Does fibronectin (Fn) stimulate the sperm capacitation process in humans? Fibronectin stimulates human sperm capacitation. Capacitation is a process that occurs in the oviduct. It has been suggested that some molecules present in the oviductal fluid and cells as well as proteins present in the cumulus oophorus could be involved in the modulation of sperm function and their acquisition of fertilizing capacity. Fibronectin is a glycoprotein that is present in the fluid and the oviduct epithelium, and its receptor (alpha 5 beta 1 integrin) is present in human sperm. When alpha 5 beta 1 (α₅β₁) integrin binds to fibronectin, intracellular signals similar to the process of sperm capacitation are activated. Human sperm were selected via a percoll gradient and were then incubated in non-capacitated medium (NCM) or reconstituted capacitated medium (RCM), in the presence or absence of fibronectin for different time periods. A total of 39 donors were used during the study, which lasted 3 years. Freshly ejaculated sperm from healthy volunteers were obtained by masturbation. All semen samples were normal according to the World Health Organization parameters. Six approaches were used to determine the effects of fibronectin on sperm capacitation: chlortetracycline (CTC) assay, heterologous co-culture of human sperm with bovine oviductal epithelial cells (BOEC), measurement of cyclic (c) AMP levels, activity of protein kinase A (PKA), phosphorylation of proteins in tyrosine (Tyr) residues, and induction of acrosome reaction with progesterone. When sperm were incubated in RCM in the presence of Fn, we observed differences with respect to sperm incubated in RCM without Fn (control): (i) a 10% increase in the percentage of sperm with the B pattern (capacitated sperm) of CTC fluorescence from the beginning of capacitation (P < 0.001); (ii) an effect on both the concentration of cAMP (P < 0.05) and PKA activity (P < 0.05) during early capacitation; (iii) an increase in the degree of phosphorylation of proteins on tyrosine residues after 60 min of capacitation (P < 0.01); (iv) an increase in the percentage of acrosome-reacted sperm in response to progesterone (P < 0.05); and (v) a decrease in the percentage of sperm attached to BOEC (P < 0.05). Moreover, we noted that the effect of Fn was specific and mediated by alpha 5 beta 1 integrin (P < 0.001). Fn by itself had no effect on sperm capacitation. This study was carried out with sperm from young adult men. Men with abnormal semen samples were excluded. The results cannot be directly extrapolated to other mammalian species. Currently, male subfertility has become a huge public health problem, which makes it imperative to develop new treatments. This is a novel discovery that extends our current knowledge concerning normal and pathological sperm physiology as well as events that regulate the process of fertilization. This study was supported by grants from FONDECYT (1130341, E.S.D. and 1120056, P.M.) and FONCYT (PIP 2011-0496, S.P.-M). The authors have no conflicts of interest.

There are few reports on the role of protein phosphatases during capacitation. Here, we report on the role of PP2B, PP1, and PP2A during human sperm capacitation. Motile sperm were resuspended in non-capacitating medium (NCM, Tyrode's medium, albumin- and bicarbonate-free) or in reconstituted medium (RCM, NCM plus 2.6% albumin/25 mM bicarbonate). The presence of the phosphatases was evaluated by western blotting and the subcellular localization by indirect immunofluorescence. The function of these phosphatases was analyzed by incubating the sperm with specific inhibitors: okadaic acid, I2, endothall, and deltamethrin. Different aliquots were incubated in the following media: 1) NCM; 2) NCM plus inhibitors; 3) RCM; and 4) RCM plus inhibitors. The percent capacitated sperm and phosphatase activities were evaluated using the chlortetracycline assay and a phosphatase assay kit, respectively. The results confirm the presence of PP2B and PP1 in human sperm. We also report the presence of PP2A, specifically, the catalytic subunit and the regulatory subunits PR65 and B. PP2B and PP2A were present in the tail, neck, and postacrosomal region, and PP1 was present in the postacrosomal region, neck, middle, and principal piece of human sperm. Treatment with phosphatase inhibitors rapidly (≤1 min) increased the percent of sperm depicting the pattern B, reaching a maximum of ∼40% that was maintained throughout incubation; after 3 h, the percent of capacitated sperm was similar to that of the control. The enzymatic activity of the phosphatases decreased during capacitation without changes in their expression. The pattern of phosphorylation on threonine residues showed a sharp increase upon treatment with the inhibitors. In conclusion, human sperm express PP1, PP2B, and PP2A, and the activity of these phosphatases decreases during capacitation. This decline in phosphatase activities and the subsequent increase in threonine phosphorylation may be an important requirement for the success of sperm capacitation.

Fibronectin (Fn) enhances human sperm capacitation via the cAMP/PKA pathway, and the endocannabinoid system participates in this process. Moreover, Fn has been linked to endocannabinoid system components in different cellular models, even though no evidence of such interactions in human sperm is available. Normal semen samples were evaluated over a 4‐year period. Our findings suggest that (a) the capacitating effects of Fn were reversed by preincubating the sperm with a cannabinoid receptor 1 (CB1) or transient receptor potential cation channel subfamily V member 1 (TRPV1) antagonist ( p < 0.001 and p < 0.05, respectively); (b) cooperation between CB1 and TRPV1 may exist ( p < 0.01); (c) the activity of specific fatty acid amide hydroxylase (FAAH) decreased after 1 min ( p < 0.01) and increased after 60 min ( p < 0.01) of capacitation in the presence of Fn; (d) the effects of Fn on FAAH activity were prevented by preincubating spermatozoa with a protein kinase A (PKA) inhibitor ( p < 0.01); (e) Fn modulated both the cyclic adenosine monophosphate concentration and PKA activity ( p < 0.05) during early capacitation; and (f) FAAH was a PKA substrate modulated by phosphorylation. These findings indicate that Fn stimulates human sperm capacitation via the cAMP/PKA pathway through modulation of the endocannabinoid system. Understanding the functional competence of human spermatozoa is essential for facilitating clinical advances in infertility treatment and for developing novel contraceptive strategies.