• 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.

• Clinical and Experimental Reproductive Medicine
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• v.29 no.1
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• pp.45-56
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• 2002

Objectives: Recently, recombinant FSH (rFSH) has been manufactured using a Chinese hamster ovary cell line transfected with the gene encoding human FSH. Both rFSH and urinary gonadotropin (uFSH) could be used for controlled ovarian hyperstimulation (COH). However, uFSH implies a number of disadvantages, such as batch-to-batch inconsistency, no absolute source control, dependence on large amounts of urine, low specific activity, and low purity. The purpose of this study was to evaluate the efficacy of rFSH in human IVF-ET program. Materials and Methods: A total of 508 infertile women was enrolled in this study. They are classified into rFSH group (n=177) or uFSH group (n=331), and all of them were matched by age and cause of infertility in same period. The $Puregon^{(R)}$ (Organon, Holland) was used as rFSH, and the Metrodin-$HP^{(R)}$ (Serono, Switzeland) and $Humegon^{(R)}$ (Organon, Holland) was used as uFSH. We subdivided the patients into three age groups. The outcomes of IVF-ET program were analyzed using the statistical package for social sciences (SPSS). Results: There was no significant differences in the level of estradiol on hCG injection day, the numbers of retrieved oocytes, matured oocytes, fertilized oocytes, transferred embryos, frozen embryos between the two groups. The total dose (IU) of gonadotropin for COH was significantly lower in the rFSH group compared to uFSH group ($1339{\pm}5491.1$ vs $2527.8{\pm}1075.2$ IU, p<0.001). Clinical pregnancy rate per embryo transfer in the rFSH group showed increasing tendency, compared to the uFSH group, but there was no statistical significance (35.2% vs 29.3%). Our results demonstrated that the relative efficiency of rFSH compared with uFSH is higher in older patients. Conclusions: The ovarian stimulatory effect and clinical outcome of recombinant FSH was similar to that of the urinary gonadotropin. The IVF-ET cycles with significantly lower dose of gonadotropin in rFSH group showed comparable results. Therefore, we suggest that recombinant FSH is more potent and effective than urinary gonadotropin.

• 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 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.

• 대한생식의학회:학술대회논문집
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• 2000.02a
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• pp.7-19
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• 2000

• Development and Reproduction
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• v.1 no.1
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• pp.25-28
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• 1997

The present study was conducted to determine the effect of recombinant human follicle stimulating hormone (rhFSH) on the estrogen synthesis by human fetal ovarian tissues. Fetal ovaries were 18-19 weeks old (18 wks:n=1, 19 wks:n=2). One case of 19-week-old fetal ovaries were obtained from anencephalic fetus. Obtained ovarieswere cleaned and diced around $600\mu\textrm{m}$ pieces, and cultured in the sandwich agar bed system for 21-23 days. Estrone ($E_{1}$) and estradiol-17 $\beta$($E_{2}$) in the medium was measured by radioimmunoassay. Amount of $E_{2}$ synthesis was greater than $E_{1}$ in both normal cases. In contrast, fetal ovaries from anencephalic fetus did not produce neither $E_{1}$ nor $E_{2}$ in the presence or absence of rhFSH. Results suggest that the fetal ovaries have a capacity of estrogen production at 18-19 weeks of gestation Existence of FSH receptor is also supposed. Different results by anecephalic fetal ovaries suggest the difference in the development between normal and anencephalic fetal ovaries.

• Journal of Aquaculture
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• v.20 no.3
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• pp.154-159
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• 2007

Manchurian trout (Brachymystax lenok) is an endangered fish species in East Asia including the Korean peninsula. To establish a method for artificial propagation and to improve our understanding of the reproduction in the species, we have produced recombinant gonadotropins, follicle-stimulating hormone (r-mtFSH) and luteinizing hormone (r-mtLH), which may play central roles in reproductive activities. In the present study, the biological activities of the recombinant hormones were analyzed by gonadosomatic index (GSI), ovarian follicle diameter, and sex steroid levels in mature rainbow trout (Oncorhynchus mykiss). In the 6th day post-injection, FSH-injected fish were slightly decreased in the GSI value, although there were no significant differences among those of control, r-mtFSH, and r-mtLH treatments. Injection of the r-mtFSH increased follicle diameters significantly as compared with those of control- and r-mtLH-injected fish. The plasma steroid levels showed wide differences in the groups at 1, 3, or 6th day post-injection. Despite the variable steroid levels, three individuals receiving either r-mtFSH or r-mtLH showed a great increase in a maturation-inducing steroid, $17{\alpha},20{beta}$-dihydroxy-4-pregnen-3-one, at 3 and 6 days. Taken together, these results suggest that biological efficacies of the recombinant FSH and LH should be further studied in the Manchurian trout.

• Journal of Microbiology and Biotechnology
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• v.15 no.2
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• pp.395-402
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• 2005

Biologically active recombinant human follicle stimulating hormone (rhFSH) was produced in Chinese hamster ovary cells and purified by a series of chromatographic steps. The chromatographic steps included anion-exchange chromatography (DEAE Sepharose F/F, Q Sepharose F/F), hydrophobic interaction chromatography (Source 15 PHE), and hydroxyapatite chromatography (Macro-Prep ceramic hydroxyapatite type I). A distinctive step of the purification process developed was the use of ZnCl$_2$ for the removal of non-glycosylated or lowly-glycosylated FSH and impurities through co-precipitation with Zn$^{2+}$. Purified rhFSH was identified and characterized by several physicochemical and biological methods such as gel electrophoresis, high-performance liquid chromatography, amino acid analysis, carbohydrate analysis, and biological activity. The overall yield of the purification was ~$30\%$. The rhFSH preparation obtained showed high purity (>$99\%$) and high in vivo potency (>16,000 IU/mg). Carbohydrate analysis suggested that the purified rhFSH contained approximately $40\%$ (w/w) carbohydrate with di­or tri-antennary structure on average, which is somewhat more heavily sialylated than commercially available rhFSH. In conclusion, the results of these analyses established an identity of the purified rhFSH with natural FSH from human pituitary glands, and furthermore, the purified rhFSH preparation showed higher in vivo potency and was slightly more heavily sialylated than commercially available rhFSH.

• Clinical and Experimental Reproductive Medicine
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• v.27 no.4
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• pp.387-392
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• 2000

Objectives: 1) To compare the efficacy of urofollitropin (Follimon) to that of recombinant human FSH (rhFSH) on the growth and maturation of mouse early preantral follicles in vitro, and 2) effect of vitrification on the growth and maturation of preantral follicles and oocytes . Methods: Isolated early preantra1 follicles (100-130 ${\mu}m$ diameter) were cultured for 12 days in 20 ${\mu}l$ ${\alpha}$-MEM media drop under the mineral oil. Follimon or rhFSH was added to the culture medium at various concentrations (0, 10, 100, and 1000 mIU/ml). Results: With Follimon, the dose of 10 mIU/ml showed the best follicle survival, growth, and MIl rate of oocyte than the other concentrations. Whereas the optimal dose of rhFSH was 100 mIU/ml. Despite the different optimal doses, the efficacy of two different FSHs on the follicle growth and maturation was similar. Isolated mouse preantral follicles were cryopreserved by vitrification and cultured in vitro for 12 days with 100 mIU/ml rhFSH. Despite the decreased follicular survival rate after thawing, the follicular growth and maturation rate of its oocyte were comparable to those of the fresh follicle. Conclusion: Results from the present study revealed that 1) the optimal doses of Follimon and rhFSH for in-vitro culture of mouse follicles are different, and 2) the frozen-thawed follicles develop normally after vitrification.

• Development and Reproduction
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• v.12 no.3
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• pp.261-266
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• 2008

In the present study, we investigated in vivo effects of Manchurian trout recombinant gonadotrophin hormone (mt-rGTH) on the induction of maturation in female eel, Anguilla japonica. The brood stock, female eel (450$\pm$50 g) were weekly injected intramuscularly with different doses of 0.1, 1, 10 ${\mu}g\m{\ell}$/fish (mt-rFSH or mt-rLH) for 10 week. The effects of r-mtGTH were analyzed by gonadosomatic index (GSI), ovarian follicle diameter and sex steroid levels. All groups did not exhibit significant differences in the GSI values. Whereas plasma testosterone (T) and estradiol-17$\beta$ (E2) levels did not change significantly in control group, plasma levels of T and E2 by injection of the r-mtFSH or r-mtLH were increased at 2 or 4 week after injection. In addition, injection of the mt-rFSH (1, 10 ${\mu}g/m{\ell}$/fish) or mt-rLH (0.1, 1, 10 ${\mu}g/m{\ell}$/fish) significantly increased follicle diameters comparing to the control group. These results demonstrate that the recombinant hormone may affect early ovary development and maturation in female eel. Taken together, these results suggest that the recombinant Manchurian trout FSH and LH are effective for reproductive activities in female eel.