Autoimmunity to the Follicle-Stimulating Hormone Receptor (FSHR) and Luteinizing Hormone Receptor (LHR) in Polycystic Ovarian Syndrome.

Hyperandrogenemia and ovulatory dysfunction are hallmarks of polycystic ovary syndrome (PCOS), pointing to a deranged hypothalamus-pituitary-ovarian (HPO) axis. An autoimmune etiology of PCOS is suspected in a subset of patients due to the relatively high concordance of PCOS with common autoimmune diseases. For this reason, we tested the hypothesis that natural autoantibodies (aAb) to the follicle-stimulating hormone receptor (FSHR) or luteinizing hormone receptor (LHR) are prevalent in PCOS. To this end, new luminometric assays for quantifying aAb to the FSHR (FSHR-aAb) or LHR (LHR-aAb) were developed using full-length recombinant human receptors as fusion proteins with luciferase as reporter. Prevalence of FSHR-aAb and LHR-aAb was determined in serum samples from healthy controls and PCOS patients. Steroid hormone profiles were compared between patients with and without FSHR-aAb or LHR-aAb. Signal linearity and detection ranges were characterized and both methods passed basic performance quality checks. The analysis revealed a relatively low prevalence, with 4 out of 430 samples positive for FSHR-aAb in the control versus 11 out of 550 samples in the PCOS group, i.e., 0.9% versus 2.0%, respectively. Similarly, there were only 5 samples positive for LHR-aAb in the control versus 2 samples in the PCOS group, i.e., 1.2% versus 0.4%, respectively. Samples positive for FSHR-aAb displayed steroid hormones in the typical range of PCOS patients, whereas the two samples positive for LHR-aAb showed relatively elevated free testosterone in relation to total testosterone concentrations with unclear significance. We conclude that the FSHR and LHR constitute potential autoantigens in human subjects. However, the prevalence of specific autoantibodies to these receptors is relatively low, both in control subjects and in women with PCOS. It is therefore unlikely that autoimmunity to the LHR or FSHR constitutes a frequent cause of hyperandrogenemia or ovulatory dysfunction in PCOS.

Immunohistochemical demonstration of LH/CG receptors in non-neoplastic human adrenal cortex and adrenocortical tumors.

INTRODUCTION: Numerous data indicate that luteinizing hormone and/or chorionic gonadotropin (LH/CG) exert direct actions on the adrenal cortex and are involved in the adrenal pathology. However, the immunohistochemical studies on the expression of LH/CG receptors (LH/CGR) in the human adrenal cortex and in the adrenocortical tumors are scarce. MATERIAL AND METHODS: Paraffin sections of samples of 6 human non-neoplastic adrenal cortex and 25 adrenocortical tumors were immunostained with anti-LH/CGR polyclonal antibody. RESULTS: All zones of the human non-neoplastic adrenal cortex present a positive immunoreaction with anti-LH/CGR antibody showing the strongest reaction in cell membranes. The LH/CGR immunostaining in the vast majority of hormonally non-functioning adenomas and in all hormone-secreting adenomas does not differ from the non-neoplastic adrenal cortex. In contrast to non-neoplastic adrenal cortex and benign adenomas, in adrenocortical cancers the immunostaining with anti-LH/CGR antibody behaves differently. The immunopositive material is almost totally filling the cytoplasm of the cells but the immunopositivity of cell membranes is weak or lacking. CONCLUSIONS: The data presented in our study show that the expression of LH/CGR in adrenocortical tumors is not ectopic but eutopic. The immunohistochemical examination of LH/CGR may be useful in the differentiation between benign and malignant lesions in the adrenal cortex. Moreover, the loss of membrane localization of LH/CGR in adrenocortical cancer suggests the alteration of receptors' function.

Triptorelin and cetrorelix induce immune responses and affect uterine development and expressions of genes and proteins of ESR1, LHR, and FSHR of mice.

CONTEXT: GnRH immunity can reduce the expression of pituitary GnRH levels, and cause the changes in reproductive behaviors. It is unclear whether triptorelin (TRI) and cetrorelix (CET) immunity influences uterine development and expression of follicle-stimulating hormone receptor (FSHR), luteinizing hormone receptor (LHR), and estradiol receptor 1 (ERS1) in the uterus. OBJECTIVE: The study investigated the effects of active immunity of GnRH agonist and antagonist on uterine development, microstructures, expression of hormone receptors mRNAs, and proteins in uteri. MATERIALS AND METHODS: One hundred and five mice were assigned into CET, TRI, and control groups (CG). Mice in CET-1, CET-2, and CET-3 (n = 15) were subcutaneously injected with 10, 20, and 40 µg CET antigens for seven days, respectively. Mice in TRI-1, TRI-2, and TRI-3 were injected with 10, 20, and 40 µg TRI antigens for seven days, respectively. The qPCR and Western blot were implemented to determine expressions of ESR1, LHR and FSHR mRNAs, and proteins. RESULTS: Compared with CG, the uterine weights of CET-1, CET-2, and CET-3 increased by 42.86, 62.86, and 10.00% on day 35 (p < 0.05), respectively. Uterine weights of TRI-2, TRI-3 reduced by 28.57% and 11.43% (p < 0.05), respectively. The uterine cavity in CET-1, CET-2, and CET-3 increased; the uterine wall became thick. The cytoplasm of endometrial epithelial cells (EEC) increased slightly. In TRI group, the uterine wall thinned. Uterine cavity became narrow slightly in TRI-1. Numbers of uterine glands reduced. The endometrium epithelial thickness (EET) in CET-1 and CET-2 increased by 68.21% and 79.46% (p < 0.05), respectively. EET in TRI-1 was decreased by 13.69%. Uterine wall thicknesses (UWT) in CET-1 and CET-2 were higher than CG, with the increment of 28.59% and 30.72%. UWT of TRI-1, TRI-2, and TRI-3 reduced by 29.35, 15.36, and 14.41%, respectively. Expressions of ESR1, FSHR, and LHR mRNAs in CET and TRI mice increased. ESR1 and FSHR protein levels increased in all experimental mice (p < 0.05), with a maximum of TRI-3. LHR protein levels of the CET decreased. LHR protein levels of TRI group increased, with a maximum of TRI-3 (p < 0.05). ESR1 protein level had significant negative correlations to mRNA expressions of ESR1, LHR, and FSHR. CONCLUSIONS: CET immunity promoted the uterine development, improved EET and UWT, and also promoted the expressions of ESR1 and FSHR protein levels. It lessened the LHR protein levels. TRI immunity blocked EET and UWT, inhibited uterine growth and development. The efficacy of CET immunity was more obvious than TRI.

Severe gastrointestinal dysmotility developed after treatment with gonadotropin-releasing hormone analogs.

BACKGROUND: Sporadic cases of abdominal pain and dysmotility has been described after treatment with gonadotropin-releasing hormone (GnRH) analogs. The aim of the present study was to scrutinize for patients with severe gastrointestinal complaints after treatment with GnRH analogs, to describe the expression of antibodies against progonadoliberin-2, GnRH1, GnRH receptor (GnRHR), luteinizing hormone (LH), and LH receptor in serum in these patients, and to search for possible triggers and genetic factors behind the development of this dysmotility. METHODS: Patients suffering from prolonged gastrointestinal complaints after treatment with GnRH analogs at the Department of Gastroenterology, Skåne University Hospital, were included. GnRHR and LH receptor (LHCGR) genes were exome-sequenced. Serum was analyzed by enzyme-linked immune sorbent assays for the presence of antibodies. Healthy blood donors and women treated with GnRH analogs because of in vitro fertilization (IVF) were used as controls. RESULTS: Seven patients with severe gastrointestinal complaints after GnRH treatment were identified, of whom six suffered from endometriosis. Several variants were found within the 11 exons of LHCGR. The minor allele G, at the single nucleotide polymorphism rs6755901, was detected in homozygosity in two patients (28.5%) who had developed chronic intestinal pseudo-obstruction and in 5.5% of the IVF controls. Three patients expressed IgM antibodies against progonadoliberin-2 and three against GnRH1 (42.9%) when cut off was set to a titer >97.5th percentile in blood donors. CONCLUSION: A high prevalence of endometriosis, polymorphism in the LHCGR and GnRH1 and progonadoliberin-2 antibodies in serum was found among the patients with severe dysmotility after treatment with GnRH analogs.

Autoantibodies and gastrointestinal symptoms in infertile women in relation to in vitro fertilization.

BACKGROUND: Prior reports suggest a link between gonadotropin-releasing hormone (GnRH) and gastrointestinal function. The aim of the study was to prospectively investigate women subjected to in vitro fertilization (IVF) using the GnRH analog buserelin, taking into account gastrointestinal symptoms and antibody development against buserelin, GnRH, luteinizing hormone (LH), and their receptors. METHODS: Gastrointestinal symptoms were registered by the Visual Analogue Scale for Irritable Bowel Syndrome (VAS-IBS) before and after IVF treatment, and five years later. Health-related quality of life was evaluated by the 36-item Short-Form questionnaire (SF-36). ELISA was used for antibody analyses before and after treatment. Data were compared with women from the general population. RESULTS: In total, 124 patients were investigated before and after IVF, and 62 were re-evaluated after five years. Buserelin treatment led to significant impairment of constipation (p = 0.004), nausea and vomiting (p = 0.035), psychological well-being (p = 0.000), and the intestinal symptoms' influence on daily life (p = 0.027). At 5-year follow-up, abdominal pain was worsened (p = 0.041), but psychological well-being was improved (p = 0.036), compared to prior treatment, and 15% had an observable deterioration in gastrointestinal symptoms. None developed severe dysmotility. Patients had higher prevalence of IgG antibodies against LH (p = 0.001) and its receptor (p = 0.016), and IgM antibodies against the GnRH receptor (p = 0.001) prior treatment compared with controls, but no antibody development was observed after IVF. CONCLUSION: Patients experience gastrointestinal symptoms during buserelin treatment, and abdominal pain is still increased after five years, but buserelin does not increase antibody formation against GnRH, LH or their receptors.

Quantitative ELISAs for serum soluble LHCGR and hCG-LHCGR complex: potential diagnostics in first trimester pregnancy screening for stillbirth, Down's syndrome, preterm delivery and preeclampsia.

BACKGROUND: Soluble LH/hCG receptor (sLHCGR) released from placental explants and transfected cells can be detected in sera from pregnant women. To determine whether sLHCGR has diagnostic potential, quantitative ELISAs were developed and tested to examine the correlation between pregnancy outcome and levels of serum sLHCGR and hCG-sLHCGR complex. METHODS: Anti-LHCGR poly- and monoclonal antibodies recognizing defined LHCGR epitopes, commerical anti-hCGbeta antibody, together with recombinant LHCGR and yoked hCGbeta-LHCGR standard calibrators were used to develop two ELISAs. These assays were employed to quantify serum sLHCGR and hCG-sLHCGR at first trimester human pregnancy. RESULTS: Two ELISAs were developed and validated. Unlike any known biomarker, sLHCGR and hCG-sLHCGR are unique because Down's syndrome (DS), preeclampsia and preterm delivery are linked to both low (less than or equal to 5 pmol/mL), and high (equal to or greater than 170 pmol/mL) concentrations. At these cut-off values, serum hCG-sLHCGR together with PAPP-A detected additional DS pregnancies (21%) which were negative by free hCGbeta plus PAPP-A screening procedure. Therefore, sLHCGR/hCG-sLHCGR has an additive effect on the current primary biochemical screening of aneuploid pregnancies. More than 88% of pregnancies destined to end in fetal demise (stillbirth) exhibited very low serum hCG-sLHCGR(less than or equal to 5 pmol/mL) compared to controls (median 16.15 pmol/mL, n = 390). The frequency of high hCG-sLHCGR concentrations (equal to or greater than 170 pmol/mL) in pathological pregnancies was at least 3-6-fold higher than that of the control, suggesting possible modulation of the thyrotropic effect of hCG by sLHCGR. CONCLUSIONS: Serum sLHCGR/hCG-sLHCGR together with PAPP-A, have significant potential as first trimester screening markers for predicting pathological outcomes in pregnancy.

The antibodies against the computationally designed mimic of the glycoprotein hormone receptor transmembrane domain provide insights into receptor activation and suppress the constitutively activated receptor mutants.

The exoloops of glycoprotein hormone receptors (GpHRs) transduce the signal generated by the ligand-ectodomain interactions to the transmembrane helices either through direct hormonal contact and/or by modulating the interdomain interactions between the hinge region (HinR) and the transmembrane domain (TMD). The ligand-induced conformational alterations in the HinRs and the interhelical loops of luteinizing hormone receptor/follicle stimulating hormone receptor/thyroid stimulating hormone receptor were mapped using exoloop-specific antibodies generated against a mini-TMD protein designed to mimic the native exoloop conformations that were created by joining the thyroid stimulating hormone receptor exoloops constrained through helical tethers and library-derived linkers. The antibody against the mini-TMD specifically recognized all three GpHRs and inhibited the basal and hormone-stimulated cAMP production without affecting hormone binding. Interestingly, binding of the antibody to all three receptors was abolished by prior incubation of the receptors with the respective hormones, suggesting that the exoloops are buried in the hormone-receptor complexes. The antibody also suppressed the high basal activities of gain-of-function mutations in the HinRs, exoloops, and TMDs such as those involved in precocious puberty and thyroid toxic adenomas. Using the antibody and point/deletion/chimeric receptor mutants, we demonstrate that changes in the HinR-exoloop interactions play an important role in receptor activation. Computational analysis suggests that the mini-TMD antibodies act by conformationally locking the transmembrane helices by means of restraining the exoloops and the juxta-membrane regions. Using GpHRs as a model, we describe a novel computational approach of generating soluble TMD mimics that can be used to explain the role of exoloops during receptor activation and their interplay with TMDs.

An attempt to induce "Graves' disease of the gonads" by immunizing mice with the luteinizing hormone receptor provides insight into breaking tolerance to self-antigens.

BACKGROUND: Gonadotropin receptors, unlike the thyrotropin receptor (TSHR), are not cleaved into disulfide-linked A- and B-subunits, nor do they shed A-subunits. Heavily glycosylated TSHR A-subunits initiate or amplify responses leading to stimulating TSHR-autoantibodies and Graves' hyperthyroidism. METHODS: To investigate the possibility that mice immunized with luteinizing hormone receptor (LHR) would develop functional antibodies, we constructed adenoviruses expressing the rat-LH holoreceptor (LHR-Ad) and an LHR A-subunit equivalent (LHR-289-Ad). Female BALB/c mice were immunized with high doses (10(11) particles) of LHR-Ad, LHR-289-Ad, or control (Con)-Ad. Sera were tested using LHR-expressing eukaryotic cells for antibody binding by flow cytometry and for bioactivity by measuring cyclic adenosine monophosphate (cAMP) stimulation. RESULTS: Elevated serum binding to LHR cells in some LHR-Ad and LHR-289-Ad immunized mice was not specific for LHR-expressing cells. Moreover, sera lacked bioactivity, consistent with unchanged serum estradiol and ovary histology. The difference between rat and mouse LHR-ectodomains is relatively small (3% at the amino-acid level). In contrast, despite amino-acid identity, immunization of mice with adenovirus expressing membrane-bound mouse thyroid peroxidase (TPO), but not soluble mouse TPO ectodomain, elicited strong TPO-specific antibodies. CONCLUSIONS: Our investigations provide insight into antibody responses to self-antigens. First, antibodies are induced to large self-antigens like mouse-TPO when membrane bound. Second, lesser amino acid homology between the immunogen and mouse protein (91% vs. 97% for the human-TSHR and rat-LHR, respectively) favors antibody induction. Finally, from previous studies demonstrating the immunogenicity of the highly glycosylated human TSHR A-subunit versus our present data for the nonimmunogenic less glycosylated rat LHR, we suggest that the extent of glycosylation contributes to breaking self-tolerance.

Effects of a thyroid-stimulating human monoclonal autoantibody (M22) on functional activity of LH and FSH receptors.

OBJECTIVE: The glycoprotein hormones luteinizing hormone (LH), follicle-stimulating hormone (FSH), and thyrotropin (TSH) show low-level cross-reactivity between their respective receptors (R). Patient serum autoantibodies to the thyrotropin receptor (TSHR) do not appear to cross-react with the luteinizing hormone receptor (LHR) or follicle-stimulating hormone receptor (FSHR), although the concentrations of autoantibody with which it is feasible to carry out experiments of this type are limited. Consequently, we have studied the effects of high doses of the thyroid-stimulating human monoclonal autoantibody (M22) on the LHR and FSHR. DESIGN: Chinese Hamster ovary (CHO) cells stably expressing the TSHR, LHR, and FSHR and purified M22 IgG preparations were used in the study. METHODS: CHO-TSHR, CHO-LHR, and CHO-FSHR cells were incubated with bovine TSH (0.1-25mU/mL), human recombinant chorionic gonadotropin (hCG; 0.5-10mU/mL) or human recombinant FSH (100-5000mU/mL) or with M22 IgG (0.001-5.0 microg/mL), and the extracellular cyclic AMP was measured by radioimmunoassay. RESULTS: Cyclic AMP levels increased in a dose-dependent manner after incubation of CHO-TSHR cells with TSH or M22 IgG, and on a molar basis the effects of TSH and M22 were similar. Cyclic AMP stimulation was not detectable in CHO-LHR and CHO-FSHR cells after incubation with M22 IgG, whereas incubation with hCG or FSH, respectively, caused dose-dependent cyclic AMP stimulation. On a molar basis, concentrations of M22 IgG approximately 100x those of FSH causing clear stimulation were ineffective with CHO-FSHR cells. Similarly, molar concentration of M22 IgG 20,000x those of hCG causing clear stimulation had no effect on CHO-LHR cells. CONCLUSIONS: This study shows that at relatively high concentrations, M22 IgG is unable to stimulate cyclic AMP levels in CHO-LHR or CHO-FSHR cells, suggesting that TSHR autoantibodies have greater specificity for the TSHR than TSH itself.

An alternative use of basic pGEX vectors for producing both N- and C-terminal fusion proteins for production and affinity purification of antibodies.

Glutathione S-transferase (GST) fusion proteins are widely used in protein production for pure immunogens, protein-protein, and DNA-protein interaction studies. Using basic pGEX vectors, foreign DNA is introduced to the C-terminus of the GST gene and the produced fusion proteins are C-terminally orientated. However, because the orientation of foreign polypeptides may have a very important role in the correct folding of the produced polypeptides, N-terminal fusion proteins are needed to express especially the N-terminus of the foreign polypeptide. Here, we introduce a novel use of the basic pGEX vectors for the production of N-terminal fusion proteins. In this procedure, PCR generated DNA fragments were cloned into the N-terminus of the GST gene in a unique EcoNI site located down-stream of the ATG initiation codon. The N-terminal fusion proteins were expressed in high quantities, easily solubilized, and affinity purified using our modification of current purification protocols. We also introduce here a new modification of the affinity purification of antibodies using covalently crosslinked GST and fusion proteins to glutathione-agarose beads. Our procedure was tested successfully for producing antibodies against both N- and C-terminus of the luteinizing hormone/chorionic gonadotropin receptor.

TSH receptor antibodies do not alter the function of gonadotropin receptors stably expressed in eukaryotic cells.

OBJECTIVE: TSH receptor (TSHr) mediates the activating action of TSH on the thyroid gland resulting in the growth and proliferation of thyrocytes and thyroid hormone production. TSHr is a major autoantigen in Graves' disease (GD) and is the target for TSHr antibodies. In GD, thyroid-stimulating antibodies (TSAb) are competitive agonists of TSH. In atrophic thyroiditis (AT), thyroid-stimulating blocking antibodies (TSHBAb) are TSH antagonists. The TSHr together with the LH receptor (LHr) and FSH receptor (FSHr) are G-protein-coupled receptors with considerable amino acid homologies in the extracellular domain. We studied the cross-reactivity of the antibodies measured in sera from patients with GD or AT on the LHr and FSHr function. METHODS: We tested the activity of TSAb and TSHBAb in cell lines expressing the LHr and the FSHr. To this purpose a pSVL-FSHr construct was transfected in CHO cells and one clone was used. RESULTS: Twenty-eight sera from patients with GD and four from patients with AT, known to contain TSHr antibodies measured with a radioreceptor assay, were selected. TSAb and TSHBAb activities were measured in CHO cells expressing the TSHr (CHO-TSHr). TSAb and TSHBAb were then tested with the cell lines expressing the LHr and the FSHr for their ability to elicit cAMP accumulation or inhibit FSH/LH-induced cAMP production. None of the TSAb identified was able to stimulate cAMP increase in CHO-LHr or CHO-FSHr. Similarly, none of the TSHBAb was able to block the cAMP response induced by FSH or LH in the respective cell lines. CONCLUSIONS: Our results confirm the notion of the organ-specific nature of the TSHr antibodies.

Functional, structural, and distribution analysis of the chorionic gonadotropin receptor using murine monoclonal antibodies.

LH and human chorionic gonadotropin (hCG) control steroid production and gametogenesis. They also function as growth factors through interaction with a specific receptor that is a member of the seven-transmembrane receptor family coupled via G proteins to signal pathways involving cAMP and phospholipase C/inositol 3 phosphate. For this study, monoclonal antibodies (mAbs) were raised against the human LH receptor (LHR)/hCG receptor (hCGR), using Chinese hamster ovary LHR-transfected cells as the immunogen. Two reagents were then selected on the basis of their ability to recognize the full-length transmembrane receptor expressed both by Chinese hamster ovary LHR-transfected cells and by a limited number of tumor cell lines. One of these mAbs reacts with the LHR/hCGR in tissue sections of both frozen and paraffin-embedded specimens. This unique feature allowed us to map the cytological distribution of LHR/hCGR in human breast tissues at different stages of development in physiological and benign pathological conditions. The same mAb proved to be agonistic: receptor ligation elicits signals that modulate the growth of selected breast tumor cell lines. This observation suggests that the mAb recognizes an epitope that is included in the domain of the receptor involved in the interaction with the natural ligand.

Effect of immunization with bovine luteinizing hormone receptor on ovarian function in cats.

OBJECTIVE: To determine the effect of immunization with bovine luteinizing hormone receptor (LH-R) on ovarian function of cats. ANIMALS: 9 adult female domestic cats. PROCEDURE: 7 cats were immunized with 0.5 mg of LH-R encapsulated in a silastic subdermal implant (3 x 10 mm); 2 served as control cats. Receptors had 80% specific binding to 125I-human chorionic gonadotropin with a binding capacity of 2,682 pM/mg. Cats received booster injections of LH-R. Cats were induced to ovulate with luteinizing hormone (LH) releasing hormone on day 345. Samples of venous blood and vaginal cells were collected through day 395. Observation of estrus behavior continued until day 516. Serum concentrations of estradiol, progesterone, thyroid gland hormones, LH, and LH-R antibody were determined. RESULTS: LH-R antibody was detected in the sera of immunized cats within 21 days after implantation. Detection of LH-R antibody was associated with suppression of serum progesterone to < or = 0.5 ng/mL during the study period, compared with concentrations of 5 to 10 ng/mL in control cats. Immunized cats did not display signs of estrus. Release of LH after administration of LH-releasing hormone indicated an intact hypothalamic-pituitary axis but poor corpus luteum function. Serum estradiol concentrations remained between 30 to 40 pg/mL in immunized and control cats. With the decrease antibody titers, hormone concentrations returned to a pattern consistent with that during fertility. CONCLUSIONS AND CLINICAL RELEVANCE: Active immunization with LH-R suppressed corpus luteum function in cats. The effect was reversible. An LH-R-based antifertility vaccine may have clinical application in other vertebrates.

Modulation of ovarian function in female dogs immunized with bovine luteinizing hormone receptor.

Adult female dogs were immunized with 0.5 mg bovine luteinizing hormone receptor (LH-R) encapsulated in a silastic subdermal implant and subsequently with four intramuscular booster injections of 0.1 mg LH-R each. Circulating LH-R antibody was detected in the sera 3 weeks post-implant. The appearance of LH-R antibody was associated with a decline in the serum progesterone concentrations to a range of 0-0.5 ng/ml until day 365 in the immunized dogs in comparison with a range of 5-10 ng in the control animals, suggesting a lack of ovulation and corpus luteum function in immunized dogs. The immunized dogs did not show signs of 'standing heat' and failed to ovulate when induced by LH-RH challenge. Serum oestradiol levels, however, remained in the range of 30-40 pg/ml in both the immunized and the control dogs. With the decline in the antibody titres, the hormonal profile and vaginal cytology returned to a fertile state and the dogs exhibited signs of 'standing heat', as well as vaginal bleeding. Dogs immunized with LH-R did not show any serious metabolic, local or systemic adverse effects. The hypothalamic--pituitary gonadal axis remained intact as indicated by little difference in pituitary LH levels between control and immunized animals, and by the release of LH by LH-RH challenge. These studies demonstrate that active immunization of female dogs with LH-R could immunomodulate ovarian function to cause a reversible state of infertility. It may be postulated that, due to extensive interspecies homology, a recombinant LH receptor-based immunocontraceptive vaccine may also be effective in other vertebrates.

Use of antibodies against LH receptor, 3beta-hydroxysteroid dehydrogenase and vimentin to characterize different types of testicular tumour in dogs.

Testicular tumours in dogs are of Sertoli cell, Leydig cell or germinal origin and mixed tumours are also frequently observed. The cellular components of mixed tumours are usually identified by histological examination but sometimes this is difficult. In this study, a panel of specific antibodies was used to identify the different cell types in testicular tumours by immunohistochemistry. Leydig cells were identified using an antibody against the LH receptor and an antibody against the steroidogenic enzyme 3beta-hydroxysteroid dehydrogenase (3beta-HSD), both of which are characteristic of Leydig cells in testes. Sertoli cells were identified using an antibody against the intermediate filament vimentin. Seminoma cells did not stain with any of these antibodies. Vimentin was used only in histologically complex cases. Eighty-six tumours, diagnosed histologically as 29 Sertoli cell tumours, 25 Leydig cell tumours, 19 seminomas and 13 mixed tumours, were studied. Feminization was observed in 17 dogs. Leydig cell tumours stained positively with the antibodies against the LH receptor and 3beta-HSD, whereas seminomas and Sertoli cell tumours were negative (unstained). The antibody against vimentin stained both Sertoli and Leydig cells, and tumours arising from these cells, but not seminomas. Immunohistochemistry revealed that three tumours identified histologically as Sertoli cell tumours were actually Leydig cell tumours. In 14 dogs the histological diagnosis appeared to be incomplete, as mixed tumours instead of pure types of tumours were identified in 11 dogs, and in three dogs mixed tumours appeared to be pure types. Hence, the histological diagnosis was insufficient in approximately 20% of dogs. Furthermore, immunohistochemical analysis of testis tumours revealed that feminization occurred in dogs with Sertoli cell tumours or Leydig cell tumours and their combinations, but not in dogs with a seminoma. In conclusion, incubation with antibodies against LH receptor and 3beta-HSD proved to be a consistently reliable method for identification of Leydig cell tumours in dogs. Vimentin can be used to discriminate between Sertoli cell tumours and seminomas. Overall, this panel of antibodies can be very useful for determination of the identity of testicular tumours in which histological characterization is complicated and the pathogenesis of feminization is not clear.

Critical relationship between glycosylation of recombinant lutropin receptor ectodomain and its secretion from baculovirus-infected insect cells.

The lutropin receptor ectodomain overexpressed under the control of the powerful polyhedrin promoter in baculovirus-infected Sf9 insect cells, is mainly found in an inactive, intracellularly-aggregated form. It is secreted in an active form under the control of the P10 promoter, a somewhat weaker and earlier promoter, at the price of a lower production. The apparent molecular masses of the two species encoded by the same cDNA are 48 kDa and 60-68 kDa, respectively. The relationship between the extent and type of glycosylation and the extracellular targeting for the recombinant lutropin receptor ectodomains was investigated precisely with endoglycosidases, lectins of various specificities, and a glycosylation inhibitor, and tested with monoclonal and polyclonal antibodies. The results indicate that the strong polyhedrin promoter probably overwhelms the processing capacity of the ER in Sf9 cells, so that only a high-mannose precursor is expressed in large amounts. Only a minute amount of protein is secreted, which has been processed by Sf9 exoglycosidases/glycosyltransferases and bears complex/hybrid oligosaccharides. The weaker P10 promoter allows secretion of a mature and active receptor ectodomain, bearing complex glycosylation. An important O-linked glycosylation is also added post-translationally on this species. In particular, beta-galactose and sialic acid residues were specifically detected in the secreted species, evidence of the induction of the corresponding glycosyltransferases or of their genes. These results suggest that Sf9 cells should eventually be engineered with chaperones and glycosyltransferases in order to improve the production of demanding glycoproteins such as the porcine lutropin ectodomain, so as to open the way to resolution of the three-dimensional structures of these receptors.

Monoclonal antibodies specific for the binding site on the LH receptor alter progesterone production in cultured granulosa cells.

Monoclonal antibodies (mAbs) specific for the LH receptor (LHR) were generated through a modified auto-anti-idiotypic approach in which human chorionic gonadotropin (hCG) was used as the immunogen followed by cyclophosphamide to induce anti-idiotypic antibodies. The purpose of this study was to investigate the effectiveness of these antibodies to alter progesterone production in porcine granulosa cells in vitro. Anti-LHR mAbs were incubated with granulosa cells in the presence or absence of a stimulatory dose of hCG. Progesterone output by treated cells was measured using a RIA procedure. Most of the mAb could inhibit stimulated progesterone production by cultured granulosa cells. It was speculated that two possible mechanisms may cause the inhibition effect observed. Several of the antibodies appeared to block hCG binding thus removing the stimulatory effects of hCG. However, the most potent inhibiting mAbs for progesterone production had little or no effect on hCG binding, suggesting that some other mechanism was responsible for the observed inhibition. In addition, several of the antibodies were found to have a stimulatory effect on progesterone production by granulosa cells even in the absence of a stimulating dose of hCG. It is proposed that these antibodies were able to mimic hCG.

Characterization of site-directed antibodies to the LH receptor in functionally active gonadal cells and their differential effects on LH-stimulated signal transduction in Leydig tumour (MA10) cells.

In this study site-directed antibodies have been used to investigate the structure/activity relationships of the LH receptor in functionally active gonadal cells. Polyclonal antibodies were raised in rabbits against synthetic peptides corresponding to regions within both the extracellular N-terminal domain (antibodies 1 and 2 against residues 48-65 and 187-206, respectively) and the cytoplasmic C-terminal domain (antibody 3 against residues 622-636) of the LH receptor. Following affinity purification by chromatography on columns of immobilised peptides the antibodies were demonstrated to be peptide specific both by ELISA and by dot-blotting assays. On Western blots of membranes proteins prepared from superovulated rat ovaries, mouse Leydig tumour (MA10) cells, and rat testes, all three antibodies recognised a single broad band of apparent M(r) 95,000-100,000 corresponding to the putative LH receptor. The protein of apparent M(r) 95,000-100,000 also bound 125I-hCG on ligand blots, and binding was displaced by excess unlabelled hCG. The binding of 125I-hCG in the ligand blots was completely inhibited by excess unlabelled hCG. The two N-terminal antibodies (antibodies 1 and 2 (10 micrograms/ml)) also inhibited 125I-hCG binding to a greater extent than the C-terminal antibody (antibody 3 (10 micrograms/ml)). Antibody 1 (1 and 10 micrograms/ml) also potently inhibited the binding of 125I-hCG to MA10 cells. A lesser but still significant inhibition of binding was produced by antibody 2 (with 10 micrograms/ml), whereas at the concentrations tested antibody 3 exerted no greater inhibition than that yielded by pre-immune IgG. At 0.1 micrograms/ml antibody 1 significantly inhibited and at 10 micrograms/ml completely inhibited LH-stimulated cAMP and progesterone production by MA10 cells. With antibody 2, 10 micrograms/ml was required to give a significant inhibition, whereas neither antibody 3 nor pre-immune IgG had a significant effect. The antibodies had no effect on cAMP or progesterone production when added to the MA10 cells in the absence of LH. These results indicate that binding of antibody 1 and, to a lesser extent, antibody 2 interferes with ligand binding which consequently affects signal transduction. In view of the ability of the antibodies to recognise the LH receptors both in the ovary and the testis and in more than one rodent species, and their greater apparent potency than previously available antisera, the anti-peptide antibodies raised in the present study will therefore be useful to study LH receptors in normal, functionally active gonadal cells.

Effect of immunization with lutropin-receptor on the ovarian function of rabbits.

Three New Zealand white adult female rabbits, designated as A, B, and C, were immunized to produce lutropin receptor antibodies. Antisera inhibited the binding of 125I-hCG to the lutropin receptor and the production of testosterone by hCG stimulated rat Leydig cells. During the study period of, approximately 10 months after the last immunization, rabbits did not ovulate in response to an injection of 75 IU of human chorionic gonadotropin or mating as revealed by laparotomy. As the antibody titers declined, induction of ovulation and laparotomy revealed restoration of ovulation and corpus luteum formation. However, no pregnancy occurred when rabbits A and B were mated and artificially inseminated. These observations indicate that lutropin receptor antibodies can cause infertility in female rabbits.

Phosphorylation-independent desensitization of the luteinizing hormone/chorionic gonadotropin receptor in porcine follicular membranes.

Phosphorylation of several G protein-coupled receptors mediates desensitization. This study determined whether LH/CG receptor was phosphorylated under conditions that promoted human CG (hCG)-induced desensitization. Cell-free desensitization of LH/CG receptor-mediated adenylylcyclase activity in porcine follicular membranes occurred in the presence of GTP and was time- and hCG dose-dependent, reaching 36-52% upon preincubation at 30 C for 40 min with 1.0 micrograms/ml hCG. However, under conditions that promoted GTP-dependent desensitization, there was no apparent phosphorylation of LH/CG receptor (obtained via immunoprecipitation) by endogenous membrane-associated protein kinases using [gamma-32P]GTP or [gamma-32P]ATP as phosphate donor. On the other hand, LH/CG receptor (88-90 kilodaltons) from both control and hCG-incubated membranes was phosphorylated in vitro by the catalytic subunit of cAMP-dependent protein kinase (protein kinase A). However, protein kinase A (in the absence of exogenous GTP) did not promote LH/CG receptor desensitization. These data demonstrate that, unlike with other G protein-linked receptors, LH/CG receptor phosphorylation by endogenous follicular membrane-associated protein kinase(s) does not mediate desensitization.