A Single-Cell Atlas of an Early Mongolian Sheep Embryo.

Cell types have been established during organogenesis based on early mouse embryos. However, our understanding of cell types and molecular mechanisms in the early embryo development of Mongolian sheep has been hampered. This study presents the first comprehensive single-cell transcriptomic characterization at E16 in Ujumqin sheep and Hulunbuir short-tailed sheep. Thirteen major cell types were identified at E16 in Ujumqin sheep, and eight major cell types were identified at E16 in Hulunbuir short-tailed sheep. Function enrichment analysis showed that several pathways were significantly enriched in the TGF-beta signaling pathway, the Hippo signaling pathway, the platelet activation pathway, the riboflavin metabolism pathway, the Wnt signaling pathway, regulation of the actin cytoskeleton, and the insulin signaling pathway in the notochord cluster. Glutathione metabolism, glyoxylate, and dicarboxylate metabolism, the citrate cycle, thyroid hormone synthesis, pyruvate metabolism, cysteine and methionine metabolism, thermogenesis, and the VEGF signaling pathway were significantly enriched in the spinal cord cluster. Steroid biosynthesis, riboflavin metabolism, the cell cycle, the Hippo signaling pathway, the Hedgehog signaling pathway, the FoxO signaling pathway, the JAK-STAT signaling pathway, and the Wnt signaling pathway were significantly enriched in the paraxial mesoderm cluster. The notochord cluster, spinal cord cluster, and paraxial mesoderm cluster were found to be highly associated with tail development. Pseudo-time analysis demonstrated that the mesenchyme can translate to the notochord in Ujumqin sheep. Molecular assays revealed that the Hippo signaling pathway was enriched in Ujumqin sheep. This comprehensive single-cell map revealed previously unrecognized signaling pathways that will further our understanding of the mechanism of short-tailed sheep formation.

Dosage Compensation of the X Chromosome during Sheep Testis Development Revealed by Single-Cell RNA Sequencing.

Dosage compensation is a mechanism first proposed by Susumu Ohno, whereby X inactivation balances X gene output between males (XY) and females (XX), while X upregulation balances X genes with autosomal gene output. These mechanisms have been actively studied in Drosophila and mice, but research regarding them lags behind in domestic species. It is unclear how the X chromosome is regulated in the sheep male germline. To address this, using single-cell RNA sequencing, we analyzed testes in three important developmental stages of sheep. We observed that the total RNA per cell from X and autosomes peaked in SSCs and spermatogonia and was then reduced in early spermatocytes. Furthermore, we counted the detected reads per gene in each cell type for X and autosomes. In cells experiencing dose compensation, close proximity to MSL (male-specific lethal), which is regulated the active X chromosome and was observed. Our results suggest that there is no dose compensation in the pre-meiotic germ cells of sheep testes and, in addition, MSL1 and MSL2 are expressed in early germ cells and involved in regulating mammalian X-chromosome inactivation and activation.

Skin transcriptome reveals the dynamic changes in the Wnt pathway during integument morphogenesis of chick embryos.

Avian species have a unique integument covered with feathers. Skin morphogenesis is a successive and complex process. To date, most studies have focused on a single developmental point or stage. Fewer studies have focused on whole transcriptomes based on the time-course of embryo integument development. To analyze the global changes in gene expression profiles, we sequenced the transcriptome of chicken embryo skin samples from day 6 to day 21 of incubation and identified 5830 differentially expressed genes (DEGs). Hierarchical clustering showed that E6 to E14 is the critical period of feather follicle morphogenesis. According to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the DEGs, two kinds of Wnt signaling pathways (a canonical pathway and a non-canonical pathway) changed during feather follicle and feather morphogenesis. The gene expression level of inhibitors and ligands related to the Wnt signaling pathway varied significantly during embryonic development. The results revealed a staggered phase relationship between the canonical pathway and the non-canonical pathway from E9 to E14. These analyses shed new light on the gene regulatory mechanism and provided fundamental data related to integument morphogenesis of chickens.

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

The distributions of ß-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 ß-defensin 1 and 2, whereas a small number of goblet cells (GCs) were immunopositive only for ß-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 ß-defensins. In the laryngeal and tracheal epithelia, a very few NCs and GCs were immunopositive for both ß-defensins. In laryngeal and tracheal glands, a very few acinar cells and a large number of ductal epithelial cells were immunopositive for both ß-defensins. In the extra-pulmonary bronchus, a small number of NCs were immunopositive for both ß-defensins. A small number of GCs were immunopositive for ß-defensin 1, whereas few GCs were immunopositive for ß-defensin 2. From the intra-pulmonary bronchus to alveoli, a very few or no epithelial cells were immunopositive for both ß-defensins. In the mucus and periciliary layers, ß-defensin 1 was detected from the nose to the extra-pulmonary bronchus, whereas ß-defensin 2 was weakly detected only in the nose and the larynx. These findings suggest that the secretory sources of ß-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.

Immunohistochemical and histoplanimetrical study on the endothelial receptor involved in transportation of minute chylomicrons into subepithelial portal blood in intestinal villi of the rat jejunum.

A portion of the minute chylomicrons less than 75 nm in diameter are transcytosed from the extravascular tissue into the subepithelial blood capillaries (sBC) in the villous apices of the rat jejunum. However, the details of the transportation mechanism have not been clarified. In this study, the endothelial receptor involved in the transportation of minute chylomicrons into the sBC's lumina was immunohistochemically and histoplanimetrically examined in intestinal villi of the rat jejunum. Immunopositivity for very low density lipoprotein (VLDL) receptor was detected on the luminal and basal surfaces of the endothelial cells of sBC in approximately 68% of those apices of jejunal villi that possessed numerous chylomicrons in the lamina propria, while VLDL receptor was detected on the endothelial cells of sBC in only approximately 8% of intestinal villi that possessed few or no chylomicrons in the lamina propria. No immunopositivity for LDL receptor was detected in the sBC of all intestinal villi. These findings suggest that VLDL receptor is expressed by the endothelial cells of the sBC in conjunction with the filling of the lamina propria of jejunal villi with many chylomicrons produced by the villous columnar epithelial cells and that the VLDL receptor mediates the transportation of minute chylomicrons, maybe VLDL, into the subepithelial portal blood from the extravascular tissue of the rat jejunal villi.

Ultrastructural demonstration of the absorption and transportation of minute chylomicrons by subepithelial blood capillaries in rat jejunal villi.

Chylomicrons from villous columnar epithelial cells are generally known to be transported only by central lymph vessels (CLV), whereas antigenic particulates derived from the intestinal lumen can also be transported by subepithelial blood capillaries (sBCs) in rat intestinal villi. The possibility of chylomicron absorption by sBCs was histoplanimetrically studied in the rat jejunum under a transmission electron microscope. The chylomicrons more abundantly presented in villous venules than in arterioles. The most frequent size (MFS) of chylomicrons was 75 to 90 nm in diameter in the areas near sBCs, while it was 45 to 60 nm in the epithelial intercellular spaces just above sBCs or the intermediate areas between sBCs. The MFS of chylomicrons was 45 to 60 nm in the intermediate areas between sBCs and in the epithelial intercellular spaces just above these areas. The MFS of chylomicrons in CLV was intermediate between that in the area adjacent to sBCs and that in the intermediate areas between sBCs. Chylomicrons were found in small vesicles in the endothelial cytoplasms of sBCs. No chylomicrons larger than 600 nm were observed in the lamina propria. These findings suggest that some of the chylomicrons smaller than 75 nm, which are probable intestinal very low-density lipoproteins (VLDL), are directly transported to the liver by hepatic portal blood in addition to CLV and that epithelial fat droplets larger than 600 nm are not discharged into lamina propria in rat jejunum under physiological conditions.

Persorption of IgG-Fc-coated particulates from intestinal lumen into portal blood via villous columnar epithelial cells in rat small intestine.

Previously, the specific antibody-mediated persorption of antigenic molecules and particulates from the small-intestinal lumen into the peripheral blood was clarified in rats, but the intermediation of the receptor for the specific antibodies was not. In this study, the existence of receptor for the specific antibody was experimentally examined in the rat small intestine. Glutaraldehyde-fixed sheep erythrocytes (SEs) coated by Fc-fragments of IgG (IgG-Fc), (Fab')(2)-fragments of IgG (IgG-Fab) or bovine serum albumin (BSA), were injected into 3 jejunal loops each 2 cm in length in non-orally pre-immunized rats, respectively. Thirty minutes after the injection, IgG-Fc-coated SEs were significantly more engulfed by villous columnar epithelial cells than Fab- or BSA-coated SEs. The most frequent absorption sites were the intestinal villous apices. The IgG-Fc-coated SEs were adhered to the striated borders and were engulfed by villous columnar epithelial cells. IgG-Fc-coated SEs passing through the epithelial cells were also detected in the subepithelial blood capillaries just beneath the villous epithelium, but not in the connective tissue and the lymph vessels. These findings suggest that the absorption of luminal antigenic particulates is probably mediated by the Fc-receptor, and that the absorbed antigenic particulates are directly transferred to the hepatic portal blood by passing through the endothelium of the subepithelial blood capillaries.

Immunohistochemical and histoplanimetrical study on the spatial relationship between the settlement of indigenous bacteria and the secretion of bactericidal peptides in rat alimentary tract.

To clarify the regulatory mechanism by bactericidal peptides secretion, the secretion of bactericidal peptides was immunohistochemically and histoplanimetrically compared with the degree of Gram-positive/negative bacterial colonization throughout the rat alimentary tract. In the associated exocrine glands from the oral cavity to the stomach, no comparable differences were observed under the changes of development of indigenous bacterial colonies. In the small intestine, immunopositive granules for lysozyme and secretory phospholipase A2 (sPLA2) were markedly decreased, whereas immunopositive vacuoles in the Paneth cells were more increased at sites with hyper-development of indigenous bacterial colonies in the intervillous spaces than at sites with no or less development. No changes in exocrine glands were observed in the large intestine because of the constant existence of large quantities of bacteria. Gram-positive bacterial colonies on the mucosal surfaces were dominant from the oral cavity to the stomach. Gram-negative bacteria were dominant in the large intestine, and the distributions of both Gram-positive and negative bacteria were intermediate in the small intestine. These findings suggest that lysozyme and sPLA2 secreted from the Paneth cells contribute to the regulation of the proliferation of indigenous bacteria in the intervillous spaces of the small intestine, and that the inversion of distributions of Gram-positive and -negative bacteria in the alimentary tract might be caused by the secretion of lysozyme and sPLA2 in the small intestine.

Immunohistochemical detection of phosphatidylserine and thrombospondin on denucleating erythroblasts in rat bone marrow.

The hypothesis that apoptotic factors play some roles in the denucleation of erythroblasts has been confirmed by the immunohistological detection of both phosphatidylserine and thrombospondin as phagocytosis-inducing factors in general apoptotic events. Both phosphatidylserine and thrombospondin were detected on the surface of cell membrane of mature erythroblasts, while thrombospondin was also detected in more immature erythroblasts. The intensities of their immune reactions increased as the erythroids matured. During denucleation, the positivities of both phosphatidylserine and thrombospondin were restricted on the surface of the cell membrane surrounding the protruding nuclei. Thus, the apoptotic process involves denucleation of erythroblasts and phosphatidylserine, and thrombospondin acts as phagocytosis-inducing factors in the denucleation event.

Immunohistochemical study on the secretory host defense system of bactericidal peptides in rat digestive organs.

To clarify the fundamental regulation mechanism against indigenous bacterial proliferation in the alimentary tract, we immunohistochemically examined the localization of 4 bactericidal peptides (BP) in the rat digestive exocrine glands. In the upper alimentary tract, lysozyme was detected in the gustatory, extraorbital lacrimal and parotid glands. Secretory phospholipase A2 (sPLA2) was detected in the extraorbital lacrimal glands. ß-defensin1 was detected in the gustatory and extraorbital lacrimal glands. ß-defensin2 was detected in the Harderian glands. In the stomach, ß-defensins were detected in the gastric superficial epithelial cells. In the small and large intestines, only lysozyme and sPLA2 were detected in the Paneth cells. In the cecum, all 4 BP were detected in the middle to apical portions of the crypts, and only sPLA2 was detected in the basal portion. No BP were localized in other exocrine glands associated with the alimentary tract. In addition, all 4 BP were also detected in the columnar epithelial cells of the apical portions of intestinal villi. In the intestinal superficial epithelial cells, lysozyme and ß-defensins were detected in the ascending colon, whereas only ß-defensin1 was detected in the descending colon and rectum. These results suggest that BP are mainly secreted from exocrine tissues in the initial portion of the digestive tract and play a role in host defense against indigenous bacteria throughout the digestive tract. Part of the BP in the chyme might be absorbed by the epithelium at the most inner sites of mucosae in the small and large intestines.

Lectin histochemical detection of special sugars on the mucosal surfaces of the rat alimentary tract.

Surfaces of the most luminal positions of mucosae are fundamental settlement sites of indigenous bacteria throughout the rat alimentary tract. In these positions, also epithelial cell-shedding sites, the special sugar expression in the glycocalyx is very important as it provides possible ligands of bacterial lectins for attachment to epithelial cells. Therefore, the sugar expression in glycocalyx of epithelial cells was lectin-histochemically surveyed using 21 lectins throughout the rat alimentary tract. From the tongue to the nonglandular part of the stomach, α-D-Man, α-D-Glc and α-D-GalNAc were detected on the surface of the keratinized stratified squamous epithelium. In the glandular part of the stomach, α-D-Man, ß-D-Gal-4GlcNAc, D-Gal, D-GalNAc, D-GlcNAc, α-L-Fuc- α-D-Gal-ß(1-4)GlcNAc and bisected triantennary N-glycans were detected on the surface of gastric superficial epithelial cells. From the duodenum to the ileum, (GlcNAc)(2-4) was expressed exclusively on the epithelial cells in the apical portions of the intestinal villi. From the cecum to the rectum, α-D-Man, ß-D-Gal-4GlcNAc, D-Gal, D-GalNAc, α-D-Gal(1-3)D-GalNAc, (GalNAc)(n) and NeuNAc were expressed on the intestinal superficial epithelial cells. These results suggest that special sugars are expressed on the most luminal portions of mucosae as exclusive epithelial cell-shedding sites, and that sugar expression differs among the various segments of the alimentary tract. These site differences might reflect differences in resident bacterial species in the rat alimentary tract.

Histoplanimetrical study on the relationship between cellular kinetics of epithelial cells and proliferation of indigenous bacteria in the rat colon.

The aim of this study was to clarify the regulatory effects of epithelial kinetics on indigenous bacterial proliferation in the large intestine. The lifespan, migration speed and proliferation rate of crypt epithelial cells in the initial 20% of the colon (proximal colon) and the 50% of the colon (middle colon) in bromodeoxyuridine-administrated rats were histoplanimetrically and chronologically compared. The proximal colon possessed well-developed mucosal folds and a large amount of indigenous bacteria which filled the crypt lumen, whereas no folds or bacteria were found to occupy the crypt lumen in the middle colon. The cell lifespans were 32.2, 42.5 and 33.6 hr in the apical and the basal parts of the mucosal folds of the proximal colon, and in the middle colon, respectively. The migration speeds were 4.2, 2.1 and 3.3 microm/hr, respectively, while the appearance frequencies of proliferating cell nuclear antigen (PCNA)-positive crypt epithelial cells were 35.0, 24.6 and 33.8%. These findings suggest that the lifespan was shortened and the migration speed increased in the most luminal mucosa of colon, contributing to the elimination of the adhered bacteria from the most luminal mucosa. By contrast, the elongation of the lifespan and deceleration of the migration of epithelial cells in the basal parts of the mucosal folds might contribute to reliable settlement of indigenous bacteria, resulting in the maintenance of a large amount of indigenous bacteria in the lumen of the proximal colon.

Histoplanimetrical study on the spatial relationship of distribution of indigenous bacteria with mucosal lymphatic follicles in alimentary tract of rat.

The spatial relationship between the distribution of indigenous bacteria (IB) and the situation of mucosal lymphatic follicles (LF) is histoplanimetrically studied in the rat alimentary tract. From the oral cavity to the nonglandular part of the stomach, IB adhered to the corneal layer of the most luminal mucosa. In the glandular part of the stomach, IB adhered only to the most luminal mucosa but not in the gastric pits. In the small intestine, IB consistently adhered around the apices of both intestinal villi and the domes, and their amounts decreased toward their basal portions. No IB entered the intestinal crypts. In the large intestine, IB consistently adhered to the most luminal mucosa. Numerous IB were suspended in the intestinal crypts of both the cecum and the proximal colon, whereas there were no IB in the crypts of the distal colon and the rectum. When IB spread over the basal portions of the intestinal villi, IB with the same morphology were detected on the neighboring LF, whereas no bacteria were detected on the neighboring LF, when IB were located in the apical to middle portions of the intestinal villi. This close relationship between the distribution of IB and mucosal LF was also observed in the large intestine. These results suggest that the most luminal mucosae are a fundamental settlement site of IB throughout the alimentary tract and that the hyperproliferation of IB's colonies might be detected by neighboring LF in the rat intestine.

Fetal and neonatal exposure to three typical environmental chemicals with different mechanisms of action: mixed exposure to phenol, phthalate, and dioxin cancels the effects of sole exposure on mouse midbrain dopaminergic nuclei.

A major question is whether exposure to mixtures of low-dose endocrine disruptors (EDs) having different action mechanisms affects neurodevelopment differently than exposure to EDs individually. We therefore investigated the effects of fetal and neonatal exposure to three typical EDs - bisphenol A (BPA), di-(2-ethylhexyl)-phthalate (DEHP), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) - on the midbrain dopaminergic system associated with functions - including motor activity, emotion, and cognition - affected by neuropsychiatric diseases such as attention-deficit/hyperactivity disorder. ICR mouse dams and their pups were orally treated with BPA (5mg/(kg day)), DEHP (1mg/(kg day)), or TCDD (8ng/kg) individually, or with mixtures thereof, to compare the effects between sole and mixed administration. We analyzed tyrosine hydroxylase (TH)- and Fos-immunoreactive (ir) neurons as markers of dopamine and neuronal activation, respectively. The numbers of TH- and/or Fos-ir neurons and the intensity of TH-immunoreactivity within midbrain dopaminergic nuclei (A9, A10, and A8) of each sole administration group significantly differed from controls at 2, 4, and 6 weeks of age. In contrast, no significant differences were detected in the mixture groups, suggesting counteractions among those chemicals. These results indicate that ED mixtures as pollution have unique and elusive effects. Thyroid hormones and/or aryl hydrocarbon receptor-related mechanisms may be responsible for this counteraction.

Histoplanimetrical study on the relationship between the cell kinetics of villous columnar epithelial cells and the proliferation of indigenous bacteria in rat small intestine.

The relationship between the kinetics of villous columnar epithelial cells and the expansion of colonies of indigenous bacteria from the narrow apical portions of intestinal villi was immunohistochemically and histoplanimetrically investigated in the small intestine of bromodeoxyuridine administred Wistar rats. As a result, the lifespan of villous columnar epithelial cells was slightly shorter in the distal ileum than in other portions of small intestine, accompanying the minimum height of the intestinal villi of the distal ileum in the small intestine. The migration speed of villous columnar epithelial cells was significantly decreased toward the distal small intestine. The migration speed in the distal ileum was about one-fourth of that in the duodenum. The migration speed of the villous columnar epithelial cells was greater and their lifespans were shorter in the sites with wide expansion of the indigenous bacterial colony from the narrow apical portions of the intestinal villi than that in sites with no or less expansion. Additionally, the expansion of the indigenous bacterial colony from narrow villous apices also immediately shortened the heights of the intestinal villi. These findings suggest that the migration speed of villous columnar epithelial cells might contribute to the regulation of the settlement of bacteria at the villous apices and the inevitable proliferation of indigenous bacteria at the intervillous spaces in the rat small intestine.

An immunohistochemical detection of actin and myosin in the indigenous bacteria-adhering sites of microvillous columnar epithelial cells in Peyer's patches and intestinal villi in the rat jejunoileum.

The mechanism of physical elimination of indigenous bacteria was ultrastructurally and immunohistochemically investigated in microvillous columnar epithelial cells of Peyer's patches and intestinal villi of the rat jejunoileum. From ultrastructural observation, the microfilaments accumulated to form several electron-dense layers beneath the bacteria adhering to the cell membrane, which was slightly invaginated in the epithelial cells of Peyer's patches and intestinal villi. As the microfilamentous layers were forming, the end portions of invaginations were deformed into a cone-shape and were finally collapsed. At the same time, the end portions of the adhered bacteria were also deformed into cone-shapes. The bacterial cells were moved back toward the invagination orifices with no morphological change in their inner structure. From immunohistochemical observation, beta-actin and nonmuscle-type myosin were detected at the thin layer just beneath the invaginated cell membrane. These findings suggest that indigenous bacteria which adhere to epithelial cells are removed by only a physical action of actin and myosin filaments, but are not killed. This bacterial cell removal system might lead to the establishment of a settlement of indigenous bacteria on host cells.

Ultrastructural study on the epithelial responses against attachment of indigenous bacteria to epithelial membranes in peyer's patches of rat small intestine.

The ultrastructure of epithelial responses against the membrane adhesion of indigenous bacteria was investigated in the follicle-associated epithelium (FAE) of rat small intestine. The most frequent adherence of the various morphological types of bacteria to the epithelial membranes was found at the apex of the FAE. The attachment sites were deeply invaginated, and their bottoms were deformed into a sharp cone shape. Four layers with different electron densities were formed just beneath the apical membranes by microfilaments which surrounded the invaginations. The electron density of each layer was gradually decreased as being apart from the invaginations. The extremities of some bacteria in the invaginations were deformed into sharpened shapes. The cell walls of the extremities of the bacteria were occasionally dissolved in the invaginations, and their cytoplasms were slightly swollen with low electron densities. In some invaginations, the attached bacteria were eliminated to leave their fragments such as filamentous debris and a part of cell walls. Finally these remnants disappeared completely. When the bacterial colonies existed in the middle region of the FAE, the attachment of bacteria resulted in the engulfment of bacteria by M cells. The degenerated bacteria whose cytoplasmic matrices were separated into high electron dense materials and cleared materials were occasionally engulfed by ordinary microvillous columnar epithelial cells or goblet cells throughout the FAE. These findings suggest that the epithelial cells reject the attachment of live indigenous bacteria and that the M cells absorb indigenous bacteria in rat Peyer's patches.

Immunohistochemical study on the delayed progression of epithelial apoptosis in follicle-associated epithelium of rat Peyer's patch.

It is well known that some caspases in apoptosis is involved in determinant of terminal differentiation and maturation of various cells. Our previous study ultrastructurally clarified the differentiation into M cells from immature microvillous epithelial cells and the redifferentiation from M cells to microvillous epithelial cells in the follicle-associated epithelium (FAE) of rat Peyer's patch. In this study, the difference of epithelial apoptosis between the FAE of Peyer's patch and intestinal villi was immunohistochemically investigated in rat jejunoileum. As a result, cleaved caspase-3 was limited to several epithelial cells at the tip of FAE, whereas almost all of the epithelial cells were cleaved caspase-3 positive in intestinal villi. Cleaved caspase-9 was detected only in a few exfoliating or exfoliated epithelial cells of both FAE and intestinal villi. Nuclear DNA-fragmentation was detected only in several epithelial cells of the tip of FAE, while it was expressed from the middle regions in the intestinal villi. The DNase I expression of the epithelial cytoplasm was much weaker in FAE than in intestinal villi. Bcl-x expression was restricted in the apical cytoplasms of epithelial cells in the FAE, whereas it was restricted in whole cytoplasms in villous epithelial cells. These findings suggest that the progression of the apoptotic process in the epithelial cells of FAE is later than in the intestinal villi, so that the possibility of epithelial differentiation might be remained in the FAE, unlike in the intestinal villi.

Ultrastructural study on the differentiation and the fate of M cells in follicle-associated epithelium of rat Peyer's patch.

The differentiation process of immature microvillous epithelial cells to M cells and the fate of M cells in the follicle-associated epithelium (FAE) of the mucosa-associated lymphoid tissues are still unclear. In this study, the differentiation process and the fate of M cells were clarified in rat Peyer's patches under a transmission electron microscope. Almost all immature epithelial cells were found to possess long, slender microvilli, which gradually shortened, thickened and dispersed as the immature epithelial cells migrated away from the crypt orifices. These morphological changes started in the centers and moved to the peripheries of the apical surfaces of epithelial cells, accompanied by the protrusion of apical cytoplasm out of the terminal web. During these changes, the bundles of microfilaments of microvilli never shortened, and both small vesicles in the apical cytoplasm and tiny invaginations of the apical membranes were found. The intraepithelial migrating cells gradually accumulated to form typical intraepithelial pockets. In all FAE, there was no morphological sign of cell death in M cells. The rearrangement of microfilament bundles, the reconstruction of microvilli and the disappearance of pockets resulted in the transformation of M cells into microvillous epithelial cells. These serial ultrastructural changes suggest that M cells are a temporal and transitional cell type caused by the active engulfment of luminal substances and that when the engulfment ceases, the M cells transform into mature microvillous epithelial cells.