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.

Luteinizing hormone-like and follicle-stimulating hormone-like activities of equine chorionic gonadotropin β-subunit mutants in cells expressing rat luteinizing hormone/chorionic gonadotropin receptor and rat follicle-stimulating hormone receptor

BYAMBARAGCHAA MUNKHZAYA,박아영,길소진,이해원,고윤정,최승희,강명화,민관식 한국통합생물학회 2021 Animal cells and systems Vol.25 No.3

To identify the specific region of eCG involved in FSH-like activity, the following mutant expression vectors were constructed targeting the amino acid residues 102–104 of the eCG β-subunit: single mutants, eCGβV102G/α, eCGβF103P/α, and eCGβR104K/α; double mutants, eCGβV102G;F103P/α, eCGβV102G;R104K/α, and eCGβF103P;R104K/α; triple mutant, eCGβV102G;F103P;R104K/α. The LH-like and FSH-like activities of eCG mutants were examined in CHO-K1 cells expressing rat LH/ CG receptor and rat FSH receptor. The levels of eCGβV102G/α, eCGβR104K/α, and eCGβV102G; R104K/α in the culture supernatant were markedly lower than those of eCGβ/α-wt. The other mutants and rec-eCGβ/α-wt were efficiently secreted into the culture supernatant. The LH-like activities of eCGV104G/α, eCGβV102G;R104K/α, and eCGβF103P;R104K/α were approximately 61%, 52%, and 54%, respectively, of those of eCG-wt. The Rmax values of the mutants were 58.9%–78.8% those of eCG-wt with eCGβR104K/α exhibiting the lowest value. The FSH-like activities of single mutants were only 16%–20% of those of eCG-wt. Additionally, the FSH-like activity of double mutants was less than 10% of that of eCG-wt. In particular, the FSH-like activities of βV102G;R104K/α and βF103P;R104K/α were 2.5–2.9% of that of eCG-wt. These results suggest that the amino acid residues 102–104 of the eCG β-subunit are dispensable and that the residue 104 of the eCG β-subunit plays a pivotal role in signal transduction through the rat FSH receptor. Thus, these mutants may aid future studies on eCG interactions with mammalian FSH receptors in vitro and in vivo.

Anticancer Potential of an Ethanol Extract of Saussurea Involucrata against Hepatic Cancer Cells in vitro

Byambaragchaa, Munkhzaya,Cruz, Joseph Dela,Kh, Altantsetseg,Hwang, Seong-Gu Asian Pacific Journal of Cancer Prevention 2014 Asian Pacific journal of cancer prevention Vol.15 No.18

Saussurea involucrata is a Mongolian medicinal plant well known for its effects in promoting blood circulation, and anti-inflammation and analgesic functions. Earlier studies reported that Saussurea involucrata has anticancer activity. The purpose of this study was to confirm the anticancer activity of an ethanol extract of Saussurea involucrata against hepatic cancer and elucidate its mechanisms of action. Hepatocellular carcinoma cells were tested in vitro for cytotoxicity, AO/EB staining for apoptotic cells, apoptotic DNA fragmentation and cell cycle distribution in response to Saussurea involucrata extract (SIE). The mRNA expression of caspase-3,-9 and Cdk2 and protein expression of caspase-3,-9, PARP, XIAP, Cdk2 and p21 were analyzed through real time PCR and Western blotting. Treatment with SIE inhibited HepG2 cell proliferation dose- and time-dependently, but SIE only exerted a modest cytotoxic effect on a viability of Chang human liver cells. Cells exposed to SIE showed typical hallmarks of apoptotic cell death. Cell cycle analysis revealed that SIE caused G1-phase arrest in HepG2 cells. In conclusion, Saussurea involucrata ethanol extract has potential cytotoxic and apoptotic effects on human hepatocellular carcinoma cells. Its mechanism of action might be associated with the inhibition of DNA synthesis, cell cycle (G1) arrest and apoptosis induction through up-regulation of the protein expressions of caspase-3,-9 a nd p21, degradation of PARP and down-regulation of the protein expression of Cdk2 and XIAP.

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.

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.

Signal Transduction of Equine Follicle-Stimulating Hormone Receptor (eFSHR) by rec-eelFSHβ/α, Natural Porcine FSH, and Natural Human FSH

Byambaragchaa, Munkhzaya,Kim, Dae-Jung,Kang, Myung-Hwa,Min, Kwan-Sik The Korean Society of Animal Reproduction 2018 Reproductive & developmental biology Vol.42 No.1

In this study, we analyzed signal transduction by equine follicle-stimulating hormone receptor (eFSHR) on sti- mulation with recombinant $eelFSH{\beta}/{\alpha}$ ($rec-eelFSH{\beta}/{\alpha}$), 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 $EC_{50}$ value of $rec-eelFSH{\beta}/{\alpha}$ was 53.35 ng/mL. The Rmax values of $rec-eelFSH{\beta}/{\alpha}$ and pFSH were 28.12 and 2.88 ng/mL, respectively. The activity of $rec-eelFSH{\beta}/{\alpha}$ 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, $rec-eelFSH{\beta}/{\alpha}$ 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 $eelFSH{\beta}/{\alpha}$ has potent activity in cells expressing eFSHR. Thus, $rec-eelFSH{\beta}/{\alpha}$ may efficiently bind to eFSHR, where as natural hFSH does not bind to eFSHR.

Functional characterization of naturally-occurring constitutively activating/inactivating mutations in equine follicle-stimulating hormone receptor

BYAMBARAGCHAA MUNKHZAYA,Ahn Tae-Young,Choi Seung-Hee,Kang Myung-Hwa,Min Kwan-Sik 아세아·태평양축산학회 2022 Animal Bioscience Vol.35 No.3

Objective: Follicle-stimulating hormone (FSH) is the central hormone involved in mammalian reproduction, maturation at puberty, and gamete production that mediates its function by control of follicle growth and function. The present study investigated the mutations involved in the regulation of FSH receptor (FSHR) activation. Methods: We analyzed seven naturally-occurring mutations that were previously reported in human FSHR (hFSHR), in the context of equine FSHR (eFSHR); these include one constitutively activation variant, one allelic variant, and five inactivating variants. These mutations were introduced into wild-type eFSHR (eFSHR-wt) sequence to generate mutants that were designated as eFSHR-D566G, -A306T, -A189V, -N191I, -R572C, -A574V, and -R633H. Mutants were transfected into PathHunter EA-parental CHO-K1 cells expressing β-arrestin. The biological function of mutants was analyzed by quantitating cAMP accumulation in cells incubated with increasing concentrations of FSH. Results: Cells expressing eFSHR-D566G exhibited an 8.6-fold increase in basal cAMP response, as compared to that in eFSHR-wt. The allelic variation mutant eFSHR-A306T was not found to affect the basal cAMP response or half maximal effective concentration (EC50) levels. On the other hand, eFSHR-D566G and eFSHR-A306T displayed a 1.5- and 1.4-fold increase in the maximal response, respectively. Signal transduction was found to be completely impaired in case of the inactivating mutants eFSHR-A189V, -R572C, and -A574V. When compared with eFSHR-wt, eFSHR-N191I displayed a 5.4-fold decrease in the EC50 levels (3,910 ng/mL) and a 2.3-fold decrease in the maximal response. In contrast, cells expressing eFSHR-R633H displayed in a similar manner to that of the cells expressing the eFSHR-wt on signal transduction and maximal response. Conclusion: The activating mutant eFSHR-D566G greatly enhanced the signal transduction in response to FSH, in the absence of agonist treatment. We suggest that the state of activation of the eFSHR can modulate its basal cAMP accumulation. Objective: Follicle-stimulating hormone (FSH) is the central hormone involved in mammalian reproduction, maturation at puberty, and gamete production that mediates its function by control of follicle growth and function. The present study investigated the mutations involved in the regulation of FSH receptor (FSHR) activation.Methods: We analyzed seven naturally-occurring mutations that were previously reported in human FSHR (hFSHR), in the context of equine FSHR (eFSHR); these include one constitutively activation variant, one allelic variant, and five inactivating variants. These mutations were introduced into wild-type eFSHR (eFSHR-wt) sequence to generate mutants that were designated as eFSHR-D566G, -A306T, -A189V, -N191I, -R572C, -A574V, and -R633H. Mutants were transfected into PathHunter EA-parental CHO-K1 cells expressing β-arrestin. The biological function of mutants was analyzed by quantitating cAMP accumulation in cells incubated with increasing concentrations of FSH.Results: Cells expressing eFSHR-D566G exhibited an 8.6-fold increase in basal cAMP response, as compared to that in eFSHR-wt. The allelic variation mutant eFSHR-A306T was not found to affect the basal cAMP response or half maximal effective concentration (EC50) levels. On the other hand, eFSHR-D566G and eFSHR-A306T displayed a 1.5- and 1.4-fold increase in the maximal response, respectively. Signal transduction was found to be completely impaired in case of the inactivating mutants eFSHR-A189V, -R572C, and -A574V. When compared with eFSHR-wt, eFSHR-N191I displayed a 5.4-fold decrease in the EC50 levels (3,910 ng/mL) and a 2.3-fold decrease in the maximal response. In contrast, cells expressing eFSHR-R633H displayed in a similar manner to that of the cells expressing the eFSHR-wt on signal transduction and maximal response.Conclusion: The activating mutant eFSHR-D566G greatly enhanced the signal transduction in response to FSH, in the absence of agonist treatment. We suggest that the state of activation of the eFSHR can modulate its basal cAMP accumulation.

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.

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.

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.