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.     

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.     

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.     

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.     

Spontaneous maturation of follicular oocytes in Rana dybowskii in vitro: seasonal influences, progesterone production and involvement of cAMP

Seasonal and hormonal influences regulating oocyte maturation (germinal vesicle breakdown, GVBD) in ovarian follicles of Rana dybowskii were investigated. During the early winter (Dec.-Jan.) GVBD occurred at a low incidence following in vitro culture of intact follicles. Addition of progesterone of frog pituitary homogenate (FPH) to such follicles induced oocyte maturation, whereas IBMX or forskolin inhibited hormone-induced oocyte maturation. The time course of spontaneous in vitro maturation varied markedly with the seasons and between animals. Follicles isolated from the ovaries in early February required 21-24 hours of culture to mature spontaneously, and addition of FPH or progesterone to the culture medium markedly accelerated the time course of GVBD. In contrast, follicles isolated in late February matured very rapidly (less than 6 hours), and FPH or progesterone were ineffective in accelerating the time course of GVBD. IBMX and forskolin separately or in combination stimulated follicular progesterone production, which resembled that seen following FPH stimulation. FPH addition to such follicles shifted the steroid peak to the left (accelerated) and increased the absolute amount of hormone detected in late-maturing follicles (50% GVBD, about 18 hours) but not in rapidly maturing follicles (50% GVBD, 3 hours). In contrast to other amphibians, a high incidence of spontaneous oocyte maturation occurred during in vitro culture. Essentially all animals exhibited spontaneous maturation during the normal breeding season, even those animals collected in the early winter and kept in artificial hibernation at 4 degrees C for extended periods.     

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.     

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.     

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.     

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.     

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.     

Induction of ovulation and oocyte maturation of amphibian (Rana dybowskii) ovarian follicles by protein kinase C activation in vitro.

We previously reported that protein kinase C (PKC) activation induced meiotic maturation (germinal vesicle breakdown, GVBD) of Rana dybowskii follicular oocytes cultured in vitro without hormone treatment. The experiments reported here were carried out to establish whether ovarian follicles ovulated in response to PKC activation during culture. A phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), was used for PKC activation. TPA addition (10 microM) to cultured ovarian fragments induced ovulation and maturation of the oocytes similar to that seen following addition of frog pituitary homogenate (FPH, 0.05 pituitary/ml) or progesterone (0.5 microgram/ml). Such changes were not observed when ovarian fragments were treated with inactive phorbol ester. The time course of TPA-induced ovulation was similar to that produced by FPH-stimulated ovulation. Both TPA- and FPH-stimulated ovulation and maturation were blocked by treatment with cycloheximide, forskolin (an adenylate cyclase stimulator), and 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine (H-7; a PKC inactivator). FPH treatment markedly increased progesterone levels in the medium during ovarian fragment culture whereas TPA treatment failed to elevate progesterone levels. Thus, TPA treatment mimics FPH and progesterone in inducing ovulation and meiotic maturation in cultured amphibian ovarian fragments. The data strongly suggest that PKC plays an important role in regulating ovulation as well as in modulating amphibian oocyte maturation during follicular differentiation.     

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.     

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.     

Vasoactive intestinal peptide stimulates oocyte maturation, steroidogenesis, and cyclic adenosine 3',5'-monophosphate production in isolated preovulatory rat follicles

Vasoactive intestinal peptide (VIP) is present in the rat ovary and has been shown to stimulate cyclic adenosine 3',5'-monophosphate (cAMP) and progesterone production in cultured rat granulosa cells. In the present study, VIP-stimulated cAMP production has been studied in relation to steroid accumulation and oocyte maturation in isolated preovulatory rat follicles. VIP stimulated resumption of meiosis (oocyte maturation) in up to 60% of the follicle-enclosed oocytes after 6 h at 1 microM (control, 1.8%; luteinizing hormone 99%). The effect was time- and dose-dependent up to 6 h and was seen with both natural and synthetic VIP. VIP also stimulated the accumulation of steroids (estrogen, 2.3-fold; testosterone, 2.0-fold; and progesterone, 1.6-fold increase after 6 h of incubation) and lactate (2.6-fold) by the follicles. VIP-increased tissue levels of cAMP in the follicle were dose- and time-dependent. This effect was potentiated by a phosphodiesterase inhibitor. When isolated oocyte-cumulus complexes were studied, VIP caused a transient inhibition of spontaneous oocyte maturation, and demonstrated no effect on denuded oocytes. These results extend earlier preliminary observations on the ability of VIP to induce meiotic maturation of follicle-enclosed oocytes. Our results also show that VIP can stimulate steroid and lactate accumulation in the isolated follicles. The pattern of steroids produced suggests an effect both on the theca- and granulosa cells. We also show that VIP can delay spontaneous oocyte maturation. These effects appeared, at least partially, to be mediated by cAMP.     

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.     

Induction and Inhibition of Meiotic Maturation in Follicle-enclosed Mouse Oocytes by Forskolin

A continuous exposure of follicle-enclosed mouse oocytes to ovine luteinizing hormone (LH, 10 μg/ml) in vitro resulted in a 3-fold elevation of CAMP levels in the follicle cells, but not the oocytes, with subsequent oocyte maturation. When follicle-enclosed oocytes were exposed to forskolin (0.01–10 μM) for 2 hr and then incubated in forskolin-free medium (transient exposure group), oocytes underwent germinal vesicle breakdown in a dose-dependent manner. In contrast, a continuous exposure of the follicles to forskolin (10 μM) for up to 10 hr failed to induce resumption of meiosis. Follicle cell cAMP levels increased within 2 hr after the initial exposure to forskolin, and thereafter decreased rapidly regardless of whether forskolin treatment was transient or continuous. A similar transient increase in oocyte cAMP levels was observed after transient or continuous treatment with forskolin. It was evident, however, that at any time examined oocyte cAMP levels were consistently higher in the continuous exposure group than in the transient exposure group. Furthermore, a continuous exposure to forskolin also blocked LH-induced meiotic maturation. These findings suggest that elevated levels of cAMP in the oocyte block meiotic maturation in mouse oocytes. The present results further suggest that an increase in follicle cell cAMP levels is essential to the LH-induced meiotic maturation.     

Induction and inhibition of in vitro oocyte maturation and production of steroids in fish follicles by forskolin

The effects of forskolin (FK) on in vitro oocyte maturation and production of steroids were examined in Oryzias latipes. When oocytes within preovulatory follicles were preincubated in the presence of FK for 2-10 hr, they matured normally after additional incubation for 10-20 hr in plain culture medium. Naked (follicle cell-free) oocytes did not mature under these conditions. FK stimulated dose-dependent production of steroids (estradiol-17 beta, E2, and 17 alpha,20 beta-dihydroxy-4-pregnen-3-one, 17 alpha,20 beta-diOHprog) and cAMP in follicle (granulosa) cells. On the other hand, exposure to FK within 2 hr after 17 alpha,20 beta-diOH prog stimulation caused reversible inhibition of gonadotropin (PMS)- or 17 alpha,20 beta-diOH prog-induced maturation of the intrafollicular oocytes in vitro. FK also significantly inhibited the 17 alpha,20 beta-diOHprog-induced maturation of naked oocytes, suggesting the existence of adenylate cyclase in fish oocytes. These data indicate that in Oryzias latipes, FK induces oocyte maturation by stimulating follicular production of maturation-inducing steroid (MIS), probably 17 alpha,20 beta-diOH prog, via an increase in cAMP, and that it may inhibit oocyte maturation by increasing ooplasmic cAMP and some inhibitory interaction between the granulosa cells and the oocyte through intercellular communication.     

Insulin induction of meiosis of rana pipiens oocytes: Relation to endogenous progesterone

The follicle wall was previously shown to be involved in insulin induction of oocyte maturation in Rana pipiens ovarian follicles. Steroidogenic involvement in insulin induction of maturation was investigated following development of a radioimmunoassay (RIA) for progesterone to measure endogenous progesterone associated with in vitro incubates. Insulin and frog pituitary homogenate (FPH) were both found to elevate progesterone levels significantly in these incubates. FPH was more effective in elevating progesterone levels than insulin and caused progesterone increase of about 2 orders of magnitude greater than insulin. Removal of the follicle wall eliminated the steroidogenic effects of insulin. Considerable interanimal variation was observed in the ability of insulin to induce oocyte germinal vesicle breakdown (GVBD) in intact follicles. The hypothesis was proposed that differences in endogenous progesterone might explain this variation. To test this hypothesis, an experiment was carried out in which hormone production and follicular sensitivity to insulin were simultaneously determined in follicles obtained from the same animals. Results of the experiment show that the ability of insulin to induce GVBD, as indicated by the effective concentration needed for 50% response (ED₅₀), was strongly correlated with the levels of endogenous progesterone as measured by RIA. The results provide direct evidence that insulin's action on the follicle wall involves steroid production. It was thus concluded that increased endogenous progesterone facilitates GVBD induction by insulin. It is unclear how the two hormones interact to produce an enhanced effect, but interactions at the receptor or postreceptor level may be involved. This follicle system may provide important insights into the mode of action and interaction of these two important hormones.     

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.     

Spontaneous oocyte maturation in Rana pipiens: Estrogen and follicle wall involvement

Immature (germinal vesicle stage) Rana pipiens oocytes typically remain arrested in prophase I of meiosis even after extended periods of in-vitro culture, if not stimulated with hormones. We have, however, sporadically observed “spontaneous” occurrences of oocyte maturation in vitro without the addition of hormones. This study documents some of our observations on this phenomenon and presents experimental results concerning the effects and possible involvement of estrogen and follicle wall components in regulating spontaneous oocyte maturation. Estrogen was found to inhibit spontaneous oocyte maturation (GVBD) in a dose-dependent fashion. Follicles in which spontaneous maturation was inhibited by estrogen retained their responsiveness (GVBD) to both frog pituitary homogenate (FPH) and progesterone stimulation. Inhibitory effects of estrogen on spontaneous maturation, however, were not reversed following incubation of washed follicles in plain culture medium without added hormones. Possible involvement of progesterone synthesis in spontaneous oocyte maturation was ascertained by simultaneously monitoring endogenous progesterone synthesis and the occurrence of spontaneous GVBD over the course of the maturation process. In spontaneous maturing follicle there was a gradual increase in basal levels of progesterone synthesis that preceded GVBD. Significantly, addition of estrogen abolished both the spontaneous progesterone production and spontaneous oocyte maturation. When FPH was added to follicles exhibiting spontaneous oocyte maturation, progesterone production was augmented and the time course of oocyte maturation was greatly accelerated. Involvement of ovarian components in the maturation process was investigated by selective removal of various follicle layers by microdissection. Removal of follicle epithelium and theca layer (defolliculation) markedly decreased spontaneous and FPH-induced maturation, whereas removal of the entire follicle wall (denudation) completely blocked it. Our results suggest that both spontaneous and FPH-induced maturation involve an estrogen sensitive process in the follicle wall. Thus, somatic follicle cells appear to serve as a common mediator for both types of maturation, which are linked by some intrafollicular mechanism involving steroidogenesis. Hence, estrogen may play an important role as an endogenous intrafollicular regulator of oocyte meiotic maturation.