The involvement of platelet-activating factor in thrombocytopenia and follicular rupture during gonadotropin-induced superovulation in immature rats

To investigate whether the platelets in the ovaries are activated by the action of platelet-activating factor (PAF) during gonadotropin-induced ovulation, we examined the changes in the platelet count in immature rats after administration of PMSG followed 48 h later by human CG (hCG). The platelet count in the inferior vena cava was significantly decreased 48 h after PMSG administration and was further decreased after hCG administration. When both ovaries of rats were extirpated, the administration of PMSG and hCG did not decrease the platelet count. Subcutaneous administration of a PAF antagonist, Y24180 (0.5-5 mg/kg.6 h), after PMSG injection decreased the number of ova shed in a dose-dependent manner. The decrease in the platelet count induced by the administration of PMSG and hCG was reversed to the level of the untreated control group by Y24180 (2.5 mg/kg.6 h). This inhibitory activity of Y24180 on ovulation and thrombocytopenia was completely reversed by the ip injection of synthetic PAF. Subcutaneous administration of indomethacin (IDM) also reduced the number of ova shed in a dose-dependent manner. However, thrombocytopenia was not reversed by IDM. Moreover, the inhibition of ovulation by IDM was not reversed by synthetic PAF. The present study suggests that: 1) platelets are activated by PAF during gonadotropin-induced ovulation in immature rats; 2) PAF is also involved in the rupture of follicles; 3) the presence of the ovary is indispensable for the generation of PAF in gonadotropin-stimulated immature rats; and 4) the mechanism of PAF action on ovulation may be different from that of prostaglandins.     

Gonadotropin-induced superovulation drives ovarian surface epithelia proliferation in CD1 mice

The ovarian surface epithelium (OSE) is a monolayer of cells that surround the ovary and accommodate repeated tear and repair in response to ovulation. OSE cells are thought to be the progenitors of 90% of ovarian cancers. Currently, the total amount of proliferation of the OSE has not been reported in response to one ovulatory event. In this study, proliferation of the OSE was quantified in response to superovulation induced by ip injection of pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) in immature 27-d-old CD1 mice using bromodeoxyuridine (BrdU). BrdU incorporation into the OSE cells was measured from the time of hCG injection for a total cumulative label of 12 h. BrdU incorporation was also measured from the time of PMSG injection for a total label of 60 h to correlate proliferation with specific gonadotropin stimulation. The OSE proliferation was significantly higher in superovulated animals compared with control mice at all time points. Proliferation was also analyzed in discrete anatomical sections and indicated that OSE covering antral follicles and corpora lutea proliferated more rapidly than OSE distal to follicular growth. Finally, apoptosis was assessed in response to ovulation, and virtually no cell death within the OSE was detected. These data demonstrate that the OSE, especially near antral follicles and corpora lutea, proliferates significantly in response to the gonadotropins PMSG and hCG. Therefore, ovarian surface cell division in response to ovulation could contribute to ovarian cancer by proliferation-induced DNA mutations and transformed cell progression.     

Tissue-type plasminogen activator in rat oocytes: expression during the periovulatory period, after fertilization, and during follicular atresia

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.     

Studies on follicular atresia: lysosomal enzyme activity and gonadotropin receptors of granulosa cells following administration or withdrawal of gonadotropins in the rat

The changes that occur during follicular growth and atresia in the lysosomal enzyme activities and in gonadotropin receptors of isolated granulosa cells were studied. At different intervals after an injection of PMSG to 21-day-old female rats, the granulosa cells (GC) were isolated and the total activity of cathepsin-D, a representative lysosomal enzyme, and FSH receptor activity in terms of binding of [125I]oFSH to GC and the steroidogenic response of GC to FSH in vitro were determined. During the period of both follicular growth and atresia there was an inverse correlation between the lysosomal enzyme activity and FSH receptor activity of granulosa cells, the former being low during follicular growth or tropic phase when the levels of FSH receptors were increasing. During the atretic phase, beyond 48 h of PMSG treatment (perhaps due to the normal metabolic clearance of the injected PMSG), the cathepsin-D activity showed a significant increase while the FSH receptors were on the decline. That lack of the hormone was responsible for atresia was confirmed when an antiserum to PMSG was injected during the tropic phase (24 h after PMSG); this resulted in a sharp increase in cathepsin-D activity. Injection of excess amounts of either FSH or LH, along with or soon after PMSG antiserum, was found to prevent the increase in cathepsin-D activity, and also resulted in the maintenance of gonadotropin receptors. This suggested that gonadotropins can 'rescue' follicles from undergoing atresia. In the unprimed immature rat FSH, but not LH or hCG, mimicked the effects of PMSG, in terms of bringing about a reduction in cathepsin-D activity of GC. Also in such rats, neutralization of endogenous FSH with an antiserum to FSH resulted in an increase in the cathepsin-D activity of GC, suggesting that even the prepubertal rat ovary is dependent on the tropic support of FSH for prevention of atresia.     

Human chorionic gonadotrophin-induced suppression of serum inhibin involves reduction in ovarian inhibin alpha-subunit messenger RNA levels in the pregnant mare serum gonadotrophin-primed female rat

We have investigated the effect of administration of human chorionic gonadotrophin (hCG) to immature female rats pretreated with pregnant mare serum gonadotrophin (PMSG) on ovarian inhibin alpha-subunit mRNA, serum inhibin and circulating gonadotrophin levels. PMSG stimulation alone caused a 5-fold increase in relative inhibin alpha-subunit mRNA levels and a 12-fold increase in serum inhibin by 48 h. However, injection of hCG at 40 h suppressed PMSG-stimulated ovarian inhibin alpha-subunit mRNA and serum inhibin to levels at 48 h not statistically significantly different from controls. Serum follicle-stimulating hormone (FSH) fell after PMSG treatment, but rose after combined PMSG-hCG treatment. Serum luteinizing hormone (LH) was unchanged after PMSG alone but also rose after combined PMSG and hCG therapy. In conclusion, hCG suppresses ovarian inhibin synthesis in PMSG-stimulated immature female rats with preservation of the reciprocal relationship between inhibin and FSH. This immature female rat model therefore provides further insight into the effects of LH or hCG on granulosa cell inhibin production just prior to ovulation.     

Follicular steroidogenesis and gonadotropin binding to ovine follicles during the estrous cycle

The interrelationships among [125I]hCG binding in thecal and granulosa cells, antral fluid steroid concentrations, follicular size, and ovarian steroid secretion were examined at three different stages of the estrous cycle. Group 1 ewes were ovariectomized during the luteal phase of the estrous cycle, and the other groups were ovariectomized before (group 2), or after (group 3) the peak of the preovulatory LH surge. Times of luteolysis and the LH surge were assessed by measurement of peripheral concentrations of progesterone and LH. Three patterns of [125I]hCG binding to follicles were noted: 1) binding to both thecal and granulosa cells (activated follicle), 2) binding to the thecal cell layer only, and 3) no observed binding. In general, there was one active follicle per ewe, or one per ovary, and the number of active follicles was not different from the number of corpora lutea in each of the three groups. The active follicles were significantly larger than the other two classes of follicles. Antral fluid estradiol concentrations were significantly greater in the active follicles and were higher in group 2 ewes than in the other two groups. In group 2, antral fluid testosterone concentrations were significantly higher in follicles with LH receptors in the thecal cell layer only. Ovarian secretion of testosterone and estradiol increased during the early follicular phase (group 2), with the major secretion coming from the ovary containing the active follicle. Ovarian progesterone secretion was high in ovaries containing active corpora lutea which prevented the assessment of ovarian follicular secretion of progesterone. The follicle with LH receptors in thecal and granulosa cells was responsible for the increased estradiol secretion observed during the preovulatory period and is presumed to be the ovulatory follicle.     

Non-invasive analysis of rat ovarian angiogenesis by MRI

Magnetic resonance imaging (MRI) was employed for non-invasive analysis of vascular remodeling during follicular maturation in the PMSG/hCG rat ovary model. Changes in water diffusion and in perfusion led us to suggest that hypoxic stress may be a component in the regulation of angiogenesis in the growing follicle. However, in contrast with solid tumors of similar size, the spatial and temporal pattern of expression of vascular endothelial growth factor (VEGF), did not match the angiogenic response. The mismatch could be explained by the role of hyaluronan as a high molecular weight suppressor of angiogenesis maintaining an avascular follicular antrum.     

Prostaglandin dehydrogenase and prostaglandin levels in periovulatory follicles: implications for control of primate ovulation by prostaglandin E2

Prostaglandin (PG) E2 produced by the periovulatory follicle in response to the midcycle LH surge is essential for successful ovulation in primates. Granulosa cells express the PG synthesis enzyme cyclooxygenase-2 in response to the LH surge, but elevated cyclooxygenase-2 mRNA levels precede rising follicular fluid PGE2 levels by 24 h. Therefore, PG metabolism may play a significant role in regulating follicular concentrations of PGE2 during the periovulatory interval. To test this hypothesis, granulosa cells, follicular fluid, and whole ovaries were obtained from adult monkeys receiving exogenous gonadotropins to stimulate development of multiple, large follicles at times spanning the 40-h periovulatory interval. Ovarian expression of the NAD+-dependent 15-hydroxy PG dehydrogenase (PGDH) was assessed by RT-PCR, Western blotting, and immunohistochemistry. PGDH mRNA levels were low in granulosa cells obtained 0 h after hCG, rose 10-fold 12 h after hCG, and were not different from 0 h by 24-36 h after hCG administration. Granulosa cell PGDH protein was present 0-12 h after hCG but was low/nondetectable 36 h after hCG administration. Follicular fluid PGE2 levels were low at 0-12 h, slightly higher at 24 h, and then rose 10-fold to peak at 36 h hCG. Levels of biologically inactive PGE2 metabolites in follicular fluid were also low at 0 h but elevated at 12-24 h after hCG, times at which PGE2 levels remain low. Therefore, PGDH is present in the primate periovulatory follicle in a pattern consistent with modulation of follicular PGE2 levels during the periovulatory interval, supporting the hypothesis that gonadotropin-regulated PGDH plays a role in the control and timing of ovulation in primates.     

Preovulatory progestins: Observation on their source in rhesus monkeys.

Progesterone (P) and 20alpha-hydroxypregen-4-en-3-one (20alpha-ol)concentrations were measured in systemic and ovarian vein plasma of adult cycling female rehsus monkeys by gas liquid chromatography with electron capture detection. Blood samples and ovaries were removed at various times during the intermenstrual period. The animals were then reclassified according to the stage of the ovarian cycle and the morphology of the ovary. Very little P or 20alpha-ol is secreted by the ovary during the follicular phase until the largest developing follicle measures 5.6 mm in diameter. In the prevular period, the concentrations of both P and 20alpha-ol in the two ovarian veins are nearly equal. As the luteal phase develops, the ratios of progestin concentrations in the ovarian vein change, more progestin being found in ovarian venous plasma of the ovary with the corpus luteum. The secretion of preovulatory progestin can be correlated with large amounts of interstitial gland cells that appear before ovulation in the ovary. In a second experiment, follicular development was estimated by a rapid radioimmunoassay for estrogen in the systemic circulation. When the systemic plasma levels of immunoreactive estrogen exceeded 150 pg/ml, ovarian venous samples of blood were collected from each ovary and analyzed by radioimmunoassay for estradiol-17beta and progesterone. Ffteen min after removal of the ovary that contained the mature Graafian follicle, blood was drawn from the contralateral ovarian vein. In this preovulatory condition, the progesterone concentrations in both ovarian veins greatly exceeded those in the systemic circulation. In 2 animals, removal of the ovary that contained the mature Graafian follicle did not lower the blood levels of progesterone in the contralateral ovarian vein after 15 min. These data suggest that both ovaries of the rhesus monkey secrete preovulatory progestin which is available for physiological action.     

Alterations in follicle development, steroidogenesis, and gonadotropin receptor binding in a model of ovulatory blockade

Immature female rats treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) before gonadotropin-induced follicle development and ovulation ovulate significantly fewer ova compared with controls. This study was designed to investigate potential ovarian-specific mechanisms of TCDD-mediated inhibition of ovulation. Immature hypophysectomized rats were treated with TCDD (32 microg/kg) or corn oil vehicle. Follicle development was initiated by injection of 10 IU PMSG 24 h after TCDD, and ovulation was induced 52 h after PMSG by injection of 10 IU hCG. The number of ova flushed from the oviduct was assessed the morning after hCG injection, and ovaries were collected at multiple times throughout the treatment schedule for histological analysis and analysis of FSH and hCG receptor binding and ovarian cAMP levels. In addition, serum levels of estradiol and progesterone were determined. Control rats ovulated 9.3 +/- 1.5 ova, whereas TCDD-treated rats ovulated 0.6 +/- 0.3. Gonadotropin receptor binding was evaluated 52 h after PMSG at the usual time of hCG injection to induce ovulation. Both FSH binding and hCG binding were significantly reduced in animals treated with TCDD. Serum estradiol levels in control animals were increased by 52 h after PMSG administration. In contrast, serum levels of estradiol in TCDD-treated animals remained low. In response to the ovulatory dose of hCG, serum levels of both estradiol and progesterone increased in control animals. Steroid levels also increased in TCDD-treated animals, but did not reach the peak levels observed in controls. TCDD treatment further resulted in lower ovarian cAMP levels at 52 h post-PMSG and at 5 h post-hCG. Together the data indicate that TCDD treatment altered the ability of the ovary to respond to PMSG, resulting in the development of follicles not comparable to controls (lower gonadotropin binding, lower estradiol production, lower levels of cAMP). It appears that critical steps in the development and maturation of follicles are disrupted by TCDD.     

CONCENTRATION OF OESTRONE AND OESTRADIOL IN FOLLICULAR FLUID AND OVARIAN VENOUS BLOOD OF WOMEN

The concentrations of oestrone and oestradiol as measured by a double isotope derivative method in fluid from Graafian follicles at different phases of the menstrual cycle were compared to the corresponding concentrations in ovarian and peripheral venous plasma collected simultaneously. In follicular fluid the concentration of oestradiol was much greater than that of oestrone. The concentrations of oestrogens were similar in fluid collected from small follicles at all phases of the cycle. The concentration of oestradiol in large follicles (greater than 1 cm diameter) was much higher in the mid-late follicular phase of the cycle (mean = 5390 plus or minus 1330 nmol/l) than in large or small follicles at other phases of the cycle. In the mid-late follicular phase of the cycle large follicles were invariably associated with high concentrations of oestradiol in venous plasma draining the corresponding ovary (mean 49 plus or minus 12 nmol/l). The mean ratio of oestradiol/oestrone in fluid from large follicles in which the concentration of oestradiol was greater than 1400 nmol/l (18.0 plus or minus 1.0, n = 10) was significantly higher than the ratio in corresponding samples of ovarian venous plasma (10.7 plus or minus 1.4, n = 11). These findings suggest that when the concentration of oestradiol in a Graafian follicle exceeds 3500 nmol/l it is likely to be functionally active.     

Dissociation between the direct stimulatory and inhibitory effects of a gonadotropin-releasing hormone analog on ovarian functions

The paradoxical effects of gonadotropin-releasing hormone (GnRH) on the ovary have hitherto been believed to result from different regimens of administration; an acute treatment was shown to stimulate the ovary while chronic administration of the hormone inhibited LH-induced responses. In the present report we demonstrate that a single injection of a GnRH analog (D-Ala6)des-Gly10-GnRH-N-ethylamide (GnRHa, 2 micrograms/rat) is sufficient to obtain a significant inhibition (75%) of hCG-induced ovulation in PMSG-primed, either intact or hypophysectomized, immature rats. Inhibition of ovarian development, in terms of growth and ovulation, by multiple injections with GnRHa (2 micrograms/rat, twice daily for 3 days) could be obtained only upon administration of the hormone at early stages of follicular development, i.e. concomitantly with the PMSG injection. When administered after PMSG, GnRHa could not inhibit the ovary but rather induced ovulation by itself in the absence of hCG. A 12-24 h delay in initiation of GnRHa treatment triggered 65% of the rats to ovulate while a delay of 48 h resulted in 100% ovulation. Under both regimes of GnRHa administration, either the inhibitory or the stimulatory, the oocytes of the treated rats were induced to resume meiotic maturation. Since under the inhibitory regime ovulation did not occur, maturation was followed by a massive degeneration of the oocytes trapped within their follicles. These findings demonstrate that the follicular stage of development rather than the dose and/or duration of GnRHa administration determines whether GnRHa inhibits ovarian growth and ovulation, while the competence of the oocytes to respond to the GnRHa stimulus and mature is independent of hormonal priming.     

Epidermal growth factor inhibits follicular response to human chorionic gonadotropin: possible role of cell to cell communication in the response to gonadotropin.

Epidermal growth factor (EGF) affects follicular steroidogenesis and expression of gonadotropin receptors. The effects of EGF on hCG-induced estradiol and progesterone secretion and ovulation were examined in the in vitro perfused rabbit ovary. We also examined the effects of EGF on hCG-induced progesterone secretion by isolated granulosa cells. In addition, distribution of hCG within the follicle was probed by immunohistochemical means 30 min after its administration to the in vitro perfused ovary. EGF significantly (P less than 0.05) reduced hCG-induced secretion of estradiol (control, 117 +/- 12 pg/min.follicle; 10 ng/ml EGF, 55 +/- 10) and progesterone (control, 18.2 +/- 1.2 ng/min.follicle; 10 ng/ml EGF, 11.9 +/- 0.8) by the perfused ovary. In contrast, EGF did not inhibit hCG-induced progesterone secretion by isolated granulosa cells. Ovulatory efficiency (number of ovulated ova per number of mature follicles x 100) when EGF was given 30 min before hCG was reduced dose-dependently from 58.2% with no EGF to 8.3% with 10 ng/ml EGF (P less than 0.001). Ovulation was not inhibited by EGF when it was given 30 min after hCG. Distribution of hCG in the preovulatory follicle was confined to the basement membrane, thecal cell layer, and a small fraction of the outer granulosa cell layer. These observations suggest that gonadotropin stimulates the follicle through the release of a secondary signal(s) from ligand-bound granulosa cells near the follicle wall to unexposed cells of the inner avascular area. EGF may inhibit the follicular response to hCG by attenuation of this cell to cell communication.     

Arginine vasopressin in human follicular fluid

Arginine vasopressin (AVP) was determined in plasma and follicular fluid in 28 women in an in vitro fertilization program. In 23 women, follicular fluid was collected by laparoscopy during general anesthesia, and in 5 women, it was collected transvaginally with no such anesthesia. Plasma AVP increased markedly from its basal (preanesthesia) value in the first group, whereas it did not change in the second group. AVP concentrations were approximately 10-fold lower in the follicular fluid than in the plasma collected simultaneously in the anesthetized women. AVP levels were not significantly different in plasma and follicular fluid in the women of the second group. AVP concentrations were similar in ovarian venous and brachial venous plasma in 4 women during surgery. These results indicate that AVP concentrations in follicular fluid are equal to or lower than those in plasma and that AVP concentrations are not higher in efferent blood from the ovary than in peripheral blood.     

Pregnancy-associated plasma protein-a is the insulin-like growth factor binding protein-4 protease secreted by human ovarian granulosa cells and is a marker of dominant follicle selection and the corpus luteum

Insulin-like growth factors (IGFs), IGF binding proteins (IGFBPs), and IGFBP proteases are important in ovarian function. IGFs stimulate granulosa steroidogenesis, an effect that is inhibited by IGFBP-4 and augmented by IGFBP-4 proteolysis. We have recently identified the IGFBP-4 protease in human ovarian follicular fluid (FF) as pregnancy-associated plasma protein-A (PAPP-A). In the current study, we identify the IGFBP-4 protease secreted by cultured human ovarian granulosa cells as PAPP-A, based on specific immunoinhibition and immunodepletion of the IGFBP-4 protease activity with PAPP-A polyclonal antibodies and immunorecognition by PAPP-A monoclonal antibodies in ELISA. PAPP-A was barely detectable in conditioned media (CM) from granulosa derived from /=9 mm, coincident with dominant follicle selection, and by luteinizing granulosa. PAPP-A levels in CM from the latter did not change in response to IGF-II or hCG (100 ng/mL). A naturally occurring inhibitor of PAPP-A, proform of eosinophil major basic protein (proMBP), was detected by ELISA in estrogen-dominant follicular fluid FF, but not in CM from granulosa or luteinizing granulosa cells treated with IGF-II (0-200 ng/mL), FSH (0-100 ng/mL) or hCG (0-100 ng/mL), suggesting an alternative source (other than granulosa) for proMBP, compared to PAPP-A. The data demonstrate granulosa cells as a source of PAPP-A in human ovary and suggest that PAPP-A is a marker of ovarian follicle selection and corpus luteum formation. In addition the data suggest complex regulation of this system in human ovary.     

Identification and measurement of testosterone in plasma and follicular fluid of the mare, using gas chromatography-mass spectrometry associated with isotope dilution

Testosterone has been identified by mass spectrometry in blood and follicular fluid aspirated from mature Graafian follicles of mares. Quantitative measurements made by gas chromatography-mass spectrometry have validated the determination of plasma testosterone made by radioimmunoassay. However, because of high levels of epitestosterone (17 alpha-hydroxyandrost-4-en-3-one) in the follicular fluid, radioimmunoassay overestimates the true concentrations of testosterone. The occurrence of testosterone in mare follicular fluid at a concentration which is two orders of magnitude higher than that in peripheral plasma suggests that the follicle may contribute to the production of circulating testosterone. A biosynthetic pathway for oestradiol-17 beta which involves testosterone is therefore likely to occur in the mare ovary as in many other mammalian species.