抗苗勒激素及生长分化因子-9对环磷酰胺体外降低大鼠卵巢储备力机制的研究

Study on the mechanism of anti-mullerian hormone and growth differentiation factor-9 on cyclophosphamide reducing in vitro ovarian reserve of rats

目的研究抗苗勒激素(AMH)和生长分化因子-9(GDF-9)对环磷酰胺(Cy)降低卵巢储备力机制中的作用。方法成年雌性SD大鼠25只,取双侧卵巢组织,并切制成500μm厚的卵巢片,将卵巢片分为7组:空白组、Cy组、Cy+卵泡刺激素(FSH)组、Cy+AMH组、Cy+GDF-9组、Cy+FSH+AMH组、Cy+FSH+GDF-9组,放入含DMEM-F12培养液的培养皿中分别培养2 d或4 d。除空白组外,其余各组于培养液中分别添加Cy 100 ng/mL、FSH 10 mIU/mL、AMH 100ng/mL、GDF-9 100 ng/mL。利用双抗体夹心酶联免疫吸附技术(ELISA)检测雌二醇(E2)、抑制素B(INHB)的量;光镜下计数始基卵泡(PMF)及闭锁卵泡;运用DNA缺口原位末端标记法(TUNEL)检测细胞凋亡情况。结果①与空白组相比,Cy组PMF数目明显降低,闭锁卵泡明显增多;Cy+AMH组及Cy+FSH+AMH组PMF数目明显多于除空白组之外的其他各组。各组培养4 d较培养2 d始基卵泡数目明显降低,闭锁卵泡数目明显增多。②与空白组相比,Cy组培养2 d和4 d时E2及INHB水平明显降低;与Cy组相比,培养2 d和4 d时Cy+FSH组、Cy+GDF-9组及Cy+FSH+GDF-9组E2、INHB明显升高。与Cy组相比,培养2 d和4 d时Cy+AMH组及Cy+FSH+AMH组E2明显升高。培养4 d时Cy+AMH组、Cy+FSH+AMH组INHB水平明显升高。③Cy组与空白组比较,凋亡细胞明显增多;Cy+FSH组、Cy+GDF-9组与Cy组相比,凋亡细胞的数量并没有显著减少。Cy+AMH组、Cy+FSH+AMH组与Cy组相比凋亡细胞的数量显著减少。结论Cy可加速PMF的募集与耗竭,从而使卵巢储备力降低乃至衰竭。FSH及GDF-9可加速PMF的募集与发育,使更多的PMF进入生长卵泡阶段而受到Cy细胞毒的损害,以致PMF库存逐渐耗竭;AMH可抑制PMF的募集与发育,从而减缓Cy对PMF的加速募集与耗竭,起到保护卵巢储备力的作用。

Objective To research the effects of anti-mullerian hormone（AMH） and growth differentiation factor-9（GDF-9） on cyclophosphamide（Cy） reducing ovarian reserve.Methods Twenty-five adult female SD rats were used to remove the bilateral ovarian tissues and cut them into 500 μm slices.The slices were divided into control,Cy,Cy ＋ follicle-stimulating hormone（FSH）,Cy ＋ AMH,Cy ＋ GDF-9,Cy ＋ FSH ＋ AMH,and Cy ＋ FSH ＋ GDF-9 groups,and put into petri dishes including DMEM-F12 for 2 or 4 d.Except the control group,the other groups were added with Cy 100 ng/mL,FSH 10 mIU/mL,GDF-9 100 ng/mL,and AMH 100 ng/mL.The contents of estradiol（E2） and inhibitant B（INHB） were detected by ELISA,the numbers of primordial follicles（PMF） and atresic follicles were counted under light microscope,and the rat ovarian cell apoptosis was detected by TUNEL.Results ① Compared with the control group,the numbers of PMF were significantly reduced and the numbers of atresic follicles were increased in Cy group.In Cy ＋ AMH and Cy ＋ FSH ＋ AMH groups,the numbers of PMF were obviously more than those in the other groups except control group.The numbers of PMF were significantly reduced and the atresic follicles were significantly increased in 4 d culture compared with 2 d culture,② Compared with the control group,the E2 and INHB contents of rats in Cy group were significantly Reduced;Those in GDF-9 groups were significantly increased,and the E2 content of rats in Cy ＋ AMH and Cy ＋ FSH ＋ AMH groups was significantly increased.At the day 4,the INHB contents of rats in Cy ＋ AMH and Cy ＋ FSH ＋ AMH groups were significantly increased.③ Compared with the control group,the apoptotic cells in Cy group were significantly increased.Compared with Cy group,the numbers of apoptotic cells in Cy ＋ FSH and Cy ＋ GDF-9 groups were not significantly reduced,and the numbers of apoptotic cells in Cy ＋ AMH and Cy ＋ FSH ＋ AMH groups were significantly reduced.Conclusion Cy could accelerate the recruitment and depletion of the PMF,so that the ovarian reserve is reduced and even failure.FSH and GDF-9 could accelerate the recruitment and the development of the PMF,leading to more PMF into the growing follicle stage and suffering from Cy cytotoxic damage,so the PMF stock is gradually depleted.AMH could restrain the PMF recruitment and development,thus slow down the acceleration of the PMF recruitment and depletion by Cy,and protect the ovarian reserve.