Toyooka Y, Tanaka S S, Hirota O, Tanaka S, Takagi N, Yamanouchi K, Tojo H, Tachi C
Developmental Biology Group, Mitsubishi Kasei Institute of Life Sciences, Tokyo, Japan.
Int J Dev Biol. 1998 Nov;42(8):1143-51.
With the aim of identifying the gene(s) located downstream from SRY, we transfected an ES cell line with XX karyotype, TMA-18, with a Sry DNA construct and established cell lines, TS18-1 and TS18-2, where the transfected Sry was expressed in the functional linear mRNA form. Among the five potential SRY-target genes examined, i.e., MIS, SF1, P450arom, Sox9 and WT1, only the expression of WT1 was induced de novo by the unscheduled expression of Sry in the transfected cell lines. No clear indication of Sry-induced enhancement of Sox9 expression was obtained in the present series of experiments. Function of a yet unidentified gene(s) located on the Y chromosome might be needed for the up-regulation of Sox 9 expression which takes place during the development of male gonads. Quantitative RT-PCR analysis of the patterns of WT1 expression in developing fetal gonads revealed that although both male and female fetal gonads express WT1, male gonads invariably expressed WT1 mRNA at higher levels than female ones after the Sry expression. Immunohistochemical analysis of the male fetal gonads between 10.5 and 13.5 dpc demonstrated the presence of strong WT1 immunoreactivity in Sertoli cells of the primordial testes. Suggestions were made in the past indicating that both SF1 and WT1 proteins might be active in a common pathway upstream from Sry. Our results showed that WT1 is located downstream, rather than upstream from Sry and behaves independently from SF1. Analysis using an appropriate in vitro system will be essential to understand the molecular mechanisms of SRY action within cells.
为了鉴定位于SRY下游的基因，我们用XX核型的ES细胞系TMA-18转染了一个携带Sry DNA构建体的细胞系，并建立了细胞系TS18-1和TS18-2，在这些细胞系中转染的Sry以功能性线性mRNA形式表达。在所检测的五个潜在的SRY靶基因中，即抗苗勒氏管激素（MIS）、类固醇生成因子1（SF1）、芳香化酶P450（P450arom）、性别决定区Y框蛋白9（Sox9）和威尔姆斯瘤蛋白1（WT1），只有WT1的表达在转染细胞系中由Sry的异常表达从头诱导产生。在本系列实验中未获得Sry诱导Sox9表达增强的明确证据。在雄性性腺发育过程中发生的Sox9表达上调可能需要位于Y染色体上的一个尚未鉴定的基因的功能。对发育中的胎儿性腺中WT1表达模式的定量逆转录聚合酶链反应（RT-PCR）分析表明，尽管雄性和雌性胎儿性腺都表达WT1，但在Sry表达后，雄性性腺中WT1 mRNA的表达水平始终高于雌性。对10.5至13.5天妊娠龄（dpc）的雄性胎儿性腺进行免疫组织化学分析，结果显示在原始睾丸的支持细胞中存在强烈的WT1免疫反应性。过去有人提出，SF1和WT1蛋白可能在SRY上游的一个共同途径中发挥作用。我们的结果表明，WT1位于SRY的下游而非上游，并且其行为独立于SF1。使用合适的体外系统进行分析对于理解SRY在细胞内的作用分子机制至关重要。
