인간 난관세포와의 체외 공동배양과정에서 혈소판 활성요소가 생쥐배의 발달에 미치는영향

민부기,김기석,이희섭,홍기연,김흥곤,신무철,이찬근,최은하,Min, Bu-Kie,Kim, Kie-Seok,Lee, Hee-Sup,Hong, Kie-Youn,Kim, Heung-Gon,Shin, Mu-Cheol,Lee, Chan-Kun,Choi, Eun-Ha 대한생식의학회 1996 Clinical and Experimental Reproductive Medicine Vol.23 No.1

There are a number of problems during the process of culture in vitro on fertilization and embryo development compared to those on in vivo counterparts. And the platelet activating factor (PAF), which is found not only in mammalian spermatozoa but also preembryos, is implicated on reproductive process. To improve the environment of culture on in vitro fertilization and embryo development, coculture using salpingeal epithelial cells has been considered to accept the better result on pregnancy rate. This study was designed to determine if two different culture systems, coculture alone and PAF treated coculture, are positive or negative influence on process of in vitro fertilization and embryo culture in mouse. The cell cleavage rate reached to 2-4 cell stage at 24 hours of culture is 56.81% (50/88) and 48.21%(54/112) respectively, in PAF treated group which is added PAF on coculture and in coculture group. But the rate of cells cleavage was similar in both group after 48 hours of culture. The rate of unfertilization after insemination of oocytes was higher in coculture group(55..53%) than in PAF treated group(42.37%). And in assessment of undeveped embryos, the rate of equalized cell block was similar on both, coculture alone (35.3%)and PAF treated coculture(35.5%). while unequalized cell block was higher rate in PAF treated coculture(19.4%) than coculture alone (11.8%). But the rate of cytoplasmic degeneration of undeveloped embryos was significantly higher in PAF treated coculture than coculture alone. In conclusion, we have observed that PAF treated coculture is superior in the rates of in vitro fertilization and early embryo cell cleavage compared to those in coculture alone, but there is no difference on the rates of embryo develpments, cell degeneration, cell quality in both PAF treated coculture and coculture alone when the embryo cells were continuosly cultured for 48 hours or more.

NO(Nitric Oxide)가 생쥐의 배 발달에 미치는 영향

민부기,김기석,이희섭,홍기연,신형도,성연경,김형민,Min, Bu-Kie,Kim, Kie-Suk,Rhee, Hee-Sub,Hong, Gi-Youn,Shin, Hyeong-Do,Sung, Yeon-Kyeong,Kim, Hyung-Min 대한생식의학회 1998 Clinical and Experimental Reproductive Medicine Vol.25 No.2

Objective: To ananlyze the direct effect of nitric oxide (NO), generated from sodium prusside (SNP) on the embryo developments in reproductive process. Design: Ova from mouse were treated to allow fertilization in in vitro culture. And the samples of fertilized ova were alloted into five alliqutos. Each alliquot was cultured in media treated with either concentration at 0 (n=92), $25{\mu}M$ (n=84), $50{\mu}M$ (n=80), $100{\mu}M$ (n=77), $500{\mu}M$ (n=54) of SNP. Main Outcome Measure: Rates of embryonal cell cleavages, viability and cell morphology were assessed during in vitro fertilization and culture. Results: As analyse the cell cleavage at 24 hours after in vitro culture of fertilised egg in variuos NO concentration, all of egg cells of each alliquot were developed to $2\sim4$ cell stage. But the alliquot of egg cells treated with $50{\mu}M$, which were totally degenerated. And also all embryonal cells of each alliquot were developed to 8 cell stage and morula stage on culture continuosly. And the embryonal cells of each alliquot were analysed at 24 and 48 hours following the in vitro culture. The rates of cell fragmentation and fusion were $4.2{\pm}3.4%$ in control group which is not treated with NO, while experimental groups was high, as rated $23.4{\pm}6.2%$ in $25{\mu}M$, $28.2{\pm}5.7%$ in $50{\mu}M$ and $32.1{\pm}6.4%$ in $100{\mu}M$ concentration of NO. Accordingly the rate of abnormal morphology of embryonal cell in control was lower significantly than that in each alliquot of experimental groups (p<0.05). And the degenerated rates of embryonal cells were 0% in control, $17.8{\pm}6.7%$ in $25{\mu}M$, $23.6{\pm}4.7%$ in $50{\mu}M$ and $26.8{\pm}11.2%$ in $100{\mu}M$ at 8 cells and morula on culture of 48 and 72 hours. On the examination of embryonal cells developed to blastocyst through in vitro culture, the rates of degenerated cells were $16.8{\pm}7.2%$ in control, $37.5{\pm}6.2%$ in $25{\mu}M$, $73.4{\pm}4.6%$ in $50{\mu}M$, 100% in $100{\mu}M$. Conclusion: This results suggeted that the NO in any concentrations is harmful on embryos in view of morphology as well as viability of cell, and the toxicity of NO on embryo is stronger at condition in higher concentration of NO.

체외배양에서 인간 난포액이 생쥐의 배 발달에 미치는 영향

민부기,최기욱,김기석,이희섭,홍기연,이봉주,이선영,박승택,Min, Bu-Kie,Choi, Ki-Wook,Kim, Kie-Suk,Lee, Hee-Sub,Hong, Ki-Yeon,Lee, Bong-Ju,Lee, Sun-Young,Park, Seung-Teak 대한생식의학회 1999 Clinical and Experimental Reproductive Medicine Vol.26 No.2

The follicular fluid (FF) of ovary contains various biological active products which affected on the growth of follicles and the fertilization of oocyte in physiological reproductive process of mammals. This study was designed to determine the effects of human FF on fertilization of oocyte and embryonal development in vitro culture. The FF was prepared as clear without blood contamination by needle aspiration from mature follicles of human at the time of oocytes retrieval for in vitro fertilization (IVF). As the medium for culture in vitro of embryonal cells, human tubal fluid (HTF) supplemented with follicular fluids at concentrations of 10%, 40% and pure FF were used. These effects were compared to control group of cultured embryos in HTF supplemented with 0.4% BSA (bovine serum albumin). For IVF, 64 eggs in control group, 67 eggs in 10% FF, 57 eggs in 40% FF and 64 eggs in pure FF were respectively allocated. And the rates of fertilization were almost similar in all groups as resulting 82.81% in control, 85.07% in 10% FF, 87.71% in 40% FF and 81.25% in pure FF. On the examination for embryonal cleavage from fertilized eggs, the rates of developing to 4 cell stage was similar in all groups, as results 98.11% in control, 98.27% in 10% FF and 98% in 40% FF but 78.84% in pure FF. And the rates of developing to 8-16 cell stage were significantly reduced as 44% in 40% FF and 44.23% in pure FF (p<0.05) compare to 71.69% in control media. As likewise, the rates of developing to morular stage were also significantly reduced to 36% (p<0.05) and 21.15% (p<0.01) respectively in 40% FF and pure FF. And the rates to blastocystic stage of embryo was lowest as 7.69% in pure FF (Table 1). The quality of embryonal cells on cleavage to the 8-16 cell stage was poorer, higher concentrations of FF. The rates of grade 1 in pure FF, as 23.07%, was lowest compare to those of other groups, in which the rates of grade 1 in control, 10% FF and 40% FF were 58.49%, 47.36% and 34% respectively. And on the contrary, the rate of grade 4 in pure FF was highest as 23.07%, while those were 5.66% in control, 8.77% in 10% FF and 20% in 40% FF (Table 2). On the viability of embryos, the rate of embryonal cell death was more rise, at the higher concentrations as well as longer exposure in the follicular fluid. At 48 hours after in vitro culture of embryos, the rate of survival embryos in pure FF was markedly lowered as 44.23%, compare to that of control (p<0.05). But there was not significant difference between the rates of survival embryos in each group beside the pure FF, which the rates were 77.35% in control, 70.17% in 10% FF and 60% in 40% FF respectively. And at 72 hours after in vitro culture, the rates of survival embryos were also significantly dropped to 21.15% in pure and 36% in 40% at concentration of FF compare to 62.26% in control (p<0.05, p<0.01). Finally, the rate of embryonal death at 96 hours after in vitro culture was highest as 82.69% in pure FF among all groups which those were 35.84 in control, 56.14% in 10% FF and 64% in 40% FF respectively (Fig. 1, 2, 3). In conclusion, this study suggests that the FF has no effects, in particular, to the in vitro fertilization of oocytes but exerted a bad effect to the cleavage, quality and viability of the embryonal cells during in vitro culture. However, the FF is harmful on embryonal development at conditions in higher concentration and especially on the embryos after $8{\sim}16$ cell stage.

Nitroprusside가 인간정자의 생존력, 운동성, Reactive Oxygen Species 발생에 미치는 영향

민부기,이희민,김기석,이희섭,김흥곤,홍기연,이봉주,Min, Bu-Kie,Lee, Hee-Min,Kim, Ki-Seok,Lee, Hee-Sup,Kim, Heung-Gon,Hong, Gi-Youn,Lee, Bong-Ju 대한생식의학회 1996 Clinical and Experimental Reproductive Medicine Vol.23 No.3

Objective: To analyze the direct effect of nitre oxide, generated from sodium nitroprusside, on sperm motility and reactive oxygen species. Design: Human sperm samples were treated to allow swim-up and washing. And the samples were devided into four aliquots. Each aliquot was incubated with either concentration at 0, 100nM, $10{\mu}M$, 1mM of nitroprusside. Intervention: Samples were measured chemiluminosence for reactive oxygen species of each aliquot with concentrations at 0, 100nM, $10{\mu}M$, 1mM of nitroprusside at allowing swim-up and washing of sperm. Main Outcome Measures: Percent motion parameters and viability were asse-ssed at 0, 3, 6, 12, 24 hours incubation. Results: The percent viablity was lower slightly in control group (50.2%) than that in sperm treated with 100nM of nitroprusside(57.5%) at 24 hours after incubation, while was reduced significantly in sperm with concentra-tion of $10{\mu}M(42.1%)$ and 1mM(21.3%)of nitroprusside at 6 hours after incubation. And the sperm treated with 1mM of nitroprusside was immotile totally at 6 hours after incubation. The straight line$(35.3{\pm}5.6%)$, the rapid forward$(37.2{\pm}6.4%)$ and the weak curvilinear velocity$(9.6{\pm}2.4%)$were more favorable comparing with those ($32.4{\pm}4.2%$, $30.0{\pm}7.8%$ and $18.0{\pm}4.6%$ respectively) in control group at 3 hours after incubation, but reduced significantly in sperm treated with $10{\mu}M$ and 1mM of nitroprusside. The levels of reactive oxygen species in control(700 c.p.m.) is lower significantly than that in each experimental groups of sperm treated with nitroprusside. And the levels of reactive oxygen species were 2200 c.p.m. in 100nM, 6200c.p.m. in $1{\mu}M$ and 12800c.p.m. in 1mM respectively. Conclusion: These results suggested that the concentration of 100nM of nitroprusside on sperm is beneficial to the maintanance of viablity and motile velocity, but detriment in high concentration of $10{\mu}M$ or 1mM of nitroprusside.

생쥐난의 체외 배양에서 인간난포액과 표피 성장 인자가 난성숙에 미치는 영향

민부기,Min, Bu-Kie 대한생식의학회 1993 Clinical and Experimental Reproductive Medicine Vol.20 No.2

The human follicular f1uids(F.F.) may be considered to contribute the maturation of the oocytes on the in vitro culture. To investigate the effects of epidermal growth factor (E.G.F.), which is present in mature and immature follicular fluids, we had experiments of mouse oocytes maturation in vitro. The endpoints assayed were rated as percentage of oocytes undergoing germinal vesicle breakdown(G.V.B.D.) and polar body(P.B.) formation at 12 hours after in vitro culture. The rates of G.B.B.D. were 87% in mature F.F. 68% in immature F.F. and 78% in Ham's F-10 medium respectively. And overall the mature F.F. seem to stimulate on in vitro oocyte maturation compared with either immature F.F. or Ham's F-10 medium. As the concentration of addition of E.G.F. in immature F.F., the rates of G.V.B.D. and P.B. formation were 82 %, 23% in addition with 2 ng/ml while 84%, 32% in addition with 4 ng/ml respectivly. And at the concentration of addition of E.G.F. in Ham's F-10 media as well, the rates of G.V.B.D. and P.B. formation were 84%, 40% and 82%, 44% in addition with each 2ng, 4ng. AccordinglY there was no influence on the oocytes maturation at the addition of E.G.F. to each immature F.F. and Ham's F-10 medium. In conclusion, the E.G.F. is not able to induce oocyte maturation independent of it's effects in immature F.F. and Ham's F-10 media.

Leukemia Inhibitory Factor가 배의 배포형성에 미치는 영향

민부기,오수미,김기석,홍기연,김훈영,심재량,박승택,Min, Bu-Kie,Oh, Soo-Mi,Kim, Kie-Suk,Hong, Gi-Youn,Kim, Hun-Young,Sim, Jea-Ryang,Park, Seung-Teak 대한생식의학회 2001 Clinical and Experimental Reproductive Medicine Vol.28 No.1

Objective: To determine the effects of leukemia inhibitory factor (LIF) on embryonal development in in vitro culture. Methods: This is designed in vitro model using eggs from mouse. The eggs from mouse were assigned 29 for control group, 53 for 20 ng/ml of LIF, 88 for 40 ng/ml of LIF, 68 for 80 ng/ml of LIF respectively for in vitro fertilization. And 26 fertilized eggs at 2 cell stage from mouse also were assigned. The mouse embryos of all groups were cultured in medium supplemented with LIF in different concentrations, whereas the eggs in control group was cultured in medium without supplement of LIF. Results: At 72 hours culture of eggs from in vitro fertilization, there was a slight increas in rate of embryonal development to morula in both LIF-20 and LIF-40 as results of 64.15% and 75% respectively, while 42.65% in inferior rate of LIF-80, compare with 51.72% in control group. But the difference between these each groups were not significant in statistically ($p{\le}0.05$). And after 96 hours culture of eggs, the rates blastocyst formation was significantly higher in both LIF-20 and LIF-40 as 56.6% and 63.63% than those in control and LIF-80 as 44.83% and 35.29% respectively. On culturing eggs from in vivo fertilization, the rates of blastocyst formation was significantly not only higher as 85% and 81.81% respectively in medium supplemented with LIF-40 and LIF-80 than 42.3% in LIF-20 but also embryonal cell viability were remakedly improved at 96 hours after culture. Conclusion: The LIF in low dose is embryotrophic, but LIF in high dose is embryotoxic on eggs from in vitro fertilization. Whereas on culturing eggs from in vivo fertilization, LIF is more beneficial with dose dependent in high concentration.

자궁내막증 치료 전후 환자의 혈청이 생쥐 난자의 수정률에 미치는 영향

김기석,민부기,이희섭,홍기연,이선영,박현진,김흥곤,Kim, Kie-Suck,Min, Bu-Kie,Rhee, Hee-Sub,Hong, Kie-Youn,Lee, Sun-Young,Park, Heon-Jin,Kim, Heung-Gon 대한생식의학회 1999 Clinical and Experimental Reproductive Medicine Vol.26 No.3

Objective: To evaluate the effect of serum obtained before and after treatment for endometriosis on in vitro fertilization and development of two cell mouse embryo. Design: Pretreatment and posttreatment comparoson of fertilization of mouse oocyte and embryo development in serum supplement from patients with endometriosis; result were compared using Stuent T-test analysis. Method: Infertility Clinic, Department of Obstetrics and Gynecology, Collage of Medicine, Won kwang university, Korea. Patients was chosed eleven consecutive women with endometriosis. Interventions was all patient underwent laparoscopic or conservative surgery. This was followed by a 6-month course of burserelin acetate $900{\mu}g/d$. Main outcome was measured total number of fertilization and embryo that was fertilization after 24 hours and reached blastocyst stage after 72 hours of incubation were compared before and after treatment. Result: Before treatment, 47% of the oocyte were fertilized and 31% of the embryo reached blastocyst stage. After treatment, Significantly more fertilized and Significantly more embryo developed to blastocyst on the stage I and II of endometriosis. Conclusion: The fertilization and embryo toxicity of serum samples from patients with endometriosis is lost after treatment.

폐경기 골다공증 여성에 있어서 Alendronate 치료에 대한 골교체의 생화학적 표지자의 평가

김기석(Kie Suck Kim),민부기(Bu Kie Min),이승필(Seung Fil Lee),김인숙(In Suk Kim),김훈영(Hun Young Kim),심재량(Jae Ryang Sim) 대한폐경학회 2000 대한폐경학회지 Vol.6 No.1

N/A Objectives: To evaluate the clinical utility of recently developed biochemical markers of bone turnover to monitor the response of osteoporotic patients to antiresorptive therapy, we compared the results of two advanced assays for markars of bone resorption and two of bone formation. Subjects and Methods: The rate of bone turnover in 37 women (mean±SD age, 58.2±4.5yr) with low boe mass and all postmenopausal women (mean±SD yr PMP, 8.3±5.2) was compared to that in 16 Premenopausal women(mean+SD age, 40.2±5.3yr) randomly selected from out-patient in our hospital and all have a normal spine bone mineral density(BMD). Perodically during the 12-month study,the level of several markers of bone turnover wen, measured. Serum osteocalcin, bone specific alkaline phosphatase mesured by RIA were used to assess bone formation. To assess bone resorption, we measured urinary excretion of Dedxypyridinoline, type l collagen cross-linked N- telopeptide, Result: All bone formation markers and all bone resorption marker were significantly increased in PMP Osteoporotic women. Under treatment with alendronate, resorption markers decreased earlier than marker of bone formation, Conclusion: This study, using biochemical markers of bone turnover, demonstrates that bone turnover is increased in PMP osteoporotic women. Alendronate treatment decreased bone turnover to the normal premenopausal range, with a steady state level reached after 1 month of therapy with 10mg for resorption markers and after 3-6 months of therapy for markers of bone formation.

Estradiol과 Medroxyprogesterone Acetate가 골아세포의 성장에 미치는 영향

김기석 ( Kie Suk Kim ),민부기 ( Bu Kie Min ),홍기연 ( Gi Youn Hong ),박승택 ( Seung Taek Park ),이승필 ( Seung Phil Lee ),김인숙 ( In Suk Kim ) 대한폐경학회 2001 대한폐경학회지 Vol.7 No.1

N/A This study was designed to clarify the hormonal effect on the growth of osteoblast. And the osteoblasts from neonatal mouse were cultured in the medium at various concentrations of 17β-estradiol(EST), medroxyprogesterone acetate(MPA) in addition 10nM of dexamethasone(DMS) and alcohol for 48 hours. And in order to detect the response of osteoblast on the those hormones, the alkaline phosphatase(ALP) activity also was measured. 1. At 48 hours after culture of osteoblasts in media at various concentrations of hormones, EST has an effect on increasing of the cell number in fashion of dose dependent, whereas MPA did not increase the cell number. 2. Dexametasone significantly promoted differentiation of osteoblasts does dependently with increasing of ALP activity. 3. In osteoblasts culture in media supplemented with alcohol in the absence of 10nM DMS, EST increased significantly ALP activity, while decreased ALP activity of osteoblast in the presence of DMS, 4. In culture of osteoblasts treated with alcohol in the absence of 10nM DMS, MPA did not show any change of ALP activity. But in the presence of DMS, MPA decreased ALP activity of osteoblast. 5. EST plus MPA stimulated the ALP activity in osteoblasts treated with alcohol in the absence of DMS. While, in the cells treated with DMS, they synergistically decreased ALP activity.

난관 세포와 공동 배양에 의한 배 세포 발달의 향상

김정호,홍기연,김기석,최정훈,민부기,Kim, Jung-Ho,Hong, Gi-Youn,Kim, Kie-Sock,Choi, Jung-Hoon,Min, Bu-Kie 대한생식의학회 1994 Clinical and Experimental Reproductive Medicine Vol.21 No.1

To improve in vitro embryonic cell development, this study was desigend to culture in vitro fertilized early embryos of mouse in two different systems; conditioned medium alone and ampullary cells co-culture. Thirty two of 83 embryos(38.6%) were blocked in the 2 cell stage by co-culture, as compared to forty of 42 embryos(95.2%) in control group for 24hours culture. And all the embryonic cells cultured for conditioned medium alone were blocked for 48 hours culture. Twenty seven of 46 embryos (58.7 %) which overcome culture block in 2 cell stage by cocultured were developed morular and expanded blastocyst, and ninteen of 46 embryos(26.1 %) underwent hatching for 96 hours culture. The cellular fragmented rates for embryo were 26.2% in medium alone; 10 fragmented blastomere were graded mild status and 1 fragmented blastomere in severe status. On the other hand, the fragmented rate for 48 hours co-cultured were 15.7%03/83); 8 fragmented embryos were graded mild status, moderate status in 3 fragmented embryos and severe in 2 fragmented embryos respectively. In conclusion, the co-culture of embryos with ampullary cells is good to improve quality of embryos and overcome of culture block as well as development of cell cleavage.