Role of Carbonic Anhydrases II and IV in pH-dependent activation of CatSper channel

• Applicant: Dr. Caroline Wiesehöfer
• Subject Area: Reproductive Medicine, Urology ; Biochemistry
• Term: from 2022 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 514808153

Infertility is a global health concern in which male infertility accounts for 40-50%. The primary causes of male infertility are defects in sperm motility as well as capacitation, the maturation process whereby sperm undergo biochemical changes in the female reproductive tract for successful fertilization. This capacitation process is not fully understood until now. Ion channels are crucial for the signal transduction mechanisms in sperm capacitation, especially CatSper, a sperm specific, pH-sensitive Ca2+-selective ion channel. EFCAB9, a CatSper auxiliary subunit in complex with the CatSper subunit CATSPERζ, seems to be essential for pH-dependent activation of the CatSper channel. Beside different ion channels regulating intracellular alkalization, also carbonic anhydrases (CA) contribute to changes in intracellular pH by catalyzing the reversible hydration of CO2 to HCO3- and protons. CAII and CAIV knockout mice show an imbalanced HCO3- homeostasis, which leads to a reduced sperm motility and swimming speed as well as HCO3- enhanced beat frequency. These results suggest that CAII and CAIV are key enzymes in the regulation of sperm motility and therefore required for normal fertility. This interdisciplinary proposal aims to gain new insights into sperm physiology and to uncover new signaling pathways that could control sperm motility. Thus, determination of the 3D distribution of CAII and CAIV within single sperm flagella will be analyzed using super-resolution (SR) fluorescence microscopy. Additionally, performing SR imaging of CatSper1 KO will demonstrate whether CAII distribution is affected by CatSper1. If incompatible distribution of CAII and CAIV with CatSper is identified, SR imaging will also be performed of spermatozoa with a KO in other pH-sensitive ion channels. If incompatible distribution of CAII and CAIV with CatSper is identified, SR imaging should also be performed of sperm with a KO in other pH-sensitive ion channels. This analysis should detect either a functional link between CAs and CatSper or other pH-sensitive ion channels. Intracellular pH regulation by CAII or CAIV will be detected using fluorescence-based intracellular pH indicators (BCECF, SNARF-5F). Finally, co-immunoprecipitation will be used to investigate whether CAII or CAIV regulates CatSper activity via interaction with EFCAB9-CatSperζ.The overall goal of this project is to elucidate if CAII, CAIV, due to intracellular pH changes, can modify CatSper function, which prolong the hyperactivated state, sperm motility and which is required for male fertility. Our experiments will inform basic biological processes of the utmost importance, but will also provide much needed insight into the causes of infertility.

• DFG Programme: WBP Fellowship
• International Connection: USA
• Host: Professorin Dr. Jean-Ju Chung