A novel CFTR-AQP7 protein complex regulates glycerol transport and motility of human sperm

Abstract STUDY QUESTION Does the interaction between CFTR and AQP7 in human spermatozoa play a role in the molecular mechanisms underlying sperm motility? SUMMARY ANSWER CFTR inhibition reduces sperm motility and AQP7-mediated glycerol permeability in human spermatozoa, and CFTR and AQP7 co-localize in the equatorial segment of the sperm head, with in silico modeling suggesting a potential interaction between these proteins. WHAT IS KNOWN ALREADY CFTR modulates the permeability of aquaglyceroporins in multiple tissues, and their interaction is mediated by the scaffolding protein NHERF1. AQP7-mediated glycerol permeability correlates with sperm motility. STUDY DESIGN, SIZE, DURATION Semen samples were collected from normozoospermic men (normal motility; n = 33) and men with asthenozoospermia (reduced motility; n = 15) at a fertility clinic between September 2020 and January 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS Isolated sperm from men with normozoospermia were used to study the effect of CFTR on sperm motility (N = 10) and glycerol permeability (N = 23). Sperm from 14 asthenozoospermic samples and 13 normozoospermic samples were used to compare the effect of CFTR on AQP7-mediated glycerol permeability, after screening for the absence of common CFTR gene variants. Sperm membrane permeability to glycerol was measured using stopped-flow light scattering, and the effect of CFTR conductance was modulated using a specific inhibitor (CFTRinh172). The interaction between CFTR and AQP7 was investigated using co-immunofluorescence, proximity ligation assay, and in silico approaches like ColabFold and GROMACS. Gaussian distribution of the data was measured by the Shapiro–Wilk normality test. Data showing non-normal distribution was treated with the Kruskal–Wallis test, whereas normal distribution data were treated with an ordinary one-way ANOVA. Comparisons between normozoospermic and asthenozoospermic groups were performed using an unpaired two-tailed Mann–Whitney U test. A P-value less than 0.05 was considered significantly different. MAIN RESULTS AND THE ROLE OF CHANCE CFTR inhibition negatively affected sperm motility (0.53 ± 0.11-fold variation to control, P < 0.05) and AQP7-mediated glycerol permeability (0.459-fold [0.314; 0.537] variation to control, P < 0.01). Despite this, the effect of CFTR dysfunction on AQP7-mediated glycerol permeability of sperm from normo- versus asthenozoospermic samples did not reach statistical significance (P = 0.068) due to low statistical power, but a tendency was apparent. A larger sample size is needed to confirm this trend. CFTR and AQP7 (the main glycerol diffuser in human sperm) co-localize and are in proximity in the midpiece and in the equatorial section of the sperm head in human sperm. In silico analysis supports the interaction of CFTR with AQP7 intermediated by NHERF1, indicating a mechanism of physical modulation of AQP7 permeability by CFTR. LIMITATIONS, REASONS FOR CAUTION Only cystic fibrosis-associated CFTR variants were screened during this study; the presence of assumed benign variants that could slightly decrease CFTR function may have impacted the results. Glycerol permeability was measured indirectly by assuming its proportionality with the change in sperm volume through time after the osmotic shock. A larger sample size would be needed to confirm the trends that did not reach statistical significance. Furthermore, pharmacological assays were conducted in a non-nutrient buffer to specify direct effects of the channel; this condition differs from physiological media and represents a specific limitation of this study. WIDER IMPLICATIONS OF THE FINDINGS Our findings suggest that a novel mechanism based on the functional and physical interaction between CFTR and AQP7 may underlie some cases of asthenozoospermia and idiopathic male infertility; the results also increase our knowledge of the molecular mechanisms governing sperm motility. STUDY FUNDING/COMPETING INTEREST(S) This research was funded by Fundação para a Ciência e a Tecnologia (FCT) to UMIB (UIDB/00215/2020, and UIDP/00215/2020), ITR-Laboratory for Integrative and Translational Research in Population Health (LA/P/0064/2020), and the post-graduate student João C. Ribeiro (UI/BD/150749/2020). The work was co-funded by FEDER through the COMPETE/QREN, FSE/POPH, and POCI-COMPETE 2020 (POCI-01-0145-FEDER-007491) funds. P.F.O. is funded by national funds through FCT, I.P., under the Scientific Employment Stimulus-Institutional Call-reference CEEC-INST/00026/2018. This work also received support and help from FCT/MCTES to LAQV-REQUIMTE (LA/P/0008/202—DOI 10.54499/LA/P/0008/2020, UIDP/50006/2020—DOI 10.54499/UIDP/50006/2020, and UIDB/50006/2020—DOI 10.54499/UIDB/50006/2020) and to iBiMed (UIDB/04501/2020—DOI 10.54499/UIDB/04501/2020 and UIDP/04501/2020—DOI 10.54499/UIDP/04501/2020), through national funds. There are no conflicts of interest to declare. TRIAL REGISTRATION NUMBER N/A.