Sperm capacitation is required for fertilization and involves several ion permeability changes. Although Cl(-) and HCO(3)(-) are essential for capacitation, the molecular entities responsible for their transport are not fully known. During mouse sperm capacitation, the intracellular concentration of Cl(-) ([Cl(-)](i)) increases and membrane potential (Em) hyperpolarizes. As in noncapacitated sperm, the Cl(-) equilibrium potential appears to be close to the cell resting Em, opening of Cl(-) channels could not support the [Cl(-)](i) increase observed during capacitation. Alternatively, the [Cl(-)](i) increase might be mediated by anion exchangers. Among them, SLC26A3 and SLC26A6 are good candidates, since, in several cell types, they increase [Cl(-)](i) and interact with cystic fibrosis transmembrane conductance regulator (CFTR), a Cl(-) channel present in mouse and human sperm. This interaction is known to be mediated and probably regulated by the Na(+)/H(+) regulatory factor-1 (official symbol, SLC9A3R1). Our RT-PCR, immunocytochemistry, Western blot, and immunoprecipitation data indicate that SLC26A3, SLC26A6, and SLC9A3R1 are expressed in mouse sperm, localize to the midpiece, and interact between each other and with CFTR. Moreover, we present evidence indicating that CFTR and SLC26A3 are involved in the [Cl(-)](i) increase induced by db-cAMP in noncapacitated sperm. Furthermore, we found that inhibitors of SLC26A3 (Tenidap and 5099) interfere with the Em changes that accompany capacitation. Together, these findings indicate that a CFTR/SLC26A3 functional interaction is important for mouse sperm capacitation.
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...
Unlike most cells of the body which function in an ionic environment controlled within narrow limits, spermatozoa must function in a less controlled external environment. In order to better understand how sperm control their membrane potential in different ionic conditions, we measured mouse sperm membrane potentials under a variety of conditions and at different external K+ concentrations, both before and after capacitation. Experiments were undertaken using both wild-type, and mutant mouse sperm from the knock-out strain of the sperm-specific, pH-sensitive, SLO3 K+ channel. Membrane voltage data were fit to the Goldman-Hodgkin-Katz equation. Our study revealed a significant membrane permeability to both K+ and Cl− before capacitation, as well as Na+. The permeability to both K+ and Cl− has the effect of preventing large changes in membrane potential when the extracellular concentration of either ion is changed. Such a mechanism may protect against undesired shifts in membrane potential in changing ionic environments. We found that a significant portion of resting membrane potassium permeability in wild-type sperm was contributed by SLO3 K+ channels. We also found that further activation of SLO3 channels was the essential mechanism producing membrane hyperpolarization under two separate conditions, 1) elevation of external pH prior to capacitation and 2) capacitating conditions. Both conditions produced a significant membrane hyperpolarization in wild-type which was absent in SLO3 mutant sperm. Hyperpolarization in both conditions may result from activation of SLO3 channels by raising intracellular pH; however, demonstrating that SLO3-dependent hyperpolarization is achieved by an alkaline environment alone shows that SLO3 channel activation might occur independently of other events associated with capacitation. For example sperm may undergo stages of membrane hyperpolarization when reaching alkaline regions of the female genital tract. Significantly, other events associated with sperm capacitation, occur in SLO3 mutant sperm and thus proceed independently of hyperpolarization.
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