• Asian-Australasian Journal of Animal Sciences
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• v.30 no.12
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• pp.1679-1683
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• 2017

Objective: An experiment was conducted to study the association between the single nucleotide polymorphisms (SNPs) in 5'-untranslated regions (5'-UTR) of equine chorionic gonadotropin (eCG) genes and the serum eCG levels. Methods: SNPs in 5'-UTR of eCG genes were screened across 10 horse breeds, including 7 Chinese indigenous breeds and 3 imported breeds using iPLEX chemistry, and the association between the serum eCG levels of 174 pregnant Da'an mares and their serum eCG levels (determined with ELISA) was analyzed. Results: Four SNPs were identified in the 5'-UTR of the $eCG{\alpha}$ gene, and one of them was unique in the indigenous breeds. There were 2 SNPs detected at the 5' end of the $eCG{\beta}$ subunit gene, and one of them was only found in the Chinese breeds. The SNP g.39948246T>C at the 5'-UTR of $eCG{\alpha}$ was associated significantly with eCG levels of 75-day pregnant mare serum (p<0.05) in Da'an mares. Prediction analysis on binding sites of transcription factors showed that the g.39948246T>C mutation causes appearance of the specific binding site of hepatocyte nuclear factor 3 forkhead homolog 2 (HFH-2), which is a transcriptional repressor belonging to the forkhead protein family of transcription factors. Conclusion: The SNP g.39948246T>C at the 5'-UTR of $eCG{\alpha}$ is associated with eCG levels of 75-day pregnant mare serum (p<0.05).

• Proceedings of the KSAR Conference
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• 2004.06a
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• pp.221-221
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• 2004

Equine chorionic gonadotropin (eCG), which consists of highly glycosylated α- and β-subunits, is a unique member of the gonadotropin family because it elicits response characteristics of both follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in other species than the horse. To determine whether α and β subunits can be synthesized as a single polypeptide chain (tethered-eCG) and also display biological activity, the tethered-eCG molecule was constructed and transfected into Chinese hamster ovary (CHO-K1) cells. (omitted)

• Development and Reproduction
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• v.25 no.4
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• pp.199-211
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• 2021

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 wild-type 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 rec-eCG glycosylation sites in equidaes.

• Journal of Life Science
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• v.11 no.2
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• pp.103-107
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• 2001

The equine chorionic gonadotropin (eCG) subunits $\alpha$ and ${\beta}$ are transcribed from different genes and associate noncovalently to form the bioactive eCG heterodimer. Dimerization is rate limiting for eCG secretion, and dissociation leads to hormone inactivation. The correct conformation of the heterodimer is alto important for efficient secretion, hormone-specific post-translational modifications, receptor binding and signal transduction. To determine whether ${\alpha}$ and ${\beta}$ subunits can be synthesized as a single polypeptide chain (tethered-eCG) and also display biological activity, the tethered-eCG molecule by fusing the carboxyl terminus of the eCG ${\beta}$-subunit to the amino terminus of the af-subunit was construe-ted and transfected into chinese hamster ovary (CHO-Kl) cells. LH- and FSH-like activities were assayed in terms of testosterone production and aromatase activity in primary cultured rat Leydig cells and granulosa cells, respectively. The tethered-eCG was efficiently secreted and showed similar LH-like activity to the dimeric eCG ${\alpha}$/${\beta}$ and native eCG. FSH-like activity of the tethered-eCG was also shown similarly in comparison with the native and wild type eCG ${\alpha}$/${\beta}$. Our data for the first time suggest that the tethered-eCG can be expressed efficiently and the produced product by the CHO-K1 cells is fully LH- and FSH-like activities in rat in vitro bioassay system. Our results also suggest that this molecular can imply particular models of FSH-like activity not LH-like activity in the eCG. Taken together, these data indicate that the constructs of tethered molecule will be useful in the study of mutants that affect subunit association and/or secretion.

• Journal of Animal Reproduction and Biotechnology
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• v.36 no.4
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• pp.169-174
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• 2021

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.

• Development and Reproduction
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• v.26 no.1
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• pp.1-12
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• 2022

This study aimed to investigate the signal transduction of phosphorylation sites at the carboxyl (C)-terminal region of equine luteinizing hormone/chorionic gonadotropin receptor (eLH/CGR). The eLH/CGR has a large extracellular domain of glycoprotein hormone receptors within the G protein-coupled receptors. We constructed a mutant (eLH/CGR-t656) of eLH/CGR, in which the C-terminal cytoplasmic tail was truncated at the Phe656 residue, through polymerase chain reaction. The eLH/CGR-t656 removed 14 potential phosphorylation sites in the intracellular C-terminal region. The plasmids were transfected into Chinese hamster ovary (CHO)-K1 and PathHunter Parental cells expressing β-arrestin, and agonist-induced cAMP responsiveness was analyzed. In CHO-K1 cells, those expressing eLH/CGR-t656 were lower than those expressing eLH/CGR wild-type (eLH/CGR-wt). The EC₅₀ of the eLH/CGR-t656 mutant was approximately 72.2% of the expression observed in eLH/CGR-wt. The maximal response in eLH/CGR-t656 also decreased to approximately 43% of that observed in eLH/CGR-wt. However, in PathHunter Parental cells, cAMP activity and maximal response of the eLH/CGR-t656 mutant were approximately 173.5% and 100.8%, respectively, of that of eLH/CGR-wt. These results provide evidence that the signal transduction of C-terminal phosphorylation in eLH/CGR plays a pivotal role in CHO-K1 cells. The cAMP level was recovered in PathHunter Parental cells expressing β-arrestin. We suggest that the signal transduction of the C-terminal region phosphorylation sites is remarkably different depending on the cells expressing β-arrestin in CHO-K1 cells.

• Journal of Life Science
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• v.27 no.8
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• pp.864-872
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• 2017

Equine chorionic gonadotropin (eCG) consists of highly glycosylated ${\alpha}-$ and ${\beta}-subunits$ and is a unique member of the gonadotropin family, because it elicits the response characteristics of follicle stimulating hormone (FSH) and luteinizing hormone (LH) in species other than the horse. To directly assess the biological function of $rec-eCG{\beta}/{\alpha}$, we constructed mammalian expressing vectors of equine luteinizing hormone/chorionic gonadotropin receptors (eLH/CGR). The activity of $rec-eCG{\beta}/{\alpha}$ in vitro assayed in transient transfected CHO-K1 cells and in stably transfected PathHunter Parental cells with eLH/CGR was investigated. $rec-eCG{\beta}/{\alpha}$ was efficiently secreted in the CHO-K1 suspension cell media, and the quantity detected was about 200 mIU/ml from 1 to 7 days after transfection. In the western blot analysis, the $rec-eCG{\beta}/{\alpha}$ protein was broadly identified to be about 40~45 kDa molecular weight. The cAMP stimulation in CHO-K1 cells expressing eLH/CGR was determined to evaluate the activity of $rec-eCG{\beta}/{\alpha}$. The cAMP concentration increased in direct proportion to the concentration of the $rec-eCG{\beta}/{\alpha}$. The $EC_{50}$ value in the transient transfected CHO-K1 cells was $8.1{\pm}6.5ng$. The stable cell lines of eLH/CGR were established in the PathHunter Parental cells expressing ${\beta}-arrestin$. We found that $rec-eCG{\beta}/{\alpha}$ had full LH activity in the PathHunter Parental cells expressing eLH/CGR. The $EC_{50}$ value in transient and stable cells was $5.0{\pm}4.7ng/ml$ and $4.5{\pm}5.2ng/ml$, respectively. These results suggest that $rec-eCG{\beta}/{\alpha}$ has a biological activity in a cell expressing eLH/CGR. These stable cells expressed in PathHunter Parental cells could be useful for elucidating the functional mechanisms of deglycosylated $rec-eCG{\beta}/{\alpha}$ mutants.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.10
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• pp.1374-1377
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• 2004

The present study was conducted to explore the possibilities of estrus induction and superovulation in a native Indian breed of goats called 'Black Bengal'. Forty-two adult non-pregnant females were divided in two groups, of which 18 goats were subjected to a superovulatory treatment comprising of equine chorionic gonadotropin (eCG), Prostaglandin (PGF2$\alpha$) and human chorionic gonadotropin (hCG) to induce superovulation. The remaining 24 goats received no treatment and served as controls for the parameter under study as well as recipients for embryo transfer studies. The average duration of estrus was found to be significantly increased in treated goats (34.2${\pm}$3.4 h) compared to controls 3.0${\pm}$2.4 h). The average duration between PGF administration and occurrence of estrus was 2.0${\pm}$5.2 h. After mid ventral laparotomy, superovulatory responses indicated a significant increase in the number of follicles, which was 8.27${\pm}$0.37 in the treatment group compared to 4.16${\pm}$0.17 in the control group. The number of corpora lutea was also significantly increased in treated animals compared to control (2.90${\pm}$0.86 vs. 0.74${\pm}$0.04) respectively per ovary per goat.

• Journal of Embryo Transfer
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• v.26 no.3
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• pp.215-217
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• 2011

This is about the successful use of eCG and hCG for producing a Siberian tiger pup born from 10-year-old, primiparous, albino Siberian tiger. From February 2010 to July 2010, natural breeding had been tried three times with no conception. During this period of five months, estrus behaviors appeared to be typically normal and a lot of matings were observed. After consecutive failures, 1000 IU eCG (equine chorionic gonadotropin) were intramuscularly injected on the day showing estrus behavior, followed with an injection of 750 IU hCG (human chorionic gonadotropin) 80 hours later. The tiger stopped recurrence of estrus, and a cub, weighed 780 gram, was born alive 104 days after hCG injection. This study is the first report showing the unique, successful use of exogenous hormones as one of artificial breeding programs in the long history of captive breeding of carnivorous zoo animals in Korea.

• Development and Reproduction
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• v.4 no.1
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• pp.87-93
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• 2000

Equine chorionic gonadotropin (eCG) consists of highly glycosylated noncovalently linked $\alpha$- and $\beta$-subunits and belongs to the glycoprotein hormone family that includes lutropin (LH), follitropin (FSH), and thyrotropin (FSH). eCG is a unique member of the gonadotropin family because it elicits response characteristics of both FSH and LH in other species than the hone. eCG is synthesized and secreted by trophoblastic cells of the endometial cups between 40 and 130 days of gestation. In the present study, mRNA expression ratio of eCG, eLH and eFSH $\alpha$- and $\beta$-subunints was investigated in the placenta and pituitary. mRNA was extracted from equine placenta on day 70 of gestation and from pituitary of male horse (27 month-old). When the expression of both subunit mRNAs of eCG in the equine placenta was compared by Northern blotting, the expression of the $\beta$ -subunit mRNA was relatively greater than that of the $\alpha$-subunit. And mRNA expression of $\alpha$-, LH $\beta$- and FSH $\beta$-subunits was analysed in the equine pituitary. An $\alpha$-subunit was revealed with a size of approximately 0.8 kb. FSH $\beta$-subunit mRNA also was detected out 1.8 kb. It is the same size of the FSH $\beta$ -subunit mRNA cloned. The intensity of $\alpha$-subunit mRNA was greater than that of the $\beta$-subunit suggesting that the expression of $\alpha$ -subunit was dominant in the equine anterior pituitary. Thus, the subunit mRNA levels seem to be independently regulated and their imbalance may account for differences in the quantities of $\alpha$- and $\beta$-subunits in the equine placenta and pituitary.