Abstract
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.
Highlights
Fertilization is the process by which two haploid gametes, the sperm and the egg, unite to produce a genetically distinct individual
One antibody recognizes the a and b isoforms of the PP2A catalytic subunit (PP2Ac, 38 kDa); a second antibody recognizes isoforms a and b of regulatory subunit A or PR65 (PP2A-A, 62 kDa); and the third antibody recognizes regulatory subunit B, the PR55 subunit belonging to the family B (PP2A-B, 52 kDa)
The experimental evidence that supports this assumption is as follows: 1) three members of the Ser/Thr phosphatase families were detected in human sperm, including PP2A; 2) when any of these three PPs were inhibited, the percentage of sperm depicting B pattern increased rapidly and significantly (#1 min of inhibitor addition); 3) all of the inhibitors used blocked phosphatase enzymatic activity; 4) the enzymatic activity of these three phosphatases decreased during capacitation; 5) phosphorylation of proteins on Thr residues increased during capacitation; and 6) all of the inhibitors used increased Thr protein phosphorylation in a very fast and dramatic manner
Summary
Fertilization is the process by which two haploid gametes, the sperm and the egg, unite to produce a genetically distinct individual. Capacitation has been divided into the following processes: a) fast and early events that comprise activation of the vigorous and asymmetric movement of the flagellum, which occurs as soon as the sperm leave the epididymis; cholesterol loss from the plasma membrane [4]; increased membrane fluidity; changes in intracellular ion concentration [5]; and hyperpolarization of the plasma membrane [6]; and b) slow and late events that comprise changes in the pattern of movement (hyperactivation), ability to carry out the acrosome reaction stimulated by a physiological agonist, and phosphorylation of proteins at Tyr [4,5] Both fast and slow events are centrally regulated by the activation of the cAMP/PKA (protein kinase A) pathway
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