Chloride Is Essential for Capacitation and for the Capacitation-associated Increase in Tyrosine Phosphorylation

Abstract

After epididymal maturation, sperm capacitation, which encompasses a complex series of molecular events, endows the sperm with the ability to fertilize an egg. This process can be mimicked in vitro in defined media, the composition of which is based on the electrolyte concentration of the oviductal fluid. It is well established that capacitation requires Na ؉ , HCO 3 ؊ , Ca 2؉ , and a cholesterol acceptor; however, little is known about the function of Cl ؊ during this important process. To determine whether Cl ؊ , in addition to maintaining osmolarity, actively participates in signaling pathways that regulate capacitation, Cl ؊ was replaced by either methanesulfonate or gluconate two nonpermeable anions. The absence of Cl ؊ did not affect sperm viability, but capacitation-associated processes such as the increase in tyrosine phosphorylation, the increase in cAMP levels, hyperactivation, the zona pellucidae-induced acrosome reaction, and most importantly, fertilization were abolished or significantly reduced. Interestingly, the addition of cyclic AMP agonists to sperm incubated in Cl ؊ -free medium rescued the increase in tyrosine phosphorylation and hyperactivation suggesting that Cl ؊ acts upstream of the cAMP/protein kinase A signaling pathway. To investigate Cl ؊ transport, sperm incubated in complete capacitation medium were exposed to a battery of anion transport inhibitors. Among them, bumetanide and furosemide, two blockers of Na؊ cotransporters (NKCC), inhibited all capacitation-associated events, suggesting that these transporters may mediate Cl ؊ movements in sperm. Consistent with these results, Western blots using anti-NKCC1 antibodies showed the presence of this cotransporter in mature sperm.Before becoming fertilization-competent, mammalian sperm must undergo a series of maturational processes in the female reproductive tract (1). The molecular, biochemical, and physiological changes that occur in sperm, whereas in the female tract are collectively referred to as capacitation. These functional changes associated with capacitation are not one event but are a combination of sequential and concomitant processes involving modifications at the molecular level occurring both in the head (i.e. preparation for the acrosome reaction) and the tail (i.e. motility changes such as hyperactivation). Molecular events implicated in the initiation of capacitation can be mimicked in vitro and have been partially defined. These include removal of cholesterol from the sperm plasma membrane; modifications in plasma membrane phospholipids; fluxes of HCO 3 Ϫ and other intracellular ions; increased protein tyrosine phosphorylation; and hyperpolarization of the sperm plasma membrane potential (E m ) in mouse and other species (for review see Ref. 2).With respect to the changes in the plasma membrane E m in mouse sperm, it is hypothesized that the capacitation-associated hyperpolarization results from changes in the activity of ion-selective channels and transporters. Consistent with this hypothesis, our studies in sperm f...

Hyperactivated Motility of Frozen-Thawed Spermatozoa from Fertile and Subfertile Japanese Black Bulls Induced by Cyclic Adenosine 3',5'-Monophosphate Analogue, cBiMPS

“…The cAMP/PKA pathway has been suggested to be involved in regulation of hyperactivation in mammalian spermatozoa (boar [4][5][6], human [24], mouse [25]), but in bull spermatozoa, cAMP does not generally induce hyperactivation [11,26]. There have so far been no reports describing either induction of hyperactivated motility in bull spermatozoa by treatment with cAMP analogue or the time course changes in the proportion of spermatozoa displaying a circular swimming pattern of hyperactivation.…”

Obesity, a serious etiologic factor for male subfertility in modern society

“…The membranal cholesterol efflux that removes off cholesterol from sperm membrane during sperm capacitation is essential for modifying the membranal fluidity and further contributes to sperm motility maintenance and normal acrosome reaction (Wertheimer et al 2008, Whitfield et al 2015. Both clinic and animal studies have revealed the significant rise in sperm cholesterol content in obese males.…”

Effect of calcium, bicarbonate, and albumin on capacitation-related events in equine sperm

“…These initial events are associated with a complex intracellular cascade modulated via the adenylyl cyclase-cAMP-protein kinase A (PKA) pathway, whose final hallmark is an overall increase in phosphorylation of tyrosine residues (PY) of a large number of proteins (Visconti et al 1999a). Loss of membrane cholesterol is a vital part of this pathway; Visconti et al (1999b) demonstrated that suppression of cholesterol efflux inhibited the increase in PY in mouse sperm, although this could be bypassed by direct activation of the cAMP pathway (Visconti et al 1999b, Wertheimer et al 2008. Once capacitated, sperm become capable of undergoing subsequent fertilization-related processes when exposed to the appropriate stimuli, including zona pellucida (ZP) binding, the acrosome reaction, oocyte penetration, and, finally, fusion with the female gamete (Visconti et al 1998(Visconti et al , 2011.…”