超排兔卵巢卵母细胞直接用于体细胞核移植的研究

Study on Oocytes Aspirated from Superovulated Rabbit Ovary Follicles Used Directly for Somatic Cell Nuclear Transfer

为了评估兔超排后卵巢残留卵泡内卵母细胞用作体细胞核移植的潜力,应用PMSG或FSH诱导母兔超排,排卵结束(hCG注射14h)后,抽吸卵巢表面卵泡回收卵母细胞(抽吸组/处理组)用于体细胞核移植(SCNT),同时与自然排卵到输卵管内的卵母细胞(排出组/对照组)进行比较。结果表明,PMSG和FSH超排获得的抽吸组成熟卵母细胞(以可见第一极体为准)的比率分别极显著和显著低于各自的排出组(PMSG:70.7%vs94.6%,P<0.01;FSH:81.5%vs94.6%,P<0.05),且PMSG和FSH抽吸组间卵母细胞的成熟率差异显著(70.7%vs 81.5%,P<0.05)。PMSG和hCG超排的抽吸组成熟卵母细胞的去核效率也显著低于各自的排出组(PMSG:抽吸vs排出=69.2%vs83.6%,P<0.05;FSH:抽吸vs排出=71.6%vs81.0%,P<0.05)。抽吸组成熟的卵母细胞注核效率以及此类卵母细胞用作胞质受体经体细胞移植后的重构胚在融合率、分裂率以及分裂胚胎发育至囊胚的比例与排出组差异均不显著(P>0.05)。另外,抽吸组卵母细胞孤雌激活后胚胎的发育能力以及囊胚细胞总数与排出组差异也不显著(P>0.05)。但是,PMSG超排后的抽吸组成熟卵母细胞体细胞核移植的总体效率显著低于相应的排出组(8.4%vs11.5%,P<0.05),而FSH超排的卵母细胞抽吸组与排出组差异则不显著(10.2%vs11.6%,P>0.05)。结果显示,抽吸自超排兔卵巢表面卵泡内的卵母细胞大多数都已经成熟,虽然去核效果要差些,但获得的重构胚与源自排出到输卵管内的卵母细胞的重构胚具有等同的发育能力,可以不经体外成熟培养而直接用于体细胞核移植,而且FSH超排获得的抽吸组卵母细胞体细胞核移植的总体效率与自然排卵到输卵管内的卵母细胞没有显著差别。

Within a common somatic cell nuclear transfer （SCNT） protocol, a comparison between oocytes recovered by direct aspiration from available ovarian follicles （treatment/aspirated） and oocytes flushed out from the oviducts （control/ovulated） ,was carried out. The aim of this study was to evaluate if oocytes aspirated directly from postovulatory ovarian follicles of rabbits superovulated with PMSG or FSH, 14 h post hCG administration could be efficiently used as ooplasm recipients for SCNT. The results showed that maturation rates of aspirated oocytes from PMSG and FSH treatments were 70.7% and 81.5%, signifi- cantly lower than 94.6% （P〈0.01） and 94.6% （P〈0.05）, respectively from ovulated oocytes. Enuclea- tion efficiency of mature oocytes from aspirated groups of both PMSG and FSH treatments was also signifi- cantly lower than their counterparts of ovulated groups （PMSG. aspirated vs ovulated=69.2% vs 83.6% , P〈0.05 ; FSH ： aspirated vs ovulated= 71.6% vs81.0 0% , P〈0.05）. Following enucleation of matured oo- cytes as ooplasm recipients for SCNT, no difference was recorded in nuclear injection efficiency,fusion and cleavage rate, as well as blastocysts development from cleaved embryos or hatching blastocysts from blas- tocysts between aspirated and ovulated groups. Additionally, some matured aspirated and flushed oocytes were also used for immediate parthenogenetic activation, and resulting embryo development was also not significantly different. However, overall SCNT efficiency of aspirated oocytes recovered from PMSG treat- ment was significantly lower than that of ovulated oocytes （8.4% vs 11.5% ,P〈0. 05）, whereas no differ- ence from FSH treatment （10.2% vs11.6% ,P〉0.05）. Results from this study showed the followings：i） the majority of oocytes aspirated from postovulatory ovarian follicles of superovulated rabbits 14 h post hCG administration are matured and can be used direct for SCNT without in vitro maturation; ii） the reconstructed embryos derived from these oocytes have similar developmental ability to those flushed from oviducts. Furthermore, the overall SCNT efficiency of aspirated oocytes recovered from FSH treatment is similar to ovulated ones.