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.     

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.     

Conservation of RNA polymerase during maturation of the Rana pipiens oocyte.

DNA-dependent RNA polymerase was extracted from oocytes of the frog, Rana pipiens. The bulk of the enzyme activity was present in the germinal vesicle and the amounts of each major form of such activity did not significantly change during oocyte maturation. Therefore, either nuclear polymerase activity is conserved after breakdown of the oocyte nucleus during maturation or, alternatively, de novo synthesis of the enzymes must occur during oocyte maturation concomitant with degradation. We have measured rates of protein synthesis in oocytes and determined a maximum rate of synthesis for RNA polymerases. Our kinetic studies show that no more than 20, 10, and 5% of RNA polymerases type I, IIa, and IIb, respectively, could be synthesized during steroid-induced oocyte maturation. These results thus show that the bulk of RNA polymerase accumulates in the germinal vesicle during oogenesis, is dispersed into the cytoplasm during maturation, and, since only limited synthesis seems to be occurring, the polymerase is available during embryogenesis.     

Inhibitory action of the gonadopeptide inhibin on amphibian (Rana pipiens) steroidogenesis and oocyte maturation.

In view of recent reports on the production of inhibin- and activin-like proteins in lower vertebrates and their important role during development, we have examined the effects of the gonadopeptide inhibin in the process of oocyte maturation using amphibian (Rana pipiens) fully grown preovulatory ovarian follicles cultured in vitro. In the presence of frog pituitary homogenate (FPH), which stimulates progesterone (P4) levels and the subsequent germinal vesicle breakdown (GVBD), purified porcine inhibin (35-50 IU) inhibited both of these responses in a dose-dependent manner. Inhibin also blocked GVBD initiated by exogenously added P4 in intact as well as denuded oocytes. Thus, inhibin seems to act at the follicle (granulosa) cells because it blocked steroidogenesis and at the oocyte because it altered the steroid-induced oocyte maturation. The P4-treated follicles were susceptible to the inhibin action during the first 3 hr of steroid stimulation, which indicates that inhibin affects some early events during the process of GVBD. Maximum inhibitory effect was observed when P4 and inhibin were added simultaneously at the beginning of the incubations. Moreover, the inhibitory effect on GVBD caused by the gonadopeptide was dependent on the length of exposure of the follicles to inhibin. The continuous presence of inhibin in the culture was required to block GVBD efficiently. Data also indicate that the inhibitory effect of inhibin was reversible. Taken together, results from this study present evidence that inhibin may be a relevant paracrine/autocrine regulator of ovarian functions.     

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.     

Cytodifferentiation in the Rana pipiens oocyte

Summary In early diplotene frog oocytes incubated to illustrate thiamine pyrophosphatase (TPPase) activity, reaction product is uniformly distributed within the compartments of the endoplasmic reticulum and nuclear envelope as well as within the saccules and small vesicles comprising the dictyosomes. With continued oocyte development the reaction product becomes concentrated in localized regions of the dictyosome saccules. Eventually, the enzyme is no longer apparent within the endoplasmic reticulum, but is concentrated in the cisternae of the inner dictyosome saccules. The variations noted suggest that the enzyme is synthesized early in diplotene by the endoplasmic reticulum and is subsequently transported to the Golgi apparatus where it is consistently observed at later developmental stages. TPPase activity is also present in the Golgi apparatus of follicle and theca cells as well as in ovarian epithelial cells. The enzyme is also detected in micropinocytotic vesicles contained within the cells comprising the follicle envelope and in intercellular spaces of the follicle. Horseradish peroxidase injected into the coelomic cavity is transported via micropinocytotic vesicles into and through the cells comprising the follicle envelope and in intercellular spaces. The exogenous protein is not found even after a prolonged time period in early diplotene oocytes. The protein is, however, present in large spherical and tubular vesicles in the cortex of vitellogenic oocytes approximately 500 microns in diameter. The possible functional role of the enzyme TPPase during oogenesis is discussed.This investigation was supported by a research grant from the National Science Foundation (GB-8736).     

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.     

Regulation of mouse oocyte maturation: involvement of cyclic AMP phosphodiesterase and calmodulin

A decrease in mouse oocyte cAMP occurs during commitment to resume meiosis (R. M. Schultz, R. R. Montgomery, and J. R. Belanoff, 1983, Dev. Biol. 97, 264-273). Experiments described in this report were performed to ascertain if oocyte cyclic nucleotide phosphodiesterase (PDE) is involved in this decrease. PDE activity was found in extracts of mouse oocytes. The activity appeared soluble and not membrane bound. For each of three different PDE inhibitors, a positive correlation was found between the ability of increasing concentrations of each compound to inhibit PDE in oocyte extracts and to inhibit germinal vesicle breakdown (GVBD). Moreover, the more potent the PDE inhibitor, the more effectively it inhibited GVBD. The possibility that calmodulin (CaM) plays a role in maturation was examined since CaM modulates PDE activity in other systems. About 0.3% of total oocyte protein is CaM as determined by radioimmunoassay and activation of exogenous PDE. A CaM-dependent step in maturation was suggested since the CaM inhibitors trifluoperazine and calmidizolium inhibited GVBD in a dose-dependent manner. In addition, the CaM inhibitors W7 and W13 inhibited GVBD at lower concentrations than the less-active corresponding congeners W5 and W12. Oocyte extracts contained a CaM-modulated PDE. Activity was inhibited about 50% by addition of EGTA, and fully restored by addition of exogenous CaM and excess calcium. cAMP hydrolysis was inhibited in a dose-dependent manner by either trifluoperazine, calmidizolium, or W7; maximal inhibition was also about 50%. CaM-modulated PDE, however, did not appear to be the target for the effects of CaM inhibitors on GVBD, since concentrations of W7 that inhibited maturation did not inhibit cAMP hydrolysis in the oocyte. Results from these studies suggest that oocyte PDE is involved in the decrease in cAMP associated with resumption of meiosis, but that the CaM-dependent step occurs subsequent to or concurrently with the drop in cAMP.     

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.     

The role of follicle cells in Rana pipiens oocyte maturation induced by delta 5-pregnenolone.

Previous studies have indicated that pituitary-initiated oocyte maturation in the amphibian is mediated by steroidogenesis in the somatic portion of the follicle. This study compares the ability of (1) oocytes surrounded by a single layer of follicle cells, (2) denuded oocytes, and (3) isolated follicle cells to metabolize Δ⁵-pregnenolone, the common precursor of the steroids. Use of radiolabeled compounds demonstrates that the conversion of Δ⁵-pregnenolone to progesterone is confined to the follicle cells, while further reduction of progesterone takes place in the oocyte. The follicle cells also convert Δ⁵-pregnenolone to a form more potent in inducing meiotic maturation. Thus, the behavior of follicle cells in isolation is consistent with the suggested site of pituitary action leading to meiotic maturation as proposed by an earlier theory.     

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.     

Development of calcium release mechanisms during starfish oocyte maturation

In response to the maturation-inducing hormone 1-methyladenine, starfish oocytes acquire increased sensitivity to sperm and inositol trisphosphate (InsP3), stimuli that cause a release of calcium from intracellular stores and a rise in intracellular free calcium. In the immature oocyte, the calcium release in response to 10 sperm entries is less than that seen with a single sperm entry in the mature egg. Likewise, the sensitivity to injected InsP3 is less in the immature oocyte. Approximately 100 times as much InsP3 is required to obtain the same calcium release in an immature oocyte as in a mature egg. However, with saturating amounts of InsP3, immature oocytes and mature eggs release comparable amounts of calcium. These results indicate that although calcium stores are well-developed in the immature oocyte, mechanisms for releasing the calcium develop fully only during oocyte maturation.     

The nature and fate of Rana pipiens proteins synthesized during maturation and early cleavage

Electrophoretic analysis of proteins synthesized during maturation, cleavage, and gastrulation indicates that patterns of protein synthesis are characteristic for each stage studied. Oocytes induced to maturein vitro markedly alter their pattern of protein synthesis during the period between 24 and 48 hours after hormone exposure. The same alteration occurs in oocytes from which the germinal vesicles were removed prior to hormone exposure, indicating that the oocyte cytoplasm during maturation is capable of regulating the nature of protein synthesis.     

Cumulus cells steroidogenesis is influenced by the degree of oocyte maturation

Background  

The possibility to predict the ability of a germ cell to properly sustain embryo development in vitro or in vivo as early as possible is undoubtedly the main problem of reproductive technologies. To date, only the achievement of nuclear maturation and cumulus expansion is feasible, as all the studies on cytoplasmic maturation are too invasive and have been complicated by the death of the cells analyzed. The authors studied the possibility to test the cytoplasmic quality of pig oocytes by evaluating their ability to produce steroidogenesis enabling factor(s). To this aim, oocytes matured under different culture conditions that allowed to obtain gradable level of cytoplasmic maturation, were used to produce conditioned media (OCM). The secretion of the factor(s) in conditioned media was then recorded by evaluating the ability of the spent media to direct granulosa cells (GC) steroidogenesis.