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.

Mammalian ovulation is a complex, hormone-dependent developmental program in which several events must take place in an ordered progression to ensure that the oocyte is competent for fertilization. This process requires the coordinated expression of many genes which must be turned on and off in the right place at the right time for proper development of the follicle. While the hormone signals from the brain that initiate ovulation are known, the master control genes which regulate this process are not well known. Homeobox proteins are potential candidates to perform as master regulators. Homeobox proteins are DNA-binding proteins that regulate the transcription of downstream genes and thereby control biological events. We recently identified a new homeobox gene cluster on the mouse X chromosome that are only expressed in reproductive tissues. These reproductive homeobox (Rhox) homeobox genes are expressed in the ovary, placenta, testis, and epididymis, and thus are good candidates to regulate both male and female reproductive tissue development and physiology. Rhox gene expression fell into three categories: Class I exhibited peak expression prior to ovulation (0-8 hours after hCG), Class II were predominantly expressed during ovulation (8-16 hours after hCG), and Class III peaked after ovulation. The slightly overlapping windows of peak Rhox gene expression suggest that these genes may regulate specific events during the ovulatory cycle. The founding member of the cluster, Rhox5, is highly expressed in granulosa cells of pre-ovulatory follicles. We previously reported that Rhox5-null female mice are viable and fertile, suggesting that RHOX5 is either not essential for ovulation, or that one of the other RHOX factors may compensate functionally in granulosa cells. In order to identify potentially redundant RHOX factors, we examined the expression patterns of all 32 Rhox genes using an eCG primed, hCG induced superovulation model, in wild-type, Rhox5-null, and heterozygous littermate mice. Expression levels of Rhox1, exhibited peak expression prior to being hormonal primed, was reduced in the Rhox5-null animal. However, Rhox8 mRNA and protein were reduced at 2h and 4h post hCG, but recovered once the follicles passed the antral stage of development. Conversely, in progesterone receptor knockout mice (PRKO), Rhox8 exhibited normal stimulation by eCG, but failed to reach its peak mRNA level at 8h post-hCG found in WT mice. This suggests a model in which Rhox8 transcription is dependent upon RHOX5 during early folliculogenesis and progesterone during the periovulatory window when RHOX5 normally wanes. Subsequent promoter analysis in granulosa cells revealed essential homeobox binding and progesterone response elements within Rhox8's 5'-flanking region. Transfection of RHOX5 and PGR expression plasmids stimulated, whereas dominant negative and mutant constructs inhibited, Rhox8 promoter activity. At present, the specific impact of misregulation of Rhox5 and Rhox8 during early folliculogenesis is not known. However, follicle counts from serially sectioned ovaries, extirpated from normal cycling animals, indicated that Rhox5-null mice possess ~50% fewer follicles than heterozygous littermates. Loss of RHOX5 in Sertoli cells results in male subfertility characterized by poor germ cell survival due in part to the misregulation of metabolism promoting genes. One of these genes, Ins2, is also stimulated by RHOX5 and RHOX8 in granulosa cells, suggesting impaired insulin signaling may contribute reduced follicle development in Rhox5-null ovaries.