• Clinical and Experimental Reproductive Medicine
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• v.21 no.2
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• pp.183-190
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• 1994

Purpose of the present study was to find the optimal culture conditions for the maturation and fertilization of immature oocytes by the use human body fluids and gonadotropins (Gn) in the mouse model. Cumulus-enclosed mouse immature oocytes were incubated in the medium containing various human body fluids with or without Gn in vitro, and examined to confirm nuclear maturation (NM) and fertilization. Female ICR mice were stimulated with 7.5 IU pregnant mares' serum gonadotropin (PMSG). Cumulus-enclosed immature oocytes were isolated at 48-52 hr post PMSG injection and cultured in TCM 199 supplemented with various concentrations (20, 50, and 70%) of human body fluids such as fetal cord serum (hCS), follicular fluid (hFF), peritoneal fluid (hPF) and amniotic fluid (hAF) in the presence or absence of 10 IU/ml PMSG and 10 IU/ml human chorionic gonadotropin (hCG) for 18 hr. Fetal calf serum (FCS) was used as a control for the supplements. Matured oocytes were fertilized with sperm collected from the epididymis of male mice. Fertilization was conducted in T6 medium containing 15 mgl ml bovine serum albumin, and confirmed at 6 hr post-insemination. Evaluation of nucler maturation and fertilization was carried out by rapid staining using fuchin. There was no significant difference between the effects of human body fluids and FCS supplements on nuclear maturation of cumulus enclosed mouse immature oocytes. When maturation medium was supplemented with 20% hPF or 20% hAF, fertilization rates were significantly (P<0.01) lower than that of 20% FCS, hCS and hFF groups. However, higher concentrations of body fluids during IVM were not more beneficial on fertilizability of oocytes. The addition of Gn significantly increased the fertilization rates in hPF and hAF groups (hPF without Gn; 51.5%, compared with 85.1% for addition of Gn, and hAF without Gn; 30.1% compared with 85.8% for addition of Gn) at 20% concentration. These results suggest that human body fluids at 20% concentration and gonadotropins can be used as supplements for the maturation of mouse immature oocytes in vitro. When gonadotropins supplemented with the human body fluids in the maturation medium, fertilizability of mouse immature oocytes was increased in hPF and hAF groups. These results can be applied to maturation of human immature oocytes in vitro.

• Development and Reproduction
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• v.6 no.2
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• pp.97-104
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• 2002

In most mammals, mature oocyte-cumulus complexer(OCCs) ovulate into the oviduct where fertilization by sperm takes place. However, the complex that fail to fertilize eventually undergoes degeneration while they reside in the oviduct. Yet there is no blown mechanism how both oocyte and cumulus cells degenerate. Using human follicular fluid (hFF), bovine oviductal tissue extract (BOX) and mouse OCC, the present study aimed to find how the oviduct influence the viability of the oocyte and cumulus cells in vitro. There was no difference of oocyte maturation rate between the control and BOX-treated groups. However, there was a significant difference in the survival of cumulus cells between two groups. Cumulus cells cultured in the presence of hFF alone underwent initially expansion and then they formed monolayer in the culture dish. Even after 72 hr, they proliferated well and showed fibroblast-like morphology. Cumulus cells cultured in the presence of both hFF and BOX also expanded after 24 hr, however, after 72 hr culture, they eventually detached and degenerated. Cumulus cells cultured in the BOX alone gave a similar drastic result. When the cumulus cells cultured in the presence of BOX were stained with DAPI, their nuclei showed partial condensation and fragmentation. After detailed analysis of these cells by TUNEL assay, many nuclei of them exhibited well stained spots indicating the signs of apoptosis. Based upon these observations, it is suggested that BOX might possess a factor that leads mouse cumulus cells to undergo apoptosis in vitro.

• Clinical and Experimental Reproductive Medicine
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• v.27 no.2
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• pp.165-172
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• 2000

Objective: In vitro fertilization (IVF) and a prolonging the time of culture may be helpful in establishing a viable pregnancy through a selection effect. Some embryos do not develop beyond the 4-cell stage and some may not develop to the blastocyst stage. We have evaluated the safety of SET and the outcomes of pregnancy. Methods: Sperms were treated with Ham's F-10 supplemented with 10% human follicular fluid (hFF). oocytes or fertilized oocytes were cultured in Dulbecco's Modified Eagle Medium (DMEM) with 10% or 20% hFF respectively. Up to five oocytes were inseminated with approximately 200,000 sperm cells/2 ml in each well. Fertilization was examined in the following morning and fertilized oocytes were co-cultured until embryo transfer. Vero cells for co-culture were prepared in Tissue Culture Medium - 199 (TCM-199) with 10% fetal bovine serum. At the two to four cell and blastocyst on day 2 and day 5, embryo and blstocyst grading were evaluated. Pregnancy rate was determined after transfer of human embryos at the two to four cell stage on day 2 (Group I) or subsequent transfer of embryos on day 2 and at the blastocyst stage on day 5 (Group II). For statistical analysis, Student's t-test and Chi-square (${\chi}^2$_test) were used. Results were considered statistically significant when p value was less than 0.05. Results: No differences was found in the fertilization between Group I (81.0%, 98/121) and Group II (81.8%, 180/220). In case of cleavage rate, no difference was found in Group I (95.9%, 94/98) and Group II (97.8%, 174/178). However, the rate of-clinical pregnancy was significantly higher (p=0.014) in Group II (66.7%, 12/18) than in Group I (26.3%, 5/19). Conclusion: The results of this study showed that SET is safe and effective, and significantly increases the pregnancy rate.

• Journal of Embryo Transfer
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• v.18 no.3
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• pp.243-248
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• 2003

Sperrnatogenesis, the process by which the male germ-line stem cells(GSCs; type A spermatogonia) divide and differentiate to produce the mature spermatozoa, occurs in the seminiferous tubules of the testis. The GSCs proliferate actively to produce two types of cells: other GSCs and differentiating spermatogonia. GSCs have unipotentcy, devoted solely to the generation of sperm. The function of GSCs has broad implications for development, disease, and evolution. Spermatogenesis is fundamental for propagation of species and the defects of this system can result in infertility or disease. The ability to identify, isolate, culture, and alter GSCs will allow powerful new approaches in animal transgenesis and human gene therapy relating to infertility. Until recently, research on stem cells in the testis has been limited because of technical difficulties in isolating and identifying these cell populations. Here, we were trying to find out optimal conditions for in vitro culture of GSCs for identifying and isolating GSCs. We collected mouse GSCs from 3-days old mouse by two-step enzyme digestion method. GSCs were plated and grown on mouse embryonic fibroblasts in Dulbecco's modified Eagle's medium (DMEM) containing 15％ fatal bovine serum, 10 mM 2-mercaptoethanol, 1％ non-essential amino acids, 1 ng/$m\ell$ bFGF, 10 $\mu$M forskolin, 1500 U/$m\ell$ human recombinant leukemia inhibitory factor (LIF). Over a period 3∼5 days, GSCs gave rise to large multicellular colonies resembling those of mouse pluripotent stem cells. After 5th passages, cells within the colonies continued to be alkaline phosphatase and Oct-4 positive and tested positive against a panel of two immunological markers(Integrin $\alpha$ 6 and Integrin $\beta$ 1) that have been recognized generally to characterize GSCs. SSEA-1, SSEA-3, and SSEA-4 also showed positive signals. Based on our data, these GSCs-derived cultures meet the criteria for GSCs itself and even other pluripotent stem cells. We reported here the establishment of in vitro cultures from mouse male GSCs.