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The joint treatment of sperm by prolactine and GTP have determined the increase of the number acrosome-reacted spermatozoa in bulls

There are contradictory opinions concerning involvement of prolactin (PRL) in the process of sperm capacitation and acrosome reaction (Vigil P. et al., 2011 Biol Res, 44:151-159). It was shown that PRL stimulates release of Ca2+ from IP3-sensitive stores, and GTP stimulates release of this ion from IP3-insensitive stores (Denisenko V. et al., 2015 Tsitologiya, 3:1-8). GTP forms a connection between IP3-sensitive and IP3-insensitive intracellular stores and promotes transition of Ca2+ between these stores (Mullaney J. et al., 1987 J. Biol. Chem. 262: 13865—13872). The aim of the present study was to examine the mobilization of Ca2+ from intracellular stores after the treatment spermatozoa by PRL and GTP and to evaluate the status of spermatozoa after these treatments. Intensity of fluorescence of membrane-bound Ca2+ was determined with a fluorescence spectrophotometer Hitachi MPF-4 (excitation: 380-400nm, emission: 530 nm) using 40 µM chlortetracycline (CTC) - (Denisenko V. et al., Tsitologiya 3:1-8, 2015). Intensity of fluorescence of membrane-bound Ca2+ was determined in Sp-TALP medium where the concentration of cells was adjusted to 1, 5 X 106 sperm/mL. The CTC assay was used to determine the functional status of spermatozoa (Ded L. et al., 2010 Reprod Biol Endocrinol, 8-87). Samples were examined with fluorescence microscope Zeiss Axo Imager M1. Ejaculates from three fertile bulls were used, and five replicates were performed for each experiment. In each sample, 200 cells were evaluated. Sperm were evaluated according to 1 of 3 CTC staining patterns: fluorescence over the entire head (precapacitated cells), fluorescence-free band in the postacrosomal region (capacitated cells) and low fluorescence over the entire head except for a thin bright fluorescent band along the equatorial segment (acrosome-reacted cells). All reagents that were used in this study were produced by Sigma-Aldrich (Moscow, Russia). Data were analyzed by Student's t-test. Treatment spermatozoa by PRL (10 ng/ml) or GTP (10 μmol) resulted in release of Ca2+ from intracellular stores (0.70±0.019 and 0.69±0.017 vs 0.85±0.016; P<0.001). There was additional release of Ca2+ with the combined effect of PRL and GTP (0.62±0.011 vs 0.70±0.019 and 0.69±0.017; P<0.001). There was no additional release of Ca2+ after the joint action by the pair of these reagents in the presence of protein kinase C inhibitor (Ro 31-8220, 10ng/ml). The average percentages of capacitated spermatozoa did not change after treatment by PRL, GTP or both these reagents. The percentage of cells that underwent acrosome reaction have increased after treatment by PRL and GTP jointly (46% vs 62%, P<0.01); there was no such effect at preliminary treatment of sperm by Ro 31-8220 (10 ng/ml). Thus, Ca2+ transition between intracellular stores in bull spermatozoa after the treatment with PRL and GTP jointly is leading to increasing in the percentage of acrosome-reacted spermatozoa.