Fertilization is the process by which male and female haploid gametes (sperm and egg) unite to produce a genetically distinct individual. In mammals, fertilization involves a number of sequential steps, including sperm migration through the female genital tract, sperm penetration through the cumulus mass, sperm adhesion and binding to the zona pellucida, acrosome exocytosis, sperm penetration through the zona and fusion of the sperm and egg plasma membranes. However, freshly ejaculated sperm are not capable of fertilizing an oocyte. They must first undergo a series of biochemical and physiological changes, collectively known as capacitation, before acquiring fertilizing capabilities. Several molecules are required for successful capacitation and in vitro fertilization; these include bicarbonate, serum albumin (normally bovine serum albumin, BSA) and Ca(2+). Bicarbonate activates the sperm protein soluble adenylyl cyclase (SACY), which results in increased levels of cAMP and cAMP-dependent protein kinase (PKA) activation. The response to bicarbonate is fast and cAMP levels increase within 60 s followed by an increase in PKA activity. Several studies with an anti-phospho-PKA substrate antibody have demonstrated a rapid increase in protein phosphorylation in human, mouse and boar sperm. The target proteins of PKA are not known and the precise role of BSA during capacitation is unclear. Most of the studies provide support for the idea that BSA acts by removing cholesterol from the sperm. The loss of cholesterol has been suggested to affect the bilayer of the sperm plasma membrane making it more fusogenic. The relationship between cholesterol loss and the activation of the cAMP/PKA pathway is also unclear. During early stages of capacitation, Ca(2+) might be involved in the stimulation of SACY, although definitive proof is lacking. Protein tyrosine phosphorylation is another landmark of capacitation but occurs during the late stages of capacitation on a different time-scale from cAMP/PKA activation. Additionally, the tyrosine kinases present in sperm are not well characterized. Although protein phosphorylation depends upon the balanced action of protein kinases and protein phosphatase, we have even less information regarding the role of protein phosphatases during sperm capacitation. Over the last few years, several reports have pointed out that the ubiquitin-proteasome system might play a role during sperm capacitation, acrosome reaction and/or sperm-egg fusion. In the present review, we summarize the information regarding the role of protein kinases, phosphatases and the proteasome during sperm capacitation. Where appropriate, we give examples of the way that these molecules interact and regulate each other's activities.

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.

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.

In human spermatozoa, protein kinases have a role in the acrosome reaction (AR) induced by a variety of stimuli. However, there is disagreement or a lack of information regarding the role of protein kinases and phosphatases in the progesterone (P)-induced increase in intracellular calcium concentration ([Ca ] ). In addition, there are no studies regarding the role of Ser/Thr and Tyr phosphatases and there are contradictory results regarding the role of Tyr kinases in the P-induced acrosome reaction. Here, we performed a simultaneous evaluation of the involvement of protein kinases and phosphatases in the P-induced acrosome reaction and in the P-induced calcium influx. Motile spermatozoa were capacitated for 18 h and different aliquots were allocated to treated or control groups and then evaluated for their ability to undergo the acrosome reaction and to increase [Ca ] in response to P. The acrosome reaction was evaluated using Pisum sativum agglutinin (PSA)-FITC, and [Ca ] was evaluated using fura 2AM. At all of the concentrations tested, PKA inhibitors significantly reduced the percentage of the P-induced acrosome reaction (p < 0.001). However, only the highest concentrations of PKA inhibitors reduced the P-induced calcium influx; lower concentrations of PKA inhibitors did not affect it. Similar results were apparent for PKC inhibitors and for tyrosine kinase inhibitors. None of the Ser/Thr phosphatase inhibitors affected the P-induced acrosome reaction or the P-induced calcium influx, except for the PP2B inhibitors that significantly reduced the P-induced acrosome reaction without affecting calcium influx. Finally, the protein tyrosine phosphatase inhibitors significantly blocked the P-induced acrosome reaction and reduced the amplitude of the P-induced calcium transient (p < 0.001) as well as the amplitude of the plateau phase (p < 0.01). The data suggest that protein kinases and possibly PP2B have a role on the acrosome reaction at some point downstream of calcium entry and that Tyr phosphatases have a role on the acrosome reaction upstream of calcium entry.

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