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.

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

Munkhzaya Byambaragchaa,So-Yun Lee,Dae-Jung Kim,강명화,민관식 한국발생생물학회 2018 발생과 생식 Vol.22 No.1

Previous studies showed that recombinant equine chorionic gonadotropin (rec-eCGβ/α) 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-eCGβ/α and native eCG. The cyclic adenosine monophosphate (cAMP) stimulation in CHO-K1 cells expressing eelLHR was determined upon exposure to differ-ent 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 re-sponse 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 pitu-itary extract.

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.

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.

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.

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.

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.

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.

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.

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.