Gonadotropin receptors of Labeo rohita: Cloning and characterization of full-length cDNAs and their expression analysis during annual reproductive cycle.

Follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh), secreted from pituitary, stimulate gonadal function by binding to their cognate receptors FSH receptor (FSHR), and LH/choriogonadotropin receptor (LHCGR). Rohu (Labeo rohita) is a commercially important seasonal breeder freshwater fish species, but till date, the regulation of expression of gonadotropins and their receptors gene during different phases of annual reproductive cycle has not been investigated. We envisaged the critical role of these molecules during seasonal gonadal development in this carp species. We cloned full- length cDNAs of fshra and lhcgrba from rohu testis using RACE (Rapid amplification of cDNA ends) and analyzed their expression along with fsh and lh by quantitative real time PCR (qRT-PCR) assay at various gonadal developmental stages of the annual reproductive cycle. Full-length rohu fshra and lhcgrba cDNA encodes 670 and 716 amino acids respectively, and in adult fish, they were widely expressed in brain, pituitary, gonad, liver, kidney, head kidney, heart, muscle, gill, fin, eye and intestine. In male, both fsh and fshra transcripts showed high level of expression during spermatogenesis, however, in female, expression level was found to be higher in the fully grown oocyte stages. The expression of rohu lh and lhcgrba mRNA increased with increment of gonadosomatic index and showed highest level during spermiation stage in male and fully matured oocyte stage in female. These results together may suggest the involvement of fshra and lhcgrba in regulating function of seasonal gonadal development in rohu.

Purification of two types of gonadotropin receptors from carp ovarian follicles: overlapping recognition by two different ligands.

Teleostean gonadotropin receptors were solubilized from the plasma membrane preparation of carp ovarian follicles by lithium diiodosalicylate and Triton X-100. Solubilization resulted fourfold increase in GTH binding activity as compared to the crude plasma membrane preparation. An addition of 25% glycerol and protease inhibitors to the solubilized receptor retained more than 90% original activity at -20 degrees C for 30 days. Gel filtration through Sephadex G 100 significantly increased the specific binding capacity from 70fmol/mg protein (soluble receptor) to 250fmol/mg protein. Peak I of gel filtration showing the receptor activity was further purified by affinity chromatography on purified salmon GTH II - Sepharose with a remarkable increase in specific binding capacity from 250fmol/mg protein (gel filtration peak) to 2300pmol/mg protein for salmon GTH I ligand and 2800pmol/mg protein for GTH II ligand. In SDS-PAGE affinity eluate the active peak showed two distinct bands corresponding to 66 and 62kDa molecular masses. These two proteins were clearly separated in FPLC Mono S chromatography, 62kDa as GTHR I and 66kDa as GTHR II. The former preferably binds to GTH I ligand, while the latter to GTH II, although both demonstrated overlapping recognition to both the ligands. GTH receptor protein I (GTHR I) and II (GTHR II) were purified 42,000- and 54,000-fold, respectively. Competitive binding inhibition studies indicate GTH I and ovine FSH a better ligand for GTHR I, while GTH II and ovine LH were preferable ligands for GTHR II. Biological relevance of these two receptor proteins was ascertained by monitoring the specific binding capacity of GTHR I and II at different stages of the annual reproductive cycle. GTHR I-GTH I was a prevailing complex during preparatory and pre-spawning stages, while GTHR II-GTH II was the dominant one at the maturational and final maturational stages. It may be concluded there are two GTH receptor proteins, each having a preferred ligand. The overlapping ligand-binding activity and profile of each receptor ligand complex suggest their link to seasonal development and maturation of carp ovarian follicle.

Cloning, functional characterization, and expression of a gonadotropin receptor cDNA in the ovary and testis of amago salmon (Oncorhynchus rhodurus).

A gonadotropin receptor was cloned from amago salmon (Oncorhynchus rhodurus) ovarian follicles. This receptor (sGTH-R) belongs to the glycoprotein hormone receptor family with a large extracellular and seven-transmembrane domains. Its sequence homology is highest with mammalian LH receptors. Phylogenetic analysis reveals that sGTH-R is grouped with mammalian and chicken FSH and LH receptors, but not with mammalian TSH receptors. sGTH-R is expressed dominantly in the ovary and testis. Functional characterization examined with transiently transfected mammalian cells revealed increased intracellular cAMP level when exposed to mammalian and fish gonadotropins; the most potent hormone was salmon GTH II. These results indicate that the cloned cDNA encodes a functional amago salmon GTH receptor protein.

Solubilization and purification of the gonadotropin (GTH II) receptor from rainbow trout (Oncorhynchus mykiss) ovaries.

The rainbow trout (Oncorhynchus mykiss) ovarian gonadotropin (GTH II) receptor was solubilized by extraction with the nonionic detergent 1% Triton X-100 in the presence of 20% glycerol. The hormone-binding characteristics of the soluble receptors were similar to those of membrane-bound receptors: the Scatchard plot of the equilibrium binding data produced a straight line, suggesting that the solubilized GTH II receptors, like membrane-bound receptors, contained a single class of high affinity 125I-sGTH II binding sites with an association constant of 2-5 x 10(10) M-1 (Ka = 1.4-2 x 10(10) M-1 for membrane-bound receptor). The maximal binding capacity was very low and varied from 7 to 17 fmol/mg proteins (about 5 fmol/mg ovarian membrane protein). The soluble receptor was purified by a simple and rapid immunoaffinity chromatography. The sGTH II-solubilized receptor complex was adsorbed to anti-sGTH II beta-subunit gammaglobulins covalently linked to Sepharose 4B and then eluted with an acidic buffer. About 50% of the binding activity present in the Triton X-100 extract was recovered in the pH 4 eluate. The other binding sites were eluted as a hormone-receptor complex and/or a damaged form. The free purified receptor presented a Ka of 1.3 x 10(10) M-1 in agreement with those found in membrane preparation and solubilized extract.

Purification of gonadotropin releasing hormone receptors using the avidin-biotin technique.

The avidin-biotin technique has been applied to the purification of gonadotropin releasing hormone (GnRH) receptors from other solubilized membrane proteins. The following steps were involved in this approach: (a) solubilization of rat pituitary GnRH receptor with the zwitterionic detergent CHAPS, 3-[(3-cholamidopropyl)-di-methylammonio]-1-propane sulfonate, (b) equilibration of the solubilized GnRH receptor with [biotinyl-D-Lys6]GnRH immobilized on avidin-agarose; and (c) elution of the receptors with high salt and GnRH analogues. Following two cycles of affinity chromatography the GnRH receptor was purified to homogeneity. The overall recovery of the purified receptor was 4-10% of the initial activity in the CHAPS extract and the calculated purification was approximately 10,000 to 15,000 fold. The development of a two step affinity chromatography for the purification of GnRH receptors can be used for detailed studies on the structure and function of the receptor. These studies will advance our understanding of the molecular basis of GnRH action.

Evidence for monomeric and oligomeric hormone-binding domains in affinity-purified gonadotropin receptor from rat ovary.

Rat ovarian lutropin/choriogonadotropin receptor was purified from a Triton X-100-solubilized membrane preparation by affinity chromatography with Affi-Gel 10 coupled to purified human choriogonadotropin. The affinity-purified receptor preparations contained a single class of high-affinity binding sites for 125I-labeled human choriogonadotropin, with an equilibrium dissociation constant (Kd) of 2.5 x 10(-9) M, which is comparable to the Kd values for membrane-bound and solubilized receptors. The purified receptor appeared as two dominant bands with molecular weights of 135,000 and 92,000 after sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) under nonreducing conditions. These two bands were also detected in subsequent direct ligand blotting analysis when the purified receptor was electrophoretically transferred to a nitrocellulose membrane after SDS/PAGE under nonreducing conditions. When the individual affinity-purified receptor bands were electroeluted from the gel and analyzed again by SDS/PAGE under nonreducing conditions, both the Mr 92,000 and the 135,000 proteins retained their original molecular form even when 8 M urea was included in the gel. However, when the electrophoretically purified Mr 92,000 and 135,000 bands were subjected to SDS/PAGE under reducing conditions, the Mr 135,000 species was almost completely converted to a Mr 92,000 band, but the Mr 92,000 species did not undergo any alteration in molecular weight. The results suggest that the lutropin/choriogonadotropin receptor from rat ovary exists in two molecular forms, and the higher molecular weight form appears to be composed of disulfide-linked Mr 92,000 subunit, which comprises the hormone-binding domain.

Glycosylation characteristics of the mouse liver lactogenic receptor.

The structural characteristics and glycosylation properties of the lactogenic receptor were examined in 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS)-solubilized plasma membranes from female mouse liver. The specific binding of the radioiodinated human growth hormone [( 125I]hGH) was displaced with an equivalent potency by both hGH and prolactin. After a mild neuraminidase treatment, this binding was increased by 40%, as a result of an increase in receptor affinity. Affinity chromatography on immobilized lectins revealed that the [125I]hGH-receptor complexes were specifically retained and eluted from ricin lectin-agarose, concanavalin A and lentil lectin, indicating the presence of N-linked glycans. Covalent cross-linking of solubilized [125I]hGH-receptor complexes with disuccinimidyl suberate, followed by analysis by sodium dodecyl sulfate-gel electrophoresis (SDS-PAGE) under reducing conditions, and autoradiography resulted in the appearance of two bands with apparent Mr approximately 62,000 and approximately 100,000. The labelling of these bands was prevented by unlabelled hGH or ovine prolactin (oPrl) but not by bovine growth hormone (bGH). Neuraminidase treatment of the two receptor forms resulted in increased electrophoretic mobility which was inhibited by simultaneous addition of sialyl-lactose, a neuraminidase substrate. The both cross-linked forms were unaffected by endoglycosidase H, while endoglycosidase F decreased the molecular weight of each of the forms by about 8000 Da, yielding bands at Mr approximately 54,000 and approximately 92,000. In conclusion, taking into account that hGH is a Mr 22,000 polypeptide, the two forms of the receptor correspond to glycoproteins of Mr approximately 40,000 and approximately 78,000, respectively. They contain polypeptide backbones of Mr approximately 32,000 and approximately 70,000, and complex N-linked oligosaccharide chains with terminal sialic acid residues which could be involved in receptor binding affinity.

[Gonadotropin receptors]. / Les récepteurs des gonadotropines.

The reproductive function of both the male and female is under the control of several pituitary hormones including lutropin (LH) and follitropin (FSH). These hormones are highly homologous glycoprotein dimers sharing a common subunit. Their polymorphism mainly resides in the microheterogeneity of their carbohydrate chains that is also responsible for the variations in biological activity. In the gonads, these hormones bind to specific receptors and regulate steroidogenesis and gametogenesis. LH receptors also bind the choriogonadotropin hormone and are located in Leydig cells in testis, interstitial and medulla cells in ovary. FSH receptors are present in Sertoli cells in the male while they control follicular maturation in the female. In both sexes, their activity is coupled to that of other receptors such as that of prolactin or one able to recognize GnRH. The purification of LH receptor has been successfully achieved in several animal species and showed a significant structural homology between the receptors of both sexes.

Hydrodynamic properties of the gonadotropin receptor from a murine Leydig tumor cell line are altered by desensitization.

The murine Leydig tumor cell line 1 (MLTC-1) contains gonadotropin receptors (GR) that are coupled to adenylate cyclase through the stimulatory guanine nucleotide binding protein (Gs). The binding of human choriogonadotropin (hCG) causes MLTC-1 cells to accumulate cAMP. With time, the ability of MLTC-1 cells to respond to hCG is attenuated by a process called desensitization. The hydrodynamic properties of GR from control and desensitized MLTC-1 cells were studied. Sucrose density gradient sedimentation in H2O and D2O and gel filtration chromatography were used to estimate the Stokes radius (a), partial specific volume (vc), sedimentation coefficient (S20,w), and molecular weight (Mr) of the detergent-solubilized hormone-receptor complex (hCG-GR). [125I]hCG was bound to MLTC-1 cells under conditions that allow (37 degrees C) or prevent (0 degree C) desensitization, and hCG-GR was solubilized in Triton X-100. In the absence of desensitization, control hCG-GR had a Mr of 213,000 (a = 6.2; vc = 0.76; S20,w = 7.3), whereas desensitized hCG-GR had a Mr of 158,000 (a = 6.1; Vc = 0.71; S20,w = 6.6). Deglycosylated hCG (DG-hCG) is an antagonist that binds to GR with high affinity but fails to stimulate adenylate cyclase or cause desensitization. [125I]DG-hCG was bound to MLTC-1 cells and DG-hCG-GR solubilized in Triton X-100. The hydrodynamic properties of DG-hCG-GR (Mr 213,000; a = 5.8; Vc = 0.77; S20;w = 7.6) were the same as that for control hCG-GR. There was no evidence for the association of adenylate cyclase or Gs with GR in Triton X-100 solubilized preparations.(ABSTRACT TRUNCATED AT 250 WORDS)

Subunits of luteinizing hormone-human chorionic gonadotropin receptor from bovine corpora lutea.

A batch of 24 mg of luteinizing hormone-human chorionic gonadotropin (LH-hCG) receptor was isolated from bovine corpora lutea. The LH-hCG receptor showed specific binding with hCG. The receptor-hCG complex activated the regulatory Ns protein isolated from rabbit liver, which in turn stimulated adenylate cyclase to convert ATP into cAMP in vitro, attesting to the biological activity of the purified LH-hCG receptor. The LH-hCG receptor was treated with 2% sodium dodecyl sulfate (SDS) to prepare the molecular weight (Mr) 280K dimer and with 50 mM dithiothreitol (DTT) to prepare the Mr 120K monomer and subunits of Mr 85K and 38K. Oligomers of various molecular weights were recovered from gel filtration columns due to the reassociation of disulfide bonds between monomers and subunits. Hence, the receptor monomer was also dissociated into subunits of Mr 85K and 38K by reduction of -S-S-bonds with 50 mM DTT in 2% SDS and alkylation of sulfhydryl groups in the presence of 100 mM N-ethyl-maleimide. The subunits were separated by gel filtration through columns of Ultrogel AcA-44 and Sephadex G-75. The yields of the purified alkylated subunits of Mr 85K and 38K were 1.8 and 1.5 mg, respectively. Each subunit migrated as a single entity in SDS-polyacrylamide gel electrophoresis. The monomer of the receptor of Mr 120K showed specific binding with 125I-hCG, suggesting it to be the minimum molecular weight functional unit of the receptor. The Mr 85K and 38K subunits bound 125I-hCG, which could not be displaced with unlabeled hCG.(ABSTRACT TRUNCATED AT 250 WORDS)

Autoradiographic localization of gonadotrophin receptors in ovaries of the common carp, Cyprinus carpio L.

Binding sites for carp gonadotrophin have been located in carp ovaries using [125I]labeled gonadotrophin and autoradiography. The radioactive gonadotrophin was displaced from tissue by unlabeled gonadotrophin or carp hypophysial homogenate in a dose-dependent fashion. No binding of gonadotrophin was found in previtellogenic oocytes but binding appeared with the first indications of vitellogenesis. In the smaller vitellogenic oocytes binding was uniformly distributed in the follicular envelope, but in the largest oocytes binding was restricted to the interstitial tissue. In these more mature oocytes gonadotrophin was also found within the oocyte and appeared to migrate toward the nucleus. The relationship between binding location, steroidogenesis, and oocyte maturation is discussed. We found no evidence for specific binding of [125I]thyroxine under comparable conditions.