• Korean Journal of Animal Reproduction
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• v.27 no.3
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• pp.197-206
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• 2003

hFSH (human follicle stimulating hormone) is heterodimer consisting of $\alpha$ and $\beta$ subunits. Since assembly of the both subunits in the cell is often the rate-limiting step in production of functional hormone, single-chain hormones have been engineered by genetically linking two different cDNA fragments with a linker sequence. Using retrovirus vector system, the resulting recombinant hFSH gene was transferred in CHO cells and chicken embryos, and the expression of the gene was investigated. In CHO cells, protein synthesis from the single-chain FSH gene was 17 fold higher than that from the heterodimeric counterpart. In the study of transgenic chickens, ten of the eleven chicks hatched from 62 embryos manupulated with recombinant retrovirus stock was determined to carry transgenic genes. RT-PCR analyses confirmed transcription of the single-chain FSH gene, however, no recombinant FSH was detected from the blood samples.

• Journal of Embryo Transfer
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• v.21 no.1
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• pp.13-20
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• 2006

The purpose of this study was to develope the transgenic cattle expressing hFSH into the urine using the nuclear transfer. To produce the interest gene in urine, the specific vector was ligated with hFSH gene undo. maUII promoter. The fetal fibroblast cells (KbFF) were isolated from a 45-day male fetus. The hFSH gene was co-transfected with pcDNA3 (neo) vector to KbFF cells by electroporation. The gene-transfected cells were cultured with G-418 selection medium for 2 weeks. Selected colonies were confirmed by PCR. For nuclear transfer, enucleated bovine oocytes were transferred with hFSH transfected or nontransfected fetal fibroblasts. The cleavage and blastocyst formation rates were significantly lower (p<0.05) in cloned embryos transfected with hFSH gene (68.7% and 15.7%) than in those non-transfected (67.6% and 24.5 %), respectively. Apoptosis analysis showed no difference between hFSH transfected and non-transfected blastocysts (p>0.05). The blastocysts were transfected to 77 (control 24, hFSH 53) recipient cows. Two calves were born (1.9%) following transfer with NT embryos transfected with hFSH gene, but they were confirmed not to be transgenic calves. This result shows that the hFSH colonies were mixed with transfected and non transfected cells. Further research will be needed for selection and establishment of gene transfected cells.

• Asian-Australasian Journal of Animal Sciences
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• v.22 no.2
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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.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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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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• v.23 no.3
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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.

• Clinical and Experimental Reproductive Medicine
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• v.25 no.3
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• pp.281-286
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• 1998

Premature ovarian failure is a condition causing amenorrhea, hypoestrogenism, and elevated gonadotropins in women younger than 40 years. Many causes of premature ovarian failure were reported, including genetic abnormalities, enzymatic defects, defects in gonadotropin secretion or action, autoimmune disorders, physical and idiopathic causes. Recently, Finnish group reported a point mutation in the follicle stimulating hormone (FSH) receptor gene in premature ovarian failure patients. But it was reported that the group from United States could not find any mutation in FSH receptor gene. So we analysed C566T point mutation of FSH receptor gene using restriction fragment length polymorphism (RFLP) and compared the result between premature ovarian failure patient with idiopathic and known causes. But we did not find 556C${\rightarrow}$T mutation in the FSH receptor gene in both groups. These findings suggest that the missense mutation in the human FSH receptor gene reported in Finnish women with premature ovarian failure is uncommon in Korean women with premature ovarian failure.

• Journal of Embryo Transfer
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• v.28 no.4
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• pp.361-371
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• 2013

The present study assessed the effect of FSH and LH on oocyte meiotic, cytoplasmic maturation and on the expression level and polyadenylation status of several maternal genes. Cumulus-oocyte complexes were cultured in the presence of FSH, LH, or the combination of FSH and LH. Significant cumulus expansion and nuclear maturation was observed upon exposure to FSH alone and to the combination of FSH and LH. The combination of FSH and LH during entire IVM increased the mRNA level of four maternal genes, C-mos, Cyclin B1, Gdf9 and Bmp15, at 28 h. Supplemented with FSH or LH significantly enhanced the polyadenylation of Gdf9 and Bmp15; and altered the expression level of Gdf9 and Bmp15. Following parthenogenesis, the exposure of oocytes to combination of FSH and LH during IVM significantly increased cleavage rate, blastocyst formation rate and total cell number, and decreased apoptosis. In addition, FSH and LH down-regulated the autophagy gene Atg6 and upregulated the apoptosis gene Bcl-xL at the mRNA level in blastocysts. These data suggest that the FSH and LH enhance meiotic and cytoplasmic maturation, possibly through the regulation of maternal gene expression and polyadenylation. Overall, we show here that FSH and LH inhibit apoptosis and autophagy and improve parthenogenetic embryo competence and development.

• Reproductive and Developmental Biology
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• v.35 no.1
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• pp.115-121
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• 2011

hFSH is a glycoprotein secreted from anterior pituitary and consists of ${\alpha}$ and ${\beta}$ subunits. Because of its major biological functions including sperm formation in the male and for follicular growth, FSH is used to cure woman's sterility. In this study we tried to produce recombinant hFSH in vitro using a retrovirus expression vector. Two major components of the vector we constructed are: ( i ) a DNA fragment containing ${\alpha}$ and ${\beta}$ genes fused by a DNA sequence coding carboxyl terminal peptide (CTP) of human chorionic gonadotropin, (ii) a DNA fragment corresponding woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). Evaluation of expression profile of the recombinant FSH using reverse transcription PCR and enzyme-linked immunosorbent assay (ELISA). Among three cell lines tested, HeLa cells were the best for hFSH expression (5,395 mIU/ml), then followed by chicken embryonic fibroblast (CEF) cells and Chinese hamster ovary (CHO) cells in the order of hFSH production. In addition to the amount, the FSH produced from HeLa cells was highest in terms of biological activity which was determined by measuring cAMP.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.42-42
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• 2003

복제기술은 기존의 형질전환 동물 생산의 효율을 향상시킬 수 있는 기술로서 인정하고 있으며 또한 이를 이용하여 형질전환 동물의 생산이 이루어지고 있다. 따라서 본 연구는 표지유전자 (GFP)와 유용유전자 (hFSH)를 이용하여 임신 45일령에 채취한 태아섬유아세포에 transfection 하고, transfection 된 세포의 효율적인 선발과 이를 이용한 형질전환 복제 수정란을 생산하고자 실시하였다. 대조구 (KbFF), GFP (79KbFF-GFP c-3) 및 hFSH (79KbFF-hFSH n-1)에 공시한 세포는 모두 동일한 태아유래의 세포 (모 79, 부 KPN178,♂)를 이용하였다. pAB-eGFP와 hFSH 유전자는 각파 electroporation 방법을 이용하여 transfection 하고, 이를 2주 동안 G418로 배양하며 selection 하였다.

• Proceedings of the KSAR Conference
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• 2004.06a
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• pp.220-220
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• 2004

The purpose of this study was to development of transgenic cow using the nuclear transfer. To secrete hFSH in urea, the vector was constructed with UPII promoter. The fetal fibroblast cells (KbFF) were constructed from pregnant day 45 male fetus. The hFSH genes were cotransfected with pcDNA3 (neo) vector to KbFF cells by electroporation. (omitted)