Response of follicle cells to ovulatory stimuli within the follicle and in primary culture.

Cultures of mural granulosa cells (mGCs) and cumulus oocyte complexes (COCs) were employed to investigate various aspects of follicle cell function and response to gonadotropins. Yet, such studies do not reveal the intricate cell-to-cell interactions in the whole follicle. Here we compare the ovulatory responses to LH/hCG or epiregulin (ER) of rat preovulatory follicles and of mGC and COC whether they were stimulated within the follicle or in primary cell cultures. The expression of TSG-6 and COX-2 mRNA varied according to the culture system and mode of stimulation. In primary cultures stimulated with LH or ER resulted in their lower expression as compared to stimulation of follicles. LH/hCG stimulated higher follicular and mGC AR, ER and EGFR mRNA levels than in primary mGC cultures. COCs stimulated by LH/hCG in vivo responded with AR, ER and EGFR mRNA expression, but not in culture where only EGFR mRNA was stimulated. The differences in gene expression of mGCs and COCs when stimulated within their intact follicle or in primary cultures revealed here underscore the important role of cell-cell interactions in follicle physiology. Therefore, results obtained in primary mGC cultures need careful validation in models reproducing such in situ interactions for revealing mGC activity within the intact follicle.

Resumption of oocyte meiosis in mammals: on models, meiosis activating sterols, steroids and EGF-like factors.

De novo synthesis of meiosis activating sterols (MAS) was stimulated by LH- and AY-9944 in rat cultured follicles and cumulus oocyte complexes (COCs), but could not be measured in denuded oocytes. Thus, MAS synthesized by the somatic compartment of the follicle could serve as a signal for the resumption of meiosis. Nevertheless, the delay in germinal vesicle breakdown (GVB) after MAS or AY-9944 stimulation as compared with gonadotropins, obtained by several groups, remains the strongest evidence against the suggested role of MAS as an essential mediator of LH in meiosis resumption. Recently several studies using mammalian COCs in culture have implied that steroids, like in fish and amphibians, serve as signals in mediating the LH/hCG stimulation of meiosis. However, in these studies there was no clear distinction between the requirement for steroids for the acquisition of meiotic competence, oocyte and follicle wellbeing or as a signal for meiotic resumption. Further, some of the authors overlooked earlier studies showing that blocking ovarian or follicular steroidogenesis does not affect GVB, the first step of meiosis resumption. Finally, in vivo and in vitro studies in the rat confirm and extend recent studies showing that locally produced and released EGF-like factors, such as epiregulin, seem to mediate at least part of the LH/hCG actions on oocyte maturation and release of ova at ovulation.

Epidermal growth factor family members: endogenous mediators of the ovulatory response.

Previous studies showed that epidermal growth factor (EGF) and TGFalpha mimic the action of LH on the resumption of oocyte maturation. We tested whether EGF-like agents, such as amphiregulin (AR), epiregulin (ER), and betacellulin (BTC), also mediate the LH stimulation of the ovulatory response in the rat. LH induced transient follicular expression of AR, ER, and BTC mRNA, reaching a maximum after 3-h incubation. Furthermore, the addition of ER, AR, and BTC to the culture medium could mimic some of LH actions. AR and ER fully simulated LH-induced resumption of meiosis in vitro, whereas BTC was less effective. To study the putative involvement of EGF-like factors in mediation of LH signal, the effect of the EGF receptor kinase inhibitor AG1478 was tested. When added with LH, AG1478, but not its inactive analog AG43, reduced EGF receptor phosphorylation and oocyte maturation compared with follicles treated with LH only. In addition to the inhibition of resumption of meiosis, AG1478 administration into the bursa (3 microg/bursa) resulted in 51% (P < 0.0005) inhibition of ovulation in the treated ovaries, compared with the untreated contralateral ones, as well as to the vehicle-treated ovaries (P < 0.02). LH, as well as ER, induced the expression of genes associated with the ovulatory response like rat hyaluronan synthase-2, cyclooxygenase-2, and TNFalpha-stimulated gene 6 mRNA, whereas AG1478 inhibited this effect of LH. Release of EGF-like factors from the membrane is dependent on activated metalloproteases. Indeed, Galardin, a broad-spectrum metalloprotease inhibitor, but not a specific matrix metalloprotease 2 and 9 inhibitor, suppressed meiotic maturation induced by LH. Conversely, meiotic maturation induced by ER was not affected by Galardin, thus, supporting the notion that LH releases follicular membrane-bound EGF-like agents. In summary, EGF-like factors such as ER, AR, and BTC seem to mediate, at least partially, the LH stimulation of oocyte maturation, ovulatory enzyme expression, and ovulation.

Role of meiosis-activating sterols in rat oocyte maturation: effects of specific inhibitors and changes in the expression of lanosterol 14alpha-demethylase during the preovulatory period.

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 P(450) lanosterol 14alpha-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 microM) 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.

Effects of ketoconazole on ovulatory changes in the rat: implications on the role of a meiosis-activating sterol.

In-vitro studies on mouse oocytes have shown that human follicular fluid and bull testes contain an activity which partially overrides the inhibitory action of hypoxanthine on meiosis. This activity was ascribed to two closely related sterols, subsequently named meiosis-activating sterols (MAS). We have used a potent inhibitor of sterol synthesis, ketoconazole, in order to test in vivo and in vitro whether MAS play a necessary physiological role in the resumption of meiosis in the rat. When administered systemically, ketoconazole (8.3-16.6 mg/rat) suppressed ovulation by 40%. Local unilateral administration of the drug into the ovarian bursa (1.25 mg/bursa) resulted in 75% inhibition of ovulation in comparison with the contralateral ovary. All the ovulated ova in the oviduct were mature. Histological examination of the ketoconazole-treated ovaries revealed mature oocytes trapped in follicles which failed to ovulate. Furthermore, extraction of oocytes from the large follicles of such ovaries revealed that 79% of them were mature. Addition of ketoconazole (0.0001-0.01 mM) to the culture medium did not affect significantly the spontaneous maturation of rat oocytes. However, ketoconazole at a higher concentration (0.1 mM) caused the degeneration of oocytes. Ketoconazole (0.01 mM) did not affect luteinizing hormone (LH)-stimulated oocyte maturation in explanted preovulatory follicles, even though it inhibited follicular progesterone production to levels below the hormone-free control follicles. At higher levels, ketoconazole caused the degeneration of follicles and the enclosed oocytes. In conclusion, using a potent inhibitor of MAS we have failed to confirm the suggested obligatory role of MAS in the resumption of meiosis in the rat both in vivo and in vitro.

Localization of preovulatory expression of plasminogen activator inhibitor type-1 and tissue inhibitor of metalloproteinase type-1 mRNAs 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.

Preovulatory changes in ovarian expression of collagenases and tissue metalloproteinase inhibitor messenger ribonucleic acid: role of eicosanoids.

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.

Production and characterization of antibodies to gonadotropin-releasing hormone receptor.

Antibodies to the gonadotropin-releasing hormone (GnRH) receptor of bovine pituitary membranes have been raised in rabbits by immunization with affinity-purified receptor preparations. These antibodies did not compete with 125I-labeled GnRH analog (Buserelin) for binding to the receptors but did precipitate rat and bovine solubilized receptors labeled with 125I-Buserelin. Binding of the antibodies to the receptors was also demonstrated by immunoprecipitation of 125I-labeled purified receptors and photoaffinity-labeled receptors. The antibodies did not have a GnRH-like activity but rather inhibited, in a dose-dependent manner, GnRH-stimulated luteinizing hormone release from cultured rat pituitary cells. In addition, the antibodies did not inhibit luteinizing hormone release stimulated by high K+ concentration. This suggests that the antibodies recognize domains of the receptor other than the binding site of the hormone and thereby inhibit the biological response. These GnRH receptor antibodies provide a useful tool for studying GnRH receptor structure, function, localization, and biosynthesis.

Onset of endogenous synthesis of epidermal growth factor in neonatal mice.

We have analyzed mouse fetuses and neonates for the presence of epidermal growth factor (EGF)-specific mRNA. No detectable EGF-specific mRNA was found in fetuses, fetal membranes, or placentae from Day 9 of gestation through birth or in the early neonatal period. While the kidneys begin to produce EGF specific transcripts by two weeks postpartum, the salivary glands begin to produce detectable levels of EGF mRNA only after weaning and even then at levels far below the adult amount. Reports of EGF and EGF-related material in rodent fetuses failed to determine whether this material was of maternal or fetal origin. We now conclude that authentic EGF in these embryos is probably of maternal origin. We have performed experiments designed to determine whether EGF can be transported into the fetus. A small percentage of 125I-EGF administered to pregnant females either systemically or directly into the uterine arteries reached the fetus itself. The uterus and the placenta attained a high level of labeling, whereas the amniotic fluid and yolk sac were virtually devoid of the tracer. In the neonatal period, milk may be the physiologically relevant source of EGF. We have found that 125I-EGF ingested by neonates was absorbed into the circulation, reached many internal organs, and was eventually excreted in the urine. Previously demonstrated EGF receptors in mouse embryonic cell types may be activated by either alpha type transforming growth factor or maternal EGF transported via the placenta.

p53 cellular tumor antigen: analysis of mRNA levels in normal adult tissues, embryos, and tumors.

The relative levels of mRNA specific for the mouse p53 cellular tumor antigen were determined in various normal adult tissues, embryos, and tumors. All tumors studied contained concentrations of p53 mRNA well above those present in most normal tissues. Normal spleen, however, had p53 mRNA levels comparable to those found in some tumors, despite the fact that they contained barely detectable p53 protein. This apparent discrepancy was found to be due to the extremely rapid turnover rate of p53 in the spleen (half-life, approximately equal to 6 min). In developing fetuses, a marked reduction of p53 mRNA levels was manifest from day 11 onwards, whereas the levels during organogenesis (days 9 to 11) were comparable to those found in undifferentiated embryonic stem cells and in some tumors.