A proteomic approach for the identification of serine protease inhibitors in seminal plasma and male sex accessory glands in mammals

ZALAZAR, L; CLEMENTI M; FORNÉS MW; DE CASTRO, R.; CESARI A

Congreso; XLVI Reunion Anual SAIB; 2010

The presence of serine-protease inhibitors in seminal plasma (SP) and accessory sexual glands has been reported in several mammals. They play a role in balancing protease activities and are believed to be important in regulating the fertilization process, acting as decapacitating factors. An important application to improve the membrane alterations in cryopreserved sperm (resembling the capacitation process) might be the addition of these inhibitors to the sperm thawing medium. The aim of the present study was to investigate the presence of serine protease inhibitors in seminal vesicle (SV) and SP through proteomic analysis and to obtain recombinant inhibitors. Bidimensional electrophoresis was performed from mouse SV and bovine SP low-Mw protein extracts. A serine protease inhibitor, named SPINK3 was identified. This protein has a known inhibitory effect against Ca+2 uptake in mouse sperm. The corresponding cDNA was obtained by reverse transcription of mouse SV RNA. The cDNA encoding the mature protein was cloned into PGEX-4T-3 vector, expressed and the protein was purified. The biological activity of the fusion protein was confirmed by evaluating the acrosomal reaction and capacitation. This is the first approach in obtaining this inhibitor with proteomic tools. Further studies are needed to analyze the ability of SPINK3 to protect against sperm cryoconservation damage.