(236) TMC5 Complex at the Spermatid Plasma Membrane is Essential for Spermiogenesis

Wenxuan (Sharon) Zheng1 , Gaizun Hu1 , Fan Lin1 , Christopher B. Geyer2, Seham Ebrahim1 

1. Department of Molecular Physiology and Biomedical Physics, School of Medicine, University of Virginia 

2. Department of Anatomy & Cell Biology, Brody School of Medicine, East Carolina University; East Carolina Diabetes and Obesity Institute, East Carolina University.

Abstract Text:

Spermiogenesis transforms round spermatids into mature spermatozoa through highly orchestrated events including nuclear condensation, cytoplasmic extrusion, and flagellum formation. Despite its biological importance, the molecular mechanisms underlying spermatid remodeling remain poorly understood, posing a barrier to advancing both infertility treatment and male contraceptive development.

We identify TMC5, a previously uncharacterized member of the Transmembrane Channel-like (TMC) family, as essential for spermatid maturation. Tmc5 is a highly conserved gene (80% sequence identity) in humans and mice, and is enriched in early and late spermatids, as well as mature sperm in both species. Tmc5 knockout (KO) male mice are infertile, exhibiting non-obstructive azoospermia (NOA) due to arrested spermiogenesis, but show no other overt phenotype. These mice displayed significant disruptions in spermatid elongation, including failures in cytosol elimination, mitochondria organization, and nucleus condensation. Using knock-in (KI) mice with an mCherry tag at the endogenous C-terminus of TMC5, we show that TMC5 localizes to the plasma membrane of early and late spermatids. Co-immunoprecipitation and subsequent mass spectrometry analysis revealed top co-purified proteins were TMC7, CIB1, and CIB4 - all of which have established roles in spermiogenesis and whose knockout in mice leads to male infertility, indicating they may be core components of the TMC5 complex. Immunofluorescence confirms that CIB1 co-localizes to the wide-type (WT) spermatid membrane. In Tmc5 KO spermatids, the CIB1 membrane localization is lost. Together these results consolidate that TMC5 and its binding partners are critical regulators of spermatid morphogenesis.

Our data also indicate that TMC5 function is critical for maintaining plasma membrane integrity, actin network organization, and calcium homeostasis. Phosphatidylserine (PS) externalization is identified using Annexin V on the plasma membrane of healthy wild-type late spermatids, which appears homogeneous in WT spermatids but becomes aberrantly patchy in Tmc5 KO cells - likely due to apoptotic membrane blebbing as they are TUNEL positive. Super-resolution imaging of Tmc5 KO spermatids reveals disassembly of the actin-rich acrosome-acroplaxome-manchette complex, crucial for nuclear shaping. Furthermore, the spermatid intracellular calcium increases accompanying the expression of TMC5 during spermiogenesis. Isolated WT spermatogenic cells had higher calcium intake response in hypotonic solution than Tmc5 KO, suggesting potential roles of TMC5 in regulating calcium.

TMC5’s role extends beyond basic biology as the understudied eligible ion channels part of the NIH “Druggable Proteome” - a subset of proteins amenable to small-molecule modulation of which only 5-10% are currently targeted by FDA-approved drugs. Its deletion results in complete male infertility without systemic side effects, making it ideal for non-hormonal male contraceptive targets. Our work not only provides new mechanistic insight into the cellular remodeling of spermatids but also establishes TMC5 complex as a promising therapeutic target. Future directions include cryo-electron microscopy to resolve the structure of the native TMC5 complex and structure-based drug design to develop small-molecular inhibitors. Together this research advances our understanding of male reproductive biology and offers a rational foundation for the development of safe, effective, and reversible non-hormonal male contraceptives.