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.     

Leptin effect on nitric oxide and GnRH-induced FSH secretion from ovine pituitary cells in vitro.

The secretion of gonadotrophins from anterior pituitary cells can be modulated by leptin and signals originating from the immune system, among others, by nitric oxide (NO). There are some studies that have demonstrated a role for leptin and NO in the regulation of FSH in rodents, however, no similar data are available in regards to ewes. Therefore, the objective of the present study was to analyse the leptin effect on GnRH-induced FSH secretion from the ovine anterior pituitary cells in vitro. Additionally, the influence of leptin on NO release and its role in the GnRH and leptin-modulated secretion of FSH from pituitary gland of ewes was investigated. The obtained results show that the influence of leptin on FSH secretion is biphasic. Leptin in concentration 10(-8) and 10(-7) M/l significantly enhances, whereas 10(-6) and 10(-5) M/l of leptin suppresses FSH secretion from the pituitary cells in comparison to the control. The secretion of FSH and NO release under the influence of leptin are in very high positive correlation (r=0.77). The inhibition of NO synthesis with L-NAME., instead, disables leptin from the stimulation of FSH secretion.     

Interactions of prostaglandin E1 (PGE1) and LRH on anterior pituitary function

Numerous biochemical pathways influence the synthesis and release of anterior pituitary hormones. Releasing factors extracted from the hypothalamus and prostaglandins (PGs) appear to alter a common biochemical activity, adenyl cyclase, in pituitary cells. Luteinizing hormone releasing hormone (LRH), prostaglandin (PGE₁), 7 oxa-13-prostynoic acid and cycloheximide were tested for individual and interacting effects on the in vitro release of FSH, LH and prolactin from hemipituitaries of 15 day old female rats. LRH (10 ng/ml) consistently released both LH and FSH in all in vitro experiments and inhibited prolactin release in 1 of 2 experiments. Lower concentrations (5 and 1 ng/ml) also stimulated LH and FSH release but did not influence prolactin release. Concurrent depletion of stored LH and FSH in the gland was observed. PGE₁ in a 6.5 hour incubation increased the storage of LH within the gland in the absence of LRH. In a 1.5 hour incubation in the presence of LRH, storage of LH was also increased. PGE₁ had no effect on LH and FSH release; however, in 1 of 2 experiments it stimulated prolactin release in the absence of LRH. Prostynoic acid stimulated LH and FSH release but did not synergize with LRH action in the same tissue. Cycloheximide did not affect LH release during the first 30 minutes of incubation; however, the release during the subsequent 1 hour was significantly inhibited. Similar tissue also exposed to cycloheximide was still responsive to LRH during the latter 1 hour incubation period. Cycloheximide had no effect on prolactin storage and release from the same tissue.     

Effect of inhibin-like activity on LH-RH-stimulated release of FSH by pituitary glands from female rats in vitro

During long-term incubation of pituitary glands from intact female rats in the presence of inhibin-like activity, LH-RH-stimulated release of FSH becomes inhibited after 4 h of incubation. However, at the same time inhibition of basal FSH release is included. Therefore, glands were at first incubated for 4 h in the presence of inhibin-like activity to block basal release completely and thereafter LH-RH was added to the medium. It was found that LH-RH still could stimulate FSH release, despite the continuous presence of inhibin-like activity. This means that LH-RH-stimulated release of FSH could be investigated separately from basal release. Using this way of incubation, it was found that part of the action of LH-RH on FSH release was independent of protein synthesis. Also part of LH-RH-stimulated FSH release was independent of the presence of extracellular Ca2+. Furthermore it was found that LH-RH, when added after 4 h of incubation did stimulate FSH synthesis, in the presence as well as absence of inhibin-like activity. The present results indicate that LH-RH-stimulated release of FSH is not affected by inhibin-like activity. Complete inhibition of basal release and synthesis of FSH does not prevent LH-RH from stimulating FSH release and synthesis. It is suggested that two separate releasing mechanisms for FSH could exist in the pituitary gland.     

Interactions of prostaglandin E1 (PGE1) and LRH on anterior pituitary function

Numerous biochemical pathways influence the synthesis and release of anterior pituitary hormones. Releasing factors extracted from the hypothalamus and prostaglandins (PGs) appear to alter a common biochemical activity, adenyl cyclase, in pituitary cells. Luteinizing hormone releasing hormone (LRH), prostaglandin (PGE₁), 7 oxa-13-prostynoic acid and cycloheximide were tested for individual and interacting effects on the $\text{in vitro}$ release of FSH, LH and prolactin from hemipituitaries of 15 day old female rats. LRH (10 ng/ml) consistently released both LH and FSH in all $\text{in vitro}$ experiments and inhibited prolactin release in 1 of 2 experiments. Lower concentrations (5 and 1 ng/ml) also stimulated LH and FSH release but did not influence prolactin release. Concurrent depletion of stored LH and FSH in the gland was observed. PGE₁ in a 6.5 hour incubation increased the storage of LH within the gland in the absence of LRH. In a 1.5 hour incubation in the presence of LRH, storage of LH was also increased. PGE₁ had no effect on LH and FSH release; however, in 1 of 2 experiments it stimulated prolactin release in the absence of LRH. Prostynoic acid stimulated LH and FSH release but did not synergize with LRH action in the same tissue. Cycloheximide did not affect LH release during the first 30 minutes of incubation; however, the release during the subsequent 1 hour was significantly inhibited. Similar tissue also exposed to cycloheximide was still responsive to LRH during the latter 1 hour incubation period. Cycloheximide had no effect on prolactin storage and release from the same tissue.     

Effects of quinestrol on reproductive hormone expression, secretion, and receptor levels in female Mongolian gerbils (Meriones unguiculatus)

Quinestrol, a synthetic estrogen with marked estrogenic effects and prolonged activity, has potential as a contraceptive for Mongolian gerbils. The objective of this study was to describe the effects of quinestrol on reproductive hormone expression, secretion, and receptor levels in female Mongolian gerbils. Serum and pituitary concentrations of follicle stimulating hormone (FSH) and luteinizing hormone (LH) were decreased, whereas serum concentrations of estradiol (E2) and progesterone (P4) were increased after quinestrol treatment; the effects were both time- and dose-dependent. Furthermore, quinestrol downregulated expression of FSHβ and LHβ mRNA in the pituitary gland, as well as FSH receptor (FSHR) and estrogen receptor (ER) β in the ovary. However, it up-regulated mRNA expression levels of ERα and progesterone receptor (PR) in the pituitary gland and uterus, as well as mRNA for LH receptor (LHR) and PR in the ovary (these effects were time- and dose-dependent). In contrast, quinestrol had no significant effects on the mRNA expression levels of ERα in the ovary, or the gonadotropin α (GtHα) subunit in the pituitary gland. We inferred that quinestrol impaired synthesis and secretion of FSH and LH and that the predominant ER subtype in the pituitary gland of Mongolian gerbils may be ERα. Overall, quinestrol disrupted reproductive hormone receptor expression at the mRNA level in the pituitary-gonadal axis of the Mongolian gerbil.     

丘脑下部促黄体素释放激素类似物(LRH-A)的作用机制 Ⅲ.对罗非鱼脑垂体促性腺激素分泌细胞超微结构的影响

前文(方永强等,1981)探讨了罗非鱼脑垂体促性腺细胞的生理活动与雌鱼性腺发育不同时期的相互关系,对比注射LRH-A后2、4和10小时对促性腺细胞的影响。根据促性腺细胞核的形态和细胞质中分泌颗粒数量的变化,以及DNA、RNA、酸碱性蛋白和酸碱性磷酸酶等一系列反应,初步阐明了LRH-A的作用机制与细胞核和细胞质的代谢变化有关。在此基础上,本文较详细观察了LRH-A对垂体促性腺细胞超微结构的影响,目的在于从亚细胞的水平上,进一步了解其作用机制。     

Relationship between serum gonadotropins and pituitary immunoreactive gonadotropins and steroid receptors during the first FSH increase of the estrous cycle and following steroid treatment in heifers

The objectives were to determine the effects of (i) time during the first FSH increase of the estrous cycle (time-course study) and (ii) exogenous steroid treatment (steroid feedback study) on the relationship between circulating serum gonadotropins, and the proportions of pituitary cells immunoreactive for gonadotropins and steroid receptors during the estrous cycle in heifers. Pituitaries were collected from heifers (n=40) slaughtered at 13h (n=8), 30h (n=24) and 66h (n=8) after estrous onset, corresponding to before, during and after the first FSH increase of the estrous cycle. Heifers slaughtered during the FSH increase (at 30h) either received no treatment (n=8), or were treated (n=16) with estradiol benzoate and/or progesterone before slaughter. During the time-course study, the proportion of pituitary cells immunoreactive for FSH increased (P<0.05) during the first transient FSH increase reflecting serum concentrations. The proportion of pituitary cells immunoreactive for LH was unaltered, a reflection of serum LH concentrations. The proportion of estrogen receptors (ER)-alpha, but not ER-beta, was decreased (P<0.05) at 30h compared with at either 13 or 66h. During the steroid feedback study, exogenous progesterone with or without estradiol suppressed (P<0.05) the proportions of pituitary cells immunoreactive for gonadotropins, serum FSH concentrations and LH pulse frequency. Steroid treatment did not alter the proportion of pituitary cells positive for estrogen receptors (alpha and beta). While progesterone receptors (PR) were not detected in the anterior pituitary by immunohistochemistry during the early estrous cycle or in response to steroid treatment, quantitative real-time PCR revealed that mRNA for progesterone receptors was expressed at very low levels. The expression of pituitary PR mRNA was decreased (P<0.05) at 30 and 66h compared with 13h, and was suppressed (P<0.05) following steroid treatments. Alterations in pituitary steroid receptors are implicated in the differential regulation of gonadotropin secretion during the first transient FSH rise, but not in response to exogenous steroids. The time-course study and steroid feedback responses support the hypothesis that LH pulse frequency is tightly linked to regulation of GnRH pulse frequency. Serum FSH is regulated by its own synthesis, as reflected by pituitary FSH content and perhaps by alterations in pituitary sensitivity to circulating steroids by changes in steroid receptor content.     

Inhibin production by Sertoli cells in culture.

Studies on the secretion of inhibin and its mode of action were carried out in vitro, utilizing cell cultures. Isolated rat Sertoli cells secreted an inhibin-like heat-labile, non-dialysable substance, Sertoli Cell Factor (SCF), which could selectively suppress FSH secretion by rat anterior pituitary cells. SCF selectively suppressed the basal and GnRH-stimulated FSH release as well as the de-novo synthesis of FSH by acting directly on the pituitary cells. In 1 out of 5 experiments, SCF also suppressed the synthesis of LH, possibly by affecting the overall protein synthesis. Under similar culture conditions, Sertoli cells isolated from animals between 18 and 90 days of age secreted comparable amounts of SCF. In contrast, anterior pituitary cells from adult rats (60-90 days old) were considerably more sensitive to SCF than pituitary cells obtained from younger (18-33 days old) animals, suggesting that decline in circulating FSH level, occurring at approximately 35 days of age, may result from increased pituitary sensitivity to inhibin. Besides identifying the Sertoli cells as the site of inhibin production in the testis, these studies demonstrated direct action of inhibin at the pituitary cell level, resulting in suppression of FSH synthesis and release.     

Adenosine regulates thrombomodulin and endothelial protein C receptor expression in folliculostellate cells of the pituitary gland

Adenosine stimulates the release of interleukin 6 (IL-6) and vascular endothelial growth factor from folliculostellate cells of the anterior pituitary gland indicating that such cells are also involved in the communication between the immune and endocrine systems during stress and inflammation. In order to understand the precise actions of adenosine on folliculostellate cells, DNA microarray analysis was used to determine global changes in gene expression. Hierarchical clusters revealed, of the genes that had altered expression, the majority were suppressed and many, such as B cell translocation gene 2 and cyclin-dependent kinase inhibitor 2b were related to cell cycle arrest or inhibition of proliferation. Several of the up-regulated genes were associated with cytokine signalling or membrane receptor activity. The most notable of these being IL-6, sulfiredoxin 1, endothelial protein C receptor (EPCR) and thrombomodulin (THBD) which can all play a role in controlling inflammation. The EPCR and THBD pathway is well known in anti-coagulation but also has anti-inflammatory and anti-apoptotic properties. Up-regulation of EPCR and THBD in folliculostellate cells was confirmed by qRT-PCR and western blotting analysis and their expression were also demonstrated in many of the hormone-secreting cells of the anterior pituitary gland. Our findings suggest that adenosine can stimulate expression of stress and inflammation related genes from folliculostellate cells of the anterior pituitary gland. These genes include EPCR and THBD, neither of which has been previously identified in the pituitary gland.     

Gonadotropins, their receptors, and the regulation of testicular functions in fish

The pituitary gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone (FSH) regulate steroidogenesis and spermatogenesis by activating receptors expressed by Leydig cells (LH receptor) and Sertoli cells (FSH receptor), respectively. This concept is also valid in fish, although the piscine receptors may be less discriminatory than their mammalian counterparts. The main biological activity of LH is to regulate Leydig-cell steroid production. Steroidogenesis is moreover modulated in an autoregulatory manner by androgens. The male sex steroids (testosterone in higher vertebrates, 11-ketotestosterone in fish) are required for spermatogenesis, but their mode of action has remained obscure. While piscine FSH also appears to have steroidogenic activity, specific roles have not been described yet in the testis. The feedback of androgens on gonadotrophs presents a complex pattern. Aromatizable androgens/estrogens stimulate LH synthesis in juvenile fish; this effect fades out during maturation. This positive feedback on LH synthesis is balanced by a negative feedback on LH release, which may involve GnRH neurones. While the role of GnRH as LH secretagogue is evident, we have found no indication in adult male African catfish for a direct, GnRH-mediated stimulation of LH synthesis. The limited available information at present precludes a generalized view on the testicular feedback on FSH.     

Effects of starvation on gonadotropin and thyrotropin subunit mRNA levels and plasma hormone levels in the male Japanese quail (Coturnix coturnix japonica)

Contents of mRNAs encoding LHbeta-, FSHbeta-, TSHbeta- and common a-subunit precursor molecules were measured in male Japanese quail deprived of food for three days. Plasma LH, FSH, thyroxine and triiodothyronine levels were also measured in the same birds. Plasma LH levels declined during the period of food deprivation. Levels in starved birds were not different from those in control birds after one day of starvation but were significantly lower after three days. Plasma FSH levels showed a similar decline, although the changes were not significant. Plasma thyroxine levels did not decrease during starvation, whilst plasma triiodothyronine levels decreased drastically and significantly soon after the start of starvation. All the hormone subunit mRNA contents in starved birds also decreased, with differences from control birds significant 3 days after the start of starvation. Plasma FSH levels showed a strong positive correlation with pituitary FSHbeta mRNA levels, while plasma LH levels had a strong positive correlation with common a mRNA levels and practically no correlation or even a negative correlation with LHbeta mRNA levels. These results suggest that starvation suppresses not only gonadotropin and thyrotropin secretion but also their synthesis in the pituitary gland. Furthermore, these results showed that FSH and LH have different synthesis and secretion dynamics in the Japanese quail. Contradicting results with TSHbeta mRNA and thyroid hormones lead us to assume that starvation affects thyroid hormone metabolism in peripheral tissue, presumably in the liver.     

A change in basal FSH release accompanies the onset of the second or selective phase of increased serum FSH in the cyclic rat

Experiments were undertaken to investigate if changes occur at the level of the anterior pituitary gland to result in selective follicle-stimulating hormone (FSH) release during late proestrus in the cyclic rat. At 1200 h proestrus, prior to the preovulatory luteinizing hormone (LH) surge in serum and the accompanying first phase of FSH release, serum LH and FSH concentrations were low. At 2400 h proestrus, after the LH surge and shortly after the onset of the second or selective phase of FSH release, serum LH was low, serum FSH was elevated about 4-fold, pituitary LH concentration was decreased about one-half and pituitary FSH concentration was not significantly decreased. During a two hour $\text{in}$$\text{vitro}$ incubation, pituitaries collected at 2400 h released nearly two-thirds less LH and 2.5 times more FSH than did pituitaries collected at 1200 h. Addition of luteinizing hormone releasing hormone (LHRH) to the incubations caused increased pituitary LH and FSH release. However, the LH and FSH increments due to LHRH in the 2400 h pituitaries were not different from those in the 1200 h pituitaries. The results indicate that a change occurs in the rat anterior pituitary gland during the period of the LH surge and first phase of FSH release which results in a selective increase in the basal FSH secretory rate. It is suggested that this change is primarily responsible for the selective increase in serum FSH which occurs during the second phase of FSH release.     

Spontaneous recovery from hypopituitarism due to postpartum hemorrhage

A rare case is presented of a woman with spontaneous recovery from hypopituitarism following postpartum hemorrhage. One month after delivery, serum thyroid hormone, TSH, LH and FSH levels were low, and their secretion from the pituitary gland responded poorly to the TRH and LH-RH tests. Pituitary TSH response was normal 3 months after delivery. In the LH-RH test, pituitary LH and FSH response returned to normal at 2 months. Pituitary GH secretion and serum cortisol levels induced by ITT already responded normally one month postpartum. Excessive secretion of pituitary PRL was observed 3 months after delivery and improved gradually thereafter. These results indicate that the secretion of pituitary tropic hormones was sensitive to pituitary ischemia in the following order: TSH, gonadotropin, GH and ACTH. The disturbance of these hormones also persisted in the same order.     

Sequence analysis and expressional regulation of messenger RNAs encoding beta subunits of follicle-stimulating hormone and luteinizing hormone in the red-bellied newt, Cynops pyrrhogaster

Two distinct cDNAs encoding beta subunits of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were cloned from the cDNA library constructed for the pituitary of the red-bellied newt, Cynops pyrrhogaster, and sequenced. The newt FSHbeta and LHbeta cDNAs encode polypeptides of 129 and 131 amino acids, including signal peptides of 20 and 19 amino acids, respectively. The number and position of cysteine and N-glycosylation in each of the beta subunits of FSH and LH, which are considered essential for assembly of the alpha subunit, are well conserved between the newt and other tetrapods. The high homology (41.6%) between the beta subunits of newt FSH and LH imply less specificity of FSH and LH in gonadal function. One cDNA encoding the common polypeptide chain alpha subunit of FSH and LH was also isolated from the newt pituitary gland. The mRNAs of FSHbeta, LHbeta, and the alpha subunit were expressed only in the pituitary gland among various newt tissues. Double-staining with in situ hybridization and immunohistochemistry revealed coexpression of FSHbeta and LHbeta in the same newt pituitary cells. Ovariectomy induced a significant increase in FSHbeta mRNA levels, but there was no significant change in LHbeta or alpha subunit mRNA levels compared with those in control animals. Taken together, these data suggest that two kinds of gonadotropins, namely FSH and LH, are expressed in the same gonadotropin-producing cells in the pars distalis of the newt as well as in other tetrapods and that the expression of FSHbeta is negatively regulated by the ovaries.     

左旋十八甲基炔诺酮对大鼠卵巢颗粒细胞和垂体细胞激素合成及分泌的影响

研究了左旋十八甲基炔诺酮对大鼠卵巢颗粒细胞和垂体细胞激素合成及分泌的直接作用。结果显示左旋十八甲基炔诺酮单独作用于无血清培养的颗粒细胞时,抑制雌二醇的合成,但刺激孕酮的分泌;在与促卵泡素合并处理时,颗粒细胞雌二醇、孕酮的分泌量随着左旋十八甲基炔诺酮浓度的增加而增加。利用促性腺激素生物测定方法证明大鼠整体用左旋十八甲基炔诺酮处理后,垂体中促卵泡素和促黄体生成素活性明显下降;同时外周血清中促卵泡素的活性亦下降。培养的垂体单纯用左旋十八甲基炔诺酮处理后,其培养液经生物测定呈现抑制颗粒细胞雌二醇和孕酮的分泌,促性腺激素释放激素可减弱左旋十八甲基炔诺酮的抑制作用。提示左旋十八甲基炔诺酮除通过垂体卵巢轴系起作用外,还能直接作用于卵巢。     

Effects of acute ovariectomy on anterior pituitary gland follicle-stimulating hormone and luteinizing hormone secretion in the metestrous rat

Changes at the anterior pituitary gland level which result in follicle-stimulating hormone (FSH) release after ovariectomy in metestrous rats were investigated. Experimental rats were ovariectomized at 0900 h of metestrus and decapitated at 1000, 1100, 1300, 1500, 1700 or 1900 h of metestrus. Controls consisted of untreated rats killed at 0900 or 1700 h and rats sham ovariectomized at 0900 h and killed at 1700 h. Trunk blood was collected and the serum assayed for FSH and luteinizing hormone (LH) concentrations. The anterior pituitary gland was bisected. One-half was used to assay for FSH concentration. The other half was placed in culture medium for a 30-min preincubation and then placed in fresh medium for a 2-h incubation (basal FSH and LH release rates). The basal FSH release rate and the serum FSH concentration rose significantly by 4 h postovariectomy and remained high for an additional 6 h. The basal FSH release rate and the serum FSH concentration correlated positively (r=0.71 with 72 degrees of freedom) and did not change between 0900 and 1700 h in untreated or sham-ovariectomized rats. In contrast, the serum LH concentration and the basal LH release rate did not increase after ovariectomy. Ovariectomy had no significant effect on anterior pituitary gland FSH concentration. The results suggest that the postovariectomy rise in serum FSH concentration is the result, at least in part, of changes which cause an increase in the basal FSH secretion rate (secretion independent of the immediate presence of any hormones of nonanterior pituitary gland origin). The similarities between the selective rises in the basal FSH release rate and the serum FSH concentration in the ovariectomized metestrous rat and in the cyclic rat during late proestrus and estrus raise the possibility that an increase in the basal FSH release rate may be involved in many or all situations in which serum FSH concentration rises independently of LH.     

The effects of testosterone propionate and estradiol benzoate on the vitro synthesis of FSH.

The effects of estradiol benzoate (EB) and testosterone propionate (TP) on pituitary and plasma concentrations of follicle stimulating hormone (FSH) and the in vitro synthesis of FSH in pituitary tissue was studied in mature male rats. By the 4th day of treatment with EB, pituitary content and concentration of FSH had declined, and content had fallen to 6% and concentration to 3% of pretreatment values. Similar results occurred during in vitro synthesis. However, serum levels of FSH did not show any decline until the 21st and 28th days of treatment. Administration of TP produced a progressive increase in pituitary content and concentration of FSH, though serum levels remained unchanged for the 1st 7 days, after which they fell slightly. The effect of TP on the in vitro synthesis of FSH showed no consistent pattern, though in no case was a decrease in the uptake of labeled leucine into immunoprecipitable FSH observed. The results suggest that EB and TP have different effects on pituitary FSH in normal adult male rats.