Search for genes involved in oocyte maturation and developmental competence in oocytes during the first wave of folliculogenesis in immature mice

在出生后的第一个月内，未成熟小鼠的卵巢中出现同步卵泡生长（第一波）。以前，我们表明，小鼠卵母细胞在第一波更有能力的发展，在老年女性，虽然成熟卵母细胞的数量并没有随着女性年龄的不同。在这项研究中，我们研究了影响小鼠卵母细胞的发育能力在第一波抑制性消减杂交（SSH）的遗传因素。从17和24日龄B6 D2 F1雌性（分别为D17和D24）收集的卵母细胞未经激素处理，在Waymouth培养基中成熟。经过17小时的培养，总RNA分离的卵母细胞的生殖囊泡已被打破（成熟卵母细胞），分别在三个独立的培养组，每个年龄（每组含有卵母细胞从四只动物）。将来自每个独立组的一些总RNA按年龄合并并用于SSH。将消减后的PCR产物克隆到pGEM-T载体中。对来自消减文库（D24 -D17）的克隆进行测序，并使用NCBI BLAST搜索检查它们的同一性。在成熟的D24卵母细胞中优先表达的候选基因的差异表达建议SSH证实与cDNA转录分别在每个年龄的三个独立的培养组，使用实时定量PCR。在测序的513个克隆中，在发育能力更强的D24卵母细胞中优先表达的前6个候选基因是spindlin、bmi-1、细胞周期蛋白B1、E330034 G19 Rik、Jagged 1和Ndfip 2。当GAPDH用作内部对照时，证实了在成熟D24卵母细胞中spindlin的表达比在D17卵母细胞中高约11倍。我们的研究结果表明，spindlin是导致小鼠卵母细胞在卵泡发育过程中获得发育能力的关键因素之一。

During the first month after birth, synchronous follicular growth occurs in the ovary of immature mice (first wave). Previously, we showed that mouse oocytes during the first wave were more competent developmentally in older females, although the numbers of mature oocytes did not differ with female age. In this study, we examined the genetic factors that affect the developmental competence of mouse oocytes during the first wave using suppression subtractive hybridization (SSH). Oocytes collected from 17-and 24-day-old B6D2F1 females (D17 and D24,respectively) without hormonal treatment were matured in Waymouth medium. After a 17-h culture, total RNA was isolated from oocytes whose germinal vesicles had broken down (mature oocytes), separately, in three independent culture groups per age (each group contained oocytes from four animals). Some of the total RNA from each independent group was pooled by age and used for SSH. The subtracted PCR products were cloned into pGEM-T vector. Clones from the subtracted library (D24 -D17) were sequenced and their identities were examined using the NCBI BLAST search. The differential expression of candidate genes preferentially expressed in mature D24 oocytes suggested by SSH was confirmed with cDNA transcribed separately in the three independent culture groups per age using real-time quantitative PCR. Of 513 clones sequenced, the top six preferentially expressed candidate genes in more developmentally competent D24 oocytes were spindlin, bmi-1,cyclin B1,E330034G19Rik, Jagged1,and Ndfip2. The expression of spindlin in mature D24 oocytes was confirmed to be approximately 11-fold higher than in D17 oocytes when GAPDH was used as an internal control. Our results suggest that spindlin is one of the key factors leading to the acquisition of developmental competence in mouse oocytes during folliculogenesis.