The cystic fibrosis transmembrane conductance regulator (CFTR) is present in mature sperm and is required for sperm motility and capacitation. Both these processes are controlled by ions fluxes and are essential for fertilization. We have shown that SLC26A8, a sperm-specific member of the SLC26 family of anion exchangers, associates with the CFTR channel and strongly stimulates its activity. This suggests that the two proteins cooperate to regulate the anion fluxes required for correct sperm motility and capacitation. Here, we report on three heterozygous SLC26A8 missense mutations identified in a cohort of 146 men presenting with asthenozoospermia: c.260G>A (p.Arg87Gln), c.2434G>A (p.Glu812Lys), and c.2860C>T (p.Arg954Cys). These mutations were not present in 121 controls matched for ethnicity, and statistical analysis on a control population of 8,600 individuals (from dbSNP and 1000 Genomes) showed them to be associated with asthenozoospermia with a power > 95%. By cotransfecting Chinese hamster ovary (CHO)-K1 cells with SLC26A8 variants and CFTR, we showed that the physical interaction between the two proteins was partly conserved but that the capacity to activate CFTR-dependent anion transport was completely abolished for all mutants. Biochemical studies revealed the presence of much smaller amounts of protein for all variants, but these amounts were restored to wild-type levels upon treatment with the proteasome inhibitor MG132. Immunocytochemistry also showed the amounts of SLC26A8 in sperm to be abnormally small in individuals carrying the mutations. These mutations might therefore impair formation of the SLC26A8-CFTR complex, principally by affecting SLC26A8 stability, consistent with an impairment of CFTR-dependent sperm-activation events in affected individuals.

The Slc26 gene family encodes several conserved anion transporters implicated in human genetic disorders, including Pendred syndrome, diastrophic dysplasia and congenital chloride diarrhea. We previously characterized the TAT1 (testis anion transporter 1; SLC26A8) protein specifically expressed in male germ cells and mature sperm and showed that in the mouse, deletion of Tat1 caused male sterility due to a lack of sperm motility, impaired sperm capacitation and structural defects of the flagella. Ca(2+), Cl(-) and HCO(3)(-) influxes trigger sperm capacitation events required for oocyte fertilization; these events include the intracellular rise of cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA)-dependent protein phosphorylation. The cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in mature sperm and has been shown to contribute to Cl(-) and HCO(3)(-) movements during capacitation. Furthermore, several members of the SLC26 family have been described to form complexes with CFTR, resulting in the reciprocal regulation of their activities. We show here that TAT1 and CFTR physically interact and that in Xenopus laevis oocytes and in CHO-K1 cells, TAT1 expression strongly stimulates CFTR activity. Consistent with this, we show that Tat1 inactivation in mouse sperm results in deregulation of the intracellular cAMP content, preventing the activation of PKA-dependent downstream phosphorylation cascades essential for sperm activation. These various results suggest that TAT1 and CFTR may form a molecular complex involved in the regulation of Cl(-) and HCO(3)(-) fluxes during sperm capacitation. In humans, mutations in CFTR and/or TAT1 may therefore be causes of asthenozoospermia and low fertilizing capacity of sperm.

These results highlight the critical role of these ICL residues in the assembly of the different domains and/or in the Cl(-) permeation pathway of CFTR.