Mizukami Takuo, Kanai Yoshiakira, Fujisawa Masahiko, Kanai-Azuma Masami, Kurohmaru Masamichi, Hayashi Yoshihiro
Department of Veterinary Anatomy, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.
Mol Reprod Dev. 2008 Jun;75(6):1002-10. doi: 10.1002/mrd.20850.
In mammals, Sry, Sox9, and M33 act as regulators at a chromatin level and promote the maturation of embryonic gonads into testes. Recently, it was shown that transcriptional regulation by DNA methylation plays crucial roles in gene expression during the differentiation and development of various cell types. To determine the involvement of DNA methylation in sex determination of the gonad, we developed and performed organ culture of gonad with the DNA methyltransferase inhibitor 5-azacytidine to induce global DNA methylation status changes. In vitro treatment with 5-azacytidine specifically inhibited testicular cord formation in a dose-dependent manner; however, no appreciable defect was observed in ovarian explants. Inhibition of testicular cord was observed only in gonads from 11.5 days post-coitus embryos. These effects were not observed in 5-azacytidine-treated gonads from 12.0 days post-coitus embryos. To determine the effect of 5-azacytidine on Sertoli and Leydig cell differentiation in the testis, we performed whole mount in situ hybridization analysis. The Leydig and stromal cell marker genes Lhx9, Mfge8, and 3beta-Hsd were normally induced in 5-azaytidine-treated testicular explants. Sertoli cell marker genes, Sox9 and MIS were normally induced, but Col9a3, encoding an extracellular matrix component, was inhibited in 5-azacytidine-treated testicular explants. Thus, our data show that DNA methylation is involved in testicular cord formation and Sertoli cell differentiation, acting directly on the gonad at 11.5 days post-coitus.
在哺乳动物中，Sry、Sox9和M33在染色质水平上发挥调节作用，促进胚胎性腺向睾丸的成熟。最近的研究表明，DNA甲基化介导的转录调控在各种细胞类型的分化和发育过程中的基因表达中起着关键作用。为了确定DNA甲基化在性腺性别决定中的作用，我们开发并进行了性腺器官培养，使用DNA甲基转移酶抑制剂5-氮杂胞苷来诱导整体DNA甲基化状态的改变。用5-氮杂胞苷进行体外处理以剂量依赖的方式特异性抑制睾丸索的形成；然而，在卵巢外植体中未观察到明显的缺陷。仅在交配后11.5天胚胎的性腺中观察到睾丸索的抑制。在交配后12.0天胚胎经5-氮杂胞苷处理的性腺中未观察到这些效应。为了确定5-氮杂胞苷对睾丸中支持细胞和间质细胞分化的影响，我们进行了全组织原位杂交分析。在经5-氮杂胞苷处理的睾丸外植体中，间质细胞标记基因Lhx9、Mfge8和3β-Hsd正常诱导表达。支持细胞标记基因Sox9和MIS也正常诱导表达，但编码细胞外基质成分的Col9a3在经5-氮杂胞苷处理的睾丸外植体中受到抑制。因此，我们的数据表明，DNA甲基化参与睾丸索的形成和支持细胞的分化，在交配后11.5天直接作用于性腺。
