摘要
目的探讨卵巢储备功能减退(DOR)患者卵泡液外泌体对小鼠卵母细胞体外成熟的影响,以期为女性因DOR导致的生育功能降低和不孕症的发生提供新的科学实验数据。方法选取SPF级4~6周龄C57BL/6J未孕雌性小鼠,收集未成熟卵母细胞并进行体外成熟培养;同时收集DOR患者卵泡液,并提取外泌体。利用Dio荧光标记外泌体后与小鼠未成熟卵丘-卵母细胞复合物(COCs)共孵育培养,观察外泌体摄入情况;通过添加不同浓度0、50、100和150μg/ml外泌体处理小鼠未成熟COCs 16 h观察卵丘扩展和第一极体排出情况,分析其对卵母细胞成熟的影响并筛选出最佳处理浓度;设置分组为体内成熟组(control)、外泌体添加组(exo+)和未添加组(exo-),各组成熟后的COCs利用qPCR检测卵丘扩展相关基因Has2、Tnfaip6和Ptgs2以及凋亡相关基因Bax、Bcl2的mRNA相对表达水平,分析各组间的差异。结果(1)Dio标记的外泌体与小鼠COCs共孵育,卵丘颗粒细胞上能观察到明显的Dio荧光信号,证实COCs能够摄入外泌体;(2)不同浓度外泌体处理组的GV期卵母细胞百分比均显著高于0μg/ml组(P<0.05),而MⅡ期卵母细胞百分比均显著低于0μg/ml组(P<0.05)。使用150μg/ml作为后续实验工作浓度;(3)qPCR的结果显示,小鼠COCs中卵丘扩展相关基因Has2的mRNA表达水平在体内成熟组及体外成熟培养组各组间的差异无统计学意义(P>0.05),exo+组和exo-组的Tnfaip6、Bcl2和Bax mRNA表达水平均显著低于control组(P<0.05或P<0.01),exo+组Ptgs2的mRNA表达水平显著低于control组(P<0.05);此外,exo+组Tnfaip6、Ptgs2和Bcl2的mRNA表达水平均显著低于exo-组(P<0.05或P<0.01)。结论DOR患者卵泡液外泌体能够抑制小鼠卵母细胞体外成熟过程中卵丘扩展和减数分裂恢复,并且显著抑制了卵丘扩展相关基因Ptgs2和Tnfaip6以及细胞凋亡抑制基因Bcl2的表达。
Objective:To investigate the effect of follicular fluid exosomes from patient with diminished ovarian reserve(DOR)on the in vitro maturation of mouse oocytes,in order to provide new scientific experimental evidence for women fertility reduction and infertility caused by DOR.Methods:The immature oocytes of SPF grade C57BL/6J non-pregnant female mice aged from 4 to 6 weeks were collected and cultured in vitro.Follicular fluid from DOR patients was collected and exosomes were extracted.Exosomes were labeled with Dio fluorescence probe and incubated with mouse immature cumulus-oocyte complexs(COCs)to observe the uptake of exosomes.By adding different concentrations of 0,50,100 and 150μg/ml exosomes were used to treat mouse immature COCs for 16 hours to observe the cumulus expansion and first polar body discharge,and then analyze the effect of exosome on oocyte maturation and select the optimal treatment concentration.Three groups were set as in vivo maturation group(control),exosome treated group(exo+)and non-treated group(exo-).qPCR was used to detect the relative mRNA expression level of cumulus expansion related genes Has2,Tnfaip6 and Ptgs2 and apoptosis-related genes Bax and Bcl2 in matured COCs of each group,and analyzed the difference among the groups.Results:Dio-labeled exosomes were co-incubated with mouse COCs,and evident Dio fluorescence signal could be observed in cumulus cells.The percentage of oocytes at GV stage in different concentration exosome treatment groups was significantly higher than 0μg/ml group(P<0.05),while the percentage of oocytes in MⅡstage was significantly lower than that in 0μg/ml group(P<0.05).150μg/ml was used as the working concentration in the following experiments.qPCR results showed that the mRNA expression level of the cumulus expansion related gene Has2 in mouse COCs was not significantly different among the in vivo maturation group and the different subgroups of in vitro maturation culture group(P>0.05).The expression levels of Tnfaip6,Bcl2 and Bax in exo+group and exo-group were significantly lower than those in the control group(P<0.05 or P<0.01).Ptgs2 mRNA expression in exo+group was significantly lower than that in the control group(P<0.05).In addition,the mRNA expression levels of Tnfaip6,Ptgs2 and Bcl2 in exo+group were significantly lower than those in the exo-group(P<0.05 or P<0.01).Conclusions:DOR patient’s follicular fluid exosomes can inhibit cumulus expansion and meiotic resumption during in vitro maturation of mouse oocytes,and significantly inhibit expression of cumulus expansion related genes Ptgs2 and Tnfaip6 and apoptosis suppressor gene Bcl2.
作者
沈开元
杨冠群
罗平
代小丽
黄萍
梁媛媛
李顺
陈立群
曲晓力
SHEN Kai-yuan;YANG Guan-qun;LUO Ping;DAI Xiao-li;HUANG Ping;LIANG Yuan-yuan;LI Shun;CHEN Li-qun;QU Xiao-li(Reproductive Medicine Center,Liuzhou People’s Hospital,Liuzhou 545006)
出处
《生殖医学杂志》
CAS
2022年第10期1422-1429,共8页
Journal of Reproductive Medicine
基金
广西自然科学基金项目(2018GXNSFBA281048)
广西壮族自治区卫生健康委员会自筹经费科研课题(Z20180308,Z20190175,Z20200270)。
