The Effect Of (S)-α-chlorohydrin On Spermatozoa Capacitation-associated Protein Phosphorylation And Its Mechanism

a-chlorohydrin, ACH, also called3-mono-chloro-propane-1,2-diol,3-MCPD, is an important intermediate in chemical industry. Human may be exposed to it from contaminated foods and drinking water which was treated with epichlorohydrin resin. As a classic post-testicular toxicant, α-chlorohydrin can cause quick reversible infertility in males of many species at low oral dose. ACH possesses a chiral carbon atom and its antifertility action effect is solely attributed to the (S)-isomer (SACH). This study focused on the relationship between SACH exposure and rodent or human sperm capacitation-associated protein phosphorylation, and tried explore the effects and its possible mechanism of SACH on the regulatory signaling pathway of sperm capacitation.Part I The effects of (S)-α-chlorohydrin on rat spermatozoa functionsTo develop a incubation condition for supporting rat spermatozoa capacitation, rat caudal epididymal sperm were incubated in a modified Biggers, Whitten, Whittingham medium (BWW),37℃and5%CO2for6h, and the rate of spontaneous acrosome reaction, protein tyrosine phosphorylation and in vitro sperm-oocyte fusion test were measured. It showed that there was no acrosome-reacted rat sperm at the beginning of the culture, and protein tyrosine phosphorylation was very low. During the period of capacitation, the spontaneous acrosome reaction rates were increasing and reached at60%at the end of6h incubation; while protein tyrosine phosphorylation was showed an increase at4.5h and got a fully phosphorylation at6h. And on average,59.1%oocytes were fused by rat sperm and two-cell-stage embryos were seen. All these validated the capacitation condition. After incubated rat caudal epididymal sperm with10μM,25μM,50μMå'Œ100μM SACH under the capacitating condition for6h, sperm velocity parameters such as VAP, VCL and VSL, and also ALH were suppressed by25μM, and the proportion of sperm with VCL≥400μm/s was significantly decreased comparing the control(P<0.05) indicating that hyperactivation was inhibited. The rates of spontaneous acrosome reaction declined by10μM and25μM SACH, however, they recovered to normal level when concentration reacted50μM or more.100μM SACH suppressed protein tyrosine phosphorylation of85kDa band by90%and55kDa by70%, and this level of SACH did not affected on IVF rate, and only1.0mM or10mM SACH diminished the two-cell rates. In summary, SACH could inhibit rat sperm motility, hyperactivation and protein tyrosine phosphorylation, but there were,no consistent evidence to prove whether sperm capacitation was effected by100μM SACH.Part II The effects of (S)-a-chlorohydrin on rat sperm capacitation-associated protein phosphorylationProtein tyrosine phosphorylation, which was mediated by cAMP/protein kinase A (PKA) pathway, is a key event for regulating mammalian spermatozoa capacitation. PKA may regulate protein tyrosine phosphorylation by phosphorylating the target protein on their Ser/Thr residues. Our study showed an increase of PKA substrates phosphorylation in rat caudal epididymal sperm during their capacitation, and phosphatidylinositol3-kinases (PI3K)85kDa/55kDa subunits were tyrosine phosphorylated at the end of capacitating culture. After rat sperm were exposed to SACH under capacitating condition, both PKA substrates phosphorylation and PI3K tyrosine phosphorylation were inhibited by50μM and100μM SACH, suggesting that PKA activity might be suppressed. The inhibition of capacitation-associated protein phosphorylation by50μM SACH could be rescued by cAMP analog dibutyryl-cAMP (dbcAMP) and phosphodiesterase inhibitor3-isobutyl-1-methylxanthine (IBMX), indicating that SACH might inhibit phosphorylation by decreasing cAMP levels in sperm. SACH was found to inhibits sperm-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDS) activities and then suppress ATP levels., and on the other hand, addition of glycerol could protect GAPDS, ATP, capacitation-associated protein phosphorylation against the inhibition of SACH, indicating that the block of glycolysis played a critical role in the effect of SACH. However, there was no direct association between inhibition of protein tyrosine phosphorylation by SACH and sperm dysfunction.Part â…¢ The effects of (S)-α-chlorohydrin and its related environmental chemicals on capacitation-associated protein phosphorylation in human or mouse spermJust as rat sperm did, both human and mouse sperm showed an increase of protein tyrosine phosphorylation during the capacitating culture. In human sperm, the most significant phosphorylation were found in110kDa and85kDa bands, and in the case of mouse sperm, it happened in83kDa band, however, all of them were inhibited by SACH. Glycerol could restore protein tyrosine phosphorylation which inhibited by SACH, and it was showed that human sperm preferred to apply the ATP derived from glycolysis, but not oxidative phosphorylation, to phosphorylate their protein during capacitation, so it may be reasonable that SACH inhibited human sperm protein tyrosine phosphorylation in the same way of that in rat. Mouse sperm showed a decrease of PKA substrates phosphorylation during capacitation, and the phosphorylation could be inhibited by SACH. SACH inhibited GAPDS activities in mouse sperm, and diminished the ability of sperm to binding oocytes. In order to investigate whether the physiological or biochemical changes that occur during capacitation or fertilization could be used as an endpoint in assessment of male reproductive toxicity, in this study we dosed human sperm with glycolytic inhibitors, including bromopyruvic acid (BrPA), ornidazole, fluoride and a kind of drinking water contaminant iodoacetic acid (IAA) and found that protein tyrosine phosphorylation were inhibited by these chemicals. These findings indicated it was hopeful that protein tyrosine phosphorylation could be developed to an in vitro test to assess male reproductive toxicity of xenobiotics.