奶牛卵泡超数同步发育及其卵母细胞的发育潜能

目的建立促奶牛卵泡超数同步发育的方法,并利用体细胞核移植和分子技术评价来自这些卵泡的卵母细胞发育潜能。方法用E2-P4对淘汰的高产奶牛进行联合处理后,分别对其实施不同组合的外源促性腺激素处理FSH12h组,FSH48h组,FSH48h-LH6h组（简称LH6h组）,FSH48h-LH26h组（简称LH26h组）]以促进其卵泡超数同步发育;然后从卵泡中回收COCs进行体外成熟,排放第一极体的卵供作优质种牛的体细胞核转移（nucleartransfer,NT）受体,以评价卵的发育潜能。结果E2-P4能成功地诱导奶牛卵巢中产生一个新的卵泡起始波,并能保证卵泡在外源促性腺激素作用下实现超数同步发育;上述四组不同组合外源促性腺激素处理的卵母细胞衍生的NT重构胚经体内培养7d后,后3组的囊胚发育率显著高于FSH12h组和对照组（P〈0.01）;将这些克隆囊胚移植同步发情的受体牛子宫后,仅LH26h组的卵所获得的克隆胚能实现全程发育（直至克隆小牛出生）。经RT-PCR分析颗粒细胞中FSHr、LHr和连接蛋白43（Connexin43,Cx43）等mRNA含量变化,以及Western印迹分析其Bcl-2和Bax蛋白表达水平的变化,证实该组卵巢提供的同步发育卵泡为早期闭锁卵泡。结论E2-P4-FSH48h-LH26h组合处理可人为调控牛卵泡的同步发育;处于早期闭锁状态卵泡的卵母细胞具备较高的发育潜能。

Developmental Competence of Ova Derived from Synchronized Follicles of Cow under Exogeneous Hormonal Control

Objective To develop a method suitable to recover available amount of developmentally synchronized follicles and using somatic cell nuclear transfer （cloning technique） to evaluate the developmental competence of ova derived from those follicles. Methods After being treated by E2-P4, donor cows were conducted by different combinations of exogeneous hormones, including FSH 12 h group,FSH 48 h group,FSH 48 h-LH 6 h （LH 6 h group） ,FSH 48 h-LH 26 h （LH 26 h group）. Follicles from ovaries treated ordy by E2-P4 served as control. All recovered COCs from follicles were cultured 22 - 24 h in maturation medium. Subsequently, ovum which excluded the first polar body were enucleated and used as cytoplast （recipient of somatic cell nuclei） in the preparation of cloned cow in order to evaluate its developmental competence. Results E2-P4 treatment successfully induced the initiation of a new generation of follicular development wave, and assured the synchronized development of follicles after a treatment of FSH. After being cultured in oviducts of Sannen goats for 7 day, the blastocyst rate of the reconstructed embryos derived from the three later groups were significantly higher than that of FSH 12 h and the control groups （ P 〈 0.01 ）. After transferring 1 or 2 blastocyts into the uterus of each synchronized surrogate cow, only the reconstructed embryos derived from LH 26 h group realized their full term development and delivered a cloned offspring. Results obtained from RT-PCR analysis of FSHr, Lhr and Connexin 43 mRNA, and from Western blot analysis of Bcl-2 and Bax proteins in the granulose cells of follicles proved that the follicles derived from LH 26 h group were classified as early follicle atresia. Conclusion E2-P4-FSH 48 h-LH 26 h treatment can regulate cow follicular development in a synchronized fashion. Oocytes in these early atresia follicles possess full competence.