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The role of cysteine rich secretory protein 4 in sperm function
thesisposted on 16.01.2017, 23:37 authored by Reddy, Thulasimala
One in eight Australian couples of reproductive age is infertile and in more than 40% of these couples, the infertility is due, at least in part, to male factors. In view of this finding, research is committed to addressing male fertility issues. Several regulatory genes have been identified as having a potential role in male infertility and animal models are widely used to establish functional activity. As a corollary of such research, many proteins are being assessed for their potential value as contraceptive targets. The mammalian cysteine-rich secretory proteins (CRISPs) are a family of four proteins exhibiting a high amino acid sequence similarity and belonging to the CAP (CRISP, Antigen-5 proteins and the plant Pathogenesis related-1 proteins) superfamily. CRISPs are predominantly expressed and localised to the male reproductive tract and were therefore investigated for their potential as fertility markers. Functional evidence of mammalian CRISPs is emerging, and data from snake venom CRISPs and from mouse CRISPs in our own lab, is suggesting that all CRISPs function as ion channel regulators. To determine the localization of all 4 members of the mouse CRISP family, I identified 16 tissues likely to express CRISPs, from EST databases and conducted RT-PCR to identify the transcripts and confirmed the localization of the proteins using immunohistochemistry. Consistent with published data, my results showed that Crisps have an expression bias to the male reproductive tract. In addition, however, my data showed that Crisps have a wider expression profile than was previously understood, and as such, raises the possibility of a role of CRISPs in the normal physiology of multiple organs. To investigate the effect of CRISP4 on sperm function, I expressed recombinant full length CRISP4 in a bacterial system but upon purification, it was found to be insoluble. After extensive efforts, CRISP4 was solubilized and refolded and is currently being characterized by other researchers. In a parallel study, I expressed and purified the CRISP domain of CRISP4 for use in sperm functional assays. At that time, our lab had determined that CRISP4 CRISP domain could regulate calcium flow through TRPM8 ion channels on the sperm plasma membrane. As such, I explored the potential for change on sperm function induced by TRPM8 activation, and the ability of CRISP4 to reverse such changes. Results from in vitro studies showed that TRPM8 activation results in a suppression of the ability of spermatozoa to undergo the progesterone-induced acrosome reaction. This effect was reversed by CRISP4. In order to extend the significance of this finding regarding the in vitro situation, I characterized the reproductive phenotype of Crisp4-deficient mice. An investigation of the function of sperm from Crisp4 null mice showed that the percentage of sperm undergoing the acrosome reaction in response to progesterone was significantly reduced compared to wild type sperm. These in vivo results are consistent with the in vitro experiments and provide the first direct evidence that endogenous CRISP4 is a regulator of ion channel (including TRPM8) function, with implications in acrosome maturation within the epididymis and potentially in the fine tuning of the timing of the acrosome reaction within the female reproductive tract. The novel findings in this study provide, for the first time, a conclusive function of endogenous mouse CRISP4 and add to the growing evidence that mammalian CRISPs are regulators of fertility. These findings have significant implications for identifying the function of human CRISPs and their potential as male contraceptive targets.