Morphology and Transcriptome Analysis of Intestinal Organoids in Bovine fetus

BackgroundIntestinal health is an important guarantee for the healthy development of mammals. Therefore, the research on intestinal development and function continues to be a hot spot. And intestinal organoids are a hopeful alternative for the intestine, especially in large animals. In this study, we selected intestine of fetus calves as a target to study the differences in intestinal development and the development and functions of organoids in vitro, which were less reported in the past. The fetus intestine tissue is considered a preferred source for obtaining organoid because of its sterility and excellent developmental potential. MethodsWe compared the morphology of crypts and epithelial cells from duodenum, jejunum, ileum and colon at 4-6 month age fetus calves by HE staining, immunohistochemistry and transmission electron microscopy. And we cultured organoids from jejunum tissue and analyzed the structure by optical and electronic microscopy, immunological methods. Subsequently, through RNA sequencing, we confirmed the characteristics of gene expression between organoids and crypts. ResultsThe results showed the villus of jejunum and ileum was significantly higher than others, but the depth of the duodenum crypt was significantly deeper than the other two (P<0.001). And a large number of fissions were observed in duodenum crypts. In addition, intestinal epithelial stem cell markers LGR5 and CTNNB1 were highly expressed in the epithelium, but there was significant difference in villus and crypt of different intestinal segments. And By electron microscopy, we noticed that crypt stem cells were typically long and arranged in single and multiple layers closely, with a big nucleus and a large number of microvilli at the top of the cells. Each cell contained a large number of organelles, such as mitochondria and ribosomes. There are more free ribosomes in jejunum, ileum and colon crypt. Some Paneth cells and goblet cells were interspersed among crypt cells. There are typical tight junctions and adherent junctions (fingers cross pattern) between crypt cells. In addition, we obtained jejunum organoids by tissue culture possessing similar traits, such as microvillus and cell tight junctions. CTNNB1, PCNA and Lyso were highly expressed in the crypt cells around the surface of the organoids which hinted at the prominent capacity of cell proliferation. Furthermore, according to RNA sequencing, we found metabolism-related genes were significantly higher expression in organoids than in crypt stem cells (Q<0.001). On the contrary, immune and disease related genes were significant higher in crypt stem cells (Q<0.001). Finally, we found metabolic genes existed significantly differential (Q<0.001): crypt stem cells were biased towards energy metabolism (fructose and mannose) and vitamin metabolism(retinol), and organoids were biased towards amino acid process(glutathione metabolic process) and cholesterol process. This also implied that enteral nutrition absorption needed to be further processed in myofibroblasts except the crypt epithelium. ConclusionsIn short, different intestinal epithelial morphology, structure and development were different and related to function and organoids from jejunum. The organoids from jejunum were recommended as an ideal model for studying nutrient metabolism in cattle.

Molecular Mechanism of Stimulation of Na-K-ATPase by Leukotriene D4 in Intestinal Epithelial Cells

Na-K-ATPase provides a favorable transcellular Na gradient required for the functioning of Na-dependent nutrient transporters in intestinal epithelial cells. The primary metabolite for enterocytes is glutamine, which is absorbed via Na-glutamine co-transporter (SN2; SLC38A5) in intestinal crypt cells. SN2 activity is stimulated during chronic intestinal inflammation, at least in part, secondarily to the stimulation of Na-K-ATPase activity. Leukotriene D4 (LTD4) is known to be elevated in the mucosa during chronic enteritis, but the way in which it may regulate Na-K-ATPase is not known. In an in vitro model of rat intestinal epithelial cells (IEC-18), Na-K-ATPase activity was significantly stimulated by LTD4. As LTD4 mediates its action via Ca-dependent protein kinase C (PKC), Ca levels were measured and were found to be increased. Phorbol 12-myristate 13-acetate (PMA), an activator of PKC, also mediated stimulation of Na-K-ATPase like LTD4, while BAPTA-AM (Ca chelator) and calphostin-C (Cal-C; PKC inhibitor) prevented the stimulation of Na-K-ATPase activity. LTD4 caused a significant increase in mRNA and plasma membrane protein expression of Na-K-ATPase α1 and β1 subunits, which was prevented by calphostin-C. These data demonstrate that LTD4 stimulates Na-K-ATPase in intestinal crypt cells secondarily to the transcriptional increase of Na-K-ATPase α1 and β1 subunits, mediated via the Ca-activated PKC pathway.

Changes in the Gut Microbiome after Galacto-Oligosaccharide Administration in Loperamide-Induced Constipation

Unbalanced dietary habits and the consumption of high protein and instant foods cause an increase in constipation. Here, we evaluated the effects of galacto-oligosaccharide (GOS) on a rat model of loperamide-induced constipation by measuring various biological markers and cecal microbiota. The fecal water content and intestinal transit ratio significantly increased in the GOS-administered (GL and GH) groups than in the control group (p < 0.05, p < 0.01, and p < 0.001, respectively). The length of intestinal mucosa (p < 0.05 and p < 0.01, respectively) and area of crypt cells were (p < 0.01, both) significantly increased in the GOS-administered groups compared to the control group. The distribution of interstitial cells of Cajal, which is related to the intestinal movement, showed a significant increase in GOS-administered groups than in the control group (p < 0.01, both). The relative abundance of lactic acid bacteria (LAB), especially Lactobacillus and Lactococcus, significantly increased in the GL group than in the control group. Furthermore, there was a positive correlation between short chain fatty acids (SCFAs) and the gut microbiota in the GL groups. These results demonstrated that GOS administration effectively alleviates constipation by increasing LAB proliferation in the intestinal microbiota and SCFA production.

Ontogenesis of the digestive gland through the planktotrophic stages of Strombus gigas

ABSTRACT The queen conch, Strombus gigas (Linnaeus, 1758), is a marine mollusc of ecological and economic importance in the Caribbean. Its populations are declining due to overexploitation. We describe ontogenesis of the digestive gland in S. gigas during the larval stages. Larvae were studied over a period of 42 d in laboratory culture, from eggs to crawling stage. Experiments were conducted at 28 ± 1°C. Veligers were reared at a density of 100 larvae l−1 in 10-l containers. Larvae were fed with the microalgae Nannochloropsis oculata at a concentration of 1,000 cells l−1. In this study, we analysed ultrastructural ontogenesis of the digestive gland in strombid larvae using light and electron microscopy. Examination for Coccidia (Apicomplexa) symbionts in the digestive gland was done by viewing sections with light and scanning electron microscopes at different larval development stages during a 42-d period. In early veligers (9 d after hatching), only digestive cells were observed in the digestive gland. By the late veliger stages (17 d old), both digestive and crypt cells were apparent in the digestive gland. Within crypt cells, spherocrystals were detected and the presence of Ca, Cl, Cu, P and Zn was identified by energy-dispersive X-ray spectroscopy. In late pediveligers (35 d old), the digestive gland still exhibited only digestive and crypt cells. Vacuolated cells (i.e. harbour the coccidian symbionts in adults) were only observed in newly settled juveniles (42 d old) and were devoid of apicomplexan structures. While coccidian symbionts were found in the digestive gland of adult S. gigas, they were not observed in the digestive gland of S. gigas larvae from hatching to settlement under laboratory conditions. This suggests that this symbiont is not vertically transmitted to new host generations in this marine gastropod species.

SEBS colonized in ileal mucosa, optimized bacterial composition, improved Se level and activated TLR2－NF-kB1 signaling pathway to regulate β-defensin 1 expression

Background Both selenium (Se) and probiotic bacillus improve immunity. Beta defensin 1 (BD1), a component of intestinal mucosal immunity, could be up-regulated in dietary selenium enriched bacillus subtilis (SEBS) supplementation. SEBS was supplemented in the culture medium of mouse intestinal crypt cells and the diets of chicks to observe the effects of SEBS on BD1 in the intestine by colonization of bacillus subtilis, its recognition and signaling pathway, bacterial composition optimization, and biological functions of Se. Results BD1 was formed in intestinal crypt cells and secreted into the lumen through the villi brush border. BD1 was up-regulated in distal ileum segments with SEBS and bacillus subtilis colonization. This occurred through the recognition of toll like receptor 2 and the NF-kB1 signaling pathway (TLR2-MyD88།NF-kB1), this increased expression was further enhanced with Se combination. Pro-inflammatory cytokines, TNF-α, IL-1β, and IL-6, were up-regulated with bacillus subtilis supplementation, while this up-regulation was inhibited with Se. Colonization of bacillus subtilis in distal segments of the ileum improved bacterial diversity, while reducing the number of endogenous Salmonella and lactobacilli sp. in ileal mucous membranes with SEBS supplementation. Species of unclassified Lachnospiraceae, uncultured Anaerosporobacter, Ruminococcaceae_UCG-014, Peptococcus, and Lactobacillus salivarius, and unclassified Butyricicoccus were substantial in ileal mucous membranes to promote BD1 concentration. Conclusion SECB, colonized in the ileal mucous membrane, optimized bacterial composition, enhanced BD1 secretion through activation of the TLR2-MyD88།NF-kB1 signaling pathway, and reduced pro-inflammatory factors. Our results suggest a new avenue for the combination of probiotic bacteria and essential micro-element selenium to improve intestinal mucosal immunity, increase defense against cold stress, and reduce illness incidence and mortality.