to mature following continuous exposure to the same doses of TPCK in the presence or absence of progesterone. Continuous treatment of follicles with lower levels of TPCK occasionally induced GVBD in the absence of progesterone and augmented maturational effects of low levels of progesterone. TPCK induced maturation of intrafollicular oocytes without stimulating progesterone production and also induced maturation of naked oocytes. Stimulation of follicular progesterone synthesis following gonadotropin stimulation or addition of pregnenolone was inhibited by TPCK, indicating that TPCK affects metabolic processes in both the somatic and germinal components of the ovarian follicle. Oocyte maturation induced by either TPCK or progesterone was inhibited by cycloheximide, calcium-deficient medium, and forskolin. Results suggest that TPCK induces oocyte maturation independent of steroidogenesis via mechanisms similar to those triggered by progesterone involving protein synthesis, formation of cytoplasmic maturation-promoting factor (MPF), and changes in cAMP levels. Our data indicate that a chymotrypsin-like protease plays a role(s) in regulating the oocyte meiotic maturation process.     

Sources of calcium and the involvement of calmodulin during steroidogenesis and oocyte maturation in follicles of Rana pipiens

In the amphibian ovarian follicle, progesterone production is thought to induce maturation of the enclosed oocyte. Intracellular mechanisms regulating these events in the somatic and germ cells are incompletely understood. However, calcium appears to play a role in the production and action of progesterone. Experiments using calcium antagonists were carried out to delineate the role of extra- and intracellular calcium during in vitro stimulation of follicular steroidogenesis and oocyte maturation. Calcium-free medium, verapamil, and La3+ were used to block Ca2+ influx and inhibited follicular progesterone accumulation in response to frog pituitary homogenate (FPH) or exogenous cAMP + IBMX. Progesterone accumulation was not impaired under identical conditions when pregnenolone was added to cultured follicles. TMB-8, an inhibitor of intracellular Ca2+ mobilization, partially inhibited progesterone levels stimulated by FPH at low doses but not higher doses of the inhibitor. However, TMB-8 inhibited FPH-induced oocyte germinal vesicle breakdown (GVBD) in a dose-dependent manner, as well as maturation due to exogenous progesterone or La3+. Calmodulin antagonists, W-7, R24571, and trifluoperazine, were used to assess the involvement of calmodulin in the responses of these two cell types. All three antagonists inhibited progesterone accumulation induced by FPH with the apparent order of potency being R24571 greater than W-7 greater than TFP. W-7 inhibited cAMP-induced progesterone elevation, but had no effect on conversion of pregnenolone to progesterone. Of these three calmodulin antagonists, only R24571 exhibited a dramatic ability to inhibit GVBD induced by exogenous progesterone and was associated with morphologic alterations in the oocytes. These data suggest that Ca2+, acting through calmodulin at some specific step(s) distal to cAMP elevation and prior to pregnenolone formation, is involved in FPH-induced progesterone accumulation, apparently with the participation of both extracellular and intracellular pools of Ca2+. In the oocyte, mobilization of Ca2+ from intracellular stores appears to be of primary importance to maturation while extracellular Ca2+ is not. These data provide further evidence that Ca2+ mediates the hormonally provoked responses in both cell types in the intact follicle, but that the source of Ca2+ may differ. Using intact follicles it seems apparent that exploiting this difference with selective inhibitors provides a means for differential modulation and functional uncoupling of these cells with regard to steroidogenesis and steroid action.     

Protein kinase C (PKC) role in bovine oocyte maturation and early embryo development

The aims of the present study were to determine the role of protein kinase C (PKC) on meiotic resumption and its effects on pronuclear formation and cleavage in the bovine. Oocytes were matured in the presence of 0, 1, 10 and 100 nM of phorbol 12-myristate 13-acetate (PMA), to evaluate the percentage of germinal vesicle breakdown. To study pronuclear formation and cleavage, oocytes were randomly distributed in four groups and matured in modified TCM-199 with LH and FSH (negative control); 10% of estrous cow serum (positive control); 100 nM of PMA (treatment); 100 nM of 4alpha-PDD (phorbol ester control). Oocytes were also matured in positive control medium, fertilized and transferred to KSOM with increasing concentrations of a PKC inhibitor. The protein profile and the presence of PKC at the end of maturation period were determined by SDS-PAGE followed by Silver Stain and Western blot, respectively. PMA stimulated meiotic resumption in a concentration-dependent manner. PKC stimulation during oocyte maturation caused an increase in pronuclear formation and did not cause parthenogenetic activation. Inhibitor of PKC (MyrPKC) inhibited cleavage in a dose-dependent and irreversible manner. A protein band around 74 kDa was not detected in PMA-treated oocytes and PKC was not detected by Western blot at the end of the maturation period. In conclusion, meiotic resumption was accelerated and the rate of oocytes with two pronuclei was increased when PKC was activated during oocyte maturation. Moreover, cleavage was inhibited in the presence of PMA.     

Role of cAMP in modulating intrafollicular progesterone levels and oocyte maturation in amphibians (Rana pipiens)

The role of cAMP in regulating follicular progesterone levels and oocyte maturation was investigated following in vitro culture of amphibian (Rana pipiens) ovarian follicles. Intrafollicular levels of cAMP were manipulated with the use of a stimulator of cAMP synthesis (forskolin) or by exogenous addition of cAMP alone or either of these in combination with an inhibitor of cAMP catabolism (3-isobutyl-1-methyl xanthine, IBMX). Follicular progesterone content was determined by RIA and oocyte maturation was assessed cytologically. In the presence of increasing doses of forskolin (0-3 microM), cAMP (0-3 mM), or dibutyryl cAMP (dbcAMP, 0-2.5 mM) increasing but low levels of progesterone were detected. Increasing doses of IBMX (0-0.09 mM) alone had no significant effect on follicular steroid content. Exogenous cAMP, dbcAMP, or IBMX (0.09 mM) suppressed hormone-induced oocyte maturation. Simultaneous exposure of follicles to increasing doses of both forskolin (0-3 microM) and IBMX (0-0.09 mM) markedly increased intrafollicular progesterone levels to those produced by frog pituitary homogenate (FPH). A marked increase in progesterone levels also occurred when follicles were exposed to exogenous cAMP (3 mM) and IBMX (0.09 mM). These results indicate that exogenous cAMP is incorporated by follicle cells and that forskolin effects are mediated through cAMP. Changes in follicular progesterone levels (increase and decrease) over time following FPH or cAMP manipulation (cAMP + IBMX or forskolin + IBMX) were essentially identical. In contrast to cAMP, cGMP was inactive in inhibiting hormone induced GVBD or stimulating follicular progesterone accumulation. Elevation of follicular and medium levels of progesterone resulting from FPH or cAMP stimulation required the presence of the somatic follicular cells. The decrease in follicular progesterone levels with prolonged culture was not associated with a corresponding increase in progesterone levels in the medium. The decrease in follicular progesterone levels appears to reflect steroid catabolism rather than loss of steroid to the culture medium. The results suggest that the level of intracellular cAMP in the follicle cells is modulated by the relative activity of the adenylate cyclase system and phosphodiesterase and that FPH can affect both components. Thus, intracellular levels of cAMP play a key role in regulating follicular progesterone levels and FPH action on the follicle cells. The steroidogenic capacity of follicle cells can be manipulated independently of FPH stimulation.     

Spontaneous and LH-induced maturation in Bufo arenarum oocytes: importance of gap junctions

It has been demonstrated in Bufo arenarum that fully grown oocytes are capable of meiotic resumption in the absence of a hormonal stimulus if they are deprived of their follicular envelopes. This event, called spontaneous maturation, only takes place in oocytes collected during the reproductive period, which have a metabolically mature cytoplasm. In Bufo arenarum, progesterone acts on the oocyte surface and causes modifications in the activities of important enzymes, such as a decrease in the activity of adenylate cyclase (AC) and the activation of phospholipase C (PLC). PLC activation leads to the formation of diacylglycerol (DAG) and inositol triphosphate (IP(3)), second messengers that activate protein kinase C (PKC) and cause an increase in intracellular Ca(2+). Recent data obtained from Bufo arenarum show that progesterone-induced maturation causes significant modifications in the level and composition of neutral lipids and phospholipids of whole fully grown ovarian oocytes and of enriched fractions in the plasma membrane. In amphibians, the luteinizing hormone (LH) is responsible for meiosis resumption through the induction of progesterone production by follicular cells. The aim of this work was to study the importance of gap junctions in the spontaneous and LH-induced maturation in Bufo arenarum oocytes. During the reproductive period, Bufo arenarum oocytes are capable of undergoing spontaneous maturation in a similar way to mammalian oocytes while, during the non-reproductive period, they exhibit the behaviour that is characteristic of amphibian oocytes, requiring progesterone stimulation for meiotic resumption (incapable oocytes). This different ability to mature spontaneously is coincident with differences in the amount and composition of the phospholipids in the oocyte membranes. Capable oocytes exhibit in their membranes higher quantities of phospholipids than incapable oocytes, especially of PC and PI, which are precursors of second messengers such as DAG and IP(3). The uncoupling of the gap junctions with 1-octanol or halothane fails to induce maturation in follicles from the non-reproductive period, whose oocytes are incapable of maturing spontaneously. However, if the treatment is performed during the reproductive period, with oocytes capable of undergoing spontaneous maturation, meiosis resumption occurs in high percentages, similar to those obtained by manual defolliculation. Interestingly, results show that LH is capable of inducing GVBD in both incapable oocytes and in oocytes capable of maturing spontaneously as long as follicle cells are present, which would imply the need for a communication pathway between the oocyte and the follicle cells. This possibility was analysed by combining LH treatment with uncoupling agents such as 1-octanol or halothane. Results show that maturation induction with LH requires a cell-cell coupling, as the uncoupling of the gap junctions decreases GVBD percentages. Experiments with LH in the presence of heparin, BAPTA/AM and theophylline suggest that the hormone could induce GVBD by means of the passage of IP(3) or Ca(2+) through the gap junctions, which would increase the Ca(2+) level in the oocyte cytoplasm and activate phosphodiesterase (PDE), thus contributing to the decrease in cAMP levels and allowing meiosis resumption.