The mode of reproductive‐derived Spink (serine protease inhibitor Kazal‐type) action in the modulation of mammalian sperm activity

Abstract

Summary The reproductive‐derived serine protease inhibitor Kazal‐type (Spink) has been identified in seminal plasma, and Spink–spermatozoa binding has been illustrated in many mammalian species including human. We used mice as experimental animal to study the mode of Spink action in the modulation of mammalian sperm activity. A Spink3‐binding zone was cytochemically stained on the sperm head at apical hook separated from intact acrosome, whether the cells were capacitated or not. The Spink3–spermatozoa binding neither changed the population of cells in the uncapacitated, capacitated and acrosome‐reacted status nor affected the capacitation‐related protein phosphorylation and cell motility enhancement. Despite that, the Spink–spermatozoa interaction resulted in decreasing the intracellular calcium concentration ([Ca2+]i) of the cell head and suppressing both the acrosome reaction induced by Ca+2 ionophore A23187 and the cell fertility. Furthermore, Spink3 seen on the head of spermatozoa in the uterine cavity after coitus could be removed by the trypsin‐like activity in the uterine fluid of oestrous females, and free Spink3 in the uterine cavity suppressed the protease activity. We integrated our data to shed light on the molecular mechanism of how Spink and its inhibiting protease are interplayed to modulate the activity of mammalian spermatozoa during their transit in the reproductive tract.

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

“…In agreement with previous results of purified caltrin I from rat (Dematteis et al 2008) and recombinant SPINK3 (Ou et al 2012), we observed an inhibition of both spontaneous and P 4 -induced AR mediated by GST-SPINK3, while viability remained invariable. However, the physiological significance of the effect over induced AR is uncertain.…”

“…Accordingly, in this work we showed that a modified recombinant SPINK3 protein that lacks trypsin inhibitory activity (GST-SPINK3) can bind sperm, reduce intracellular Ca 2C concentration, and modulate AR and protein phosphorylation, while it has no effect on sperm motility. All these data taken together show that the serine protease inhibitory activity of SPINK3 might not be necessary in sperm signaling which is consistent with the results obtained by Ou et al (2012) with the recombinant SPINK3 carrying the point mutation R19L.…”

“…The reduction of Ca 2C was mainly observed at the sperm head ( Fig. 3B) as recently reported (Ou et al 2012).…”

“…Capacitation is a Ca 2C -dependent process and is known to be prevented by intercellular and extracellular chelators (Leclerc et al 1998), so one is prompt to hypothesize that SPINK3 should inhibit capacitation. However, previous work with the native rat protein or the recombinant protein showed no effect on protein phosphorylation (Dematteis et al 2008, Ou et al 2012. In this work, the change in sperm to capacitating medium triggered the typical increase of tyrosine phosphorylation of sperm proteins of 105 and 81 kDa observed when the assay is performed without the phosphatase inhibitor orthovanadate (Leclerc et al 1996, Herrero et al 1999, Kulanand & Shivaji 2001, coincidently with a detectable endogenous NO production (see Figs 4 and 6).…”

“…By this method we wanted to know whether GST-SPINK3 affects the signaling pathways activated by incubation of sperm in a capacitating media. It has been reported that rat caltrin I (Dematteis et al 2008) and recombinant SPINK3 (Ou et al 2012) had no effect on protein phosphorylation. Contrary to those reports in this work, even when capacitated sperm with GST-SPINK3 did not reestablish the pattern of tyrosine-phosphorylated proteins observed in untreated noncapacitating sperm, GST-SPINK3 reduced the intensity of the 82 and 105 kDa bands (Fig.…”