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대한생식의학회지 제26권 제3호 2010년
난포세포가 생쥐 난자의 chymotrypsin에 대한 내성에 미치는 영향
이화여자대학교 의과대학, 의학과1 및 의과학연구소 분자생물학부1, 성신여자대학교 생물학과2, 서울여자대학교 생물학과3
김성임1, 배인하2, 김해권3, 김성례1,
Effects of follicle cells on the chymotrypsin resistance of mouse oocytes
Kim, si, Bae ih2, Kim hk3, Kim sr1
Mammalian follicle cells are the most important somatic cells which help oocytes grow, mature and ovulate and thus are believed to provide oocytes with various functional and structural components. In the present study we have examined whether cumulus or granulosa cells might play a role in establishing the plasma membrane structure of mouse oocytes during meiotic maturation. In particular the differential resistances of mouse oocytes against chymotrypsin treatment were examined following culture with or without cumulus or granulosa cells, or in these cell-conditioned media.. When mouse denuded oocytes, freed from their surrounding cumulus cells, were cultured in vitro for 17-18 hr and then treated with 1 % chymotrypsin, half of the oocytes underwent degeneration within 37.5 min(t50 = 37.5 ¡¾ 7.5 min) after the treatment. In contrast cumulus-enclosed oocytes showed t50 = 207.0. Similarly, when oocytes were co-cultured with cumulus cells which were not associated with the oocytes but present in the same medium, the t50 of co-cultured oocytes was 177.5 ¡¾ 13.1 min. Furthermore, when oocytes were cultured in the cumulus cell-conditioned medium, t50 of these oocytes was 190.0 ¡¾10.8 min whereas t50 of the oocytes cultured in M16 alone was 25.5 ¡¾ 2.9 min. Granulosa cell-conditioned medium also increased the resistance of oocytes against chymotrypsin treatment such that t50 of oocytes cultured in granulosa cell-conditioned medium was 152.5 ¡¾ 19.0 min while that of oocytes cultured in M16 alone was 70.0 ¡¾ 8.2min. To see what molecular components of follicle cell-conditioned medium are involved in the above effects, the granulosa cell-conditioned medium was separated into two fractions by using Microcon-10 membrane filter having a 10kDa cut-off range. When denuded oocytes were cultured in medium containing the retentate, t50 of the oocytes was 70.0 ¡¾ 10.5 min. In contrast, t50 of the denuded oocytes cultured in medium containing the filtrate was 142.0 ¡¾ 26.5 min. T50 of denuded oocytes cultured in medium containing both retentate and filtrate was 188.0 ¡¾ 13.6 min. However, t50 of denuded oocytes cultured in M16 alone was 70.0 ¡¾ 11.0 min and that of oocytes cultured in whole granulosa cell-conditioned medium was 156.0 ¡¾ 27.9 min. When surface membrane proteins of oocytes were electrophoretically analyzed, no difference was found between the protein profiles of oocytes cultured in M16 alone and of those cultured in the filtrate. Based upon these results, it is concluded that mouse follicle cells secrete a factor(s) which enhance the resistance of mouse oocytes against a proteolytic enzyme treatment. The factor appears to be a small molecules having a molecular weight less than 10 kDa.
키워드 : Mouse oocyte, Maturation, Chymotrypsin resistance
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