Klinger Francesca Gioia, De Felici Massimo
Department of Public Health and Cell Biology, University of Rome Tor Vergata, Rome, 00133, Italy.
Dev Biol. 2002 Apr 1;244(1):85-95. doi: 10.1006/dbio.2002.0592.
The development of follicles in the mammalian ovary involves a bidirectional communication system between the follicular cells and oocyte that is now beginning to be characterized. Little is known about the mechanisms underlying the beginning of the oocyte growth and the acquisition of the competence to resume meiosis by the growing oocyte. In the present study, we devised a multistep culture system for mouse oocytes obtained from 15.5- to 16.5-days postcoitum embryos (mean diameter +/- SEM, 9.7 +/- 1.3 microm), allowing three stages of the oocyte growth to be identified: (i) an early stage in which the oocyte growth is induced by direct stimulation of a soluble growth factor, namely stem cell factor (SCF), independent of the formation of gap junctions with granulosa cells; (ii) a second phase in which the oocyte growth depends on the combined action of SCF and contacts with granulosa cells; and (iii) a third phase of granulosa cell-dependent, SCF-independent growth. At each stage, key events of oocyte development and differentiation, such as the c-kit reexpression, the early zona pellucida assembly, and the beginning of follicologenesis, were observed to occur independently by the presence of SCF. At the end of the in vitro growing phases, lasting 18-20 days, oocytes reached a size (50 +/- 2.5 microm) and a chromatin differentiation (stage I-II) equivalent to those of 9- to 10-day-old preantral oocytes and were unable to complete the growth phase. About 50% of the in vitro-grown oocytes were induced to resume meiosis by okadaic acid (OA) treatment. However, a significant fraction of them (48%) showed inability to maintain the chromosome condensation in M-phase. When in vitro-grown oocytes were treated with UO126, a specific MEK inhibitor that prevents activation of mitogen-activated protein kinases (ERK-1 and ERK-2), for 1 h before, during, and following OA treatment, only 22% of oocytes underwent germinal vesicle breakdown after 24 h from the OA treatment. These studies demonstrate that SCF alone can induce the onset of the oocyte growth. This is, however, not sufficient to fully activate the mechanisms governing the acquisition of the meiotic competence previously described as a 15-day oocyte-autonomous clock starting at the onset of growth. The inability of oocytes to progress into the last stages of growth and the lack of synchrony between nuclear and cytoplasm maturation showed by a subset of them resemble the characteristics of oocytes from connexin-37- and -43-deficient mice and indicate the preantral/antral transition point as a critical stage of oocyte development requiring the coordinated differentiation of the oocyte with granulosa cells and the maintenance of adequate communication between these two cell types to assure the correct oocyte meiotic maturation.
哺乳动物卵巢中卵泡的发育涉及卵泡细胞与卵母细胞之间的双向通信系统，目前这一系统正开始得到表征。关于卵母细胞生长起始以及生长中的卵母细胞恢复减数分裂能力的潜在机制，我们所知甚少。在本研究中，我们为从妊娠15.5至16.5天胚胎获取的小鼠卵母细胞设计了一个多步骤培养系统（平均直径+/-SEM，9.7+/-1.3微米），从而能够识别卵母细胞生长的三个阶段：（i）早期阶段，其中卵母细胞生长由可溶性生长因子即干细胞因子（SCF）的直接刺激诱导，独立于与颗粒细胞形成间隙连接；（ii）第二阶段，其中卵母细胞生长取决于SCF与颗粒细胞接触的联合作用；以及（iii）颗粒细胞依赖、SCF非依赖的生长第三阶段。在每个阶段，观察到卵母细胞发育和分化的关键事件，如c-kit重新表达、早期透明带组装以及卵泡发生起始，通过SCF的存在独立发生。在体外生长阶段结束时，持续18至20天，卵母细胞达到了与9至10日龄前卵泡期卵母细胞相当的大小（50+/-2.5微米）和染色质分化（I-II期），但无法完成生长阶段。约50%的体外生长卵母细胞通过冈田酸（OA）处理被诱导恢复减数分裂。然而，其中很大一部分（48%）显示在M期无法维持染色体凝聚。当在OA处理前、处理期间和处理后用UO126（一种阻止丝裂原活化蛋白激酶（ERK-1和ERK-2）激活的特异性MEK抑制剂）处理体外生长的卵母细胞1小时后，从OA处理24小时起，只有22%的卵母细胞发生生发泡破裂。这些研究表明，单独的SCF就能诱导卵母细胞生长起始。然而，这不足以完全激活控制减数分裂能力获得的机制，先前将其描述为从生长起始时开始的15天卵母细胞自主时钟。卵母细胞无法进入生长的最后阶段以及其中一部分所显示的核与细胞质成熟缺乏同步性，类似于连接蛋白-37和-43缺陷小鼠的卵母细胞特征，并表明前卵泡/卵泡期转变点是卵母细胞发育的关键阶段，需要卵母细胞与颗粒细胞的协调分化以及这两种细胞类型之间保持充分的通信，以确保正确的卵母细胞减数分裂成熟。
