330 CORRELATION BETWEEN TWO APOPTOTIC MARKERS IN FRESH BOAR SPERMATOZOA

Abstract

Apoptosis is a carefully regulated process of cell death that occurs as a normal part of development. It is a well-characterized mechanism that allows eukaryotes to eliminate unneeded, senescent, or aberrant cells, but the significance of apoptosis in ejaculated animal sperm is still unresolved. In this experiment, we designed 2 methods to detect early changes in the membranes of boar spermatozoa based on the slight increase in membrane permeability (Vybrant Apoptosis Assay Kit #4; Molecular Probes, Eugene, OR, USA) and on translocation of the phospholipid phosphatidyserine (PS) from the inner to the outer leaflet of the plasma membrane (Annexin-V-FLUOS Staining Kit; Roche, Mannheim, Germany). Detection of early changes in the sperm plasma is very important when designing storage protocols. Three ejaculates from 3 boars were used in the experiment. After collection and separation of the gel, the semen was analyzed using the following assays: (1) Annexin-V-FITC/PI assay: Sperm (2 × 10⁶) were washed and diluted in 100 µL of HEPES buffer; 6 µL of Annexin-V-FITC and 4 µL of PI were added to the sample. The tubes were incubated for 15 min at room temperature in the dark. (2) YO-PRO-1/PI assay: YO-PRO-1 stock solution (1 µL) and 1 µL of PI stock solution were added to the samples. The tubes were gently mixed and incubated for 20 min at room temperature in the dark. After the incubation period, the sperm cell suspensions were analyzed under a fluorescence microscope at 40× magnification. At least 200 spermatozoa per sample were evaluated. Using the YO-PRO-1/PI assay, we observed 3 groups of sperm: apoptotic sperm showed green fluorescence (2–8%), necrotic sperm showed red and green fluorescence (9–37%), and live sperm showed no fluorescence (58–89%). Using the Annexin V-PI assay, 4 sperm subpopulations were easily detectable: apoptotic sperm showed green fluorescence (0.5–7%), early necrotic sperm showed red and green fluorescence (10–35%), necrotic sperm showed only red fluorescence (2–6%), and live sperm showed no fluorescence (57–87%). The results were compared by a chi-squared test. Significant differences (P < 0.01) in the percentage of all sperm subpopulations (apoptotic, necrotic, and live sperm) among boars and among ejaculates from the same boar were observed. We also observed a strong correlation between these 2 methods. Using the Annexin-V-FITC/PI assay, we detected more sperm subpopulations, and this method seems to be more sensitive than the YO-PRO-1/PI assay. However, these 2 methods detect changes in membrane spermatozoa but in different aspects of apoptosis, and this may also cause differences in the frequencies of apoptotic cells found by the different assays.

This study was supported by the Polish Research Committee, grant no. 2 P06D 023 30.