Inhibition of follicle-stimulating hormone receptor binding by circulating immunoglobulins

We have characterized a circulating inhibitor of FSH receptor binding found in two patients with hypergonadotropic amenorrhea and myasthenia gravis. The inhibitor behaves as an immunoglobulin according to several criteria, including precipitation by 30% ammonium sulfate, migration on DEAE-cellulose chromatography, specific binding to protein A-Sepharose, characterization as a 7S protein in sucrose density gradients, and immunoprecipitation with specific antihuman immunoglobulin G. Evidence suggests that these antibodies are directed at determinants on or near the FSH receptor, and they may be responsible for the observed clinical FSH resistance.     

Glycerol prevents loss of immunoreactive follicle-stimulating hormone and luteinizing hormone from frozen urine

First morning urine samples were collected from both menstruant and post-menopausal women and stored at -25 degrees C. Immunoreactive FSH disappeared from these samples (t1/2 = 30 days), ultimately stabilizing at about 20% of the initial value. The loss was more rapid at -20 degrees C and less rapid at -55 degrees C and +4 degrees C. Immunoreactive LH was also lost from frozen urine, but more slowly than FSH. The addition of glycerol to urine (0.52 mol/l) stored at -25 degrees C prevented loss of immunoreactive FSH and LH for at least 105 days.     

Inhibition of follicle-stimulating hormone secretion from gonadotroph adenomas by repetitive administration of a gonadotropin-releasing hormone antagonist

As a preliminary step in searching for a pharmacological treatment for gonadotroph adenomas, we administered the GnRH antagonist analog Nal-Glu GnRH to five patients, four men and a woman, with FSH-secreting gonadotroph adenomas in order to determine its effect on FSH secretion. Administration of a single 10-mg dose of Nal-Glu GnRH to four of the patients produced a significant decrease in the serum FSH concentration in two patients and returned the FSH level to normal in only one. Administration of 5 mg Nal-Glu every 12 h for 7 days, however, produced a significant (P less than 0.001) decrease, and to within the normal range, in four of the five patients (mean +/- SEM, 32.7 +/- 5.6 IU/L during the 3 days before treatment and 9.8 +/- 1.4 IU/L during the last 3 days of treatment). Also, in response to the 7-day treatment, LH fell significantly in all five patients, alpha-subunit fell in three, and testosterone fell in all four men. Administration for 6 weeks of the GnRH agonist analog leuprolide did not decrease the serum FSH concentration of one of the patients whose serum FSH did decrease in response to Nal-Glu GnRH. We conclude that repetitive administration of Nal-Glu GnRH may often inhibit FSH secretion by gonadotroph adenomas and that FSH secretion by gonadotroph adenomas may be dependent on endogenous GnRH secretion.     

Inhibition of growth hormone and thyrotropin release by growth hormone-release inhibiting hormone

Addition of increasing doses of synthetic growth hormone-release inhibiting hormone (GH-RIH) leads to a progressive decrease of the basal and N⁶monobutyryl cyclic AMP-,theophylline- and prostaglandin E₂-induced release of immunoreactive growth hormone (GH) and thyrotropin (TSH) release from rat anterior pituitary cells in monolayer culture. A halfmaximal effect is measured at 3 × 10^?9 M GH-RIH while a maximal inhibition to 10–20% of the control level is found at 1 × 10^?7 M. Using rat hemipituitaries and measurement of GH release by both polyacrylamide gel electrophoresis and radioimmunoassay, a maximal effect of GH-RIH was found in the first 5 min of incubation. The inhibitory effect of GH-RIH on GH release remained constant for at least 3 h. GH-RIH does not affect the basal or induced release of prolactin and luteinizing hormone nor the high K ⁺-induced release of GH and TSH.     

Platelet-derived growth factor enhances granulosa cell luteinizing hormone receptor induction by follicle-stimulating hormone and serum

Platelet-derived growth factor potentiated FSH-dependent LH receptor induction in serum-free and serum-containing cultures of rat granulosa cells. Levels of LH receptor binding comparable to pre-ovulatory levels could be achieved in vitro using a combination of FSH, platelet-derived growth factor, and serum. Receptor sites induced under these conditions were capable of mediating an hCG-stimulated increase in progestin secretion. Our results in vitro suggest a possible role for platelet-derived growth factor or related compounds in granulosa cell differentiation in vivo.     

Dynamic response to follicle-stimulating hormone of secretion of progesterone by superfused rat ovarian granulosa cells

Granulosa cells from immature female rats, pretreated with pregnant mare's serum gonadotropin, were cultured with microcarrier beads for 24 h, and superfused with culture medium. Progesterone was transiently released following a 10-min pulse of FSH (100 ng/ml), and there was a self-priming effect of FSH. 10-min pulses of 8-bromo-adenosine 3',5'-cyclic monophosphate (8Br-cAMP) (1 mg/ml) mimicked the effects of follicle-stimulating hormone (FSH). Continuous superfusion with FSH induced biphasic secretion of progesterone, which was composed of a parabolic (the first) and a plateau (the second) phase. By contrast, the pattern of secretion induced by continuous superfusion with 8Br-cAMP was monophasic. FSH-stimulated secretion of progesterone was rapidly inhibited by the addition of 10 microM cycloheximide (CX), but secretion recovered upon removal of this inhibitor. In the second phase, the recovery of secretion was accompanied by an overshoot of the plateau value. The present results suggest that: (1) the generation of the time-related biphasic pattern of secretion cannot be interpreted by cAMP alone; (2) FSH stimulates the secretion of progesterone by a mechanism that involves newly synthesized protein.     

Identification of ovarian genes regulated by follicle-stimulating hormone (Fsh) in vitro during early secondary oocyte growth in coho salmon

Follicle-stimulating hormone (Fsh) function in fishes is poorly understood. This study aimed to reveal Fsh-regulated genes in coho salmon previtellogenic ovarian follicles in vitro. Four suppression subtractive hybridization libraries were generated with RNA isolated from Fsh-treated and control follicles or follicle cell-enriched tissue fractions. Fsh induced steroidogenesis and dynamically upregulated several genes predominantly expressed in follicle cells, including WAP domain-containing protease, connexin 34.3, clusterin (clu1, clu2), fibronectin, wilms tumor 2-like, and influenza virus NS1A-binding protein a. Genes downregulated by Fsh included connective tissue growth factor, alcohol dehydrogenase 8-like, and serine/threonine-protein kinase pim-1. This study demonstrates for the first time in fishes that Fsh influences the expression of a unique suite of ovarian genes involved in processes like cell communication, survival and differentiation, and extracellular matrix remodeling. Collectively, these findings suggest that Fsh and/or steroids induce differentiation of granulosa cells and remodeling of the follicle in preparation for onset of vitellogenesis.     

Androgen and follicle-stimulating hormone interactions in primate ovarian follicle development.

We have previously shown that androgens stimulate early stages of follicular development and that granulosal androgen receptor (AR) gene expression is positively correlated with follicular growth. The present study was aimed at elucidating potential interactions between FSH and androgens in follicular development. Study groups included eight normal cycling rhesus monkeys (five follicular and three luteal-phase), eight testosterone (T)-treated, and four FSH-treated animals. Examination of sequential ovary sections revealed selective colocalization of AR and FSH receptor (FSHR) messenger RNAs (mRNAs) in healthy, growing follicles. Moreover, individual follicles demonstrate a highly significant (P < 0.001) positive correlation between FSHR and AR mRNA levels in all study groups. Androgen treatment significantly increased granulosa cell FSHR mRNA abundance (by approximately 50-100%, depending on follicle size). FSH treatment increased granulosa AR mRNA levels only in primary follicles. The finding that T augments follicular FSHR expression suggests that androgens promote follicular growth and estrogen biosynthesis indirectly, by amplifying FSH effect, and may partially explain the enhanced responsiveness to gonadotropin stimulation noted in women with polycystic ovary syndrome.     

Analysis of ovarian gene expression in follicle-stimulating hormone beta knockout mice.

FSH is a heterodimeric glycoprotein hormone that is produced in the gonadotroph cells of the anterior pituitary. It acts on Sertoli cells of the testis and granulosa cells of the ovary. We previously demonstrated that FSHbeta knockout female mice are infertile due to a block in folliculogenesis preceding antral stage development. To investigate aberrations of ovarian gene regulation in the absence of FSH, we analyzed the expression of several important marker genes using Northern blot and in situ hybridization techniques. Key findings are as follows: 1) Follicles of FSHbeta knockout mice develop a well organized thecal layer, which is positive for P450 17alpha-hydroxylase and LH receptor messenger RNAs (mRNAs). This indicates that theca recruitment is completed autonomously with respect to FSH. 2) Granulosa cells in FSH-deficient mice demonstrate an increase in FSH receptor mRNA, and decreases in P450 aromatase, serum/glucocorticoid-induced kinase, and inhibin/activin subunit mRNAs. These data support studies that implicate FSH signaling cascades in the expression of these genes. 3) In contrast to the thecal layer, granulosa cell populations in FSHbeta knockout mice do not accumulate LH receptor mRNA. This suggests that although the granulosa cells have a block in proliferation at the antral follicle stage in the absence of FSH, they do not initiate programs of terminal differentiation as seen in luteinizing cells of wild-type ovaries. 4) Ovaries of FSH-deficient mice demonstrate a modest decrease in cyclin D2 mRNA, without up-regulation of cell cycle inhibitor mRNAs associated with luteinization (i.e. p15, p27, and p21). Although components of the FSH null phenotype may be caused by partial cyclin D2 loss of function, these findings indicate that the mechanisms of granulosa cell cycle arrest in FSHbeta knockout mice are distinct from those of cycle withdrawal at luteinization. Underscoring the usefulness of the FSH-deficient mouse model, this study clarifies aspects of gonadotropin-dependent folliculogenesis, thecal layer development, cycle control in granulosa cells, and luteinization.