Screening for inhibitors in seminal vesicle which regulate sperm serine proteases

CLEMENTI M; BOARELLI PV; FORNÉS MW; CESARI A

Mendoza

Congreso; XXVII Reunión Científica Anual de la Sociedad de Biología de Cuyo.; 2009

Sociedad de Biología de Cuyo

The regulation of serin protease activity in spermatozoa was attributed to inhibitors found in seminal plasma (SP). SP is mainly composed by secretions of the seminal vesicles (SV). The aim of this work, was to find and identify serine protease inhibitors in murine SV. We obtained protein extracts from SV of CF-1 mice. Small molecules were selected by filtration membranes (30-6kDa). Total activity in this fraction was quantified by the ability to reduce trypsin and endogenous protease activity from capacitated sperm, showing both 26% and 35% inhibitory activity, respectively. Proteins with inhibitory activity were detected by reverse zymography. By means of MS, SV-IV precursor was identified. Although the inhibitory activity of this protein has not been demonstrated, it evolved from a cluster of genes containing the WFDC motif, present in some serine proteases inhibitors. An enriched fraction was obtained by affinity chromatography of trypsin agarose. MS of the retained fraction identified SPINK3. In conclusion, a low Mw protein fraction from SV containing putative serine protease inhibitors is able to inhibit both trypsin and endogenous sperm proteases. However, we look forward to elucidate which proteases would be their substrates.