• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2002년도 국제심포지엄
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• pp.100-100
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• 2002

As an preliminary experiment for making transgenic animals producing human follicle stimulating hormone (hFSH), we tried to express recombinant hFSH gene in vitro. hFSH is a heterodimeric glycoprotein hormone produced in the anterior pituitary gland. The hormone is essential in the regulation of reproductive processes, such as follicular development and ovulation. Genes encoding the common gonadotrophin alpha subunit and FSH-specific beta subunit were inserted into retroviral vectors under the control of the rat beta actin promoter. Gene transfer to the Chinese hamster ovary (CHO) cells was done by infection of the retroviruses harvested from PT67 packaging cells transfected with recombinant retrovirus vector DNA. After selection with G4l8, PCR and RT-PCR analyses of the G4l8-resistant CHO cells showed successful transfer and expression of both ${\alpha}$ and ${\beta}$ fragments of the FSH gene.

• Asian-Australasian Journal of Animal Sciences
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• 제22권2호
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• pp.168-173
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• 2009

Human follicle-stimulating hormone (hFSH) is a pituitary glycoprotein that regulates follicular development and ovulation. Clinically, hFSH has been used to induce follicular growth in infertile women. The hormone is composed of heterodimers, including a common ${\alpha}$ subunit among the gonadotropin family and a hormone-specific ${\beta}$ subunit. Since assembly of the heterodimer is a rate-limiting step in the production of functional hFSH, transgenic clone cows carrying a single-chain hFSH transgene may efficiently produce functional hormone. Genes encoding the ${\alpha}$ and ${\beta}$ subunits of hFSH were linked using the C-terminal peptide sequence from the ${\beta}$ subunit of human chorionic gonadotropin. Bovine fetal fibroblasts were transfected with the gene construct, including the goat ${\beta}$-casein promoter and a single-chain hFSH coding sequence. Transfected fibroblasts were transferred into enucleated oocytes, and individual nuclear transfer (NT) embryos developed to the blastocyst stage were analyzed for the transgene by polymerase chain reaction. Seventy eight blastocysts (30.8%) were developed from 259 reconstructed embryos. Among these blastocysts, the hFSH gene was detected in 70.8% (34/48) of the embryos. Subsequent transfer of hFSH-transgenic clone embryos to 31 recipients results in 11 (35.5%) early pregnancies. However, all fetuses were lost before reaching day 180 of gestation. The results from this study demonstrated that bovine NT embryos carrying single-chain hFSH could be produced, and further extensive studies in which NT embryos are transferred to more recipients may give rise to single chain hFSH-transgenic cows for biomedical applications.

• Reproductive and Developmental Biology
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• 제34권1호
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• pp.33-40
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• 2010

Equine chorionic gonadotropin (eCG) is a heavily glycosylated glycoprotein composed of non-covalently linked $\alpha$- and $\beta$-subunits. To study the function and signal transduction of tethered recombinant-eCG (rec-eCG), a single chain eCG molecule was constructed, and the rec-eCG protein was prepared. In this study, we constructed 5 mutants (${\Delta}1$, ${\Delta}2$, ${\Delta}3$, ${\Delta}4$, and ${\Delta}5$) of rec-eCG using data about known glycoprotein hormones to analyze the role of specific follicle stimulating homone (FSH)-like activity. Three amino acids of certain specific sites were replaced with alanine. The expression vectors were transfected into CHO cells and subjected to G418 selection for 2~3 weeks. The media were collected and the quantity of secreted tethered rec-eCGs was quantified by ELISA. The LH- and FSH-like activities were assayed in terms of cAMP production by rat LH/CG and rat FSH receptors. Then, the metabolic clearance rate analyzed by the injection of rec-eCG (5 IU) into the tail vein was analyzed. The mutant eCGs (${\Delta}l$, ${\Delta}4$, and ${\Delta}5$) were transcripted, but not translated into proteins. Rec-eCG A2 was secreted in much lower amounts than the wild type. Only the rec-eCG ${\Delta}3$ ($\beta$-subunit: $Gln^{94}-Ile^{95}-Lys^{96}{\rightarrow}Ala^{94}-Ala^{95}-Ala^{96}$) was efficiently secreted. Although activity is low, its LH-like activity was similar to that of tethered $eCG{\beta\alpha}$. However, the FSH-like activity of rec-$eCG{\beta\alpha\Delta}3$ was completely flat. The result of the analysis of the metabolic clearance rate shoed the persistence of the mutant in the blood until 4 hours after the injection. After then, it almost disappeared at 8 hours. Taken together, these data suggest that 94~96 amino acid sequences in eCG $\beta$-subunit appear to be of utmost importance for signal transduction of the FSH receptor.

• 한국발생생물학회지:발생과생식
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• 제24권3호
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• pp.149-158
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• 2020

Kisspeptin, expressed mainly in the hypothalamus, stimulates gonadotropin-releasing hormone neurons to facilitate reproduction. In some model animals, the kisspeptin is also expressed in the pituitary. Recently, a pathway has been suggested in which kisspeptin acts directly on the pituitary to secretion of gonadotropin in mammals. In the present study, pituitaries of the Nile tilapia (Oreochromis niloticus) were cultured at different concentrations of kisspeptin-10 (Kp-10, FNYNPLSLRF) for 3 hours to observe the effect of kisspeptin on the expression of follicle-stimulating hormone β subunit (fshβ) gene and luteinizing hormone β subunit (lhβ) gene. Pituitary tissues were cultured with 0.1 μM of Kp-10, luteinizing hormone releasing hormone (LHRH), or LHRH+Kp-10 for 3, 6, 12, and 24 hours to investigate changes in the expression of fshβ and lhβ mRNA. Pituitaries cultured with high concentration of Kp-10 more than 0.1 μM for 3 hours exhibited a significant increase of fshβ mRNA expression, but not lhβ mRNA. The expression of both fshβ and lhβ mRNA increased after 6 hours in 0.1 μM of Kp-10 medium in comparison with that in the control medium. Tissues cultured in the LHRH medium however exhibited increased expression of both genes not only at 6 but also 12 hours. There were no significant differences of fshβ and lhβ gene expression in tissues cultured with LHRH+KP-10 medium compared with the control. These results suggested that although kisspeptin plays an important role in fshβ and lhβ expression in the pituitary of Nile tilapia, its action is far more complicated than expected.

• 한국가축번식학회지
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• 제27권3호
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• pp.197-206
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• 2003

hFSH는 $\alpha$와 $\beta$ subunit으로 구성된 heterodimer로서 두 subunit의 조합은 활성을 지닌 호르몬의 생산에 있어서 매우 중요한 단계이다. 이 조합과정의 효율을 증대하기 위하여 본 연구에서는 hFSH를 단일사슬의 단백질로 구축하고자 하였으며, 이의 일환으로 각 subunit 대한 cDNA단편을 연결하는 서열로 CTP linker를 도입하였다. 재조합한 hFSH-CTP 유전자는 pseudotype의 retrovirus vector system을 이용하여 CHO 세포와 닭의 배로 각각 전이되었다. CHO 세포에서의 FSH의 생산은 $\alpha$와 $\beta$를 각각 전이한 경우에 비해 hFSH-CTP를 전이한 경우에서 17배 이상 높은 것으로 나타났다. 닭에서는 유전자 전이를 시도한 62개체 중에서 11마리가 부화하였으며 그 중 10마리가 형질전환된 닭인 것으로 RT-PCR을 통하여 확인되었다. 그러나 개체의 혈중 FSH의 생산은 확인하지 못하였다. 이상의 실험 결과를 바탕으로 하여 재조합된 hFSH-CTP는 FSH의 발현에 매우 효율적인 구조로 생각되며, 또한 retrovirus를 이용한 유전자 전이 방법은 형질전환 가금의 생산에 있어서 매우 적절한 방법으로 사료된다.

• 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.

• Asian-Australasian Journal of Animal Sciences
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• 제23권3호
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• pp.292-301
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• 2010

Follicle-stimulating hormone (FSH) is a pituitary glycoprotein hormone that is encoded by separate alpha- and betasubunit genes. It plays a key role in stimulating and regulating ovarian follicular development and egg production in chicken. FSH signal transduction is mediated by the FSH receptor (FSHR) that exclusively interacts with the beta-subunit of FSH, but characterization of prokaryotic expression of the FSHb gene and its effect on the expression of the FSHR gene in local chickens have received very little attention. In the current study, the cDNA fragment of the FSHb gene from Dagu chicken was amplified using reverse transcription polymerase chain reaction (RT-PCR), and inserted into the pET-28a (+) vector to construct the pET-28a-FSHb plasmid. After expression of the plasmid in E. coli BL21 (DE3) under inducing conditions, the recombination protein, $FSH{\beta}$ subunit, was purified and injected into the experimental hens and the effect on the mRNA expression levels of the FSHR gene was investigated. Sequence comparison showed that the coding region of the FSHb gene in the local chicken shared 99%-100% homology to published nucleotides in chickens; only one synonymous nucleotide substitution was detected in the region. The encoded amino acids were completely identical with the reported sequence, which confirmed that the sequences of the chicken FSHb gene and the peptides of the $FSH{\beta}$ subunit are highly conserved. This may be due to the critical role of the normal function of the FSHb gene in hormonal specificity and regulation of reproduction. The results of gene expression revealed that a recombinant protein with a molecular weight of about 19 kDa was efficiently expressed and it was identified by Western blotting analysis. After administration of the purified $FSH{\beta}$ protein, significantly higher expression levels were demonstrated in uterus, ovary and oviduct samples (p<0.05). These observations suggested that the expressed $FSH{\beta}$ protein possesses biological activity, and has a potential role in regulation of reproductive physiology in chickens.

• 한국발생생물학회지:발생과생식
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• 제22권2호
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• pp.175-182
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• 2018

In order to examine the effects of four different light spectra (white, red, green, and blue) on the oocyte maturation, the change of reproductive parameters, via brain-pituitary-gonad (BPG) axis in grass puffer, were investigated. After exposure four different light spectra for 7 weeks, the abundance of gonadotropin-releasing hormone (GnRH) mRNA which is a type of seabream (sbGnRH) and two different subunit of gonadotropin hormones mRNAs, follicle-stimulating hormone ($fsh{\beta}$) mRNA and luteinizing hormone ($lh{\beta}$) mRNA, were analyzed in the brain and pituitary. Histological analysis showed that the mature oocyte ratio in the green spectrum was higher than other light spectra-exposed groups. Gonadosomatic index (GSI) and oocyte developmental stage were also investigated in the gonad based on histological observations. GSI value with the presence of yolk stage oocytes was significantly increased in the green spectrum-exposed group when compared to that of the other light-exposed groups (white, red, and blue) (p<0.05). The abundances of sbGnRH mRNA and $fsh{\beta}$ mRNA in the green spectrum-exposed group were also significant higher than those of the other light spectra-exposed groups (p<0.05). These results indicate that the maturation of oocyte in grass puffer can be accelerated by exposure to the spectrum of green. To better understand the molecular mechanism for the maturation of oocyte in grass puffer, further study examining the relationship between oocyte development and its related genes is required.

• Clinical and Experimental Reproductive Medicine
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• 제38권1호
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• pp.37-41
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• 2011

Objective: To compare the effectiveness and convenience of a pen device for the self-administration of follitropin ${\beta}$ with a conventional syringe delivering follitropin ${\beta}$ solution in patients undergoing IVF-ET. Methods: GnRH agonist long protocol was used for controlled ovarian stimulation (COS) in all subjects. A total of 100 patients were randomized into the pen device group or the conventional syringe group on the first day of COS. Local tolerance reactions were assessed within 5 minutes, at 1 hour and at 3 hours after each injection. On the day of hCG injection, patients were asked to rate their overall pain and convenience experienced with self-injection on a visual anlaogue scale (VAS). Results: There were no differences in patients' characteristics between the two groups. The duration of COS was significantly shorter in the pen device group than in the conventional syringe group. Patients included in the pen device group needed a significantly smaller amount of follitropin ${\beta}$. However, no differences between the two groups were found in IVF results and pregnancy outcome. The incidence of local pain within 5 minutes, at 1 hour and at 3 hours after the injection was significantly lower in the pen device group. VAS scores indicated that injections using the pen device were significantly less painful and more convenient. Conclusion: The pen device for self-administration of follitropin ${\beta}$ is less painful, safer and more convenient for the patients, and can be more effective because of the shorter duration and smaller dose of follitropin ${\beta}$ when compared with the conventional syringe.