• Clinical and Experimental Reproductive Medicine
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• 제27권1호
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• pp.23-29
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• 2000

Objective: Zona pellucida (ZP) has been thought to be the barrier of egg to sperm penetration before and after fertilization. The phenomenon of ZP hardening has been considered as a post-fertilization event until now, and it is generally accepted that it is caused by the secretory products of cortical granules released during the cortical reaction. Hardening of ZP could occur "spontaneously" in mammalian oocytes in standard culture conditions, and that it is probably not a consequence of cortical reaction. The purpose of our study was to investigate the effect of human amniotic fluid (HAF) on nuclear maturation (NM) and fertilization ability of mouse immature oocytes. Methods: HAF was obtained from patients undergoing amniocentesis at $16{\sim}20$ weeks of gestation. HAF from five to ten patients was centrifuged and the supernatants was pooled. Cumulusenclosed mouse immature oocytes were incubated in the medium containing HAF, and examined to confirm NM and fertilization. Female ICR mice (about 3 weeks old) were stimulated with 7.5 IU PMSG. Immature oocytes were isolated at $48{\sim}52$ hrs post PMSG injection and cultured in TCM-199 supplemented with 20% HAF for 18 hrs. FBS was used as a control for the examination. Matured oocytes (MII) were fertilized with sperms collected from the epididymis of male mice (over 10 weeks old). Fertilization was in conducted T6 medium containing 15 mg/ml BSA, and confirmed at 6 hrs post-insemination. Fertilization rate was assessed in zona-intact or zona-free oocytes (denuded by trypsin). Evaluation of NM and fertilization was carried out by rapid staining method. ZP hardening was evaluated by incubating cumulus cell-free mature oocytes in 0.001% chymotrypsin at $37^{\circ}C$ for 10 min. Results: There was no significant difference between the effects of HAF (86.6%) and FBS (87.7%) supplements on NM of immature oocytes. When maturation medium was supplemented with HAF, total fertilization rates (7%) were significantly lower (p<0.01) than that of FBS (85.1%). In HAF group, fertilization rate was increased (p<0.01) in zona-free oocytes (7% versus 100%). The resistance of mouse oocyte ZP to digestion by chymotrypsin after maturation in vitro was significantly higher (p<0.01) in HAF group (86.7%) than in FBS (6.7%). To culture oocytes in FBS were very effective in preventing ZP hardening. However cultured oocytes in HAF showed high rate of ZP hardening (p<0.01). Conclusions: These results suggest that HAF can be used as a supplement for the NM of mouse immature oocytes in vitro. However, HAF induces spontaneous hardening of ZP of mouse immaure oocytes during maturation in vitro.

• 한국가축번식학회지
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• 제23권2호
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• pp.133-139
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• 1999

본 연구는 생쥐 미성숙란올 초자화 동결-융해하였올 때, 체외/체내 배발달능을 검토하고자 실시하였다. 생쥐 미성숙란은 동해제인 EFS40(40% ethylene glycol, 18% ficoll, 0.5 M sucrose)으로 초자화동결되었으며, 융해 후 16 시간동안 체외성숙을 유도하여, 제 1 극체가 나타난 성숙된 난자를 1~2$\times$$10^{6}$$m\ell$ 농도의 정자로 체외수정시킨 다음, 난할율 ($\geq$ 2- 세포기)과 체외 / 체내 발달율을 조사하였다. 쥐 미성숙란을 초자화 동결 융해하였던 군 (63.1%)의 체외성숙율은 동해제 노출군 (67.5%)과 대조군(66.3%)에 유사하게 나타났으나, 초자화 동결군의 난할율과 배반포형성율 (64.9, 59.0%)은 동해제노출군 (83.7, 74.7%)과 대조군 (90.7, 83.7%) 에 비해 유의하게 감소하였다 (p＜0.05). 그러나, 초자화 동결 융해하였던 생쥐 미성숙란으로부터 얻어진 배반포기배를 가임신 생쥐에 이식하였을 때, 체내발달율인 전체착상율 (31.3%)과 착상된 배로부터 발달된 산자형성율 (66.7%)은 대조군의 결과 (40.8%, 58.1%)와 각각 비교하였을 때 유의차가 인정되지 않았다. 따라서, 생쥐 미성숙란을 초자화 동결-융해하였을 때, 체외발달율은 유의하게 감소하였지만 생성된 배반포기배로부터의 산자발달율은 대조군과 유사하게 나타나, EFS40을 이용한 초자화 동결 방법은 생쥐 미성숙란 동결에 유용하게 이용될 수 있다는 것을 알 수 있었다.

• Clinical and Experimental Reproductive Medicine
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• 제21권2호
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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.

• Clinical and Experimental Reproductive Medicine
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• 제26권3호
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• pp.363-368
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• 1999

연구목적: 본 연구는 동해방지제인 EFS40을 이용한 초자화동결이 생쥐 미성숙란의 cytoskeleton과 염색체의 성상에 미치는 영향을 indirect immunocytochemistry방법과 염색체 분석법으로 알아보고자 실시하였다. 연구재료 및 방법: 본 실험은 생쥐 미성숙란을 EFS40 (40% ethylene glycol, 18% ficou과 0.5 M sucrose가 들어있는 M2배양액)으로 초자화 동결하여 융해한 후 16시간동안 체외 성숙을 유도하여, 제 1극체가 나타난 성숙된 난자를 기준으로 동해제노출군 또는 대조군과 비교 조사하였다. 결과: 초자화동결된 미성숙란의 응해 후 생존율과 체외성숙율은 90.3%과 64.7%로써, 동해제노출군 (86.7%, 69.2%)과 대조군 (100%, 58.3%)에 유사하였다. 초자화동결이 미성숙란의 microtubule과 microfilament에 미치는 영향을 조사하였던 바, 동결군의 microtubule과 micro-filament의 정상적인 형성율 (93.9%, 100.0%)은 동해 제노출군 (94.4%, 100.0%)과 대조군 (100.0%, 100.0%)의 성적과 유사하게 나타났다. 또한, 초자화동결군에서 정상적인 염색체수를 가진 난자의 비율도 65.8%로써, 대조군(79.6%)과 노출군 (69.0%)의 결과와 유의한 차이가 없었다. 결론: 생쥐 미성숙란을 EFS40에 노출하고 동결하는 것이 미성숙란의 cytoskeleton과 염색체성상에 영향을 미치지 않으며, 본 연구에서 사용된 EFS40을 이용한 초자화동결법은 생쥐 미성숙란 동결에 적합하다는 것을 알 수 있었다.

• Clinical and Experimental Reproductive Medicine
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• 제18권1호
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• pp.55-61
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• 1991

In order to increase the pregnancy rate by means of cryopreservation of the excess oocytes in IVF-ET program, the survival rate of the frozen-thawed oocytes of mouse was examined according to the stages of maturation, cryoprotectants and their treatment. The results were summarized as follows. First, during the continuous treatment with cryoprotectant media, the survival rate of oocytes was higher in DMSO than in PROH, and higher at low temperature($4^{\circ}C$) than at room temperature($25^{\circ}C$). Second, as regard with the maturation of immature(GV-intact) oocytes after treatment with cryoprotectant media, the rate of maturation in DMSO-treated group(52%) was higher than in PROH-treated group(35%). Third, according to the treatment of cryoprotectant media, the survival rate of frozen-thawed oocytes in DMSO-treated group (45%) was higher than in PROH-treated group(29%), and that of oocytes in DMSO 4-step treated group was higher than any other groups. Finally, in the post-thaw oocytes frozen at various stage of maturation, the survival rate of immature oocytes with GV was the highest in all groups. These results suggest that in the cryopreservation of mouse oocytes, DMSO was better than PROH as cryoprotectant, in treatment of cryprotectant the multi-step treatment was better than single-step, and the post-thaw survival rate of oocytes was closely related to the maturity of oocytes. It is assumed that the highest survival rate of mouse oocytes with GV is due to the stability of the structures in nucleus and intracelluar organelles, and of physiological function.

• Clinical and Experimental Reproductive Medicine
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• 제20권2호
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• pp.157-160
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• 1993

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.

• 한국동물학회지
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• 제31권4호
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• pp.265-272
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• 1988

세포융합방법을 사용하여 성장증인 포유동물의 난자에 들어있는 성숙억제요인(maturation inhibiting activity, 1연Al에 대해 조사하였다. 성장중인 생쥐난자와 성장한 미성숙난자를 1:1로 융합하여 배양했을 서 (14-17시간)에는 거의 모두 핵붕괴를 일으키었으나(90oyo), 2:1로 융합했을 때는 대부분(약 64%) 3개의 핵을 모두 간직하고 있었다. 돼지난자의 경우는 성장중인 것깎 성장한 것을 1:1로 융합하여 배양했을 때에도 융합체들은 모두 핵을 간직하고 있었으며 돼지의 성장중인 난자와 생쥐의 성장한 난자를 융합했을 때에도 모두 핵을 보존하고 있었다. 이에 반하여 돼지와 생쥐 모두에서 성장한 난자끼리 융합했을 때에는 예외없이 핵붕괴가 일어났다. 이러한 결과는 성장중인 생쥐나 돼지의 난자에 각IA가 존재한다는 열과 이종간에도 효과가 있다는 것을 보여주고 있다. 또한 이는 MIA와 성숙촉진요인(maturation promoting factor, MPH의 상대적인 양의 변화가 난자의 성숙조절에 증요한 9f할을 한다는 것을 시사해주고 있다.In an attempt to elucidate the nature of maturation inhibiting activity (MIA) in growing mamma-lian oocvtes, growing mouse and pig oocytes incompetent to resume meiosis were fused with fully grown immature oocvtes in various combinations and cultured for 14-17 hours. In slant cells composed of two mouse growing ooh임es and one large immature oocyte (2:기, their GVs remained well conserved (about 64%) after culture, but not in the ceils composed of one by one pairs. In giant cells of pig composed of one growing and onto large immature oocytes, both GVs remained conserved. In the cells composed of one pig growing and one mouse large oocytes, both GVs were also conserved. In contrast to this, pairs of large mouse oocvtes or those of large pig oocvtes had no CVs after culture. Thus, we could acertain the existEnce of MIA and none-pecificty of it in the mouse and pig growing oocvtes. The results also suggest that the relative amount of substances showlns MfA or MPF activity may be important in the regulation of oocyte amount of substances showing MIA or MPF activity may be important in the regulation of oocyte maturation.

• Clinical and Experimental Reproductive Medicine
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• 제31권3호
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• pp.155-168
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• 2004

Objective: Melatonin, which is secreted by pineal gland play an important role in the regulation of ovarian function via seasonal rhythm and sleep in most mammals. It also has a role in the protection of cells by removing toxic oxygen free radicals brought about by metabolism. In the present study, effects of melatonin on the mouse oocyte maturation were examined using two different culture conditions provided with 5% or 21% oxygen concentration. Material and Method: Immature mouse oocytes were obtained from the ovarian follicles of $3{\sim}4$ weeks old ICR strain mice intraperitoneally injected with 5 I.U. PMSG 44 hour before. Under stereomicroscope, morphologically healthy oocytes with distinct germinal vesicle (GV) were liberated from the graafian follicles and collected using mouth-controlled micropipette. They were then cultured for 17 hour at $37^{circ}C$, 5% $CO_2$ and 21% $O_2$ (95% air) or 5% $CO_2$, 5% $O_2$ and 90% $N_2$. New modified Hank's balanced salt solution (New MHBS) was used as a culture medium throughout the experiments. Effects of melatonin were examined at a concentration of $0.0001{\mu}M$, $0.01{\mu}M$ or $1.0{\mu}M$. For the prevention of spontaneous maturation of immature oocytes during culture, dibutyryl cyclic AMP (dbcAMP) and/or hypoxanthine were included in the medium. Results: Under 21% oxygen condition, oocytes cultured in the presence of $0.01{\mu}M$ melatonin showed a significantly higher maturation rates, in terms of germinal vesicle breakdown (95.0% vs 89.0%) and polar body formation (88.1% vs 75.4%), compared to those cultured with $0.0001{\mu}M$ or $1.0{\mu}M$ melatonin. However, no difference was observed in oocytes cultured under 5% oxygen whether they were treated with melatonin or not. In the presence of $0.01{\mu}M$ melatonin, oocytes either cultured under 21% or 5% oxygen exhibited no difference in the polar body formation (85.6% vs 86.7%). However, in the absence of melatonin, oocytes cultured under 21% oxygen exhibited lower polar body formation (74.7%). When oocytes were cultured in the presence of dbcAMP alone or with varying concentrations of melatonin, those treated with both compounds always showed better maturation, i.e., germinal vesicle breakdown and polar body formation, compared to those cultured with dbcAMP alone. At the same concentration of melatonin, however, oocytes exposed to 21% oxygen showed poor maturation than those to 5% oxygen. Similar results were obtained from the experiments using hypoxanthine instead of dbcAMP. Conclusion: Based upon these results, it is suggested that melatonin could enhance the meiotic maturation of mouse oocytes under 21% oxygen concentration, and release oocytes from the meiotic arrest by dbcAMP or hypoxanthine regardless of the concentration of oxygen, probably via the removal of oxygen free radicals.

• 한국동물학회지
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• 제30권1호
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• pp.89-98
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• 1987

생쥐 미성숙 난자를 재료로 하여 난자 성숙 억제제인 dbcAMP 존재하에서 GVBD 난자와의 융합에 따른 체외성숙 양상을 조사하였다. 기본 배양액 내에서 3시간이 경과하였을 때 GVBD 난자와 융합된 미성숙 난자 뿐만이 아니라 미성숙 난자 뿐만이 아니라 미성숙 난자끼리 융합된 것들도 모두 성숙을 재개하였다. 그러나 dbcAMP가 함유된 배양액 내에서 3시간 경과 하였을 때는, 미성숙 난자끼리 융합된 것들은 모두가 GV상태로 성숙이 억제된 채로 있었고 반면에 GVBD 난자와 융합된 미성숙 난자들은 비록 dbcAMP가 존재하더라도 보두 성숙을 재개하였다. dbcAMP가 함유된 배양액 내에서 20시간이 경과하였을 때 미성숙 난자끼리 융합된 것들은 여전히 성숙이 억제되어 있었으나 GVBD 난자와 융합된 미성숙 난자들은, 기본 배양액 내에서 배양된 융합 난자들과 마찬가지로 다수가 하나 혹은 두개의 극체를 형성하는 제2 감수분열 중기에 진입하였다. 두 개의 극체를 방출한 융합난자들 중에는 하나의 세포질 내에 감수분열 중기방추사가 두 곳에서 형성되는 것이 관찰되었다. 이 실험 결과로 보아 이미 성숙이 재개된 난자내에는 난자 성숙 억제제인 dbcAMP의 억제효과보다 더 영향이 큰 난자 성숙 유도 물질이 있으며, 이 물질이 융합하고 있는 미성숙 난자내로 이전하여 dbcAMP에 의해서 그 성숙이 억제된 미성숙 난자의 성숙을 유도한다는 것을 알 수 있다.

• 한국동물학회:학술대회논문집
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• 한국동물학회 2001년도 한국생물과학협회 학술발표대회
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• pp.186.3-186
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• 2001

No Abstract, See Full Text