Date of Award

12-1-2012

Degree Name

Master of Science

Department

Molecular Cellular and Systemic Physiology

First Advisor

MacLean, James

Abstract

The Reproductive Homeobox X-linked, Rhox, genes encode transcription factors that are expressed exclusively in the testis, epididymis, placenta, and ovary and are therefore good candidates to regulate postnatal reproductive events. The founding member of this 33 gene cluster Rhox5, previously known as Pem, is important in male mice for both spermatogenesis and sperm maturation. This is supported by the findings that Rhox5-null mice are hypofertile due to increased apoptosis of meiotic germ cells in the testis as well as motility defects in epididymal sperm. Rhox5 was also the first homeobox gene to exhibit stage specific androgen sensitivity exclusive to Sertoli cells during spermatogenesis. Within the Rhox family, only RHOX8 shows expression in Sertoli cells at time intervals similar to that of RHOX5. Interestingly, while Rhox8 exhibits high expression in Sertoli cells it does not appear to be dependent upon androgens for transcription. The co-localization of these genes supports the hypothesis that RHOX5 and RHOX8 may exhibit partially redundant functions in the testis, and potentially explain why Rhox5-null animals are subfertile and not infertile. However, due to the androgen dissimilarity between Rhox5 and Rhox8 aforementioned it is possible that RHOX8 also exhibits functionally unique expression in the male gonad as well. Therefore, we sought to ablate Rhox8, and potentially derive Rhox5/Rhox8 double knockouts to better assess these questions. However, due to the proximity of Rhox5 and Rhox8 on the X chromosome, recombination of individual knockout lines produced by traditional strategies would be unlikely to succeed. To circumvent this issue, we used a novel tissue-specific RNAi approach to knockdown RHOX8 in vivo. Our knockdown system used the Rhox5 proximal promoter which contains regulatory elements for expression of the Rhox8-siRNA transgene in Sertoli cells. Candidate siRNAs were evaluated for knockdown of RHOX8 in Sertoli cell lines, and the most efficient was used to produce 11 independent founder lines. Among these founders the majority of the males failed to produce any subsequent litters. However, backcrossed littermates from female founder lines have exhibited knockdown of RHOX8 in total testis lysates using Western blot analysis. Immunohistochemical analysis confirmed Sertoli cell specific RHOX8 protein knockdown as well as knockdown of Sertoli cell marker, SOX9. However, other control targets, GATA1, AR, and RHOX5; maintain normal expression patterns. Rhox8-knockdown and Sox9-regulation was also observed at the mRNA level via real-time qPCR, showing significant knockdown of both transcripts when compared to wild-type animals. Further characterization revealed that this single knockdown of RHOX8 sufficiently yields a subfertile phenotype. Therefore, the central hypothesis of this study is that RHOX8 is imperative for normal sperm output, an essential gene regulator in the adult testis, and possibly employs an important role during embryonic testis development.

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