Development of the signalling pathways associated with sperm capacitation during epididymal maturation

Abstract

As spermatozoa mature within the epididymis they acquire the potential for capacitation and ultimately fertilization. In biochemical terms, the former is reflected in the progressive activation of a signal transduction pathway characterized by cAMP-mediated induction of phosphotyrosine expression on the sperm tail. In this study, we have examined the cellular mechanisms controlling this maturational event. Caput epididymal spermatozoa exhibited tyrosine phosphorylation on the sperm head that was largely unresponsive to cAMP and not significantly impaired by removal of extracellular HCO(3) (-). In contrast, caudal epididymal spermatozoa exhibited low levels of phosphorylation on the sperm head, yet responded dramatically to cAMP by phosphorylating a new set of proteins on the sperm tail via mechanisms that were highly dependent on extracellular HCO(3) (-). The impact of extracellular HCO(3) (-) depletion on caudal cells was not associated with a significant change in the redox regulation of cAMP but could be fully reversed by buffering the intracellular pH with N-Tris[Hydroxymethyl]methyl-3-amino-propanesulfonic acid (TAPS). The pattern of tyrosine phosphorylation was also profoundly influenced by the presence or absence of added extracellular calcium. In the presence of this cation, only caudal spermatozoa could respond to increased extracellular cAMP with tyrosine phosphorylation of the sperm tail. However, in calcium-depleted medium, this difference completely disappeared. Under these conditions, caput and caudal spermatozoa were equally competent to exhibit phosphotyrosine expression on the sperm tail in response to cAMP. These results emphasize the pivotal role played by calcium and HCO(3) (-) in modulating the changes in tyrosine phosphorylation observed during epididymal maturation.

Bicarbonate Is Required for Migration of Sperm Epididymal Protein DE (CRISP-1) to the Equatorial Segment and Expression of Rat Sperm Fusion Ability1

“…This result differs Ϫ HCO 3 from that of Baker et al [20], who found that, in the absence of , protein tyrosine phosphorylation in the rat Ϫ HCO 3 did not return to control levels even after exposure of spermatozoa to a maximal dbcAMP-plus-PTX stimulus. Thus, although these authors proposed that the factor responsible for this failure is low intracellular pH, our results support the notion that, in the rat, like in the mouse, the effect of on protein tyrosine phosphorylation likely results Ϫ HCO 3 from the ability of the anion to regulate the sperm adenylyl cyclase [17].…”

“…Adenylyl cyclase, the enzyme responsible for cAMP synthesis, is markedly stimulated by physiologically relevant Ϫ HCO 3 concentrations [11][12][13], and it likely is soluble testicular bicarbonate-dependent adenylyl cyclase [14][15][16]. In several species, the -dependent increase in cAMP that occurs Ϫ HCO 3 during capacitation is implicated in the regulation of a phosphorylation cascade leading to the increase in protein tyrosine phosphorylation [17][18][19][20].…”

Expression of two-pore domain potassium channels in nonhuman primate sperm

“…Although the full biologic and clinical significance of the differential distribution of ion channels in mammalian sperm remains to be elucidated fully, the importance of potassium and calcium-mediated signaling to sperm physiology remains undisputed (1,(32)(33)(34). Recent experimental evidence has linked murine sperm membrane hyperpolarization mediated by voltage-gated potassium channels to the acquisition of calcium signaling required for normal sperm function (4,35,36).…”

Identification of Cytochrome-b5 Reductase as the Enzyme Responsible for NADH-Dependent Lucigenin Chemiluminescence in Human Spermatozoa1

“…All Western blot analyses were essentially performed as previously described [6]. Anti-GFP antibody was used at 1/1000 dilution, and the goat anti-rabbit FITC conjugated secondary antibody was used at 1/2000 dilution.…”

“…Within the reproductive field, ROS have been implicated in male infertility and the redox regulation of tyrosine phosphorylation during sperm capacitation in a variety of species, including humans [4][5][6][7].…”