Hormonal modulation of structural alteration of rat ovarian luteinizing/human chorionic gonadotropin receptors

The structure-stabilizing effect of homologous and heterogeneous desensitization and albumin on rat ovarian LH/hCG receptors was analyzed by thermal perturbation technique. HCG-induced down-regulation shifted the heat inactivation profile of hCG-binding sites to a temperature lower by about 7 degrees C (T50 values). In heterogeneous desensitization, which also involves uncoupling of receptors from adenylyl cyclase system, only follicle stimulating hormone (FSH) changed the stability of ovarian LH/hCG receptors. Stimulation of other hormonal receptors, which belong to the family of membrane spanning G protein-linked receptors, i.e. beta-adrenergic, glucagon, serotonin and prostaglandin E (PGE) had no effect on the stability of the LH/hCG receptor. Reduction of the stability of the LH/hCG receptor by about 3 degrees C after PGF2alpha injection to luteinized rats may be connected with specific process of luteolysis. On the other hand, albumin had a stabilizing effect on the receptor. The receptor destabilizing action of oleic acid incorporated into ovarian membranes along with calcium stimulation of endogenous phospholipase A (PLA) activity and reversal of these effects when BSA was used as fatty acid scavenger, may indicate that free fatty acids are responsible for the thermal instability of hCG-binding sites. Fluorescence quenching studies indicated that extraction of free fatty acids by albumin elevated the accessibility of fluorophores for acrylamide, and suggest that modificated lipid-protein interactions may affect the stability of the LH/hCG receptor structure.     

Targeted disruption of luteinizing hormone/human chorionic gonadotropin receptor gene

LH/hCG receptors were disrupted by gene targeting in embryonic stem cells. The disruption resulted in infertility in both sexes. The gonads contained no receptor mRNA or receptor protein. Serum LH levels were greatly elevated, and FSH levels were moderately elevated in both sexes; estradiol and progesterone levels decreased but were not totally suppressed in females; testosterone levels were dramatically decreased and estradiol levels moderately elevated in males. The external and internal genitalia were grossly underdeveloped in both sexes. Abnormalities included ambiguous vaginal opening, abdominal testes, micropenis, dramatically decreased weights of the gonads and reproductive tract, arrested follicular growth beyond antral stage, disarray of seminiferous tubules, diminished number and hypotrophy of Leydig cells, and spermatogenic arrest beyond the round spermatid stage. LH/hCG receptor gene disruption had no effect on FSH receptor mRNA levels in ovaries and testes, progesterone receptor (PR) levels in ovaries and androgen receptor (AR) levels in testes. However, it caused a dramatic decrease in StAR and estrogen receptor-alpha (ERalpha) mRNA levels and an increase in ERbeta mRNA levels in both ovaries and testes. Estradiol and progesterone replacement therapy in females and testosterone replacement in males, to determine whether phenotype and biochemical changes were a consequence of decreased gonadal steroid levels or due to a loss of LH signaling, revealed complete restoration of some and partial restoration of others. Nevertheless, the animals remained infertile. It is anticipated that the LH receptor knockout animals will increase our current understanding of gonadal and nongonadal actions of LH and hCG.     

Biological actions of monoclonal luteinizing hormone/human chorionic gonadotropin receptor antibodies.

Several recent studies have elucidated the structure of the mammalian LH/hCG receptor; as reported in the present work, we have developed a series of monoclonal antibodies (mAbs) against the rat ovarian LH/hCG receptor using highly purified receptor as immunogen and by screening hybridomas with purified LH/hCG receptors. The mAbs were able to specifically immunoprecipitate LH/hCG receptors from solubilized preparations of rat ovarian membranes as well as from partially purified preparations. Western blotting with mAb P1B4 detected a probable receptor dimer and a receptor fragment in rat and porcine ovarian tissue but not in other tissues. This mAb also partially inhibited hCG binding to rat and porcine ovarian tissues. The receptor mAbs were able to inhibit hCG-induced progesterone synthesis in cultured human and porcine granulosa cells without affecting cAMP- and FSH-induced progesterone synthesis. The mAb P1B4 was used to demonstrate that the majority of ovarian receptors are internalized after hCG treatment and that in pseudopregnant rats receptors are present in the rough endoplasmic reticulum and in microvesicles. Bovine corpus luteal cells also contained P1B4 binding sites, as detected by immunohistochemical technique. Taken together, these results suggest that the mAbs are specific for the LH/hCG receptor, mAb P1B4 recognizes an epitope that is highly conserved among mammals, and this epitope is probably in the extracellular domain.     

Specificity of heterotrimeric G protein regulation by human chorionic gonadotropin and low-molecular agonist of luteinizing hormone receptor

Luteinizing hormone (LH) and its homologue, human chorionic gonadotropin (hCG), are very important regulators of the reproductive system. These hormones stimulate various types of G proteins—primarily, G_s and G_q proteins—by binding to the specific LH-hCG receptor, which leads to the activation of adenylate cyclase (AC) and phospholipase C, respectively. It has been suggested that many side effects of LH and hCG are associated with low selectivity of their effect on G proteins. Low-molecular agonists of LH-hCG receptor developed on the basis of thienopyrimidine derivatives do not cause these side effects, and differences in the interaction with G proteins may be ones of the cause for this. To test this, a comparative study of the effect of hCG and synthesized by us thienopyrimidine derivative, 5-amino-N-tert-butyl-2-(methylsulfanyl)-4-(3-(nicotinamido)phenyl)thieno[2,3-d]pyrimidine-6-carboxamide (TP03) on the AC activity and GTP binding of G proteins in plasma membranes isolated from the rat ovaries and testes was performed. Cholera toxin (CT) and pertussis toxin (PT) were used to selectively switch off the signal transduction via G_s and G_i/o proteins, the peptide corresponding to the C-terminal segment 349–359 of the Gα_q subunit was used to suppress G_q-dependent cascades. It was shown that treatment of ovarian and testicular membranes with CT resulted in suppression of TP03 and hCG stimulatory effects on the AC activity, but in different ways influenced the GTP binding stimulation: it completely blocked the effect of 10^–6 M TP03 and reduced by 45–46% the effect of hCG (10^–8 M). Preincubation of membranes with the peptide 349–359 reduced the hCG stimulatory effect on GTP binding by 34 (ovaries) and 45% (testes), but did not affect the corresponding effect of 10^–6 M TP03. Preincubation with the peptide 349–359 also reduced the GTP stimulatory effect of 10^–4 M TP03, but to a small extent. The obtained data indicate that, in contrast to hCG, the targets of which in the ovaries and testes are G_s and G_q proteins, the action of TP03 is realized mainly via G_s proteins. Only at a concentration that exceeds EC₅₀ by two orders TP03 is capable to relatively weakly activate G_q proteins. The PT treatment of the membranes did not affect the effects of TP03 and hCG, which indicates the lack of their effective interaction with G_i/o proteins. Thus, the selectivity of activation of G_s-dependent cascades responsible for the synthesis and production of steroid hormones is a significant advantage of low-molecular agonists of LH-hCG receptor over gonadotropins.     

A model system for the biochemical study of luteinizing hormone/chorionic gonadotropin receptor synthesis

A model system for the biochemical study of LH/CG receptor synthesis has been developed. Culture conditions for porcine granulosa cells were adapted that maximized the selective induction of LH/CG receptors by cAMP-inducing stimuli with an elimination of background LH/CG receptor appearance. It was found that the addition of FSH (1.5 μg/ml) or cholera toxin (10 ng/ml) 1 day after plating resulted in optimal induction of the LH/CG receptor (20–60 pg [¹²⁵I]CG bound/μg DNA 72 h after addition) with virtually no LH/CG receptor appearance in the absence of added stimuli. Later additions of FSH or cholera toxin required insulin (1.0 μg/ml) which alone caused background LH/CG receptor appearance in the absence of any additional stimuli. Furthermore, insulin increased the general rate of cellular protein synthesis, whereas FSH or cholera toxin each decreased it. Thus, the use of FSH or cholera toxin, without insulin, may enable one to detect the synthesis of the LH/CG receptor by metabolic labeling techniques where background protein synthesis has been lowered.     

Induction of ovarian follicular cysts in the pregnant rat by human chorionic gonadotropin.

Prolonged stimulation by human chorionic gonadotropin (hCG) induces ovarian follicular cysts in progesterone-synchronized immature rats [Bogovich, Endocrinology 1989; 124:1646-1653]. To determine if unabated stimulation by hCG has a similar effect on follicular development in adult ovaries, pregnant rats were given either 0 (control), 1, or 3 IU hCG twice daily for 9 days beginning on Day 13 of pregnancy. By Day 22 of pregnancy, rats treated with 1 IU hCG possessed large antral follicles at least 1 mm in diameter: approximately 33% larger than the diameters of preovulatory follicles observed in control rats (0 IU hCG). In contrast, rats treated with 3 IU hCG displayed ovarian follicular cysts up to 5 mm in diameter, with well-developed thecae and just a remnant of granulosa cells. Progesterone, androstenedione, and estradiol accumulation was greater in follicular incubates from hCG-treated rats than in incubates from control rats. Progesterone increased in response to cAMP in incubates from all treatment groups on all days tested. Androstenedione increased in response to cAMP on Day 22 of pregnancy for follicles from control animals, on all days tested for follicles from rats treated with 1 IU hCG, and on Days 15-19 for follicles from rats treated with 3 IU hCG. Androstenedione production in the presence of 300 ng of exogenous testosterone was significantly greater in follicular incubates from animals treated with 1 and 3 IU hCG than incubates from control animals on Days 19-22 of pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activin A and gonadotropin regulation of follicle-stimulating hormone and luteinizing hormone receptor messenger RNA in avian granulosa cells.

Activin A regulation of the expression of mRNA for the LH receptor, FSH receptor, and the inhibin alpha subunit as well as the effect of activin A on the secretion of progesterone were investigated in chicken granulosa cell cultures. Granulosa layers were isolated from the F(1) and F(3) + F(4) follicles from five hens, pooled according to size, dispersed, and cultured for 48 h. In experiment 1 (n = 3 replications), granulosa cells were cultured with or without highly purified ovine (o) FSH at 50 ng/ml and in the presence of 0, 10, or 50 ng/ml of recombinant chicken activin A. Experiment 2 (n = 4 replications) followed the same protocol as experiment 1, except that oFSH was replaced with oLH. Results from these experiments showed that addition of activin A to the granulosa cell cultures had no effect on the expression of mRNA for the inhibin alpha subunit or the FSH receptor, but it did affect the expression of mRNA for the LH receptor. Treatment of F(3) + F(4) granulosa cells with LH stimulated the expression of mRNA for the LH receptor; however, when LH was combined with either dose of activin A, this induction was prevented. The highest dose of activin A with or without LH resulted in decreased expression of the LH receptor compared to the untreated controls in the F(3) + F(4) cell cultures. Progesterone secretion by the granulosa cells from both follicle sizes was not altered by activin A. In experiment 3 (n = 3 replications), the effect of activin A on the growth of granulosa cells was examined with the following treatments: 0, 10, or 50 ng/ml of activin A; 50 ng/ml of either oLH or oFSH; and oLH or oFSH combined with 10 ng/ml of activin A. The highest dose of activin reduced the rate of granulosa cell proliferation in both follicle types. Growth of F(1) and F(3) + F(4) granulosa cells was stimulated by the addition of either gonadotropin, and the presence of 10 ng/ml of activin A with either gonadotropin did not alter this proliferation, except for the LH-treated F(3) + F(4) granulosa cells, in which the increase in proliferation was prevented. The results suggest that activin A could act as a local factor that regulates follicular maturation by preventing excessive or untimely LH receptor expression.     

Effects of hyper- and hypoprolactinemia on gonadotropin secretion, rat testicular luteinizing hormone/human chorionic gonadotropin receptors and testosterone production by isolated Leydig cells

The effect of prolactin (Prl) on gonadotropin secretion, testicular luteinizing hormone (LH)/human chorionic gonadotropin (hCG) receptors, and testosterone (T) production by isolated Leydig cells has been studied in 60-day-old rats treated for 4 days, 4 and 8 weeks with sulpiride (SLP), a dopaminergic antagonist, or for 4 days and 4 weeks with bromocriptine (CB), a dopaminergic agonist. Plasma Prl concentrations were significantly greater in the SLP groups (204 +/- 6 ng/ml) and lower in the CB groups (3.0 +/- 0.2 ng/ml) than those measured in the control groups (54 +/- 6 ng/ml). The plasma concentrations of gonadotropin were not affected by a 4-day treatment with SLP or CB, nor were they after a 4-week treatment with CB. However, the hyperprolactinemia induced by an 8-week treatment with SLP was associated with a reduced secretion of gonadotropin (LH, 16 +/- 4 vs. 35 +/- 6 ng/ml; FSH, 166 +/- 12 vs. 307 +/- 14 ng/ml). In SLP-induced hyperprolactinemia, a 30% increase in the density of the LH/hCG testicular binding sites was observed (178 +/- 12 fmol/mg protein), whereas a 60% decrease was measured in hypoprolactinemia (55 +/- 5 vs. control 133 +/- 5 fmol/mg protein). Plasma T levels were increased in 4-day and 4-week hyperprolactinemic animals (4.3 +/- 0.4 and 3.9 +/- 0.4 ng/ml, respectively), but returned to normal levels in the 8-week group (3.0 +/- 0.5 vs. C: 2.3 +/- 0.2 ng/ml). No T modifications were observed in hypoprolactinemic animals. Two distinct populations of Leydig cells (I and II) were obtained by centrifugation of dispersed testicular cells on a 0-45% continuous Metrizamide gradient. Both possess LH/hCG binding sites. However, the T production from Leydig cells of population II increased in the presence of hCG, whereas that of cell population I which also contain immature germinal cells did not respond. The basal and stimulated T secretions from cell populations I and II obtained from CB-treated animals were similar to controls, whereas from 4 days to 8 weeks of hyperprolactinemia, basal and hCG induced T productions from cell population II decreased progressively. These data show that hyperprolactinemia causes, in a time-dependent manner, a trophic effect on the density of LH/hCG testicular receptors; reduces basal and hCG-stimulated T production from isolated Leydig cells type II; and results in an elevated plasma T concentration which decreases with time. The latter suggests a slower T catabolism and/or an impaired peripheral conversion of T into 5 alpha-dihydrotestosterone (DHT). Although hypoprolactinemia is associated with a marked reduction in testicular LH receptors, it does not affect T production.     

Palmitoylation of the luteinizing hormone/human chorionic gonadotropin receptor regulates receptor interaction with the arrestin-mediated internalization pathway.

The luteinizing hormone/human chorionic gonadotropin receptor (LH/hCGR) undergoes palmitoylation at cysteine residues 621 and 622 located in the carboxyl terminal tail of the receptor. This study examined the biological function of palmitoylation with respect to its effect on receptor internalization. Coexpression of wild-type (WT) or C621/622G mutant receptors with arrestin-2 increased receptor internalization in 293T cells. Furthermore, measurements of rate enhancement upon overexpression of arrestin indicate that the palmitoylation deficient mutant receptor is more prone to utilizing the arrestin mediated internalization pathway than the WT receptor. Coexpression of G-protein-coupled receptor kinase 4 (GRK4) with wild type receptor resulted in an increase in internalization, while coexpression with the mutant receptor did not result in further enhancement of internalization. Additionally, 293T cells expressing mutant receptor were responsive to hCG with respect to production of inositol phosphates. Taken together, these results suggest that the palmitoylation state of the receptor governs internalization by regulating the accessibility of the receptor to the arrestin-mediated internalization pathway.     

Hormone interactions to Leu-rich repeats in the gonadotropin receptors. I. Analysis of Leu-rich repeats of human luteinizing hormone/chorionic gonadotropin receptor and follicle-stimulating hormone receptor

The luteinizing hormone receptor (LHR) and follicle-stimulating hormone receptor (FSHR) have an approximately 350-amino acid-long, N-terminal extracellular exodomain. This exodomain binds hormone with high affinity and specificity and contains eight to nine putative Leu-rich repeat (LRR) sequences. LRRs are known to assume the horseshoe structure in ribonuclease inhibitors, and the inner lining of the horseshoe consists of the beta-stranded Leu/Ile-X-Leu/Ile motif. In the case of ribonuclease inhibitors, these beta strands interact with ribonuclease. However, it is unclear whether the putative LRRs of LHR and FSHR play any role in the structure and function. In this work, the beta-stranded Leu/Ile residues in all LRRs of the human LHR and FSHR were Ala-scanned and characterized. In addition, the 23 residues around LRR2 of LHR were Ala-scanned. The results show that beta-stranded Leu and Ile residues in all LRRs are important but not equally. These Leu/Ile-X-Leu/Ile motifs appear to form the hydrophobic core of the LRR loop, crucial for the LRR structure. Interestingly, the hot spots are primarily in the upstream and downstream LRRs of the LHR exodomain, whereas important LRRs spread throughout the FSHR exodomain. This may explain the distinct hormone specificity despite the structural similarity of the two receptors.     

Permeability of rat ovarian follicles to LH during development and luteinization

A 'double isotope' technique has been used to describe the temporal relationship between plasma and follicular concentrations of LH after injection of 51Cr and 125I-rat LH into immature rats. Radiolabelled LH was detectable in all follicles 1 min after injection. Concentrations in small antral and large preovulatory follicles were not significantly different at any time and reached a maximum of 34.2 +/- 3.0% of plasma concentrations at 40 min. Concentrations of LH in preovulatory follicles exposed to an ovulatory dose of hCG 4 h previously were significantly greater (P less than 0.05) than those in small antral and preovulatory follicles at all times, and reached a maximum of 46.2 +/- 1.7% of plasma concentrations after 1 h. Polyacrylamide gel electrophoresis and immunoprecipitation with an antibody specific for rat LH indicated that radioactivity in plasma and follicular fluid represented radio-iodinated LH. Steroidogenic activities, light microscopy and measurements of follicular volume of each class of follicle confirmed that small antral, preovulatory follicles and preovulatory follicles exposed to an ovulatory dose of hCG in vivo could be isolated specifically. Based on these findings it is possible to calculate that, during an endogenous pulse of LH secretion, follicular concentrations of LH never exceed 20% of peak plasma concentrations. Pronounced increases in functional activities during antral growth were not correlated with increased follicular permeability. Only after acute exposure to an ovulatory dose of hCG in vivo was permeability significantly increased. We conclude that entry of LH into antral follicles is restricted and that exposure to an ovulatory dose of hCG results in greater amounts of LH entering preovulatory follicles.