• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2004년도 춘계학술발표대회
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• pp.210-210
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• 2004

Equinechorionic gonadotropin(eCG) is a member of the glycoprotein hormone family which includes FSH, hCG, TSH. These hormone family is characterized by a heterodimeric structure composed a common α-subunit noncovalently linked to a hormone specific β-subunit. To determine a and β-subunits can be synthesized as a single polypeptide chain (tethered-eCG) and also display biological activity, the tethered-molecule by fusing the carboxyl terminus of the eCG β-subunit to the amino terminus of the α-subunit was constructed and transfected into chinese hamster ovary (CHO-K1) cells. (omitted)

• 한국가축번식학회지
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• 제26권3호
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• pp.299-308
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• 2002

eCG(말 융모성성선자극 호르몬)$\beta$와 eLH(말 황체형성호르몬)$\beta$는 하나의 유전자로 코드 되어 있으며, eCG와 eLH는 당쇄의 구조에 있어서 차이가 있는데, LH는 sulfate가 CG는 sialylate가 수식되어 있다. eCG는 다른 동물에 있어서 강력한 FSH (난포자극)와 LH의 이중활성을 나타내어 아주 특이하고, 많은 탄수화물로 수식되어 있는 당단백질 호르몬이다. eCG의 이러한 이중활성은 성선 자극호르몬의 구조, 기능 및 수용체와 이들 호르몬과의 특이결합에 대하여 분자 생물학적인 관점에서 연구하는데 아주 흥미롭다 따라서, eCG는 당쇄의 구조와 생화학적인 기능에서 아주 특이한 분자이다. 이러한 중요점을 당쇄첨가부위의 돌연변이를 통하여 분석한 결과, LH의 활성에서는 eCG$\alpha$의 56번 당쇄가 필수불가결한 역할을 하지만, eCG$\beta$의 카르복실기 말단의 O-linked 당쇄는 중요하지 않은 것으로 관찰되었다. 한편, N- 및 O-linked 당쇄 모두는 FSH활성에는 중요한 기능을 가지고 있는데, 양쪽 당쇄의 제거는 오히려 FSH 활성을 증가시켰다. 따라서, eCG의 LH와 FSH의 이중활성은 $\alpha$의 N-linked 당쇄의 제거와, $\beta$의 O-linked 당쇄를 제거함으로써 완전히 분리할 수 있으며, eCG에 있어서 당쇄는 생화학적 활성에 대하여 아주 중요하게 작용한다는 새로운 사실이 밝혀졌다. 단일체인 eCG($\beta$의 C-terminal에 u를 연결한)도 eCG$\alpha$/$\beta$ 및 천연형 eCG와 비교한 결과 효율적으로 분비되어지고 완전한 LH와 FSH 활성을 나타내었다. 이러한 결과들은 eCG 분자에 있어서 지금까지 문제시되어왔던 LH활성을 나타내지 않고, 높은 FSH 활성만을 나타내는 특이한 모델을 만들 수가 있으며, 현재 단일체인 분자에 있어서 당쇄의 기능에 대한구축은 각 단체의 결합, 분비에 영향을 미치는 당쇄 돌연변이 연구에 아주 유용할 것으로 사료된다.

• Reproductive and Developmental Biology
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• 제33권4호
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• pp.229-236
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• 2009

The glycoprotein hormone family represents a class of heterodimers, that includes the placental hormone equine chorionic gonadotropin (eCG) and the anterior pituitary hormones- follitropin (FSH), lutropin (LH), and thyrotropin (TSH). The 4 hormones are heterodimers, with a common $\alpha$-subunit and unique $\beta$-subunits. eCG is the most heavily glycosylated of the known pituitary and placental glycoprotein hormones. Recent observations using single chain glycoprotein hormone analogs in which, the $\beta$-and $\alpha$-subunits are linked, implied that heterodimeric-like quaternary configuration is not a prerequisite for receptor/signal transduction. To study the function and signal transduction of tethered rec-eCG, a single chain eCG molecule was constructed and rec-eCG protein was produced. Molecular mass of the single chain is about 45 kDa. All mice were ovulated by tethered rec-eCG treatment. The dual activity of tethered rec-eCG was determined in receptor cell lines of nonequid species; in fact, this dual activity was proven in species other than horse. Tethered rec-eCG in equids does not bind to FSH receptors, suggesting that eCG is primarily an LH-like hormone in the horse. Taken together, these data suggest that tethered rec-eCG has dual activity in nonequid species in vitro. However, it has only LH-like activity in equid species in vitro.

• 한국가축번식학회지
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• 제24권4호
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• pp.357-364
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• 2000

Members of the glycoprotein family, which includes CG, LH, FSH and TSH, comprise two noncovalently linked $\alpha$- and $\beta$-subunits. Equine chorionic gonadotropin (eCG), known as PMSG, has a number of interesting and unique characteristics since it appears to be a single molecule that possesses both LH- and FSH-like activities in other species than the horse. This dual activity of eCG in heterologous species is of fundamental interest to the study of the structure-function relationships of gonadotropins and their receptors. CG and LH $\beta$ genes are different in primates. In horse, however, a single gene encodes both eCG and eLH $\beta$ -subunits. 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 placenta and pituitary. The dual activities of eCG could be separated by removal of the N-linked oligosaccharide on the $\alpha$-subunit Asn 56 or CTP-associated O-linked oligosaccharides. The tethered-eCG was efficiently secreted and showed similar LH-like activity to the dimeric eCG. Interestingly, the FSH-like activity of the tethered-eCG was increased markedly in comparison with the native and wild type eCG. These results also suggest that this molecular can implay particular models of FSH-like activity not LH-like activity in the eCG/indicate that the constructs of tethered molecule will be useful in the study of mutants that affect subunit association and/or secretion. A single-chain analog can also be constructed to include additional hormone-specific bioactive generating potentially efficacious compounds that have only FSH-like activity. The LH/CG receptor (LH/CGR), a membrane glycoprotein that is present on testicular Leydig cells and ovarian theca, granulosa, luteal, and interstitial cells, plays a pivotal role in the regulation of gonadal development and function in males as well as in nonpregnant and pregnant females. The LH/CGR is a member of the family of G protein-coupled receptors and its structure is predicted to of a large extracellular domain connected to a bundle of seven membrane-spanning a-helices. The LH/CGR phosphorylation can be induced with a phorbol ester, but not with a calcium ionophore. The truncated form of LHR also was down-regulated normally in response to hCG stimulation. In contrast, the cell lines expressing LHR-t631 or LHR-628, the two phosphorylation-negative receptor mutant, showed a delay in the early phase of hCG-induced desensitization, a complete loss of PMA-induced desensitization, and an increase in the rate of hCG-induced receptor down-regulation. These results clearly show that residues 632~653 in the C-terminal tail of the LHR are involved in PMA-induced desensitization, hCG-induced desensitization, and hCG-induced down-regulation. Recently, constitutively activating mutations of the receptor have been identified that are associated with familial male-precocious puberty. Cells expressing LHR-D556Y bind hCG with normal affinity, exhibit a 25-fold increase in basal cAMP and respond to hCG with a normal increase in cAMP accumulation. This mutation enhances the internalization of the free and agoinst-occupied receptors ~2- and ~17- fold, respectively. We conclude that the state of activation of the LHR can modulate its basal and/or agonist-stimulated internalization. Since the internalization of hCG is involved in the termination of hCG actions, we suggest that the lack of responsiveness detected in cells expressing LHR-L435R is due to the fast rate of internalization of the bound hCG. This statement is supported by the finding that hCG responsiveness is restored when the cells are lysed and signal transduction is measured in a subcellular fraction (membranes) that cannot internalize the bound hormone.

• Reproductive and Developmental Biology
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• 제34권1호
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• pp.47-53
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• 2010

Glycoprotein hormones have a common $\alpha$-subunit that is involved in the signaling pathway together with G protein, adenylcyclase and cAMP induction; however, it is an unclear how this common structure is related to hormonal action. To determine the biological functions of the COOH-terminal amino acids in the $\alpha$-subunit of these glycoprotein hormones, a tethered-molecule was constructed by fusing the $NH_2$-terminus of the $\alpha$-subunit to the COOH-terminus of the $\beta$-subunit of equine chorionic gonadotropin (eCG). The following deletion mutants were created by PCR; Ile was inserted at position 96 to form ${\Delta}96$, Lys was substituted at position 95 to form ${\Delta}95$, His was inserted at position 93 to form ${\Delta}93$ and Tyr was substituted at position 87 to form ${\Delta}87$. Each mutant was transfected into CHO-K1 cells. Tethered-wt eCG, and ${\Delta}96$, ${\Delta}95$, and ${\Delta}93$ mutants were efficiently secreted into the medium but the ${\Delta}87$ mutant was not secreted. Interestingly, the RT-PCR, real-time PCR, and northern blot analyses confirmed that the RNA was transcribed in the ${\Delta}87$ mutant. However, the ${\Delta}87$ mutant protein was not detected in the medium or the intracellular fraction of the cell lysates. The LH- and FSH-like activities of the recombinant proteins were assayed in terms of cAMP production using rat LH/CG and rat FSH receptors. The metabolic clearance rate (MCR) was determined by injecting rec-eCG (2 IU) into the tail vein. The ${\Delta}95$ and ${\Delta}93$ mutants were completely inactive in both the LH- and FSH-like activity assays. The ${\Delta}96$ mutant showed slight activity in the LH-like activity assay. In comparison to the wild type, the activity of the ${\Delta}96$ mutant in the FSH-like activity assay was the highest among all the mutants. The MCR assay in which rec-eCG was injected showed a peak at 10 min in all the treatment groups, which disappeared 4 h after injection. These results imply a direct interaction between the receptor and the COOH-terminal region of the a-subunit. The data also reveal a significant difference in the mechanism by which the eCG hormone interacts with the rLH and rFSH receptors. The COOH-terminal region of the $\alpha$-subunit is very important for the secretion and functioning of this hormone.

• 한국발생생물학회지:발생과생식
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• 제21권2호
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• pp.111-120
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• 2017

Equine chorionic gonadotropin (eCG) is a unique molecule that elicits the response characteristics of both follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in other species. Previous studies from this laboratory had demonstrated that recombinant eCG (rec-eCG) from Chinese hamster ovary (CHO-K1) cells exhibited both FSH- and LH-like activity in rat granulosa and Leydig cells. In this study, we analyzed receptor internalization through rec-eCGs, wild type eCG ($eCG{\beta}/{\alpha}$) and mutant eCG ($eCG{\beta}/{\alpha}{\Delta}56$) with an N-linked oligosaccharide at $Asn^{56}$ of the ${\alpha}-subunit$. Both the rec-eCGs were obtained from CHO-K1 cells. The agonist activation of receptors was analyzed by measuring stimulation time and concentrations of rec-eCGs. Internalization values in the stably selected rat follicle-stimulating hormone receptor (rFSHR) and rat luteinizing/chorionic gonadotropin receptor (rLH/CGR) were highest at 50 min after stimulation with 10 ng of $rec-eCG{\beta}/{\alpha}$. The dose-dependent response was highest when 10 ng of $rec-eCG{\beta}/{\alpha}$ was used. The deglycosylated $eCG{\beta}/{\alpha}{\Delta}56$ mutant did not enhance the agonist-stimulated internalization. We concluded that the state of activation of rFSHR and rLH/CGR could be modulated through agonist-stimulated internalization. Our results suggested that the eLH/CGRs are mostly internalized within 60 min by agonist-stimulation by rec-eCG. We also suggested that the lack of responsiveness of the deglycosylated $eCG{\beta}/{\alpha}{\Delta}56$ was likely because the site of glycosylation played a pivotal role in agonist-stimulated internalization in cells expressing rFSHR and rLH/CGR.

• 한국가축번식학회지
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• 제24권2호
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• pp.125-142
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• 2000

임신초기 말의 태반으로부터 분비되는 융모성성선자극 호르몬 (eCG)은 황체형성 (LH), 난포자극 (FSH) 및 갑상선자극 (TSH) 호르몬과 같이 알파 및 베타 단체의 비공유결합으로 구성되어져 있는 당단백질 호르몬이다. 알파단체의 아미노산 배열은 동물종내에서 동일하지만, 베타단체는 호르몬에 따라 작용의 특이성을 가지고 있다고 알려져 있다. 말의 융모성 성선자극 호르몬은 모체의 자궁내막에 침입한 태아 유래의 융모조직 (자궁내막배)에서 합성ㆍ분비되어진다. eCG 는 당단백칠 호르몬중 탄수화물 함량이 40% 이상으로 가장 많이 함유되어 있으며, 알파단체는 두 개의 N-linked 당쇄첨가 부위 (아미노산 56 및 82번), 베타단체는 13번에 1개의 N -linked 당쇄첨가 부위와 카르복실기 말단부위에 적어도 11개의 O-linked 당쇄첨가 부위를 가지고 있는 것이 특징이다. 또한, eCG 는 다른 동물에 있어서 강력한 난포자극 및 황체형성 호르몬의 기능을 가지고 있는 아주 특이한 호르몬이다. 말의 태반과 뇌하수체 조직으로부터 eCG $\alpha$ 및 $\beta$ 단체와 eFSH $\beta$ 단체의 cDNA를 cloning 하였으며, 각 단체의 mRNA 발현은 태반과 뇌하수체에서 독립적으로 조절되어진다. 따라서, eCG 의 기능 및 수용체에 대한 호르몬의 특이한 작용을 분자생물학, 생화학적인 측면에서 연구하는데 아주 흥미로운 호르몬이다. 왜 eCG 가 이러한 이중활성을 가지고 있는지에 대해서는 아직까지 구체적으로 연구된 바가 없지만, 지금까지의 eCG 연구를 종합하면, eCG 의 알파 및 베타 단체 의 cDNA 의 유전자 구조 (알파단체는 96개 아미노산 ; 베타단체는 149개아미노산) 가 밝혀짐으로서 각각의 당쇄첨가 부위에 대한 기능연구에 박차를 가하게 될 것으로 보인다. 따라서 Site-directed mutagenesis 를 활용 어느 특정부위의 당쇄 수식이 없는 유전자 재조합 eCG 에 대한 연구로 이들 당단백질 호르몬에 대한 생물학적 특성에 대해서 확실하게 밝혀질 것으로 기대하고, 이러한 연구가 계속 진행되고 있으며, 가까운 미래에 eCG 에 있어서 지금까지 의문으로 남아있는 난포자극 및 황체형성의 이중활성에 대한 당쇄의 기능이 완전히 해결될 것으로 기대한다. eCG 의 황체형성에 대한 당쇄의 기능은 본 연구팀에 의해 알파단체의 56 번 N-linked 당쇄첨가 부위가 필수불가결하다는 결과를 얻었지만, 앞으로 난포자극 활성에 미치는 당쇄의 중요성에 관해서는 현재 연구 중에 있다.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2000년도 국제심포지움
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• pp.10-12
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• 2000

Members of the glycoprotein family, which includes CG, LH, FSH and TSH, comprise two noncovalently linked $\alpha$- and $\beta$-subunits. Equine chorionic gonadotropin (eCG), known as PMSG, has a number of interesting and unique characteristics since it appears to be a single molecule that possesses both LH- and FSH-like activities in other species than the horse. This dual activity of eCG in heterologous species is of fundamental interest to the study of the structure-function relationships of gonadotropins and their receptors. CG and LH $\beta$ genes are different in primates. In horse, however, a single gene encodes both eCG and eLH $\beta$-subunits. 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 placenta and pituitary. The dual activities of eCG could be separated by removal of the N-linked oligosaccharide on the $\alpha$-subunit Asn 56 or CTP-associated O-linked oligosaccharides. The tethered-eCG was. efficiently secreted and showed similar LH-like activity to the dimeric eCG. Interestingly, the FSH-like activity of the tethered-eCG was increased markedly in comparison with the native and wild type eCG. These results also suggest that this molecular can implay particular models of FSH-like activity not LH-like activity in the eCG/indicate that the constructs of tethered molecule will be useful in the study of mutants that affect subunit association and/or secretion. A single-chain analog can also be constructed to include additional hormone-specific bioactive generating potentially efficacious compounds that have only FSH-like activity. The LH/CG receptor (LH/CGR), a membrane glycoprotein that is present on testicular Leydig cells and ovarian theca, granulosa, luteal, and interstitial cells, plays a pivotal role in the regulation of gonadal development and function in males as well as in nonpregnant and pregnant females. The LH/CGR is a member of the family of G protein-coupled receptors and its structure is predicted to consist of a large extracellular domain connected to a bundle of seven membrane-spanning a-helices. The LH/CGR phosphorylation can be induced with a phorbol ester, but not with a calcium ionophore. The truncated form of LHR also was down-regulated normally in response to hCG stimulation. In contrast, the cell lines expressing LHR-t63I or LHR-628, the two phosphorylation-negative receptor mutant, showed a delay in the early phase of hCG-induced desensitization, a complete loss of PMA-induced desensitization, and an increase in the rate of hCG-induced receptor down-regulation. These results clearly show that residues 632-653 in the C-terminal tail of the LHR are involved in PMA-induced desensitization, hCG-induced desensitization, and hCG-induced down-regulation. Recently, constitutively activating mutations of the receptor have been identified that are associated with familial male-precocious puberty. Cells expressing LHR-D556Y bind hCG with normal affinity, exhibit a 25-fold increase in basal cAMP and respond to hCG with a normal increase in cAMP accumulation. This mutation enhances the internalization of the free and agonist-occupied receptors ~2- and ~17-fold, respectively. We conclude that the state of activation of the LHR can modulate its basal and/or agonist-stimulated internalization. Since the internalization of hCG is involved in the termination of hCG actions, we suggest that the lack of responsiveness detected in cells expressing LHR-L435R is due to the fast rate of internalization of the bound hCG. This statement is supported by the finding that hCG responsiveness is restored when the cells are lysed and signal transduction is measured in a subcellular fraction (membranes) that cannot internalize the bound hormone.

• 한국수산과학회지
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• 제50권6호
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• pp.770-776
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• 2017

This study investigated the effects of recombinant eel gonadotropin hormone (rJeGTH) produced in silkworms, with and without a carboxyl-terminal peptide from equine chorionic gonadotropin (eCG), on the induction of sexual maturation in female eels Anguilla japonica. Experiments were conducted both in vivo and in vitro. In in vitro trials, germinal vesicle breakdown (GVBD) induction did not significantly differ between rJeFSH and $rJeFSH{\cdot}eCG$ treatments and the control group. However, previous studies did find that rJeLH and $rJeLH{\cdot}eCG$ treatments induced GVBD in female eels. Our in vitro exploration of $estradiol-17{\beta}$ ($E_2$) levels in immature ovarian tissues revealed significantly higher $E_2$ levels in the group treated with $rJeFSH{\cdot}eCG$ $1{\mu}g/mL$ than in the control group. In contrast, the in vivo experiments showed no effect of recombinant hormones on the sexual maturation of feminized eels. Previous studies and our own in vitro results have clearly shown that rJeGTH and $rJeGTH{\cdot}eCG$ have a positive effect on the sexual maturation of feminized eels. To develop the activity of rJeGTH in vivo, further studies should confirm circulation time and activity of these hormones in eels' bloodstream, modify the structure of the recombinant gene, and implement additional glycosylation.

• Journal of Animal Science and Technology
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• 제54권3호
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• pp.165-174
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• 2012

For horse breeders or managers, it is critical to understand the estrous cycle of mares. Breeding of mares cannot be successfully achieved throughout the whole year as mares breed seasonally. Mares are only able to breed when day length is more than 16 h, and this period is known as the breeding season. Their estrous cycle is approximately 21 days with 5-7 days of estrus and 14 to 15 days of a diestrus period. The estrous cycle of the mare is mainly controlled by gonadotropins, which control follicular development and ovulation. Mares exhibit unique ovulatory events which are not observed in other species. A LH surge occurs for several days, with levels of LH reaching their peak after ovulation. The LH level at the time of LH peak is lower than most other species. The unique anatomical structure of the ovaries of mares is known to limit the number of eggs ovulated. Several attempts have been made to develop chemical/hormonal agents which might be used to manipulate the timed ovulation of mares. Agents that have been tested include hCG, native GnRH, Deslorelin (Ovuplant, GnRH-agonist), Buserelin (GnRH analogue), equine pituitary extracts and equine chorionic gonadotropin (eCG or PMSG). However, the function, purity or stability of these agents is not reliable. Recombinant equine LH, an alternative agent for the timed ovulation, has been developed and tested for its biological activities, through the use of both in vitro and in vivo experiments. The reLH was suggested to be a reliable agent in inducing ovulation within 48 h after being administered through injection, when the size of dominant follicle is 35 mm in diameter.