Oocyte maturation inhibitor

The preovulatory surge of gonadotropins induces within the mature Graafian follicle a series of changes culminating in the release of a fertilizable ovum. These include resumption of the meiotic division, a process held in abeyance from a short time after birth, and the progression of the oocyte from the dictyate stage to the metaphase of the second meiotic division. Here the role of a follicular factor, oocyte maturation inhibitor (OMI), in preventing resumption of meiosis by ova of antral follicles prior to the surge of gonadotropins has been reviewed. The suggested involvement of OMI in regulation of meiosis is based on the following observations: (1) fully grown mammalian oocytes explanted from their follicles undergo meiotic maturation spontaneously, whereas follicle-enclosed ova remain immature until stimulated; (2) co-culture of oocytes isolated from their follicles with follicular granulosa cells, granulosa cell extract and follicular fluid inhibits the spontaneous maturation; (3) the inhibition of oocyte maturation by OMI is reversible and in several of the models employed can be removed by the addition of the physiological trigger of meiosis, luteinizing hormone (LH). The current state of OMI characterization and purification has been described and the involvement of additional factors, such as cyclic AMP, in the regulation of meiosis discussed.     

Effects of transforming growth factors and inhibin-related proteins on rat preovulatory graafian follicles in vitro

In view of recent reports on ovarian production and action of transforming growth factors (TGFs) and inhibin-related proteins (inhibin, activin, and follistatin), we have examined the effects of these hormones on the function of preovulatory follicles in vitro. Individual preovulatory follicles were obtained from PMSG-treated rats and incubated with these hormones in the absence or presence of LH. Oocyte maturation and progesterone production were monitored. Treatment with TGF alpha alone, but not with TGF beta or inhibin-related proteins, mimicked the action of LH on oocyte maturation by inducing the resumption of meiosis in follicle-enclosed oocytes (56.6% and 80.6% oocytes resumed meiosis in the presence of 0.5 and 1.0 microgram/ml TGF alpha, respectively). In follicle cultures treated with LH to induce oocyte maturation, cotreatment with inhibin and TGF beta (30-50 ng/ml), but not other related hormones, partially inhibited LH-induced meiosis in follicle-enclosed oocytes (from 82% mature ova in the presence of LH to 51% and 55% mature ova with TGF beta and inhibin, respectively). In contrast to follicle cultures, none of the hormones tested significantly affected the spontaneous maturation of rat oocytes explanted from their follicles and cultured within their cumulus mass for 4 h. Treatment with TGF alpha, but not with TGF beta, inhibin, activin, or follistatin, stimulated progesterone production. The present study demonstrated that TGF alpha, like LH, induces oocyte maturation and progesterone production in preovulatory rat follicles. Furthermore, inhibin and TGF beta suppressed LH-induced resumption of meiosis in follicle-enclosed oocytes. Because these growth factors and inhibin-related proteins are synthesized by follicle cells, they may play important roles in regulating follicular development and activity.     

Epidermal growth factor induces maturation of rat follicle-enclosed oocytes

Gonadotropin-induced differentiation of ovarian granulosa cells in culture is inhibited by epidermal growth factor (EGF). The present study was undertaken to test a possible inhibitory effect of EGF on LH-induced maturation of rat follicle-enclosed oocytes. We have found that EGF not only failed to affect LH action but served by itself as an inducer of maturation of follicle-enclosed oocytes. EGF action on the oocytes was dose and time dependent and could be prevented by (Bu)2 cAMP. The response of the oocytes was specific to EGF and could not be elicited by other growth factors such as nerve growth factor and insulin. The response to EGF was not limited to the large antral follicles, as oocytes enclosed by small antral follicles (less than 0.4 mm) were induced to mature by EGF as well. In addition, we have demonstrated that oocytes, induced to mature by EGF, are concomitantly uncoupled from the follicular cells. Based on these results we suggest that EGF may terminate the transfer of a follicular inhibitor to the oocyte. It is also possible, however, that EGF induces oocyte maturation by a mechanism independent of its effect on communication between the cellular components of the follicle.     

Midcycle administration of a progesterone synthesis inhibitor prevents ovulation in primates.

Progesterone receptors appear in granuloma cells of preovulatory follicles after the midcycle gonadotropin surge, suggesting important local actions of progesterone during ovulation in primates. Steroid reduction and replacement during the gonadotropin surge in macaques was used to evaluate the role of progesterone in the ovulatory process. Animals received gonadotropins to induce development of multiple preovulatory follicles, followed by human chorionic gonadotropin (hCG) administration (day 0) to promote oocyte (nuclear) maturation, ovulation, and follicular luteinization. On days 0-2, animals received no further treatment; a steroid synthesis inhibitor, trilostane (TRL); TRL + R5020; or TRL + dihydrotestosterone propionate (DHT). On day 3, ovulation was confirmed by counting ovulation sites and collecting oviductal oocytes. The meiotic status of oviductal and remaining follicular oocytes was evaluated. Peak serum estradiol levels, the total number of large follicles, and baseline serum progesterone levels at the time of hCG administration were similar in all animals. Ovulation sites and oviductal oocytes were routinely observed in controls. Ovulation was abolished in TRL. Progestin, but not androgen, replacement restored ovulation. Relative to controls, progesterone production was impaired for the first 6 days post-hCG in TRL, TRL + R5020, and TRL + DHT. Thereafter, progesterone remained low in TRL but recovered to control levels with progestin and androgen replacement. Similar percentages of mature (metaphase II) oocytes were collected among groups. Thus, steroid reduction during the gonadotropin surge inhibited ovulation and luteinization, but not reinitiation of oocyte meiotic maturation, in the primate follicle. The data are consistent with a local receptor-mediated role for progesterone in the ovulatory process.     

Naturally occurring steroids in Xenopus oocyte during meiotic maturation. Unexpected presence and role of steroid sulfates

In the ovary, oocytes are surrounded by follicle cells and arrested in prophase of meiosis I. Although steroidogenic activity of follicle cells is involved in oogenesis regulation, clear qualitative and quantitative data about the steroid content of follicles are missing. We measured steroid levels of Xenopus oocytes and follicles by gas chromatography-mass spectrometry. We show that dehydroepiandrosterone sulfate is the main steroid present in oocytes. Lower levels of free steroids are also detected, e.g., androgens, whereas progesterone is almost undetectable. We propose that sulfatation is a protective mechanism against local variations of active steroids that could be deleterious for follicle-enclosed oocytes. Steroid levels were measured after LH stimulation, responsible for the release by follicle cells of a steroid signal triggering oocyte meiosis resumption. Oocyte levels of androgens rise slowly during meiosis re-entry whereas progesterone increases abruptly to micromolar concentration, therefore representing the main physiological mediator of meiosis resumption in Xenopus oocyte.     

Are steroids obligatory mediators of luteinizing hormone/human chorionic gonadotropin-triggered resumption of meiosis in mammals?

Steroids mediate the gonadotropic stimulus of oocyte maturation in fish and amphibians. Such a role of steroids in mammals has not been confirmed until recently. A series of studies presented data suggesting that steroids might be involved in meiosis of mouse oocytes. Here we examined this suggestion using in vitro cultures of rat and mouse follicle-enclosed oocytes (FEOs) and cumulus-enclosed oocytes (CEOs). In FEOs that mature only in response to gonadotropins or other stimuli, we tested the ability of steroids to trigger meiosis and whether addition of steroid receptor antagonists blocks LH/human chorionic gonadotropin stimulation of meiosis. In CEOs that mature spontaneously, we tested whether steroid antagonists block maturation and whether steroids overcome the inhibition of maturation by hypoxanthine (Hx), a mild inhibitor of meiotic resumption. The progesterone antagonists mifepristone (RU 486) and Organon 31710 as well as the estrogen antagonist faslodex did not prevent LH-triggered maturation of rat or mouse FEOs or the spontaneous maturation of CEOs. In accordance, the progesterone agonist promegestone (R5020) and estradiol did not stimulate the resumption of meiosis in rat and mouse FEOs, and both did not overcome the Hx inhibition of meiosis in rat and mouse CEOs. Flutamide, an androgen antagonist, did block meiosis in rat FEOs, but this action could not be affected by adding dihydrotestosterone, suggesting that it was not androgen receptor mediated. Flutamide did not affect spontaneous maturation of rat CEOs, and dihydrotestosterone could not stimulate meiosis inhibited by Hx. Thus, in contrast to lower vertebrates, in mammals, steroids do not seem to serve as an obligatory signal by which the somatic cells of the follicle transfer the gonadotropic stimulation of meiosis to the oocyte.     

Hormonal activation of ionic currents in follicle-enclosed Xenopus oocytes.

Membrane currents were recorded, using the voltage clamp technique, from Xenopus laevis oocytes still surrounded by their enveloping follicular and epithelial cells. Exposure of the follicles to mammalian gonadotropins elicited a current generated largely by an increase in membrane K+ conductance. The gonadotropin response resembled responses elicited by adenosine and catecholamines in the same follicle, but was not blocked by purinergic or catecholaminergic antagonists. The gonadotropin-induced currents were potentiated by the adenylate cyclase activator forskolin and by phosphodiesterase inhibitors; similar currents were elicited in the same follicle by intraoocyte injection of cAMP, which indicates a role for this second messenger in the response mechanism. Gonadotropin responses were either abolished or substantially reduced after treatments that remove the ovarian epithelial and follicular cells. Our experiments suggest that the gonadotropin receptors, and the K+ channels they regulate, reside in the follicular cells.     

The roles of follicular envelopes in the initiation of Xenopus oocyte maturation

Gonadotropins and progesterone induce in vitro meiotic maturation of the follicleenclosed Xenopus laevis oocyte. The kinetics of maturation are identical for both hormones. The enzymatic removal of the follicular envelopes which suppresses gonadotropin activity does not modify the efficiency of progesterone (1 μM)-induced maturation. It was, however, shown that defolliculation decreases the kinetic of maturation: GVBD₅₀ (the time necessary to obtain 50% of germinal vesicle breakdown or maturation) was 8.08 ± 1.25 hr when follicles are induced to mature in the presence of progesterone and 4.30 ± 0.89 hr in the case of defolliculated oocytes. This result demonstrates that the follicular envelopes play a role in inhibition of steroid-induced maturation. When oocytes are defolliculated in the presence of aminogluthetimide, an inhibitor of steroid synthesis, the kinetic of oocyte maturation is also decreased (GVBD₅₀: 4.98 ± 1.75 hr) indicating that the biosynthesis of steroid at the level of follicular envelopes is not involved in the phenomenon.     

The effects of forskolin and LH on cAMP changes and maturation in the follicle-enclosed oocytes of hamsters

Graafian follicles from mature pro-oestrous hamsters were incubated with LH, various concentrations of forskolin, or forskolin plus LH. The incubations were either terminated at different time periods for analysis of follicular or oocyte cAMP levels or incubated for the entire 6 h and the oocytes examined to determine maturational status. Incubations with LH (1 microgram/ml) produced a short transient rise in follicular and oocyte cAMP concentrations, while forskolin (60 microM, 20 microM and 10 microM) produced cAMP values which remained elevated for longer periods of time. The 1 microM concentration of forskolin initiated oocyte maturation (28%) but at a level which was significantly below that stimulated by LH (74%). When LH was included with forskolin, a dramatic rise in follicular cAMP occurred which was approximately 2 times greater than levels seen with LH alone. A significant percentage of oocytes matured when 100 nM forskolin (45%) was included with LH (1 microgram/ml) but not with any other concentration of forskolin tested. Maturation percentages for follicle-enclosed oocytes exposed to 1 microM forskolin plus 1 microgram/ml of LH (3.8%) were not different from the controls (7%). However, when 1 microM forskolin was combined with 100 ng/ml of LH a significant percentage of oocytes matured (47%). While continuous incubations with forskolin did not stimulate a high percentage of oocytes to mature, oocytes from follicles exposed to forskolin (60 microM and 20 microM) for short periods (5 min-30 min) with a change to plain medium did mature. The results of these studies indicate that, in the hamster, long term exposure to forskolin inhibits maturation in follicle-enclosed oocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dissociation between the inhibitory and the stimulatory action of cAMP on maturation of rat oocytes

The possible mediatory role of cAMP in the induction of oocyte maturation by luteinizing hormone (LH) is not yet clear since evidence for both inhibitory and stimulatory actions of the nucleotide on the oocyte has been provided. To elucidate the role of cAMP in regulation of oocyte meiosis we tried in the present study to dissociate between the inhibitory and stimulatory action of this nucleotide on oocyte maturation. To induce maturation, oocytes enclosed by their follicles were transiently exposed to either dibutyryl cAMP (dbcAMP) or to the phosphodiesterase inhibitor methylisobutylxanthine (MIX). Inhibition of maturation was obtained by the addition of the above agents to either follicle-enclosed oocytes incubated in the presence of LH or isolated cumulus-free oocytes that mature spontaneously in vitro. We found that inhibition of oocyte maturation is obtained by a relatively low dose of either dbcAMP or MIX while higher concentrations of these agents are required to induce oocyte maturation. Coupling of the oocyte to the cumulus cells, as expressed by the fraction of labeled uridine transferred from the cumulus cells to the oocyte following exposure of the follicle-enclosed cumulus-oocyte complex to MIX, was also determined. We found that uncoupling of the oocyte from the cumulus cells corresponded with the induction, but not inhibition of oocyte maturation, both by its concentration dependence and time-course. We suggest that cAMP has a dual role in regulation of oocyte maturation. Lower levels of the nucleotide act to maintain meiotic arrest, while elevated levels of cAMP mediate LH action to induce meiosis resumption.     

Stimulatory role of cyclic adenosine monophosphate as a mediator of meiotic resumption in rabbit oocytes.

The present study was undertaken to assess the role of alterations of intraoocyte cAMP concentrations in the meiotic maturation of isolated and follicle-enclosed oocytes. In isolated oocyte culture, the intracellular cAMP content of denuded oocytes declined within 15 min of incubation, whereas the cAMP content of cumulus-enclosed oocytes did not change substantially for 1.5 h of incubation, and then declined abruptly. Commitment to meiotic maturation was preceded by reduced concentrations of intraoocyte cAMP. Forskolin inhibited the spontaneous maturation of cumulus-enclosed oocytes in a dose-dependent manner. However, this inhibition was attenuated as the duration of incubation increased. Forskolin significantly stimulated the intracellular cAMP content of denuded and cumulus-enclosed oocytes, but intraoocyte cAMP returned to pretreatment values within 4 h. The decline in intraoocyte cAMP was followed by the meiotic maturation of isolated oocytes. In in vitro perfused rabbit ovaries, exposure to forskolin at 10(-4) M, as well as to 50 IU human CG, accelerated the meiotic maturation of follicle-enclosed oocytes. The intraoocyte cAMP content increased significantly within 30 min and reached its maximum 2 h following exposure to forskolin. Thereafter, cAMP decreased abruptly and returned to pretreatment levels by 6 h. These alterations of intraoocyte cAMP contents following exposure to forskolin paralleled those observed in human CG-treated ovaries. The decline in cAMP content of follicle-enclosed oocytes was followed by their meiotic maturation. In conclusion, the sustained elevation of intraoocyte cAMP levels inhibits the initiation of meiotic maturation in isolated and follicle-enclosed oocytes. Within the follicle, resumption of meiosis is triggered via a transient increase in intraoocyte cAMP, but commitment to meiosis must await the decline of intraoocyte cAMP.     

Disruption of gap junctional communication within the ovarian follicle induces oocyte maturation

Meiotically arrested mammalian oocytes are stimulated to resume meiosis by LH. This response, which can be reversed by elevation of intraoocyte cAMP levels, is associated with interruption of gap junctional communication (GJC) within the ovarian follicle. In the present study, we examined the hypothesis that disruption of GJC within the ovarian follicle is sufficient for induction of oocyte maturation. For this purpose, we incubated rat follicle-enclosed oocytes with carbenoxolone (CBX), a known blocker of gap junctions. We found that this selective disruptor of GJC promoted maturation of almost all the follicle-enclosed oocytes after 5 h of incubation; this response was also obtained by a transient (2 h) exposure to this agent. CBX-induced oocyte maturation was accompanied by a substantial decrease in intraoocyte concentrations of cAMP that was not associated with elevated activity of type 3A phosphodiesterase (PDE3A). The effect of CBX on reinitiation of meiosis was blocked by isobutylmethylxanthine, a phosphodiesterase inhibitor. Unlike LH, CBX did not activate MAPK in the follicular cells, and inhibition of the MAPK signaling pathway by means of UO126 did not prevent the resumption of meiosis. Injection of CBX into the ovarian bursa of intact animals stimulated maturation in 30% of the oocytes, whereas no maturation was observed in the contralateral ovary injected with PBS. We conclude that, because experimentally induced breakdown of communication within the ovarian follicle is associated with a drop in intraoocyte cAMP concentrations and results in resumption of meiosis, this could be the physiological mechanism employed by LH to stimulate oocyte maturation.     

Involvement of estradiol-17β and its membrane receptor, G protein coupled receptor 30 (GPR30) in regulation of oocyte maturation in zebrafish, Danio rario

The orphan G protein coupled receptor, GPR30, has the characteristics of a high affinity, specific estrogen membrane receptor on Atlantic croaker oocytes and mediates estrogen inhibition of oocyte maturation in this perciform fish. In order to determine the broad applicability of these findings to other teleosts, similar experiments were conducted in a cyprinid fish, zebrafish, in the present study. GPR30 mRNA expression was detected in zebrafish oocytes but not in the ovarian follicular cells. Both spontaneous and 17, 20β-dihyroxy-4-pregnen-3-one (DHP)-induced maturation of follicle-enclosed zebrafish oocytes was significantly decreased when they were incubated with either estradiol-17β, or the GPR30 agonists, ICI 182 780 and tamoxifen, or with the GPR30 specific agonist G-1. On the other hand spontaneous oocyte maturation increased two-fold when zebrafish ovarian follicles were incubated with an aromatase inhibitor, ATD. Moreover, the stimulatory effects of ATD on germinal vesicle breakdown (GVBD) were partially reversed by co-treatment with 100 nM of E2 or G-1. These results suggest that endogenous estrogens acting through GPR30 are involved in maintaining meiotic arrest of zebrafish oocytes.     

Involvement of estradiol-17beta and its membrane receptor, G protein coupled receptor 30 (GPR30) in regulation of oocyte maturation in zebrafish, Danio rario

The orphan G protein coupled receptor, GPR30, has the characteristics of a high affinity, specific estrogen membrane receptor on Atlantic croaker oocytes and mediates estrogen inhibition of oocyte maturation in this perciform fish. In order to determine the broad applicability of these findings to other teleosts, similar experiments were conducted in a cyprinid fish, zebrafish, in the present study. GPR30 mRNA expression was detected in zebrafish oocytes but not in the ovarian follicular cells. Both spontaneous and 17, 20beta-dihyroxy-4-pregnen-3-one (DHP)-induced maturation of follicle-enclosed zebrafish oocytes was significantly decreased when they were incubated with either estradiol-17beta, or the GPR30 agonists, ICI 182 780 and tamoxifen, or with the GPR30 specific agonist G-1. On the other hand spontaneous oocyte maturation increased two-fold when zebrafish ovarian follicles were incubated with an aromatase inhibitor, ATD. Moreover, the stimulatory effects of ATD on germinal vesicle breakdown (GVBD) were partially reversed by co-treatment with 100 nM of E2 or G-1. These results suggest that endogenous estrogens acting through GPR30 are involved in maintaining meiotic arrest of zebrafish oocytes.     

Rat oocyte maturation in vitro: Relief of cyclic AMP inhibition by gonadotropins

The hormone-independent, spontaneous maturation that rat oocytes undergo in vitro can be inhibited by derivatives of cyclic AMP and inhibitors of cyclic nucleotide phosphodiesterase. In this study, we have shown that this inhibition of maturation can be partially relieved by preparations of ovine and rat luteinizing hormone or follicle-stimulating hormone. The ability of gonadotropins to foster the resumption of maturation in cultures of cyclic AMP-inhibited oocytes suggests that this system is suitable for studies of the hormonal control of oocyte development. The dose and time dependency of the response to gonadotropins has been examined in order to study the role of these hormones in oocyte maturation and to compare this effect to other known responses of the cumulus-oocyte complex. These studies show that highly purified preparations of rat gonadotropins are less effective inducers of maturation than the more commonly used, but considerably less purified, preparations of ovine gonadotropins. Almost complete relief of inhibition is observed, however, when the oocytes are exposed to a combination of rat luteinizing hormone and follicle-stimulating hormone. Oocyte maturation was not influenced by the sex steroids progesterone or 17beta-estradiol. Our results suggest that: (i) cyclic AMP is involved in the intrafollicular inhibition of oocyte maturation; (ii) both gonadotropins are required for maximal stimulation of the resumption of oocyte meiosis; and (iii) steroids are not involved in this response to gonadotropins.     

Transforming growth factor-beta stimulates meiotic maturation of the rat oocyte

The effect of transforming growth factor-beta (TGF beta) on meiotic maturation was analyzed in oocytes from immature rats treated with PMSG. TGF beta accelerated the maturation of both follicle-enclosed oocytes and cumulus-oocyte complexes, as measured by an increase in the percentage of oocytes with germinal vesicle breakdown. Concentrations of the growth factor as low as 1 pM (25 pg/ml) increased oocyte maturation by 50% above control values, and 100 pM TGF beta caused a maximal 2-fold rise in the maturation rate. Germinal vesicle breakdown was significantly increased by TGF beta during the first 4 h of incubation, and stimulatory effects were observed as early as 1 h. However, by 8 h over 90% of the oocytes showed maturation in the absence or presence of TGF beta, indicating that the growth factor enhanced the spontaneous rate of oocyte development. TGF beta had no effect in denuded oocytes, demonstrating that the growth factor altered maturation through an action on the surrounding cumulus cells. Oocyte maturation was not accelerated by TGF beta in the presence of inhibitors of germinal vesicle breakdown, such as cAMP and hypoxanthine. Other growth factors, including IGF-I (50 ng/ml) and IGF-II (50 ng/ml), also stimulated oocyte maturation, while platelet-derived growth factor (100 ng/ml) and insulin (1 microgram/ml) had minimal effects on germinal vesicle breakdown. Although epidermal growth factor (EGF; 100 ng/ml) also increased the maturation of oocytes, lower concentrations of TGF beta (1-10 pM) suppressed EGF action by up to 30%. TGF beta, EGF, and insulin-like growth factors had minimal effects on cAMP production by cumulus-oocyte complexes. These results demonstrate that TGF beta and other growth factors are potent in vitro stimulators of oocyte maturation in the rat. Such effects of growth factors in vivo, in the presence of endogenous follicular factors and gonadotropic hormones, may regulate the selection and meiotic maturation of oocytes during follicular development. The rapidity of growth factor action in the oocyte provides a defined model to study signal transduction pathways of growth factors in relationship to their biological activity.     

Evidence that androgens are the primary steroids produced by Xenopus laevis ovaries and may signal through the classical androgen receptor to promote oocyte maturation.

Steroid-induced maturation of Xenopus oocytes has long served as a model for studying meiosis. Progesterone has been considered the relevant steroid controlling maturation, perhaps through interactions with classical progesterone receptors. In this study, we provide evidence that androgens, rather than progesterone, are the physiologic mediators of Xenopus oocyte maturation. Androgens were equal or more potent activators of maturation in vitro relative to progesterone and were significantly more abundant in the serum and ovaries of beta-human chorionic growth hormone-stimulated frogs. Androgen action appeared to be mediated by classical androgen receptors (ARs) expressed in oocytes, as androgen-induced maturation and signaling was specifically attenuated by AR antagonists. Interestingly, we found that progesterone was rapidly converted to the androgen androstenedione in isolated oocytes by the enzyme CYP17, suggesting that androgens may be promoting maturation even under conditions typical for "progesterone-mediated" maturation assays. Androgens are thought to play an important role in ovarian development as well as pathology, and signaling through the AR may prove to be a major regulatory mechanism mediating these processes.