Phosphoproteomic Study on Mechanisms of Silymarin Modulating Fungicide Induced Sperm Dysfunction

Mammalian sperms are required to undergo a process known as capacitation before they can undertake the fertilization process. Capacitated spermatozoa exhibit the acrosome reaction and hyperactivation. Bovine serum albumin and trace hydrogen peroxide promotes capacitation and hence induced cAMP/protein kinas A activation and serial phosphorylations. Experimental exposure to estrogenic or antiandrogenic endocrine disrupters (ED) has been shown to alter male functions. However, little is known about the molecular impact of exposure to excess hydrogen peroxide and distinct EDs. The purpose of this proposal is to determine the modulation effects of excess hydrogen peroxide or endocrine disrupters on albumin-induced capacitated mouse sperms. The mechanisms of those test chemicals on phosphorylated protein expressions will be further investigated. During first study year, identification of phosphoprotein expression patters response to excess hydrogen peroxide will be established by phosphoproteome in capacitated sperms from two different strains of mice. The involvement of cAMP pathways in the influence of excess hydrogen peroxide will be investigated while compared to the capacitated sperm proteins. The types and specific sites of phosphorylated proteins including outer dense fiber 1 (ODF1), CG031, and A-kinase anchoring protein 4 (AKAP4) will be the research targets. Techniques including immunoprecipitation, two-dimensional gel-electrophoresis and immunocytochemistry will be utilized for phosphoprotein identification especially for CG031 protein. During the second year, regulations of target phosphoproteins in mouse sperms exposure to various insults including excess H2O2, vinclozolin or silymarin will be analyzed. EDs include vinclozolin, a fungicide known to cause AKAP4 precursor overexpressions, and silymarin, possess dual modulation of glucose and estrogen modulator. The involvement of Cdk5 for excess hydrogen peroxide induced decreases of ODF1 phosphorylation will be tested by Cdk5 activation. The involvement of PKCα pathway for silymarin-modulated phosphoryaltions will be investigated by PKCα inhibitors. Parameters of sperm structures and functions (capacitation/acrosome reactions) will be also determined accordingly. Potential protective effects of silymarin will be also confirmed in vivo. This phosphoproteomic approach is expected to function as a non-invasive experimental tool in the diagnosis of male infertility and in evaluating a fertility-restoring pharmacotherapy.