Reproduction Abstracts

The reproductive homeobox X-linked, Rhox, genes encode transcription factors that are expressed selectively in reproductive tissues. While there are 33 testis-expressed Rhox genes in mice, only Rhox5 and Rhox8 are produced by Sertoli cells, suggesting that they alone regulate somatic-cell gene products crucial for germ-cell development. We used the Rhox5 promoter to drive tissue-specific RNAi to knockdown (KD) RHOX8 in vivo. Western and immunohistochemical analysis confirmed Sertoli-specific KD of RHOX8. However, other Sertoli markers, Gata1, Ar, and Rhox5; maintained normal expression patterns, suggesting the KD affect was specific. Male RHOX8-KD animals showed reduced fecundity in timed breeding experiments, ~50% decline in spermatogenic output, and 30% decrease in sperm motility, in four independent Rhox8-KD lines. Increased germ cell apoptosis and a delay in spermatogenesis stages VI–VII transition contributed to the phenotype. Analysis of established RHOX5-regulated genes found some apoptosis related factors similarly misregulated in RHOX8-KD mice. However, Rhox7, Rhox10, and Rhox11 were uniquely downregulated in RHOX8-KD testes. At present the function of these genes in germ cells is unknown. In postnatal Sertoli cells, Sox8 (partially) and Sox9 (strongly) were downregulated in RHOX8-KD testes. These genes are essential factors for Sertoli development and function and are likely key mediators of RHOX8 action for maximal sperm production. We are currently investigating this hypothesis, as well as other genetic interactions; through the characterization of a conditional Cre-LoxP activated shRNA KD model that can be employed to elucidate Rhox8’s role in embryonic testis development as well as confirm our present postnatal results.