Signal Transduction of Eel Luteinizing Hormone Receptor (eelLHR) and Follicle Stimulating Hormone Receptor (eelFSHR) by Recombinant Equine Chorionic Gonadotropin (rec-eCG) and Native eCG

Byambaragchaa, Munkhzaya,Lee, So-Yun,Kim, Dae-Jung,Kang, Myung-Hwa,Min, Kwan-Sik The Korean Society of Developmental Biology 2018 발생과 생식 Vol.22 No.1

Previous studies showed that recombinant equine chorionic gonadotropin ($rec-eCG{\beta}/{\alpha}$) exhibits both follicle-stimulating 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 $rec-eCG{\beta}/{\alpha}$ 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 $rec-eCG{\beta}/{\alpha}$ and native eCG. The $EC_{50$ values of $rec-eCG{\beta}/{\alpha}$ and native eCG were 172.4 and 786.6 ng/mL, respectively. The activity of $rec-eCG{\beta}/{\alpha}$ 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 $rec-eCG{\beta}/{\alpha}$ and native eCG. We concluded that $rec-eCG{\beta}/{\alpha}$ and native eCG were completely active in cells expressing eelLHR, similar to the activity in the mammalian cells expressing LHRs. However, $rec-eCG{\beta}/{\alpha}$ 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 $rec-eCG{\beta}/{\alpha}$ can induce eel maturation by administering gonadotropic reagents (LH), such as salmon pituitary extract.

Mutations that induce constitutive activation or inactivation in lutropin/choriogonadotropin receptors

Munkhzaya Byambaragchaa,Kwan-Sik Min 한국수정란이식학회 2018 한국수정란이식학회 학술대회 Vol.2018 No.11

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.

Functions of Eel Luteinizing Hormone Receptor Mutants (Activating and Inactivating Receptors)

Munkhzaya Byambaragchaa,Jeong-Soo Kim,Hun-Ki Seong,Kwan-Sik Min 한국동물생명공학회(구 한국동물번식학회) 2017 Reproductive & Developmental Biology(Supplement) Vol.41 No.2

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 and receptor activation system on eel. To assess the functional effects of 5 receptor mutations directly, wt and mutant eel- LHRs were transiently expressed in CHO-K1 cells, and basal and recombinant eel LH-stimulated cAMP and IP-1 accumulations were measured. Rec-eelLH (0.076~1,200 ng/mL) produced a concentration-dependent increase in cAMP production in wt eelLHR expressing cells with an EC50 of 160 ng/mL and basal cAMP level of 2.6 nM. In contrast, the L469R activation mutant had most elevated (16.88 fold higher than wt) basal cAMP production (basal cAMP level=43.9 nM). Compared with the wt eelLHR, all the activation mutant receptors produced higher basal levels of cAMP (18.4 nM for D590Y and 7.9 nM for M410T). However, eelLH-stimulated (0.076~1,200 ng/mL) basal cAMP levels in the constitutive inactivation mutants D383N and Y546F did not obviously altered from that in wt eelLHR. D383N mutation increased the EC50 value to 185 ng/mL (inhibited receptor activity to 86%), while Y546F mutation increased that to 170 ng/mL which implies that receptor activity was inhibited to 94% only. As seen in IC50 values in IP-1 accumulation, activity for M410T mutant was 19% higher than that for wt receptor, but other activation mutants did not show any difference in IP-1 production. In case of inactivation mutants, there were no significant differences in IP-1 production and only 7% decreased activity was identified in D383N mutant. In summary, we have demonstrated 3 mutations that are responsible for constitutive activation of eelLHR. Although predicted 2 inactivation mutations led to slightly diminished activation of receptor, those could not impair signal transduction of eelLHR.

Molecular Characterization of Constitutively Active and Inactive Equine FSH Receptor

Munkhzaya Byambaragchaa,Tae-Young Ahn,Kwan-Sik Min 한국동물생명공학회(구 한국동물번식학회) 2018 발생공학 국제심포지엄 및 학술대회 Vol.2018 No.06

Equine follicle-stimulating hormone (eFSH) is a member of the glycoprotein family with luteinizing hormone (LH), thyroid stimulating hormone (TSH), and equine chorionic gonaodotropin (eCG). These receptors called G protein-coupled (GPCR) receptor were synthesized in granulosa/theca and Sertoli/Leydig cells in ovary/testis. GPCRs are key signaling proteins that regulate nearly every aspect of cell function. GPCRs relay information from extracellular stimuli to intracellular responses in a wide range of physiological and pathological processes. All of the receptors in this superfamily transverse the plasma membrane with seven highly conserved a-helices oriented with an extracellular amino terminus and an intracellular carboxy terminus. To access the functional effect of the activation mutant (D566G) and inactivations (A189V, N191I, R572C, A574V, and R633H) in the eFHSR, wild type and the mutated eFSHR were transiently expressed in CHO-K1 cells and cAMP accumulation was measured. The activity of activation mutant at residue 556 exhibits a 9.4-fold increase in basal cAMP accumulation. In the inactivation mutants, EC50 values of the 189 site was 73.2% compared to that of wild type receptor. The other site (191) does not affect in the cAMP responsiveness. Other 3 sites (572, 574, and 633), the EC50 values were detected 109.7, 118.9, and 115.3%, respectively. The mutation is localized in a crucial region of the transmembrane domain, highly conserved in all glycoprotein hormone receptors, and within the FSH receptor of different species. The same site (Asp-Gly) has been previously reported to lead to constitutive activation of these receptors (LHR, and TSHR). Those receptors were found in patients with pseudoprecocious puberty and thyroid adenoma, respectively. In summary, we have identified constitutively activating point mutations and inactivating mutations of eFSHR in CHO-K1 cells. Knowledge of those mutations will facilitate genetic counseling and diagnosis, as well as provide the basis to study the three-dimensional conformation of the receptor domains involved in ligand-binding and G protein activation. To our knowledge, this remains the sole example of a naturally occurring activating mutation of the FSH receptor described so far. Thus, the significance of naturally occurring polymorphisms in the FSH receptor is still unknown. We insist that some receptor variant or combination of variants is related to a higher incidence of reproductive disorders.

Anti-metastatic Potential of Ethanol Extract of Saussurea involucrata against Hepatic Cancer in vitro

Byambaragchaa, Munkhzaya,de la Cruz, Joseph,Yang, Seung Hak,Hwang, Seong-Gu Asian Pacific Journal of Cancer Prevention 2013 Asian Pacific journal of cancer prevention Vol.14 No.9

The rates of morbidity and mortality of hepatocellular carcinoma (HCC) have not lessened because of difficulty in treating tumor metastasis. Mongolian Saussurea involucrata (SIE) possesses various anticancer activities, including apoptosis and cell cycle arrest. However, detailed effects and molecular mechanisms of SIE on metastasis are unclear. Thus, the present study was undertaken to investigate antimetastatic effects on HCC cells as well as possible mechanisms. Effects of SIE on the growth, adhesion, migration, aggregation and invasion of the SK-Hep1 human HCC cell line were investigated. SIE inhibited cell growth of metastatic cells in dose- and time-dependent manners. Incubation of SK-Hep1 cells with $200-400{\mu}g/mL$ of SIE significantly inhibited cell adhesion to gelatin-coated substrate. In the migration (wound healing) and aggregation assays, SIE treated cells showed lower levels than untreated cells. Invasion assays revealed that SIE treatment inhibited cell invasion capacity of HCC cells substantially. Quantitative real time PCR showed inhibitory effects of SIE on MMP-2/-9 and MT1-MMP mRNA levels, and stimulatory effects on TIMP-1, an inhibitor of MMPs. The present study not only demonstrated that invasion and motility of cancer cells were inhibited by SIE, but also indicated that such effects were likely associated with the decrease in MMP-2/-9 expression of SK-Hep1 cells. From these results, it was suggested that SIE could be used as potential anti-tumor agent.

Identification of sperm mRNA biomarkers associated with sex-determinant reagent treatment in semen of Korean Native Cattle

Munkhzaya Byambaragchaa,Kwan-Sik Min,Hyun-Kim,Myung-Hum Park 한국수정란이식학회 2018 한국수정란이식학회 학술대회 Vol.2018 No.11

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.

Biochemical Characterization of 20α-Hydroxysteroid Dehydrogenase

Byambaragchaa, Munkhzaya,Min, Kwan-Sik The Korean Society of Animal Reproduction 2018 Reproductive & developmental biology Vol.42 No.2

In this review, we have tried to summarize the evidence and molecular characterization indicating that $20{\alpha}$-hydroxysteroid dehydrogenase ($20{\alpha}$-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 $20{\alpha}$-HSD in cows, pigs, deer, and monkeys. The specific antibody against bovine $20{\alpha}$-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 $20{\alpha}$-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 $20{\alpha}$-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.

Specific Biological Activity of Equine Chorionic Gonadotropin (eCG) Glycosylation Sites in Cells Expressing Equine Luteinizing Hormone/CG (eLH/ CG) Receptor

Munkhzaya Byambaragchaa,Seung-Hee Choi,Hyo-Eun Joo,Sang-Gwon Kim,Yean-Ji Kim,Gyeong-Eun Park,Myung-Hwa Kang,민관식 한국발생생물학회 2021 발생과 생식 Vol.25 No.4

Equine chorionic gonadotropin (eCG), produced by the endometrial cups of the placenta after the first trimester, is a specific glycoprotein that displays dual luteinizing hormone (LH)- like and follicle-stimulating hormone (FSH)-like effects in non-equid species. However, in equidaes, eCG exhibits only LH-like activity. To identify the specific biological functions of glycosylated sites in eCG, we constructed the following site mutants of N- and O-linked glycosylation: eCGβ/αΔ56, substitution of α-subunit56 N-linked glycosylation site; eCGβ-D/ α, deletion of the O-linked glycosylation sites at the β-subunit, and eCGβ-D/αΔ56, double mutant. We produced recombinant eCG (rec-eCG) proteins in Chinese hamster ovary suspension (CHO-S) cells. We examined the biological activity of rec-eCG proteins in CHO-K1 cells expressing the eLH/CG receptor and found that signal transduction activities of deglycosylated mutants remarkably decreased. The EC50 levels of eCGβ/αΔ56, eCGβ-D/ α, and eCGβ-D/αΔ56 mutants decreased by 2.1-, 5.6-, and 3.4-fold, respectively, compared to that of wild-type eCG. The Rmax values of the mutants were 56%-80% those of wildtype eCG (141.9 nmol/104 cells). Our results indicate that the biological activity of eCG is greatly affected by the removal of N- and O-linked glycosylation sites in cells expressing eLH/ CGR. These results provide important information on rec-eCG in the regulation of specific glycosylation sites and improve our understanding of the specific biological activity of receCG glycosylation sites in equidaes.

Biochemical Characterization of 20α-Hydroxysteroid Dehydrogenase

Munkhzaya Byambaragchaa,Kwan-Sik Min 한국동물번식학회 2018 Reproductive & developmental biology Vol.42 No.2

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.

Constitutive Activating Eel Luteinizing Hormone Receptors Induce Constitutively Signal Transduction and Inactivating Mutants Impair Biological Activity

Munkhzaya Byambaragchaa,Seung-Hee Choi,Dong-Wan Kim,민관식 한국발생생물학회 2021 발생과 생식 Vol.25 No.3

In contrast to the human lutropin receptor (hLHR) and rat LHR (rLHR), very few naturally occurring mutants in other mammalian species have been identified. The present study aimed to delineate the mechanism of signal transduction by three constitutively activating mutants (designated M410T, L469R, and D590Y) and two inactivating mutants (D383N and Y546F) of the eel LHR, known to be naturally occurring in human LHR transmembrane domains. The mutants were constructed and measured cyclic adenosine monophosphate (cAMP) accumulation via homogeneous time-resolved fluorescence assays in Chinese hamster ovary (CHO)-K1 cells. The activating mutant cells expressing eel LHR-M410T, L469R, and D590Y exhibited a 4.0-, 19.1-, and 7.8-fold increase in basal cAMP response without agonist treatment, respectively. However, inactivating mutant cells expressing D417N and Y558F did not completely impaired signal transduction. Specifically, signal transduction in the cells expressing activating mutant L469R was not occurred with a further ligand stimulation, showing that the maximal response exhibited approximately 53% of those of wild type receptor. Our results suggested that the constitutively activating mutants of the eel LHR consistently occurred without agonist treatment. These results provide important information of LHR function in fish and regulation with regard to mutations of highly conserved amino acids in glycoprotein hormone receptors.