How, where and when is SPINK3 bound and removed from mouse sperm?

Sperm capacitation in mammals is a fundamental requirement to acquire their fertilizing capacity. Little is known about the action mechanism of the molecules that prevent capacitation from occurring prematurely. These molecules are known as decapacitation factors (DFs) and they must be removed from the sperm surface for capacitation to occur successfully. Serine protease inhibitor Kazal type 3 (SPINK3) has been proposed as one of these DFs. Here, we evaluate how this protein binds to mouse sperm and its removal kinetics. We describe that SPINK3 is capable of binding to the membrane of mature epididymal sperm through protein-lipid interactions, specifically to lipid rafts subcellular fraction. Moreover, cholera toxin subunit b (CTB) avoids SPINK3 binding. We observe that SPINK3 is removed from the sperm under in vitro capacitating conditions and by the uterine fluid from estrus females. Our ex vivo studies show the removal kinetics of this protein within the female tract, losing SPINK3 formerly from the apical region of the sperm in the uterus and later from the flagellar region within the oviduct. The presence of acrosome-reacted sperm in the female duct concurs with the absence of SPINK3 over its surface.

Recombinant TrxAFNIIx4His6 improves post-thaw motility of ram sperm measured by a sperm motility tracker software.

The aim of the present study was to evaluate a freezing extender supplemented with recombinant TrxAFNIIx4His6, a reported decapacitating factor. Semen samples were diluted in tris-egg yolk medium with 0, 1.5 µM and 3.0 µM of TrxAFNIIx4His6. Computer-assisted sperm motility tracking and subpopulations evaluation showed that addition of TrxAFNIIx4His6 improved post-thaw total and progressive motility at both concentrations evaluated. TrxAFNIIx4His6 increased the sperm subpopulation with the highest progressiveness and great velocity and decreased the subpopulation of poorly motile and almost non-progressive sperm. Incorporation of TrxAFNIIx4His6 to freezing extender shows potential for the development of cryoprotection media which may lead to improved fertility after artificial insemination.

Male Decapacitation Factor SPINK3 Blocks Membrane Hyperpolarization and Calcium Entry in Mouse Sperm.

Mammalian sperm acquire ability to fertilize through a process called capacitation, occurring after ejaculation and regulated by both female molecules and male decapacitation factors. Bicarbonate and calcium present in the female reproductive tract trigger capacitation in sperm, leading to acrosomal responsiveness and hyperactivated motility. Male decapacitating factors present in the semen avert premature capacitation, until detached from the sperm surface. However, their mechanism of action remains elusive. Here we describe for the first time the molecular basis for the decapacitating action of the seminal protein SPINK3 in mouse sperm. When present in the capacitating medium, SPINK3 inhibited Src kinase, a modulator of the potassium channel responsible for plasma membrane hyperpolarization. Lack of hyperpolarization affected calcium channels activity, impairing the acquisition of acrosomal responsiveness and blocking hyperactivation. Interestingly, SPINK3 acted only on non-capacitated sperm, as it did not bind to capacitated cells. Binding selectivity allows its decapacitating action only in non-capacitated sperm, without affecting capacitated cells.

Recombinant SPINK3 improves ram sperm quality and in vitro fertility after cryopreservation.

Capacitation-like changes affect sperm of several species, such as ram, reducing cell survival and fertilizing competence. Proteins from seminal plasma stabilize sperm plasma membranes, being an interesting focus to develop strategies for improving cryopreserved ram semen performance. To date, biotechnologies are focused to reduce damage in frozen-thawed ram spermatozoa through the addition of bioactives. Serine Protease Inhibitor Kazal-type 3 (SPINK3) is a little protein synthesized by mouse seminal vesicle and secreted to seminal plasma. While attached to the sperm, this protein binds to non-capacitated sperm and blocks calcium entry, avoiding a premature physiological capacitation and consequently, acrosome reaction. Due to these characteristics, SPINK3 has been proposed as a decapacitating factor. The aim of this work was to assess whether heterologous SPINK3 is able to protect ram sperm from the well-known cell damages produced by freezing/thawing and to understand the mechanisms by which it is acting. Sperm were supplemented with 13 µM SPINK3 before freezing in an egg yolk-based extender or after thawing and selection. Under both conditions, SPINK3 decreased intracellular calcium content (p < 0.05) and reduced the 25 kDa tyrosine phosphorylated protein demonstrating a decapacitating effect, although the addition of the protein before cryopreservation was not enough to improve other sperm parameters. However, the addition of SPINK3 post thawing was able to significantly ameliorate viability, motility, mitochondrial status and to avoid the increase of lipid peroxidation (p < 0.05). Moreover, sperm treated with SPINK3 was not only still capable to fertilize, but also improved it, as evidenced by an increase in the oocyte cleavage rate (p < 0.05) although, the embryo development and embryo quality were not affected. Our findings would contribute to develop a strategy for improving sperm quality by using decapacitating proteins. In fact, the outcomes of this work demonstrate that SPINK3 is able to reduce sperm cryo-injuries when is added after thawing, improving functionality and thus in vitro fertilization results.

Recombinant peptide reverses cryo-capacitation in ram sperm and improves in vitro fertilization.

Semen cryopreservation is a very important technique for assisted reproduction; however, the cryopreservation process is harmful because it results in a reduction in sperm motility and viability, and leads to premature signals of capacitation, resulting in lesser than desirable fertility rates after artificial insemination. A fraction of seminal plasma, enriched in proteins that contain type II fibronectin domains (FNII) can reverse molecular indicators of cryo-capacitation. The beneficial effects of these proteins, however, depend on the relative abundance in seminal plasma. To create a safe additive for improving frozen sperm functionality, in the present study there was cloning and expression of a recombinant peptide containing four FNII domains (named TrxA-FNIIx4-His6) and evaluation of its effect after addition to frozen/thawed ram sperm. The cDNA for this protein was expressed in E. coli and after denaturation and re-naturalization of the protein, toxicity and binding capacity were assessed. By fluorescent labelling assessment, there was binding of the protein to the thawed sperm. At the two doses used (0.15 and 0.3 µM), TrxA-FNIIx4-His6 had the capacity to reverse the molecular indicators of cryo-capacitation as indicated by the reduction on phosphorylated substrates of PKA. Furthermore, the supplementation with this protein resulted in a normal capacitation process as evidenced by the increase in the in vitro fertilization rate when the greatest concentration of the protein was evaluated (73.25 ±â€¯2.95; 40.13 ±â€¯11.82 for 0.3 µM and control, respectively). There was no effect of protein supplementation on sperm objective motility compared to untreated sperm. In conclusion, the use of TrxA-FNIIx4-His6 is a promising biotechnological approach for cryopreserving ram sperm and maintaining sperm viability.

Seminal plasma proteins modify the distribution of sperm subpopulations in cryopreserved semen of rams with lesser fertility.

Any physiological mechanism involved in sperm selection and semen improvement has effects on heterogeneous sperm populations. This is mainly due to the fact that sperm populations within a single ejaculate have considerable heterogeneity for many variables, such as motility which is meaningful in terms of understanding how some sperm cells possess fertility advantages as compared with other cells. In the present research, initially there was a multivariate and clustering analysis used to assess sperm motility data from cryopreserved ram semen to identify subpopulations and compare the distribution of these clusters between rams with lesser and greater fertility. There were four classifications made of sperm subpopulations (clusters): CL1 fast/linear/progressive sperm; CL2 fast/non-linear sperm; CL3 very fast/linear sperm with vigorous beating and CL4 slow/non-linear sperm. Rams with greater fertility had a lesser proportion of sperm considered as "hyperactivated" (CL2) and a greater proportion of slow and non-linear sperm (CL4) than sperm of rams with lesser fertility. In addition, the effects were assessed for the capacity of seminal plasma (SP) and interacting SP proteins (iSPP) that were present during different seasons of the year to improve the distribution of sperm within subpopulations of semen from rams with lesser fertility. The iSPP and SP were obtained by artificial vagina (AV) and electroejaculation (EE) during breeding and non-breeding seasons and added to thawed semen. All the aggregates had a significant effect on the distribution of sperm subpopulations and effects differed among seasons of the year and depending on collection method used. Even though, future studies are needed to assess the contribution of each subpopulation on ram sperm fertility, it is important that a multivariate analysis be used to evaluate the effect of a treatment on sperm quality variables.

Novel inhibitory activity for serine protease inhibitor Kazal type-3 (Spink3) on human recombinant kallikreins.

Kallikrein-related peptidases (KLKs) are trypsin-like and chymotrypsin-like serine proteases which are expressed in several tissues. Their activity is tightly controlled by inhibitors including members of the serine protease Kazal-type (SPINK) family. These enzymes are promising targets for the treatment of skin desquamation, inflammation and cancer. Spink3 or caltrin I is expressed in mouse pancreas and males accessory glands and the resulting mature protein has been associated with different activities such as an inhibitor of trypsin and acrosin activity, calcium transport inhibitor in sperm and inhibitor of cell proliferation during embryogenesis. In this study, we produced a soluble recombinant Spink3 from mouse seminal vesicle (rmSpink3) that inhibited the activity of human KLKs. Using FRET substrates, rmSpink3 exhibited a potent inhibitory activity against human KLK2, KLK3, KLK5 (Ki ranging from 260 to 1500 nM), and to a lesser extent against KLK6, KLK1 and KLK7 (Ki around 3000 nM). As shown by mass spectrometry analysis of rmSpink3 incubated with trypsin, the inhibitor was not truncated by the target enzyme. Based on the in silico analysis of the expression of Spink3/SPINK1 and KLKs it is speculated that some KLKs may be natural targets of Spink3/SPINK1, however experimental confirmation using both proteins from mouse or human origin is needed. This work shows that rmSpink3 is a potent inhibitor of various human KLK members suggesting the potential of this molecule in the diagnosis/prevention of several human diseases.