Fan Heng-Yu, Shimada Masayuki, Liu Zhilin, Cahill Nicola, Noma Noritaka, Wu Yun, Gossen Jan, Richards JoAnne S
Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA.
Development. 2008 Jun;135(12):2127-37. doi: 10.1242/dev.020560.
Activation of the RAS family of small G-proteins is essential for follicle stimulating hormone-induced signaling events and the regulation of target genes in cultured granulosa cells. To analyze the functions of RAS protein in granulosa cells during ovarian follicular development in vivo, we generated conditional knock-in mouse models in which the granulosa cells express a constitutively active KrasG12D. The KrasG12D mutant mice were subfertile and exhibited signs of premature ovarian failure. The mutant ovaries contained numerous abnormal follicle-like structures that were devoid of mitotic and apoptotic cells and cells expressing granulosa cell-specific marker genes. Follicles that proceeded to the antral stage failed to ovulate and expressed reduced levels of ovulation-related genes. The human chorionic gonadotropin-stimulated phosphorylation of ERK1/2 was markedly reduced in mutant cells. Reduced ERK1/2 phosphorylation was due, in part, to increased expression of MKP3, an ERK1/2-specific phosphatase. By contrast, elevated levels of phospho-AKT were evident in granulosa cells of immature KrasG12D mice, even in the absence of hormone treatments, and were associated with the progressive decline of FOXO1 in the abnormal follicle-like structures. Thus, inappropriate activation of KRAS in granulosa cells blocks the granulosa cell differentiation pathway, leading to the persistence of abnormal non-mitotic, non-apoptotic cells rather than tumorigenic cells. Moreover, those follicles that reach the antral stage exhibit impaired responses to hormones, leading to ovulation failure. Transient but not sustained activation of RAS in granulosa cells is therefore crucial for directing normal follicle development and initiating the ovulation process.
小G蛋白RAS家族的激活对于促卵泡激素诱导的信号转导事件以及培养的颗粒细胞中靶基因的调控至关重要。为了分析RAS蛋白在体内卵巢卵泡发育过程中颗粒细胞的功能，我们构建了条件性敲入小鼠模型，其中颗粒细胞表达组成型激活的KrasG12D。KrasG12D突变小鼠生育力低下，并表现出卵巢早衰的迹象。突变卵巢含有许多异常的卵泡样结构，这些结构缺乏有丝分裂和凋亡细胞以及表达颗粒细胞特异性标记基因的细胞。进入窦状期的卵泡未能排卵，且排卵相关基因的表达水平降低。在突变细胞中，人绒毛膜促性腺激素刺激的ERK1/2磷酸化明显降低。ERK1/2磷酸化降低部分归因于ERK1/2特异性磷酸酶MKP3表达的增加。相比之下，即使在未进行激素处理的情况下，未成熟KrasG12D小鼠的颗粒细胞中磷酸化AKT水平也明显升高，并且与异常卵泡样结构中FOXO1的逐渐减少有关。因此，颗粒细胞中KRAS的不适当激活阻断了颗粒细胞分化途径，导致异常的非有丝分裂、非凋亡细胞而非致瘤细胞持续存在。此外，那些达到窦状期的卵泡对激素的反应受损，导致排卵失败。因此，颗粒细胞中RAS的短暂而非持续激活对于指导正常卵泡发育和启动排卵过程至关重要。
