Assesment of intracellular homocysteine concentration in sperms



Year of publication 2011
Type Conference abstract
MU Faculty or unit

Faculty of Medicine

Description The aim of this study was to evolve an effective procedure for the sperm lysis, obtention of intracellular content and subsequent assesment of Hc concentration and, in consequence, determine the correlations between blood plasma and seminal fluid Hc concentration, spermiogram parameters and DNA injury. Firstly, we used the method of repeated freezing and melting according to Ebisch et al., 2006 for the sperm lysis. Because the microscopic control of the samples didn’t show any markable sperm injury, we projected and compared other methods of the cell lysis – ultrasound, melting in ultrasound and freezing 10 times, methanol, distilled water, Triton X-100 solution, and lysis solution + 1% Triton X-100 solution. We used the method of high-performance liquid chromatography with fluorescence detection (HPLCFD) for the determination of Hc concentration and comet analysis for the assesment of DNA injury. Correlation coefficient was used to determine the correlations between the intracellular Hc concentration and the other parameters. The most effective methods of sperm lysis were resuspending of sperms in distilled water or 80% methanol, one-hour incubation and subsequent fivefold freezing and melting in ultrasound bathe. Distilled water causes osmotic shock and membrane craze, methanol causes protein denaturation. Measured Hc concentrations ranged between 85.8–528.4 nmol/106 of sperms (median 233.4 nmol/106 sp., mean 265.4 nmol/106 sp., N = 8). We found positive correlation between intracellular concentration of Hc and rate of sperms with abnormal neck morphology (R = 0.818, p < 0.05) and weak positive correlation between seminal fluid and blood plasma Hc (R = 0.645 and R = 0.574, respectively). We measured the intracellular Hc concentrations which are three orders of magnitude higher than published. We attribute it to really effective breaking of sperms and getting intracellular content. The results show possible correlations with some spermiogram parameters.

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