N-linked glycosylation facilitates processing and cell surface expression of rat luteinizing hormone receptor

The extracellular domain of the luteinizing hormone (LH) receptor has six potential N-linked glycosylation sites. Although previous studies have shown that mutation of the first three sites results in decreased ligand binding at the cell surface, the role of glycosylation in LH receptor processing is not understood. In the present study, we examined whether mutation of the first three sites has any affect on receptor synthesis, processing, and degradation of the mutant receptors. The data show that mutation of N77, N152, or N173 did not affect receptor synthesis, but did significantly reduce processing of the receptor precursor to the mature, cell surface form. Furthermore, defective processing was due to increased degradation of the precursor rather than increased turnover of cell surface receptors. Thus, lack of glycosylation decreases LH receptor processing and targets the receptors for degradation thereby leading to decreased cell surface expression. These results show that glycosylation of the LH receptor plays an important role in receptor processing and cell surface expression.     

Specificity of the immunocytochemical luteinizing hormone-releasing hormone receptor reaction.

Affinity-purified anti-LHRH, affinity-purified LHRH-anti-LHRH complex, as well as antisera depleted of anti-LHRH by solid phase immunoabsorption were used to test the specificity of the immunocytochemical approach for detection of receptor-bound LHRH. The solid phase immunoabsorbent was prepared by attaching LHRH to Sepharose via an RNase spacer. Purified anti-LHRH was eluted from the immunoabsorbent by acidification. Unabsorbed antisera as well as purified antibody conferred moderate immunocytochemical staining to the secretion granules of rat pituitary gonadotrophs. Staining intensity became greatly increased upon treatment of the electron microscopic sections with LHRH before immunocytochemical staining. All gonadotroph staining was abolished when absorbed antisera were used. The specificity of the immunoabsorbent was tested on mixtures of anti-LHRH and anti-ACTH17-39. On absorption of such mixtures with insolubilized LHRH, all gonadotroph staining disappeared, but the optical density indices of ACTH staining remained unaffected. It is concluded that gonadotroph secretion granules contain, in addition to an LHRH receptor reacting with LHRH in vitro, stable complexes of endogenous LHRH with receptor.     

Transgenic and knockout mouse models for the study of luteinizing hormone and luteinizing hormone receptor function

The main functions of luteinizing hormone (LH) are concerned with regulation of gonadal function, and these functions are today well delineated through previous physiological studies. However, novel information of less well-known aspects of actions of this hormone is currently emerging from studies on genetically modified mouse models, with either enhanced or suppressed LH/LH receptor (LHR) function. The novel functions of LH include its role, in specific situations, as promoter of formation and growth of gonadal and extragonadal tumors. Chronically elevated LH levels in transgenic (TG) mice can also induce responses to this hormone in extragonadal tissues. The knockout (KO) mouse for the LHR has elucidated various less well-known details in the function of LH during ontogeny and adult life. Finally, studies on LHR promoter function have revealed that the expression of this gene occurs in age, sex and tissues-specific fashion. The purpose of this brief review is to summarize some of our recent findings upon studies of TG and KO mice with altered function of LH or its receptor.     

Steroid priming of the luteinizing hormone response to luteinizing hormone releasing hormone.

Perifusion experiments were performed to study the stimulatory effects of luteinizing hormone releasing hormone (LH-RH) on the release of LH from anterior pituitary tissue. Exposure of pituitary tissue from normal male rats to LH-RH (5 ng/ml for 5 min) induced a small release of LH; in tissue from ovariectomized rats receiving no pretreatment, the release was more than three times greater and in tissue from gonadectomized male or female rats pretreated with oestradiol benzoate and progesterone, the release was six times greater than that observed in normal rats. Further exposure of pituitary tissue from gonadectomized steroid-pretreated male and female rats to LH-RH (5 ng/ml) induced an increase in the level of LH even greater than that seen after the initial exposure (priming action of LH-RH); in tissue from ovariectomized rats receiving no pretreatment, less LH was released than after the first exposure to LH-RH and in tissue from normal male rats the response was unchanged.     

Differential control of luteinizing hormone and follicle-stimulating hormone by luteinizing hormone releasing hormone in the ram.

Adult Soay rams with low concentrations of gonadotrophins in the circulation as a result of 12 weeks of exposure to long daylengths (16 h light : 8 h darkness) were given small doses (100 ng) of synthetic luteinizing hormone releasing hormone (LH-RH) into the jugular vein two, four or seven times/day for 10 days. Each injection of LH-RH induced a transitory increase in the concentration of LH and testosterone in the plasma, whereas the concentration of FSH showed little immediate change. After repeated treatment with pulses of LH-RH, the responses of LH and testosterone became slightly enhanced and the plasma concentration of FSH became permanently raised; these changes were most conspicuous in the animals receiving the most frequent injections. At the end of the study when the injections of LH-RH were stopped, the concentrations of LH and testosterone remained low but the concentrations of FSH continued to be maintained at a high level for at least 24 h.     

Effects of luteinizing hormone releasing hormone on plasma follicle-stimulating hormone and luteinizing hormone levels in the ferret.

Heterologous radioimmunoassays for FSH and LH were employed to examine the effect of synthetic LH-RH upon gonadotrophin secretion in the ferret. Intravenous injection of 4 microng LH-RH induced a surge of FSH and of LH secretion in male and in female animals. In intact and in castrated males, the rise of LH was much more marked than that of FSH. The gonadotrophin response to LH-RH was greater in anoestrous than in oestrous females; FSH secretion was not enhanced during oestrus. Ovariectomized females behaved as anoestrous females with respect to LH secretion, while FSH secretion remained unchanged. Treatment of ovariectomized females with progesterone did not alter the pattern of response to LH-RH, but oestradiol treatment depressed the reaction to match that seen in oestrous females. Repetitive injections of LH-RH induced repetitive surges of FSH and LH in anoestrous females, but only of LH during oestrus: slow i.v. infusion of LH-RH induced a sustained elevation of plasma LH levels both in oestrous and in anoestrous females; again FSH levels rose only in anoestrous females. Injection of synthetic TRH did not alter gonadotrophin secretion in corresponding groups of male or female ferrets.     

Suppression of the luteinizing hormone releasing effect of luteinizing hormone releasing hormone by arginine-vasotocin.

The concentrations of 17-oxosteroids in the spermatic venous blood of anaesthetized dogs were used as an index of LH release to assess the effects of arginine-vasotocin on the response of the canine pituitary gland to exogenous luteinizing hormone releasing hormone (LH-RH). When injected into the carotid artery, arginine-vasotocin (1.0 microgram/kg body wt) caused no significant alterations in the testicular output of 17-oxosteroids. The administration of LH-RH (5 microgram/kg body wt, a standard dose) into the carotid artery produced typical stimulation of testicular 17-oxosteroid secretion. Administration of arginine-vasotocin (0.01, 0.1 or 1.0 microgram/kg body wt) into the carotid artery 3 h before the administration of a standard dose of LH-RH inhibited the testicular secretion of 17-oxosteroids normally induced by LH-RH. However, pretreatment with arginine-vasotocin (1.0 microgram/kg body wt) did not affect the testicular response to i.v. administration of human chorionic gonadotrophin (5 i.u./kg body wt). These results indicate that in the dog, arginine-vasotocin inhibits the LH-RH-induced release of LH by acting acting directly on the anterior pituitary gland.     

Characterization of an inhibitor for luteinizing hormone receptor site binding.

An inhibitor for luteinizing hormone (LH) receptor site binding has been partially purified from aqueous extracts of ovaries from pseudo-pregnant or pregnant rats. The LH receptor binding inhibitor (LH-RBI) was heat-resistant, partially inactivated by trypsin or pronase digestion, and had a molecular weight of approximately 3800 daltons. The LH-RBI was not found in the ovary of mature non-pregnant rats or immature rats, nor was it found in testis or liver extracts. The LH-RBI strongly inhibited the in vitro binding of 125I-labeled ovine LH to ovarian LH receptors but did not inhibit 125I-labeled ovine prolactin binding to ovarian PRL receptors.     

Effect on luteinizing hormone secretion of GABA receptor modulation in the medial preoptic area at the time of proestrous luteinizing hormone surge.

Using a bilateral medial preoptic area (MPOA) infusion system in conscious female rats we have investigated the role of the GABA system in this area on the proestrous luteinizing hormone (LH) surge. Fifteen-minute blood samples for LH estimation were taken throughout the afternoon of proestrus from female rats exhibiting 4-day oestrous cycles and implanted at least 2 weeks prior with cerebral guide cannulae. Between 15:00 and 17:00 h rats received an infusion (1 microliter/30 min) of artificial cerebrospinal fluid (n = 7), 10 microM GABA (n = 6) or 10 microM bicuculline methiodide (BMI, n = 6). Animals infused with GABA failed to exhibit an LH surge, while BMI-treated animals displayed an LH surge which was not significantly different to controls. These data show that on the afternoon of proestrus, there are no tonic modulatory actions of the GABA system, acting through the GABAA receptor, on neural elements controlling the LH surge in the MPOA. If, however, GABA levels are elevated in the MPOA at this time then the LH surge is blocked. In conjunction with data from correlative studies showing a decrease in endogenous GABA release prior to the LH surge, we suggest that this fall in activity is an essential component of the LH surge mechanism.     

Activation of the luteinizing hormone receptor in the extracellular domain

Glycoprotein hormone receptors have ligand-binding ectodomains consisting of leucine-rich repeats, followed by a conserved cysteine-rich hinge region to the seven transmembrane (TM) region. Based on constitutively active mutations at Ser-281 in the hinge region of the thyroid-stimulating hormone receptor, we mutated the conserved serine in the luteinizing hormone (LH) receptor (S277I) and observed increased basal cAMP production and ligand affinity by mutant receptor. Conversion of Ser-277 to all natural amino acids led to varying degrees of receptor activation. Hydropathy index analysis indicated that substitution of neutral serine with selective nonpolar hydrophobic residues (Leu>Val>Met>Ile) confers constitutive receptor activation. Furthermore, mutation of the angular proline near Ser-273 to flexible Gly also led to receptor activation. The findings suggest the ectodomain of LH receptor constrains the TM region. Point mutations in the hinge region or ligand binding could cause conformational changes in the TM region that result in Gs activation.     

Regulation of follicle-stimulating and luteinizing hormone receptor signaling by

Follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) belong to the super-family of G protein-coupled receptors (GPCR); GPCRs are negatively regulated by RGS ("regulators of G protein signaling") proteins. In this study we evaluated the effects of RGS3 and RGS10 on FSHR and LHR ligand binding and effector coupling. FSHR and LHR ligand binding were unchanged in the presence of RGS3 or RGS10. However, signaling by FSHR and LHR was diminished by RGS3 but not by RGS10. This constitutes the first demonstration of an interaction between RGS proteins and LH and FSH signaling pathways and identifies a mechanism for negative regulation of RGS3 on FSHR and LHR signaling.     

Ovarian follicular development in the rat: hormone receptor regulation by estradiol, follicle stimulating hormone and luteinizing hormone.

The effects of estradiol, FSH and LH on ovarian follicular development and granulosa cell differentiation were examined in the immature rat hypophysectomized on day 24 of age. Administration of estradiol to hypophysectomized rats for 4 days stimulated the growth of large preantral follicles with a concomitant 1.5-fold increase in FSH receptor content and a 4-fold decrease in LH receptor content in the granulosa cells. When highly purified hFSH was administered alone, receptor content for FSH increased progressively for 4 days while receptor for LH remained essentially unchanged. However, when rats were pretreated with estradiol, the response of follicles to FSH was markedly enhanced as indicated by the appearance of large, antral follicles and elevated receptor content for both FSH and LH. Receptor content for FSH increased markedly in response to hFSH following only one day of estradiol pretreatment, while receptor content for LH increased most rapidly in response to hFSH after 3 days of estradiol pretreatment. LH administered to rats possessing large preovulatory follicles caused luteinization of granulosa cells and a marked decline in receptor content for both gonadotropins within 24 h. Receptor content remained low even 48 h after LH administration when granulosa cells were fully luteinized. These results indicated that follicular development and granulosa cell differentiation are dependent on steroid-protein hormone regulation of hormone specific receptors.     

Developmental changes in levels of luteinizing hormone receptor and androgen receptor in rat Leydig cells.

To further assess the hormonal response capabilities of Leydig cell progenitors (PLC) from 21-day-old rats, their levels of LH and androgen receptors (LH-R and AR) were measured and compared to those of isolated immature (ILC) and adult Leydig cells (ALC) from 35- and 90-day-old rats, respectively. Levels of LH receptor were estimated by Scatchard analysis of binding to [125I]hCG, and levels of LH receptor mRNA were determined by Northern blot analysis using a rat LH receptor antisense RNA probe. The numbers of LH receptors per cell measured by the binding study were 2,623 +/- 1,110 in PLC, 9,024 +/- 1,992 in ILC, and 39,896 +/- 15,234 in ALC (mean +/- SEM of four replicate experiments; ALC significantly greater than either PLC or ILC at P less than 0.05). The Northern blotting revealed three major bands [6.7, 2.6, and 2.3 kilobases (kb)] that were present in Leydig cells at all three ages and were not detected in HepG2 cells. When the steady state levels of the predominant 6.7-kb species were normalized to actin mRNA, PLC were 6.3-fold lower than ILC and 1.7-fold lower than ALC (n = 3 replicate isolations of poly(A) RNA). The 2.6- and 2.3-kb species exhibited similar trends. Levels of AR were estimated by immunoblotting using a polyclonal antibody against a synthetic peptide of the receptor (residues 14-32) that detected a 110-kilodalton AR protein. Levels of AR mRNA were estimated by Northern blot analysis, using a rat AR antisense RNA probe that detected a single 10-kb AR mRNA. The relative levels of AR protein were 1.0, 1.5, and 0.5 in PLC, ILC, and ALC, respectively (n = 3). Similar trends were observed for AR mRNA (n = 3). The observation that both LH and AR levels were lower in PLC compared to ILC is consistent with the hypothesis that the former are progenitors of Leydig cells.     

On the structure of the luteinizing hormone/chorionic gonadotropin receptor

In this review we have tried to argue that the evidence indicating that the LH/CG receptor is composed of a single polypeptide is stronger than the evidence indicating that the LH/CG receptor is a more complex structure composed of several subunits. Clearly, however, this issue has not been resolved and probably will not be resolved by performing additional experiments similar to those summarized here. It is our opinion that this issue will be resolved only by 1) reconstitution experiments in which the ability of the purified LH/CG receptor to bind hCG and activate adenylyl cyclase activity is tested; and/or 2) isolation and expression of a full length complementary DNA (cDNA) for the LH/CG receptor and the demonstration of hCG binding and adenylyl cyclase activation by the expressed receptor. Similar experiments will also clarify the proposed structures for the FSH and TSH receptors. As the second decade of work on the LH/CG receptor draws to an end it appears that these experiments are now possible, and hopefully a resolution of the existing controversy will be forthcoming in the near future.