SPINK3 modulates mouse sperm physiology through the reduction of nitric oxide level independently of its trypsin inhibitory activity.
Serine protease inhibitor Kazal-type (SPINK3)/P12/PSTI-II is a small secretory protein from mouse seminal vesicle which contains a KAZAL domain and shows calcium (Ca(2+))-transport inhibitory (caltrin) activity. This molecule was obtained as a recombinant protein and its effect on capacitated sperm cells was examined. SPINK3 inhibited trypsin activity in vitro while the fusion protein GST-SPINK3 had no effect on this enzyme activity. The inactive GST-SPINK3 significantly reduced the percentage of spermatozoa positively stained for nitric oxide (NO) with the specific probe DAF-FM DA and NO concentration measured by Griess method in capacitated mouse sperm; the same effect was observed when sperm were capacitated under low Ca(2+) concentration, using either intracellular (BAPTA-AM) or extracellular Ca(2+) (EDTA) chelators. The percentage of sperm showing spontaneous and progesterone-induced acrosomal reaction was significantly lower in the presence of GST-SPINK3 compared to untreated capacitated spermatozoa. Interestingly, this decrease was overcome by the exogenous addition of the NO donors, sodium nitroprusside (SNP), and S-nitrosoglutathione (GSNO). Phosphorylation of sperm proteins in tyrosine residues was partially affected by GST-SPINK3, however, only GSNO was able to reverse this effect. Sperm progressive motility was not significantly diminished by GST-SPINK3 or BAPTA-AM but enhanced by the addition of SNP. This is the first report that demonstrates that SPINK3 modulates sperm physiology through a downstream reduction of endogenous NO concentration and independently of SPINK3 trypsin inhibitory activity.
Instituto de Investigaciones Biológicas, Universidad Nacional de Mar del Plata, CCT-Mar del Plata, CONICET, Funes 3250, Mar del Plata 7600, Argentina.
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Zalazar
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Saez Lancellotti
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Clementi
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C
Lombardo
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L
Lamattina
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De Castro
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MW
Fornés
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Cesari
D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D015536:Down-Regulation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D006023:Glycoproteins / Q000235:genetics / Q000494:pharmacology / Q000502:physiology*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008954:Models, Biological; D009569:Nitric Oxide / Q000032:analysis / Q000378:metabolism*; D009994:Osmolar Concentration; D029584:Prostatic Secretory Proteins / Q000235:genetics / Q000494:pharmacology / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000494:pharmacology; D055101:Semen Analysis; D013075:Sperm Capacitation / Q000187:drug effects / Q000502:physiology; D013094:Spermatozoa / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D014357:Trypsin / Q000378:metabolism / Q000494:pharmacology; D014359:Trypsin Inhibitor, Kazal Pancreatic
D004791:Enzyme Inhibitors; D006023:Glycoproteins; D029584:Prostatic Secretory Proteins; D011993:Recombinant Fusion Proteins; C487134:Spink1 protein, mouse; D009569:Nitric Oxide; D014359:Trypsin Inhibitor, Kazal Pancreatic; D014357:Trypsin