Date of Award
Master of Science
Molecular Cellular and Systemic Physiology
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.
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