Recombinant growth differentiation factor-9 (GDF-9) enhances growth and differentiation of cultured early ovarian follicles
Recombinant growth differentiation factor-9 (GDF-9) enhances growth and differentiation of cultured early ovarian follicles
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DOI：
10.1210/en.140.3.1236
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发表时间：
1999-03-01
期刊：
影响因子：
4.8
通讯作者：
Hsueh, AJW
中科院分区：
文献类型：
--
作者：
Hayashi, M;McGee, EA;Hsueh, AJW
缺失 GDF-9（生长分化因子-9）基因的转基因小鼠的特点是卵泡发育在初级阶段停滞。基于 GDF-9 对早期卵泡发育很重要的假设，我们分离了大鼠 GDF-9 互补 DNA (cDNA) 并生成重组 GDF-9 蛋白以研究其生理作用。使用细菌衍生的 GDF-9-谷胱甘肽 S-转移酶 (GST) 融合蛋白，生成了针对成熟形式的 GDF-9 的特异性抗体。卵巢切片的免疫组织化学染色表明GDF-9蛋白定位于初级、次级和腔前卵泡的卵母细胞中，而免疫印迹表明用GDF-9 cDNA转染的哺乳动物细胞分泌GDF-9。重组 GDF-9 被证明是一种 N-糖基化蛋白，能够刺激早期卵泡发育。通过 GDF-9 或 FSH 治疗，从未成熟大鼠中分离出的腔前卵泡的生长得到增强，而联合治疗则显示出相加效应。此外，与毛喉素一样，用 GDF-9 治疗也可以刺激新生儿卵巢外植体中抑制素醇的含量。相比之下，GDF-9 的刺激作用并没有被氨基末端标记的 GDF-9 所模仿，后者显然没有生物活性。因此，本研究证明了 GDF-9 在早期卵泡生长和分化中的重要作用。重组生物活性 GDF-9 的出现使得未来能够研究 GDF-9 在体内卵巢发育中的生理作用。
Transgenic mice with deletion of the GDF-9 (growth differentiation factor-9) gene are characterized by the arrest of ovarian follicle development at the primary stage. Based on the hypothesis that GDF-9 is important for early follicle development, we isolated rat GDF-9 complementary DNA (cDNA) and generated recombinant GDF-9 protein to study its physiological role. Using bacteria-derived GDF-9-glutathione S-transferase (GST) fusion protein, specific antibodies to the mature form of GDF-9 was generated. Immunohistochemical staining of ovarian sections indicated the localization of GDF-9 protein in the oocyte of primary, secondary and preantral follicles, whereas immunoblotting demonstrated the secretion of GDF-9 by mammalian cells transfected with GDF-9 cDNAs. Recombinant GDF-9 was shown to be an N-glycosylated protein capable of stimulating early follicle development. Growth of preantral follicles isolated from immature rats was enhanced by treatment with either GDF-9 or FSH whereas the combined treatment showed an additive effect. In addition, treatment with GDF-9, like forskolin, also stimulated inhibin-ol content in explants of neonatal ovaries. In contrast, the stimulatory effects of GDF-9 were not mimicked by amino-terminal tagged GDF-9 that was apparently not bioactive. Thus, the present study demonstrates the important role of GDF-9 in early follicle growth and differentiation. The availability of recombinant bioactive GDF-9 allows future studies on the physiological role of GDF-9 in ovarian development in vivo.