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.     

Decline of follicular oocyte maturation inhibitor coincident with maturation and achievement of fertilizability of oocytes recovered at midcycle of gonadotropin-treated women.

To examine whether a decline in follicular oocyte maturation inhibitor (OMI) is associated with attainment of oocyte maturation and fertilizability, OMI was measured in follicular fluid (FF) of 39 follicles of 20 normal women given human menopausal gonadotrophin and human chorionic gonadotrophin to induce follicular growth and maturation. Oocytes were aspirated per laparoscope, the fluid was saved, and the egg was observed, incubated, and inseminated with the husband's sperm. Concepti that developed to the 4- to 8-cell stage were transferred to the uterus and the women were followed for pregnancy. OMI activity in each FF was measured by using cultured cumulus-enclosed porcine oocytes (30-40 oocytes per FF sample). Estrogen, progesterone, oocytes (30-40 oocytes per FF sample). Estrogen, progesterone, and delta 4-androstenedione were measured in FF by radioimmunoassay. The FF of 13 preovulatory follicles yielding oocytes that were mature and fertilizable had significantly less OMI activity (mean +/- SEM) (0.58 +/- 0.10 unit/ml) compared to follicles yielding immature oocytes (2.8 +/- 0.56 units/ml; n = 9), atretic oocytes (5.5 +/- 2.5 units/ml; n = 7), or preovulatory oocytes with fractured zonae (1.9 +/- 0.63 units/ml; n = 7). The estrogen concentration (mean +/- SEM) of preovulatory follicles yielding mature fertilizable eggs or mature eggs with fractured zonae was greater (396 +/- 34 ng/ml; n = 20) compared to follicles yielding immature or atretic eggs (203 +/- 59 ng/ml; n = 9 and 97 +/- 47 ng/ml; n = 7, respectively; P less than 0.05). Progesterone concentration (mean +/- SEM; ng/ml) of FF was generally elevated in all preovulatory follicles (635 +/- 53) compared to immature or atretic follicles (230 +/- 64 and 76 +/- 17, respectively; P less than 0.05). It may be concluded that in normal follicle maturation there is a decline in OMI in the follicle containing an oocyte that becomes mature and fertilizable. There is also an increase in estrogen, progesterone, and follicle size. It is also possible to have an abnormal follicle maturation when there is an increase in size as well as FF, estrogen, and progesterone, but withut a decline in OMI--a situation which can lead to production of a nonfertilizable oocyte.     

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.     

Melatonin accelerates maturation inducing hormone (MIH): induced oocyte maturation in carps

The present communication is an attempt to demonstrate the influence of melatonin on the action of maturation inducing hormone (MIH) on the maturation of oocytes in carps. The oocytes from gravid female major carp Labeo rohita were isolated and incubated separately in Medium 199 containing (a) only MIH (1 microg/ml), (b) only melatonin (at concentrations of 50, 100 or 500 pg/ml), and (c) both melatonin and MIH, but at different time intervals. In the latter group, melatonin was added to the incubating medium either (i) 4 h before addition of MIH, (ii) 2 h before addition of MIH, (iii) co-administered with MIH (0 h interval) or (iv) 2 h after addition of MIH. In each case, oocytes were further incubated for 4, 8, 12 or 16 h post- administration of MIH, and the effects of treatment on oocyte maturation were evaluated by considering the rate (%) of germinal vesicle breakdown (GVBD). Incubation of oocytes in a medium containing only melatonin did not result in GVBD of any oocyte. Nearly all the oocytes underwent GVBD when incubated with MIH for 16 h. Administration of melatonin along with MIH (at 0 h interval) or 2 h after addition of MIH did not result in any significant change in the rate of GVBD compared to that in a medium containing only MIH. However, it was quite interesting to observe that incubation of oocytes with melatonin especially 4 h prior to addition of MIH in the medium, led to an accelerated rate of GVBD in the oocytes. Experiments with the oocytes of another major carp Cyprinus carpio following an identical schedule depicted similar results except a difference in the optimum melatonin dose. In L. rohita, 50 pg/ml melatonin had maximum acceleratory effect on MIH-induced GVBD of oocytes, while it was 100 pg/ml in C. carpio. Further study revealed that pre-incubation with melatonin accelerates the action of MIH on the formation of a complex of two proteins (MPF), a regulatory component called cyclin B and the catalytic component protein kinase known as cyclin-dependent kinase, Cdk1. Densitometric analysis of the immunoblot data collected from the melatonin pre-treated MIH incubated oocytes showed that cyclin B level continued to increase even after 4 h of incubation, and reached the peak after 12 h. Moreover, determination of H1 kinase activity as an indicator of MPF activity in oocytes revealed that melatonin pre-incubation considerably increased MIH stimulation of histone H1 phosphorylation as compared to MIH alone. Thus, the present study demonstrates for the first time that prior incubation with melatonin accelerates the action of MIH on carp oocyte maturation.     

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)     

Müllerian inhibiting substance as oocyte meiosis inhibitor

Müllerian inhibiting substance (MIS) inhibited the resumption of meiosis in both denuded and cumulus cell-enclosed rat oocytes in vitro. Spontaneous germinal vesicle breakdown was prevented in both types of oocytes treated by a purified MIS preparation at protein concentrations of 15 micrograms to 150 pg/ml. The inhibiting effect of MIS on the resumption of meiosis was dose dependent, reversible and cyclic AMP independent. Neither follicular-stimulating hormone, luteinizing hormone, progesterone, estradiol, nor testosterone acted significantly to influence MIS-mediated inhibition of rat oocyte maturation. In contrast, MIS had no influence on meiosis in the mouse, where other protein has been reported to inhibit the cumulus cell-enclosed oocyte in a cyclic AMP-dependent fashion. Thus MIS may be yet another inhibitor of oocyte meiosis, acting in the rat by a mechanism different from those inhibitors known, in the mouse ovary, to exert their effect in a cyclic AMP-dependent manner.     

Response of mouse graafian follicles in organ culture to varying doses of follicle-stimulating hormone and luteinizing hormone.

The response of mouse ovaries maintained in organ culture to follicle-stimulating hormone (FSH), LUTEINIZING HORMONE (LH) and human chorionic gonadotrophin (HCG) was assessed using quantitative histological and radioimmunoassay techniques. In terms of the induction of preovulatory maturation in follicular oocytes, 1 mugFSH/ml medium was as effective as 10 mugLH/ml. The lowest doses of HCG and LH used (0.2 i.u./ml and 1 mug/ml respectively) had no effect on oocyte maturation, whereas the response to FSH WAS VIRTUALLY UNCHANGED IRRESPECTIVE OF DOSE (1-10 MUG/ML). When the level of progesterone in the medium at the end of organ culture was used as an index of ovarian response, LH was more effective than FSH and HCG, although all the hormones induced a significant increase, irrespective of dose. These results are discussed in terms of the mode of action of gonadotrophins in the processes culminating in ovulation.     

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.     

Human follicular fluid insulin concentrations

In mammals, insulin stimulates granulosa cell aromatase activity and steroid production and is a regulating factor of oocyte maturation. To assess the role of insulin in human follicular and oocyte maturation, human follicular fluid was obtained 32-36 h after hCG administration at the time of oocyte recovery for in vitro fertilization. Follicular fluid insulin levels, measured by RIA, ranged from undetectable (less than 2 microU/ml) to 65.4 microU/ml. In women treated with human menopausal gonadotropin (n = 21), clomiphene citrate (n = 4), and human menopausal gonadotropin/clomiphene citrate (n = 14), follicular fluid insulin concentrations were 18.0 +/- 4.3 (+/- SE), 10.2 +/- 4.2, and 12.0 +/- 3.8 microU/ml, respectively (P = NS). Similarly, there was no significant difference in follicular fluid insulin concentrations in follicles with mature (n = 33) or immature (n = 6) oocytes (13.3 +/- 2.7 vs. 24.7 +/- 9.5 microU/ml) or in oocytes which eventually did (n = 35) or did not (n = 4) fertilize (16.4 +/- 3.0 vs. 3.2 +/- 0.8 microU/ml). Follicular fluid insulin levels (n = 30) correlated positively with follicular fluid progesterone levels (P less than 0.05), but not with follicular fluid estradiol or androstenedione levels or the estradiol to androstenedione ratio. The relationship of follicular fluid insulin and progesterone levels suggests that, as in other mammals, follicular fluid insulin may have a physiological role in follicular maturation.     

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.     

Basic fibroblast growth factor induction of granulosa cell tissue-type plasminogen activator expression and oocyte maturation: potential role as a paracrine ovarian hormone

Gonadotropin-induced ovulation is associated with oocyte maturation and preovulatory increases of tissue plasminogen activator (tPA) expression. Basic fibroblast growth factor (bFGF), an angiogenic factor found in many organs including the ovary, modulates steroidogenesis in granulosa cells and increases PA activity in endothelial cells. Here studies were performed to examine the possible roles of bFGF as an intragonadal regulator of tPA expression and oocyte maturation. In cultured granulosa cells, bFGF caused a time-dependent (onset at 24 h) and dose-dependent (ED50 = 0.6 nM) increase (up to 5-fold) in tPA enzyme activity as measured by the fibrin overlay technique. Northern blot hybridization also revealed that treatment of cells with bFGF (2 nM) increased the level of the 22S tPA messenger RNA. Slot blot analysis indicated that the effects of bFGF were time dependent and dose dependent; tPA message levels increase before tPA activity levels. bFGF (0.6 nM) also significantly increased granulosa cell cAMP production in both the absence and presence of a phosphodiesterase inhibitor. In follicle-enclosed oocytes incubated for 24 h in media with or without increasing concentrations of LH or bFGF, germinal vesicle breakdown was observed in only 1.6% of controls, but 85% of LH (1 microgram/ml)-treated oocytes underwent maturation. Likewise, bFGF induced germinal vesicle breakdown (10-80%) over a dose range of 0.6 to 333 nM. In the same follicles, bFGF, like LH, also stimulated prostaglandin E production. These results, coupled with the identification of bFGF in growing follicles, suggest that bFGF acts as an intraovarian inducer of granulosa cell tPA gene expression and oocyte maturation.     

Melatonin protects porcine oocyte in vitro maturation from heat stress

Melatonin is a pleiotropic molecule which plays an important role in animal reproductive activities. Because of the increased global warming, the impact of heat stress (HS) on stockbreeding has become an inevitable issue to be solved. To investigate the potential effects of melatonin on the in vitro maturation of porcine oocyte under the HS, a HS model for porcine oocyte maturation has been used in this study and the different concentrations of melatonin (10^?6–10^?9 m ) were also tested for their protective effects on oocytes. The polar body rate, the index of the nuclear maturation of the oocytes, and the cleavage rate as well as the blastocyst rate were measured to evaluate the developmental competence of the oocytes after parthenogenetic activation (PA). The results showed that HS [in vitro maturation (IVM) 20–24 hr, 42°C] significantly reduced the polar body rate of oocytes and the blastocyte rate of porcine PA embryos, while melatonin (10^?7 m ) application not only improved polar body rate and blastocyte rate, but also preserved the normal levels of steroid hormone which is disrupted by HS. The presence of melatonin (10^?7 m ) during the oocyte maturation under the HS reduced reactive oxygen species (ROS) formation, enhanced glutathione (GSH) production, inhibited cell apoptosis, and increased the gene expressions of SIRT1, AKT2, and Polg2. Importantly, the endogenously occurring melatonin of cumulus–oocyte complexes was significantly induced by HS. The results indicated that melatonin application effectively protected the oocytes from HS. These observations warranted the further studies in vivo regarding to improve the reproductive activities of animals under the global warming environment.     

A prospective, randomized, double-blind study to compare two doses of recombinant human chorionic gonadotropin in inducing final oocyte maturity and the hormonal profile during the luteal phase

CONTEXT: Different doses of human chorionic gonadotropin (hCG) have been used in various in vitro fertilization (IVF) treatment protocols to achieve final oocyte maturation. There is as yet no agreement on the optimum dose required. OBJECTIVES: The objective of this study was to compare the effectiveness of 250 and 500 microg recombinant hCG (r-hCG), which represented the lower and upper limits of the dose range, in inducing final oocyte maturation during IVF and intracytoplasmic sperm injection cycles. DESIGN: This was a prospective, randomized, double-blind study. SETTING: This study was performed at an IVF clinic in a teaching hospital. PATIENTS: Sixty patients with an indication for intracytoplasmic sperm injection were studied. INTERVENTION: The treatment dose used was 250 or 500 microg r-hCG. MAIN OUTCOME MEASURES: The percentage of metaphase II oocytes retrieved per patient, as an indicator of oocyte maturation, and the hormone profiles of the treatment cycle starting from the day of hCG up to hCG+10 d were the main outcome measures. RESULTS: The percentage of metaphase II oocytes was similar in the two groups (89.3% vs. 86.0%; P = 0.326) despite higher serum and follicular fluid hCG levels on hCG+2 and hCG+4 d, as was the follicular fluid to serum hCG ratio in the 500-microg r-hCG group. Serum estradiol and progesterone levels were comparable initially, but became significantly higher in the 500-microg r-hCG group on hCG+10 d. CONCLUSION: The two doses of r-hCG were equally effective in inducing final oocyte maturation. It remains unclear whether the higher midluteal estradiol and progesterone levels in the 500-microg r-hCG group confer any benefit.     

Protein kinase C, rather than protein kinase A is involved in follicle-stimulating hormone-mediated meiotic resumption of mouse cumulus cell-enclosed oocytes in hypoxanthine-supplemented medium

It has been reported that protein kinase C (PKC) activation participated in the porcine and bovine oocyte maturation, but not in mouse oocyte maturation in vitro. In the present study, the activators and inhibitors of protein kinase A (PKA) (forskolin, CDPKI and MDL-12230A) or PKC (PMA, staurosporine and sphingosine) were used to investigate the in vitro effect of PKA or PKC on spontaneous murine oocyte maturation, oocyte resumption of meiosis from HX inhibiting medium (medium+HX), and follicle stimulating hormone (FSH)-induced oocyte maturation. The results showed that when cumulus cell enclosed oocytes (CEOs) or denuded oocytes (DOs) were cultured for 24 h in the medium supplemented with forskolin (5 microM), an activator of adenylate cyclase, the spontaneous oocyte maturation were inhibited. A transient exposure (2 h) to forskolin (2-10 microM) in the medium+HX, and then transferred to a new medium+HX for the further culture, stimulated CEO resumption of meiosis. CDPKI (10(-10)-10(-6) M), an inhibitor of PKA, also stimulated oocyte meiotic maturation of CEO in the medium+HX, but not on DO. However, MDL-12230A (10(-12)-10(-9) M), an inhibitor of adenylate cyclase, did not promote oocyte maturation in HX arrested CEO. CDPKI (10(-10)-10(-6) M) or MDL-12230A (10(-12)-10(-9) M) had no effect on FSH-stimulated oocyte meiotic resumption, except at high doses of CDPKI (10(-7)-10(-6) M) or MDL-12230A (10(-9) M) which inhibited the FSH-induced formation of the first polar body (PB1). An activator of PKC, PMA (10(-11)-10(-7) M) dose-dependently inhibited spontaneous oocyte maturation of CEO or DO. Inhibitors of PKC, staurosporine (10(-9)-10(-6) M) or sphingosine (10(-8)-10(-5) M) induced oocytes in CEOs to resume meiosis in the presence of HX in a dose dependent manner, but had no effect on DOs. FSH (50IU/L) stimulated mouse oocytes in CEOs to override the arrest of HX and resume meiosis, while PMA, at the level of 10(-8)-10(-6) M, dramatically inhibited the stimulatory effect of FSH. These results indicate that PKC or PKA may be implicated in the regulation of mouse oocyte maturation. Thus while sustained high level of cAMP or PKA inhibit the resumption of meiosis, a transient rise in cAMP or PKA levels promotes oocyte maturation. The activation of PKC can also block oocyte meiotic resumption. Thus the inactivation of PKC, instead of the transient rise of PKA activity, appears to be involved in the process of FSH-mediated oocyte meiotic maturation.     

Loss of oocytes in Dazl knockout mice results in maintained ovarian steroidogenic function but altered gonadotropin secretion in adult animals

Within 2 days of birth, the mouse ovary is mainly composed of oocytes surrounded by a few pregranulosa cells forming primordial follicles that remain quiescent until they are recruited by intraovarian or other unknown factors to initiate growth of the oocyte and proliferation of the attendant granulosa cells. However, the role of the oocyte in this early development and organization of the follicle is poorly understood. The Dazl knockout (-/-) mouse in which there is total ablation of oocytes in fetal life has allowed us to address this issue. Ovaries from -/- females lack any follicular structure and have no cells positive for either Mullerian inhibiting factor or sulfated glycoprotein-1, indicating a lack of small follicles or corpora lutea. However, by immunocytochemistry, there are cells positive for 3beta-hydroxysteroid dehydrogenase, 17alpha-hydroxylase, and aromatase, indicating the presence of steroidogenically active cells capable of producing estrogen. This was confirmed by the presence of hypertrophied uterine endometrium expressing both estrogen receptor alpha (ER alpha) and ER beta together with normal levels of plasma estradiol. In addition, these steroidogenically active cells contain ER beta, inhibin alpha, and betaB-subunits, and -/- mice have low measurable plasma inhibin A and B levels. The ovarian steroids and inhibins had no significant effect on either plasma or pituitary gonadotropin levels, with significantly (P < 0.01) lower LH and FSH in intact +/+ and +/- females. However, significantly (P < 0.05) increased plasma inhibin B together with significantly (P < 0.05) lower FSH were observed in the +/- females. In conclusion, our data showed that despite oocyte loss in fetal life, the adult ovaries contained steroidogenically active cells capable of producing estradiol and inhibin. Furthermore, in the +/- mice, the enhanced plasma inhibin B implies a role for Dazl protein within the oocyte either from more small follicles or increased inhibin B production from each follicle.     

Rat oocytes induced to mature by epidermal growth factor are successfully fertilized.

Epidermal growth factor (EGF), which is a known mitogen, can also induce resumption of meiosis in the rat oocyte. The present study was designed in an attempt to elucidate whether oocytes, induced to mature by EGF in a follicle-enclosed oocyte culture, are fertilizable and can further develop into two-cell embryos. For further clarification of the effect of EGF on steroidogenesis in the ovarian follicle, progesterone concentrations were determined by radioimmunoassay. We found that oocytes matured by EGF (100 ng/ml) were successfully fertilized. Even though their rate of fertilization was relatively lower as compared to that of oocytes stimulated by luteinizing hormone (LH) both in vitro and in vivo (61%, 79%, and 83% respectively), once fertilized they exhibit an equal potential for further development (EGF: 48%, LH: 45%). On the other hand, EGF-induced progesterone production was very poor. These findings strongly support the idea that both mitogenesis and meiogenesis can be mediated by common signals. The results further suggest that progesterone production and oocyte maturation, in the rat, are independent events.     

Role of meiosis activating sterols, MAS, in induced oocyte maturation

Meiosis of follicle enclosed oocytes is maintained in the prophase of the first meiotic division and oocytes do not spontaneously resume meiosis during oocyte growth and follicle development. Arrest of the meiotic process is most likely secured by the presence of follicular purines, e.g. hypoxanthine, which maintain high levels of cAMP in the oocyte and which also in vitro prevent oocytes from resuming meiosis. Only in response to the mid-cycle surge of gonadotropins will oocytes of preovulatory follicles overcome the meiosis arresting effect of hypoxanthine and resume meiosis proceeding to the metaphase of the second meiotic division. Morphologically, resumption of meiosis is observed by the disappearance of the oocyte's nuclear membrane (germinal vesicle), a process called germinal vesicle breakdown (GVB). The molecular mechanism down-stream to receptor activation by which the mid-cycle surge of gonadotropins induces oocytes to resume meiosis is, however, only partly understood. The oocyte itself lacks gonadotropin receptors and its action is mediated through the attached cumulus cells. In vitro it has been shown that FSH induces synthesis of a signal in the cumulus cells, which overcomes the meiosis arresting effect of hypoxanthine. We have shown that a group of sterols, meiosis activating sterols (MAS), induces oocyte maturation in vitro even in oocytes depleted of cumulus cells. MAS were identified as intermediates in the cholesterol biosynthesis between lanosterol and cholesterol. The two best characterized members of the MAS family are FF-MAS purified from human follicular fluid (4,4-dimethyl-5alpha-cholest-8,14,24-triene-3beta-ol) and T-MAS purified from bull testicular tissue (4,4-dimethyl-5alpha-cholest-8,24-diene-3beta-ol). The synthesis, quantification, localization and tissue-accumulation of MAS are reviewed. Several publications have documented the pharmacological effect of MAS in different species, including oocytes from mouse, rat and human. Conflicting results obtained by the use of sterol synthesis inhibitors, which prevent MAS-accumulation, are also discussed. Whether FSH actually uses MAS as a signal transduction molecule for inducing oocyte maturation and the mechanism by which MAS induce resumption of meiosis is currently unknown, but data to support that MAS is part of the FSH induced signal transduction pathway are presented.     

Follicle-stimulating hormone and somatomedin-C stimulate inhibin production by rat granulosa cells in vitro

The direct effect of somatomedin-C (Sm-C) and FSH on inhibin production by rat granulosa cells in vitro has been examined. FSH stimulated accumulation of inhibin in culture media in a dose-dependent manner with maximal stimulation (6-fold) being observed at a dose of 300 ng FSH/ml. Addition of Sm-C (30 ng/ml) either alone or in the presence of FSH (3-300 ng/ml) increased inhibin production (up to 5-fold). Sm-C alone was effective over the physiological dose range of 3-100 ng/ml. Concomitant addition of FSH (100 ng/ml) and Sm-C (3-100 ng/ml) resulted in a significant increase in inhibin production at all doses of Sm-C. The dose-dependent effects of FSH and Sm-C were also time dependent with a synergistic effect apparent after 48 h of culture. The Sm-C induced FSH inhibitory activity of granulosa cell culture media was confirmed as authentic inhibin by the demonstration of a dose-dependent neutralization of this activity by a monoclonal antibody raised against purified bovine inhibin. The data indicate a direct role for both FSH and Sm-C in ovarian inhibin production and provide additional evidence for an autocrine-paracrine role for Sm-C in granulosa cell differentiation.