Luteinizing Hormone Chorionic Gonadotropin Research Articles

Tracking conformational transitions of the gonadotropin hormone receptors in a bilayer of (SDPC) poly-unsaturated lipids from all-atom molecular dynamics simulations.

Glycoprotein hormone receptors [thyrotropin (TSHR), luteinizing hormone/chorionic gonadotropin (LHCGR), and follicle stimulating hormone (FSHR) receptors] are rhodopsin-like G protein-coupled receptors. These receptors display common structural features including a prominent extracellular domain with leucine-rich repeats (LRR) stabilized by β-sheets and a long and flexible loop known as the hinge region (HR), and a transmembrane (TM) domain with seven α-helices interconnected by intra- and extracellular loops. Binding of the ligand to the LRR resembles a hand coupling transversally to the α- and β-subunits of the hormone, with the thumb being the HR. The structure of the FSH-FSHR complex suggests an activation mechanism in which Y335 at the HR binds into a pocket between the α- and β-chains of the hormone, leading to an adjustment of the extracellular loops. In this study, we performed molecular dynamics (MD) simulations to identify the conformational changes of the FSHR and LHCGR. We set up a FSHR structure as predicted by AlphaFold (AF-P23945); for the LHCGR structure we took the cryo-electron microscopy structure for the active state (PDB:7FII) as initial coordinates. Specifically, the flexibility of the HR domain and the correlated motions of the LRR and TM domain were analyzed. From the conformational changes of the LRR, TM domain, and HR we explored the conformational landscape by means of MD trajectories in all-atom approximation, including a membrane of polyunsaturated phospholipids. The distances and procedures here defined may be useful to propose reaction coordinates to describe diverse processes, such as the active-to-inactive transition, and to identify intermediaries suited for allosteric regulation and biased binding to cellular transducers in a selective activation strategy.

GnRH antagonist treatment of malignant adrenocortical tumors

Aberrantly expressed G protein-coupled receptors in tumors are considered as potential therapeutic targets. We analyzed the expressions of receptors of gonadotropin-releasing hormone (GNRHR), luteinizing hormone/chorionic gonadotropin (LHCGR) and follicle-stimulating hormone (FSHR) in human adrenocortical carcinomas and assessed their response to GnRH antagonist therapy. We further studied the effects of the GnRH antagonist cetrorelix acetate (CTX) on cultured adrenocortical tumor (ACT) cells (mouse Cα1 and Y-1, and human H295R), and in vivo in transgenic mice (SV40 T-antigen expression under inhibin α promoter) bearing Lhcgr and Gnrhr in ACT. Both models were treated with control (CT), CTX, human chorionic gonadotropin (hCG) or CTX+hCG, and their growth and transcriptional changes were analyzed. In situ hybridization and qPCR analysis of human adrenocortical carcinomas (n = 11-13) showed expression of GNRHR in 54/73%, LHCGR in 77/100% and FSHR in 0%, respectively. CTX treatment in vitro decreased cell viability and proliferation, and increased caspase 3/7 activity in all treated cells. In vivo, CTX and CTX+hCG (but not hCG alone) decreased ACT weights and serum LH and progesterone concentrations. CTX treatment downregulated the tumor markers Lhcgr and Gata4. Upregulated genes included Grb10, Rerg, Nfatc and Gnas, all recently found to be abundantly expressed in healthy adrenal vs ACT. Our data suggest that CTX treatment may improve the therapy of human adrenocortical carcinomas by direct action on GNRHR-positive cancer cells inducing apoptosis and/or reducing gonadotropin release, directing tumor cells towards a healthy adrenal gene expression profile.

GnRH antagonist treatment of malignant adrenocortical tumors.

Aberrantly expressed G protein-coupled receptors in tumors are considered as potential therapeutic targets. We analyzed the expressions of receptors of gonadotropin-releasing hormone (GNRHR), luteinizing hormone/chorionic gonadotropin (LHCGR) and follicle-stimulating hormone (FSHR) in human adrenocortical carcinomas and assessed their response to GnRH antagonist therapy. We further studied the effects of the GnRH antagonist cetrorelix acetate (CTX) on cultured adrenocortical tumor (ACT) cells (mouse Cα1 and Y-1, and human H295R), and in vivo in transgenic mice (SV40 T-antigen expression under inhibin α promoter) bearing Lhcgr and Gnrhr in ACT. Both models were treated with control (CT), CTX, human chorionic gonadotropin (hCG) or CTX+hCG, and their growth and transcriptional changes were analyzed. In situ hybridization and qPCR analysis of human adrenocortical carcinomas (n = 11–13) showed expression of GNRHR in 54/73%, LHCGR in 77/100% and FSHR in 0%, respectively. CTX treatment in vitro decreased cell viability and proliferation, and increased caspase 3/7 activity in all treated cells. In vivo, CTX and CTX+hCG (but not hCG alone) decreased ACT weights and serum LH and progesterone concentrations. CTX treatment downregulated the tumor markers Lhcgr and Gata4. Upregulated genes included Grb10, Rerg, Nfatc and Gnas, all recently found to be abundantly expressed in healthy adrenal vs ACT. Our data suggest that CTX treatment may improve the therapy of human adrenocortical carcinomas by direct action on GNRHR-positive cancer cells inducing apoptosis and/or reducing gonadotropin release, directing tumor cells towards a healthy adrenal gene expression profile.

Genetic Polymorphisms in Pakistani Women With Polycystic Ovary Syndrome

Polycystic ovary syndrome (PCOS) is the major cause of anovulatory infertility. Although the genetic basis of PCOS is not well understood, it is a common metabolic and endocrine disorder. This study investigates the possible genomic variants associated with PCOS in Pakistani women from the Punjab region. DNA samples from 96 patients with genetically unrelated PCOS and 96 controls were analyzed by direct sequencing to determine the polymorphisms of different loci on follicle-stimulating hormone receptor (fshr), follicle-stimulating hormone β (fshrβ), luteinizing hormone chorionic gonadotropin (lhcgr), luteinizing hormone β (lhβ), estrogen receptor α (esr1), and estrogen receptor β (esr2) genes. Significant associations were observed within the genotype frequencies, allele frequencies, and multi-single-nucleotide polymorphism (SNP) haplotype analysis of most polymorphisms studied. This study identified new SNPs at positions 605+52 Del/T in lhcgr genes occurring in this particular subpopulation. The strong r (2) value suggests that polymorphisms in the fshr and esr1 genes were in linkage disequilibrium. Our study provides evidence of statistically significant associations between susceptibility to PCOS in Pakistani women and the gene polymorphisms mentioned earlier. This suggests that the susceptible loci for PCOS lie within or very close to the chromosomal regions spanning these genes.

Pharmacokinetics and pharmacodynamics of Phor21-βCG(ala), a lytic peptide conjugate

Phor21-betaCG(ala), a 36-amino acid peptide comprised of a lytic peptide (Phor21) conjugated to a modified 15-amino acid segment of the beta-chain of chorionic gonadotropin (betaCG(ala)), selectively kills cancer cells that over-express luteinizing hormone/chorionic gonadotropin (LH/CG) receptors by disrupting cellular membrane structure. These studies were designed to further characterize its in-vitro inhibition and in-vivo destruction of prostate cancer cells, biostability and pharmacokinetics to determine its pharmacokinetic and pharmacodynamic profile. Inhibitory effects of Phor21-betaCG(ala) were tested in PC-3 and Caco-2 cells as well as in nude mice bearing PC-3 cells transfected with the luciferase gene (PC-3.luc). Plasma stability, protease hydrolysis and pharmacokinetics of Phor21-betaCG(ala) were measured by using liquid chromatography mass spectrometry (LC/MS/MS). Phor21-betaCG(ala) selectively inhibited proliferation in-vitro and in-vivo metastases of PC-3 cells. Phor21-betaCG(ala) was relatively stable in mouse, rat, dog and human plasma. Its degradation was partially due to protease hydrolysis and thermodynamic catalysis. Intravenous administration of Phor21-betaCG(ala) showed its blood C(max) and AUC(0-->infinity) around the in-vitro effective levels. In the tested rodents, Phor21-betaCG(ala) displayed a moderate volume of distribution at steady state (Vd(ss)) and slow clearance (Cl) in the rodents. In conclusion, Phor21-betaCG(ala) displayed promising in-vitro and in-vivo anti-cancer activity with favourable pharmacokinetics, and may offer a novel approach to metastatic cancer chemotherapy.

Human chorionic villous macrophages as a fetal biological shield from maternal chorionic gonadotropin

In addition to its most well characterized biological role in the rescue and maintenance of corpus luteum function, human chorionic gonadotropin (hCG) also stimulates the onset of fetal gonadal steroidogenesis. However, excess hCG is teratogenic to fetal gonadal tissues, and therefore hCG must be tightly regulated. Although there is an anatomical barrier between the fetal vessels and maternal blood, other mechanisms may regulate hCG levels. In the present study, we investigated whether human chorionic villous macrophages degraded maternal hCG. Isolated human macrophages incorporated and degraded hCG in a time-dependent manner. Human placental villous macrophages and phorbol myristate acetate (PMA)-treated THP-1 cells expressed the gene encoding an exon 9-deleted form of the luteinizing hormone/chorionic gonadotropin (LH/CG) receptor; expression of the full-length receptor was not determined. While both PMA-treated or untreated THP-1 cells could uptake hCG into their cytoplasms, hCG degradation and excretion of its byproducts only progressed in PMA-treated THP-1 cells. In conclusion, hCG internalization and degradation are different processes in macrophages that protect fetal gonadogenesis from excess hCG. The exon 9-deleted LH/CG receptor, but not the full-length receptor, is involved in the degradation of cytoplasmic hCG by organ-specific, dominant-negative interactions.

LHRH-conjugated Magnetic Iron Oxide Nanoparticles for Detection of Breast Cancer Metastases

Targeted delivery of superparamagnetic iron oxide nanoparticles (SPIONs) could facilitate their accumulation in metastatic cancer cells in peripheral tissues, lymph nodes and bones and enhance the sensitivity of magnetic resonance imaging (MRI). The specificities of luteinizing hormone releasing hormone (LHRH) and luteinizing hormone/chorionic gonadotropin (LH/CG)- bound SPIONs were tested in human breast cancer cells in vitro and were found to be dependent on the receptor expression of the target cells, the time of incubation and showed saturation kinetics. In incubations with MDA-MB-435S.luc cells, the highest iron accumulation was 452.6 pg Fe/cell with LHRH-SPIONs, 203.6 pg Fe/cell with beta-CG-SPIONs and 51.3 pg Fe/cell with SPIONs. Incubations at 4 degrees C resulted in 1.1 pg Fe/cell. Co-incubation with the same ligands (betaCG or LHRH) decreased the iron accumulation in each case. LHRH-SPIONs were poorly incorporated by macrophages. Tumors and metastatic cells from breast cancer xenografts were targeted in vivo in a nude mouse model. LHRH-SPION specifically accumulated in cells of human breast cancer xenografts. The amount of LHRH-SPION in the lungs was directly dependent on the number of metastatic cells and amounted to 77.8 pg Fe/metastastic cell. In contrast, unconjugated SPIONs accumulated in the liver, showed poor affinity to the tumor, and were not detectable in metastatic lesions in the lungs. LHRH-SPION accumulated in the cytosolic compartment of the target cells and formed clusters. LHRH-SPIONs did not accumulate in livers of normal mice. In conclusion, LHRH conjugated SPIONs may serve as a contrast agent for MR imaging in vivo and increase the sensitivity for the detection of metastases and disseminated cells in lymph nodes, bones and peripheral organs.

Targeting Breast and Prostate Cancers Through Their Hormone Receptors1

A targeted treatment that effectively destroys human breast, prostate, ovarian, and testicular cancer cells that express luteinizing hormone/chorionic gonadotropin (LH/CG) receptors has been developed. The treatment consists of a conjugate of a membrane-disrupting lytic peptide (Hecate, Phor14, or Phor21) and a 15-amino acid segment of the beta chain of CG. Because these conjugates act primarily by destroying cell membranes, their effects are independent of cell proliferation. The conjugates are relatively small molecules, are rapidly metabolized, and are not antigenic. In a series of independent experiments conducted in three different laboratories, the validity of the concept has been established, and it has been shown that the LH/CG receptor capacity of the cancer cells is directly related to the sensitivity of the lytic peptide conjugates. Sensitivity to the drugs can be increased by pretreating prostate or breast cancer cells with FSH or estradiol to up-regulate LH/CG receptors. A series of 23 in vivo experiments involving a total of 1630 nude mice bearing xenografts of human prostate or breast cancer cells showed convincingly that all three lytic peptide-betaCG compounds were highly effective in destroying tumors and reducing tumor burden. Hecate-betaCG was less effective in mice bearing ovarian epithelial cancer cell xenografts, but was highly effective in treating granulosa cell tumors in transgenic mice. In addition, Hecate-betaCG and Phor14-betaCG were highly effective in targeting and destroying prostate and breast cancer cell metastases in the presence or absence of the primary tumors. Although effective in vitro, neither Hecate nor Phor14 alone were effective in reducing primary tumor volume or burden in nude mice bearing prostate or breast cancer xenografts.

Expression of variant luteinizing hormone/chorionic gonadotropin receptors and degradation of chorionic gonadotropin in human chorionic villous macrophages

Human gonads and non-gonadal organs/tissues express luteinizing hormone/chorionic gonadotropin (LH/CG) receptors. This study aimed to identify the LH/CG receptors and to clarify their function in human placental chorionic villous macrophages. Macrophages as well as syncytiotrophoblasts of human chorionic villous tissues were immunohistochemically positive for LH/CG receptor throughout gestation. By reverse transcription-nested polymerase chain reaction methods, villous macrophages were shown to express a variant type of LH/CG receptor, the sequencing of which revealed a deletion of exon 9. For experiments in vitro, a monocyte-macrophage cell line, THP-1, was transfected with vector alone, wild-type LH/CG receptor, and exon 9-deleted LH/CG receptor after phorbol 12-myristate 13-acetate (PMA) treatment. Non-PMA-treated THP-1 cells transfected with vector alone were also examined. THP-1 cells expressed exon 9-deleted LH/CG receptor after treatment with PMA. After the cells of the four groups were cultured in medium containing intact human CG (hCG), the concentrations of hCG and its beta-core fragment (beta-CF) were measured in the supernatant of the culture medium and in the cell cytosol. Time-dependent hCG uptake was observed in both non-PMA-treated and PMA-treated THP-1 cells, suggesting that the variant receptor is not directly involved in the ingestion of hCG. The degradation of hCG and excretion of beta-CF were progressed in PMA-treated cells but not in the un-treated cells. In the cell cytosol, the ratio of beta-CF and hCG concentrations (beta-CF/hCG) was significantly higher in the PMA-treated cells than in non-PMA-treated cells; however, it did not differ between the PMA-treated cells transfected with exon 9-deleted receptor and those transfected with vector alone. Macrophages may express the variant receptor in order to recognize the intracytoplasmic hCG and transport it to the lysosome. Among the two PMA-treated cells, the ratio was lower in those transfected with wild-type receptor. The expression of the variant receptor may modulate the degradation of hCG but be reduced by expression of the wild-type receptor in its lacking macrophages. Our data suggest a potentially important role for exon 9-deleted LH/CG receptors expressed in human placental villous macrophages in the local metabolism of hCG.

Expression of the full-length and alternatively spliced equine luteinizing hormone/chorionic gonadotropin receptor mRNAs in the primary corpus luteum and fetal gonads during pregnancy.

The full-length equine luteinizing hormone/chorionic gonadotropin (LH/CG) receptor (eLH/CG-RA) cDNA and two alternatively spliced isoforms (eLH/CG-RB,C) were isolated from luteal tissue and characterized using a combination of reverse transcription-polymerase chain reaction (RT-PCR) and 5'-rapid amplification of cDNA ends. The 680-amino acid full sequence of eLH/CG-RA displayed 87-92% homology with other mammalian LH/CG-Rs. The eLH/CG-RB and eLH/CG-RC cDNA isoforms were truncated from the 3'-end of exon X: eLH/CG-RB spliced out of frame into the last exon whereas eLH/CG-RC contained an in-frame stop codon within a divergent sequence. Consequently, both eLH/CG-RB and eLH/CG-RC cDNA isoforms encoded putative proteins without transmembrane and intracellular domains. In order to study the responsiveness of the primary corpus luteum (CL) and fetal gonads to eCG, the expression of eLH/CG-R mRNAs was examined by RT-PCR and Northern blot analysis during early and mid-pregnancy. All three eLH/CG-R cDNA isoforms (eLH/CG-RA,B,C) were expressed from day 14 to day 83 of pregnancy in the primary CL and from day 44 to day 222 in fetal gonads. Interestingly, the primary CL at days 89 and 151 expressed only truncated eLH/CG-R cDNA isoforms. The relative values of Northern hybridized major 7, 5.7, 3.9 and 1.8 kb eLH/CG-R mRNA transcripts tended to decrease in the primary CL whereas the unique major 1.8 kb eLH/CG-R mRNA was steadily expressed in fetal gonads during pregnancy. These results show that the expression of eLH/CG-R mRNAs occurs in the fetal gonads before ceasing in the primary CL and suggest that eCG may be involved in the gradual transition from a luteal to a feto-placental output of steroids during equine pregnancy.

Partial characterization of chorionic gonadotropin-like binding sites from the bacteria Xanthomonas maltophilia.

The gram-negative bacterium, Xanthomonas maltophilia, has low- and high-affinity luteinizing hormone/chorionic gonadotropin (LH/CG)-binding sites, similar to the LH/CG receptor found in mammals. Although the low-affinity site binds both LH and human CG (hCG), the high-affinity site is specific for hCG. In the current investigation, these two binding sites were independently isolated from X. maltophilia for further characterization. To isolate functional binding sites, we developed a solubilization method using the detergent zwittergent 3,14 and high glycerol concentrations that allowed for the maintenance of ligand-binding integrity. Gel filtration experiments established molecular weights of 170 and 11.5 kDa for the two binding sites, which were supported by data from photoaffinity labeling and ultracentrifugation experiments. Gel filtration data also suggested the presence of a third binding site of 5.4 kDa. The 170-kDa site had a binding affinity of Kd = 12 x 10(-6) and bound both LH and hCG. The small molecular weight site had an affinity of Kd = 9.4 x 10(-8) and was CG specific. Collectively, these data demonstrate the presence of multiple hormone binding sites in X. maltophilia that differ in molecular size, binding affinity, and ligand specificity.

Glycoprotein hormone receptors: determinants in leucine-rich repeats responsible for ligand specificity.

Glycoprotein hormone receptors [thyrotropin (TSHr), luteinizing hormone/chorionic gonadotropin (LH/CGr), follicle stimulating hormone (FSHr)] are rhodopsin-like G protein-coupled receptors with a large extracellular N-terminal portion responsible for hormone recognition and binding. In structural models, this ectodomain is composed of two cysteine clusters flanking nine leucine-rich repeats (LRRs). The LRRs form a succession of beta-strands and alpha-helices organized into a horseshoe-shaped structure. It has been proposed that glycoprotein hormones interact with residues of the beta-strands making the concave surface of the horseshoe. Gain-of-function homology scanning of the beta-strands of glycoprotein hormone receptors allowed identification of the critical residues responsible for the specificity towards human chorionic gonadotropin (hCG). Substitution of eight or two residues of the LH/CGr into the TSHr or FSHr, respectively, resulted in constructs displaying almost the same affinity and sensitivity for hCG as wild-type LH/CGr. Molecular dynamics simulations and additional site-directed mutagenesis provided a structural rationale for the evolution of binding specificity in this duplicated gene family.

The prevalence and clinical significance of blocking thyrotropin receptor antibodies in untreated hyperthyroid Graves' disease.

The goal of this study was to evaluate the clinical significance of the blocking thyrotropin receptor antibodies (TSHRAb) in Graves' disease. The amount of blocking and stimulating TSHRAb were measured in 200 patients with untreated hyperthyroid Graves' disease using several cell lines carrying different TSHR chimera. Stimulating TSHRAb were measured in Chinese hamster ovary (CHO) cells with wild-type human TSHR (CHO-hTSHR) or a TSHR chimera with residues 90-165 (Mc2) or 8-165 (Mc1+2) substituted by equivalent residues of rat luteinizing hormone/chorionic gonadotrophin (LH/CG) receptor or in FRTL-5 cells. Blocking TSHRAb were measured in Mc2 cells. The activities of different TSHRAb were assessed and clinical features were compared to patients who were positive or negative for blocking TSHRAb antibodies. Blocking TSHRAbs were detected in 18.5% of patients (37/200) with hyperthyroid Graves' disease. Patients with blocking antibodies had significantly lower mean stimulating TSHRAb activities than those without blocking antibodies in wild-type CHO-hTSHR cells (301 +/- 179 vs. 446% +/- 537%, p = 0.005). Mean stimulating TSHRAb activities measured by FRTL-5, Mc1+2, or Mc2 cells and mean thyrotropin receptor inhibitor immunoglobulin (TBII) activities were not different between the two groups. The patients with blocking antibodies were not different from those without blocking antibodies in age, gender ratio, initial serum free thyroxine (T4) levels, or goiter size. However, the prevalence of exophthalmos was higher (35.1% vs. 17.5%, p = 0.024) in the patients with blocking antibodies than those without. In summary, the presence of blocking TSHRAb is not rare in patients with hyperthyroid Graves' disease when measured with chimeric receptor expressing cells. Blocking TSHRAb in Graves' sera do not strongly antagonize the action of stimulating TSHRAb in vivo, but could be a major factor responsible for underestimation of stimulating TSHRAb activities measured by CHO-hTSHR. The association of blocking TSHRAb with ophthalmopathy suggests that the TSHRAb repertoire of Graves' patients is different in those who do and who do not have ophthalmopathy.

Generating FSH antagonists and agonists through immunization against FSH receptor N-terminal decapeptides.

Follicle-stimulating hormone (FSH) via interaction with G-protein coupled specific receptors plays a central role in the control of gametogenesis in mammals of both sexes. In females, FSH is crucial for follicle growth, follicle maturation and ovulation. FSH receptors, together with luteinizing hormone-chorionic gonadotropin and thyrotropin receptors belong to a subfamily of structurally related receptors within the seven transmembrane receptor family. Among several other regions, the N-terminus of these receptors is believed to be responsible for important specific hormone-receptor contact sites. Recombinant filamentous phages displaying at their surface three overlapping N-terminal decapeptides of the FSH receptor, peptides A18-27, B25-34 and C29-38 were constructed. Ewes and female mice were immunized against the three FSH receptor (FSHR) recombinant phages. Immunoglobulins purified from immunized animals were analyzed for their biochemical properties on a Chinese hamster ovary cell line expressing the porcine FSH receptor. AntiA and antiB immunoglobulins (IgGs) behave as antagonists for 125I-FSH binding and for FSH-dependent cAMP production, while antiC IgGs did not compete for hormone binding. By contrast, antibodies against the C29-38 peptide displayed FSH agonist activity and stimulated the FSH receptor, whereas antiA and antiB IgGs did not. Furthermore, when the FSHR phages were used as peptidic vaccines, they induced a reversible inhibition of ovulation rate in ewes, and impaired fertility in female mice.

In vivo responses of the primate corpus luteum to luteinizing hormone and chorionic gonadotropin.

Although it is well established that the secretory activity of the corpus luteum absolutely depends on the presence of pituitary-derived luteinizing hormone (LH), it is unknown why the life span of the corpus luteum is extended during early pregnancy by the placental production of chorionic gonadotropin (CG) but regresses in the presence of LH despite the fact that CG and LH have similar actions on the corpus luteum. To compare the responses of the corpus luteum to LH and human CG (hCG), cynomolgus monkeys whose endogenous gonadotropin secretion was blocked during the luteal phase of the menstrual cycle with a gonadotropin-releasing hormone antagonist were i.v. infused with either LH or CG. Infusion of LH at a constant rate overcame the gonadotropin-releasing hormone antagonist-mediated premature luteal regression but failed to prolong the functional life span of the corpus luteum. Continuous infusions of hCG did not effect a pregnancy-like pattern of gonadotropin secretion, but the functional life span of the corpus luteun was extended in two of three animals. Infusion of either LH or hCG in an exponentially increasing manner prolonged the functional life span of the corpus luteum beyond its normal duration. These results indicate that luteal regression at the termination of nonfertile menstrual cycles is caused by a large reduction in the responsiveness of the aging corpus luteum to LH, which can be overcome by elevated concentrations of either LH or CG.

Cleavage and shedding of the TSH receptor.

The structure of the thyroid stimulating hormone receptor (TSHR) has attracted an unusual amount of discussion. Different authors have described a wide variety of receptor forms, with molecular masses varying from 90 to 500 kDa, and comprising one to three subunits (reviewed in 1). A lack of precise structural information prevented the use of specific probes to clone the receptor. Two approaches to the problem were adopted. In one, the luteinizing hormone/chorionic gonadotrophin (LH/CG) receptor cDNA was initially cloned (2) and allowed the description of a novel family of G-protein-coupled receptors. This receptor was used as a probe in a crosshybridization experiment to clone the TSHR (3). The second approach involved the use of low-specificity PCR with probes derived from more distant G-protein-coupled receptors (4, 5). The discovery of the homology of the TSHR with other G-protein-coupled receptors at the mRNA level led to the conclusion that the mature receptor was also a single subunit protein, but different molecular masses between 90 and 210 kDa were nevertheless proposed by different authors. This was mainly the result of a lack of adequate immunological tools with which to immunopurify the receptor before studying its structure. Because of the high hydrophobicity of the receptor and its tendency to aggregate with contaminating proteins, aberrant migration of receptor may have occurred on western blots. Another reason for the discrepant observations was the low concentration of the receptor in human thyroid glands, which has prompted most authors to study non-physiological systems, e.g. heterologous transfected cells. Cloning of the TSHR cDNA has allowed the structure of the protein to be deduced, with a calculated molecular mass of 85 kDa that is highly homologous to the LH and follicle-stimulating hormone (FSH) receptors. All three receptors strikingly display the same division into regions according to their respective homology (6). The most homologous domain is the seven transmembrane domain (75% homology). Highly divergent regions within the extraor intracellular domains can be demonstrated, however, and may be involved in more specific functions for each receptor. Contrasting with this high homology at the mRNA level, the mature structure of TSH and gonadotropin receptors is different. The LH (7) and FSH (8) receptors are expressed in target cells as glycosylated monomers. Unexpectedly, western blots of receptor immunopurified from thyroid extracts, performed with different monoclonal antibodies raised against the extracellular or the intracellular parts of the receptor, showed that the TSHR is in fact a heterodimer (9). It comprises an a extracellular subunit (,53 kDa) and a broad b transmembrane and intracellular subunit (30–42 kDa), held together by disulphide bridges. These subunits were named a and b by analogy with the insulin receptor, which is also post-translationally cleaved. The maturation of the TSHR by post-translational cleavage is unique among G-protein-coupled receptors; it has to be differentiated from ligand-mediated enzymatic cleavage of the thrombin receptor during the process of receptor activation (10). In thyroid tissue, the cleavage is almost complete. Only traces of precursors can be detected. This is reminiscent of the model suggested by Rees Smith et al. (11), but different from many others proposed at the same period on the basis of studies using similar methodological approaches. Contrasting with the situation found in the thyroid, a much more complex pattern is observed in transfected cells in which mixed mature and immature forms of the receptor accumulate (12). This is probably due to the overexpression of the receptor in these cells and to the saturation of the cellular machinery necessary for the processing of the receptor. Monomeric precursors of 95 kDa and 120 kDa accumulate in these cells. Two steps are involved in the glycosylation of proteins: first, the addition of high mannose residues in the endoplasmic reticulum, and secondly the trimming of the residues and addition of complex oligosaccharides in the Golgi apparatus. Endoglycosidase H, specific for precursor high-mannose moieties of glycoproteins, can be used to identify precursor species. Indeed, the 95 kDa monomer that accumulates mainly in transfected cells is sensitive to that enzyme and corresponds to a highmannose precursor of the TSHR. It is also the first species to be observed on pulse–chase studies and undergoes subsequent mature glycosylation and cleavage (12). A second 120 kDa monomer is observed on western blots in transfected cells. It corresponds to the fully glycosylated, but uncleaved, monomer. q European Journal of Endocrinology (1997) 137 599–602 ISSN 0804-4643

Establishment of an in vitro bioassay and radio receptor assay for LH/CG in human sera using immortalized granulosa cells transfected with LH/CG receptor

Levels of gonadotropic hormones in human sera or urine are routinely measured by radioimmunoassay or by enzyme-linked immunoassay (ELISA), which determine the immunoactivity of the hormone, but not its biological activity. We have utilized immortalized stable steroidogenic granulosa cells, which express 5-10 times more of the luteinizing hormone/chorionic gonadotropin (LH/CG) receptors than the primary cells, to develop a biological assay and radioreceptor assay for this hormone. We found that stimulation of cells expressing LH/CG receptor with increasing doses of human LH or human CG resulted in a dose-dependent increase of cAMP and progesterone with an ED(50) of 30 and 57 mlU/mL, respectively. These dose-response data served as calibration curves for measuring the gonadotropin bioactivity in human serum samples at concentrations as low as 1-5 mlU/mL. We found a close correlation between LH levels measured by enzyme immunoassay (EIA) and the in vitro bioassay in normal cycling and menopausal women, as well as in normal adult men. Also, a close correlation was found between the EIA and the in vitro biological assay of hCG in pregnant women. In addition, we have developed a radioreceptor assay (RRA) for this hormone using enriched cell membranes of the appropriate cell line, which corresponds well to both the EIA and the bioassay in human sera. Deglycosylated hCG was fully active in RRA, but failed to activated cAMP response in these cells, demonstrating the importance of the bioassay in the biologically inactive form of gonadotropins. We believe this novel in vitro bioassay of gonadotropic hormones will serve as a useful tool for a more comprehensive set of assays that will determine not only the amount, but also the possible modulation in bioactivity of the gonadotropin associated with gonadal failure and miscarriage.

Novel expression of luteinizing hormone/chorionic gonadotropin receptor gene in rat prostates

The reverse transcription-polymerase chain reaction (RT-PCR) amplified an expected 255 bp luteinizing hormone/chorionic gonadotropin (LH/CG) receptor sequence from rat prostates. Northern blotting demonstrated that prostates contain 4.3, 3.3, 2.6, 1.8, 0.8 and 0.2 kb LH/CG receptor transcripts. Western immunoblotting and ligand blotting demonstrated that prostates also contain an 80 kDa receptor protein which can bind 125I-labeled hCG and this binding was inhibited by excess unlabeled hCG. In situ hybridization and immunocytochemistry revealed that while the receptors are most abundant in epithelial cells, they are scarcely found in the stroma. The ventral lobe contained more receptors than the lateral lobe and the receptors in peripheral acini of the ventral lobe are higher which progressively decreased towards central acini. In summary, prostate glands express LH/CG receptor gene. The cellular, topographical and lobular distribution of receptors suggest that LH may directly regulate prostate functions.

Mechanisms of hormone action

The binding of luteinizing hormone/chorionic gonadotropin (LH/CG) to its receptor leads to an orderly cascade of events leading to activation of cytoplasmic effector molecules. The mechanism of action of LH includes stimulation of multiple signal transduction effector systems, including adenylyl cyclase (AC) and inositol phospholipid-specific phospholipase-C (PLC). These effector systems operate by activating families of protein kinases (PKs) which regulate cell metabolism, secretion, gene induction, cellular proliferation and differentiation. The signaling pathways activated by LH interact or cross-talk with the signaling pathways activated by growth factors. The diversity of cellular signaling mechanisms elicited by LH and the potential for interactions with signals generated by growth factors may allow fine tuning of cellular responses during the process of follicular development, ovulation, and corpus luteum formation.

Transient and cell-specific expression of tissue-type plasminogen activator and plasminogen-activator-inhibitor type 1 results in controlled and directed proteolysis during gonadotropin-induced ovulation.

Proteolytic activity generated by the plasminogen-activator system (PA system) is associated with many biological processes. However, it is not known how the proteolytic activity is regulated in vivo in order to obtain directed proteolysis while, at the same time, protecting unrestrained tissue destruction. Using gonadotropin-induced ovulation as a model, we have studied how two components of the PA system, tissue-type plasminogen activator (tPA) and plasminogen-activator-inhibitor type 1 (PAI-1), are regulated temporally and spatially by gonadotropins, leading to the initiation and termination of a well-directed proteolytic process. In-situ hybridization and in-situ zymography were used to analyze the expression of tPA and PAI-1 mRNA and PA-activity in specific ovarian cell types. Both tPA and PAI-1 were found to be regulated and to have a distinct expression pattern in different ovarian compartments. tPA was expressed in both granulosa and thecal-interstitial cells; the highest levels of tPA mRNA were found in the granulosa cells of preovulatory follicles, just prior to ovulation. Consistent with a role for luteinizing hormone/chorionic gonadotropin (LH/CG) in triggering ovulation, the cells and follicles that actively expressed tPA also contained high levels of LH-receptor mRNA while cumulus cells that contain undetectable amounts of tPA mRNA were devoid of LH-receptor expression. The highest levels of PAI-1 mRNA were found about 6 h before ovulation and mainly in the thecal-interstitial cells and ovarian stroma tissue which encapsulate the follicle. Preovulatory follicles, protruding onto the surface of the ovary with less surrounding stroma tissue, expressed less PAI-1 compared to small non-ovulatory follicles embedded in inner part of the ovary. In-situ zymography also revealed that the PA activity was colocalized to the surface of the ovary just prior to ovulation. Our studies suggest that a proteolytic activity provided by tPA and modulated by PAI-1 is responsible for a controlled and directed proteolysis leading to rupture of selected follicles during ovulation.