The Rhox3 Homeobox Gene Paralogs Are Essential for Spermatogenesis In Vivo.

The X-linked Rhox gene cluster encodes homeobox transcription factors selectively expressed in reproductive tissues. The founding member of the mouse Rhox gene cluster, Rhox5, is highly expressed in Sertoli cells, where it promotes the survival of the adjacent germ cells and modestly promotes fertility. The in vivo functions of the other 32 genes in the mouse Rhox cluster have not been elucidated. Here, we report that a set of highly related Rhox genes (encoding proteins with >96% sequence identity) that have been duplicated eight times in mice - Rhox3a to Rhox3h - are essential for spermatogenesis. Because these eight Rhox3 paralogs are interspersed with other Rhox genes, a knockout strategy to determine their in vivo function was not feasible, and so we employed a conditional RNA interference (RNAi) approach instead. Towards this end, we first identified a short interfering RNA (siRNA) displaying 100% sequence complementary with all eight Rhox3 paralogs that efficiently downregulated RHOX3 level in cell lines (by 90%). This siRNA sequence was inserted (in the form of a short hairpin [sh]) downstream of a ubiquitously expressed Pol III promoter rendered non-functional by a DNA insertion flanked by loxP sites. Several transgenic lines harboring this Rhox3 shRNA construct were obtained and mated with transgenic mice lines expressing Cre recombinase to remove the insert and activate Rhox3 shRNA expression. Double-transgenic mice lines expressing Cre from a male germ cell promoter (Stra8) exhibited reduced RHOX3 protein levels in the testes (up to 90%) and had a profound block in post-meiotic germ cell progression and >1% of normal sperm count. These Rhox3-knockdown mice also had reduced expression of some meiotic genes, suggesting that the Rhox3 paralogs also have a role in meiosis. Given that the Rhox3 paralogs encode transcription factors, we propose that they regulate key genes essential for spermatogenesis, which we are currently in the process of identifying. The conditional RNAi approach that we used to identify the in vivo function of the Rhox3 paralogs has the potential to be a generally applicable and efficient means to elucidate the cell type-specific functions of genes. (platform)