Hsu S Y, Kubo M, Chun S Y, Haluska F G, Housman D E, Hsueh A J
Department of Gynecology and Obstetrics, Stanford University School of Medicine, California 94305-5317, USA.
Mol Endocrinol. 1995 Oct;9(10):1356-66. doi: 10.1210/mend.9.10.8544844.
WT1, a gene deleted in some Wilms' tumors, encodes a transcription factor with zinc fingers and shares homology with proteins in the early growth response gene family. Although defects in the WT1 gene are associated with nephroblastoma and genitourinary malformation, the specific function of WT1 in the gonads remains unclear. We investigated the expression of WT1 transcripts in rat ovary during follicle development by Northern blotting, RNase protection assay, and in situ hybridization. Abundant WT1 transcripts were found in the ovary, testis, uterus, and kidney, with lower levels in the heart and pancreas. Treatment with estrogen or gonadotropins did not affect the concentration of ovarian WT1 mRNA. In situ hybridization analysis indicated that ovarian WT1 mRNA is expressed exclusively in the surface epithelium and granulosa cells of primordial, primary, and secondary follicles, and its levels decrease during follicle growth. Although RNase protection assay suggested the presence of four alternatively spliced forms of WT1 mRNA, the ratio of these transcripts remains constant during ovarian growth. Developmental changes in the expression of two granulosa cell differentiation marker genes, inhibin-alpha and FSH receptor, were found to be inversely correlated with WT1 levels. Because potential WT1-binding sites were found in the promoter of inhibin-alpha gene, we further tested whether WT1 might regulate the expression of this gene. Cotransfection of a WT1 expression vector with a promoter reporter plasmid of inhibin-alpha resulted in the repression of promoter activities in CHO cells in a dose-dependent manner. These results suggest that WT1 is expressed in high levels in granulosa cells of primordial, primary, and secondary follicles but decreases with follicle development. This transcription factor might be a repressor of ovarian differentiation genes in the granulosa cells and play a role in arresting the differentiation of immature follicles.
WT1基因在一些肾母细胞瘤中发生缺失，它编码一种带有锌指结构的转录因子，与早期生长反应基因家族中的蛋白质具有同源性。尽管WT1基因缺陷与肾母细胞瘤和泌尿生殖系统畸形相关，但WT1在性腺中的具体功能仍不清楚。我们通过Northern印迹法、核糖核酸酶保护分析和原位杂交技术，研究了大鼠卵泡发育过程中卵巢内WT1转录本的表达情况。结果发现，WT1转录本在卵巢、睾丸、子宫和肾脏中大量存在，而在心脏和胰腺中的水平较低。用雌激素或促性腺激素处理并不影响卵巢中WT1 mRNA的浓度。原位杂交分析表明，卵巢WT1 mRNA仅在原始卵泡、初级卵泡和次级卵泡的表面上皮细胞和颗粒细胞中表达，且其水平在卵泡生长过程中降低。尽管核糖核酸酶保护分析表明存在四种WT1 mRNA的可变剪接形式，但这些转录本的比例在卵巢生长过程中保持恒定。研究发现，两种颗粒细胞分化标记基因抑制素α和促卵泡激素受体表达的发育变化与WT1水平呈负相关。由于在抑制素α基因的启动子中发现了潜在的WT1结合位点，我们进一步测试了WT1是否可能调节该基因的表达。将WT1表达载体与抑制素α的启动子报告质粒共转染，导致CHO细胞中启动子活性呈剂量依赖性抑制。这些结果表明，WT1在原始卵泡、初级卵泡和次级卵泡的颗粒细胞中高水平表达，但随着卵泡发育而降低。这种转录因子可能是颗粒细胞中卵巢分化基因的抑制因子，并在阻止未成熟卵泡的分化中发挥作用。
