Publications

Gonadotropic stimulation of meiotic resumption in mice is dependent upon mitogen-activated protein kinase (MAPK) activation in the somatic compartment of the follicle. By contrast, spontaneous resumption of meiosis is independent of MAPK activation. In view of the suggested role of meiosis-activating sterol (MAS) in oocyte maturation we have (i) compared MAPK activation in rat preovulatory follicles stimulated by LH or by accumulation of endogenous MAS by using an inhibitor of MAS conversion, AY9944; (ii) examined whether stimulation of meiosis by MAS is dependent upon MAPK activation using denuded oocytes (DO) of Mos-null mice (hereafter Mos^-/-) with oocytes unable to activate MAPK. Rat preovulatory follicles responded to LH or AY9944 stimulation by MAPK activation. Inhibition of MAPK phosphorylation blocked both LH- and AY9944 triggered resumption of meiosis. In mouse cumulus-enclosed oocytes (CEOs) and DOs AY9944 stimulated GVB in wild-type and Mos^-/- mouse CEOs cultured with hypoxanthine (Hx). Addition of MAS or AY9944 to mouse DOs cultured with Hx induced resumption of meiosis only in wild-type and Mos^+/- oocytes, but they were ineffective in Mos^-/- oocytes. The observed sluggish activation of MAPK induced by AY9944 in rat follicle-enclosed oocytes (FEO) may cause the delay in meiotic resumption in response to MAS and AY9944 stimulation. Further, it is incompatible with the suggested role of MAS as an obligatory mediator of LH in the induction of meiotic maturation. MAPK/MOS activation, whether in the somatic compartment or in denuded oocytes, is required for MAS- like LH-, FSH-, or EGF-induced resumption of meiosis.

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.

Gonadotropin releasing hormone (GnRH) has been shown to mimic the actions of LH/hCG on oocyte maturation and ovulation. Recent studies demonstrated that induction of ovulation by LH/hCG is mediated, at least in part, by transactivation of epidermal growth factor receptors (EGFR) by autocrine/paracrine EGF-like factors activated by metalloproteases. Here we have examined whether the action of GnRH on the preovulatory follicles is exerted through similar mechanisms involving activation of EGFR. The EGFR kinase inhibitor, AG1478, inhibited GnRH-induced oocyte maturation in explanted follicles in vitro. Its inactive analog, AG43, did not affect GnRH-stimulated resumption of meiosis. GnRH, like LH, stimulated transient follicular expression of EGF-like agents, as well as rat cycloxygenase-2 (rCOX-2), rat hyaluronan synthase-2 (rHAS-2), and rat tumor necrosis factor-α-stimulated gene 6 (rTSG-6) mRNAs, known ovulatory enzymes. Likewise, GnRH stimulated follicular progesterone synthesis. Conversely AG1478 inhibited all these actions of GnRH. Furthermore, Galardin, a broad-spectrum metalloprotease inhibitor, blocked GnRH-induced oocyte maturation and follicular progesterone synthesis. In conclusion, we have demonstrated that follicular EGF-like factors mediate also the GnRH-stimulation of ovulatory changes, like these of LH/hCG.

Meiosis-activating sterol (MAS) was shown to overcome the inhibitory effect of hypoxanthine on spontaneous maturation of mouse oocytes and was suggested to mediate the stimulation of meiosis by gonadotropins. Follicular fluid (FF)-MAS is synthesized by cytochrome P450 lanosterol 14α-demethylase (LDM). Follicular LDM was preferentially localized in oocytes by immunohistochemistry. Using [³H]acetate or R-[5-³H]mevalonate as precursors as well as high-performance liquid chromatographic and thin-layer chromatographic separation, we have measured the concentrations of de novo-synthesized lanosterol, FF-MAS, and cholesterol in rat graafian follicles, cumulus-oocyte complexes (COCs), and denuded oocytes (DOs) treated with LH, AY-9944 (an inhibitor of Δ14-reductase, which was anticipated to increase FF-MAS levels by inhibiting its metabolism), or both after 8 h of culture. In follicles, both LH and AY-9944 increased the accumulation of FF-MAS as compared to controls. In COCs, AY-9944 caused a marked increase in FF-MAS, but we were unable to detect accumulation of FF-MAS in DOs. Neither the endogenous increases in FF-MAS accumulation nor the addition of FF-MAS to the culture medium could overcome the inhibition on resumption of meiosis by phosphodiesterase inhibitors. Compared to LH-induced resumption of meiosis in follicles, that induced by AY-9944 was much delayed. These results call into question any role of FF-MAS as an obligatory mediator of LH activity on germinal vesicle breakdown. The discrepancy between the positive staining for LDM in oocytes and our inability to detect de novo synthesized FF-MAS in DOs may relate to the sensitivity of the methodology employed and either the number of oocytes used or a deficiency in LDM synthetic activity in such oocytes. Further studies are required to confirm any of these alternatives.

In vitro studies on mouse oocytes have shown that two closely related sterols, subsequently named meiosis-activating sterols (MAS), can overcome the inhibitory effect of hypoxanthine on the resumption of meiosis. These sterols are synthesized by cytochrome P₄₅₀ lanosterol 14α-demethylase (LDM), a key enzyme in cholesterol biosynthesis. We have used specific inhibitors of LDM, azalanstat (RS-21607) and RS-21745, to test whether MAS is an obligatory mediator in the resumption of meiosis in the rat. Addition of azalanstat and RS-21745 (1-200 μM) to culture medium of rat isolated cumulus-enclosed oocyte and preovulatory follicle-enclosed oocyte stimulated by LH/hCG did not allow separation between their inhibition of the resumption of meiosis and the degeneration of oocytes. In both models, doses of the drug that inhibited oocyte maturation also increased oocyte degeneration. The inhibitors only partially suppressed follicular progesterone production. We have examined by reverse transcriptase-polymerase chain reaction, Western blotting, and immunocytochemistry the ovarian expression of LDM mRNA and protein during the preovulatory period. We did not find evidence for the stimulation of this enzyme by LH/hCG. The strongest staining by LDM antiserum was obtained in primordial and primary oocytes, and the staining was reduced with oocyte growth. In addition, strong LDM staining could be observed in some of the granulosa cells, especially of the corona radiata localized in close proximity to the oocyte. In conclusion, our results with specific inhibitors and molecular approaches do not reveal evidence to support the hypothesis that MAS is an obligatory step in the stimulation of the resumption of meiosis. Specific inhibitors of MAS synthesis did not prevent spontaneous or LH-stimulated meiosis at doses that have previously been shown to effectively suppress LDM activity. Much higher concentrations of the inhibitors, which affected meiosis, were detrimental to oocytes, leading to their degeneration. The timing of LDM expression in the ovary was incompatible with a role for MAS in meiosis. Finally, the preferential localization of LDM protein to the oocytes suggests MAS production in oocytes rather than its transport from the somatic compartment as implied by the proposed role of MAS as a cumulus-oocyte signal molecule.

The present study was designed to determine whether progesterone might have a role in gonadotrophin-induced pituitary adenylate cyclase-activating polypeptide (Pacap) gene expression in rat ovary. Northern blot analysis revealed that treatment of pregnant mare's serum gonadotrophin (PMSG)-primed immature rats with the progestin antagonist RU486 or an inhibitor of 3β- hydroxysteroid dehydrogenase epostane, 1 h before HCG, resulted in a dose- dependent inhibition of the HCG-induced Pacap gene expression. In-situ hybridization demonstrated that the number of pre-ovulatory follicles expressing Pacap mRNA in their granulosa cells was greatly reduced in ovaries treated with RU486. Moreover, the suppressive effect of RU486 or epostane on the LH-induced Pacap gene expression in cultured pre-ovulatory follicles was reversed by co-treatment with the synthetic progestin R5020. We further cloned the 5'-flanking region of the rat Pacap gene and identified the presence of a consensus progesterone receptor element. When luciferase fusion genes containing Pacap gene promoter were transiently transfected into granulosa cells of pre-ovulatory follicles, luciferase activity was markedly stimulated by LH. Treatment with RU486 or epostane resulted in partial suppression of LH-stimulated PACAP promoter activity. Taken together, these results indicate that progesterone, acting through progesterone receptors, plays a role in gonadotrophin induction of Pacap gene expression in granulosa cells of pre-ovulatory follicles, and thereby may be involved in the process of ovulation.

During each reproductive cycle, a preovulatory surge of gonadotropins induces meiotic maturation of the oocyte in the preovulatory follicle followed by ovulation. Although gonadotropins stimulate cAMP production in somatic cells of the follicle, a decrease in intra-oocyte cAMP levels is required for resumption of meiosis in oocytes. Based on the observed compartmentalization of the cAMP-degrading enzyme, phosphodiesterase, in follicular somatic and germ cells, inhibitors of phosphodiesterase 3 were used to block meiosis in ovulating oocytes in rodents. By this strategy, we demonstrated that fertilization and pregnancy could be prevented without disturbing follicle rupture and normal estrous cyclicity. In contrast to conventional contraceptive pills that disrupt ovarian steroidogenesis and reproductive cycles, the present strategy achieves effective contraception by selective blockage of oocyte maturation and development without alterations in ovulation and reproductive cyclicity.

Atretic demise rather than ovulation is the ultimate fate of the vast majority of ovarian follicles in mammals, affecting 70-99.9% of the follicles in various species. Recent studies have established that atretic degeneration of follicles is an apoptotic process, heralded by endonuclease degradation of DNA at internucleosomal sites, which generates DNA fragments in size multiples of 185-200 bp that are seen as distinct ladder bands after agarose gel electrophoresis. Using the well-characterized model of inducing atresia of preovulatory follicles in vivo by hypophysectomy and analyzing DNA fragmentation by autoradiography of size-fractionated DNA labeled at the 3' ends by [³²P] dideoxy-ATP, we have examined the timing of atretic changes. DNA degradation was related to morphological signs of atresia, ovulability, and changes in follicular steroidogenesis. Rats were hypophysectomized on the morning of the day of proestrus, after which largest follicles were collected at various times. DNA fragmentation was analyzed in groups of five follicles. The increase in DNA fragments of low molecular weight up to 4 h after hypophysectomy was negligible (101 ± 10%; 0 h time = 100%) but progressed 8, 12, 24, 48, and 72 h after hypophysectomy (143 ± 20%, 168 ± 27%, 235 ± 29%, 3299 ± 1075%, and 2249 ± 805%, respectively; p

Ovarian collagenases are necessary for the process of ovulation, and they are believed to be activated by the preovulatory LH surge. This information is largely based on in vitro investigations in which the balance between inhibitory and stimulatory principles involved in the activation of collagenase are largely disrupted. Therefore, we developed a simple and reliable method to measure collagenolytic activity in vivo in freely moving rats. By the use of a microdialysis system, a peptide coupled with methyl-coumarin is perfused into the bursa of the ovary. Collagenolytic enzymes cleave this peptide, and the cleaved fragments rediffuse into the microdialysis system. The effluent is collected in frections, and the peptide-methyl-coumarin complex is cleaved, which results in liberation of fluorescent methyl-coumarin. This assay is linear over a wide range of collagenolytic activity, and other proteases, such as trypsin or plasmin, do not give any fluorescent signal. In proestrous rats, collagenolytic activity increases after the onset of the preovulatory LH surge. In animals in which the LH surge was disrupted by the surgical procedure but had a normal proestrous PRL surge, neither progesterone nor collagenolytic activity increased in the perfusate fluid. This indicates that it is only LH, not PRL, that activates follicular collagenolytic enzymes. Similar results were obtained in immature PMSG/hCG-treated animals. Using a well established zymographic assay, these results were confirmed, and it was further demonstrated that type I and type IV collagenase are active in the rat ovary.

Proteinases and their inhibitors control follicular connective tissue remodeling associated with follicular rupture. We examined the regulation and cellular localization of plasminogen activator inhibitor type-1 (PAI-1) and tissue inhibitor of metalloproteinase type-1 (TIMP-1) mRNAs by in situ hybridization. [³⁵S]UTP-labeled RNA probes were hybridized to ovarian sections of eCG-primed immature rats treated with hCG. Before hCG stimulation of ovulation, very low expression of PAI-1 mRNA was observed in theca cells. After hCG administration, expression of PAI-1 mRNA was increased in theca cells of most antral follicles, whereas expression in granulosa cells was limited to preovulatory follicles and only to areas where the basal membrane was dissociated. Before hCG treatment, low expression of TIMP-1 mRNA was observed in theca cells, but not in granulosa cells. After hCG treatment, TIMP-1 mRNA was greatly stimulated in theca cells irrespective of follicle size, while the expression in granulosa cells was limited to large antral follicles. The present study demonstrates cell-specific expression of PAI-1 and TIMP-1 mRNAs in the LH/hCG-stimulated ovary, thus confirming the localized control of preovulatory proteolysis by coexpression of both enzymes and their respective inhibitors.

The preovulatory surge of gonadotropins activates a cascade of proteolytic enzymes resulting in the rupture of the follicular wall and the release of a fertilizable ovum during ovulation. In the rat the process is initiated by a rise in follicular tissue-type plasminogen activator, produced predominantly in granulosa cells. Recent studies revealed a preovulatory increase in ovarian collagenolytic activity in vivo and an increase in activatable collagenase in vitro. In view of the complicated control of mammalian collagenase synthesis and activity by local inhibitors and activators, we examined the expression of ovarian interstitial and type IV collagenases and tissue inhibitor of metalloproteinase (TIMP) mRNA after an ovulatory stimulus. Ovarian mRNA was isolated from immature PMSG-treated rats 3, 6, and 9 h after hCG stimulation. Northern blot analyses revealed a mRNA of 1.7 kilobases (kb) hybridizing with the human interstitial collagenase cDNA probe. The levels of this mRNA showed a 25-fold increase between 3-6 h after hCG stimulation. The human cDNA probe of collagenase IV hybridized with a mRNA of 3.1 kb, which showed only a 4-fold increase 9 h after hCG treatment. The interstitial collagenase mRNA was expressed in both granulosa cells of preovulatory follicles and the residual ovarian tissue, whereas the expression of collagenase IV mRNA was limited to the residual tissue. Inhibitors of eicosanoid synthesis, previously shown to block ovulation and the LH/hCG-induced rise in ovarian collagenolysis, suppressed the gonadotropic stimulation of interstitial collagenase mRNA, but slightly stimulated that of collagenase IV. The mouse cDNA probe of TIMP hybridized with a 0.9-kb mRNA, which was stimulated by hCG to reach a maximum (7- to 8-fold increase) between 6-9 h after stimulation. TIMP was expressed and stimulated in both the granulosa cells and the residual tissue. Inhibitors of eicosanoid synthesis did not affect the gonadotropic stimulation of TIMP mRNA. These data support the suggested role of interstitial collagenase in follicle rupture and the essential role of eicosanoids in the mediation of gonadotropic stimulation of interstitial collagenase production and action. The observed stimulation of TIMP mRNA expression by the gonadotropin and the lack of any effect of eicosanoid synthesis inhibitors on this action of LH/hCG offer an additional mechanism by which these inhibitors may block ovulation. Thus, the suppression of ovulation by inhibitors of eicosanoid synthesis may result from selective inhibition of interstitial collagenase expression and undisturbed gonadotropin-stimulated TIMP expression.

Ovulation in mammals is preceded by surges of the two pituitary gonadotropins, LH and FSH. Although previous studies have shown that purified FSH induces ovulation when administered to hypophysectomized rats, proof that FSH has inherent ovulatory potential is lacking because all FSH preparations have varying degrees of residual LH. To determine if FSH alone can induce ovulation, we generated LH-free recombinant FSH (RCFSH) by culturing eukaryotic cells transfected with the human common α- and FSH β-subunit genes. Immature hypophysectomized rats were implanted with estrogen and then primed with PMSG (15 IU, sc). Fifty-two hours later, either RCFSH or hCG was injected (sc) to induce ovulation. A dose-dependent increase in the ovulation rate was stimulated by RCFSH, reaching 100% ovulation at 18 IU/rat, comparable to that achieved with 12 IU hCG. The maximum number of oocytes ovulated per ovary was similar for both groups. Ovulation induced by either RCFSH or hCG was time dependent and associated with a periovulatory increase in the ovarian activity and message levels of tissue-type plasminogen activator, a protease important in the preovulatory degradation of the follicle wall. Because PMSG has inherent LH-like activity in rats, we also implanted hypophysectomized rats with a minipump (sc) that released RCFSH (4 IU/day) to induce follicle growth. Fifty-two hours later, a single sc injection of a surge dose (20 IU) of RCFSH also induced ovulation, further indicating the ability of FSH alone to induce both follicle growth and ovulation. To test whether FSH can also induce ovulation in adult animals, rats were hypophysectomized on proestrous morning and treated with increasing doses of RCFSH (ip) to induce ovulation. At 7.8 IU RCFSH, all rats ovulated, with about 10 oocytes/rat. These results demonstrate that RCFSH is capable of inducing ovulation in hypophysectomized immature and adult rats, with associated increases in ovarian tissue-type plasminogen activator gene expression. Thus, FSH may be involved in follicular rupture in addition to its role in follicle recruitment and maturation. The preovulatory surges of both LH and FSH may represent a protective mechanism to ensure an optimal ovulatory stimulus. The present finding also serves as the basis to formulate new ovulation induction protocols.

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.

The ability of immunopurified, biologically potent anti-Müllerian hormone (AMH) to inhibit the spontaneous resumption of meiosis of rat oocytes was tested in vitro. Two different batches of AMH in the range of 0.75-9.0 micrograms/protein did not suppress the spontaneous resumption of meiosis. Neither did AMH (6 micrograms/ml) induce meiotic resumption of follicle-enclosed oocytes in culture. It is concluded, therefore, that AMH has no oocyte meiosis-inhibiting activity.

In order to establish criteria for selection of the best ova in in-vitro fertilization-embryo transfer (IVF-ET) programmes we have examined the follicular fluid (FF) levels of plasminogen activator (PA), collagenolytic activity, progesterone (P) and α₂ macroglobulin (α₂M) and related them to the success of pregnancy. PA activity was similar in FF of pregnant and nonpregnant cycles, 13.8 ± 3.9 mU/ml versus 14.6 + 2.9 (mean ± SEM) respectively. By contrast, FF from pregnant cycles exhibited lower collagenolytic activity (49.6 ± 3.9% versus 67.9 ± 3.0; P 2M, only 18.4% of the aspirates from pregnant cycles showed a precipitation line, whereas 76.8% of those from non-pregnant cycles were positive. Levels of P in aspirates from pregnant cycles were in the intermediate range, as compared with those from non-pregnant cycles (0.06-5.5 μg/ml versus 0.02-12.0 μg/ml). All these assays can be completed before ET and performed in IVF-ET programmes. In conclusion, it seems that a combination of follicular α₂M levels and collagenolytic activity, and to a lesser extent addition of P assay, may serve as good criteria for selecting the best embryos for establishment of pregnancy.

Vasoactive intestinal peptide (VIP) has recently been detected in rat ovaries and has been shown to stimulate steroidogenesis by cultured rat granulosa cells. In this study we investigated whether the VIP-messenger RNA (mRNA) can be detected in the ovaries, thus suggesting local synthesis of the peptide. To study VIP-gene expression, a sensitive RNA detection assay which uses in vitro transcribed RNA probes corresponding to specific exons of the VIP gene was developed. Using this method, an approximately 2000-base RNA band containing the coding sequences for VIP was detected in rat ovaries. This RNA also contains the coding sequences for the VIP-related peptide (peptide-histidine-methionine). An identical VlP-encoding RNA was previously identified in the rat cerebral cortex. However, the VIP-mRNA quantity in the cortex was 12-foldhigher as compared to the ovaries. These results may reflect the differences in VIP concentration in the two organs. The finding of VIP-encoding mRNA in the rat ovaries suggests a local synthesis of VIP in the ovaries.

In our previous study of a dose-dependent blockage of follicular rupture at ovulation by inhibitors of lipoxygenase was demonstrated. Here the presence of 5-lipoxygenase activity in the whole ovary and in the Graafian follicle is estimated by a chemiluminescence assay using unlabeled arachidonic acid as susbtrate in the presence of luminol and by conversion of ¹⁴C-arachidonic acid into lipoxygenase products as separated by HPLC. Both approaches demonstrated lipoxygenase activity in whole ovarian homogenates and in homogenates of preovulatory Graafian follicles. Furthermore, within 6 h after stimulation in vivo with hCG, lipoxygenase activity was increased by 2-fold in the whole ovarian homogenate and by 5-fold in the follicular homogenate. These results confirm the presence of lipoxygenase in rat ovaries, and its stimulation by gonadotropin and thus corroborate the suggested involvement of lipoxygenase products in follicular rupture at ovulation.

The first meiotic maturation division of mammalian oocytes is initiated in the embryo or during the early postnatal period. However, when the germ cells reach the diplotene stage the meiotic process is arrested. Meiosis is normally kept in abeyance up to a short period prior to ovulation, when the process is resumed in preovulatory follicles. Resumption of meiosis is studied in mammalian oocytes mainly in two dissimilar in vitro models, isolated oocytes maturing spontaneously in culture and hormone-induced maturation of follicle-enclosed oocytes. A third approach, namely, co-culture of oocytes with follicular constituents was adopted in order to test the role of follicular components in the control of meiosis. Such studies demonstrated an inhibitory action of granulosa cells, granulosa-cell conditioned medium and of follicular fluid (FF1) upon the spontaneous maturation of co-cultured oocytes. By contrast, theca tissue was without effect on meiosis. Addition of luteinizing hormone (LH) to co-cultures of rat granulosa cells and rat oocytes induced resumption of meiosis, as it does in vivo or in vitro in follicle-enclosed oocytes. It is therefore suggested that within antral follicles meiosis is held in abeyance by a granulosa cell product, the inhibitor of oocyte maturation (OMI). Further studies led to the conclusion that OMI is not species specific, that its production by granulosa cells is enhanced by follicle stimulating hormone (FSH) and that its concentration in FF1 is dependent upon the development of the follicle and not the stage of the oestrous cycle. OMI appears to be a peptide of less than 2000 Da. Its action on the oocyte appears to be mediated, at least partially, by cumulus cells and is potentiated by cyclic AMP. Since OMI activity has been demonstrated only in antral follicles, we examined the development of the ability of rat oocytes to undergo spontaneous maturation during their growth phase in preantral follicles. We have found that the ability of rat oocytes to resume maturation ('meiotic competence') is acquired between days 20-26 post partum. By the use of hypophysectomy on day 15 of life and by treatment with hormones and inhibitors we demonstrated that the acquisition of meiotic competence is dependent upon FSH stimulation and that it is mediated, at least partially, by ovarian oestrogen production. The findings that oocytes from preantral follicles are meiotically incompetent suggests that the physiological role of follicular OMI is limited only to antral follicles i.e. when the oocytes acquire meiotic competence.(ABSTRACT TRUNCATED AT 400 WORDS)

The possible involvement of products of the lipoxygenase pathway of archidonic acid cascade in ovulation was tested by intrabursal injection of nordihydroguaiaretic acid (NDGA); 5,8,11-eicosatriynoic acid (5,8,11-ETYA), 3 amino-1-(3 trifluromethyphenyl)-2-pyrazoline hydrochloride (BW755c) and (FPL 55712). All these drugs reduced the number of ova released from the treated ovaries in a dose-dependent manner, without affecting ovulation from contralateral ovaries. NDGA was most potent since it completely blocked ovulation from the treated ovaried in 17/38 rats receiving a dose higher than 0.15 mg/ bursa. This effect og NDGA cannot be ascribed to its inhibition of ovarian PGE synthesis. Conversion of labeled arachidonic acid and via the lipoxygenase pathway by preovulatory rat follicles was demonstrated by TLC chromatography. Collectively, these results suggest the involvement of products of lipoxygenase pathway of arachidonic acid in ovulation in the rat.

delta 1-Tetrahydrocannabinol (delta 1-TCH), the major psychoactive constituent of marihuana, was found to suppress the preovulatory surge of gonadotropins and thereby to prevent ovulation in rats, rabbits and rhesus monkeys. These studies suggested that the drug acts primarily on the hypothalamus to suppress luteinizing hormone releasing hormone (LHRH) secretion. The aim of the present study was to examine the direct effect of delta 1-THC, the psychoactive constituent of marihuana and cannabidiol (CBD), one of its nonpsychoactive constituents, on preovulatory rat follicles in vitro. Both cannabinoids inhibited follicular steroidogenesis in a dose-dependent manner. Basal accumulation of progesterone (P), testosterone (T) and estradiol-17 beta (E2) was reduced up to 60% by the highest doses examined (100-200 microM). The luteinizing hormone (LH)-stimulated increase in P and T was inhibited by 75-88% by the highest doses of both cannabinoids (50-200 microM), while E2, accumulation was inhibited by only 40%. It appears that the inhibitory action of cannabinoids is exerted beyond LH binding and activation of adenylate cyclase and prior to pregnenolone formation in the gonadal steroidogenic pathway. In addition to this anti-steroidogenic effect, both cannabinoids induced resumption of meiosis in follicle-enclosed oocytes cultured in hormone-free medium; 200 microM delta 1-THC resulted in 80% maturation and CBD in 75%. It seems that the action of cannabinoids on rat follicles in vitro is unrelated to their psychotropic activity.

Oocytes were removed from the follicles of rats at 15 to 31 days of age, and their ability to resume meiosis (\u201cmeiotic competence\u201d) in vitro was correlated with their diameter and the stage of follicular development. The majority of oocytes explanted on day 15 did not resume meiosis when placed in culture, but the percentage of competent oocytes increased from 14.1% ± 3.0% on day 20 to 67.6% ± 3.3% on day 26 of age. This ability to resume maturation correlated well (r = 0.98) with the increase in diameter of oocytes and coincided with the development of antral follicles. Hypophysectomy on day 15 of age, but not on day 20, reduced the percentage (P 2 increased the number (P

Administration of PMSG to 26-day-old rats did not increase the total number (non-atretic and atretic) of preantral (Type 4) and antral (Types 5 and 6) follicles but changed the proportion between non-atretic and atretic follicles. By 12 h after PMSG administration only 7% of Type 5 follicles were atretic, and no atretic follicles of Type 6 were observed, as compared to 52 and 62% atretic follicles, respectively, in the saline-treated controls. PMSG did not decrease the percentage of atresia in preantral (Type 4) follicles. The treatment was associated with a sharp fall by 12 h in the pyknotic index of antral (Types 5 and 6) follicles. It is concluded that PMSG causes superovulation in the rat by rescuing follicles of Types 5 and 6 from atresia and allowing them to reach ovulation.

Resumption of meiotic maturation was induced in follicleenclosed ratoocytes by treatment with the divalent cationophore A23187 (10⁻⁵M). However, the same effect was attained by incubation in Ca⁺⁺ deficient medium, even in the presence of EDTA or EGTA (lmM). The stability of the first polar body was increased under Ca⁺⁺ deficient conditions. Neither the ionophore nor Ca⁺⁺ deficient medium interfered with the spontaneous maturation of isolated oocytes of the rat. The experiments with cultured follicles suggest that redistribution of divalent cations may participate in the physiological control of meiosis in mammalian oocytes.

Follicles vary greatly in their response to a hormonal stimulus. Some of the factors that influence the sensitivity of the rat follicle to ovulatory hormones are discussed in this review. The responsiveness of the rat ovary to gonadotrophins undergoes striking augmentation during the 2nd week of postnatal development with respect to cyclic AMP formation, cAMP-stimulated protein kinase activity, inducibility of ornithine decarboxylase and oestrogen secretion. FSH, probably in concert with oestrogen, sensitizes the follicle to subsequent stimulation of adenylyl cyclase by LH. This synergism, based on heterologous receptor induction, operates during prepubertal as well as cyclic follicular maturation. The ovarian LH-stimulated adenylyl cyclase possesses a guanine nucleotide regulatory site. Occupation of this site by GTP or Gpp(NH)p reduces the concentration of LH required for half-maximal stimulation of the enzyme and increases its activity (Vmax). Androgen synergizes FSH in stimulating progestin secretion by cultured immature granulosa cells, though the steroid is known to antagonize the FSH action on granulosa cell proliferation. Thecal androgen may be required for the maturation of the FSH response mechanism in granulosa cells of preantral follicles, while promoting atresia in large antral follicles. Continued exposure of the follicle to high concentrations of LH, FSH, or PGE-2 results in refractoriness of adenylyl cyclase to further stimulation by the same hormone. Desensitization may be transient (PGE-2) or protracted (LH) and seems to depend on the synthesis of a macromolecular inhibitor that affects the coupling between hormone receptor and adenylyl cyclase. This mechanism may account for the shut-down of follicular steroid production at ovulation, which depends on cAMP, and permit the expression of processes that are inhibited by cAMP.

Reference preparations of ovine follicle-stimulating hormone (NIH-FSH-S8 and S9; 10-50 mug/ml) induced ovum maturation and stimulated cyclic AMP formation, as well as progesterone and 17beta-estradiol secretion, by rat Graafian follicles in vitro. These actions of NIH-FSH were retained after immunoabsorption of any contaminating luteinizing hormone (LH) present in the preparations, by treatment with an antiserum to the beta-subunit of purified ovine LH (anti-betaLH). In contrast, the corresponding biological actions of NIH-LH-S18 (0.5-10 mug/ml) were abolished by treatment with this anti-betaLH serum. A highly purified FSH preparation (64-96 CD, 0.25 mug/ml) also triggered oocytic meiosis and increased follicular progesterone secretion in vitro. Intraperitoneal (ip) administration of anti-betaLH-treated NIH-FSH-S9 (50 mug/rat at 1430 h) consistently induced ovulation in proestrous rats in which the endogenous gonadotropin surge had been blocked by ip injection of either Nembutal (1345 h) or antiserum to the LH-releasing hormone (1200 h). Injection (ip) of anti-betaLH serum on its own into proestrous rats at 1200 h prevented ovum maturation and follicular rupture. We conclude that currently available reference preparations of ovine FSH possess the capacity to stimulate follicular adenylate cyclase, steroidogenesis, and ovum maturation in vitro, as well as ovulation in vivo, in the rat, and that this capacity cannot be attributed to contamination with material immunochemically identical with LH. However, it is inferred that the physiological triggering of ovulation and related events in this species depends principally on LH.

Graafian follicles explanted from proestrous rats before the preovulatory gonadotropin surge secreted predominantly 17beta-estradiol, and only small amounts of progestins and androgens, during 12 h of culture in hormone-free medium. Addition of ovine luteinizing hormone (NIH-LH-S18; 0.1-1.0 mug/ml) to the medium stimulated within 1-2 h the rate of accumulation of these steroids. However, accumulation of androstenedione and estradiol ceased after 4-6 h in the LH-treated cultures, whereas progesterone continued to accumulate and became the major secretory product at 6-12 h. Incubation of the follicles with LH for only 5 min resulted in significant stimulation of the accumulation of progesterone, androstenedione and estradiol during a subsequent 6-h culture period in hormone-free medium containing antibodies to LH; 30 min exposure to the hormone sufficed to elicit a maximal steroidogenic response and to induce ovum maturation in 95% of the follicles. Addition of actinomycin D (act D; 8 mug/ml) within the first hour after exposure of follicles to LH suppressed the LH-effect on progesterone but augmented the LH effect on estradiol accumulation; when addition of this inhibitor was delayed until 2 h, progesterone accumulation continued unabated for a further 10 h. By contrast, puromycin (80 mug/ml) inhibited progesterone accumulation when added to the medium at any time (1, 2 or 3 h) after the hormone. It is suggested that (i) a short-lived protein is essential for the stimulatory effect of LH on follicular steroidogenesis; (ii) the act D-sensitive product (mRNA?) required for the production of this protein is synthesized in adequate amount during the first 2 h of LH action, and is stable for at least 10 h; and (iii) LH may stimulate the production of an additional act D-sensitive regulatory protein that inhibits enzymes engaged in the cleavage of the 17-side-chain of progesterone, or cells equipped with these enzymes.