N OCIETY' - the Society for Reproductive Biology

Identification of proteins with seasonal patterns of secretion in ram seminal plasma.J. F. Smith, R. S-F. Lee, R. M. McDonald, G. R. Murray, J. Parr.AgResearch, Ruakura Research Centre, Private Bag 3123, Hamilton, New Zealand.INTRODUCTION: Ram semen collected and frozen inthe non-breeding season is of lower fertility than semenfrom the same animals in the autumn. These changes in thefertility of frozen semen cannot be readily explained bychanges in sperm numbers alone, although associatedalterations in maintenance of sperm motility after freezinghave been reported (1). Differences in field fertility afterinsemination with frozen bull semen have been linked toconcentrations of specific seminal plasma proteins (2).This observation raises the possibility that certain seminalplasma proteins could influence the ability of sperm toretain their fertility after cryopreservation; thus, seasonalchanges in fertility of ram semen could be related toalterations in the composition of seminal plasma. Thepresent experiment was conducted to examine the seasonalvariations in protein content and composition of ramseminal plasma.METHODS: Ten rams of proven fertility were used in thisstudy. On one day each month two or three ejaculates werecollected by artificial vagina from each ram. Semensamples were kept on ice after collection and evaluated forvolume and density, then processed. Proteolytic inhibitors(benzamine and PMSF) were added to. a finalconcentration of 1 roM each and the seminal plasmaseparated by centrifugation (2x 10 min) at 1l,600g. Equalvolumes of seminal plasma from each ejaculate collectedon the same day for an individual ram were pooled,aliquoted and stored at -20°C until assay. Protein contentof the seminal plasma was determined using a modifiedLowry method (3). Seminal plasma proteins were analysedby electrophoresis in 12.5 % denaturing acrylamide gelsafter 5-fold dilution in 10 rnM Tris-Cl, pH 8.0, containing1 mM EDTA. After electrophoresis and staining withCoomassie-blue, the protein banding patterns were visuallyappraised. Samples from individual rams for each of the 12months were compared within the same gel and the proteinloading was normalised across all the lanes to correct forvariation in protein concentration between the samples.Protein identification. Five proteins that showed seasonalvariation were selected for identification by peptidesequencing. Briefly, seminal plasma proteins (40 Ilg perlane) were electrophoresed in denaturing 12.5 %polyacrylamide gels (20 x 20 x 0.075 cm). After stainingwith Coomassie-blue and destaining, the bands of interestwere excised, and depending on the abundance of theprotein, either bands from 5 lanes or 10 lanes werecombined and the gel pieces washed extensively withwater, then 50 roM Tris-HCI, pH 8.5, containing 1 roMEDTA. The gel pieces were pulverized and suspended in0.4 mI of the same buffer and Endoproteinase Lys C(sequencing grade, Promega), added to a finalconcentration of 0.1 mg/mI. The mixture was incubated at37°C overnight with shaking and the supernatant was thenseparated from the gel slurry and volume reduced to < 100III by lyophillization. After acidication with trifluoroaceticacid to a final concentration of 1 % (v/v), the peptideswere resolved by reverse-phase HPLC. Individual peptidepeaks eluting from the column were collected and selected40peak samples were subjected to ~no acid sequencingusing the Applied Biosystems PrOCIse Sequencer (ABI).RESULTS: A seasonal change in the total proteinconcentration was detected in seminal plasma, with higherlevels (36.9 vs 23.0 mg/mI; P