Mutations in the luteinizing hormone/chorionic gonadotropin receptors (LH/CGRs), representatives of the G protein-coupled receptor family, have been rapidly identified over the last 20 years. This review aims to compare and analyze the data reported the activating and inactivating mutations of the LH/CGRs between human, rat, equine and fish, specifically (Japanese eel Anguilla japonica). Insights obtained through detailed study of these naturally-occurring mutations provide a further update of structure-function relationship of these receptors. Specifically, we present a variety of data on eel LH/CGR. These results provide important information about LH/CGR function in fish and the regulation of mutations of the highly conserved amino acids in glycoprotein hormone receptors.

Equine chorionic gonadotropin (eCG) is a member of the glycoprotein hormone. eCG, over 40%, is a heavily glycosylated glycoprotein than other glycoprotein hormones. eCG is composed of non-covalently linked α and β subunit. The α subunit is common to all glycoprotein hormones, whereas the β subunit was known distinct sequences and specific receptor binding. Unusually, eCG shows both FSH and LH activities in other species. eCG α subunit has two N-glycosylation sites (Asn56, Asn82) and β subunit has one N-glycosylation site (Asn13) and about 13 O-glycosylation sites in the C-terminal region. We constructed 3 type mutants (βα△56: α-subunit Asn56→ Gln56; β-Da: β-subunit C-terminal deletion; β-Dα△56: β C-terminal deletion & α Asn56→Gln56) in the tethered eCGβα (wild type) and all mutants included myc-tag between first and second amino acid of β subunit. The plasmid DNAs cloned into pcDNA3 mammalian expressing vector were transiently transfected into CHO-Suspension cells. We also constructed rat LH/CG receptor and rat FSH receptor into pcDNA3 expression vector. These receptors were transiently transfected into CHO-K1 cell. Each receptor cells were used for further assays at 3 days after transfection. cAMP and IP-one were evaluated by CISBIO cAMP HiRange and IP-one kits using the rec-eCGβα mutants. According to cAMP assay results, IC50 values of 4 type ligand treatment in the rat FSH receptor cells were: eCGβα: IC50_16.8841; eCGβα56: IC50_95.6099; eCGβ-Dα: IC50_395.0087; eCGβ-Dα56: IC50_1439.8702. In the rat LH/CG receptor cells of 4 types ligand treatments, cAMP results were: eCGβα: IC50_0.9760; eCGβα56: IC50_8.3884; eCGβ-Dα: IC50_9.2550; eCG β-Dα56: IC50_45.9439. As seen in these data, β C-terminal region and α Asn56 play an important role in rat FSHR and rat LH/CGR, respectively. And rat LHCG receptor cells was remarkably stronger than rat FSH receptor cells. According to IP-one assay, IC50 values in rat FSH receptor cells, the results were: eCGβα: IC50_561.4490; eCGβα56: IC50_361.3005; eCGβ-Dα: IC50_911.8577; eCGβ-Dα56: IC50_139.1193. And in rat LH/CG receptor cells, IP-one results were: eCGβα: IC50_422.7315; eCGβα56: IC50_406.4915; eCGβ-Dα: IC50_537.8300; eCGβ-Dα56: IC50_254.2004. As shown in these data, IP-one result was a little different to cAMP result. The β eCGβ-Dα56 of IC50 value was shown generally high signal. Now we are trying to analyse role of C-terminal region of eLH/CGR with cAMP, IP-one and ERK signal transduction assays.

Glycoprotein hormones have a common α-subunit that is involved in the signaling pathway together with G protein, adenylcyclase and cAMP induction; however, it is an unclear how this common structure is related to hormonal action. To determine the biological functions of the COOH-terminal amino acids in the α-subunit of these glycoprotein hormones, a tethered-molecule was constructed by fusing the NH2-terminus of the α-subunit to the COOH-terminus of the β-subunit of equine chorionic gonadotropin (eCG). The following deletion mutants were created by PCR; Ile was inserted at position 96 to form Δ96, Lys was substituted at position 95 to form Δ95, His was inserted at position 93 to form Δ93 and Tyr was substituted at position 87 to form Δ87. Each mutant was transfected into CHO-K1 cells. Tethered-wt eCG, and Δ96, Δ95, and Δ93 mutants were efficiently secreted into the medium but the Δ87 mutant was not secreted. Interestingly, the RT-PCR, real-time PCR, and northern blot analyses confirmed that the RNA was transcribed in the Δ87 mutant. However, the Δ87 mutant protein was not detected in the medium or the intracellular fraction of the cell lysates. The LH- and FSH-like activities of the recombinant proteins were assayed in terms of cAMP production using rat LH/CG and rat FSH receptors. The metabolic clearance rate (MCR) was determined by injecting rec-eCG (2 IU) into the tail vein. The Δ95 and Δ93 mutants were completely inactive in both the LH- and FSH-like activity assays. The Δ96 mutant showed slight activity in the LH-like activity assay. In comparison to the wild type, the activity of the Δ96 mutant in the FSH-like activity assay was the highest among all the mutants. The MCR assay in which rec-eCG was injected showed a peak at 10 min in all the treatment groups, which disappeared 4 h after injection. These results imply a direct interaction between the receptor and the COOH-terminal region of the α-subunit. The data also reveal a significant difference in the mechanism by which the eCG hormone interacts with the rLH and rFSH receptors. The COOH-terminal region of the α-subunit is very important for the secretion and functioning of this hormone.

Equine chorionic gonadotropin (eCG) is a heavily glycosylated glycoprotein composed of non-covalently linked α- and β-subunits. To study the function and signal transduction of tethered recombinant-eCG (rec-eCG), a single chain eCG molecule was constructed, and the rec-eCG protein was prepared. In this study, we constructed 5 mutants (Δ1, Δ2, Δ3, Δ4, and Δ5) of rec-eCG using data about known glycoprotein hormones to analyze the role of specific follicle stimulating homone (FSH)-like activity. Three amino acids of certain specific sites were replaced with alanine. The expression vectors were transfected into CHO cells and subjected to G418 selection for 2～3 weeks. The media were collected and the quantity of secreted tethered rec-eCGs was quantified by ELISA. The LH- and FSH-like activities were assayed in terms of cAMP production by rat LH/CG and rat FSH receptors. Then, the metabolic clearance rate analyzed by the injection of rec-eCG (5 IU) into the tail vein was analyzed. The mutant eCGs (Δ1, Δ4, and Δ5) were transcripted, but not translated into proteins. Rec-eCG Δ2 was secreted in much lower amounts than the wild type. Only the rec-eCG Δ3 (β-subunit: Gln94-Ile95-Lys96→Ala94-Ala95-Ala96) was efficiently secreted. Although activity is low, its LH-like activity was similar to that of tethered eCGβα. However, the FSH-like activity of rec-eCGβαΔ3 was completely flat. The result of the analysis of the metabolic clearance rate shoed the persistence of the mutant in the blood until 4 hours after the injection. After then, it almost disappeared at 8 hours. Taken together, these data suggest that 94～96 amino acid sequences in eCG β-subunit appear to be of utmost importance for signal transduction of the FSH receptor.

The objective of this study was to examine the effect of eCG and various concentrations (20, 40, and 80 ) of porcine FSH on nuclear maturation and intracellular glutathione (GSH) level of oocytes, and embryonic development after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) in pigs. Immature pig oocytes were matured in TCM-199 supplemented with porcine follicular fluid, cysteine, pyruvate, EGF, insulin, and hormones (10 IU/ml hCG and 10 IU/ml eCG or FSH) for the first 22 h and then further cultured in hormone-tree medium for an additional 22 h. Nuclear maturation of oocytes () was not influencem foreCG and various concentrations FSH. Embryonic development to the cleavage stage () and mean number of cells in blastocyst ( cells) after PA were not altered but blastocyst formation e-treignificaddlor(p<0.05) improvem forthe supplementation eith 80 FSHr(64%) compared to 47%, io8%, iand 47% in oocytes that were treated with eCG, 20,i and 40 FSH,i numectivelo. In SCNT, fusion () of cell-cytoplast couplets and siosequent embryo cleavage () were not influencem fordifferent gonadotropins but blastocyst formation tended to increase forthe supplementation eith 80 FSHr(25% vs. ). Our nuults demonstrated that oocyte maturation and embryonic development after PA and SCNT e-frinfluencem fortype of gcem fortype of gits concentration. In this study, supplementation of maturation medium eith 80 FSHrimproved preimplantation development of PA and SCNT pig embryos, probably by increasing intracellular GSH concentration of matured oocytes.

Previous studies showed that recombinant equine chorionic gonadotropin (rec-eCGβ/α) exhibits both folliclestimulating hormone (FSH) and luteinizing hormone (LH)-like activities in rat LHR- and FSHR-expressing cells. In this study, we analyzed signal transduction by eelFSHR and eelLHR upon stimulation with rec-eCGβ/α and native eCG. The cyclic adenosine monophosphate (cAMP) stimulation in CHO-K1 cells expressing eelLHR was determined upon exposure to different doses (0–1,450 ng/mL) of rec-eCGβ/α and native eCG. The EC50 values of rec-eCGβ/α and native eCG were 172.4 and 786.6 ng/mL, respectively. The activity of rec-eCGβ/α was higher than that of native eCG. However, signal transduction in the CHO PathHunter Parental cells expressing eelFSHR was not enhanced by stimulation with both agonist rec-eCGβ/α and native eCG. We concluded that rec-eCGβ/α and native eCG were completely active in cells expressing eelLHR, similar to the activity in the mammalian cells expressing LHRs. However, rec-eCGβ/α and native eCG did not invoke any signaling response in the cells expressing eelFSHR. These results suggest that eCG has a potent activity in cells expressing eelLHR. Thus, we also suggest that rec-eCGβ/α can induce eel maturation by administering gonadotropic reagents (LH), such as salmon pituitary extract.