Studies on the synergistic effect of androgen on the stimulation of progestin secretion by FSH in cultured rat granulosa cells: a search for the mechanism of action.

Cultures of granulosa cells from immature hypophysectomized DES-treated rats were unable to maintain progestin production of more than 48 h in medium without hormone supplementation or in the presence of FSH only. Production of progestin (20alpha-dihydroprogesterone, as measured by radioimmunoassay) remained unimpaired in the presence of androstenedione (Ad) and was markedly increased in the presence of both Ad and FSH. The combined treatment with FSH and Ad during the first 48 h of culture brought about persistent changes in the cultured cells, since progestin accumulation did not decline upon subsequent removal of these hormones during days 3 and 4 of culture. Dibutyryl cyclic AMP (DBC) was able to mimic the changes in steroidogenic capability induced by the combined action of FSH and Ad. The extent of [125I]-FSH binding, FSH-stimulable cAMP accumulation and cyclic 3',5'-nucleotide phosphodiesterase activity were not affected by addition of Ad to the culture medium. Ad synergized with DBC in the stimulation of progestin accumulation in granulosa cell cultures. It is suggested that androgen acts at a step in the regulation of progestin biosynthesis distal to cAMP production.     

On the synergistic action of androgen and FSH on progestin secretion by cultured rat granulosa cells: Cellular and mitochondrial cholesterol metabolism

The effect of FSH and androgen on the conversion of cholesterol into progesterone by cultured rat granulosa cells (GC) was studied in intact cells or mitochondrial preparations. Culture of GC from immature hypophysectomized diethylstilbestrol-treated rats for 48 h in the presence of ovine FSH (5 μg/ml) alone, or FSH + testosterone (Te; 0.5 Mg/ml) caused a slight increase in the activity of the mitochondrial marker enzyme succinic dehydrogenase, while Te had no effect. Culture with the hormones for 48 h had no significant effect on the levels of free and esterified cellular cholesterol. GC monolayers after 48 h with or without FSH and Te converted [³H]cholesterol into 4 major metabolites, 3 of which were secreted into the medium and, in thin-layer Chromatographie behavior, resembled pregnenolone, progesterone and 20α-dihydroprogesterone. The total amount of the 3 C-21 steroids was higher (p < 0.01) in FSH- or Te-treated than in control cells, and combined treatment had a synergistic effect. The uptake of labeled cholesterol (4–10%) was significantly higher (p < 0.01) in cells pretreated with FSH or Te, whereas a combined FSH and Te treatment had an additive effect.Mitochondria isolated from GC monolayers took up cholesterol in a temperature-dependent fashion, but this uptake was not affected by hormonal pretreatment. In the presence of cyanoketone, the mitochondrial fractions actively converted cholesterol into pregnenolone. This activity was enhanced by FSH or Te (p < 0.01), and further enhancement was observed with FSH + Te; the combined effect appeared to be more than additive (p = 0.05).The results suggest that both FSH and Te enhance the activity of cholesterol side-chain cleavage, but do not affect the transport of cholesterol into the mitochondria. A possible hormonal effect on a pre-mitochondrial step is discussed.     

Modulation of prolactin receptors in cultured rat granulosa cells by FSH,LH and GnRH

The hormonal modulation of prolactin (PRL)-binding capacity of rat granulosa cells was studied. Granulosa cells obtained from immature, hypophysectomized, estrogen-treated rats were cultured for 2 days in a serum-free medium in the presence of various hormones. FSH treatment in vitro stimulated granulosa cell PRL-binding capacity by ~ 4–6-fold in a dose-dependent manner. Concomitant treatment with 10^?8 M GnRH inhibited the FSH-induced increase in PRL-binding capacity by 64%. In contrast, the inhibitory effect of GnRH was blocked by concomitant treatment with 10^?6 M of a GnRH antagonist, [D-pGlu¹, D-Phe², D-Trp^3,6]GnRH. PRL-binding capacity was also increased (~2-fold) by in vitro treatment with cholera toxin (10 μg/ml). In granulosa cells pre-treated with FSH in vitro for 2 days, hCG treatment for 2 additional days stimulated PRL-binding capacity in a dose-dependent manner (~ 2-fold). Likewise, treatment with LH (100 ng/ml) also stimulated PRL-binding capacity by ~ 2-fold. These in vitro studies demonstrated that gonadotropins (FSH, LH and hCG) directly enhanced PRL binding by granulosa cells, whereas GnRH inhibited FSH action.     

Luteinizing hormone acts directly at granulosa cells to stimulate periovulatory processes: modulation of luteinizing hormone effects by prostaglandins

The midcycle surge of luteinizing hormone (LH) triggers events within the primate periovulatory follicle that culminate in follicle rupture and luteinization of the follicle wall; these events include the shift from primarily estrogen to primarily progesterone production, vascularization of the granulosa cell layer, and expression of matrix metalloproteinases and their inhibitors (MMPs and TIMPs) thought to be necessary for follicle rupture. However, it is unknown if LH acts directly at granulosa cells to regulate these important periovulatory processes. The ovulatory LH surge also stimulates the production of prostaglandins (PGs) by the follicle just before follicle rupture, suggesting that LH may have both PG-dependent and PG-independent actions. To address these questions, gonadotropins were administered to adult female rhesus monkeys to stimulate the development of multiple, large preovulatory follicles. Granulosa cells were aspirated and maintained in vitro for up to 48 h in serum-free, chemically defined medium. Granulosa cells were cultured with LH alone or in combination with PGs to determine if these hormones act directly at granulosa cells to induce the production of factors implicated in periovulatory processes. LH treatment increased media progesterone (p<0.05) and vascular endothelial growth factor (VEGF; p<0.05) levels as well as stimulating expression of mRNAs for MMP-1 (p=0.05), MMP-9 (p<0.05), and TIMP-1 (p<0.05), similar to the effects of an ovulatory dose of gonadotropin in vivo. PGE2 alone elevated media progesterone levels but decreased LH stimulation of MMP-1 mRNA (p<0.05). PGF2α reduced LH-stimulated TIMP-1 mRNA (p<0.05) levels. These studies suggest a direct action of LH on granulosa cells to stimulate the processes involved in tissue remodeling and neovascularization, i.e., MMPs/TIMPs and angiogenic factors, as well as steroidogenesis. LH-stimulated PGs may have a regulatory role to modulate some effects of the LH surge, such as MMP/TIMP expression.     

Effect of pregnant mare serum gonadotropin on the induction and degradation of FSH and LH receptors in the granulosa cell of the immature rat

The relative induction of FSH and LH receptors in the granulosa cells of immature rat ovary by pregnant mare serum gonadotropin (PMSG) has been studied. A single injection of PMSG (15 IU) brought about a 3- and 12-fold increase in FSH and LH receptor concentration, respectively, in the granulosa cells. Maximal concentration was reached by 72 h but the receptor levels showed a sharp decline during the next 24-48 h. The kinetic properties of the newly formed FSH receptors were indistinguishable from the pre-existing ones. The induced FSH receptors were functional as demonstrated by an increase in the in vitro responsiveness of the cells to exogenous FSH in terms of progesterone production. Treatment of immature rats with cyanoketone, an inhibitor of delta 5,3 beta-hydroxysteroid dehydrogenase, prior to PMSG injection effectively reduced the PMSG-stimulated increase in the serum estradiol, uterine weight and LH receptors but had no effect on the FSH receptor induction. The ability of PMSG to induce gonadotropin receptors can be arrested at any given time by injecting its antibody, thereby suggesting a continuous need for the hormonal inducer. Estrogen in the absence of the primary inducer was unable to maintain the induced LH and FSH receptor concentration. Inhibition of prostaglandin synthesis using indomethacin aslo had no effect on either the induction or degradation of gonadotropin receptors. Administration of PMSG antiserum, 48 h after PMSG injection, brought about a rapid decline in the induced receptors over the next 24 h, with a rate constant and t 1/2 of 0.078 h-1 and 8.9 h for FSH receptors and 0.086 h-1 and 8.0 h for the LH receptors, respectively.     

Influence of follicular atresia on LH-induced cAMP and steroid synthesis by bovine thecae interna

The aim of the present study was to examine the interrelationships between the luteinizing hormone (LH) receptor, the LH-induced changes in adenosine cyclic 3', 5'-monophosphate (cAMP) and steroid synthesis in theca interna tissue of large antral follicles (greater than or equal to 8 mm diameter) from oestrous cycling cows. Three distinct types of theca interna were identified (types I, II and III), all of which contained an LH receptor: type I was capable of secreting increased amounts of cAMP dehydroepiandrosterone, androstenedione and testosterone when exposed to LH; type II was capable of secreting increased amounts of cAMP and progesterone but not the androgens when exposed to LH; type III was incapable of cAMP or steroid synthesis when exposed to LH. Follicles with type I thecae contained: a full complement of granulosa cells; high intrafollicular concentrations of oestradiol; and granulosa cells with a high capacity to metabolise testosterone to oestradiol. These follicles were considered to be non-atretic structures. Follicles with types III thecae contained: fewer granulosa cells; low intrafollicular concentrations of oestradiol; and granulosa cells with a low capacity to metabolise testosterone to oestradiol. Moreover, follicles with type III thecae contained the highest concentrations of progesterone and the lowest concentrations of androstenedione and testosterone. These follicles were considered to be severely atretic structures. Follicles with type II thecae contained granulosa cell populations and progesterone, and androgen concentrations which were intermediate between those with thecae of types I and III. These follicles were considered to be at an intermediate stage of atresia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of gonadotropin-sensitive adenylate cyclase activity in human testis: uncoupling of the receptor-cyclase complex by specific hormonal antagonist

Basal and gonadotropin stimulated adenylate cyclase activity was assessed in testicular tissues obtained from men (20-80 years). A disparity was observed in the gonadotropin responsiveness of the human testicular adenylate cyclase system to hFSH and hCG stimulation. Of the tissues analyzed, 61% were FSH responsive and 22% showed low response to hCG. Forskolin, a diterpene which activates adenylate cyclase by a receptor independent mechanism, stimulated adenylate cyclase activity in the gonadotropin unresponsive tissues. This suggests that the tissue unresponsiveness is due to an uncoupling of the catalytic subunit of the adenylate cyclase. Several functional properties of the FSH responsive human testicular adenylate cyclase were investigated. hFSH and oFSH stimulated the enzyme activity in a concentration dependent manner. However, the hormone (DG-oFSH) in which 80% of the carbohydrate residues had been removed was inactive, despite its good binding ability to the FSH receptor. hFSH stimulated adenylate cyclase activity was inhibited by DG-oFSH but not by DG-hCG (deglycosylated hCG). The data demonstrates the existence of specific FSH and LH(hCG) receptors in human testicular membranes. The FSH receptors in some tissues are coupled to adenylate cyclase. The link between the FSH receptor and adenylate cyclase may be uncoupled in the presence of the deglycosylated form of oFSH resulting in a loss of hormone response.     

Studies on pituitary follitropin. XI. Induction of hormonal antagonistic activity by chemical deglycosylation

Chemically deglycosylated ovine follitropin (DG-FSH) retained its binding ability to bovine and rat testicular membrane fractions but was completely inactive in stimulating cyclic AMP accumulation in immature rat seminiferous tubular suspensions. The native hormone induced cyclic AMP accumulation in a dose-dependent manner but no significant response was detectable at any dose of DG-FSH even in the presence of a potent phosphodiesterase inhibitor. By virtue of these different biological characteristics all preparations of DG-FSH exerted potent inhibitory action when added to the cells along with the native hormone. Complete inhibition of FSH action was observed at equivalent concentrations of the DG-FSH. It was also effective against hFSH. DG-FSH had no effect on hCG action in rat interstitial cells. The accumulation of cyclic AMP induced by a non-hormonal agent such as cholera toxin in the tubular suspensions was unaffected by the concomitant presence of DG-FSH indicating that its antagonistic activity is mediated through its binding to specific follitropin receptors.     

Mechanism of action of luteinizing hormone and follicle-stimulating hormone on the ovary in vitro.

The mechanism of action of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) upon various cell types of the mammalian ovary is reviewed. Emphasis is placed upon in vitro studies using organ and cell culture as well as short-term incubations. FSH and LH actions upon the following ovarian functions are discussed: steroidogenesis and metabolism of the ovary as a whole and of the isolated follicle and its component cell types, the granulosa and thecal cells, as well as folliculogenesis and follicular growth, oocyte maturation, follicular rupture, and corpus luteum maintenance and steroidogenesis. The roles of gonadotropin receptors, AMP, prostaglandins, protein kinase, and protein synthesis in these LH and FSH actions are discussed. Intra-ovarian regulation of LH and FSH action is reviewed, including a discussion of the possible roles of follicular fluid inhibitors upon oocyte maturation and granulosa cell luteinization.     

Determination of different putative allosteric binding pockets at the lutropin receptor by using diverse drug-like low molecular weight ligands

The lutropin/choriogonadotrophin receptor (LHCGR) is a family A G protein-coupled receptor (GPCR) which binds the endogenous hormone-ligands at the large extracellular domain. In contrast, several drug-like low-molecular-weight ligands (LMWs) have been reported to interact allosterically within the seven transmembrane domain (7TMD) of the LHCGR. Here, we were interested to study the putative allosteric LHCGR binding region with focus on the determination of two pockets for LMW ligands. A library of compounds was screened for their ability to modify the binding of an allosteric radiolabeled LMW agonist [3H]Org 43553. Further experimental and computational studies revealed that the putative binding pocket for a newly identified allosteric enhancer (LUF5419) and a previously described allosteric inhibitor (LUF5771) are overlapping and that this site is different from the Org 43553 binding site. The present study showed that these compounds are useful tools to reveal details on different allosteric binding sites located within the 7TMD of the LHCGR.