Rat Caltrin Protein Modulates the Acrosomal Exocytosis During Sperm Capacitation1

Abstract

Caltrin is a small and basic protein of the seminal vesicle secretion that inhibits sperm calcium uptake. The influence of rat caltrin on sperm physiological processes related to fertilizing competence was studied by examining its effect on 1) spontaneous acrosomal exocytosis, 2) protein tyrosine phosphorylation, and 3) sperm-egg interaction. Results show that the presence of caltrin during in vitro capacitation both reduced the rate of spontaneous acrosomal exocytosis without altering the pattern of protein tyrosine phosphorylation, and enhanced the sperm ability to bind to the zona pellucida (ZP). The significantly higher proportion of sperm with intact acrosome observed in the presence of caltrin was accompanied by a strong inhibition in the acrosomal hyaluronidase release. Enhancement of sperm-ZP binding was evident by the increase in the percentage of eggs with bound spermatozoa as well as in the number of bound sperm per egg. Similar results were obtained when the assays were performed using spermatozoa preincubated with caltrin and then washed to remove the unbound protein, indicating that the sperm-bound caltrin was the one involved in both acrosomal exocytosis inhibition and sperm-ZP binding enhancement. Caltrin bound to the sperm head was partially released during the acrosomal exocytosis induced by Ca-ionophore A23187. Indirect immunofluorescence and immunoelectron microscopy studies revealed that caltrin molecules distributed on the dorsal sperm surface disappeared after ionophore exposure, whereas those on the ventral region remained in this localization after the treatment. The present data suggest that rat caltrin molecules bound to the sperm head during ejaculation prevent the occurrence of the spontaneous acrosomal exocytosis along the female reproductive tract. Consequently, more competent spermatozoa with intact and functional acrosome would be available in the oviduct to participate in fertilization.

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

“…5A), indicating that the recombinant protein affects AR as described for purified caltrin I (Dematteis et al 2008). P 4 -induced AR was also significantly affected by GST-SPINK3 compared to untreated capacitated sperm (Fig.…”

“…It can be considered as a multifunctional protein since, in addition to its antitrypsin activity (Luo et al 2004), inhibits Ca 2C transport in sperm , Dematteis et al 2008. Because of this latter activity it has been named as caltrin protein.…”

“…It has been reported that NO donors cause an elevation of human sperm [Ca 2C ]i coming from Ca 2C internal stores by sperm protein S-nitrosylation (Machado-Oliveira et al 2008). However, SPINK3 cannot cross the sperm plasma membrane, it binds to the sperm surface during ejaculation and comes off during AR (Dematteis et al 2008), so one could speculate that the reduction of [Ca 2C ]i observed in sperm upon treatment with GST-SPINK3, and also reported for caltrin I (San Agustin et al 1987, Dematteis et al 2008, should act upstream of NO. This speculation is consistent with the reduction of NO observed by us and other authors in capacitated sperm treated with either GST-SPINK3 (Fig.…”

“…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.…”

“…A well-known Ca 2C -transport inhibitor I (caltrin; Chen et al 1998, Luo et al 2004, also known as P12 and pancreatic secretory trypsin inhibitor II, is a secretory serine protease inhibitor Kazal-type (SPINK3, NCBI ID: NP_033284.1) that is constitutively expressed in pancreas and its expression is androgen dependent in seminal vesicle and prostate of mice, rat and human (Mills et al 1987, Novella et al 1999. This protein, as other secretory proteins from the accessory glands, attaches to the sperm surface during sperm transit along the male duct and consequently, it is found on the surface of ejaculated sperm (Dematteis et al 2008). Caltrin-like proteins are known to modulate Ca 2C influx (Clark et al 1993, Coronel et al 1993.…”