Abstract
Primary cilia are small antenna-like sensory organelles, found on virtually all vertebrate cell types. They can express specialised receptor profiles, which define their key mechano- and chemo-sensory functions within a wide range of cells and tissues. Primary cilia coordinate extracellular signals by activating specific transduction pathways, vital for the appropriate function of their resident tissue. Their distribution in bovine mammary tissue has not yet been assessed, despite the prospect that they could attribute specialised perceptive and regulatory functions to this acutely-responsive gland. Therefore, the objectives of this study were to develop a procedure for detecting primary cilia within bovine mammary tissue, and subsequently assess primary cilia distribution and morphology during active lactation, milk stasis and early involution.
A time-course of bovine mammary tissue samples was analysed, in which involution was induced by the abrupt cessation of milking in non-pregnant Friesian dairy cows at mid lactation. Routinely fixed, wax-embedded and sectioned alveolar tissue (of 5 – 8 μm thickness) was obtained at intervals of 6, 12, 18, 24, 36, 72 and 192 hours after the last milking (n = 3 animals per group). Post-milking time points were assigned to active lactation (6 – 12 hours), milk stasis (18 – 36 hours) and early involution (72 – 192 hours) stages. Fluorescent immunohistochemistry was performed using antibodies to identify primary cilia (anti-acetylated α-tubulin), centrioles (anti-γ-tubulin) and myoepithelial cells (anti-α-smooth muscle actin (SMA)). Primary cilia cell-type distribution and morphology were examined by confocal microscopy across the entire range of time points, and ciliary incidence was assessed at active lactation.
Primary cilia were distributed on the apical aspect of luminal alveolar and ductal epithelia, on the alveolar-facing cell membrane of sub-luminal SMA-positive myoepithelial cells, and on stromal cells of inter-alveolar and inter-lobular regions. Primary cilia in all three cell types were short, often tapered at the distal tip, and projected in varying orientations in relation to the alveolar lumen. In secretory epithelial cells (SECs) some primary cilia appeared deflected around the apical cell membrane, and the proportion of those deflected was greatest in sections at milk stasis (72.7%) compared to active lactation (52.4%) (p = 0.0027), suggesting their orientation may be influenced by alveolar events relating to milk stasis. Ciliary length remained consistent throughout the time-course sections in SECs and SMA-positive myoepithelial cells (p > 0.05). Primary cilia alveolar incidence during active lactation was between 11% and 44% in luminal SECs, and between 14% and 46% in total alveolar cells (myoepithelial cells and SECs).
Findings show that, during active lactation, milk stasis and early involution, all cell types involved in the regulation of bovine lactation can express a primary cilium. Primary cilia were suitably-placed in three important cell types to potentially coordinate various forms of ciliary transduction relying on both mechano- and chemo-sensation according to the physical and physiological state of the gland. Their presence provides new research directions in the study of mammary regulation within the dairy cow and other mammalian species, to enhance the understanding of how various mammary-specific cellular responses may be influenced by local factors.