Monthly variations in ovine seminal plasma proteins analyzed by two-dimensional polyacrylamide gel electrophoresis.

This study was conducted to evaluate monthly changes in the ram seminal plasma protein profile using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) with a polyacrylamide linear gradient gel. Likewise, comparative analyses of the protein composition of ovine seminal plasma (SP) from ejaculates obtained along the year, and its relationship with sperm motility, viability and concentration of ejaculate were carried out. Western-blot analysis was performed to specifically detect P14, a ram SP protein postulated to be involved in sperm capacitation and gamete interaction [Barrios B, Fernández-Juan M, Muiño-Blanco T, Cebrián-Pérez JA. Immunocytochemical localization and biochemical characterization of two seminal plasma proteins which protect ram spermatozoa against cold-shock. J Androl 2005;26:539-49], and its variations along the year have also been established. The experiment was carried out from May 2003 to April 2004, with nine Rasa Aragonesa rams. Ejaculates obtained every 2 days were pooled and used for each assay, to avoid individual differences, and three two-dimensional SDS-PAGE gels were run for each month. The high resolution of the gradient gel allowed the image analysis software to detect around 252 protein spots, with pIs ranging from 4.2 to 7.6, and molecular weight (M(r)) from 12.5 to 83.9 kDa. Four protein spots (1, 2, 3 and 4) of low M(r) (15.1, 15.7, 15.9 and 21.0 kDa) and acidic pI (5.9, 5.3, 5.7 and 6.6), respectively, had the highest relative intensity in the SP map (11.2, 9.3, 4.7 and 7.7%, respectively). Spot 3 was more abundant (P<0.05) from May to December, and negatively correlated (P<0.05, r=-0.34) with sperm viability and concentration (P<0.05, r=0.36). Another 12 protein spots also had significant quantitative differences (P<0.05) along the year, and 17 protein spots, which correlated with some seminal quality parameter, did not show quantitative monthly changes. Western-blot analysis indicated that spots 1 and 2 reacted with the anti-P14 antibody, raised against the P14 band (approximate M(r) 14 kDa) of ram SP. This indicates that spots 1 and 2 are similar to RSP15 [Bergeron A, Villemure M, Lazure C, Manjunath P. Isolation and characterization of the major proteins of ram seminal plasma. Mol Reprod Dev 2005;71:461-70], bovine PDC-109 [Esch FS, Ling NC, Bohlen P, Ying S, Guillemin R. Primary structure of PDC-109, a major protein constituent of bovine seminal plasma. Biochem Biophys Res Commun 1983;113:861-7] (also called BSP A1/A2 [Manjunath P, Sairam MR. Purification and biochemical characterization of three major acidic proteins (BSP-A1, BSP-A2 and BSP-A3) from bovine seminal plasma. Biochem J 1987;241:685-92]) and goat GSP-14/15 kDa [Villemure M, Lazure C, Manjunath P. Isolation and characterization of gelatine-binding proteins from goat seminal plasma. Reprod Biol Endocrinol 2003;1:39], based on our previous results on the P14 amino acid sequence [Barrios B, Fernández-Juan M, Muiño-Blanco T, Cebrián-Pérez JA. Immunocytochemical localization and biochemical characterization of two seminal plasma proteins which protect ram spermatozoa against cold-shock. J Androl 2005;26:539-49].