Hideto Yuasa

Peculiar Composition of Epithelial Cells in Follicle-Associated Intestinal Crypts of Peyer's Patches in the Rat Small Intestine

ABSTRACT The epithelial cell composition was investigated in the follicle-associated intestinal crypt (FAIC) of rat Peyer’s patches. The epithelium of the FAIC mainly consisted of columnar epithelial cells, goblet cells and Paneth cells. The characteristics of secretory granules in Paneth cells and goblet cells of both the FAIC and ordinary intestinal crypts (IC) were almost the same in periodic acid-Schiff (PAS) reaction, Alcian blue (AB) staining and the immunohistochemical detection of lysozymes and soluble phospholipase A2. Both goblet cells and Paneth cells were markedly less frequent on the follicular sides than on the anti-follicular sides of the FAIC. Goblet cells were also markedly less frequent in the follicle-associated epithelium (FAE) than in the ordinary intestinal villi (IV). Indigenous bacteria were more frequently adhered to FAE than to follicle-associated intestinal villi or IV. These findings suggest that the host defense against indigenous bacteria is inhibited on the follicular sides of FAIC, which might contribute to the preferential settlement of indigenous bacteria on the FAE; they also suggest that differentiation into secretory cells is inhibited in the epithelium of the follicular sides of FAIC, so that differentiation into M cells might be admitted in the FAE of rat Peyer’s patches. Furthermore, intermediate cells possessing characteristics of both Paneth cells and goblet cells were rarely found in the FAIC, but not in the IC. This finding suggests that the manner of differentiation into Paneth cells in the FAIC differs from that in the IC.

Ontogenic and morphological study of gonadal formation in genetically-modified sex reversal XY(POS) mice.

Mammalian sexual fate is determined by the presence or absence of sex determining region of the Y chromosome (Sry) in the “bipotential” gonads. Recent studies have demonstrated that both male and female sexual development are induced by distinct and active genetic pathways. Breeding the Y chromosome from Mus m. domesticus poschiavinus (POS) strains into C57BL/6J (B6J) mice (B6J-XYPOS) has been shown to induce sex reversal (75%: bilateral ovary, 25%: true hermaphrodites). However, our B6N-XYPOS mice, which were generated by backcrossing of B6J-XYPOS on an inbred B6N-XX, develop as males (36%: bilateral testis with fertility as well as bilateral ovary (34%), and the remainder develop as true hermaphrodites. Here, we investigated in detail the expressions of essential sex-related genes and histological features in B6N-XYPOS mice from the fetal period to adulthood. The onsets of both Sry and SRY-box 9 (Sox9) expressions as determined spatiotemporally by whole-mount immunohistochemistry in the B6N-XYPOS gonads occurred 2–3 tail somites later than those in B6N-XYB6 gonads, but earlier than those in B6J-XYPOS, respectively. It is possible that such a small difference in timing of the Sry expression underlies testicular development in our B6N-XYPOS. Our study is the first to histologically show the expression and ectopic localization of a female-related gene in the XYPOS testes and a male-related gene in the XYPOS ovaries. The results from these and previous experiments indicate that the interplay between genome variants, epigenetics and developmental gene regulation is crucial for testis development.

Immunohistochemical analysis of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity on the developmental dentate gyrus and hippocampal fimbria in fetal mice

Dioxins are widespread persistent environmental contaminants with adverse impacts on humans and experimental animals. Behavioral and cognitive functions are impaired by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure. TCDD exerts its toxicity via the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor. The hippocampus, which plays important roles in episodic memory and spatial function, is considered vulnerable to TCDD-induced neurotoxicity, because it contains the AhR. We herein investigated the effects of TCDD toxicity on hippocampal development in embryonic mice. TCDD was administered to dams at 8.5 days postcoitum with a single dose of 20, 200, 2,000 and 5,000 ng/kg body weight (groups T20, T200, T2000 and T5000, respectively), and the brains were dissected from their pups at embryonic day 18.5. Immunohistochemical analysis demonstrated that the Glial Fibrillary Acidic Protein (GFAP) immunoreactivities in the dentate gyrus (DG) were reduced in the T5000 group. Granular GFAP immunoreactivity was observed in the hippocampal fimbria, and the number of immunoreactive fimbria was significantly decreased in the T5000 group. The number of Proliferating Cell Nuclear Antigen (PCNA)-positive cells was decreased in all TCDD-exposed groups and significantly reduced in the T20, T200 and T5000 groups. Together, these results demonstrate that maternal TCDD exposure has adverse impacts on neural stem cells (NSCs), neural precursor cells (NPCs) and granular cells in the DG and disrupts the NSC maintenance and timing of differentiation in the hippocampal fimbria, which in turn interrupt neuronal development in future generations of mice.

Ultrastructural and histochemical study on the Paneth cells in the rat ascending colon.

Paneth cells (PCs) contribute to the host defense against indigenous bacteria in the small intestine. We found Paneth cell‐like cells (PLCs) in the rat ascending colon, but the nature of PLCs is never clarified. Therefore, the present study aimed to clarify the cytological characteristics of PLCs and discuss their cellular differentiation. PLCs were localized in the bases of intestinal crypts, especially follicle‐associated intestinal crypts in proximal colonic lymphoid tissue, but were very seldom found in the ordinary intestinal crypts of the ascending colon. PLCs possessed specific granules with highly electron‐dense cores and haloes, as well as PCs in the small intestine. The secretory granules of PLCs were positive for PAS reaction, lysozyme and soluble phospholipase A2, but negative for Alcian blue staining, β‐defensin‐1 and ‐2, as well as the ones of PCs. Furthermore, intermediate cells possessing both the PLC‐specific granules and the mucus granules similar to those of goblet cells (GCs) were occasionally found in the vicinity of PLCs. Intermediate cells ranged from goblet cell‐like cells rich in mucus granules to PLC‐like cells with few mucus granules. The cellular condensation and fragmentation were exclusively found in PLCs but never seen in intermediate cells or GCs. The PLCs, which were identified as PC, were suggested to be transformed from GCs through intermediate cells and finally to die by apoptosis in intestinal crypts of proximal colonic lymphoid tissue in the rat ascending colon. Anat Rec, 297:1462–1471, 2014.

Immunohistochemical study on the distribution of β -defensin 1 and β -defensin 2 throughout the respiratory tract of healthy rats

The distributions of β-defensin 1 and 2 in secretory host defense system throughout respiratory tract of healthy rats were immunohistochemically investigated. In the nasal epithelium, a large number of non-ciliated and non-microvillous cells (NCs) were immunopositive for both β-defensin 1 and 2, whereas a small number of goblet cells (GCs) were immunopositive only for β-defensin 1. Beta-defensin 2-immunopositive GCs were few. In the nasal glands, a small number of acinar cells and a large number of ductal epithelial cells were immunopositive for both β-defensins. In the laryngeal and tracheal epithelia, a very few NCs and GCs were immunopositive for both β-defensins. In laryngeal and tracheal glands, a very few acinar cells and a large number of ductal epithelial cells were immunopositive for both β-defensins. In the extra-pulmonary bronchus, a small number of NCs were immunopositive for both β-defensins. A small number of GCs were immunopositive for β-defensin 1, whereas few GCs were immunopositive for β-defensin 2. From the intra-pulmonary bronchus to alveoli, a very few or no epithelial cells were immunopositive for both β-defensins. In the mucus and periciliary layers, β-defensin 1 was detected from the nose to the extra-pulmonary bronchus, whereas β-defensin 2 was weakly detected only in the nose and the larynx. These findings suggest that the secretory sources of β-defensin 1 and 2 are mainly distributed in the nasal mucosa and gradually decrease toward the caudal airway in healthy rats.

Ultrastructural study on the morphological changes in indigenous bacteria of mucous layer and chyme throughout the rat intestine

Indigenous bacteria in the alimentary tract are exposed to various bactericidal peptides and digestive enzymes, but the viability status and morphological changes of indigenous bacteria are unclear. Therefore, the present study aimed to ultrastructurally clarify the degeneration and viability status of indigenous bacteria in the rat intestine. The majority of indigenous bacteria in the ileal mucous layer possessed intact cytoplasm, but the cytoplasm of a few bacteria contained vacuoles. The vacuoles were more frequently found in bacteria of ileal chyme than in those of ileal mucous layer and were found in a large majority of bacteria in both the mucous layer and chyme throughout the large intestine. In the dividing bacteria of the mucous layer and chyme throughout the intestine, the ratio of area occupied by vacuoles was almost always less than 10%. Lysis or detachment of the cell wall in the indigenous bacteria was more frequently found in the large intestine than in the ileum, whereas bacterial remnants, such as cell walls, were distributed almost evenly throughout the intestine. In an experimental control of long-time-cultured Staphylococcus epidermidis on agar, similar vacuoles were also found, but cell-wall degeneration was never observed. From these findings, indigenous bacteria in the mucous layer were ultrastructurally confirmed to be the source of indigenous bacteria in the chyme. Furthermore, the results suggested that indigenous bacteria were more severely degenerated toward the large intestine and were probably degraded in the intestine.

Ultrastructural and Immunohistochemical Study on the Lamina Propria Cells Beneath Paneth Cells in the Rat Ileum: LAMINA PROPRIA CELLS BENEATH PANETH CELLS

Paneth cells secrete bactericidal substances in response to bacterial proliferation on the mucosal surface without directly contacting bacteria. However, the induction mechanism of this transient secretion has not been clarified, although nervous system and/or immunocompetent cells in the lamina propria (LP) might be involved. In this study, we ultrastructurally and immunohistochemically investigated which LP cells are localized beneath Paneth cells and examined the relationship between the Paneth cell‐derived cellular processes which extended into the LP and the LP cells. The results showed that various cells—including blood capillary, subepithelial stromal cell, and nerve fiber—were present in the LP beneath Paneth cells. Endothelial cells of blood capillary were the cells most frequently found in this location; they were situated within 1 μm of the Paneth cells and possessed fenestration on the surfaces adjacent to Paneth cells. The Paneth cells rarely extended the cellular processes toward the LP across the basal lamina. Most of the cellular processes of Paneth cells contacted the subepithelial stromal cells. Immunohistochemistry revealed that the CD34+CD31−αSMA− stromal cells preferentially localized in the LP beneath the intestinal crypt base, while PDGFRαhiαSMA+ stromal cells mainly localized around the lateral portions of the intestinal crypt and PDGFRαhiαSMA− stromal cells localized in the intestinal villus. From these findings, the existence of blood capillaries beneath Paneth cells might reflect the active exocrine function of Paneth cells. Furthermore, subepithelial stromal cells, probably with a CD34+CD31−αSMA− PDGFRα−/lo phenotype, beneath the crypt base might affect Paneth cell activity by interacting with their cellular processes. Anat Rec, 301:1074–1085, 2018.

Mechanism of M-cell differentiation accelerated by proliferation of indigenous bacteria in rat Peyer's patches

The mechanism by which indigenous bacteria on the follicle-associated epithelium (FAE) of lymphatic follicles (LFs) accelerate the differentiation of microvillous columnar epithelial cells (MV) into M-cells was immunohistochemically investigated in rat Peyer’s patches. The results showed that the number of Toll-like receptor (TLR) -4+ M-cells was greater in the FAE with expansion of bacterial colonies (LFs with bacterial colonies on the FAE: b-LF) than the FAE without expansion of bacterial colonies (nb-LF). TLR-4 was also expressed in the striated borders of MV upstream next to M-cells in the FAE of the b-LF. TLR-4+ vesicles were frequently detected in the cytoplasms of MV with TLR-4+ striated borders upstream next to TLR-4+ M-cells in the FAE of b-LF. These findings suggest that TLR-4+ MV take up TLR-4 ligands and differentiate into M-cells in the b-LF. Neither the distribution of RANK nor that of RANKL was coincident with that of M-cells in the b-LF. Moreover, RANK, but not RANKL, was expressed in intestinal villi, whereas cleaved caspase-3 was immunonegative in the MV and M-cells of the FAE, unlike in villous epithelial cells. Therefore, RANK/RANKL signaling in the LF might contribute to the down-regulation of epithelial apoptosis to facilitate the differentiation of MV into M-cells in rat Peyer’s patches.

Immunohistochemical study on the secretory host defense system with lysozyme and secretory phospholipase A2 throughout rat respiratory tract

The host defense system with lysozyme and secretory phospholipase A2 (sPLA2) was immunohistochemically investigated in rat respiratory tract under healthy conditions. In the nasal epithelium, a large number of non-ciliated and non-microvillous cells (NC) and a small number of goblet cells (GC) were immunopositive for lysozyme and sPLA2. A few acinar cells and almost all epithelial cells of intercalated ducts were immunopositive for both bactericidal substances in the nasal glands. In the laryngeal and tracheal epithelia, few NC and GC were immunopositive for both bactericidal substances. In the laryngeal and tracheal glands, a few acinar cells and most ductal epithelial cells were immunopositive for both bactericidal substances. In extra-pulmonary bronchus, small numbers of NC and GC were immunopositive for lysozyme and sPLA2, whereas few NC and no GC were immunopositive in the intra-pulmonary bronchus. No secretory source of either bactericidal substance was located in the bronchioles. In the alveolus, many glandular epithelial cells and alveolar macrophages were immunopositive for lysozyme but immunonegative for sPLA2. Moreover, lysozyme and sPLA2 were detected in the mucus layer and in the periciliary layer from the nose to the extra-pulmonary bronchus. These findings suggest that secretory sources of lysozyme and sPLA2 are distributed in almost all the respiratory tract. Their secretory products are probably transported to the pharynx and contribute to form the first line of defense against inhaled bacteria throughout the respiratory tract.

Differential expression of Toll-like receptor-2, -4 and -9 in follicle-associated epithelium from epithelia of both follicle-associated intestinal villi and ordinary intestinal villi in rat Peyer’s patches

The expressions of Toll-like receptor (TLR) -2, -4 and -9 were immunohistochemically investigated in the follicle-associated epithelium (FAE), and epithelia of the follicle-associated intestinal villus (FAIV) and ordinary intestinal villus (IV) in rat Peyer’s patch regions with no bacterial colonies on the mucous membranes. TLR-2 was expressed in the striated borders of microvillous columnar epithelial cells (MV) in both FAIV and IV except in the apices. However, TLR-2 expression in the striated borders was weaker in the epithelium of the follicular side of FAIV (f-FAIV) than in epithelia of IV and the anti-follicular side of FAIV. TLR-4 and -9 were not expressed in the FAIV and IV. In the FAE, TLR-2, -4 and -9 were not expressed in the striated borders of MV, but the roofs of some typical M-cells were immunopositive for all TLRs. Especially, no TLR-positive MV were found at the FAE sites where M-cells appeared most frequently. In the follicle-associated intestinal crypt (FAIC), immunopositivity for all TLRs was observed in the striated borders of MV and the luminal substances. In conclusion, the lower levels of TLR-2 in both FAE and the epithelium of f-FAIV probably reduce recognition of indigenous bacteria. TLR-2, -4 and -9 appear not to participate directly in differentiation of MV into M-cells, because TLRs were not expressed in any MV in the upstream region of M-cells in FAE with no settlement of indigenous bacteria in the rat Peyer’s patches.