Protein Phosphatases Decrease Their Activity during Capacitation: A New Requirement for This Event

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.

Distinct segment‐specific functions of calyculin A‐sensitive protein phosphatases in the regulation of cAMP‐triggered events in ejaculated bull spermatozoa

“…Serine/threonine protein phosphatases are involved in the regulation of various sperm functions, including progressive motility (Goto and Harayama, ; Krapf et al, ), capacitation (Visconti et al, ; Signorelli et al, ), and hyperactivation (Suzuki et al, ). In this study, calyculin A‐sensitive protein phosphatases suppressed serine/threonine phosphorylation of AGC‐kinase substrates in the connecting piece, leading to protein tyrosine phosphorylation and full‐type hyperactivation in ejaculated bull spermatozoa.…”

Detection, Localization and Tyrosine Phosphorylation Status of Ser/Thr Protein Phosphatase1γ in Freshly Ejaculated, In Vitro Capacitated and Cryopreserved Buffalo Spermatozoa

“…; Signorelli et al. ). Previous reports have shown that the high protein phosphatase activity is correlated with low sperm motility, whereas low catalytic activity is associated with vigorous motility in bovine and monkey spermatozoa (Smith et al.…”

“…; Signorelli et al. ). It has been reported that Ser/Thr phosphatase inhibitors induce capacitation signalling (Goto and Harayama ; Krapf et al.…”

“…Among them, PP1, PP2A and PP2B were identified in mammalian spermatozoa (Signorelli et al. ). In addition, PP1 was expressed in three different isoforms encoded by different genes like PP1α, PP1β and PP1γ (Cohen ).…”

Signalling Events and Associated Pathways Related to the Mammalian Sperm Capacitation

“…Protein Phosphatase activity is decreased during capacitation; this has significance because they may dephosphorylate and thus deactivate proteins required for capacitation (Signorelli et al. ). Multidrug resistance‐associated protein 4 (MRP4) is localized in the mid‐piece of bovine spermatozoa (Osycka–Salut et al.…”