Effects of luteinizing hormone and follicle-stimulating hormone and insulin-like growth factor-I on aromatase activity and P450 aromatase gene expression in the ovarian follicles of red seabream,Pagrus major

To clarify the mechanism of estradiol-17beta production in the ovarian follicle of red seabream, in vitro effects of luteinizing hormone (LH), follicle-stimulating hormone (FSH), and insulin-like growth factor (IGF-I) on aromatase activity (conversion of testosterone to estradiol-17beta) and cytochrome P450 aromatase (P450arom) mRNA expression in ovarian fragments of red seabream were investigated. Of the growth factors used in the present study, only IGF-I stimulated both aromatase activity and P450arom gene expression in the ovarian fragments of red seabream. LH from red seabream pituitary, but not FSH, stimulated both aromatase activity and P450arom gene expression. IGF-I slightly enhanced the LH-induced aromatase activity and P450arom gene expression. These data and our previous results indicate that LH, but not FSH, stimulates estradiol-17beta production in the ovarian follicle of red seabream through stimulation of aromatase activity and P450arom gene expression and IGF-I enhances the LH-stimulated P450arom gene expression.     

Ovarian steroidogenesis and the role of sex steroid hormones on ovarian growth and maturation of the Japanese eel

Three sex steroid hormones, estradiol-17β (E2), 11-ketotestosterone (11-KT), and 17α,20β-dihydroxy-4-pregnen-3-one (DHP), are well established as primary estrogen, androgen, and progestin, respectively, in teleost fish. Japanese eel, Anguilla japonica, would be a suitable candidate to study ovarian steroid physiology of fish because the ovarian growth and steroidogenesis is dormant under laboratory condition but can be induced by administration of exogenous gonadotropic reagents. In this review, we summarized our work on the function and production of sex steroid hormones in the ovary of the Japanese eel during ovarian growth and oocyte maturation artificially induced by treatment with extract of salmon pituitary. In vitro and in vivo assays suggest that 11-KT and E2 play primary roles in previtellogenic and vitellogenic growth of oocytes, respectively, whereas DHP is essential for induction of final oocyte maturation. We also reviewed the correlation between ovarian steroidogenesis to produce these sex steroid hormones, serum titers and gene expression.     

Regulation of follicle-stimulating hormone secretion by estradiol and dimeric inhibins in the infantile female rat.

Plasma and ovarian levels of the dimeric forms of inhibin and plasma estradiol-17beta were investigated and compared with changes in plasma gonadotropins from Postnatal Day (PND) 5 to PND 30 in the female rat. The inhibin subunit proteins were localized in follicular granulosa cells of the ovary. Plasma immunoreactive inhibin levels were low until PND 15 and increased thereafter. Plasma levels of inhibin B (alpha and beta(B) subunits) remained very low until PND 15 and then increased by approximately 24-fold. In contrast, plasma levels of inhibin A (alpha and beta(A) subunits) were relatively low and steady until PND 20, then increased by approximately 3-fold at PND 25. Changes in ovarian inhibin A and B levels closely resembled those in plasma levels. Plasma FSH levels were low at PND 10 but started to peak from PND 15 and remained high until PND 20, followed by a remarkable reduction at PNDs 25 and 30. This dramatic fall in FSH coincided with the rise of inhibin A. A significant inverse correlation was observed between plasma FSH and plasma inhibin A (r = -0.67, P < 0.0002), ovarian inhibin A (r = -0.48, P < 0.01), plasma inhibin B (r = -0.48, P < 0.05), and ovarian inhibin B (r = -0.54, P < 0.01). Plasma estradiol-17beta levels were elevated from PND 5 through PND 15, then fell sharply through PND 30. Plasma estradiol-17beta was significantly and positively (r = 0.75, P < 0.0002) correlated with plasma FSH. Plasma LH rose to higher levels at PND 15 and tended to be lower thereafter. The inhibin alpha, beta(A), and beta(B) subunits were localized to primary, secondary, and antral and large antral follicles, but the types of these immunopositive follicles varied with age. It appeared that, at PND 25 and afterward, all three subunits were mainly confined to large antral follicles in the ovary. We conclude that estradiol-17beta likely is the major candidate in stimulation of FSH secretion in the infantile female rat. We also conclude that inhibin regulation of pituitary FSH secretion through its negative feedback in the infantile female rat begins to operate after PND 20. We suggest that this negative feedback is achieved by increases in plasma levels of the two dimeric forms, and that inhibin A appears to be the major physiological regulator of FSH secretion at the initiation of this mechanism. We also conclude that large antral follicles in the ovary are the primary source of these bioactive inhibins that are secreted in large amounts into the circulation after PND 20.     

Regulation of ovarian steroidogenesis in vitro by follicle-stimulating hormone and luteinizing hormone during sexual maturation in salmonid fish

The regulation of ovarian steroidogenesis in vitro by coho salmon FSH and LH was investigated in intact coho salmon follicles and isolated follicular layers at various stages of oocyte maturation, from late vitellogenesis until the completion of germinal vesicle breakdown (GVBD). In granulosa layers from all stages, LH, but not FSH, stimulated 17alpha,20beta-dihydroxy-4-pregnen-3-one (17, 20beta-P) production. In theca-interstitial layers from all stages, FSH and LH stimulated steroid production, LH being more potent than FSH. The basal steroid output of intact follicles was significantly lower than that of isolated follicular layers, and their response to FSH and LH also differed. First, the intact follicles produced 17alpha-hydroxyprogesterone in response to FSH during the central germinal vesicle stage while theca-interstitial layers did not. Second, estradiol-17beta production was not inhibited by LH during final oocyte maturation in intact follicles, as observed for granulosa layers. Our results indicate that LH is the determining factor regulating the production of the maturation-inducing steroid, 17,20beta-P, and the induction of GVBD in the salmonid ovary. In summary, we have provided evidence for maturation-associated changes in the effects of FSH and LH in the salmonid ovary, which further supports the hypothesis that FSH and LH have distinct functions in the teleost ovary.     

Temporal relations between plasma concentrations of luteinizing hormone, follicle-stimulating hormone, estradiol-17beta, progesterone, prolactin, and alpha-melanocyte-stimulating hormone during the follicular, ovulatory, and early luteal phase in the bitch

Compared with other domestic animals, relatively little is known about the changes in, and temporal relations between, reproductive hormones around the time of ovulation in the domestic bitch. Therefore, plasma concentrations of luteinizing hormone (LH), follicle-stimulating hormone (FSH), estradiol-17beta, progesterone, prolactin (PRL), and alpha-melanocyte-stimulating hormone (alpha-MSH) were determined one to six times daily from the start of the follicular phase until 5 days after the estimated day of ovulation in six Beagle bitches. In all bitches, the pre-ovulatory LH surge was accompanied by a pre-ovulatory FSH surge. A pre-ovulatory PRL or alpha-MSH surge was not observed. The pre-ovulatory FSH and LH surges started concomitantly in four bitches, but in two bitches the FSH surge started 12 h earlier than the LH surge. The FSH surge (110+/-8 h) lasted significantly longer than the LH surge (36+/-5 h). In contrast with the pre-ovulatory FSH surge, the pre-ovulatory LH surge was bifurcated in four of six bitches. The mean plasma LH concentrations before (1.9+/-0.4 microg/L) and after (1.9+/-0.3 microg/L) the LH surge were similar, but the mean plasma FSH concentration before the FSH surge (1.6+/-0.3 U/L) was significantly lower than that after the FSH surge (3.1+/-0.2 U/L). In most bitches the highest plasma estradiol-17beta concentration coincided with or followed the start of the pre-ovulatory LH surge. In five of the six bitches the plasma progesterone concentration started to rise just before or concurrently with the start of the LH surge. In conclusion, the results of this study provide evidence for the differential regulation of the secretion of LH and FSH in the bitch. In addition, the interrelationship of the plasma profiles of estradiol-17beta and LH suggests a positive feedback effect of estradiol-17beta on LH surge release. The start of the pre-ovulatory LH surge is associated with an increase in the plasma progesterone concentration in this species.     

In vitro fertilization and development of bovine oocytes matured in serum-free medium

This study was undertaken to investigate the effects of supplementation of serum (fetal calf serum), gonadotropins (LH, FSH, prolactin) and estradiol-17 beta (E2) to culture medium during in vitro maturation of bovine cumulus oocyte complexes on subsequent fertilization and development to the blastocyst stage in vitro. Serum supplementation during bovine oocyte maturation was not required but hormonal supplementation, gonadotropins (LH + FSH) and E2, enhanced the fertilizability and developmental ability of bovine oocytes matured in vitro. The addition of prolactin to maturation medium containing LH, FSH, and E2 did not further enhance frequencies of fertilization and development.     

Effect of estrogen administration on endogenous and luteinizing hormone-releasing-hormone-induced luteinizing hormone secretion and follicular development in the lactating sow

The objectives of this study were to investigate whether estradiol treatment during lactation modifies 1) the patterns of endogenous LH, FSH, and prolactin (PRL) release; 2) the sensitivity of the pituitary to exogenous injections of LHRH; and 3) the responsiveness of the ovarian follicles to gonadotropin. Plasma LH, FSH, and PRL were determined in samples taken repeatedly from 18 sows on Days 24-27 of lactation. Ovaries were then recovered, and follicular development was assessed by measuring the follicular diameter (FFD) and follicular fluid estradiol-17 beta concentration (FFE) of the ten largest follicles dissected from each ovary. Sows were randomly allocated to one of four treatments: 1) Group C (4 sows) received no treatment; 2) Group LHRH (5 sows) received 800 ng of LHRH every 2 h throughout the sampling period; 3) Group E2 (4 sows) received subcutaneous implants containing estradiol-17 beta 24 h after start of sampling; 4) Group LHRH + E2 (5 sows) were administered a combination of LHRH and estradiol-17 beta implants. Between-animal variability for plasma LH, FSH, and PRL was considerable. LH concentration and LH pulse frequency increased (p less than 0.05) after LHRH treatment in the LHRH and LHRH + E2 groups; however, an acute inhibition of LH secretion was observed in the latter group immediately after estradiol implant application. In the absence of LHRH treatment, estradiol caused chronic inhibition of LH secretion. Follicular development was greater in the LHRH and LHRH + E2 groups compared to the C and E2 groups (p less than 0.05 for both FFD and FFE).(ABSTRACT TRUNCATED AT 250 WORDS)     

Induced ovulation and changes in pituitary responsiveness to continuous infusion of gonadotropin-releasing hormone during the ovarian cycle in the bullfrog, Rana catesbeiana

Chronic (2-4 days) constant-rate infusions of mammalian gonadotropin releasing hormone (GnRH) were performed in female bullfrogs, Rana catesbeiana. The magnitude and temporal relationship of profiles of plasma follicle-stimulating hormone (FSH), luteinizing hormone (LH) and sex steroids [testosterone (T), estradiol-17 beta (E2) and progesterone (P)] during GnRH infusion were dependent on ovarian stage. However, in all females, the same biphasic increase in plasma gonadotropins was apparent and initial elevations in gonadotropins were accompanied by correlated increments in plasma T and E2. Complete pituitary "desensitization" to chronic GnRH infusion was not observed. Females in early follicular stages were relatively unresponsive to infusions of 1.0-10.0 micrograms/h GnRH; elevations in plasma LH were marginal and FSH was unchanged. Females with fully developed (preovulatory) ovaries were more responsive: infusion of 1.0 micrograms/h GnRH produced significant elevations in plasma LH by 2 h followed by even larger increases ("surges") after 12 h. This LH "surge" was preceded by a decline in plasma T and E2 and was accompanied by abrupt elevations in plasma P and by ovulation. Postovulatory females showed a more gradual and smaller increase in plasma LH. Infusion of GnRH in the female bullfrog establishes a clear relationship between pituitary responsiveness and the ovarian cycle not evident from acute GnRH injection; GnRH was most effective immediately before ovulation. These data are also the first to detail periovulatory changes in plasma gonadotropins and ovarian steroids in an amphibian.     

Mechanisms regulating the ovarian cycle in the frog Rana cyanophlyctis: effect of unilateral ovariectomy, season, feeding, human chorionic gonadotrophin, and estradiol-17 beta

Effects of 30-day unilateral ovariectomy (ULO) on compensatory ovarian hypertrophy (COH) was studied in adult frog Rana cyanophlyctis in relation to reproductive phase/season, feeding, and treatment with human chorionic gonadotrophin (HCG) or estradiol-17 beta. Compensatory growth of the remaining ovary was assessed in terms of weight as well as changes in the dynamics of the pool sizes of different oocytes viz., first growth phase (FGP), medium-sized second growth phase (MSGP), large-sized second growth phase (LSGP), and atretic oocytes. The frogs were fed with live guppies 6 days a week. The effect of underfeeding on COH was studied in the frogs fed once a week. The ovaries removed at operation and those of the sham-operated frogs were used for comparison (controls). COH occurred in both pre- and postbreeding phases (February and November, respectively). In both instances follicular atresia was greatly reduced. In February, COH was due to recruitment of both MSGP and LSGP oocytes. The number of these oocytes increased significantly over controls, and the ovarian weight nearly doubled. However, in November COH occurred because of an increase in FGP oocytes and therefore total oocytes, but there were no changes in the ovarian weights. Administration of 20 IU HCG (6 days a week) had no influence on the basic pattern of COH-response exhibited by the remaining ovary in relation to reproductive phase/season, but it increased the recruitment of oocytes, i.e., FGP oocytes in November and SGP oocytes in February. Follicular atresia was drastically reduced with HCG. Underfeeding or treatment with estradiol-17 beta abolished the COH in both February and November.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibitory role of sex steroid in the regulation of ovarian follicle-stimulating hormone receptors during pregnancy.

The present study was conducted to determine the role of sex steroids in the regulation of FSH receptors in pregnant rats. In the normal physiological condition, FSH bindings per unit ovarian weight (density of binding) and per 2 ovaries (total binding) increased during days 14-21 gestation. Scatchard plot analyses of the binding suggested that the increase in FSH binding was due to an increase in the number of FSH-binding sites. The plasma FSH concentration in pregnant rats was stable during the receptor change. In contrast, the plasma estradiol-17 beta concentration continuously increased from gestation day 14 to 21, and the testosterone level showed a large peak on gestation day 18. Estradiol-17 beta (one silastic plate containing 13 mg crystal)-implanted pregnant rats during 14-21 days of gestation induced significant decreases in the total FSH binding and ovarian weight on gestation day 21. Estradiol administration increased the plasma estradiol level 2.3-fold but did not change the FSH level. Testosterone or 5 alpha-dihydrotestosterone, a nonaromatizable androgen, did not influence the binding level under the same dose treatment. In contrast, continuous treatment with aminoglutethimide (2 plates containing 20 mg crystal), an inhibitor of adrenocortical steroidogenesis, for 7 days significantly increased the total FSH binding without a significant change in the ovarian weight. The plasma titers of estradiol and testosterone in pregnant rats treated with aminoglutethimide were reduced by 37% and 51%, respectively. Aminoglutethimide did not influence plasma FSH levels. These results suggest that circulating estradiol acts as a negative factor in the regulation of ovarian FSH receptors, at least during the second half of pregnancy. Other factor(s) that is (are) independent of sex steroids and FSH may contribute to FSH receptor induction.     

Influence of equine growth hormone,insulin-like growth factor-I and its interaction with gonadotropins on in vitro maturation and cytoskeleton morphology in equine oocytes

In horses, successful in vitro fertilization procedures are limited by our inability to consistently mature equine oocytes by in vitro methods. Growth hormone (GH) is an important regulator of female reproduction in mammals, playing an important role in ovarian function, follicular growth and steroidogenesis. The objectives of this research were to investigate: the effects of equine growth hormone (eGH) and insulin-like growth factor-I (IGF-I) on the in vitro maturation (IVM) of equine oocytes, and the effects of eGH in addition to estradiol (E₂), gonadotropins (FSH and LH) and fetal calf serum (FCS) on IVM. We also evaluated the cytoskeleton organization of equine oocytes after IVM with eGH. Equine oocytes were aspirated from follicles <30 mm in diameter and matured for 30 h at 38.5°C in air with 5% CO₂. In experiment 1, selected cumulus–oocyte complexes (COCs) were randomly allocated as follows: (a) control (no additives); (b) 400 ng/ml eGH; (c) 200 ng/ml IGF-I; (d) eGH + IGF-I; and (e) eGH + IGF-I + 200 ng/ml anti-IGF-I. In addition to these treatment groups, we also added 1 μg/ml E₂, 5 IU/ml FSH, 10 IU/ml LH and 10% FCS in vitro (experiment 2). Oocytes were stained with markers for microtubules (anti-α-tubulin antibody), microfilaments (AlexaFluor 488 Phalloidin) and chromatin (TO-PRO₃-iodide) and assessed via confocal microscopy. No difference was observed when eGH and IGF-I was added into our IVM system. However, following incubation with eGH alone (40%) and eGH, E₂, gonadotropins and FCS (36.6%) oocytes were classified as mature v. 17.6% of oocytes in the control group (P < 0.05). Matured equine oocytes showed that a thin network of filaments concentrated within the oocyte cortex and microtubules at the metaphase spindle showed a symmetrical barrel-shaped structure, with chromosomes aligned along its midline. We conclude that the use of E₂, gonadotropins and FCS in the presence of eGH increases the number of oocytes reaching oocyte competence.     

Testosterone inhibits 11-ketotestosterone-induced spermatogenesis in African catfish (Clarias gariepinus).

Male fish produce 11-ketotestosterone as a potent androgen in addition to testosterone. Previous experiments with juvenile African catfish (Clarias gariepinus) showed that 11-ketotestosterone, but not testosterone, stimulated spermatogenesis, whereas testosterone, but not 11-ketotestosterone, accelerated pituitary gonadotroph development. Here, we investigated the effects of combined treatment with these two types of androgens on pituitary gonadotroph and testis development. Immature fish were implanted for 2 wk with silastic pellets containing 11-ketotestosterone, testosterone, 5alpha-dihydrotestosterone, or estradiol-17beta; cotreatment groups received 11-ketotestosterone in combination with one of the other steroids. Testicular weight and pituitary LH content were higher (two- and fivefold, respectively) in the end control than in the start control group, reflecting the beginning of normal pubertal development. Treatment with testosterone or estradiol-17beta further increased the pituitary LH content four- to sixfold above the end control levels. This stimulatory effect on the pituitary LH content was not modulated by cotreatment with 11-ketotestosterone. However, the stimulatory effect of 11-ketotestosterone on testis growth and spermatogenesis was abolished by cotreatment with testosterone, but not by cotreatment with estradiol-17beta or 5alpha-dihydrotestosterone. Also, normal pubertal testis development was inhibited by prolonged (4 wk) treatment with testosterone. The inhibitory effect of testosterone may involve feedback effects on pituitary FSH and/or on FSH receptors in the testis. It appears that the balanced production of two types of androgens, and the control of their biological activities, are critical to the regulation of pubertal development in male African catfish.     

Opposing effects of androgen and estrogen on pituitary-adrenal function in nonpregnant primates

Maternal administration of androstenedione produces a sustained fall in maternal plasma adrenocorticotropic hormone (ACTH) concentrations in the pregnant nonhuman primate. We hypothesize a negative feedback influence on the maternal hypothalamo-pituitary-adrenal (HPA) axis by androgens in primates. This may reflect an important maternal adaptation during pregnancy in primates preventing premature induction of labor by maternal stress. However, androstenedione is precursor for placental estradiol-17beta synthesis, and infusion of androstenedione into pregnant primates elevates maternal plasma estradiol-17beta to term concentrations. Thus, it could be argued that 1) the effects attributed to androstenedione on the maternal HPA axis are mediated by estrogen rather than by androgen and 2) the negative influence of androgens may be on placental ACTH rather than, or in addition to, pituitary ACTH. To discriminate between androgenic and estrogenic effects of androstenedione on pituitary and/or placental ACTH function in primates we measured plasma ACTH, cortisol, and dehydroepiandrosterone sulfate (DHEAS) concentrations in nonpregnant baboons after treatment with either androstenedione or estradiol-17beta. Nine female baboons were studied between 14 and 22 days postpartum prior to estrous cycling. After 2 days of baseline, a continuous i.v. infusion of androstenedione (1.5 mg/kg per h in 10% intralipid, IL) was started at 0900 h and maintained for 9 days in 3 baboons. A similar protocol was carried out in another 3 baboons that received a continuous i.v. infusion of estradiol-17beta (10 microg/kg per h in 10% IL) instead of androstenedione. Three additional baboons received continuous i.v. IL vehicle alone and served as controls. Arterial blood samples (0.5 ml) for measurement of plasma hormones were taken during baseline and after 1, 3, 5, 7, and 9 days of infusion. Baseline plasma ACTH, DHEAS, and cortisol concentrations were similar among all groups. Plasma ACTH did not change during IL, increased following estradiol-17beta, and fell during androstenedione treatment. Accordingly, plasma cortisol and DHEAS concentrations were also unaltered by IL, and both steroids increased during estradiol-17beta treatment. In contrast, plasma cortisol and DHEAS remained unaltered from baseline during androstenedione treatment, despite the fall in plasma ACTH measured at this time. These data in the nonpregnant baboon 1) are consistent with negative feedback on pituitary ACTH by androgens and 2) demonstrate a positive influence on pituitary-adrenal function by estrogen in primates.     

The effect of short-term nutritional supplementation of ewes with lupin grain (Lupinus luteus), during the luteal phase of the estrous cycle on the number of ovarian follicles and the concentrations of hormones and glucose in plasma and follicular fluid

An experiment was conducted using 16 cyclic, Welsh Mountain ewes during the luteal phase of the estrous cycle to determine the effect of a 5-day period of feeding a high-energy high-protein diet (lupin grain; 500 g/day) on folliculogenesis and on the plasma concentrations of glucose, insulin, follicle stimulating hormone (FSH) and estradiol-17beta, and on the follicular fluid concentrations of glucose, inhibin A, estradiol-17beta, androstenedione and progesterone. Average weight did not differ between lupin-fed and control groups during the experiment. There was a trend for the number of small and large follicles to increase in the lupin-fed group. The plasma concentrations of glucose (P=0.012) and insulin (P=0.007) were higher during the feeding period in lupin-fed ewes. The plasma concentrations of FSH and estradiol-17beta were not significantly different. The mean follicular fluid concentration of glucose (small follicles; <3.5 mm) from lupin-fed ewes was elevated (P=0.010) and progesterone lowered (P=0.034) compared to controls. The follicular fluid concentrations of estradiol-17beta, androstenedione and inhibin A were not significantly different. The follicular fluid concentration of estradiol-17beta was positively correlated with androstenedione (r=-0.241; P=0.001) and inhibin A (r=0.734; P< or =0.001) and glucose was negatively correlated with inhibin (r=-0.241; P=0.01), but not estradiol (r=0.075; P=0.410) or androstenedione (r=0.050; P=0.564). The lupin grain supplement increased the number of follicles as expected, but this increase was not significant. These changes were reflected in follicular fluid where lupin feeding increased the concentration of glucose and decreased the concentration of progesterone in follicles less than 3.5mm in diameter. These data suggest that the local ovarian actions of nutrients have a role in the mediation of nutritional influences on folliculogenesis.     

Effect of insulin-like growth factor-I during preantral follicular culture on steroidogenesis, in vitro oocyte maturation, and embryo development in mice

Insulin-like growth factor-I (IGF-I) is involved in the regulation of ovarian follicular development and has been shown to potentiate the FSH responsiveness of granulosa cells from preantral follicles. The aim of the present study was to investigate the effect of IGF-I during preantral follicular culture on steroidogenesis, subsequent oocyte maturation, fertilization, and embryo development in mice. Preantral follicles were isolated mechanically and cultured for 12 days in a simplified culture medium supplemented with 1% fetal calf serum, recombinant human FSH, transferrin, and selenium. In these conditions, follicles were able to grow and produce oocytes that could be matured and fertilized. The first experiment analyzed the effect of different concentrations of IGF-I (0, 10, 50, or 100 ng/ml) added to the culture medium on the follicular survival, steroidogenesis, and the oocyte maturation process. The presence of IGF-I during follicular growth increased the secretion of estradiol but had no effect on the subsequent oocyte survival and maturation rates. In the second experiment, IGF-I (0 or 50 ng/ml) was added to the culture medium during follicular growth, oocyte maturation, or both, and subsequent oocyte fertilization and embryo development rates were evaluated. Oocyte fertilization rates were comparable in the presence or absence of IGF-I. However, the blastocyst development rate was enhanced after follicular culture in the presence of IGF-I. Moreover, the total cell number of the blastocysts observed after differential labeling staining was also higher when follicles were cultured or matured in the presence of IGF-I.     

Inhibin activity and secretion of gonadotropin during the period of follicular maturation

To evaluate the relative contributions of the ovarian inhibin and estradiol-17 beta (E) on the regulation of FSH secretion, inhibin and E in ovarian venous plasma (OVP) and FSH and LH in peripheral plasma were simultaneously measured using superovulating rats with special reference to follicular maturation. By the transplantation of a pituitary gland from adult male rats under the kidney capsule between 1100 and 1200 hr on diestrus-1 in cyclic rats, superovulation was successfully induced on the morning of the next estrus without any additional treatment with human chorionic gonadotropin (hCG). The number of maturing follicles capable of ovulating in response to hCG significantly increased at 12 hours after the grafting as compared with sham-operated controls and further increases occurred until the afternoon of proestrus. In the superovulating rat, first and second surges of FSH were completely blocked and an LH surge was also partially suppressed during the periovulatory period when surges of FSH and LH were normally observed in controls. Contents of FSH as well as LH in the animal's own pituitary gland were suppressed significantly after the grafting as compared with controls. A marked increase in inhibin activity in OVP of rats with a pituitary transplant occurred concomitantly with an increase in the number of follicles capable of ovulating whereas E levels in OVP did not so. Inhibin activity in OVP at each point was much higher in the pituitary grafted rats than in controls but this was not true for E levels. These results suggest that ovarian inhibin derived from the maturing follicles rather than E may be a primary factor for regulation of FSH secretion, and high levels of endogenous inhibin can suppress synthesis of LH as well as FSH in the pituitary gland of the female rat.     

Specificity of inhibition by steroids of porcine oocyte maturation in vitro.

In order to examine the influence of several steroids on the process of oocyte maturation, denuded (adherent cumulus granulosa cells mechanically removed) and intact (cumulus granulosa cells left attached) porcine oocytes were cultured in the presence or absence of estradiol-17 beta, estradiol-17 alpha, testosterone, cortisol, progesterone, or the nonsteroidal estrogen diethyl stilbestrol (all at 10 microgram/ml) in defined medium that contained either BSA or dextran. Estradiol-17 beta was the only steroid to exert a significant inhibitory effect on the maturation of denuded oocytes, and did so only in BSA supplemented medium. The inhibition was reversible in that oocytes, cultured in steroid-free medium after initial culture in estradiol-17 beta medium, resumed meiotic maturation. Oocytes took up 3H-estradiol-17 beta in both media, although less radiolabel entered oocytes in BSA supplemented medium. The majority of label in the oocytes, when cultured with either medium, was not displaced by excess radioinert estradiol-17 beta or progesterone, nor were the oocytes saturated even when cultured in 10(-6) M estradiol-17 beta. Autoradiography of sectioned oocytes after culture in 3H-estradiol-17 beta has shown that there was no selective accumulation of silver grains over the germinal vesicle as was the case with granulosa cell nuclei. This observation suggests that estradiol-17 beta may not act at the level of the oocyte nucleus.