Date of Award

5-1-2014

Degree Name

Master of Science

Department

Biological Sciences

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. While there are 33 Rhox genes in mice, only Rhox5 and Rhox8 are expressed in Sertoli cells, suggesting that they alone regulate the expression of somatic-cell gene products crucial for testicular metabolism and germ-cell development. Targeted deletion of Rhox5, the founding member of the Rhox gene cluster, in mice, results in decreased expression of the insulin-2 gene (Ins2) and other metabolic genes, male subfertility via reduced sperm number, increased germ-cell apoptosis and a reduced proportion of sperm with normal motility. Davis et al. developed a Rhox8 siRNA knockdown transgenic model to study possible functional similarities between Rhox5 and Rhox8 and reveal compensatory actions via the breeding of Rhox5/Rhox8 double knockout mice. They observed that loss of Rhox8 results in downregulation of the sex-determining region Y gene (Sox9). Further analysis of the role of Rhox5 in testicular metabolism regulation was completed by development of mutant constructs encoding combinations of Rhox5 functional domains and subsequent analysis via qRT-PCR, luciferase assay and immunohistochemistry in cell lines transfected with expression plasmids containing these mutants. Our results indicated direct interaction of RHOX5 with the Ins2 promoter. The homeodomain and amino-terminal domain construct being sufficient for promoter activation albeit at a lower level than the full-length RHOX5 construct. MacLean et al. conducted qRT-PCR analysis of cells transfected with plasmids encoding the other Rhox genes revealed that Rhox8 and Rhox11 were also capable of upregulating Ins2 expression at a lower level than Rhox5. Our analysis of metabolic gene expression in the Rhox8 knockdown model also revealed decreased expression of Ins2 as well as insulin receptor-1 (InsR1). Continued analysis of the Rhox8-KD model and Rhox5/Rhox8 double knockout mice in young and aged (~12 months) male mice revealed a subfertility phenotype characterized by reduced litter frequency and size, reduced total spermatozoa and reduced sperm forward motility. This was reflected by a decrease in RHOX8 and SOX9 protein expression maintained in the aged mice that was more severe in the double knockout animals. qRT-PCR analysis of altered gene expression in the Rhox8 single knockdown male mice revealed significant expression downregulation of fellow Rhox genes Rhox5 and Rhox10 and expression upregulation of the growth differentiation factor-9 (Gdf9) gene normally expressed in the somatic cells of the ovary. Based on the regulation of sex specific genes Sox9 and Gdf9 in the Rhox8 knockdown model as well as data published by Daggag et al. revealing that Rhox8 is the only Rhox member expressed in the somatic cells of the embryonic testis, we sought to elucidate the possible role of Rhox8 in sex determination and testicular differentiation/development. Due to the limitations of our current knockdown model whereby siRNA production is initiated postnatally due to its androgen-dependent promoter, we elected to develop a new Rhox8 knockdown model to characterize the embryonic functions of Rhox8. Rhox8 siRNA were cloned into a Cre-recombinase activated expression vector in which a highly active U6 promoter drives expression of the shRNA. The constitutively active form of this vector exhibited knockdown of Rhox8 in stable Rhox8 overexpressing cell lines developed for this purpose. This developed vector is being used to develop a new transgenic line. While waiting for the development of the new Rhox8 knockdown transgenic mice, we chose to characterize the mRNA and protein expression of Rhox8 in the embryonic testis. Using qRT-PCR and immunohistochemistry, expression of Rhox8 was confirmed and exhibited continually increased expression in the Sertoli cells over the course of embryonic day ~13.5-18.5 (E13.5-E18.5). To examine possible functions of Rhox8 in the embryonic testis prior to receipt of the new knockdown model, we adapted a protocol to transfect the new constitutively active Rhox8 knockdown into embryonic gonads and cultured them up to 72 hours after electroporation. This protocol yielded successful expression of GFP from a GFP-expression plasmid and this was maintained for up to 72 hours in E13.5 gonads. qRT-PCR analysis of gonads transfected with the Rhox8 knockdown expression plasmid revealed significant knockdown of Rhox8 and Sox9 mRNA expression at the 48-hour and 72-hour time points.

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