Jean-Jacques Remy

Towards understanding the glycoprotein hormone receptors.

Lutropin (LH), follitropin (FSH) and thyrotropin (TSH), as well as choriogonadotropin (CG, which binds to the LH receptor) constitute the glycoprotein hormone family. Their 3 receptors have been cloned during the last few months. They belong to the large group of G-protein coupled membrane proteins, with their specific N-terminal domain likely to bind the hormone and the characteristic 7 membrane-spanning segments in their C-terminal moiety. The present review discusses the main results of amino acid sequence analysis performed on the glycoprotein hormone receptors. The putative extracellular head exhibits less than 45% homology over the 3 receptors, while approximately 70% residue conservation is found in the transmembrane moiety. Here only, limited sequence homologies (approximately 20%) can be found with other G-protein coupled receptors. The secondary structure predictions performed on the 3 receptors revealed that the polypeptide sequence predicted as ordered (either alpha-helix or beta-strand) were repeated evenly throughout the extracellular head with a period of approximately 25 amino acids. This analysis helped to define the intervening loops between this ordered stretches as potential candidates for bearing at least part of the binding site of the hormones. Some of the perspectives opened by the cloning of the receptors are described, like the production of the extracellular head of the porcine LH receptor in baculovirus-infected insect cells, and the exploration of the LH receptors mechanism of functioning as a dimer.

The porcine follitropin receptor: cDNA cloning, functional expression and chromosomal localization of the gene.

The porcine follitropin receptor-encoding cDNA (pFSHR) was cloned using reverse transcription-polymerase chain reaction (RT-PCR). Total RNA from porcine granulosa cells was used as template. Two overlapping cDNA fragments encoding, respectively, aa 1 to 290 and aa 191 to 694 of the pFSHR were obtained. Taken together, the two fragments represented the whole coding sequence, assuming a comparable length for the FSHR from the porcine, rat and human species. Functionality of the cloned receptor was assessed by expression experiments; COS cells transfected with the pFSHR cDNA exhibited high-affinity specific binding for [125I]hFSH and FSH-dependent cAMP production. The primary sequence of the porcine FSHR N-terminal hormone-binding domain showed high percentages of identity with the sequences from ovine, human, and rat origins. A truncated form of the pFSHR cDNA, lacking aa 75 to 124 in the N-terminal domain, was also cloned and sequenced. A PCR-derived cDNA fragment of 1.45 kb was used as gene-specific hybridisation probe to map the pFSHR-encoding gene by radioactive in situ hybridization. This gene was found co-localized (as in human) with the porcine lutropin hormone receptor (pLHR)-encoding gene on the q2.2-q2.3 region of pig chromosome 3.

Immunization against exon 1 decapeptides from the lutropin/choriogonadotropin receptor or the follitropin receptor as potential male contraceptive

Pituitary gonadotropin hormones lutropin (LH) and follitropin (FSH) control steroidogenesis and gametogenesis in male and female gonads through interaction with G protein-coupled receptors, LHR and FSHR. In the male, LH acts on leydig cells and is mostly responsible for the acquisition of puberty and the production of androgens while FSH, together with androgens, regulates spermatogenesis within Sertoli cells. We have engineered filamentous phages displaying mouse LHR and human FSHR decapeptides chosen in hormone binding regions. Peptides from both receptors displayed on phages belong either to the receptor specific exon 1 (amino acids 18-27) or to the homologous exon 4 (amino acids 98-107). Vaccination of prepubertal BALB/c male mice with hybrid phages using sub-cutaneous or intraperitoneal injections induced immunity against receptors. Anti-receptor immunization produced agonist or antagonist effects depending only on the circulating levels of the antibodies. Both anti-LHR and anti-FSHR vaccines induced efficient as well as reversible male contraception, through different mechanisms: targeting LH receptors inhibited or hyperstimulated Leydig cell testosterone production while targeting FSH receptors did not affect testosterone levels.

Suppression of fertility in male mice by immunization against LH receptor.

We have investigated the potential contraceptive effects of immunization against the luteinizing hormone (LH) receptor in male mice at the prepubertal stage. Two N-terminal fragments of the porcine LH receptor encoding amino acids 1-297 and 1-370 were produced in large quantities through the Baculovirus insect cell system. We have immunized three-week-old mice from two Balb/c stocks of differing fecundity with Sf9 insect cells producing the short (1-297) or long (1-370) recombinant LH receptor. A booster injection was performed at six weeks using purified antigens. Ten days later, the immunized male mice were mated over a period of two weeks with adult untreated females. After weaning of the first litters, the same partners were mated once again under the same conditions. There was no decrease in the antiserum titers against the antigens over a two-month period. The circulating testosterone decreased as the anti-LH receptor antibodies increased. The fertility of the treated male mice was reduced up to 75%, depending on the mouse stock, the antigen used and the time separating immunization and mating. The impaired fertility was mostly due to male sterilization (up to 60% of sterile mates). The delay between mating and birth was enhanced by the treatment, reflecting delayed fertility and/or delayed male behaviour acquisition.

Immunomodulation of rodent male fertility by vaccination against the luteinizing hormone receptor

Abstract Luteinizing hormone (LH) controls, through interaction with its specific receptor, the steroidogenic activity of the testicular Leydig cells. The possibility of antagonizing LH activities by means of immunization against its receptor was investigated. Immunization of prepubertal male rodents against the N-terminal extracellular binding domain of the porcine LH receptor impaired fertility. Male rats were less affected than male mice. Circulating maternal anti-LH receptor antibodies supressed fertility of the young rats to the same extent as did active immunization. In view of designing a more efficient and specific antagonist of the LH receptor, potentially usable as an immunocastration tool, a peptide involved in hormone-receptor interaction (LH receptor peptide 20–36) was used as the antigen. Immunization of prepubertal male mice against the peptide qualitatively reproduced the effects of immunization against the whole binding domain of the receptor: antifertility was mostly due to sterilization and/or delayed initiation of fertility, rather than to impaired fecundity.