Organotypic Cultures of Prepubertal Mouse Testes: A Method to Study Androgen Action in Sertoli Cells while Preserving their Natural Environment
Organotypic Cultures of Prepubertal Mouse Testes: A Method to Study Androgen Action in Sertoli Cells while Preserving their Natural Environment
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DOI：
10.1095/biolreprod.109.078360
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发表时间：
2009-12-01
影响因子：
3.6
通讯作者：
Verhoeven, Guido
中科院分区：
文献类型：
--
作者：
De Gendt, Karel;McKinnell, Chris;Verhoeven, Guido
在出生后第8-20天，对雄激素受体支持细胞选择性敲除（SCARKO）小鼠中推定的雄激素调节基因进行聚类分析，确定了三个基因（Spinlw 1，Gpd 1，Drd 4）的表达模式与Rhox 5（永久性支持细胞（SC）雄激素调节基因）的表达模式非常相似。我们使用来自第8天小鼠的器官型睾丸培养物来研究（抗）雄激素和促卵泡激素（FSH）对这些基因的控制。将所研究基因的睾丸形态和雄激素诱导保存48 h。与酮康唑预孵育24小时，以阻止内源性雄激素的产生，然后与合成雄激素R1881孵育24小时，导致Rhox 5，Spinlw 1，Gpd 1和Drd 4的mRNA水平分别诱导45，5，19和6倍。然而，观察到所研究的基因的控制的显著差异。Rhox 5和Spinlw 1完全诱导R1881在连续（48小时）存在的酮康唑，而只有边际效应，观察到Gpd 1和Drd 4的表达。同样，FSH仅轻微影响Rhox 5和Spinlw 1的表达，而它显著增加Gpd 1和Drd 4的表达。SCARKO睾丸的外植体培养证实了FSH对所研究基因的差异效应，并且对于Gpd 1，表明该效应不依赖于SC中的功能性雄激素受体，而这对于FSH对Drd 4的影响至关重要。总之，器官型培养代表了第一个在体外方法，以保持雄激素反应的推定SC表达的基因。这种方法有助于以体内不可能的方式详细分析它们的调节。
Cluster analysis at Postnatal Day 8-20 of putative androgen-regulated genes in mice with Sertoli cell-selective knockout of the androgen receptor (SCARKO) has pinpointed three genes (Spinlw1, Gpd1, Drd4) with an expression pattern strongly resembling that of Rhox5, the definitive Sertoli cell (SC) androgen-regulated gene. We used organotypic testis cultures from Day 8 mice to study control of these genes by (anti) androgens and follicle-stimulating hormone (FSH). Testis morphology and androgen induction of the studied genes were preserved for 48 h. Preincubation with ketoconazole for 24 h to block endogenous androgen production, followed by 24-h incubation with the synthetic androgen R1881, resulted in 45-, 5-, 19-, and 6-fold induction of mRNA levels of Rhox5, Spinlw1, Gpd1, and Drd4, respectively. However, noticeable differences in control of the studied genes were observed. Rhox5 and Spinlw1 were fully induced by R1881 in the continuous (48 h) presence of ketoconazole, whereas only marginal effects were observed on expression of Gpd1 and Drd4. Similarly, FSH only marginally affected expression of Rhox5 and Spinlw1, whereas it markedly increased Gpd1 and Drd4 expression. Explant cultures of SCARKO testes confirmed the differential effects of FSH on the studied genes and, for Gpd1, showed that the effect did not depend on a functional androgen receptor in SC, whereas this was essential for the effects of FSH on Drd4. In conclusion, organotypic cultures represent the first in vitro approach to preserving androgen responsiveness of putative SC-expressed genes. This approach facilitates detailed analysis of their regulation in ways not possible in vivo.