SPINK3 modulates mouse sperm physiology through the reduction of nitric oxide level independently of its trypsin inhibitory activity

ZALAZAR, L.; SAEZ LANCELLOTTI, T.E.; CLEMENTI, M.; LOMBARDO, M.C.; LAMATTINA, L.; DE CASTRO, R.E.; FORNÉS, M.; CESARI, A.

REPRODUCTION

BIOSCIENTIFICA LTD

Año: 2011

1470-1626

A small secretory protein from mouse seminal vesicle named as SPINK3/P12/PSTI-II which contains a Kazal type serine protease inhibitor domain and has calcium-transport-inhibitory-activity (caltrin I) was obtained as a recombinant protein. SPINK3 inhibited trypsin proteolytic activity in vitro while GST-SPINK3 lost this activity. Calcium and nitric oxide (NO) play central roles in the regulation of many key activities of mature sperm (capacitation, motility and acrosome reaction). NO production was observed in capacitated mouse sperm using a fluorescent probe (DAF-FM), and both SPINK3 and SPINK3-GST significantly reduced the percentage of sperm positively stained for NO. This effect was also observed when sperm were treated with two calcium chelators (EDTA and BAPTA-AM) during capacitation. The inactive protease inhibitor SPINK3-GST significantly reduced the percentage of sperm showing acrosomal reaction during capacitation and this decrease was overcome by the exogenous addition of the NO donor sodium nitroprusiate (SNP). Capacitation, evaluated as phosphorylation of sperm proteins in tyrosine residues, was partially affected by the inactive protease inhibitor SPINK3-GST, however SNP was not able to reverse this effect. Our results demonstrate that incubation of capacitated mouse sperm cells with recombinant SPINK3 modulates sperm physiology through a downstream reduction of endogenous NO concentration. The effect of SPINK3 can be reversed by the exogenous addition of NO and is independent of SPINK3 anti-trypsin activity.