猪卵泡期中等卵泡发育和闭锁的相关基因表达及激素水平分析  

Analysis of Gene Expression and Hormone Levels Related with Medium Follicular Development and Atresia during Pig Follicular Phase

作　　者：张家庆[1] 高彬文[1] 王献伟 马强[1] 高原 任巧玲[1] 白献晓[1] 邢宝松[1] 

机构地区：[1]河南省农业科学院畜牧兽医研究所,河南郑州450002 [2]河南省畜牧总站，河南郑州450008 [3]新县畜牧局,河南新县465550

出　　处：《河南农业科学》2016年第11期110-115,共6页Journal of Henan Agricultural Sciences

基　　金：河南省科技攻关项目(162102110036)

摘　　要：研究猪卵泡期内有腔卵泡发育和闭锁中颗粒细胞自噬与凋亡、卵泡内调控因子、类固醇激素及相关酶类的变化,为提高排卵前卵泡发育数量提供理论依据.从猪卵泡期卵巢中分离3-5mm有腔卵泡,根据发育变化分为健康卵泡、早期闭锁和晚期闭锁3类,应用HE染色观察内部形态结构变化,应用酶免法检测印泡液中雕二醇（E2）和孕酮（P4）的浓度变化,采用Real-time PCR方法检测自噬相关基因（Beclin,LC3B,ATG3,ATG5,ATG7）、凋亡相关基因（Caspase-3,Bim,Bcl-2和Bax）、类固醇合成酶基因（CYP11A1,3βHSD,CYP17A1,CYP19A3）、激素受体和卵泡内关键调控基因（FSHR,ERα,CART,SMAD4）在不同类型卵泡中的表达变化.结果表明,健康卵泡中颗粒细胞层完整,有少量凋亡细胞,早期和晚期闭锁卵泡中颗粒细胞层分散,且凋亡细胞明显增多.早期和晚期闭锁卵泡中P4/E2显著高于健康卵泡,自噬和凋亡相关基因的表达水平在早期和晚期闭锁的卵泡中显著高于健康卵泡,类固醇合成酶基因CYP11A1和3βHSD的表达量在早期和晚期闭锁卵泡中显著高于健康卵泡,CYP17A1、CYP19A3、FSHR、ERα和SMAD4的表达量在早期和晚期闭锁卵泡中显著低于健康卵泡,而CART的表达量则呈现相反的趋势.由此可见,颗粒细胞自噬和凋亡是卵泡闭锁的主要诱因,而类固醇合成酶基因CYP11A1和3βHSD通过提高卵泡液中孕酮的水平加速了卵泡闭锁的进程.The aim of this study was to analyze the changes of autophagy and apoptosis,intraovarian regulatory factors,steroid hormones and steroidogenic enzymes in antral follicle development and atresia during porcine follicular phase,so as to provide theoretical basis for increasing the preovulatory follicle numbers. Antral follicles about 3—5 mm in diameter were isolated from porcine ovaries in follicular phaseand classified as three groups: healthy follicles,early atretic follicles and progressed atretic follicles. HE staining was used to observe the follicular morphological structure,and enzyme-linked immunoabsorbent assay( ELISA) method was applied to examine the changes of estradiol( E2) and progesterone( P4)concentrations in follicular fluid. The relative expression levels of autophagy related genes( Becline,LC3 B,ATG3,ATG5 and ATG7),apoptosis related genes( Caspase-3,Bim,Bcl-2 and Bax),steroidogenic enzyme genes( CYP11A1,3βHSD,CYP17A1 and CYP19A3),hormone receptor and intraovarian regulatory genes( FSHR,ERα,CART and SMAD4) in different types of follicles were detected by real-time PCR method. The results showed that there were very few apoptotic cells and intact granulosa cell layer in healthy follicles,and the percentage of the apoptotic cells were remarkably increased in atretic follicles,especially in progressed atretic follicles. The ratio of P4 to E2 was significantly decreased in healthy follicles as compared to early atretic follicles and progressed atretic follicles. The relative expression levels of autophagy and apoptosis related genes were increased in early atretic follicles and progressed atretic follicles compared to healthy follicles. The mRNA levels of CYP11A1 and 3βHSD were enhanced in early atretic follicles and progressed atretic follicles compared with healthy follicles. The relative expression levels of FSHR and ERα in early atretic follicles and progressed atretic follicles were significantly lower than that of healthy follicles. The mRNA levels of SMAD4 in healthy follicles were signif

关 键 词：猪 有腔卵泡 卵泡发育 卵泡闭锁 

分 类 号：S814[农业科学—畜牧学]