Effect of seasonal variance on intestinal epithelial barriers and the associated innate immune response of the small intestine of the Chinese soft-shelled turtles.

It is conceivable that pathological conditions can cause intestinal barrier disruption and innate immune dysfunction. However, very limited information has been reported on the effect of seasonal variance on intestinal barriers and innate immunity. The present study was designed to investigate the seasonal variance in intestinal epithelial barriers and the associated innate immune response of turtle intestines during hibernation and nonhibernation periods. Goblet cells (GCs) demonstrated dynamic actions of the mucosal barrier with strong Muc2 protein expression during hibernation. However, weak Muc2 expression during nonhibernation was confirmed by immunohistochemistry, immunofluorescence and immunoblotting. Furthermore, light and transmission electron microscopy revealed that the hypertrophy of GCs resulted in the hypersecretion of mucus granules (MGs) and created a well-developed mucosal layer during hibernation. The absorptive cells (ACs), forming a physical barrier of tight junctions, and desmosomes were firmly anchored during hibernation. Conversely, during nonhibernation, the integrity of tight junctions, adherence junctions and desmosomes was noticeable expanded, causing increased paracellular permeability. As further confirmation, there was strong zonula occluden-1 (ZO-1) and connexins 43 (Cx43) protein expression during hibernation and weak ZO-1 and Cx43 expression during nonhibernation. Moreover, the expression level of the innate immune response proteins Toll-like receptors 2 and 4 (TLR2 and 4) were enhanced during hibernation and were reduced during nonhibernation. These results provide rich information about the seasonal fluctuations that interrupt intestinal epithelial barriers and innate immune response, which might be essential for protection and intestinal homeostasis.

Cellular Evidence of CD63-Enriched Exosomes and Multivesicular Bodies within the Seminiferous Tubule during the Spermatogenesis of Turtles.

The seminiferous tubule (ST) is the location of spermatogenesis, where mature spermatozoa are produced with the assistance of Sertoli cells. The role of extracellular vesicles in the direct communication between Sertoli-germ cells in the ST is still not fully understood. In this study, we reported multivesicular bodies (MVBs) and their source of CD63-enriched exosomes by light and ultrastructure microscopy during the reproductive phases of turtles. Strong CD63 immunopositivity was detected at the basal region in the early and luminal regions of the ST during late spermatogenesis by immunohistochemistry (IHC), immunofluorescence (IF), and western blot (WB) analysis. Labeling of CD63 was detected in the Sertoli cell cytoplasmic processes that surround the developing germ cells during early spermatogenesis and in the lumen of the ST with elongated spermatids during late spermatogenesis. Furthermore, ultrastructure analysis confirmed the existence of numerous MVBs in the Sertoli cell prolongations that surround the round and primary spermatogonia during acrosome biogenesis and with the embedded heads of spermatids in the cytoplasm of Sertoli cells. Additionally, in spermatids, Chrysanthemum flower centers (CFCs) generated isolated membranes involved in MVBs and autophagosome formation, and their fusion to form amphiosomes was also observed. Additionally, autophagy inhibition by 3-methyladenine (after 24 h) increased CD63 protein signals during late spermatogenesis, as detected by IF and WB. Collectively, our study found MVBs and CD63 rich exosomes within the Sertoli cells and their response to autophagy inhibition in the ST during the spermatogenesis in the turtle.

Seasonal exploration of ultrastructure and Na+/K+-ATPase, Na+/K+/2Cl- cotransporter of mitochondria-rich cells in the small intestine of turtles.

Despite the exploration of mitochondria-rich cells (MRCs) in different animal classes, very limited information has been documented about MRCs in reptiles. The present study was designed to investigate the effect of seasonal variation on the cell ultrastructure and ion transport protein activity of MRCs during hibernation and non-hibernation of Chinese soft-shelled turtle's intestine. Transmission electron microscopy revealed that, during hibernation the high-density cytoplasm of MRCs occupied large cross-sectional area and showed heterogeneous abundance of mitochondria and an expanded extensive tubular system as compared to non-hibernation. During hibernation the cytoplasm of MRCs exhibited more mitochondrial vacuolization, autophagosomes, phagophore formation and well-structured endoplasmic reticulum. During hibernation, MRCs connected with absorptive cells through wide interdigitation, and created tight junction and more desmosomes as compared to non-hibernation. Immunohistochemistry and immunofluorescence showed, the strong immunopositive reactions and immunosignaling of Na+/K+-ATPase (NKA) and Na+/K+/2Cl- cotransporter (NKCC) at basolateral region of mucosal surface of intestine during hibernation. However, weak immunopositive reactions and immunosignaling of NKA and NKCC during non-hibernation. The statistical analysis showed that the number and size of MRCs with NKA-associated immunoreactivity were significantly increased during hibernation. NKA and NKCC mRNA expression was significantly increased during hibernation via qPCR. Further confirmed, the intensity of NKA and NKCC proteins was more elevated during hibernation than non-hibernation shown by immunobloting. However, the concentrations of the plasma ions Na+ and Cl- were significantly higher during hibernation; conversely, K+ concentration was significantly higher during non-hibernation. The findings suggest that the potential role of MRCs is affected by seasonal fluctuations, during which intestinal homeostasis and hydromineral balance are essential for turtles.