Publications
2008
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(2008) Molecular Reproduction and Development. 75, 10, p. 1533-1541 Abstract
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.
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(2008) Molecular and Cellular Endocrinology. 282, 1-2, p. 26-31 Abstract
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.
2007
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(2007) Endocrinology. 148, 9, p. 4458-4465 Abstract
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.
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(2007) Endocrinology. 148, 4, p. 1717-1726 Abstract
Atresia and luteolysis are well-documented processes in which most of the growing ovarian follicles and all corpora lutea, respectively, are eliminated by apoptosis. We have previously reported that LH and FSH enhance caspase-3 and -7 activity and apoptosis in the theca-interstitial cells of rat preovulatory follicles in culture. Here we have used cultured follicles to examine whether LH-induced caspase activation is related to the ability of LH to stimulate steroid production. In these studies, we used three inhibitors of enzymes involved in steroid production: aminoglutethimide and ketoconazole, acting on cytochrome P450 side-chain cleavage (P450scc) located at the mitochondria, and epostane, acting on 3β-hydroxysteroid dehydrogenase located at the endoplasmic reticulum. We found that treatment with either aminoglutethimide or ketoconazole, but not with epostane, significantly reduced LH-induced caspase-3 and -7 activation and apoptosis, suggesting the mediation of LH-induced caspase activation by P450scc. Supplementing pregnenolone, the product of P450scc catalysis, to follicles treated with aminoglutethimide did not restore LH-induced caspase activation. On the other hand, treatment with antioxidants inhibited LH-induced caspase activation. Moreover, LH treatment was associated with an increase in reactive oxygen species which was inhibited by aminoglutethimide. Thus, P450scc catalysis results in an increase in reactive oxygen species, which in turn may trigger/facilitate caspase-3 activation. Finally, we found that in rat corpora lutea in vivo, an increase in steroidogenesis was accompanied by an increase in caspase activity. Thus, this study reveals a linkage between two seemingly distinct processes in which LH-induced caspase activation in cultured rat preovulatory follicles is coupled to mitochondrial steroidogenesis via P450scc.
2006
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(2006) Molecular Reproduction and Development. 73, 10, p. 1271-1276 Abstract
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.
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(2006) Human Reproduction. 21, 6, p. 1368-1379 Abstract
BACKGROUND: Anti-cancer therapies frequently lead to ovarian damage and impaired fertility. To preserve fertility, cryopreservation and subsequent transplantation of the ovaries have been suggested. One of the challenges in ovarian graft transplantation is overcoming the initial ischaemic damage that depletes a significant fraction of the oocyte pool. METHODS AND RESULTS: Follicular survival in ovarian grafts was examined by magnetic resonance imaging (MRI) and fluorescence microscopy in a model system in which rat ovaries were transplanted into nude mice. Transplantation into angiogenic granulation tissue created during wound healing shortened the ischaemic period by 24 h and significantly increased the pool of healthy primordial follicles and the perfused area of the transplanted grafts. Functional blood vessels were detected within the grafts as early as 2 days after transplantation. Gain of function was demonstrated both by growth of the grafts and by the hormonal influence on the host uteri. CONCLUSION: Implantation of ovarian grafts into an angiogenic granulation tissue improved graft vascularization and follicular survival. This procedure/treatment may be used for reducing the ischaemic damage in ovarian transplants, thus prolonging graft functionality and increasing the yield of oocytes that can be easily recovered for fertilization.
2005
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(2005) Biology of Reproduction. 73, 1, p. 20-28 Abstract
Heparanase (HPSE) is an endoglycosidase that cleaves heparan sulfate proteoglycans (HSPGs), major components of the basement membrane (BM) and extracellular matrix (ECM). Heparanase activity results in release of HSPC-bound molecules, including basic fibroblast growth factor (FCF2). Structural and functional development of the corpus luteum (CL) involves tissue remodeling, active angiogenesis, and steroid production. Heparanase-induced ECM and BM breakdown as well as FGF2-stimulated endothelial proliferation may have an important role in the regulation of luteal function. Heparanase mRNA was detected by reverse-transcription-polymerase chain reaction in granulosa cells recovered from follicular fluid of in vitro fertilization patients. Using sulfate-labeled ECM, heparanase enzymatic activity was determined in human luteinized granulosa cells. Employing immunohistochemistry, heparanase protein was localized predominantly in the theca interna cell layer of the mature antral follicle, whereas in human corpora lutea, both luteinized granulosa and theca cells were immunostained for heparanase. During luteolysis, heparanase was identified in macrophages surrounding the forming corpus albicans. In serially sectioned ovaries from unstimulated rats as well as from eCG-treated rats, expression of heparanase was noted exclusively in the ovarian steroid-producing interstitial tissue. Following an ovulatory dose of hCG, heparanase was immunostained also in lutein cells of the forming corpora lutea. Temporal expression of heparanase in granulosa cells during the luteal phase and in macrophages during luteal regression supports the hypothesis that heparanase plays a role in human ovarian ECM remodeling and may potentiate cellular migration and growth factor bioavailability.
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(2005) Molecular and Cellular Endocrinology. 234, 1-2, p. 37-45 Abstract
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.
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(2005) Endocrinology. 146, 1, p. 77-84 Abstract
Previous studies showed that epidermal growth factor (EGF) and TGFα 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 μg/bursa) resulted in 51% (P
2004
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(2004) Biology of Reproduction. 71, 6, p. 1807-1812 Abstract
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.
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(2004) Magnetic Resonance in Medicine. 52, 4, p. 741-750 Abstract
It has been suggested that ovarian cryopreservation and xenotransplantation can be used to preserve oocytes from damage during anticancer treatments. The main obstacle to subsequent ovarian grafting is loss of oocytes due to impaired perfusion. The aim of this study was to characterize angiogenic events following ovary xenotransplantation. Rat ovaries were transplanted into or onto the muscle of immunocompromised CD1-nude mice. Ovariectomy (OVX) of host mice prior to transplantation supported the resumption of follicular development, as manifested by the prevalence of antral follicles and corpora lutea. Two days after transplantation, the grafts were devoid of blood supply. Functional vessels within the graft were detected by MRI and histology from day 7 and on. By 2-3 weeks, both blood volume fraction and permeability in the graft, as measured with the use of albumin-based MR contrast material, were significantly elevated relative to the adjacent muscle. Extravasation of contrast material from the graft neovasculature was followed by interstitial convection in the muscle surrounding the graft, and draining toward the proximal popliteal lymph node. Development of the vasculature was monitored noninvasively, providing a time scale for revascularization and recovery of ovarian function following xenotransplantation of ovarian grafts.
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(2004) Endocrinology. 145, 4, p. 1943-1951 Abstract
Apoptosis causes the elimination of ovarian germ cells and the atretic degeneration of ovarian follicles. Here we have used cultured rat preovulatory follicles to examine the regulation of effector caspase-3 and -7 in follicles undergoing apoptosis in the presence or absence of gonadotropins or IGF-I. Culturing follicles in the presence or absence of serum resulted in the induction of apoptosis of granulosa cells (GC), which was accompanied by effector caspase activation. Surprisingly, the addition of the survival factors LH or FSH, but not IGF-I, further increased caspase-3 and -7 activity. Immunohistochemistry studies of the LH- and FSH-treated follicles indicated that cleaved caspase-3 was predominantly localized to the peripheral theca-interstitial cells (TIC). Western blot analysis and caspase-3 and -7 activity assays of the separated follicular compartments confirmed that both LH and FSH treatments significantly enhance caspase-3 and -7 activity in TIC. The elevation in caspase-3 and -7 activity in TIC was accompanied by an increase in apoptosis. Interestingly, LH and FSH also induced an increase in caspase-3 and -7 activity in GC; however, this increase was accompanied by a decrease in apoptosis. Finally, we demonstrate that in freshly isolated preovulatory follicles and in antral follicles in intact ovaries, the expression level of procaspase-3 is significantly higher in TIC than in GC. Thus, LH and FSH have a dual effect on the cultured rat preovulatory follicle: an antiapoptotic effect on GC and a proapoptotic effect on TIC.
2003
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(2003) Biology of Reproduction. 68, 6, p. 2055-2064 Abstract
Cancer patients, treated by either chemo- or radiotherapy, frequently suffer from ovarian failure and infertility. One of the new emerging techniques to preserve reproductive potential of such patients is cryopreservation of ovarian fragments prior to treatment and their retransplantation after healing. A major obstacle in survival of the ovarian implants is vascular failure, which leads to tissue necrosis. In order to investigate the role of angiogenesis in implant preservation, we used a xenograft model in which rat ovaries were transplanted into immunodeficient mice. Graft reception and maintenance were monitored by magnetic resonance imaging (MRI) and histology. Two transplantation sites were explored, i.e., subcutaneous and intramuscular. Comparison between these two transplantation sites revealed the importance of vascular smooth muscle cells and pericytes in sustaining vascular and tissue integrity. Histological examination of the grafts, at different time points and sizes, revealed that loss of perivascular cells preceded damage to endothelial cells and was closely correlated with loss of follicular and oocyte integrity. Intramuscular implantation provided better maintenance of implant perivascular cells relative to subcutaneous implantation. Accordingly, follicular integrity was superior in the intramuscular implants and the number of damaged follicles was significantly lower compared with the subcutaneous transplantation site. These results suggest that improving ovarian implant maintenance should be directed toward preservation of perivascular support.
2002
2001
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(2001) Biology of Reproduction. 64, 1, p. 299-309 Abstract
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.
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(2001) Biology of Reproduction. 65, 5, p. 1444-1451 Abstract
It is generally accepted that cyclic nucleotides are key signaling molecules in the control of oocyte meiotic resumption. Given the role of phosphodiesterases (PDEs) in cyclic nucleotide degradation, this study was undertaken to investigate the properties and regulation of PDEs expressed in rat oocytes. Cilostamide-sensitive PDE3 was the major activity detected in denuded oocytes, whereas no PDE3 activity could be detected in cumulus cells. Moreover, comparable levels of PDE3 activity were measured in cumulus-oocyte complexes (COCs) and in denuded oocytes. The oocyte PDE was recovered in the soluble fraction of the homogenate and immunoprecipitated with a specific PDE3A antibody. A significant and transient increase (P
2000
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(2000) Molecular Human Reproduction. 6, 3, p. 238-245 Abstract
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.
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Involvement of progesterone in gonadotrophin-induced pituitary adenylate cyclase-activating polypeptide gene expression in pre-ovulatory follicles of rat ovary(2000) Molecular Human Reproduction. 6, 3, p. 238-45 Abstract
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 3beta-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.
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(2000) Biology of Reproduction. 63, 4, p. 1214-1218 Abstract
Growth differentiation factor-9 (GDF-9) was shown recently to be essential for early follicular development, including the appearance of the theca layer. Theca cells provide the androgen substrate for aromatization and estrogen production by granulosa cells. Using biologically active recombinant GDF-9 (rGDF-9) and an androgen-producing immortalized theca-interstitial cell (TIC) line or primary TIC, we have examined the action of this paracrine hormone on theca cell steroidogenesis. The effect of GDF-9 on TIC progesterone synthesis was marginal and inconsistent in the primary cultures. In immortalized theca cells, GDF-9 attenuated the forskolin-stimulated progesterone accumulation. More significantly, this oocyte-derived growth factor enhanced both basal and stimulated androstenedione accumulation in the primary and transformed TIC cultures. The effects of GDF-9 on steroidogenesis by preovulatory follicles were relatively modest. Likewise, it did not affect the maturation of follicle-enclosed oocytes. The effect of GDF-9, an oocyte product, on TIC androgen production suggests a regulatory role of the oocyte on theca cell function and hence on follicle development and differentiation. This direct effect of GDF-9 on thecal steroidogenesis is consistent with its recently demonstrated actions on thecal cell recruitment and differentiation.
1999
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(1999) Experimental And Clinical Endocrinology & Diabetes. 107, 1, p. 1-11 Abstract
Ovulation, recurring every reproductive cycle of the mammalian female and triggered by a surge of luteinizing hormone (LH) released from the pituitary is an essential prerequisite for fertilization and subsequent embryonic development. Here we shall review two of the biological responses leading to follicle rupture vascular changes and proteolysis. Naturally, our present knowledge is based mainly on work in a few species, such as the rat, the mouse and, to lesser extent the pig and monkeys and observations in the human. Therefore any generalizations to other mammals, should be considered as a working hypothesis yet to be confirmed. The LH surge stimulates, in the preovulatory follicles, a cascade of proteolytic enzymes, including plasminogen activator (PA), plasmin and matrix metalloproteinases (MMPs). These enzymes bring about the degradation of perifollicular matrix and, most notably, the decomposition of the meshwork of collagen fibers which provides the strength to follicular wall. Pharmacological blockage of any of these enzymes resulted in the reduction of ovulation rate. The increased ovarian proteolytic activity associated with ovulation is controlled by locally produced specific inhibitors, plasminogen activator inhibitor-1 (PAI-1) and tissue inhibitor of metalloproteases-1 (TIMP-I). The increased synthesis of these two specific proteinase inhibitors in the theca of growing follicles ensures their development by protecting them from enzymes diffusing from ovulatory follicles. The stimulation of ovulation by the gonadotropin results in an increase in follicular blood flow, hyperemia, increase in vascular permeability and a marked increase in follicular volume. These vascular changes and the proteolytic activity are triggered either directly by LH or by local mediators and factors produced in response to the gonadotropic stimulus. These mediators allow the tight coordination of these two cascades culminating in the rupture of follicle wall. We shall review here, briefly, the various mediatory systems that have been implicated in follicle rupture. These include steroids, vascular endothelial growth factor (VEGF), cytokines, eicosanoids, platelet activating factor (PAF), nitric oxide and nitric oxide synthase (NO/NOS), kinins and oxygen radicals.
1998
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(1998) Journal of Clinical Investigation. 102, 3, p. 532-537 Abstract
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.
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(1998) Molecular Human Reproduction. 4, 5, p. 483-489 Abstract
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.
1997
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(1997) Biology of Reproduction. 57, 5, p. 990-998 Abstract
The stimulatory effects of gonadotropins on antral and preovulatory follicles are well known, but conflicting results have been reported regarding the gonadotropin responsiveness and dependency of preantral follicles. Taking advantage of the relatively uniform development of the first wave of follicles in the postnatal rat ovary, we evaluated the role of endogenous and exogenous gonadotropins on preantral follicle development. Reduction of the high levels of gonadotropins present in juvenile rats by either hypophysectomy (at Day 15) or GnRH antagonist treatment (staffing from Day 11 of age) resulted in decreased ovarian weight at Day 19 of age that was associated with a reduced number of developing follicles and increased atresia of remaining follicles. In contrast, treatment with FSHctp (a long- acting FSH agonist) in intact (Days 5-19 of age), hypophysectomized (Days 15- 19), or GnRH antagonist-treated (Days 11-19) animals resulted in increased ovarian weight and follicle development as determined histologically and by inhibin-α expression. A dose-dependent stimulatory effect of hCG on ovarian weight was seen when animals were cotreated with FSHctp and the GnRH antagonist. At low doses of hCG, augmentation of antral follicle formation occurred, whereas higher doses of hCG led to morphological signs of luteinization. These findings demonstrate the important role of endogenous gonadotropins in preantral follicle development and indicate that preantral follicles are highly responsive to exogenous gonadotropins.
1996
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(1996) Biology of Reproduction. 55, 5, p. 1075-1080 Abstract
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
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(1996) Developmental Biology. 178, 2, p. 393-402 Abstract
The second messenger cAMP has been implicated in the regulation of mammalian and amphibian oocyte maturation. Although a decrease in intraoocyte levels of cAMP precedes germinal vesicle breakdown (GVBD), the gonadotropin induction of ovulation and oocyte maturation is associated with major increases of cAMP in ovarian follicles. In the mammalian system, isolated oocytes undergo spontaneous maturation in vitro but this process is blocked by treatment with a phosphodiesterase (PDE) inhibitor, IBMX, which increases intraoocyte cAMP levels. In contrast, the same inhibitor, when added to cultured follicles for a brief time, increases follicle cAMP levels, followed by the induction of GVBD. To resolve the paradoxical actions of this PDE inhibitor on the maturation of isolated and follicle-enclosed oocytes, we hypothesized that meiotic maturation requires opposing fluctuations of cAMP levels in the somatic granulosa and germ cells. Such opposing fluctuations may result from selective expression and regulation of PDEs in the somatic and germ cell compartments of the follicle. To test this hypothesis, PDE activity was manipulated in different follicular cells using type-specific inhibitors. The impact of the ensuing changes in cAMP levels in the two compartments was monitored by the induction of GVBD. In isolated oocytes, spontaneous GVBD was blocked by two inhibitors of type 3 PDE (cGMP-inhibited: CGI-PDE), milrinone and cilostamide. In contrast, treatment with an inhibitor for type 4 PDE (cAMP-specific), rolipram, was ineffective. These findings suggest that the oocyte expresses type 3 but not type 4 PDE and that increases in intraoocyte cAMP suppress GVBD. This hypothesis was confirmed by in situ hybridization studies with PDE3 and PDE4 probes. PDE3B mRNA was concentrated in oocytes while PDE4D was mainly expressed in granulosa cells. In cultured follicles, LH treatment induced oocyte maturation but the gonadotropin action was blocked by inhibitors of type 3 but not the type 4 PDE inhibitors. Furthermore, treatment with the type 4, but not the type 3, PDE inhibitor mimics the action of LH and induces oocyte maturation, presumably by increasing cAMP levels in granulosa cells. Our findings indicate that PDE subtypes 4 and 3 are located in follicle somatic and germ cells, respectively. Preferential inhibition of PDE 3 in the oocyte may lead to a delay in oocyte maturation without affecting the cAMP-induced ovulatory process in the somatic cells. Conversely, selective suppression of granulosa cell cAMP-PDE may enhance the gonadotropin induction of ovulation and oocyte maturation. Thus, in addition to the well-recognized differential expression and regulation of adenylate cyclase in the somatic and germ cell compartments of the follicle, we suggest that selective regulation and expression of PDEs may be involved in the regulation of cAMP levels and control of oocyte maturation in the preovulatory mammalian follicle.
1995
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(1995) Advances in experimental medicine and biology. 377, p. 121-140 Abstract
Ovulation, recurring every midcycle of the mammalian female and triggered by a surge of luteinizing hormone (LH) released from the pituitary, is an essential prerequisite for fertilization and subsequent embryonic development. Here we shall describe two of the biological components of the ovulatory response, cumulus expansion (frequently denoted as cumulus maturation) and the rupture of follicular wall, both crucial for the release of a fertilizable ovum. The role of a proteolytic cascade and its regulation by eicosanoids will be emphasized in relation to follicle rupture. The new data implicating cumulus maturation as an essential step for the release of the ovum and the apparent mediatory role of interleukin-1 in this process will be presented. LH/hCG stimulates, in the preovulatory follicles, a cascade of proteolytic enzymes, including plasminogen activator (PA), plasmin and matrix metalloproteinase 1 (MMP-1). These enzymes bring about the degradation of perifollicular matrix and, most notably, the decomposition of the meshwork of collagen fibers which provides the strength to follicular wall. Furthermore, pharmacological blockage of any of these enzymes resulted in inhibition of follicle rupture. LH/hCG stimulates, in addition, an increase in ovarian production of eicosanoids. These include prostaglandins, obtained from arachidonic acid via the cyclooxygenase pathway and leukotrienes, the products of lipoxygenase. Previous studies from our and other laboratories have demonstrated the ability of inhibitors of cyclooxygenase and of lipoxygenases to suppress ovulation in several mammalian species. MK-886, which inhibits the translocation of 5-lipoxygenase (5-LO) from the cytosol and its binding to the membranal 5-LO activating enzyme, suppressed dose-dependently follicular rupture from the treated ovary. Zymographic analysis of ovarian extracts from PMSG/hCG-stimulated rats revealed a band of collagenolytic activity at 52kD, corresponding to human MMP-1 and at 72kD, corresponding to human MMP-2. Both activities were markedly stimulated by administration of hCG and were significantly inhibited by indomethacin, NDGA or MK-886. Thus, eicosanoids seem to mediate LH stimulation of follicular collagenase. Interleukin-1 (IL-1) has been recently implicated in ovulation. The ability of an IL-1 receptor antagonist (ra) to block ovulation in vivo and in vitro has been demonstrated recently. Morphological examination of the ovulatory follicles failing to ovulate suggests that this effect is exerted by inhibiting cumulus oophorus expansion and detachment from mural granulosa cells. In vitro, IL-1ra attenuated the action of hCG and FSH on cumulus expansion and follicular hyaluronic acid synthesis. Thus, IL-1 seems to mediate and/or facilitate gonadotropin action on cumulus expansion, and hence on ovulation.
1994
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(1994) Endocrinology. 135, 5, p. 1845-53 Abstract
Although the majority of ovarian follicles undergo atresia through a mechanism involving apoptotic cell death, in vivo studies concerning the hormonal regulation of atresia have been difficult due to the presence of heterogeneous population of follicles in the ovary. In the present study, the regulation of follicle apoptosis by gonadotropins, insulin-like growth factor I (IGF-I), and IGF-binding protein 3 (IGFBP-3) was examined using a serum-free culture of preovulatory follicles. Immature rats at 26 days of age received a single dose of PMSG. Two days later, the largest preovulatory follicles were collected for in vitro culture with or without hormones. After 24 h of culture, follicular apoptotic DNA fragmentation was analyzed by autoradiography of size-fractionated DNA labeled at 3'-ends by [32P]dideoxy-ATP. A spontaneous increase in apoptotic DNA fragmentation occurred after 24 h of culture in the absence of hormones, whereas treatment with human CG (hCG) or FSH suppressed follicular apoptosis in a dose-dependent manner, with 0.1 microgram/ml causing maximal suppression by 60-62%. Cotreatment with hCG and FSH had no additional effect. Like gonadotropins, treatment with IGF-I and insulin also suppressed the spontaneous onset of apoptosis, with IGF-I being more effective than insulin. Cotreatment with IGFBP-3 and hCG dose-dependently reversed the suppressive effect of hCG on apoptosis by 42%, suggesting a mediatory role of endogenously produced IGF-I. The addition of IGFBP-3 also blocked the suppressive action of IGF-I by 49%, whereas it did not affect the suppressive action of an IGF-I agonist or insulin. Treatment with IGFBP-3 alone had no effect on apoptotic DNA fragmentation. Estrogen and progesterone production by the cultured follicles were also analyzed by RIA. Gonadotropin treatment resulted in a marked stimulation of the production of both steroid productions. In contrast, treatment with IGF-I caused a small increase in estrogen but decreased progesterone production. Although treatment with IGFBP-3 alone decreased both estrogen and progesterone production, cotreatment with IGFBP-3 and hCG resulted in a slight decrease in estrogen production but an increase in progesterone production. Furthermore, IGFBP-3 did not affect IGF-I action on steroid production. To further substantiate the hypothesis that IGFBP-3 blocks the suppressive effect of hCG on apoptosis by neutralizing endogenously produced IGF-I, solution hybridization analysis was performed, and hCG treatment was shown to increase IGF-I messenger RNA levels in cultured follicles by 1.9-fold.(ABSTRACT TRUNCATED AT 400 WORDS)
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(1994) Endocrinology. 135, 5, p. 2287-2290 Abstract
Nitric Oxide (NO) is now recognized as a mediator of several biological functions. In the present study we examined the effects of NO synthase (NOS) inhibitors on the ovulatory process in vivo, and whether this effect can be reversed by a NO generator. Immature eCG-hCG treated rats were injected intraperitonealy tip) or unilaterally into the periovarian sac (intrabursal injection; ib) with inhibitors of the inducible form of NOS. Aminoguanidine (AG) suppressed ovulation in a dose-dependent manner, reaching a 54% inhibition at a dose of 20 mg/kg when injected ip (p
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(1994) Biology of Reproduction. 51, 4, p. 662-7 Abstract
Indirect evidence has implicated the interleukin-1 (IL-1) system in ovulation. Thus, the ability of IL-1 beta to induce ovulation in rat and rabbit perfused ovaries has been demonstrated. In the present study, the involvement of the IL-1 system in ovulation was directly tested in vivo, in the rat model. For this purpose, the natural inhibitor of the IL-1 system, interleukin-1 receptor antagonist (IL-1ra), was administered locally by use of an intrabursal injection route. Twenty-six-day-old Sprague-Dawley rats received injections of eCG (10 IU), followed 56 h later by hCG (15 IU). IL-1ra (75 micrograms/bursa) was administered locally into the periovarian sac, 6 h (n = 5), 2 h (n = 11), and 0 h (n = 5) before hCG administration. Control animals (n = 10) received injections of the same volume (50 microliters) of vehicle (PBS). IL-1ra administered locally into the periovarian sac inhibited ovulation from the treated ovary, reaching 40% inhibition (p
1993
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(1993) Endocrinology. 133, 6, p. 2761-2765 Abstract
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.
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Localization of interstitial collagenase gene expression in gilt preovulatory follicles(1993) Endocrine. 1, 6, p. 481-486 Abstract
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(1993) Biology of Reproduction. 48, 4, p. 905-909 Abstract
This study was initiated in order to examine the involvement of leukocytes in follicular rupture in the rat. To evaluate changes in ovarian neutrophil population, ovaries from eCG-primed (15 IU s.c. on Days 25-26) rats were collected 0, 3, 6, and 9 h after hCG (4 IU) administration, and ovarian content of neutrophils was estimated by assaying myeloperoxidase (MPO) activity. The stimulation of hCG increased ovarian MPO activity within 6 h (p
1992
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(1992) Biology of Reproduction. 47, 2, p. 245-253 Abstract
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.
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(1992) Biology of Reproduction. 46, 3, p. 379-385 Abstract
The aim of this study was to evaluate morphometrically the influence of ovulation-inhibiting doses of indomethacin, an inhibitor of the cyclooxygenase pathway, and esculetin and caffeic acid, inhibitors of the lipoxygenase pathway, on the dilatation of the perifollicular capillary network in the theca interna. The development of the perifollicular capillary network as a function of follicular size and the changes in the vascular lumen were examined by light microscopy on a series of semithin cross sections of rat ovaries. The number of capillaries in the theca interna increased linearly with increasing follicle diameter. Thus, the relative number of capillaries in the theca interna supplying the avascular stratum granulosum remained constant. This indicates that follicular function is not regulated through changes in the number of capillaries in the theca interna. After hCG injection, an increase in the capillary area could be observed in follicles having a diameter of more than 600-mu-m. Indomethacin administration increased the capillary area of the ovulatory follicles as compared to the untreated side only at 6 h after treatment. By contrast, treatment with inhibitors of lipoxygenase resulted in a significant decrease in the capillary area of large follicles at all times examined (3, 6, and 9 h after hCG injection). Nevertheless, since both types of eicosanoid inhibitors suppressed follicle rupture, in spite of their opposing actions on the capillary area, it seems unlikely that their action on ovulation is primarily due to their effect on this parameter.
1991
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Preovulatory Changes In Ovarian Expression Of Collagenases And Tissue Metalloproteinase Inhibitor Messenger-Ribonucleic-Acid - Role Of Eicosanoids: Role of eicosanoids(1991) Endocrinology. 129, 4, p. 1869-1875 Abstract
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.
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The action of transforming growth factors and inhibin-related proteins on oocyte maturation(1991) Morphologie. 75, 228, p. 109-13 Abstract
1990
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Recombinant Follicle-Stimulating-Hormone Induces Ovulation And Tissue Plasminogen-Activator Expression In Hypophysectomized Rats(1990) Endocrinology. 127, 6, p. 3023-3028 Abstract
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.
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Basic Fibroblast Growth-Factor Induction Of Granulosa-Cell Tissue-Type Plasminogen-Activator Expression And Oocyte Maturation - Potential Role As A Paracrine Ovarian Hormone: Potential role as a paracrine ovarian hormone(1990) Endocrinology. 127, 5, p. 2357-2363 Abstract
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 μg/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.
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(1990) Endocrinology. 126, 1, p. 376-83 Abstract
hCG is a member of a family of glycoprotein hormones which share a common alpha-subunit, but differ in their hormone-specific beta-subunits. The CG beta-subunit is unique in that it contains a hydrophilic carboxyl-terminal extension with four serine O-linked oligosaccharides. To examine the role of the O-linked oligosaccharides and the carboxyl-terminal extension of hCG beta on receptor binding, steroidogenesis in vitro, and ovulation induction in vivo, site-directed mutagenesis and gene transfer methods were used. Wild-type hCG alpha and hCG beta expression vectors were transfected into an O-glycosylation mutant Chinese hamster ovary cell line to produce intact dimer hCG lacking the beta-subunit O-linked oligosaccharide units. In addition, a mutant hCG beta gene (CG beta delta T) was generated which contained a premature termination signal at codon 115. This gene was cotransfected with the hCG alpha gene into Chinese hamster ovary cells to produce hCG dimer which lacked the carboxyl-terminal amino acids 115-145 of hCG beta (truncated hCG). The O-linked oligosaccharide deficient or truncated hCG derivatives were examined for their ability to bind to the mouse LH/hCG receptor and stimulate cAMP and steroidogenesis in vitro. These studies show that the O-linked oligosaccharides and carboxyl-terminal extension play a minor role in receptor binding and signal transduction. In contrast, comparison of the stimulatory effects of truncated and wild-type hCG in a rat ovulation assay in vivo via either intrabursal or iv injection revealed that the truncated derivative was approximately 3-fold less active than wild-type hCG. These findings indicate that the carboxyl-terminal extension of hCG beta and associated O-linked oligosaccharides are not important for receptor binding or in vitro signal transduction, but are critical for in vivo biological responses.
1989
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(1989) Endocrinology. 125, 4, p. 1857-62 Abstract
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.
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Human Follicular-Fluid Protease And Antiprotease Activities - A Suggested Correlation With Ability Of Oocytes To Undergo Invitro Fertilization(1989) Fertility and Sterility. 52, 2, p. 274-280 Abstract
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(1989) Science. 243, 4889, p. 381-383 Abstract
Follicle rupture during ovulation is associated with inflammation-like changes. Because platelet activating factor (PAF) participates in the inflammatory process, the effect of a PAF-specific antagonist, BN52021, on the ovulatory response was tested in rats. BN52021, administered locally, inhibited follicle rupture in rats stimulated to ovulate with human chorionic gonadotropin (hCG). In addition to suppressing rupture of the follicles, this antagonist suppressed the hCG-stimulated increase in ovarian collagenolysis and vascular permeability. The inhibition of ovulation of BN52021 could be reversed by simultaneous administration of PAF. Furthermore, PAF partially reversed the blockage of ovulation by inhibitors of eicosanoid synthesis. Collectively, these results suggest the involvement of PAF in ovulation. Its role seems to be closely related to the metabolism of arachidonic acid. Thus, modulation of PAF action may serve as an additional target for regulation of reproduction via its action on ovulation.
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(1989) Endocrinology. 124, 1, p. 415-21 Abstract
Indirect evidence has suggested a role for plasminogen activator (PA) in ovulation. Our recent studies demonstrated that 1) tissue-type PA (tPA) is the predominant PA produced by preovulatory rat follicles in response to gonadotropins or GnRH; and 2) several inhibitors of the serine proteases, to which PA and plasmin belong, block ovulation. Here, the role of tPA and plasmin in ovulation was examined directly by the use of specific antibodies to tPA and alpha 2-antiplasmin (alpha 2AP). Immature female rats at 25-26 days of age were treated (sc) with 15 IU PMSG to induce multiple preovulatory follicles. Fifty-four hours later, tPA antibodies and alpha 2AP were injected into one of the ovarian bursae to check their ability to block ovulation, which was initiated with an ovulatory dose (4 IU) of hCG. The data are expressed as percent inhibition of ovulation in the treated vs. the untreated ovaries. A significant decrease in the ovulation rate was obtained by administration of 500 micrograms antibodies to tPA (39.6%) or 1-50 micrograms alpha 2AP (36-44%), whereas minimal inhibition (12%) was found at lower doses of anti-tPA (10 micrograms) or alpha 2AP (0.1 micrograms). Furthermore, nonimmune immunoglobulin G (500 micrograms) and heat-inactivated alpha 2AP were not effective. Anti-tPA and alpha 2AP suppressed ovulation only when injected at the time of hCG administration; later injections (4-h delay) were ineffective, suggesting that PA and plasmin are involved in the early follicular responses to the ovulatory stimulus. Histological observation of the ovaries did not reveal any pathological changes associated with the anti-tPA and alpha 2AP treatment. Suppression of ovulation, as evidenced by decreased number of tubal ova, was frequently accompanied with intraovarian release of the eggs into the follicular thecal compartment. Thus, these results provide direct evidence for an essential role of tPA and plasmin in ovulation.
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(1989) Endocrinology. 124, 1, p. 187-94 Abstract
The regulation of tissue-type plasminogen activator (tPA) in rat oocytes during the periovulatory period, in early embryos, and in oocytes during induced follicular atresia was studied using a quantitative chromogenic substrate assay. Oocytes and early embryos were collected from three ovulation models: 1) intact immature female rats treated with PMSG, followed by hCG 48 h later; 2) hypophysectomized immature rats treated with PMSG, followed by a GnRH agonist (GnRHa) 56 h later; and 3) adult cyclic rats on the mornings of proestrus and estrus and up to 5 days after fertilization. In addition, follicular atresia was induced by either withdrawal of diethylstilbestrol (DES) for 2 days or injection of GnRHa for 2 days in hypophysectomized DES-implanted immature rats. Treatment with PMSG alone did not increase oocyte tPA content (5-20 microIU/oocyte) in either immature rat model, but treatment with either hCG or GnRHa induced meiotic maturation and ovulation and increased tPA activity to 80 and 140 microIU/oocyte 24 h after hCG and GnRHa treatment, respectively. Northern blot analysis of total RNA extracted from oocytes of PMSG-treated rats indicated the presence of a specific tPA message at 22S. tPA levels were low in preovulatory oocytes obtained on proestrus morning and increased in ovulated oocytes on estrus morning. After fertilization, tPA levels remained high in the embryos on days 1-4 of pregnancy, but dropped dramatically on day 5. Furthermore, oocytes from atretic follicles of hypophysectomized DES-implanted rats after either DES withdrawal or GnRHa treatment contained elevated levels of tPA, coincident with germinal vesicle breakdown (GVBD). Immunohistochemical staining revealed tPA antigen only in those oocytes that had undergone apparent meiotic maturation, as confirmed by GVBD. Thus, oocytes contain tPA mRNA and synthesize the active protease under a variety of stimuli which result in GVBD. The observed periovulatory increase in oocyte tPA activity, its maintenance until day 5 of pregnancy, and expression of tPA in nonovulatory oocytes of atretic follicles suggest diverse functions for the oocyte and embryo tPA.
1988
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(1988) Biology of Reproduction. 38, 2, p. 481-5 Abstract
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.
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1987
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(1987) Human Reproduction. 2, 6, p. 505-510 Abstract
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.
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(1987) Journal of Steroid Biochemistry. 27, 1-3, p. 359-363 Abstract
The preovulatory surge of gonadotropins stimulates follocular steroidogenesis and changes from estrogen as the major product to progesterone. We shall overview the studies dealing with the role of ovarian steroidogenesis in follicular rupture at ovulation. Several inhibitors of steroidogenesis blocked follicular rupture in vivo. Likewise, RU 38486 partially blocked ovulation triggered by hCG. Collectively, these data support the knowledge that follicular steroidogenesis is required for ovulation. Recent studies confirmed the essential role of plasminogen activator (PA) in follicular rupture. The LH stimulation of PA activity was partially blocked by several inhibitors of steroidogenesis and it could be restored by the addition of progesterone, testosterone and estradiol-17β, but not the non-aromatizable 5α-dihydrotestosterone. Gonadotropic stimulation enhanced only the synthesis of tissue type PA (t-PA) and not that of urokinase. Likewise, inhibition of steroidogenesis, reduced only the synthesis of t-PA and was reversed by addition of estradiol-17β. It seems, therefore, that follicular steroids, most probably estrogen, are involved in the preovulatory rise in follicular t-PA activity.
1986
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(1986) Endocrinology. 119, 6, p. 2606-2610 Abstract
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.
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(1986) Endocrinology. 119, 4, p. 1588-93 Abstract
Recent studies from our laboratory corroborated the suggested role of plasminogen activation in follicular rupture at ovulation, and its involvement in the activation process of collagenolysis in the follicle. In the present study, the molecular types and cellular source of plasminogen activator (PA) were examined. Explanted preovulatory follicles produced in vitro both urokinase type and tissue type (t-PA) activators. Upon gonadotropin stimulation a highly significant increase in t-PA, but not in urokinase type, was observed. Separation of the follicle into granulosa cells and residual tissue, mainly theca, revealed that both compartments produce both types of PA. The granulosa compartment was found to produce 80-90% of the total follicular PA activity. Gonadotropins stimulated predominantly t-PA. Most of the gonadotropin-enhanced PA activity produced by granulosa cells was secreted into the culture medium, whereas that from thecal origin remained in the tissue. Likewise, in whole follicles only about 10% of PA was secreted into the medium. Gonadotropin-induced PA activity in vitro was reduced by inhibitors of steroidogenesis. This inhibition was overcome by the addition of estradiol-17 beta. The inhibition of steroidogenesis affected predominantly the t-PA type of PA. In conclusion, the granulosa cells contribute most of the follicular PA activity, and t-PA is predominantly enhanced by gonadotropin and estrogen. It seems, therefore, that t-PA is the activator involved in the processes leading to follicular rupture.
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(1986) Human Reproduction. 1, 6, p. 353-356 Abstract
In view of the studies demonstrating the involvement of eicosanoids (prostaglandins and hydroxyperoxides, including leukotrienes) in ovulation in several mammalian species, we have examined the activity of the two enzyme systems, lipoxygenase and cyclooxygenase in human granulosa cells obtained from women undergoing in-vitro fertilization-embryo transfer. The activity of cyclooxygenase was assessed by radio-immunoassay of prostaglandin E and of 6-keto-prostaglan-din F1α, the conversion product of prostacyclin, accumulated in the culture medium of granulosa cells. Lipoxygenase activity was detected by the conversion of [14C]arachidonic acid into its products (hydroxyperoxides and leukotrienes) separated by reversed phase high-performance liquid chromatography. The results confirmed the activity of cyclooxygenase in human granulosa cells, production in vitro of prostaglandin E and prostacyclin and demonstrated the presence of active lipoxygenase enzymes. These results support the possible involvement of eicosanoids in ovulation of the human.
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(1986) Clinics in Endocrinology and Metabolism. 15, 1, p. 157-170 Abstract
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.
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(1986) Endocrinology. 118, 4, p. 1266-1270 Abstract
Androstenedione synthesis was studied in isolated rat preovulatory follicles and compared with that of rat testicular tissue using [14C]progesterone together with 17α-hydroxy-[3H]progesterone as substrates in the presence of NADH or NADPH as cofactors. The amount of androstenedione formed was measured by addition of carrier, reisolation, and crystallization to constant specific activity. The labeling patterns of androstenedione and 17α-hydroxyprogesterone (17-OHP) confirmed that both tisues preferentially catalyzed the synthesis of androstenedione from progesterone rather than from 17-OHP. It appears, therefore, that free 17-OHP was not an obligatory intermediate in this reaction. When hCG (5 IU) was administered sc and the follicles were isolated 3 h later, androstenedione synthesis was inhibited whether NADH or NADPH was added as cofactors. By contrast, 17-hydroxylase activity was inhibited only with NADH as cofactor. Hence, the gonadotropin, with NADH as cofactor, specifically reduced progesterone incorporation into androstenedione without affecting incorporation of 17-OHP. Thus, hCG appears to affect androstenedione production from progesterone at two different sites of the lyase complex.
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(1986) Gamete Research. 13, 1, p. 39-46 Abstract
Hypophysectomy of 15‐day‐old rats (hypox) markedly reduced the normal development of meiotic competence and abolished the development of antral follicles between days 21 and 31 postpartum (pp). Here the correlation among age of the rats, stage of follicular development, and meiotic competence was examined. Administration of pregnant mare's serum gonadotropin (PMSG‐3IU) or insertion of an estradiol‐17β (E2) capsule to hypox rats induced the development of meiotic competence provided the treatment started after day 20 pp. Hormonal treatments at an earlier age were not effective in inducing meiotic competence in hypox rats. The induction of meiotic competence by PMSG or E2 was associated with an increase in the number of granulosa cells and formation of follicular antrum. The finding that PMSG and E2 failed to induce meiotic competence when administered prior to day 21 pp suggests that the development of meiotic competence is an age‐dependent process. When the hormonal treatments commenced after day 21, both follicular development and meiotic competence were induced.
1985
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(1985) Prostaglandins. 30, 4, p. 581-590 Abstract
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.
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(1985) Endocrinology. 116, 2, p. 522-527 Abstract
Collagenolytic activity in ovarian follicles was previously demonstrated by using synthetic peptides and reconstituted collagen fibers. However, attempts to demonstrate degradation of ovarian collagen and to correlate collagenase activity with ovulation were not successful. By administration of L-(5- 3H) proline, we have labeled ovarian and follicular collagen and followed collagenolytic activity by separation of 3H-hydroxyproline (3H-Hyp) from acid hydrolyzates of ovarian tissue by HPLC. The level of ovarian and follicular 3H-Hyp decreased by about 40% on the afternoon of proestrus or after exogenous stimulation of ovulation by human CG (hCG), and this decrease was abolished by blocking the surge of gonadotropins with Nembutal. To verify that the observed reduction in 3H-Hyp was due to the action of a typical collagenase, the collagenous fraction was prepared from ovarian tissue and from preovulatory follicles before and after the ovulatory stimulus. The extracts were treated with trypsin (25 min, 25 C, 0.01 mg/ml) plasmin and pamino- phenyl-mercuric acetate to fully activate the collagenase extracted along with collagen. Both, enzymatic and chemical activation of collagenase in vitro resulted in degradation of collagen. This degradation could be inhibited by cysteine and EDTA; both are classic inhibitors of mammalian collagenases. The activity of ovarian collagenase increased within 3 h after hCG-stimulation, peaked at 5-fold 6 h after hCG, and declined afterwards. Administration of cysteine (0.001-0.01 mmol) into the bursal cavity of proestrous rats blocked ovulation and breakdown of ovarian collagen in a dose-dependent manner. Cysteine effectively inhibited ovulation even when injected 7 h after the hCG stimulus. Inhibitors of arachidonic acid metabolism prevent ovulation. Indomethacin (inhibitor of cyclooxygenase) and nordihydroguaiaretic acid (inhibitor of lipoxygenase) blocked ovulation and inhibited hCG-induced ovarian collagenolysis. Collectively, these results corroborate the essential role of collagenolysis in follicular rupture in mammals.
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(1985) Endocrinology. 116, 2, p. 516-521 Abstract
Production of plasminogen activator (PA) by granulosa cells (GC) and its stimulation by gonadotropins led to the suggestion that PA is involved in ovulation. However, whereas only LH may be regarded as the ovulation-inducing hormone in the rat, FSH was found to be much more potent than LH in enhancing PA production by GC. Assuming that the entire follicular wall, rather than isolated GC, is involved in follicular rupture, we have examined activity of PA in intact follicles. LH (NIH-LH-S23) was 5-fold more potent than FSH (NIH-FSH-S14), and purified ovine LH and FSH were equally potent in enhancing follicular PA activity. Furthermore, injection into the ovarian bursa of proestrous rats of e-amino-caproic acid and benzamidine (0.05-0.25 mmol), inhibitors of.serine proteases, including PA and plasmin, resulted in a dose-dependent inhibition of ovulation without causing changes discernible by histological examinations of the ovaries. Whereas steroids did not change basal follicular PA production in culture, addition of estradiol-17β [(E2) 1 µg/ml] but not progesterone or testosterone, further enhanced LH-stimulated PA. Aminoglutethimide phosphate (10-3 M) and 17β-formamidoandrost-4-en-3-one inhibited LH-induced increase in follicular PA and this inhibition was reversed by addition of E2. Intrabursal injection of indomethacin, an inhibitor of cyclooxygenase, and of nordihydroguaiaretic acid, an inhibitor of lipoxygenase pathway of arachidonic acid metabolism at doses which effectively blocked ovulation (0.3 mg/bursa) had no effect on PA content of the follicles. Likewise, indomethacin (10 µM) and nordihydroguaiaretic acid (100 µM) did not affect LH-stimulated PA in vitro. In conclusion, LH, the physiological trigger of ovulation is, at least, as potent as FSH in stimulating follicular PA activity. The role of serine proteases, most probably of PA and plasmin, in ovulation is further corroborated by a pharmacological approach. LH stimulation of follicular PA appears to be enhanced by E2 but is not mediated by arachidonic acid metabolites.
1984
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Regulation of the development of meiotic competence and of the resumption of oocyte maturation in the rat(1984) Symposia of the Society for Experimental Biology. 38, p. 25-43 Abstract
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)
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Experimental approaches to atresia in mammals(1984) Oxford reviews of reproductive biology. 6, p. 226-65 Abstract
1983
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(1983) Prostaglandins. 26, 6, p. 1011-1020 Abstract
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.
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(1983) Journal of Steroid Biochemistry. 19, 1 PART 3, p. 965-971 Abstract
The ability of rat oocytes to resume meiotic maturation upon isolation ("meiotic competence") was shown to be abolished by hypophysectomy on day 15 post partum (p.p.). FSH (NIAMD-rat FSH-B-1; 10-20 μg/day), but not LH (NIAMD-oLH-21; 2-20 μg/day) was able to induce meiotic competence in rats hypophysectomixed on day 15 pp. Implantation of a capsule of oestradiol-17β, but not of progesterone or androstenedione, partially restored meiotic competence in hypophysectomized rats within 24 h of treatment. When inhibitors of steroidogenesis (aminoglutethimide, 17β-formamidoandrost-4-en-3-one, 1,4,6-androstatriene-3,17-dione or 4-hydroxy-4-androstene-3,17 dione) were coadministered with FSH they inhibited its effect on the development of meiotic competence. Furthermore, when oestradiol-17β was administered together with these inhibitors of steroidogenesis, the effect of FSH on the development of meiotic competence was not disturbed. These results strongly suggest that FSH, but not LH, is involved in the development of meiotic competence. Further, this action of FSH appears to be mediated, at least partially, by follicular oestrogen production.
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(1983) Journal of Reproduction and Fertility. 68, 1, p. 247-52 Abstract
Oocyte maturation inhibitor (OMI), inhibin, progesterone and oestradiol 17 beta concentrations were measured in fluid collected from small (less than 3 mm), medium size (3-6 mm) and large (greater than 6 mm) porcine ovarian follicles, which were obtained on Days 5, 10, 15 and 18 of the oestrous cycle and at 24 h after the onset of oestrus. Concentrations of OMI decreased with increasing follicle diameter (P less than 0.05), independent of the stage of the oestrous cycle. Concentrations of inhibin showed a tendency to decrease with increasing follicle diameter on Days 10, 15 and 18, but not on Day 5 of the cycle. Concentrations of OMI and inhibin in the largest follicles were low before the onset of oestrus, and were essentially unaltered 24 h later. A positive correlation was found between OMI and inhibin concentrations, whereas the correlation between inhibin concentration and log (progesterone concentrations) was negative.
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(1983) Biology of Reproduction. 28, 1, p. 161-166 Abstract
The mechanism of action of a gonadotropin releasing hormone (GnRH) agonistic analog ([D-Ala6]GnRH) on the rat ovary has been studied in comparison to similar effects of luteinizing hormone (LH). Stimulation of meiosis resumption in vitro in follicle-enclosed oocytes by both LH and [D-Ala6]GnRH, was blocked by elevated levels of cAMP as demonstrated when either dibutyryl cAMP or the phosphodiesterase inhibitor methylisobutylxanthine was present in the culture medium. In vivo, the prostaglandin synthase inhibitor indomethacin, which blocks LH-induced ovulation, also inhibited ovulation induced by the GnRH analog in hypophysectomized rats. On the other hand, the potent GnRH-antagonist [D-pGlu1, pClPhe2, D-Trp3,6]GnRH which blocked the stimulatory effect of the agonist on oocyte maturation and ovulation had no effect on LH action. It is concluded that while a GnRH-like peptide does not seem to mediate LH action on the ovarian follicles, both LH and GnRH agonist share some common mechanistic pathways at a post-receptor locus.
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(1983) The Ovary. Serra G. B.(eds.). New York: . p. 83-94 (trueComprehensive Endocrinology). Abstract
Several reviews dealing with different aspects of oocyte maturation in mammals have been published recently. Therefore, this is not intended to be an exhaustive review of mammalian oocyte maturation, but a brief account with a special emphasis on the follicular factors involved in the regulation of the meiotic process.
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(1983) Unknown Journal. 97, 1, p. 43-49 Abstract
The aim of this study was to search for direct biochemical effects of highly purified FSH on isolated ovarian follicular theca in vitro. Granulosa cells (GC; approximately 1 x 105 cells per follicle) were flushed from isolated follicles of pro-oestrous rats. The remaining theca layer and the isolated GC were incubated with highly purified ovine FSH. Prostaglandin E (PGE) accumulation was measured by radioimmunoassay. Follicle-stimulating hormone induced a 15-fold increase in PGE accumulation over the basal level in the follicular theca, the stimulated rate exceeding threefold that observed in the GC fraction derived from the same follicle. Follicle-stimulating hormone caused no significant increase in cyclic AMP level or steroidogenesis in the theca layer, but was active on these parameters in the GC. In contrast, LH increased the accumulation of cyclic AMP, progesterone and testosterone, as well as of PGE, in follicular theca. Exogenous 8-bromo cyclic AMP or cyclic GMP also stimulated PGE production in follicular theca or GC, but FSH was without any effect on the level of endogenous cyclic GMP in GC or follicular theca. Antibodies to FSH prevented the effect of FSH (but not that of LH) on PGE formation by follicular theca and GC, while antibodies to the β-subunit of LH blocked the effect of LH but not of FSH. We conclude that highly purified FSH has a stimulatory effect on PGE formation by the follicular theca.
1982
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(1982) Biology of Reproduction. 27, 1, p. 223-31 Abstract
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.
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(1982) Endocrinology. 110, 2, p. 457-461 Abstract
The purpose of the present study was to measure norepinephrine (NE) in Graafian follicles and correlate changes in its concentration with circulating gonadotropins secreted endogenously or administered exogenously. Graafian follicles were removed from the ovaries of adult cycling rats. The follicles were pooled in groups of 10-13, and NE was determined by high performance liquid chromatography. Follicular NE (picograms per jug protein) did not change between 0900 h (3.61 ± 0.34) and 1300 h (3.12 ± 0.25) on proestrus, but was reduced significantly to 1.45 ± 0.16 at 2100 h, which is 4 h after the peak of the gonadotropin surge. There was a further reduction to 0.83 ± 0.08 in fresh corpora lutea taken on estrus at 0900 h. The decrease in follicular NE was prevented in estrous rats which were either hypophysectomized 24 h previously or treated with sodium pentobarbital at 1330 h on proestrus. To determine which pituitary hormone was responsible for follicular NE depletion, rats were injected at 0900 h on proestrus with LH (5 μg), FSH (20 μg), LH plus FSH (5 and 20 αg, respectively), or PRL (20 αg), and follicular NE was determined 4 h later. FSH reduced follicular NE significantly to 1.86 ± 0.16 compared to both the control (3.12 ± 0.25) and the PRL-injected group (2.92 ± 0.32), whereas LH caused a small but nonsignificant decrease (2.49 ± 0.2). Both LH and FSH doses used resulted in ovulation, as determined by counting tubal ova 12 h after hormonal treatment. We conclude that 1) NE in Graafian follicles is markedly reduced within 4 h after the preovulatory gonadotropin surge in the normal cycling rat; this reduction is prevented when the surge is abolished; 2) the hormone responsible for follicular NE depletion is FSH rather than LH or PRL; and 3) finally, it is suggested that follicular NE may be involved with the formation and/or functioning of the corpus luteum.
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(1982) Journal of Reproduction and Fertility. 64, 2, p. 541-551 Abstract
Pincus & Enzmann suggested that meiotic maturation in mammals is held in abeyance by a follicular factor. Recently, OMI from porcine follicular fluid and granulosa cells was partly purified and characterized. However, OMI is only one of the factors involved in the control of the meiotic process. Other factors, such as oocyte-cumulus cell communication, cyclic AMP and steroids are probably involved in controlling meiosis. Some of the putative mechanisms involved in the control of meiosis are depicted. Meiosis is prevented in antral follicles by OMI produced by granulosa cells. The inhibitory action of OMI is apparently exerted through the mediation of cumulus cells and the signal conveyed through cumulus cell-oocyte gap junctions. It is not known whether cAMP or OMI are transferred to the oocyte as the inhibitory signal. LH induces the resumption of meiosis, possibly through the mediation of mural granulosa and cumulus cells. The mode of action of LH in inducing meiosis remains to be elucidated: LH may act either by eliminating the inhibitory signal, by blocking its transfer via the cumulus cell-oocyte junctions or by terminating OMI production. Alternatively, LH may generate a yet unspecified positive signal. More studies are needed to understand the intricate mechanism involved in the control of meiotic maturation. Purification of OMI to homogeneity is required for assessing the precise physiological role of this factor in the meiotic process.
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(1982) Biology of Reproduction. 27, 2, p. 463-471 Abstract
The effect of clomiphene citrate was tested on rat preovulatory follicles in culture. Clomiphene inhibited both basal and luteinizing hormone (LH)-stimulated steroid accumulation. The dose-dependent effect of clomiphene was much more pronounced on follicles cultured with LH; while 0.01 mM had no effect, 2 mM clomiphene decreased progesterone accumulation by 97%, estradiol-17β by 90% and testosterone by 65% (P
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1981
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(1981) Gamete Research. 4, 5, p. 463-472 Abstract
Oocytes were removed from the follicles of rats at 15 to 31 days of age, and their ability to resume meiosis (“meiotic competence”) 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
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1980
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(1980) Journal of Reproduction and Fertility. 59, 2, p. 267-272 Abstract
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.
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(1980) Journal of Reproduction and Fertility. 59, 2, p. 259-265 Abstract
Graafian follicles from rats treated for 1 or 2 days with pentobarbitone sodium (Nembutal) were similar in appearance to pro-oestrus preovulatory follicles, but after 3 or 4 days of treatment early atretic changes were recognized. Ovulatory efficiency decreased to 88, 70, 52 and 31% after 1, 2, 3 and 4 days of treatment, respectively. The mean ± s.e.m. rate of accumulation (ng/follicle 24 hr) of progesterone, androstenedione and oestradiol was 3.6 ± 0.7, 4.0 ± 0.3 and 18.9 ± 3.9, respectively, in preovulatory follicles and 10.2 + 1.7, 0.9 ± 0.1 and 1.9 ± 0.4 respectively in follicles explanted from rats treated for 4 days with Nembutal. Addition of LH (5 μg/ml) to the culture median stimulated steroid accumulation by both types of follicles. Thus atretic follicles are characterized by impaired androgen and oestradiol formation. Addition of testosterone (1 μg/ml) to the culture medium increased the accumulation of oestradiol by atretic follicles. It is inferred that the early stages of atresia of rat follicles are distinguished by a deficiency in the activity of enzymes responsible for the conversion of progesterone to androgens that can serve as substrates for aromatization.
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Ovarian follicular and luteal physiology(1980) International review of physiology. 22, p. 117-201 Abstract
Follicular maturation and development is a complex process of interrelated intra- and extraovarian events that ultimately lead to ovulation of a mature oocyte and transformation of the ruptured follicle into a corpus luteum. The primordial follicle consists of an immature oocyte arrested in the dictyate stage of meiosis, surrounded by a single layer of relatively undifferentiated granulosa cells. The oocyte remains in the immature state because of many factors, one of which is the oocyte maturation inhibitor (OMI) secreted by granulosa cells. The oocyte subsequently increases in size, and as the antrum forms it becomes surrounded by cumulus cells. The cumulus cells may be intimately involved in the action of O,I to arrest the oocyte in the immature state within the follicle, as well as the resumption of meiosis during the LH surge. The compartments of the follicle that change most dramatically during follicular maturation are the cells lining the follicle--the granulosa and thecal cells. Under the influence of estrogen and FSH, the granulosa cells proliferate and also acquire FSH receptors. At this time, the thecal compartment differentiates and surrounds the granulosa cells, but remains separated from them by a basement membrane. Steroid secretion by the antral follicle involves the interplay of androgens, estrogens, and progestins. Both the granulosa and thecal cell compartments contribute to follicular fluid and serum levels of steroids; the interaction of both cell types may be necessary for estrogen and progesterone secretion in some species. As a consequence of the presence of an elevated number of FSH receptors, the granulosa cells of the small antral follicle are able to respond to FSH in many ways, including increased cyclic AMP accumulation, activation of the aromatase system, and induction of LH receptors, which permits the granulosa cells to later respond to LH. The mechanism by which thecal cells acquire their LH receptors is presently unknown. The granulosa cells of the follicle may indirectly control their own maturation and the number of follicles maturing through the secretion of follicular inhibin, which decreases the pituitary output of FSH. Even though the granulosa cells have acquired large numbers of LH receptors, they are prevented from luteinizing prematurely by factors in follicular fluid, including estrogen and a luteinizing inhibitor (LI). As serum LH levels increase during the preovulatory LH surge, a number of events occur: resumption of oocyte meiosis, transformation of the steroid enzyme complex from estrogen to progesterone secretion, follicular rupture, and formation of the corpus luteum. Granulosa cells form the bulk of the corpus luteum, which secretes elevated amounts of progesterone for a fixed time period depending on the species. Before ovulation the preovulatory follicle must be exposed to and respond to adequate LH and FSH levels in order for the eventual corpus luteum to secrete elevated amounts of progesterone for its normal lifespan...
1978
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(1978) Journal of Experimental Zoology. 205, 2, p. 293-300 Abstract
Resumption of meiotic maturation was induced in follicleenclosed rat‐oocytes 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.
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(1978) Annales de Biologie Animale, Biochimie, Biophysique. 18, 2 B, p. 523-528 Abstract
In vitro studies of mammalian oocytes led to the suggestion that a follicular factor, derived from granulosa cells (GC) and accumulated in follicular fluid (FFI), prevents the resumption of meiosis within the large Graafian follicle(s). The objectives of the present study were to test the effect upon the maturation of rat oocytes of (a) porcine FFI and a low molecular weight fraction thereof (PFFI), and (b) coculture with rat GC. Both FFI and PFFI inhibited the resumption of meiosis (germinal vesicle breakdown, GVB) of rat oocytes cultured with adherent cumulus cells. Coculture of oocytes with freshly isolated rat GC did not affect the spontaneous resumption of maturation, but GC cultured for 24 hrs before adding the oocytes inhibited maturation. Culture of oocytes in a medium in which GC had been cultured for 48 hrs resulted in similar inhibition of meiosis. Addition of LH (5 μg/ml) overcame the inhibitory action of coculture with GC but only partially reversed the effect of GC conditioned medium and of PFFI. These studies demonstrate that the maturation inhibiting action of porcine FFI is not confined to oocytes of that species; rat granulosa cells in culture produce a similar inhibitor of maturation; and LH at least partially counteracts these inhibitory activities. These findings add support to the suggested role of GC in the control of oocyte maturation.
1977
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(1977) Biology of Reproduction. 17, 1, p. 9-15 Abstract
The oxygen consumption of isolated, mechanically denuded rat oocytes in different stages of meiosis was analyzed by a highly sensitive microspectrophotometric technique. The oocytes were either induced to mature in vivo by LH or matured spontaneously when cultured. In both cases there was a gradual increase in respiration of the oocytes following the onset of morphological nuclear maturation. When oocytes were isolated from preovulatory follicles after LH injection the respiration of maturing oocytes with germinal vesicle breakdown (GVB) was 0.173±0.005 nl O2/h/oocyte (n=25). This is significantly higher than the respiration of dictyate oocytes taken from rats not treated with LH, which was 0.135±0.003 nl O2/h/oocyte (n=38). Oocytes with polar body (PB) consumed 0.206±0.007 nl O2/h/oocyte (n=14) and oocytes recovered from the oviduct after ovulation consumed 0.203±0.004 nl O2/h/oocyte (n=7). For oocytes maturing spontaneously in culture the oxygen consumption was 0.171±0.004 nl O2/h/oocyte (n=33) (oocytes with GVB) and 0.190±0.013 nl O2/h/oocyte (n=4) (oocytes with PB), in both cases a significant increase as compared to control, i.e., not cultured oocytes, which consumed 0.142±0.003 nl O2/h/oocyte (n=18). Neither after LH injection nor after cultivation was the respiration of oocytes still containing germinal vesicle different from control. It is concluded that the resumption of oocyte meiosis is accompanied by an increase in oxygen consumption and that this increase seems to be related to the meiotic process rather than to hormonal stimulation.
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(1977) Metabolism-Clinical And Experimental. 26, 4, p. 413-68 Abstract
The mechanism of action of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) upon various cell types of the mammalian ovary is reviewed. Emphasis is placed upon in vitro studies using organ and cell culture as well as short-term incubations. FSH and LH actions upon the following ovarian functions are discussed: steroidogenesis and metabolism of the ovary as a whole and of the isolated follicle and its component cell types, the granulosa and thecal cells, as well as folliculogenesis and follicular growth, oocyte maturation, follicular rupture, and corpus luteum maintenance and steroidogenesis. The roles of gonadotropin receptors, AMP, prostaglandins, protein kinase, and protein synthesis in these LH and FSH actions are discussed. Intra-ovarian regulation of LH and FSH action is reviewed, including a discussion of the possible roles of follicular fluid inhibitors upon oocyte maturation and granulosa cell luteinization.
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(1977) Journal of Endocrinology. 75, 2, p. 285-291 Abstract
The inhibitory action of porcine follicular fluid (FFl) on the spontaneous maturation of isolated rat oocytes was studied. Both FFl and a low-molecular-weight fraction thereof (PFFl) inhibited the maturation of oocytes in culture. When the oocytes were scrutinized for maturation after cultures for 6 hr in a medium containing 50% (v/v) FFl, the incidence of germinal vesicle breakdown (GVB) was reduced from 75 to 53% (P 0.05). Inhibition of GVB by PFFl occurred at concentrations ≥0.085 mg protein/ml; the extent of inhibition was related directly to the concentration of inhibitor and inversely to the duration of culture and the developmental stage of the follicles from which the oocytes were derived. The inhibition of the resumption of meiosis by FFl or PFFl was overcome by ovine LH (5 μg/ml). However, GVB was delayed even in the presence of LH, compared with controls cultured in the absence of the inhibitor. These results demonstrate that the maturation-inhibiting action of porcine follicular fluid is not species specific and that the sensitivity of the rat oocyte to the inhibitor changes during the course of follicular develpment.
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Intraovarian Factors In Ovulation - Determinants Of Follicular Response To Gonadotropins(1977) Journal of Reproduction and Fertility. 51, 1, p. 215-& Abstract
1976
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(1976) Endocrinology. 98, 3, p. 655-61 Abstract
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.
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(1976) European Journal of Endocrinology. 81, 2, p. 362-6 Abstract
Luteinizing hormone (NIH-LH-S18; 5 mug/ml) stimulated aerobic glycolysis in cultured Graafian follicles explanted from pro-oestrous rats before the preovulatory gonadotrophin surge: lactate accumulation in the medium was 70% above control levels during 6 h incubations. Iodoacetate (2.5 x 10(-5) M) prevented this effect, without impairing the ability of LH to induce resumption of oocytic meiosis. Enrichment of the medium with pyruvate (3.3 x 10(-4) M) or lactate (2.5 x 10(-2) M) did not in itself cause ovum maturation. The results do not support the hypothesis that termination of meiotic arrest by LH is due to stimulation of glycolytic activity in the follicle cells, resulting in increased availability of an energy source readily urilizable by the oocyte.
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(1976) Unknown Journal. 83, 1, p. 151-157 Abstract
Steroid release by cultured Graafian follicles explanted from rat ovaries on the morning of pro oestrus was measured by radioimmunoassay. Addition of cyanoketone (10-4M) or aminoglutethimide (10-3M) to the medium reduced the basal level of steroid secretion (progesterone, androstenedione and oestradiol 17β) and abolished the steroidogenic effects of luteinizing hormone (LH; 5μg/ml) and prostaglandin E2 (PGE2; 10 μg/ml). However, the induction of oocyte maturation by either LH or PGE2 was not impaired by total suppression of the steroidogenic response of the follicles to these hormones by cyanoketone or aminoglutethimide. It is concluded that the meiosis inducing action of LH on the mammalian egg is not mediated by the effect of the hormone on the rate and pattern of follicular steroidogenesis.
1975
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(1975) Endocrinology. 96, 6, p. 1533-42 Abstract
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.
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Influence of follicular maturation and culture conditions on the meiosis of pig oocytes in vitro(1975) Journal of Reproduction and Fertility. 43, 1, p. 149-52 Abstract
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An inhibitory influence of granulosa cells and follicular fluid upon porcine oocyte meiosis in vitro(1975) Endocrinology. 96, 4, p. 922-7 Abstract
Isolated oocytes will resume meiosis spontaneously in vitro whereas follicle-enclosed oocytes will remain in the dictyate stage when cultured unless they have been exposed to gonadotropins in vivo or in vitro. To examine the source of the follicular inhibitory influence, porcine oocytes have been cultured alone, with hemisections of follicle wall, granulosa cells, or with follicular fluid. Oocytes isolated from medium-sized (3-5 min) follicles resumed meiosis when cultured; 77.5 plus or minus 3.4 percent matured beyond the dictyate stage. When oocytes were cultured in the presence of follicle wall hemisections of medium and large (6-12 mm) follicles, the percentage of maturing oocytes was significantly reduced. The maturation of oocytes cultured in a medium containing 50 percent follicular fluid from small or large follicles was significantly inhibited. Resumption of meiosis was completely inhibited by co-culture of isolated oocytes with 10-7 granulosa cells from small, medium or large follicles. Addition of serially diminishing amounts of granulosa cells from 10-7 to 10-4 cells reduced the inhibitory influence. It is concluded that the granulosa cells are responsible for the maintenance of the oocytes in the dictyate stage within the follicle. The granulosa cells appear to exert their inhibitory influence upon meiosis by secretion of a chemical message into follicular fluid.
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1974
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Gonadotropin action on cultured graafian follicles: Mechanism of induction of maturation of the mammalian oocyte(1974) Recent Progress in Hormone Research. Vol. 30, p. 79-138 Abstract
The organ culture system described in this paper affords a model in which the maturation inducing action of gonadotropins on the mammalian oocyte can be demonstrated in vitro and analyzed in a quantitative manner. In addition to luteinizing (LH) the follicle stimulating hormone (FSH) appears to have an intrinsic maturation (inducing capability. In the hormone intact rat, however, FSH is not physiologically secreted in adequate amount to perform this function without the cooperation of LH. Prostaglandin E2 is also effective in inducing maturation of the follicle enclosed oocyte in vitro and in vivo. A common denominator of these chemically dissimilar agents is the ability to promote cyclic adenine monophosphate (cAMP) formation in the follicle. Exogenous cAMP, under special experimental conditions, could induce ovum maturation. Theophylline (10-3M), while potentiating the stimulatory action of threshold doses of LH on cAMP and progesterone accumulation, prevented the action of the hormone on ovum maturation. Isolated follicles saturable tissue and hormone specific binding of LH to receptors located on the cell membrane and coupled to adenylate cyclase, but the cellular target of LH in the follicle still requires definition. The follicle contains a cAMP sensitive protein kinase. The relevant endogenous substrate of this kinase remains unknown. Responsiveness of this kinase to cAMP, and of ovarian adenylate cyclase to LH, is acquired toward the end of the second week of postnatal development. LH, FSH, and prostaglandin E2 induce an early and dramatic rise in the rate of progesterone release from the follicle, but several independent lines of evidence indicate that the meiosis inducing action of LH on the mammalian oocyte is not mediated by a steroid. Conversely, the steroidogenic action of LH on the follicle is unaffected by the oocyte. Although LH does stimulate lactate formation by the cultured follicle, it would appear that LH does not terminate meiotic arrest by augmenting the supply of a substrate readily utilizable by the oocyte. LH induced ovum maturation requires de novo synthesis of specific protein that appears to be under translational control. The authors do not believe that prostaglandin E2 is an obligatory mediator or even an important modulator of the actions of LH on ovarian adenyl cyclase, steroidogenesis, and ovum maturation, but would entertain the possibility that it may have a permissive role in LH induced follicular rupture. Thus the 3 major functional components of ovulation ovum maturation, differentiation of luteal cells, and ovum release although all initiated by LH, appear to be mediated by disparate mechanisms and thus are susceptible, experimentally, to selective inhibition. This may have practical implications. (110 references).
1973
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(1973) Biochemistry. 12, 16, p. 3072-3076 Abstract
Intraperitoneal injection of 30 μg of luteinizing hormone (LH) into 20- to 22-day-old rats resulted in an increase in the specific activity of ornithine decarboxylase in the 38,000gmax supernatant fraction of ovarian extracts. A significant increase was apparent 1 hr after injection; the peak value (a 10-16-fold increase) occurred at 4 hr, and thereafter activity declined but at 8 hr was still significantly above the basal level. Adult rats also responded to exogenous LH by an increase in specific activity of ornithine decarboxylase. This rise was seen at every stage of the estrous cycle, even between 1400 and 1800 hr on the day of proestrus when there is a physiological rise in ornithine decarboxylase activity following the endogenous secretion of LH. The spontaneous cyclic rise in ornithine decarboxylase activity was prevented by injection of pentobarbital at 1400 hr on the day of proestrus. The ability of the ovary to respond to LH injection with increased ornithine decarboxylase activity first appears in the second week after birth and continues to increase at least until day 20. In prepuberal rats (20-day old) the effect of LH on ornithine decarboxylase activity was inhibited by injection of puromycin (180 Mg/g), cycloheximide (6 μg/g), or actinomycin D (8 μg/g). These results indicate that the action of LH results in de novo synthesis of ornithine decarboxylase, and that enhanced RNA synthesis may be required for this response.
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(1973) Journal of Endocrinology. 57, 2, p. 217-33 Abstract
The actions of luteinizing hormone (LH) and of prostaglandin E2 (PGE2) on the intact ovary or isolated components of the ovary from adult or prepubertal rats were studied in vitro with respect to the rate of incorporation of [3H]adenine via ATP into cyclic adenosine 3',5' monophosphate (cAMP), or the level of cAMP measured by a competitive protein binding assay; protein kinase activity in the 27,000 g supernatant, with calf thymus histone as the substrate; and rate of oxidation of D [6 14C] glucose. In addition, ornithine decarboxylase activity was assayed in the ovary in vitro after hormone treatment in vivo. When ovaries from adult or pubertal rats were incubated in Krebs Ringer bicarbonate buffer, addition of PGE2 to the medium resulted within 1 min in a doubling of the rate of cAMP formation; the rate was about fourfold after 5 min. PGE2 was 25 times more potent in this respect than prostaglandin F(2α). Both isolated Graafian follicles and corpora lutea responded to either PGE2 or LH with increased cAMP producton. During the perinatal period and until the age of 10 days the ovaries were unresponsive to LH, but responded to PGE2 with increased cAMP formation. Follicles cultured with LH for 18 hr and then washed were refractory to subsequent stimulation by LH, yet remained fully responsive to PGE2. A prostaglandin analogue, 7 oxa 13 prostynoic acid, did not inhibit LH stimulated or PGE2 stimulated cAMP formation in vitro in whole ovaries. Indomethacin, a substance reported to inhibit prostaglandin synthesis in other tissues, likewise failed to inhibit this action of LH. Simultaneous addition of LH and PGE2 to the incubation medium augmented cAMP production to a significantly greater extent than did either agonist when added at maximally effective concentrations on their own, though this augmentation was short of a full additive effect. The latter finding provides presumptive evidence against the view that PGE2 is an obligatory mediator of LH action on the ovary, but this question remains open. PGE2 mimicked the action of LH in stimulating glucose oxidation by ovaries in vitro, and in causing a 15 fold increase in ovarian ornithine decarboxylase activity within 4 hr of injection into prepubertal rats. Ovarian protein kinase activity in pubescent (28 day old) rats was markedly enhanced by incubation of intact ovaries with LH or PGE2 or by exogenous cAMP added to the 27,000 g supernatant. The stimulatory action of LH or PGE2 on the enzyme was attended by a significant decrease (to 10% of control value) in the binding of exogenous cyclic [3H]AMP to protein in the 2000 g supernatant fraction of the ovary, presumably reflecting saturation of the cAMP binding regulatory subunit of protein kinase by enhanced endogenous generation of the cyclic nucleotide. Stimulation of ovarian protein kinase by exogenous cAMP was demonstrable in 12 day old rats, but insignificant at the age of 7 days. It thus appears that the competence of ovarian protein kinase to respond to cAMP, and of ovarian adenylate cyclase to respond to LH, are both acquired during the 2nd wk of postnatal development.
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(1973) Prostaglandins. 3, 4, p. 461-467 Abstract
Serum LH levels were determined by radioimmunoassay at the normal time of the proestrous LH peak (17.30 - 18.00) and ovulatory performance was examined on the morning of estrus in rats treated with indomethacin, an inhibitor of prostaglandin synthesis. When the drug was administered at 14.30 on the day of proestrus, only 21% of the rats ovulated and the total number of ova shed was reduced to 4% of that found in the untreated control group, but there was no significant change in peak serum LH level (1122 ± 184 vs. 975 ± 240 ng/ml ± S.E., treated vs. control). Prostaglandin E2 (PGE2) given late on the day of proestrus (25 to 750 μ g/rat at 24.00) was effective in overcoming this antiovulatory action of indomethacin: 71-90% of the rats ovulated, though the number of eggs shed was low (24-55% of control value). Indomethacin was still effective in blocking ovulation when given at 20.00, that is after completion of the proestrous LH surge, but not at 24.00. Administration of PGE2 (2 × 750 μ g/rat) in the early afternoon of proestrus elicited a rise in serum LH levels in rats in which the cyclic LH surge had been blocked with Nembutal (470 ± 87 vs. 106 ± 17 ng/ml ± S.E.) and induced ovulation in two-thirds of these animals. The results confirm, by direct measurement, that indomethacin does not block LH release but interferes with a late phase of the ovulatory process. PGE2 reverses this action of indomethacin on the ovary. In addition, PGE2 has a central effect causing LH release.
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Suppression Of Cyclic Surge Of Luteinizing-Hormone Secretion And Of Ovulation In Rat By Delta1-Tetrahydrocannabinol(1973) Nature. 244, 5408, p. 470-471 Abstract
1972
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(1972) Prostaglandins. 2, 1, p. 1-10 Abstract
The administration of prostaglandin E2 at the dose levels of 0.7, 1.0 and 1.5 mg/rat on the afternoon of proestrus to adult rats in which the pre-ovulatory surge of LH was prevented by Nembutal induced ovum maturation in 58, 70 and 90% and ovulation in 42, 60 and 81% of the animals, respectively; the incidence of persistent uterine distension was reduced by the prostaglandin treatment, suggesting that ovarian progesterone secretion was stimulated. Injection of indomethacin, an inhibitor of prostaglandin synthesis, on its own at 14.30 on the day of proestrus (5 - 10 mg/rat) prevented follicular rupture in 78 - 89% of the animals, but maturation of the oocytes retained in the follicles was unimpaired. Concomitant treatment with indomethacin and Nembutal prevented both follicular rupture and ovum maturation. Administration of LH at a dose level adequate to induce ovulation in Nembutal-blocked rats (2.5 μg/rat), failed to overcome the indomethacin-induced block of ovulation, but prostaglandin E2 brought about follicular rupture in the majority of the indomethacin-treated animals. It is concluded that (i) indomethacin, under the conditions studied, does not block LH release, but exerts its anti-ovulatory action directly on the follicle: it prevents follicular rupture, but not ovum maturation; (ii) prostaglandins have an essential role in the mechanism by which LH brings about follicular rupture; (iii) though prostaglandin E2 is able to induce ovum maturation, prostaglandins are not indispensible for this action of LH.
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Functional relations between cyclic AMP, prostaglandins, and luteinizing hormone in rat pituitary and ovary(1972) Advances in cyclic nucleotide research. 1, p. 503-20 Abstract
1971
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LH-induced meiotic division in follicle-enclosed ova in culture(1971) Proceedings of the VII World Congress on Fertility and Sterility. 278, p. 404-410 Abstract