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 follicle stimulating hormone receptor (eFSHR) has a large extracellular domain and an intracellular domain containing approximately 10 phosphorylation sites within the G protein-coupled receptor. This study was conducted to analyze the function of phosphorylation sties at the eFSHR C-terminal region. We constructed a mutant of eFSHR, in which the C-terminal cytoplasmic tail was truncated at residue 641 (eFSHR-t641). This removed 10 potential phosphorylation sites from the C-terminal region of the intracellular loop. The eFSHR-wild type (eFSHR-wt) and eFSHR-t641 cDNAs were subcloned into the pCMV-ARMS1-PK2 expression vector. These plasmids were transfected into PathHunter CHO-K1 Parental cells expressing β-arrestin 2 enzyme acceptor fusion protein and analyzed for agonist-induced cAMP response. The cAMP response in cells expressing eFSHR-t641 was lower than the response in cells expressing eFSHR-wt. EC50 values of eFSHR-wt and eFSHR-t641 were 1079 ng/mL and 1834 ng/mL, respectively. eFSHR-t641 was approximately 0.58-fold compared with that of eFSHR-wt. The maximal response in eFSHR-wt and eFSHR-t641 was 24.7 nM and 16.7 nM, respectively. The Rmax value of phosphorylation sites in eFSHR-t641 was also decreased to approximately 68.4% of that in eFSHR-wt. The collective data implicate that the phosphorylation sites in the eFSHR C-terminal region have a pivotal role in signal transduction in PathHunter CHO-K1 cells, and indicate that β-arrestin is involved in coupling the activated receptors to the internalization system.

This study aimed to investigate the function of the constitutively activating mutation D540G on eel FSHR activity by in vitro functional studies. Site-directed mutagenesis was carried out to generate the D-to-G mutation at position 540 of the pcDNA3-eel FSHR construct. Vectors expressing either wild type or mutant receptor were transfected into Chinese hamster ovary (CHO-K1) cells. The functional characteristics of both the wild type and mutant receptors were analyzed by a cAMP assay. cAMP accumulation was highly increased in cells transfected with the D540G mutant receptor in a dose-dependent manner. Of note, basal cAMP levels were remarkably increased (~13.1-fold) with expression of this mutant when compared to wild type receptor. These findings suggest that the D540G mutation in the eel FSHR may contribute to ovulation during eel sex maturation as well as play a pivotal role in inducing FSHR activity.

This study was conducted to analyze the specific genes associated with sex-determination in Korean native cow. The highly organized spermatogenesis requires accurate spatial and temporal regulation of gene expression, which is governed by transcriptional, post-transcriptional, and epigenetic processes. Recently, farmers have been interested in determining the sexual identity of the calves in their farm. We analyzed the sperm of Korean native and Holstein cows, which were supplied from Hanwoo Improvement Center. We evaluated sperm motility and expression of sperm-specific genes after treating semen with both male- and female reagents. Sperm motility in Korean native cows decreased by approximately 10% in the first 30 minutes after treatment with sex-determination reagent. However, sperm motility of Holstein cows decreased to 60-70% after 15 minutes and to 20-30% after 30 minutes. We selected six specific genes expressing in the spermatozoa to analysis the gene expression level. The Real-time PCR results suggest that the selected genes (Gimap4, Tmeff1, Rac2, Abi2, Rac1, and Clu) were highly expressed in the group treated with the male reagent compared to the group treated the female reagent and to the untreated-group (control). In the present study, we suggest that the selected genes play a pivotal role in sex-determination.

The lutropin/chorionicgonadotropin receptor (LHR) is a member of the rhodopsin-like subfamily of G protein-coupled receptors (GPCRs) that have been shown to mediate the internalization of its five (activation: three; inactivation: two) naturally occurring mutation. Gonadotropin receptors are members of the seven transmembrane (TM) receptor families. Several point mutations in TM II, III, V and VI have been identified in the luteinizing hormone receptor (LHR) gene, leading to constitutive activation and inactivation of the receptor. In eelLHR, we generated 3 types of constitutive activating mutations (M410T, L469R and D590Y) and 2 types of constitutive inactivating mutations (D383N and Y546F) to investigate how they work on hormone-receptor interaction. To assess the functional effects of 5 receptor mutations directly, wild-type (WT) and mutant receptors were transiently expressed in CHO-K1 cells. We evaluated the basal and cAMP stimulation by rec-LH hormone. The activity was shown to be a dose-dependent increase in cAMP production in LHR-WT expressing cells with an EC50 of 24.3 ng/ml and basal cAMP level of 2.6 nM. However, three activation mutants (D590Y, L469R and M410T) was most elevated the basal cAMP response at 12.8, 21.7 and 6.1 nM, respectively. In two inactivation mutants (D383N and Y546F) are very low in the basal cAMP activation. The EC50 was also considerably decreased to 42.3 ng/ml and 1181 ng/ml, respectively.

This study was conducted to analyze the specific genes associated with sex-determination in semen of Korean Native cow. Male fertility is dependent upon the successful perpetuation of spermatogenesis that is a highly organized process of germ cell differentiation occurring within the seminiferous tubules in the testis. The highly organized spermatogenesis requires accurate, spatial and temporal regulation of gene expression governed by transcriptional, post-transcriptional and epigenetic processes. Recently, the farmers have been interesting in the male or female of calves in the their farm. In first, we analyzed the semen supplied from Hanwoo Improvement Center, NongHyup. The sperm motility in Hanwoo was decreased approximately 10 % in the 30 min after sex-determinant reagent. However, Holstein' sperm motility was decreased to 60-70% after 15 min and the motility was considerably decreased to 20-30 % after 30 min. Next, we analyzed the sperm specific expression genes both male- and female reagents treated-group. The real-time PCR results suggest that the selected genes (GIMP4, TMEFF1, RAC2, ABI2, RAC1, and CLUS) were highly expressed in the group treated with the male reagent compared to female reagent treated group and untreated-group. In the present study, although X or Y gene is play a key role in the sex-determination of mammalian, we suggest that the selected genes may be involved in the sex-determination.

In this review, we have tried to summarize the evidence and molecular characterization indicating that 20α-hydroxysteroid dehydrogenase (20α-HSD) is a group of the aldo-keto reductase (AKR) family, and it plays roles in the modulation and regulation of steroid hormones. This enzyme plays a critical role in the regulation of luteal function in female mammals. We have studied the molecular expression and regulation of 20α-HSD in cows, pigs, deer, and monkeys. The specific antibody against bovine 20α-HSD was generated in a rabbit immunized with the purified recombinant protein. The mRNA expression levels increased gradually throughout the estrous cycle, the highest being in the corpus luteum (CL) 1 stage. The mRNA was also specifically detected in the placental and ovarian tissues during pregnancy. The 20α-HSD protein was intensively localized in the large luteal cells and placental cytotrophoblast villus, glandular epithelial cells of the endometrium, syncytiotrophoblast of the placenta, the isthmus cells of the oviduct, and the basal part of the primary chorionic villi and chorionic stem villus of the placenta and large luteal cells of the CL in many mammalian species. Further studies are needed to determine the functional significance of the 20α- HSD molecule during ovulation, pregnancy, and parturition. This article will review how fundamental information of these enzymes can be exploited for a better understanding of the reproductive organs during ovulation and pregnancy.

In this study, we analyzed signal transduction by equine follicle-stimulating hormone receptor (eFSHR) on stimulation with recombinant eelFSHβ/α (rec-eelFSHβ/α), natural porcine FSH (pFSH), and natural human FSH (hFSH). cAMP stimulation in CHO-K1 cells expressing eFSHR was determined upon exposure to different doses (0-1450 ng/mL) of these hormones. The EC50 value of rec-eelFSHβ/α was 53.35 ng/mL. The Rmax values of rec-eelFSHβ/α and pFSH were 28.12 and 2.88 ng/mL, respectively. The activity of rec-eelFSHβ/α was much higher than that of natural pFSH. However, signal transduction in CHO PathHunter Parental cells expressing eFSHR was not enhanced by stimulation with natural hFSH. Thus, rec-eelFSHβ/α was completely active in cells expressing eFSHR. However, natural hFSH did not invoke a signal response in cells expressing eFSHR. Particularly, natural pFSH was weakly active in the same cells. These results showed that eelFSHβ/α has potent activity in cells expressing eFSHR. Thus, rec-eelFSHβ/α may efficiently bind to eFSHR, where as natural hFSH does not bind to eFSHR.

In all mammalian species, progesterone is essential in the preparation for and maintenance of pregnancy, if it occurs. Progesterone primes the endometrium for possible implantation and inhibits uterine contraction until birth. 20-alpha hydroxysteroid dehydrogenase (20α-HSD; EC.1.1.1.149) enzyme belongs to the family of aldo-keto reductases. 20α-HSD predominantly converts progesterone into its biologically inactive form 20α-hydroxyprogesterone (20α-OHP), and plays a crucial role in the termination of pregnancy and initiation of parturition. In addition, the activity of 20α-HSD during the luteal phase known to be inhibited by prolactin. In this study, we focused on the analysis of transgenic mice expressing EGFP under control of monkey 20α-HSD promotor in mice testis. The protein expression and localization were detected by Western blotting and Immunohistochemical analysis, respectively. 20α-HSD protein was detected at molecular weight of 37-kDa by Western blotting analysis and EGFP was found at 27-kDa in the testis of TG mice. Also EGFP and 20a-HSD protein expression on 1, 2, 4, 6 and 8 weeks after birth were assessed. Both of them were increased the expression level time-dependently. 20α-HSD were strongly expressed in seminiferous tubule from 1 week after birth as seen in Immunohistochemical analysis. However, EGFP was strongly expressed in the seminiferous epithelial cells. Then, we determined the expression of EGFP mRNA in mice testis. Using primers specific for mouse EGFP, mRNA expression levels were analyzed by RT-PCR. The EGFP molecular weights is 400bp, qRT-PCR results using EGFP primer, The Cq value of the ratio decreased as the age increased. On this basis, mRNA were increased the expression level time-dependently. In conclusion, these observations suggest that the 20α-HSD in testis could be play a pivotal role in the spermatogenesis.

In all mammalian species, progesterone is essential in the preparation for and maintenance of pregnancy, if it occurs. Progesterone primes the endometrium for possible implantation and inhibits uterine contraction until birth. Aldo-keto reductases (AKRs) belong to a superfamily of NADPH-dependent reductases that act on a wide range of substrates, including simple carbohydrates, steroid hormones, and endogenous prostaglandins. 20-alpha hydroxysteroid dehydrogenase (20α-HSD; EC.1.1.1.149) enzyme belongs to the family of aldo-keto reductases. 20α-HSD predominantly converts progesterone into its biologically inactive form 20α-hydroxyprogesterone (20α -OHP), and plays a crucial role in the termination of pregnancy and initiation of parturition. In addition, the activity of 20α-HSD during the luteal phase is known to be inhibited by prolactin. We have been reporting on the molecular characterizations of placental and ovarian 20α-HSD in the bovine, pig, deer and monkey. In this study, we focused on the 20α-HSD expression in testis(6, 9, 12, 18 and 21 days after birth) of miniature pig. The protein expression and localization were detected by Western blotting and Immunohistochemical analysis. 20α-HSD protein was detected at molecular weight of 37-kDa by Western blot analysis. Also the RNA expression were detected by Reverse Transcription-PCR and quantification PCR. Additionally, We are going to analysis the function and role of 20α-HSD in the pig testis.

The glycoprotein hormone family consists of follicle-stimulating hormone (FSH; GTH1), luteinizing hormone (LH; GTH2), and thyroid-stimulating hormone (TSH), which are secreted by the pituitary gland in all mammalian species, and chorionic gonadotropin, which is secreted by placental trophoblast cells in primates and equids. These hormones consist of non-covalently associated α-, β- subunits. Within a species, the amino acid sequence of α-subunit is identical across all glycoprotein hormones and is encoded by a single gene. The αβ dimer is the active form of the hormone, and biological specificity is conferred by the β-subunit. Both of α and β subunit of eel FSH has two N-glycosylation sites (α-subunit: Asn56 and Asn79; β -subunit: Asn5 and Asn22, respectively). In the present study, we constructed deglycosylated mutants at single and double sites in each subunits of eel FSH for identification of Asn linked oligosaccharides' biological role. Mutant cDANs were cloned into pcDNA3 expression vector and transiently transfected into CHO suspension cells. The quantity of rec-eelFSHs were quantified by sandwich ELISA system, using monoclonal antibodies produced in our lab. The wild type rec-FSH protein was detected at the predicted molecular weight of 34 kDa by western blot. The molecular weight of deglycosylated mutants at single site decreased with about 4 kDa and of mutants at double sites decreased with 8 kDa. After PNGase treatment in the rec-eel FSH proteins, molecular weight also decreased to 7-8 kDa. We generated stably parental cell lines, engineered to express a β-arrestin 2EA fusion protein, expressing eel FSHR and C-terminal deleted mutant. 2 out of 5 receptor cells each were selected by G-418 and we tested these cell lines in a receptor functionality using PathHunter β arrestin assay (DiscoverX). Follicle stimulating hormone acts through binding to its specific receptor. Binding of ligand to the receptor activates the adenosine 3',5'-cyclic monophosphate (cAMP) pathway (McFarland et al., 1989; Ji and Ji, 1991a; Rose, 1998) and the inositol 1phosphate (IP1) the second messenger systems. After stimulation of eelFSH receptor stably transfected Parental CHO cells with FSH wild type and mutant hormones as a ligand, production of cAMP and IP-1 were evaluated (Cisbio). cAMP IC-50 values by eelFSHwt; αΔ56; αΔ79; αΔ56_79; and βΔ5 were 33.1; 1154.7; 22; 410 and 311.9 ng/ml, respectively. IP-1 IC-50 values by eelFSHwt; αΔ56; αΔ79; αΔ56_79 and βΔ5 were 6.8; 7.1; 4.4; 3.8 and 10.2 ng/ml, respectively too. The cAMP activation was greatly decreased in the αΔ56αmutant. Thus, the site of α56 oligosaccharide in the eel plays an pivotal role for the cAMP stimulation using eel FSH receptor cell lines. In the IP-one assay, the activity in the αΔ56 and βΔ5 mutants was a little decreased than the wt. The biological roles of N-linked oligosaccharides in GPCR internalization are going to be estimated by measuring β arrestin recruitment system.

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.

The glycoprotein hormone family consists of luteinizing hormone (LH), follicle stimulating hormone (FSH) and thyroid stimulating hormone, which are secreted by the pituitary gland in all mammalian species, and choriogonadotropin (CG), which is secreted by the placenta in primates and equids. The hormones are composed of a common α subunit and a hormone specific β subunit which are non-covalently associated. Recent advances in biotechnology, particularly in the production of recombinant proteins, have provided opportunities to produce sufficient quantities of recombinant fish GTHs using various expression hosts. Japanese eel Anguilla japonica is one of the most important fish species being aquacultured in Japan but is hampered from the fact that this species does not reproduce in captivity. Artificial induction of gonadal maturation has been successful by administration of pituitary extracts or human chorionic gonadotropin, but the understanding the regulatory mechanism of gonadal development moderated by follicle stimulating hormone (FSH) and luteinizing hormone (LH) remains elusive due to lack of suitable amounts of eel gonadotropins (GTHs). In the present study, we produced tethered rec-eel LH and deglycosylated mutants (56, 79 and 56-79 of α subunit; 10 of β-subunit) of Asn-linked oligosaccharides in CHO suspension cells. Luteinizing hormone acts through binding to its specific receptor. Binding of ligand to the receptor activates the adenosine 3',5'-cyclic monophosphate (cAMP) pathway (McFarland et al., 1989; Ji and Ji, 1991a; Rose, 1998) and the inositol 1 phosphate (IP1) secondary messenger systems. After stimulation of eelLH/CG receptor transfected CHO cells with rec-LH wild type (wt) and mutant hormones as a ligand, production of cAMP and IP-1 were evaluated (Cisbio). cAMP IC-50 values by rec-eelLH wt; αΔ56; αΔ79; αΔ56_79 and βΔ10 were 606.2; 374.9; 100.3; 14.2 and 210.9 ng/ml, respectively. IP-1 IC-50 values by rec-eelLH wt; αΔ56; αΔ79; αΔ56.79 and βΔ10 were 28.3; 16.04; 4.3; 2.1 and 3.6ng/ml, respectively too. As seen in both of the second messenger production, general stimulatory pattern is analogous. cAMP and IP-1 stimulation by wild type and αΔ56, as well as αΔ79 and βΔ10 were approximate, but the stimulating effect of double mutant (αΔ56_ 79) was drastically higher. According to the data, deglycosylated eelLH may bind to the receptor with high affinity and cAMP production is gradually increased. Furthermore, receptor activation by tethered rec-eel mutant ligands (FreeStyle CHO-MAX Expression System) will be evaluated with β arrestin recruitment and GPCR internalization for N-linked oligosaccharides’ biological role in activation of eelLH/CGR.

The large extracellular domain of glycoprotein hormone receptors is a unique feature within the G protein-coupled receptors (GPCRs) family. After interaction with the hormone, the receptor becomes coupled to Gs, which, in turn stimulates adenylyl cyclase and the production of cAMP. Potential phosphorylation sites exist in the C-terminal region of GPCRs. The experiments described herein represent attempts to determine the functions of the eel follicle-stimulating hormone receptor (eelFSHR). We constructed a mutant of eelFSHR, in which the C-terminal cytoplasmic tail was truncated at residue 614 (eelFSHR-t614). The eelFSHR-t614 lacked all potential phosphorylation sites present in the C-terminal region of eelFSHR. In order to obtain the eelFSHR ligand, we produced recombinant follicle-stimulating hormone (rec-eelFSHβ/α) in the CHO-suspension cells. The expression level was 2-3 times higher than that of the transient expression of eelFSH in attached CHO-K1 cells. The molecular weight of the rec-eelFSHβ/α protein was identified to be approximately 34 kDa. The cells expressing eelFSHR-t614 showed an increase in agonist-induced cAMP responsiveness. The maximal cAMP responses of cells expressing eelFSHR-t614 were lower than those of cells expressing eelFSHR-wild type (eelFSHR-WT). The EC50 following C-terminal deletion in CHO-K1 cells was approximately 60.4% of that of eelFSHR-WT. The maximal response in eelFSHR-t614 cells was also drastically lower than that of eelFSHR-WT. We also found similar results in PathHunter Parental cells expressing β-arrestin. Thus, these data provide evidence that the truncation of the C-terminal cytoplasmic tail phosphorylation sites in the eelFSHR greatly decreased cAMP responsiveness and maximal response in both CHO-K1 cells and Path-Hunter Parental cells expressing β-arrestin.

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.