Tubulogenesis of bovine uterine glands by epidermal growth factor and collagen Ⅰ in 3D culture systems

Uterine glands are endometrial exocrine epithelia that support early embryo development. Their secretions are particularly essential for conceptus elongation in cattle. Uterine glands develop from the luminal epithelium and elongate into the stromal layer toward the myometrium. This process is regulated by growth factors, WNT proteins, and the surrounding extracellular matrix (ECM); however, the precise mechanisms that govern bovine uterine gland morphogenesis remain unclear. In this study, we determined how these signaling factors and ECM components affect the tubular formation of bovine uterine gland fragments in 3D culture systems. Uterine gland fragments were enzymatically isolated from bovine endometria and 3D-cultured in Matrigel with or without growth factors (EGF, FGF1, FGF2, FGF7, FGF10, and IGF-1) and WNT (WNT3A, WNT5A, and WNT7A) proteins. Of these, only EGF stimulated the elongation of uterine gland fragments and eventually induced the formation of uterine gland-like structures. EGF-induced tubulogenesis was accompanied by a rapid increase in cell proliferation and alterations in cell-ECM interactions. The supplementation of collagen Ⅰ with Matrigel further promoted the elongation of the tubular structures. Although the addition of collagen Ⅰ did not alter the gene expression profiles of the uterine gland-like structures, the integrin-ROCK pathway contributed to the collagen-induced enhancement of elongation. Our findings clarified that EGF and collagen Ⅰ, but not FGFs, IGF-1, or WNTs, are key regulators for the tubular formation of 3D-cultured bovine uterine gland fragments. This 3D culture system provides a new platform to examine the cellular and molecular mechanisms underlying bovine uterine gland morphogenesis.