The reaction of CO2 with H2O to form HCO3– and H+ controls sperm motility and fertilization via HCO3--stimulated cAMP synthesis and alkalization. A complex network of signaling proteins has been proposed to participate in this reaction. Here, we identify key players that regulate intracellular pHi and HCO3- in human sperm by quantitative mass spectrometry, kinetic fluorometry, and patch-clamp recording. pHi is set by an amiloride-sensitive Na+/H+ exchanger of the SLC9B type; the sperm-specific putative Na+/H+ exchanger SLC9C1 is not involved in voltage-gated H+ transport. Many transporters and channels previously implied in HCO3- are absent. Instead, HCO3- is produced by rapid CO2 diffusion into the cell and readjustment of the CO2/HCO3-/H+ equilibrium by carbonic anhydrase. The proton channel Hv1 serves as a unidirectional valve that blunts the ensuing acidification. This work calls for substantial revision of signaling concepts in human sperm and provides a new framework for the study of male infertility.

PRM-MS in combination with AQUA peptides was used to determine copy numbers of protein of interest in human sperm, additionally, limit-of-detection values were determined for proteins that were not detected in human sperm. For both purposes, we used internal calibration curves, which means that we spiked different concentrations of AQUA peptides into peptides derived from a specified cell number.

We worked with human sperm cells that were cleaned up by a swim up essay. After clean up, the samples were submitted to a in-solution digest and after that, samples were prepared for the PRM measurements.