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대한생식의학회지 제27권 제1호 2010년
인간양수에 의한 생쥐 난자 투명대의 정자수용능력 억제의 관찰
경북대학교병원 산부인과학교실1, 대구대학교 축산학과2
박기상12, 이택후1, 송해범2, 전상식1,
Human Amniotic Fluid Induces Spontaneous Hardening of the Zona Pellucida of Mouse Immature Oocytes During Maturation In Vitro
Kee Sang Park12, Taek Hoo Lee1, Hai Bum Song2, Sang Sik Chun1
1Department of OB/Gyn, Kyungpook National University Hospital and 2Department of Animal Science, Taegu University
Objective: Zona pellucida (ZP) has been thought to be the 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 16~20 weeks of gestation. HAF from five to ten patients was centrifuged and the supernatants was pooled. Cumulus-enclosed 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 48~52 hr% 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 of 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 0.0001% chymotrypsin at 37C˚ 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.
키워드 : Mouse immature oocyte, Human aminotic fluid (HAF), Hardening of zona pellucida.
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