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The Relationship between Proliferation and Maturation of Sertoli cells and Development of Male Genitalia and Spermatogenesis

支持细胞增殖、成熟与男性生殖器发育和精子发生的关系

基本信息

批准号：
21592081
负责人：
HAYASHI Yutaro
金额：
$ 2.91万
依托单位：
Nagoya City University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2009
资助国家：
日本
起止时间：
2009 至 2011
项目状态：
已结题

项目摘要

Molecular mechanisms of sex differentiation have been unraveled by gene expression cascade triggered by the SRY gene, but the whole picture still remains unclear. In the present study, to clarify the genetic characteristics of the XX testis without SRY, we histologically analyzed the testicular tissue of patients with 46, XX testicular DSD and attempted to identify the genes with differential expression compared with the XY testis by using PCR-based subtractive hybridization technique. We used specimens obtained from testicular biopsy in boys with 46, XX testicular DSD. Age-matched XY testis derived from hydrocele were used as the control. After PCR-based subtractive hybridization, the subtracted cDNAs were sequenced and analyzed. Further, distribution of candidate genes in the testicular tissue was clarified by immunohistochemistry. The present study was approved by the institutional review board in our university. We finally identified 13 upregulated and 7 downregulated genes in the XX testis. Among the candidate genes, we focused on ROCK1, PQBP1, and UCP2 in the upregulated-gene group and on EEF1A1 in the downregulated-gene group. In immunohistochemistry, ROCK1 protein was detected in the germ cells and Leydig cells, PQBP1 protein in the Leydig cells, and UCP2 protein in the Sertoli cells. From previous reports and in vitro inhibitory assay, it is feasible that ROCK1 phosphorylates and activates SOX9 in Sertoli cells. It is suggested that testis formation is initiated by an alternative signaling pathway attributed to ROCK1 activation, and not SRY activation, in XX testis.

通过SRY基因引发的基因表达级联反应已经阐明了性别分化的分子机制，但整体情况仍不清楚。本研究通过对46，XX例睾丸发育不良患者的睾丸组织进行组织学分析，并利用基于PCR的消减杂交技术，对XX例睾丸发育不良患者与XY例睾丸发育不良患者的差异表达基因进行鉴定，以阐明XX例睾丸发育不良患者的遗传学特征。我们使用了从46，XX睾丸DSD男孩的睾丸活检中获得的标本。取睾丸鞘膜积液标本，取同侧XY睾丸作为对照。经PCR消减杂交后，对消减后的cDNA进行测序和分析。此外，通过免疫组化阐明了候选基因在睾丸组织中的分布。本研究已获得我校机构审查委员会的批准。我们最终确定了XX睾丸中13个上调和7个下调的基因。在候选基因中，我们集中在ROCK1，PQBP1和UCP2的上调基因组和EEF1A1的下调基因组。免疫组化结果显示，ROCK 1蛋白在生殖细胞和Leydig细胞中表达，PQBP 1蛋白在Leydig细胞中表达，UCP 2蛋白在Sertoli细胞中表达。从以前的报道和体外抑制实验来看，ROCK 1在Sertoli细胞中磷酸化并激活SOX 9是可行的。这表明，睾丸的形成是由一个替代的信号转导途径，ROCK 1激活，而不是SRY激活，在XX睾丸。