pH Regulation in Human Sperm

Abstract

In the female reproductive tract, sperm undergo a maturation process, called capacitation, which enables sperm to fertilize the oocyte. The complex cellular mechanisms underlying capacitation are not well understood. Besides other cellular events, the sperm cytosol alkalizes and the intracellular calcium concentration rises, which changes the flagellar beating. Remarkably, during their journey through the female reproductive tract, human sperm face drastic changes in the extracellular pH (pHo): in the vagina, pHo is below 5; in the oviduct, at the site of fertilization, pHo is above 7.5. Signaling cascades that regulate sperm swimming depend on the intracellular pH (pHi): a rise in the cAMP concentration in sperm increases the flagellar beat frequency. cAMP, in turn, is produced by a HCO₃^- activated, soluble adenylyl cyclase, thus connecting its activity to pHi via the CO₂/HCO₃ /H⁺ equilibrium. In addition, the sperm-specific, alkaline-activated calcium channel CatSper allows calcium influx that is crucial for a particular flagellar beating called hyperactivation. It has been suggested that changes in pHo translate to changes in pHi via transmembrane channels or exchangers. Several anion exchangers of the SLC family and CFTR were suggested to propagate an HCO₃^- influx, while sperm-specific Na⁺/H⁺ exchangers and Hv1 were suggested to facilitate proton extrusion. <br> In my thesis, I used electrophysiological, fluorometric, and pharmacological methods to decipher the cellular mechanisms that regulate pHi in human sperm. I showed that upon extracellular alkalization, mimicking the increasing pHo in the female reproductive tract, pHi slowly follows pHo. The pHi is set by an amiloride-sensitive Na⁺/H⁺ exchange. Exposing sperm to physiological CO₂/HCO₃^- concentrations leads to rapid and persistent acidification caused by CO₂ diffusion into the cell and subsequent hydration to HCO₃^- + H⁺ by carbonic anhydrase; HCO₃^- in turn activates the sAC. In contrast, HCO₃^- transport into the cell seems to play if any only a minor role in pHi regulation. Thus, CO₂ diffusion over the membrane dominates over HCO₃^- transport. Further, I show that Hv1 is the only voltage-dependent pH regulator in human sperm. Using patch-clamp fluorometry, I was able to record the intracellular alkalization induced by depolarizing Hv1 currents, which were potentiated in presence of CO₂/HCO₃^-. Alkalization by Hv1 is, however, only transiently present for the duration of the depolarization. My results are corroborated by a recently assembled sperm proteome, which also suggests that fewer proteins than previously thought are involved in pH regulation in human sperm.