CAJ | 학술논문

背景与目的:初级卵母细胞生长过程中对理化因子反应敏感,拟建立小鼠腔前卵泡体外培养成熟(IVG)-染色体分析技术,为生殖毒理学提供有效的检测模型.材料与方法:采用机械分离法从12～14日龄昆明小鼠的卵巢中分离出直径为140～160 μm的腔前卵泡,单个卵泡放入微液滴培养体系中,隔天半量换液,连续培养10 d,对成活卵泡体外诱导超排,于16～24 h后,检测卵母细胞成熟情况,分别计数停滞在生发泡期(GV)、生发泡期破裂(GVBD)和排出第一极体(PB)的卵母细胞数,对卵母细胞进行染色体数目与结构分析,并与体内发育体外成熟卵母细胞(IVM)及体内超排成熟卵母细胞(IVC)进行比较分析. 结果:从4只小鼠卵巢中分离得到332个腔前卵泡,培养早期卵泡直径增长显著,随后呈"摊鸡蛋"样生长,部分形成假腔.培养10 d后存活率为95.78%(318/332).激素诱导排卵后,卵丘.卵母细胞复合体排出率72.01%(229/318),其中11.80%(27/229)停滞在GV期,39.30%(90/229)发生GVBD,47.16%(108/229)发育成熟,排出第一极体(PB),1.74%(4/229)发生孤雌活化.IVG与IVM、IVC两种方法获得的卵母细胞染色体对比分析,未见明显的染色体数目与结构异常. 结论:小鼠腔前卵泡体外培养成熟,可获得与体内生长相同的成熟卵母细胞,染色体分析未见异常,可成为一种良好的生殖毒理学体外检测与卵泡发育机制研究模型.
배경여목적:초급란모세포생장과정중대이화인자반응민감,의건립소서강전란포체외배양성숙(IVG)-염색체분석기술,위생식독이학제공유효적검측모형.재료여방법:채용궤계분리법종12～14일령곤명소서적란소중분리출직경위140～160 μm적강전란포,단개란포방입미액적배양체계중,격천반량환액,련속배양10 d,대성활란포체외유도초배,우16～24 h후,검측란모세포성숙정황,분별계수정체재생발포기(GV)、생발포기파렬(GVBD)화배출제일겁체(PB)적란모세포수,대란모세포진행염색체수목여결구분석,병여체내발육체외성숙란모세포(IVM)급체내초배성숙란모세포(IVC)진행비교분석. 결과:종4지소서란소중분리득도332개강전란포,배양조기란포직경증장현저,수후정"탄계단"양생장,부분형성가강.배양10 d후존활솔위95.78%(318/332).격소유도배란후,란구.란모세포복합체배출솔72.01%(229/318),기중11.80%(27/229)정체재GV기,39.30%(90/229)발생GVBD,47.16%(108/229)발육성숙,배출제일겁체(PB),1.74%(4/229)발생고자활화.IVG여IVM、IVC량충방법획득적란모세포염색체대비분석,미견명현적염색체수목여결구이상. 결론:소서강전란포체외배양성숙,가획득여체내생장상동적성숙란모세포,염색체분석미견이상,가성위일충량호적생식독이학체외검측여란포발육궤제연구모형.
BACKGROUND AND AIM: In view of the follicles proliferation is sensitive to the physical and chemical stimuli, to establish the follicles culture in vitro growth system, as an efficient biological model of reproductive toxicity. MATERIALS AND METHODS: 140-160 μm preantral follicles were selected to culture in vitro for ten days, the diameter of follicles was measured each other day. The ovulation of survival follicles was induced by HCG hormone. About 16-24 hours later, the matured oocytes, germinal vesicle breakdown (GVBD) and germinal vesicle (GV) oocytes were calculated under the stereomicroscope. Chromosomal abnormalities of matured oocytes including numerical and structural aberrations were investigated and recorded. RESULTS: Total of 332 follicles were separated from ovaries of 4 female mice, the mean diameter of follicles was increased at the early culture time in vitro, then follicles grow as ' Frangipani', some of them formed the antral-like cavity. On the 10 day of culture, the survival rate was 95.78% (318/332),the rate of ovulation induced by hormone in vitro was 72.01% (229/318) ,including 11.80% oocytes arrested at germinal vesicles stage, 39.30% oocytes showed germinal vesicle breakdown, 47.16% oocytes emitted the first polar body and 1.74% oocytes were parthenogenesis. The chromosomal abnormalities of matured oocytes have no significant difference comparing with that of oocytes in vitro maturation (IVM) and oocytes superovulation (FVC) . CONCLUSION: The oocytes matured from preantral follicles cultured in vitro were similar to those matured from in vivo growth. Therefore, the preantral follicles cultured in vitro growth system maybe a good testing model of reproductive toxicology or follicular developmental mechanism in vitro.