Effects of Epigallocatechin-3-gallate (EGCG) on boar sperm

Studies in animals and humans have suggested that green tea extracts, and particularly its main polyphenol, epigallocatechin-3-gallate (EGCG), exert positive effects on human and animal health and exhibit antioxidant activities. Although high concentrations of reactive oxygen species (ROS) may cause sperm damages, an accumulating body of evidence indicates that a mild and controlled generation of ROS plays a physiological role in the acquisition of sperm fertilizing ability. Basing on these information, two experiments were conducted to study the effects of EGCG on boar sperm. In a first experiment, spermatozoa were incubated for 1 h in Brackett & Oliphant's medium supplemented with 12 % FCS and 0.7 mg/ml caffeine, in order to induce sperm capacitation, in presence of 0, 5, 10, 25 µg/ml EGCG. The degree of sperm capacitation was assessed on the basis of Hsp70 immunolocalization, that has been demonstrated to be strictly related to the functional status of sperm. Hydrogen peroxide production was measured by an Amplex Red Hydrogen Peroxide Assay Kit (Molecular Probes). When sperm capacitation was induced in presence of 25 μg/ml EGCG, the percent of spermatozoa displaying the Hsp70 capacitated pattern was significantly lower as compared to controls (62.8±5.6 vs. 79.8±3.6, P<0.05) and sperm H2O2 was significantly reduced (1.7±0.1 μM vs. 2.6±0.2, P<0.05). In a second experiment spermatozoa were coincubated for 1 h with IVM oocytes in presence of 0, 5, 10, 25 µg/ml EGCG in order: 1) to evaluate the occurrence of spontaneous acrosome reaction (AR) in fertilization medium by using FITC-PSA; 2) to examine the effect of EGCG on both sperm-ZP binding and ZP-induced AR by staining sperm-oocyte complexes with FITC-PNA and Hoechst 33342. EGCG 10 μg/ml significantly (P<0.05) inhibited the onset of spontaneous AR in sperm suspension (6.6 ± 0.5 vs.16.3 ± 1.4). Treatment with 25 µg/ml of EGCG significantly (P<0.05) increased the number of sperm bound to ZP after 1 h of coculture compared with the control (38.5± 16.9 vs. 7.1± 2.5, P<0.05) without influencing the incidence of AR in ZP-bound spermatozoa. These results demonstrate that EGCG inhibits sperm capacitation (probably by reducing sperm H2O2) and spontaneous AR while enhancing ZP binding as only acrosome intact sperm are able to initiate binding to ZP; the subsequent true AR, induced by ZP glycoproteins, does not seem to be influenced by this catechin.