Results were obtained from five different donors and are expressed as the meanSEM of the percentage of the B pattern. the meanSEM of the percentage of the B pattern. * p 0.05; *** p 0.001.(TIF) pone.0081286.s003.tif (290K) GUID:?E93B1CE5-46E0-4283-862D-D1FD110CA99B Physique S4: Effects of endothall and dimethylendothall (DME) around the capacitation of human sperm. Sperm resuspended in CM were treated with different concentrations of endothall and with 90 nM DME. The percent of capacitated cells was evaluated at various occasions. Results were obtained from five different Rabbit polyclonal to GAL donors LY2922470 and are expressed as the meanSEM of the percentage of the B pattern. *** p 0.001.(TIF) pone.0081286.s004.tif (331K) GUID:?0C20CAC1-50F2-4472-979D-2FC993237F51 Abstract There are few reports around the role of protein phosphatases during capacitation. Here, we report around 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 comparable 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 LY2922470 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. Introduction Fertilization is the process by which two haploid gametes, the sperm and the egg, unite to produce a genetically distinct individual. In mammals, fertilization involves a number of sequential actions, including sperm migration through the female genital tract, sperm penetration through the cumulus mass, sperm adhesion and binding LY2922470 to the zona pellucida, acrosomal exocytosis, sperm penetration through the zona pellucida, and fusion of the gamete plasma membranes [1]. However, freshly ejaculated sperm are not capable of fertilizing an oocyte. First, they must undergo a cascade of biochemical and physiological changes that facilitate the binding and penetration of the sperm into the oocyte. This time-dependent acquisition of fertilization competence has been defined as capacitation [2], [3]. Capacitation normally occurs in the female genital tract; however, it can also be achieved by incubating the sperm in an appropriate culture medium. The study of capacitation has shown this process to be a combination of sequential and parallel events, which occur both in the sperm head (preparation for the acrosome reaction) and tail (hyperactivation). Recently, 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.