Sheep rumen and omasum primary cultures and source epithelia: barrier function aligns with expression of tight junction proteins

Abstract

SUMMARYThe forestomachs of cows and sheep have historically served as important models for the study of epithelial transport. Thus, the ruminal epithelium was among the first tissues in which absorption of chloride against an electrochemical gradient was observed, requiring a tight paracellular barrier to prevent back-leakage. However, little is known about ruminal barrier function, despite the considerable implications for ruminant health. The tight junction proteins of the omasum have never been investigated, and no cell culture model exists. We present a new method for the isolation of cells from forestomach epithelia. Protein expression of cells and source tissues of sheep were studied using western blot, PCR and confocal laser scanning microscopy. Supplementary material available online at

A high-grain diet causes massive disruption of ruminal epithelial tight junctions in goats

“…The primary antibodies employed were rb-anti-claudin-1, m-anti-claudin-4, manti-occludin, and rb-anti-ZO-1 (Invitrogen, dilutions of 1:500 for claudin-1 and ZO-1, 1:800 for claudin-4 and occludin). The specificity of the antibodies was previously verified by Stumpff et al (38) and was also confirmed in our preliminary experiment. After several washes with TBST, membranes were incubated in a 1:20,000 dilution of an anti-rabbit or anti-mouse HRP-conjugated secondary antibody (Sunshine Biotechnology, Nanjing, China).…”

“…In addition, the prominent histological alterations during HG feeding strongly suggest an impaired barrier function (35-37) that may provide the opportunity for the translocation of toxins and bacteria from the rumen into the blood, ultimately affecting animal health and productivity (28). Although there is fundamental knowledge about the consequences of HG feeding on ruminal epithelial barrier function, remarkably little information is currently available about the underlying molecular changes in ruminal epithelial barrier function during HG diet feeding.The rumen epithelium (RE) of sheep and cattle has extensive barrier-forming properties, depending on the multicellular structure (stratum corneum, granulosum, spinosum, and basale) and the junctional complex that establish a barrier between sometimes hostile external environments and the internal milieu (12,27,38). Tight junctions (TJs) located in the middle layers (stratum granulosum and spinosum) play a key role in maintaining the polarity of epithelial cells, regulating the permeability of the epithelial barrier and preventing the translocation of LPS and other toxins (12,27).…”

“…The rumen epithelium (RE) of sheep and cattle has extensive barrier-forming properties, depending on the multicellular structure (stratum corneum, granulosum, spinosum, and basale) and the junctional complex that establish a barrier between sometimes hostile external environments and the internal milieu (12,27,38). Tight junctions (TJs) located in the middle layers (stratum granulosum and spinosum) play a key role in maintaining the polarity of epithelial cells, regulating the permeability of the epithelial barrier and preventing the translocation of LPS and other toxins (12,27).…”

“…In monogastrics, the TJ proteins in gastrointestinal epithelia with highly dynamic structures regulate the degree of sealing, according to external stimuli and physiological and pathological conditions, and they place the mucosal epithelium at the center of interactions between the mucosal immune system and gastrointestinal contents, including dietary antigens and microbial products (11). A recent study by Stumpff et al (38) determined the full mRNA coding sequence of claudins 1, 4, and 7 in sheep. The expression and distribution patterns of the key TJ proteins claudin-1, claudin-4, claudin-7, occludin, and ZO-1 have been previously investigated in RE using Western blot analysis and immunofluorescence (12,38).…”

“…A recent study by Stumpff et al (38) determined the full mRNA coding sequence of claudins 1, 4, and 7 in sheep. The expression and distribution patterns of the key TJ proteins claudin-1, claudin-4, claudin-7, occludin, and ZO-1 have been previously investigated in RE using Western blot analysis and immunofluorescence (12,38). To date, no studies known to the authors have investigated the changes in TJ proteins at the mRNA and protein expression levels during HG diet feeding.…”