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Abstract
The reaction of CO2 with H2O to form bicarbonate (HCO3−) and H+ controls sperm motility and fertilization via HCO3−-stimulated cAMP synthesis. A complex network of signaling proteins participates in this reaction. Here, we identify key players that regulate intracellular pH (pHi) and HCO3− in human sperm by quantitative mass spectrometry (MS) and kinetic patch-clamp fluorometry. The resting pHi is set by amiloride-sensitive Na+/H+ exchange. The sperm-specific putative Na+/H+ exchanger SLC9C1, unlike its sea urchin homologue, is not gated by voltage or cAMP. Transporters and channels implied in HCO3− transport are not detected, and may be present at copy numbers < 10 molecules/sperm cell. Instead, HCO3− is produced by diffusion of CO2 into cells and readjustment of the CO2/HCO3−/H+ equilibrium. The proton channel Hv1 may serve as a unidirectional valve that blunts the acidification ensuing from HCO3− synthesis. This work provides a new framework for the study of male infertility.
Highlights
We show that CO2/HCO3− acidifies rather than alkalizes sperm, and that synthesis of HCO3− from CO2 dominates HCO3− entry via HCO3− solute carriers
Identification of signaling molecules by mass spectrometry Several independent proteomes have been assembled from human sperm[48–51] that differ markedly in size and in key signaling components (Supplementary Table 1)
Wang et al (2013) reported an overlap of 82% of proteins identified in their analysis; of note, each of their replicates consists of sperm samples mixed from several donors, whereas we have analyzed proteomes from single donors
Summary
We applied a targeted MS approach, i.e., PRM-MS, to quantitatively scrutinize whether CFTR, SCNN1A, SLC4A4, and SLC26A6, four signaling proteins that have been invoked in HCO3− transport (Fig. 1), are absent from human sperm. In agreement with these physiological results in mouse, we could not detect CFTR and ENaC proteins in human sperm, and, according to our LOD values, maximally between 10 and
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