Background: Focal adhesion proteins act as cellular mechanosensors and molecular anchors. They form macromolecular complexes that link the actin cytoskeleton to the extracellular matrix and are vital for processes underpinning male fertility, including blood-testis barrier maintenance, spermiation, acrosome remodelling, and fertilisation. Focad is a testis‑enriched gene associated with the focal adhesion network, but its molecular role and contribution to mammalian fertility remains unknown. Here, we tested the hypothesis that FOCAD participates in focal adhesion-mediated processes during spermatogenesis and is required for sperm function in mice.
Methods: A whole‑body Focad knockout (Focad-/-) mouse was generated by CRISPR/Cas9 and the colony was maintained via heterozygotes. Male fertility phenotyping followed our established pipeline with a minimum n = 3 mice/genotype for each assay. Where appropriate, comparisons used unpaired t‑tests or non‑parametric equivalents.
Results: Focad-/- males were infertile but testis weights were comparable to wild-type (Focad+/+) mice. Histology identified delayed spermatozoa release from Focad-/- seminiferous tubules, and consistent with defective spermiation, Focad-/-epididymis weights were reduced by 25.0% (p = 0.0216). Functional analyses revealed that sperm from Focad-/- mice exhibited a 46.7% reduction in total motility versus Focad+/+ mice (p<0.0001), and ongoing confocal/electron microscopy suggests this is due to flagellar abnormalities. Preliminary assays also indicate increased rates of spontaneous acrosome reaction in Focad-/- mouse sperm, consistent with a role for FOCAD in actin remodelling at the acrosome. Collectively, these data indicate that FOCAD is required for spermiation, flagellum integrity, and aspects of acrosome function.
Conclusion: FOCAD is indispensable for the late stages of spermatogenesis and for sperm function in mice. Loss of FOCAD likely impairs transit through the female reproductive tract and fertilisation competence. Ongoing work will map FOCAD’s interactions within the focal adhesion network in male germ cells, quantify flagellar structural defects, and test fertilisation capacity in vitro to define mechanism of action.