小鼠卵泡膜细胞成熟过程中Dnmts表达变化的研究

Study on the Variation of Dnmts Expression During Follicular Theca Cells Maturation in Mouse

【目的】揭示小鼠卵泡膜细胞成熟过程中DNA甲基转移酶(Dnmts)表达的变化,并初步探讨其可能的机制。【方法】对新生昆明白小鼠第3、5及8天(days post parturition,dpp3、dpp5和dpp8)的卵巢进行切片,经H.E染色,分别观察原始、初级、次级卵泡膜细胞的形成情况,统计各级卵泡的比例并对卵泡外缘的膜细胞进行计数;半定量PCR检测成熟膜细胞特异性表达基因CYP17A1及Dnmts转录水平在dpp3、dpp5、dpp8卵巢中的变化;免疫组化观察Dnmt1蛋白在dpp3、dpp5、dpp8小鼠卵巢中的分布情况。【结果】小鼠dpp3卵巢中的初级卵泡外缘存在"梭形细胞";随出生后时间的推移,原始卵泡比例持续下降(P<0.01),次级卵泡比例持续升高(P<0.01),dpp3、dpp5初级卵泡比例无显著差异(P>0.05),而dpp8下降(P<0.05);初级卵泡周围"梭形细胞"数量的平均水平在dpp3和dpp5无显著差异(P>0.05),在dpp8上升(P<0.05);次级卵泡周围"梭形细胞"数量的平均水平在dpp3和dpp5无显著差异(P>0.05),在dpp8上升(P<0.01)。CYP17A1转录水平在dpp3、dpp5极低且无显著差异(P>0.05),在dpp8升高(P<0.01);Dnmt1s转录水平在dpp3、dpp5、dpp8持续升高(P<0.01);Dnmt1o转录水平在dpp5升高(P<0.05),在dpp8下降(P<0.01);Dnmt3a转录水平在dpp5升高(P<0.01),dpp3和dpp8转录水平无显著差异(P>0.05)。Dnmt1免疫组化显示,卵泡颗粒细胞在dpp5、dpp8卵巢中着色呈阳性,dpp3则呈阴性;同时,"梭形细胞"在dpp3、dpp5卵巢中着色呈阴性,dpp8则呈阳性。【结论】膜细胞成熟可能导致Dnmt3a转录水平下降。Dnmt1s转录及表达水平在膜细胞成熟前较低,分化成熟后上调从而在膜细胞增殖过程中维持新建立的甲基化模式。

【Objective】The variation of Dnmts expression during follicular theca cells maturation in mouse was studied,and its possible mechanisms were discussed.【Method】 Ovary sections that made from mice on days post parturition 3,5 and 8（dpp3,dpp5,dpp8） were treated with H.E staining,then,the theca cells of primordial follicles,primary follicles,secondary follicles were observed and the proportions of each developmental stage follicles as well as the number of theca cells around primary and secondary follicle were counted.The transcriptional variation of theca cells maker gene CYP17A1 and Dnmts in dpp3,5,and 8 ovaries were tested by semi-quantitative PCR.The protein Dnmt1 distribution in dpp3,5,and 8 ovaries were observed through immunohistochemistry.【Result】＂Shuttling cells＂ were found around primary follicles in dpp3 mice ovaries.With the lapse of time after birth,the proportion of primordial follicles in mice ovaries maintained reducing during post-neonatal dpp3,5 and 8（P0.01）,while the proportion of secondary follicles maintained increasing（P0.01）,the proportion of primary follicles had no statistical difference between dpp3 and dpp5,but it reduced at dpp8（P0.05）.Mean number of ＂Shuttling cell＂ around primary follicles was found no statistical difference between dpp3 and dpp5（P0.05）,and it increased at dpp8（P0.05）.Mean number of ＂Shuttling cell＂ around secondary follicles was found no statistical difference between dpp3 and dpp5（P0.05）,and it increased at dpp8（P0.01）.The transcription level of CYP17A1 was extremely low at dpp3 and dpp5,and there was no statistical difference between the two stages（P0.05）,but it up-regulated at dpp8（P0.01）.The transcriptional level of Dnmt1s continually up-regulated during dpp3,5 and 8（P0.01）.The transcriptional level of Dnmt1o up-regulated at dpp5（P0.05）,and down-regulated at dpp8（P0.01）.Dnmt3a reached the highest transcription level at dpp5（P0.01）,and there was no statistical difference between dpp3 and dpp8（P0.05）.Immunohistochemistry staining of Dnmt1 showed that the granulosa cells stained positive in dpp5 and dpp8 ovaries,but it was negative at dpp3.Meanwhile,the ＂Shuttling cells＂ at dpp3 and dpp5 ovaries stained negative,but it was positive at dpp8.【Conclusion】The down-regulated transcription level of Dnmt3a may caused by theca cells maturation.The transcription and expression level of Dnmt1s maintained low before theca cells maturation and up-regulated after the maturation,which contribute to maintain the re-established methylation pattern during theca cells proliferation.