Changes in expression of inhibin subunits in the cyclic golden hamster (Mesocricetus auratus) and the regulation of inhibin alpha subunit production by luteinizing hormone

In the present study, changes in localization of each inhibin subunit in the ovary were investigated during the estrous cycle of the golden hamster. The effect of LH surge on changes in localization in inhibin alpha subunit in the ovary was also investigated. Inhibin alpha subunit was localized in granulosa cells of various stages of follicles throughout the estrous cycle. Inhibin alpha subunit was also present in numerous interstitial cells on days 1 and 2 (day 1 = day of ovulation), but the number of positive interstitial cells was fewer on days 3 and almost disappeared on day 4 of the estrous cycle. Newly formed luteal cells were also positive for inhibin alpha subunit on days 1 and 2. On the other hand, positive reactions for inhibin beta A and beta B subunits were only present in the granulosa cells of healthy antral follicles. However, a positive reaction for inhibin beta B subunit in peripheral mural granulosa cells disappeared on days 3 and 4 of the estrous cycle. Treatment with LHRH-AS at 1100 h on day 4 completely blocked the luteinizing hormone (LH) surge and ovulation, although relatively high concentrations of plasma follicle-stimulating hormone (FSH) were maintained throughout the experiment. There were few positive reactions for inhibin alpha subunit in theca and interstitial cells 24 hr after LHRH-AS injection. The effect of LHRH-AS treatment was blocked by a single injection of 10 IU human chorionic gonadotropin. These results suggest that the major source of dimeric inhibin in the cyclic hamster was granulosa cells of healthy antral follicles. Different distribution pattern of inhibin beta A from beta B subunits in large antral follicles on days 3 and 4 of the estrous cycle suggests different secretion patterns of inhibin A from B on these days. Furthermore, the LH surge may be an important factor to induce production of inhibin alpha subunit in interstitial cells of the cyclic hamster.     

Equine luteinizing hormone possesses follicle-stimulating hormone activity in hypophysectomized female rats

The ability of equine luteinizing hormone (eLH) to promote follicular growth and maturation in hypophysectomized rats has been assessed. A single injection of equine LH has been shown to promote the growth of a large number of antral and preovulatory follicles. In addition, equine LH markedly increased serum estrogen levels and uterine weight. Furthermore, equine LH, like equine chorionic gonadotropin (eCG; PMSG) was able to significantly enhance the incorporation of [3H]thymidine into ovarian DNA, an activity shown to be specific to hormones having follicle-stimulating hormone (FSH) activity. Equine LH treated with an FSH antibody immunoaffinity column to remove any possible contamination still exhibited the above activity, demonstrating that the FSH activity is intrinsic to the eLH molecule. Equine LH has also been shown to be capable of inducing LH receptors in granulosa cells of ovaries of hypophysectomized rats, an activity specific to FSH-like hormones. From the doses required of eLH and the degree of response observed, it is concluded, however, that eLH in the hypophysectomized rat is less active than eCG as an FSH.     

Regulation of gonadal androgen secretion

The various mechanisms regulating testicular and ovarian androgen secretion are reviewed. Testicular androgen secretion is controlled by luteinizing hormone (LH) and follicle stimulating hormone (FSH), which influence the Leydig cell response to the LH. The contribution of prolactin, growth hormone and thyroid hormones to the Leydig cell function is discussed. The ovarian androgen secretion is regulated in a very similar fashion as the Leydig cell of testis. Prolactin, however, has an inhibitory effect on androgen secretion in the ovary. The intratesticular action of androgens is linked to spermatogenesis. Sertoli cells, by producing the androgen-binding protein, contribute to the intratubular androgen concentration. Inhibin production of the Sertoli cell is stimulated by androgens. In the ovary, androgens produced by the theca interna are used as precursors for the aromatization of estradiol, which stimulates together with FSH the mitosis of granulosa cells. The feedback control of androgen secretion is complicated, as the direct feedback mechanisms are joined by indirect feedback regulations like the peptide inhibin, which can be stimulated by androgens. Intragonadal mechanisms regulating androgen production are the cybernins for testicles and ovaries. In the testicle, estrogens from the Sertoli cells regulate the Leydig cell testosterone biosynthesis. In the ovary, nonaromatizable androgens are potent inhibitors of the aromatization activity in the granulosa cell. A peptide with a FSH receptor binding inhibiting activity is found in male and female gonads. Finally, LH-RH-like peptides have been found in the testicle, which are capable of inhibiting steroidogenesis. These gonadocrinins are similarly produced in granulosa cells of the ovary.     

Effects of anterior pituitary hormones on hepatic corticosterone metabolism in rats

Experiments were conducted to determine the effects of anterior pituitary hormones on hepatic corticosterone metabolism in rats. Hypophysectomy lowered A-ring reduction but did not affect sidechain metabolism. Administration of prolactin, FSH, LH or FSH + LH to hypophysectomized rats affected neither process. Similarly, ACTH or growth hormone, when given alone, did not affect corticosterone metabolism. However, combined treatment with ACTH and growth hormone significantly reduced the rate of ring A metabolism, suggesting that hormonal interactions may be important in the control of hepatic steroid metabolism.     

Hormonal regulation of ovarian cellular proliferation

The steroid hormone estradiol, and the glycoprotein hormones follicle-stimulating hormone (FSH) and luteinizing hormone (LH), are known to be essential for the growth and differentiation of follicles in the ovary. The present study was conducted to determine quantitatively the effects of estradiol, FSH and LH on proliferation of different ovarian cell types (granulosa and theca cells). The immature female hypophysectomized rate sequentially primed with estradiol, FSH and LH was used as the experimental model. Proliferation was assessed by examining changes in total DNA, incorporation of 3H-thymidine into DNA and labeling index in specific cell types. Estradiol and FSH each acted on follicles at different stages of development to stimulate proliferative activity of both granulosa and theca cells. Continued administration of either hormone caused a decrease in the proliferative activity of both cell types. These observations have been interpreted to indicate that estradiol and FSH can each alter the length of the specific phases of the cell cycle. A luteinizing dose of LH caused a cessation of proliferation in luteinizing granulosa cells while stimulating a limited proliferation of theca cells. Absence of the appropriate hormonal stimulus caused both granulosa and theca cells to stop proliferating and the follicles to undergo atresia. These results indicate that, depending upon the state of differentiation of granulosa and theca cells, estradiol, FSH and LH can stimulate or inhibit the ability of these cells to proliferate.     

Pituitary regulation of preovulatory estrogen secretion in the rat

The factors stimulating estrogen secretion in the preovulatory phase and an attempt to explain the mechanism of termination of estrogen secretion are discussed. Female Wistar rats, hypophysectomized at 1 p.m. in proestrus, were injected with rat pituitary extracts. Ovarian venous blood was collected and the estrogen activity of the plasma was measured. The estrogen secretion was minimized within 3 hours after hypophysectomy. The rat pituitary extract caused an 11-fold increase of estrogen concentration in the ovarian venous blood within 1 hour. Either LH or FSH alone was able to restore the estrogen secretion: LH took 1 hour to reach maximal response, FSH 2 hours. In the 1-hour test, the minimal effective dose for LH appeared to be less than .25 mcg per rat, for FSH, 2.5 mcg per rat. The total ability of the two preparations to produce estrogen appeared to be the same. 10 I.U. of prolactin slightly stimulated estrogen secretion, but 20 mU of ACTH was quite negative. These results demonstrate the pituitary gonadotropin dependency of estrogen secretion from the ovary having ripened follicles. It also showed that the ovary, after completion of ovulatory surge of LH, abolished its reactivity to the pituitary extract containing sufficient amount of substances in promoting estrogen secretion. Either LH or FSH was able to terminate estrogen secretion even at minute doses as small as 10 mcg. This shows that both FSH and LH provide a dual effect on ovarian estrogen secretion at the preovulatory stage, promotion and suppression. Promotion is an acute and direct action of hormones on steroidogenesis and suppression probably a delayed and indirect action of ovulation-inducing hormone, the release of which initiates the differentiation of estrogen-forming cells towards ovulation unfavorable to estrogen synthesis.     

Effect of diverse mammalian gonadotropins on estrogen and progesterone production by cultured rat granulosa cells

The ability of gonadotropins from six mammalian species to stimulate estrogen and progesterone production was investigated in granulosa cells of hypophysectomized estrogen-primed immature female rats. Granulosa cells were cultured for 2 days in the presence of delta 4-androstenedione (10(-7) M) with or without various gonadotropin preparations. Treatment with follitropin (follicle-stimulating hormone, FSH) from human, rat, ovine, porcine, equine, and bovine origins resulted in dose-dependent increases in steroidogenesis from negligible amounts to maximal levels of approximately 4-8 and 12-30 ng/10(5) cells for estrogen and progesterone, respectively. The ED50 values of the FSH preparations for stimulation of steroidogenesis were: human: 1-4 ng/ml; ovine: 2.5-30 ng/ml; rat: 1.6-4.0 ng/ml; porcine: 7.5-20 ng/ml; equine 2.5-6 ng/ml; and bovine greater than 100 ng/ml. Lutropin (luteinizing hormone, LH) from rat, ovine, bovine, and porcine origins, human chorionic gonadotropin (hCG), the alpha-subunit of human FSH and the beta-subunit of human LH were ineffective in stimulating steroidogenesis, indicating the specificity of the assay system for FSH. In a high concentration (600 ng/ml), the beta-subunit of human FSH-stimulated steroidogenesis to a small extent. Furthermore, pregnant mare serum gonadotropin and equine LH also caused a dose-dependent stimulation of estrogen and progesterone production, the half-maximal response values (ED50) being 1.8-4 and 7.5-10 ng/ml, respectively. This is consistent with previous in vivo and in vitro findings, showing the potent FSH activities of these hormones. Thus, the cultured rat granulosa cell system provides a sensitive assay for measuring FSH activities of gonadotropins from various mammalian species.     

Pituitary hormone control of implantation in the mouse

In hypophysectomized pregnant mice replacement therapy designed to mimic the normal physiological situation showed that FSH in combination with either prolactin or LH, or prolactin plus LH, could initiate implantation in the absence of the pituitary gland. No pituitary hormone was by itself capable of achieving this result. The combination of prolactin with FSH gave better results than a combination of LH with FSH. Prolactin from sheep, cattle or rats was equally effective in combination with rat FSH in initiating implantation. In mice exhibiting suckling-induced delay of implantation this delay was terminated by injection of FSH. GH by itself or in conjunction with other hormones had no significant effect on implantation or on any of the other parameters associated with implantation that were measured. On the basis of these experimental results it is suggested that prolactin and LH are involved with progesterone production and FSH with oestrogen production, both of which are required for implantation in the mouse.     

Prolactin modulates steroidogenesis by rat granulosa cells: I. Effects on progesterone

Either testosterone or follicle-stimulating hormone (FSH) stimulates progesterone secretion by granulosa cells from rats but the combination of the two hormones increases progesterone production in a synergistic manner. We have investigated the effects of graded doses of prolactin (0, 0.02, 0.2, 2, or 10 micrograms/ml) alone or in combination with testosterone (0.5 microM), FSH (300 ng/ml), or FSH + testosterone on progesterone secretion by granulosa cells at two stages of differentiation. Relatively undifferentiated granulosa cells from immature, diethylstilbestrol-treated, hypophysectomized (HPX) rats were cultured in defined (serum-free) medium for 3 days. More highly differentiated granulosa cells were obtained on the morning of proestrus from the preovulatory follicles of 30-day-old rats induced to undergo an estrous cycle by injection with 4 IU pregnant mare's serum gonadotropin; these cells were cultured in medium containing 10% fetal bovine serum. Prolactin alone did not enhance the negligible secretion of progesterone by cells from HPX rats, but increased progesterone secretion by cells from proestrous rats. Prolactin significantly enhanced the stimulatory effects of testosterone or FSH alone on cells from both HPX and proestrous rats. When cultures containing both FSH + testosterone were treated with prolactin, progesterone secretion by cells from proestrous rats was significantly enhanced, whereas secretion by cells from HPX rats was significantly depressed. Therefore when cells from HPX rats were cultured with both FSH and testosterone, the direction of the effect of prolactin was reversed from that observed with prolactin + FSH or testosterone alone, and from that observed when cells from proestrous rats were cultured with prolactin + FSH + testosterone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution of ganglioside GM3 in the rat ovary after gonadotropin stimulation.

Gangliosides are ubiquitous membrane components in mammalian cells and are suggested to play important roles in various cell functions, such as cell-cell recognition, differentiation and transmembrane signalling. Ovaries have been shown to contain GM3 as a major ganglioside. To study GM3 distribution during gonadotropin stimulation in the hypophysectomized rat ovary, ovarian sections and cultured granulosa cells were stained with specific monoclonal antibody against GM3. Interstitial cells of follicles of immature hypophysectomized rat ovary expressed ganglioside GM3. Theca cells of early antral follicles but not primary follicles expressed GM3. No granulosa cells of these follicles expressed GM3. When a surge dose of FSH/LH was injected, Graafian follicles were formed and GM3 expression was detected in granulosa cells of these follicles. After ovulation, cumulus cells kept expressing GM3 in the ampulla region of ovulated oviduct. The follicles did not show GM3 expression in their granulosa cells after an ovulatory dose of FSH/LH. At 48 h after in vitro culture with FSH/LH of granulosa cells from preantral follicles, GM3 was expressed to a detectable extent on the outer part of the granulosa layer. Finally, at 72 h after culture, all granulosa cells became positive to anti-GM3 antibody. These data suggest that the expression of ganglioside GM3 in the hypophysectomized rat ovary is spatiotemporally regulated by FSH/LH during follicular development and ovulation.     

Gonadotropin receptor of a mouse luteoma: interactions with luteinizing hormone (LH) and its and subunits

A radioligand-receptor system for luteinizing hormone (LH), USING transplantable mouse luteoma, was used to investigate the interactions of LH, other peptide hormones, and LH subunits. Since tumor size decreased as did production of androgenic hormones following hypophysectomy, the luteoma is believed to have been dependent on pituitary tropic hormones; posthypophysectomy histologic changes supported this conclusion. An homogenate was prepared from 1-4 gm luteomas, which had been borne by mice for 4-10 months. Ovine LH, bovine LH, and human chorionic gonadotrophin reduced the binding of iodine-125 human luteinizing hormone (125-I-hLH). Growth hormone, adrenocorticotrophic hormone, and prolactin had no capacity to interfere with binding of 125-I-hLH. Though follicle-stimulating hormone (FSH) and thyroid-stimulating hormone (TSH) reduced the binding somewhat, the reductions were consistent with the known presence of contaminating amounts of LH in the FSH and TSH. The accumulated results of a number of experiments suggest that binding to the luteoma LH receptor requires a particular polypeptide structural conformation, one found in the native hormone but found in neither alpha nor beta subunit alone.     

Estrogen regulation of progestin biosynthetic enzymes in cultured rat granulosa cells

The mechanism by which estrogens enhance gonadotropin-stimulated ovarian progestin production was investigated by studying the modulation of pregnenolone biosynthesis as well as the activities of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) in cultured rat granulosa cells. Cells from immature hypophysectomized, estrogen-treated rats were cultured for 3 days with follicle-stimulating hormone (FSH) and/or estrogens. Pregnenolone production was measured in the presence of cyanoketone which inhibits 3 beta-HSD activity. Activities of 3 beta-HSD and 20 alpha-HSD were determined in cell homogenates by direct enzyme assays. Some cells were also primed with FSH to induce luteinizing hormone (LH) receptors for studies on the effects of estrogens on LH-modulated parameters. Pregnenolone production by cultured granulosa cells was stimulated by FSH, while treatment with diethylstilbestrol (DES) or estradiol further enhanced the gonadotropin action. Treatment with FSH increased 3 beta-HSD activity. Similarly, concomitant treatment with DES further enhanced 3 beta-HSD activity in a dose-dependent manner with an apparent ED50 of 10(-8) M. Also, treatment with estrogens alone increased 3 beta-HSD activity. The increases in enzyme activity induced by estrogen alone or in combination with FSH were not associated with changes in the apparent Km values. FSH also stimulated 20 alpha-HSD activity by 2-fold in these cells, while concomitant treatment with DES did not affect the FSH action. In FSH-primed cells, LH stimulated pregnenolone production while the LH action was enhanced by concomitant treatment with the estrogens. Likewise, LH stimulated the activity of 3 beta-HSD, while concomitant DES treatment further augmented LH action. LH did not stimulate 20 alpha-HSD activity when added alone or in combination with DES. Thus, the estrogen enhancement of the gonadotropin-stimulated progesterone production in cultured rat granulosa cells is associated with increases in pregnenolone biosynthesis and the activity of the 3 beta-HSD enzyme, without affecting the 20 alpha-HSD activity.     

Vasoactive intestinal peptide: a novel stimulator of steroidogenesis by cultured rat granulosa cells

Vasoactive intestinal peptide (VIP) and VIPergic nerve fibers are present in the ovaries of several mammalian species, suggesting a possible ovarian action of VIP. We have investigated the direct effects of synthetic porcine VIP on rat granulosa cell steroidogenesis in vitro. The cells were obtained from immature, hypophysectomized, estrogen-primed rats, and cultured in a serum-free medium for 24 h in the absence or presence of varying amounts of VIP. Medium steroids were then determined by specific radioimmunoassay. Vasoactive intestinal peptide dose-dependently stimulated progesterone, 20 alpha-hydroxypregn-4-ene-3-one (20 alpha-OH-progesterone), and estrogen production with an approximate ED50 value of 3 X 10(-8) M. Maximum steroid production induced by VIP ranged from 15% to 28% of that seen with maximal follicle-stimulating hormone (FSH) stimulation. In contrast to the ability of FSH to induce luteinizing hormone (LH) receptor formation, treatment with VIP did not increase [125I]iodo-human chorionic gonadotropin (hCG) binding to granulosa cells. The ability of several gastrointestinal peptides, having 17-44% sequence identity to VIP, to stimulate granulosa cell steroidogenesis was also tested. The most closely related peptide, PHM-27 was less effective than VIP, and the least closely related, secretin and glucagon, were ineffective at 10(-6) M. Vasoactive intestinal peptide seems to act at least partly through cyclic 3',5'-adenosine monophosphate (cAMP)-dependent processes: addition of a phosphodiesterase inhibitor significantly potentiated the VIP stimulation of granulosa cell steroidogenesis, and VIP was capable of producing a dose- and time-dependent increase in both intracellular and medium cAMP levels. Vasoactive intestinal peptide stimulation of estrogen production seemed to be a result of increased aromatase activity. The increased progesterone production was associated with increased pregnenolone production, increased rate of conversion of pregnenolone to progesterone via 3 beta-hydroxysteroid dehydrogenase, and decreased metabolism of progesterone via 20 alpha-hydroxysteroid dehydrogenase. These results indicate that VIP exerts a specific action on granulosa cells to increase estrogen and progestin production. The observed direct effects of VIP, coupled with its identification in the ovary, suggest that VIP may be a physiologically important regulator of ovarian activity.     

Temporal relationship of the prolactin-dependent LH-induced LH receptor to the LH stimulus

The time course for LH induction of luteinizing hormone (LH) receptors as reflected in binding of ¹²⁵l-labeled hCG was investigated in hypophysecto-mized adult male rats. A low dose of oLH (10 μg) was administered to hypophysectomized adult male rats following pretreatments with prolactin, follicle-stimulating hormone (FSH), growth hormone (GH), or saline. Testicular binding of hCG was determined at different times following the LH injection using Leydig cell membrane preparations from a testicular homogenate. Seven days after hypophysectomy, hCG binding was at a nadir of 19 ± 7% (mean ± SD) of control values. Pretreatment with prolactin (100 μg/day) for 7 days was associated with a nonsignificantly different hCG binding that was 30 ± 5% of control values. Prolactin pretreatment plus a single 10 μg LH i.p. injection increased ¹²⁵l hCG binding up to 56 ± 10% of control values within 30 minutes of the LH injection. Luteinizing hormone-induced hCG binding persisted at a high level (51 ± 4% of control values) for 2 hours but returned to hypophysectomized control levels 6 hours after the i.p. LH injection. Seven days pretreatment with FSH or GH at 100 μg/day plus 10-μg LH injections was also tested. Neither FSH nor GH had a statistically significant effect on hCG binding nor could they mimic the ability of prolactin to allow for LH induction of hCG binding in the hypophysectomized adult male rats. These studies suggest that the induction or “up-regulation” of Leydig cell hCG binding by ovine LH is rapid and specifically dependent upon pre-exposure to prolactin.     

Epidermal growth factor and gonadotropin-releasing hormone inhibit cyclic AMP–dependent luteinizing hormone receptor formation in ovarian granulosa cells

The induction of luteinizing hormone (LH) receptors was studied in granulosa cells prepared from the ovaries of hypophysectomized diethylstilbestrol-treated immature rats. Incubation of granulosa cells for 48 h with increasing concentrations of follicle-stimulating hormone (FSH) or choleragen caused parallel rises in cAMP levels and LH receptors. These observations, with the finding that 8-Bromo-cAMP also induced LH receptor formation, indicate that hormonal stimulation of LH binding sites is mediated by cAMP. Peptide hormones that inhibited FSH-stimulated cAMP production, such as epidermal growth factor (EGF) and a gonadotropin-releasing hormone agonist (GnRHa), also prevented LH receptor formation. GnRHa and EGF had negligible effects on FSH-stimulated cAMP production from 0 to 24 h of culture, but reduced cAMP accumulation by 80% and 90%, respectively, from 24 to 48 h when the majority of LH receptors appeared. FSH-sensitive adenylate cyclase activity, as measured by the conversion of (³H)-ATP to (³H)-cAMP, was inhibited by GnRHa and EGF at 48 h of culture. EGF and GnRHa also reversed the inhibition of ectophosphodiesterase activity caused by FSH in granulosa cells between 48 and 72 h of culture. Both EGF and GnRHa inhibited induction of LH receptors by 8-Bromo-cAMP, suggesting that their effects are also on cAMP action. Addition of GnRHa, but not EGF, between 36 and 48 h of culture completely prevented further increases in LH receptors induced by 8-Bromo-cAMP, indicating that the inhibitory action of GnRHa can be initiated at later times during granulosa cell differentiation, whereas full expression of EGF action requires a longer period. These results demonstrate that EGF and GnRH inhibit FSH-induced LH receptor formation in the granulosa cell by reducing hormone-dependent cAMP production and also by impairing the ability of cAMP to stimulate LH receptor formation.     

Alpha-inhibin gene expression occurs in the ovine adrenal cortex, and is regulated by adrenocorticotropin

Inhibin is a glycoprotein hormone composed of two nonidentical subunits. It is produced by the ovary and testis and plays a vital role in gonadal function by inhibiting the secretion of FSH. More recently, additional activities associated with inhibin peptides have been identified. Inhibin heterodimers (alpha-beta) are reported to act directly on ovarian granulosa cells and inhibit estrogen production induced by FSH. Furthermore, homodimers of beta-inhibin subunits stimulate the secretion of FSH, an activity that is directly opposite to that of inhibin. Each of these inhibin-related activities are concerned with the hypothalamic-pituitary-gonadal axis. We have investigated further the complexity of inhibin activity by determining whether inhibin genes are expressed in nongonadal tissue. RNA hybridization experiments demonstrate that the alpha-inhibin gene is expressed in the sheep adrenal cortex and hybridization histochemistry shows that this gene is expressed in each of the functional zones within the cortex. Dot blot analysis showed that the level of alpha mRNA within the adrenal is influenced by ACTH, one of the major regulators of adrenal cortex function. These observations imply that there are inhibin-related peptides not directly associated with the gonads. beta-inhibin gene expression was not clearly detected in the adrenal and we conclude that if expression occurs then it does so at extremely low levels.     

The effects of gonadotropins on metabolism of isolated rat granulosa cells

FSH $\text{in vitro}$, but not LH, increased the O₂ uptake of isolated granulosa cells from 23 day old rats previously treated with DES or with DES and FSH. Dose response studies showed that the cells were most sensitive to FSH when the cellular binding of FSH was highest. LH increased the O₂ uptake of granulosa cells of untreated 30 day old rats. DES treatment inhibited the LH induced rise in O₂ uptake when the rats were implanted with DES capsules unless FSH was injected to induce LH receptors. Addition of dbcAMP $\text{in vitro}$ increased O₂ uptake of granulosa cells from 30 day old rats at concentrations 10X lower than those required to stimulate O₂ uptake in cells from 23 day old rats treated with DES alone.FSH $\text{in vitro}$ increased lactate formation in the absence of added substrates but did not do so when glucose was added to the media. In contrast, LH greatly increased lactate formation with added glucose. Dose response studies showed that less than 0.6 ug/ml LH S21 was effective in increasing lactate above control levels. These data suggest that FSH affects aerobic pathways while LH affects anaerobic pathways in the process of the differentiation of granulosa cells toward luteal cells.It is well known that FSH and LH interact with their target cells in the ovary by binding to specific receptors and that FSH stimulates LH-receptor production (1). Receptor binding by either hormone activates adenylate cyclase (2) raising cyclic adenosine monosphosphate (cAMP) levels (3) and increasing protein kinase activity (4). Such changes probably trigger changes in the major metabolic pathways that support follicular development because cells of corpora lutea have glycogen (5) which is not present in follicular granulosa cells (6–9). Several studies suggest that FSH and LH may regulate metabolic processes in the ovary. LH increases lactate in whole prepuberal ovaries (10,11,12) and also increases the uptake of glucose (13). FSH increases oxygen uptake in chick ovaries (14), rat ovaries (15) and prairie dog ovaries (16). However, only one study has been done using isolated ovarian cells. Hamberger (17) has reported that FSH increased the oxygen uptake of thecal cells of immature rats while LH increased the oxygen uptake of granulosa cells. Since granulosa cells from immature rats are reported to have FSH receptors while theca cells have LH receptors the effects of these hormones appear unclear.The present studies were undertaken to more accurately characterize the actions of FSH, LH, and dibutyryl cAMP (dbcAMP) on the oxygen uptake of isolated granulosa cells and remaining tissues of immature ovaries and to determine the effects of FSH and LH on the production of lactate by granulosa cells.     

Expression of alpha subunit and luteinizing hormone beta genes in the ovine anterior pituitary. Estradiol suppresses accumulation of mRNAS for both alpha subunit and luteinizing hormone beta

Bovine cDNA clones containing coding sequences for growth hormone, prolactin, alpha subunit, and luteinizing hormone beta (LH beta) have been used to quantitate their respective mRNA concentrations in anterior pituitary glands isolated from ovariectomized ewes, or from ovariectomized ewes treated for three weeks with estradiol. Concentrations of mRNAs for prolactin or growth hormone remained unchanged in either physiological state. In contrast, treatment with estradiol resulted in a 98% decrease of mRNA for LH beta, relative to untreated animals. This change in mRNA was associated with a similar decrease in the concentrations of pituitary and serum LH. Administration of estradiol also led to a reduction (86%) of alpha subunit mRNA. These results suggest that estrogen regulates the expression of the genes encoding both the alpha and LH beta subunit prior to translation. Furthermore, the pronounced effect of estradiol on the concentrations of mRNAs for alpha subunit and LH beta suggest that the assembly of mature glycoprotein hormones may not be limited solely by the rate of accumulation of the beta subunit.     

Induction of G protein-coupled estrogen receptor (GPER) and nuclear steroid hormone receptors by gonadotropins in human granulosa cells

Estradiol and progesterone mediate their actions by binding to classical nuclear receptors, estrogen receptor α (ERα) and estrogen receptor β (ERβ) and progesterone receptor A and B (PR-A and PR-B) and the non-classical G protein-coupled estrogen receptor (GPER). Several animal knock-out models have shown the importance of the receptors for growth of the oocyte and ovulation. The aim of our study was to identify GPER in human granulosa cells (GC) for the first time. Moreover, the effect of different doses of gonadotropins on estrogen and progesterone receptors in the human ovary should be investigated as follicle stimulating hormone (FSH) and luteinizing hormone (LH) are also responsible for numerous mechanisms in the ovary like induction of the steroid biosynthesis. Human GC were cultured in vitro and stimulated with different doses of recombinant human FSH or LH. Receptor expression was analyzed by immunocytochemistry and quantitative real-time RT-PCR. GPER could be identified for the first time in human GC. It could be shown that high concentrations of LH increase GPER protein expression. Furthermore FSH and LH increased ERβ, PR-A and PR-B significantly on protein level. These findings were verified for high doses of FSH and LH on mRNA level. ERα was not affected with FSH or LH. We assume that gonadotropins induce GPER, ERβ and PR in luteinized granulosa cells.     

Effect of testosterone on serum immunoactive inhibin concentrations in intact and hypophysectomized male rats

Intact and hypophysectomized rats were treated with graded doses of testosterone via subcutaneous Silastic implants over a 13-week period. Serum inhibin concentrations fell 50% (P less than 0.001) after 2 weeks of hypophysectomy, remaining suppressed at this level for 13 weeks. The administration of testosterone to hypophysectomized rats (serum testosterone values 2-12 ng/ml; control values 5.5 ng/ml) was without effect on serum inhibin values. In contrast, administration of testosterone to intact animals for 7 weeks resulted in an initial fall (P less than 0.05) in inhibin levels to 50-70% of controls then increasing to reach control levels at higher doses. Serum FSH concentrations were similarly biphasic with increasing dose of testosterone and values for these two hormones were significantly correlated (r = 0.44, P less than 0.01). Segments of seminiferous tubules in culture from rats after various times of hypophysectomy showed a partly suppressed secretion of inhibin. The administration of testosterone did not modify inhibin production although inhibin production was sensitive to FSH. It is concluded that (1) serum inhibin concentrations are partly suppressed after hypophysectomy and testosterone has no effect on serum inhibin values; and (2) the suppression of serum inhibin in intact rats treated with increasing doses of testosterone is attributable to the concomitant fall in serum FSH concentrations.