Membrane protein-mediated signaling as a driver of spermiogenesis- Investigating new molecules and mechanisms

The complete absence of sperm in the ejaculate is the most severe form of male infertility and termed azoospermia. Azoospermia is remarkably prevalent, affecting 10-20% of infertile men. The majority (~70%) of cases are classified as non-obstructive azoospermia (NOA), an irreversible condition caused by impaired spermatogenesis. NOA is of additional clinical interest due to its association with higher rates of testicular cancer and increased risk of death. Yet, our understanding of NOA is limited, largely because knowledge of the molecules and mechanisms underlying each stage of spermatogenesis is incomplete. Based on my previous research on the transmembrane channel-like (TMC) protein family, my new laboratory recently identified a novel gene, Tmc5, whose ablation in mice caused no overt phenotype except for male infertility. Strikingly, Tmc5 knockout (KO) mice are azoospermic due to a block in the spermiogenesis program, during which haploid round spermatids undergo a dramatic transformation by completing processes including nuclear condensation, cytoplasmic loss, and flagellar formation, to ultimately form condensed spermatids. To begin to elucidate the role of TMC5 in spermiogenesis, we developed knockin (KI) mice with mCherry inserted at the C-terminus of the endogenous Tmc5 gene, so that these mice express an mCherry-TMC5 fusion protein in spermatids. Using these mice we determined, using super-resolution microscopy, that TMC5 is enriched in the plasma membranes of round, elongating, and condensing spermatids as well as sperm. These results are consistent with the reported enrichment of TMC5 in human sperm and reported links to male infertility. Here we are proposing to take a multi-scale approach to elucidate the function of TMC5 at the molecular level, and its specific role in spermiogenesis at the whole organism level. Based on substantial preliminary data, our central hypothesis is that TMC5 on the spermatid plasma membrane modulates intracellular calcium to regulate changes in F-actin architecture and induce membrane lipid remodeling essential for spermiogenesis. We will use novel tools including Tmc5 KO and KI mice, as well as unique heterologous cell lines expressing membrane-targeted TMC5 (which has not previously been achieved) for mechanistic investigation of TMC5 function. These will be combined with innovative techniques including spermatogenesis synchronization, super-resolution imaging and machine learning, in vitro functional assays, and interactomics to test this hypothesis. Project Number: 1R01HD118160-01 | Fiscal Year: 2025 | NIH Institute/Center: Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) | Principal Investigator: Seham Ebrahim | Institution: UNIVERSITY OF VIRGINIA, CHARLOTTESVILLE, VA | Award Amount: $484,967 | Activity Code: R01 | Study Section: Special Emphasis Panel[ZRG1 EMS-C (02)] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R01HD11816001