Department of Biological Sciences, University of Denver, 2190 East Iliff Ave., Denver, CO 80208-9010, USA.
Biol Reprod. 2010 Mar;82(3):591-8. doi: 10.1095/biolreprod.109.080937. Epub 2009 Nov 18.
Insulin-like growth factor 1 (IGF-1)-stimulated amphibian oocyte maturation has been studied extensively by a number of laboratory groups, but in previous studies possible effects of IGF-1 on ovarian follicle cells had not been tested directly. In the study reported here, biochemical and immunofluorescent techniques were used to test Xenopus ovarian follicle cells for the presence of hormone-sensitive IGF-1 receptor. Anti-xIGF-1 receptor beta-subunit antibodies detected a 90- and 98-kDa protein doublet in manually dissected oocyte cortices (plasma membrane-vitelline envelope complexes) by protein immunoblotting both before and after removal of follicle cells from oocytes by sandpaper rolling. The 90-kDa IGF-1 receptor beta-subunit was also detected in follicle cell pellets. Tyrosine phosphorylation of the receptor beta-subunits was increased by treatment of cortices with 10 nM IGF-1 both in the presence and absence of associated follicle cells, was reduced by removal of follicle cells, and was detected in follicle cell pellets. Treatment of follicle cell pellets with nanomolar concentrations of IGF-1 stimulated receptor tyrosine phosphorylation in a dose-dependent fashion that correlated with dose-dependent stimulation of oocyte maturation. IGF-1 receptor was also detected in cultured follicle cells by immunofluorescence. Removal of follicle cells significantly reduced the IGF-1-stimulated oocyte maturation response. These results offer the first direct evidence for hormone-responsive IGF-1 receptors in Xenopus laevis ovarian follicle cells and demonstrate that follicle cells somehow support IGF-1-stimulated oocyte maturation.
胰岛素样生长因子 1（IGF-1）刺激的两栖类卵母细胞成熟已被多个实验室小组广泛研究，但在之前的研究中，IGF-1 对卵巢滤泡细胞的可能影响尚未直接测试。在本报告的研究中，使用生化和免疫荧光技术来测试 Xenopus 卵巢滤泡细胞是否存在激素敏感的 IGF-1 受体。抗 xIGF-1 受体β亚基抗体通过蛋白质免疫印迹检测到手动分离的卵母细胞皮质（质膜-卵黄 envelope 复合物）中存在 90-和 98-kDa 蛋白二聚体，在去除滤泡细胞之前和之后均如此用砂纸滚动从卵母细胞中去除。在滤泡细胞沉淀中也检测到 90-kDa IGF-1 受体β亚基。用 10 nM IGF-1 处理皮质可增加受体β亚基的酪氨酸磷酸化，无论是否存在相关的滤泡细胞，去除滤泡细胞均可降低受体β亚基的酪氨酸磷酸化，并可在滤泡细胞沉淀中检测到。用纳摩尔浓度的 IGF-1 处理滤泡细胞沉淀可刺激受体酪氨酸磷酸化呈剂量依赖性，与卵母细胞成熟的剂量依赖性刺激相关。免疫荧光也在培养的滤泡细胞中检测到 IGF-1 受体。去除滤泡细胞可显著降低 IGF-1 刺激的卵母细胞成熟反应。这些结果首次提供了 Xenopus laevis 卵巢滤泡细胞中激素反应性 IGF-1 受体的直接证据，并证明滤泡细胞以某种方式支持 IGF-1 刺激的卵母细胞成熟。
