SPINK3 regulates sperm physiology in mouse through a downstream reduction of nitric oxide concentration.

ZALAZAR L; SAEZ LANCELLOTTI TE; CLEMENTI M ; LOMBARDO C; LAMATTINA L ; DE CASTRO R; FORNÉS MW; CESARI A

Mar del Plata

Encuentro; VI Encuentro Biólogos en red; 2011

Asociación de Jóvenes Investigadores en Formación (AJIF)

Many serine protease inhibitors were reported as decapacitated factors in male reproductive tract. SPINK3 is a small secretory mouse protein from seminal vesicle (SV) with a Kazal type serine protease inhibitor domain. It attaches to the sperm surface during epididymal transit and has an inhibitory effect against extracellular Ca +2 uptake maintaining sperm in a decapacitated state before entering the female duct. The relationship between its serine protease inhibitory activity and the way in which this protein modulates signaling cascades in sperm is still unknown. In this work, mature SPINK3 protein was obtained purify as a recombinant fusion protein (GST-SPINK3) in E. colicells which lost its serine protease inhibitory activity. To study whether calcium transport inhibitory activity of SPINK3 could affect sperm intracellular signaling by reducing nitric oxide (NO) concentration, different physiological events relevant for fertilization (acrosome reaction, tyrosine phosphorylated proteins pattern and sperm motility) were evaluated. NO production by sperm was studied. Results show that GST-SPINK3 significantly reduced both the percentage of sperm positively stained for NO detected with the fluorescent probe DAF-FM DA and the NO concentration in capacitated mouse sperm quantifiedby Griess method. When sperm physiological events were analyzed, GST-SPINK3 reduced significantly spontaneous and progesterone-induced acrosomal reaction, and sperm progressive motility. However, these decreases were overcome by the exogenous addition of the NO donor sodium nitroprusiate (SNP). Phosphorylation of sperm proteins in tyrosine residues was partially affected by GST-SPINK3, nevertheless SNP was unable to reverse this effect. This is the first report that demonstrates that SPINK3, independently of its anti-trypsin activity, modulates sperm physiology through a downstream reduction of endogenous NO concentration.