Spatial mapping of Notch signaling components along the bull genital tract and in epididymal and vas deferens vesicles.

During transit in the genital tract until ejaculation, spermatozoa (SPZ) protein content and spatial distribution are remodeled through the cargo channeled by extracellular vesicles, namely epididymosomes (EpS), and the soluble luminal content. This remodeling confers the maturational competence for fertilization. This study evaluated the expression patterns of Notch components (NOTCH1-4, DLL1, 3 and 4, JAGGED1-2) in the bull genital tract tissues and their respective lumen, and their presence in EVs and SPZ, by immunocytochemistry and western blot. Results evidenced that NOTCH proteins are differentially expressed from the testis to the epididymis, the vas deferens and the accessory glands. In the testis, expression is mainly linked to acrosome biogenesis, while in post-testicular tissues is mainly localized in the adluminal cytoplasm of epithelial cells and the lumen. NOTCH1-3, DLL3 and JAGGED2 are released into the lumen via EVs, namely EpS, and are detected in SPZ of the corresponding genital tract segments. The spatial relationships support a model of region-specific acquisition of NOTCH proteins by SPZ through both testicular and EpS origins, with additional contributions from accessory glands at ejaculation. This prompts the involvement of NOTCH proteins in intercellular communication within the male reproductive tract, leading to SPZ maturation and competence for signaling in the female reproductive tract and during gamete interaction.