Primary cilia distribution and orientation during involution of the bovine mammary gland

Abstract

The regulation of mammary gland involution occurs through multiple levels including environmental factors, hormones, and local intramammary signals. Primary cilia (PC) are signaling organelles that sense biochemical and biophysical extracellular stimuli and are vital for cellular and tissue function. The aim of this study was to examine the distribution, incidence, and orientation of PC. Furthermore, we determined changes in expression levels of the signal transducer and activator of transcription (STAT)6 at the onset of bovine mammary gland involution. Mammary tissue was collected from pasture-fed, primiparous, nonpregnant Friesian dairy cows at mid lactation (n=5 per group) killed 6-h after milking (lactating controls) and during involution after 7 and 28 d of nonmilking (NM). Fluorescent immunohistochemistry and confocal microscopy of tissue sections showed that PC were present on luminal secretory epithelial cells (SEC), myoepithelial cells (MEC), and stromal fibroblast cells (SFC). Furthermore, in all 3 experimental groups, different PC positions or orientations relative to the cell surface were identified on SEC and MEC, which projected toward the lumen and were either straight, bent, or deflected against the apical cell surface, whereas PC in SFC were confined to the interalveolar space. However, by 28-d NM, fewer PC projected into the luminal space and most appeared deflected or projected toward the interalveolar space. Furthermore, by 28-d NM, with the increase in stromal connective tissue, more PC were detected within the interalveolar and interlobular stroma. At 28-d NM, we observed a decrease in luminal cilia relative to the total number of cilia. The number of ciliated cells in the total fraction (SEC, MEC, and SFC) was the same for all 3 groups, although in the luminal fraction (SEC and MEC), PC per nuclei increased by 28-d NM relative to lactation. At all 3 stages, we detected variations in shape and orientation of PC within the same alveolus, with some PC projecting directly into lumen, whereas others appeared to be bent or deflected flat against the cell surface. Within each treatment, the average number of bent cilia was low, whereas the average number of deflected cilia was higher than the average number of cilia projecting directly into the lumen. Quantitative real-time reverse transcription PCR analysis showed that expression levels of milk protein genes (αS1-casein, α-lactalbumin, and κ-casein) declined and that of lactoferrin increased in the involuted mammary tissue following NM, compared with lactating controls. Although STAT6 mRNA levels did not change following NM, STAT6 protein levels did increase following 28-d NM compared with the control lactation group. In conclusion, PC were detected in all cell types in the mammary gland, and changes in orientation during involution suggest the potential for PC to play a role in signal transduction through both mechanosensation and chemosensation. Furthermore, the STAT6-mediated signaling pathway may have a role during involution of the mammary g...

Stromal-Epithelial Interactions during Mammary Gland Development

“…Re-emergence of adipose tissue is an important feature of involution associated with infiltration of macrophages into the gland form [14]. In the mouse mammary gland, gene expression profiling during postlactational tissue regression showed an increase in genes linked to the immune system, which coincides with increasing levels of interleukins: IL-4 and IL-13 acting as macrophage chemoattractants [76]. Furthermore, ECM can fragment into matrikines and matricryptins that also serve as attractants for the peripheral immune cells [14].…”

Tight Junction Protein Abundance and Apoptosis During Involution of Rat Mammary Glands

“…In support of mechanotransduction, mammary cell stretch in vitro induces apoptotic pathways (Quaglino et al, ) and in vivo accelerates the induction of apoptosis and loss of the ECM factor β1‐integrin and the TJ protein occludin (Phyn et al, ). Furthermore, primary cilia, key signalling organelles for sensing biochemical and biophysical extracellular stimuli, have been implicated in initiating involution of the mammary gland (Millier et al, ; Biet et al, ).…”

Temporal and spatial heterogeneity in milk and immune-related gene expression during mammary gland involution in dairy cows

“…Possibilities include the accumulation of casein fractions (Shamay et al, 2003) or serotonin (Matsuda et al, 2004;Collier et al, 2012) in milk, or physical distension of the mammary gland, which results in changes in cell shape that may activate mechanotransduction pathways (Davis et al, 1999;Stelwagen, 2001). In support, cell-cell communication (Cooper et al, 2004), cell-extracellular matrix communication (Singh et al, 2005), and the primary cilia of mechanosensors (Millier et al, 2013;Biet et al, 2016) were disrupted following milk engorgement at the onset of involution of the bovine mammary gland. Furthermore, acute physical distension of rat mammary glands with sucrose solution in vivo accelerated the onset of apoptosis of MEC, disruption of cell-cell and cell-extracellular matrix communication, and activation of STAT3 (Phyn et al, 2007).…”