Histopathological features of the proper gastric glands in FVB/N-background mice carrying constitutively-active aryl-hydrocarbon receptor.

BACKGROUND: Aryl-hydrocarbon receptor (AhR) is a multiple ligand-activated transcription factor that has important roles in xenobiotic, physiological, or pathological functions. Transgenic mice systemically expressing constitutively-active AhR (CA-AhR) have been created to mimic activated AhR signaling in vivo. However, their detailed histopathological features are unclear. In the present study, we generated CA-AhR-expressing FVB/N mice (FVB-CA-AhR mice) and clarified their phenotypes in detail. METHODS: Male and female FVB-CA-AhR and wild-type mice were histopathologically examined from 6 to 33 weeks of age. RESULTS: Among the systemic organs, only the stomachs in FVB-CA-AhR mice showed pathological changes including cystic structures beneath the serosa; in addition, stomach weights increased with age. Histopathologically, cystic structures and alcian blue-positive metaplasia were observed in the mucosa of the proper gastric glands, and these two histometric parameters were positively correlated. Furthermore, proliferating cells shifted from the isthmus to the base of the glands, and parietal cells decreased. Age-related histopathological changes were clearer in females than in males. Importantly, in FVB-CA-AhR mice, intramucosal cysts developed as extramucosal cysts beneath the serosa, penetrating the lamina muscularis mucosae and the muscularis propria. Their incidence reached 100% in 28-week-old male mice and 33-week-old female mice. Extramucosal cysts contained alcian blue-, Griffonia simplicifolia lectin II-, or trefoil factor 2-positive cells, suggesting a stomach origin for the cysts and spasmolytic polypeptide-expressing metaplasia-like lesions. CONCLUSIONS: Disease onset occurred earlier in FVB-CA-AhR mice than previously reported in C57BL/6-derived CA-AhR mice. Importantly, the histopathological features were partly similar with gastritis cystica profunda in humans and animals. Excessive activation of AhR signaling aggravated abnormalities in the gastric mucosa and were affected by both genetic- and sex-related factors.

Altered morpho-functional features of bones in autoimmune disease-prone BXSB/MpJ- Yaa mice.

IMPACT STATEMENT: Bone disease, such as osteoporosis and rheumatoid arthritis, increases because of the progression of an aging society. Autoimmune disease are important and predisposing factors for the pathogenesis of the bone disease; however, the pathological mechanism is unclear. We have demonstrated that systemic autoimmune disease in BXSB/MpJ- Yaa is closely associated with the morpho-functional abnormalities of bones including bone marrow and has complicated pathology. The abnormalities are characterized by altered regulations of serum calcium, anemia tendency, and hematopoiesis with increased WBCs and decreased PLs, short length and low mass of long bones, imbalance in the populations of osteoclasts and osteoblasts, and increased expression of candidate genes for causing and/or exacerbating their phenotypes. Therefore, BXSB/MpJ- Yaa serves as a model to elucidate bone phenotypes in systemic autoimmune disease that would be affected by the factors in the bone as well as the other immune and/or mineral metabolism organs both in human and experimental medicine.

MRL/MpJ mice produce more oocytes and exhibit impaired fertilisation and accelerated luteinisation after superovulation treatment.

MRL/MpJ mice exhibit distinct phenotypes in several biological processes, including wound healing. Herein we report two unique phenotypes in the female reproductive system of MRL/MpJ mice that affect ovulation and luteinisation. We found that superovulation treatment resulted in the production of significantly more oocytes in MRL/MpJ than C57BL/6 mice (71.0±13.4 vs 26.8±2.8 respectively). However, no exon mutations were detected in genes coding for female reproductive hormones or their receptors in MRL/MpJ mice. In addition, the fertilisation rate was lower for ovulated oocytes from MRL/MpJ than C57BL/6 mice, with most of the fertilised oocytes showing abnormal morphology, characterised by deformation and cytolysis. Histological tracing of luteinisation showed that MRL/MpJ mice formed corpora lutea within 36h after ovulation, whereas C57BL/6 mice were still at the corpora haemorrhagica formation stage after 36h. The balance between the expression of matrix metalloproteinases and their tissue inhibitors shifted towards the former earlier after ovulation in MRL/MpJ than C57BL/6 mice. This result indicates a possible link between accelerated extracellular matrix remodelling in the ovulated or ruptured follicles and luteinisation in MRL/MpJ mice. Together, these findings reveal novel phenotypes in MRL/MpJ mice that provide novel insights into reproductive biology.

Ovarian mast cells migrate toward ovary-fimbria connection in neonatal MRL/MpJ mice.

MRL/MpJ mice have abundant ovarian mast cells (MCs) as compared with other strains at postnatal day 0 (P0); however, they sharply decrease after birth. These ovarian MCs, particularly beneath the ovarian surface epithelium (SE), which express mucosal MC (MMC) marker, might participate in early follicular development. This study investigated the changes in spatiotemporal distribution of MCs in the perinatal MRL/MpJ mouse ovaries. At P0 to P7, the MCs were densely localized to the ovary, especially their caudomedial region around the ovary-fimbria connection. The neonatal ovarian MCs showed intermediate characteristics of MMC and connective tissue MC (CTMC), and the latter phenotype became evident with aging. However, the expression ratio of the MMC to CTMC marker increased from P0 to P4 in the MRL/MpJ mouse ovary. Similarly, the ratio of MCs facing SE to total MC number increased with aging, although the number of ovarian MCs decreased, indicating the relative increase in MMC phenotypes in the early neonatal ovary. Neither proliferating nor apoptotic MCs were found in the MRL/MpJ mouse ovaries. The parenchymal cells surrounding MCs at ovary-fimbria connection showed similar molecular expression patterns (E-cadherin+/Foxl2-/Gata4+) as that of the ovarian surface epithelial cells. At P2, around the ovary-fimbria connection, c-kit- immature oocytes formed clusters called nests, and some MCs localized adjacent to c-kit- oocytes within the nests. These results indicated that in postnatal MRL/MpJ mice, ovarian MCs changed their distribution by migrating toward the parenchymal cells composing ovary-fimbria connection, which possessed similar characteristics to the ovarian surface epithelium. Thus, we elucidated the spatiotemporal alterations of the ovarian MCs in MRL/MpJ mice, and suggested their importance during the early follicular development by migrating toward the ovary-fimbria connection. MRL/MpJ mice would be useful to elucidate the relationship between neonatal immunity and reproductive systems.

Cotton rats (Sigmodon hispidus) possess pharyngeal pouch remnants originating from different primordia.

Pharyngeal pouches in mammals develop into specific derivatives. If the differentiation of the pharyngeal pouches is anomalous, their remnants can result in cysts, sinuses, and fistulae in the differentiated organs or around the neck. In the present study, we found several pharyngeal pouch remnants, such as cystic structures in thymus and parathyroid gland and fossulae extended from the piriform fossa, in the inbred cotton rats maintained at Hokkaido Institute of Public Health (HIS/Hiph) and University of Miyazaki (HIS/Mz). In HIS/Hiph, the fossulae extended from the apex of the piriform fossa into the thyroid glands and were lined with stratified squamous and cuboidal epithelium. Calcitonin-positive C-cells were present within their epithelium in HIS/Hiph. In contrast, the fossulae of HIS/Mz ran outside the thyroid glands toward the parathyroid glands; they were lined with columnar ciliated epithelium and a few goblet cells, but had no C-cells, which was consistent with the cystic structures in the thymus and the parathyroid gland. These results indicated that the fossulae were a remnant of the ultimobranchial body in HIS/Hiph and of the thymopharyngeal duct in HIS/Mz. Thus, the fossulae of the piriform fossa resembled the piriform sinus fistula in human. In conclusion, cotton rats frequently possessed pharyngeal pouch remnants, including the piriform sinus fistula, and therefore, might serve as a novel model to elucidate the mechanisms of pharyngeal pouch development.

Hydronephrosis with ureteritis developed in C57BL/6N mice carrying the congenic region derived from MRL/MpJ-type chromosome 11.

Inbred MRL/MpJ mice show several unique phenotypes in tissue regeneration processes and the urogenital and immune systems. Clarifying the genetic and molecular bases of these phenotypes requires the analysis of their genetic susceptibility locus. Herein, hydronephrosis development was incidentally observed in MRL/MpJ-derived chromosome 11 (D11Mit21-212)-carrying C57BL/6N-based congenic mice, which developed bilateral or unilateral hydronephrosis in both males and females with 23.5% and 12.5% prevalence, respectively. Histopathologically, papillary malformations of the transitional epithelium in the pelvic-ureteric junction seemed to constrict the ureter luminal entrance. Characteristically, eosinophilic crystals were observed in the lumen of diseased ureters. These ureters were surrounded by infiltrating cells mainly composed of numerous CD3+ T-cells and B220+ B-cells. Furthermore, several Iba-1+ macrophages, Gr-1+ granulocytes, mast cells and chitinase 3-like 3/Ym1 (an important inflammatory lectin)-positive cells were detected. Eosinophils also accumulated to these lesions in diseased ureters. Some B6.MRL-(D11Mit21-D11Mit212) mice had duplicated ureters. We determined >100 single nucleotide variants between C57BL/6N- and MRL/MpJ-type chromosome 11 congenic regions, which were associated with nonsynonymous substitution, frameshift or stopgain of coding proteins. In conclusion, B6.MRL-(D11Mit21-D11Mit212) mice spontaneously developed hydronephrosis due to obstructive uropathy with inflammation. Thus, this mouse line would be useful for molecular pathological analysis of obstructive uropathy in experimental medicine.

Degenerative and regenerative features of myofibers differ among skeletal muscles in a murine model of muscular dystrophy.

Skeletal muscle myofibers constantly undergo degeneration and regeneration. Histopathological features of 6 skeletal muscles (cranial tibial [CT], gastrocnemius, quadriceps femoris, triceps brachii [TB], lumbar longissimus muscles, and costal part of the diaphragm [CPD]) were compared using C57BL/10ScSn-Dmd mdx (mdx) mice, a model for muscular dystrophy versus control, C57BL/10 mice. Body weight and skeletal muscle mass were lower in mdx mice than the control at 4 weeks of age; these results were similar at 6-30 weeks. Additionally, muscular lesions were observed in all examined skeletal muscles in mdx mice after 4 weeks, but none were noted in the controls. Immunohistochemical staining revealed numerous paired box 7-positive satellite cells surrounding the embryonic myosin heavy chain-positive regenerating myofibers, while the number of the former and staining intensity of the latter decreased as myofiber regeneration progressed. Persistent muscular lesions were observed in skeletal muscles of mdx mice between 4 and 14 weeks of age, and normal myofibers decreased with age. Number of muscular lesions was lowest in CPD at all ages examined, while the ratio of normal myofibers was lowest in TB at 6 weeks. In CT, TB, and CPD, Iba1-positive macrophages, the main inflammatory cells in skeletal muscle lesions, showed a significant positive correlation with the appearance of regenerating myofibers. Additionally, B220-positive B-cells showed positive and negative correlation with regenerating and regenerated myofibers, respectively. Our data suggest that degenerative and regenerative features of myofibers differ among skeletal muscles and that inflammatory cells are strongly associated with regenerative features of myofibers in mdx mice.

MRL/MpJ mice show unique pathological features after experimental kidney injury.

Clarification of the renal repair process is crucial for developing novel therapeutic strategies for kidney injury. MRL/MpJ mice have a unique repair process characterized by low scar formation. The pathological features of experimentally injured MRL/MpJ and C57BL/6 mouse kidneys were compared to examine the renal repair process. The dilation and atrophy of renal tubules were observed in folic acid (FA)-induced acute kidney injury (AKI) in both strains, and the histopathological injury scores and number of interleukin (IL)-1F6-positive damaged distal tubules and kidney injury molecule 1 (KIM-1)-positive damaged proximal tubules drastically increased 1 day after AKI induction. However, KIM-1-positive tubules and the elevation of serum renal function markers were significantly fewer and lower, respectively, in MRL/MpJ mice at days 2 and 7 after AKI. After traumatic kidney injury (TKI) via needle puncture, severe tubular necrotic lesions in the punctured area and fibrosis progressed in both strains. Indices for fibrosis such as aniline blue-positive area, number of alpha smooth muscle actin-positive myofibroblasts, and messenger RNA expression levels of Tgfb1 and Mmp2 indicated lower fibrotic activity in MRL/MpJ kidneys. Characteristically, only MRL/MpJ kidneys manifested remarkable calcification around the punctured area beginning 7 days after TKI. The pathological features of injured MRL/MpJ and C57BL/6 kidneys differed, especially those of kidneys with mild proximal tubular injuries after FA-induced AKI. Lower fibrotic activity and increased calcification after TKI were observed in MRL/MpJ kidneys. These findings clarified the unique pathological characteristics of MRL/MpJ mouse kidneys and contribute to understanding of the renal repair process after kidney injury.

Testicular oocytes in MRL/MpJ mice possess similar morphological, genetic, and functional characteristics to ovarian oocytes.

In general, mammalian males produce only spermatozoa in their testes and females produce only oocytes in their ovaries. However, newborn MRL/MpJ male mice produce oocytes within their testes. In this study, we examined the initiation and progression of oogenesis in fetal and neonatal MRL/MpJ mouse testes and evaluated the characteristics of testicular oocytes. Germ cells with positive reactions to oogenesis markers such as NOBOX oogenesis homeobox and synaptonemal complex protein 3 were observed in the MRL/MpJ fetal testes on embryonic day 18.5. These fetal testicular oocytes possessed maternal-specific methylation patterns of histone and DNA. The level of DNA methylation was still low in postnatal testicular oocytes at day 14 after birth. Additionally, the postnatal testicular oocytes contained both X and Y chromosomes and had the ability to fuse with sperm. These results suggest that some XY germ cells in fetal testes of MRL/MpJ mice enter meiosis prematurely, undergo oogenesis, and differentiate into oocytes. In addition, MRL/MpJ testicular oocytes have the ability to carry on oogenesis before and shortly after birth until they obtain some of the morphological, epigenetic, and functional characteristics of oocytes.

MRL/MpJ-Fas(lpr) mice show abnormalities in ovarian function and morphology with the progression of autoimmune disease.

The immune system is known to affect reproductive function, and maternal-fetal immune tolerance is essential for a successful pregnancy. To investigate the relationship between autoimmune disease and female reproductive function, we performed a comparative analysis of the ovarian phenotypes for C57BL/6 mice, autoimmune disease-prone MRL/MpJ (MRL/+) mice and congenic MRL/MpJ-Fas(lpr) (MRL/lpr) mice harboring a mutation in the Fas gene that speeds disease onset. Both MRL-background strains showed earlier vaginal opening than C57BL/6 mice. The estrous cycle became irregular by 6 and 12 months of age in MRL/lpr mice and mice of the other two strains, respectively. Histological analysis at 3 months revealed that the number of primordial follicles was smaller in MRL-background mice than in C57BL/6 mice after 3 months. In addition, MRL/lpr and MRL/+ mice displayed lower numbers of ovarian follicles and corpora lutea at 3 and 6 months, and 6 and 12 months, respectively, than that in age-matched C57BL/6 mice. MRL/lpr and MRL/+ mice developed ovarian interstitial glands after 3 and 6 months, respectively. In particular, MRL/lpr mice showed numerous infiltrating lymphocytes within the ovarian interstitia, and partially stratified ovarian surface epithelia with more developed microvilli than that observed in C57BL/6 mice at 6 months. No significant differences in serum hormone levels were observed between the strains. In conclusion, MRL/lpr mice display altered ovarian development, morphology and function consistent with the progression of severe autoimmune disease, as these findings are less severe in MRL/+ counterparts.

Morphological characteristics observed during early follicular development in perinatal MRL/MpJ mice.

In perinatal mice, the ovary undergoes drastic morphological changes, as clusters of oocytes called nests break into smaller cysts and subsequently form individual follicles. We studied perinatal oocyte development in MRL/MpJ mice, and compared it to that observed in C57BL/6 mice between embryonic day 18.5 and postnatal day 4. Throughout the observation period, compared to C57BL/6 mice, MRL/MpJ mice displayed significantly fewer oocytes in their ovaries. Morphologically, there were no clear differences between the strains at embryonic day 18.5. However, the beginning of folliculogenesis, as evidenced by the expression of NOBOX oogenesis homeobox (Nobox) transcript and protein, was more enhanced in MRL/MpJ mice than in C57BL/6 mice at embryonic day 18.5 and postnatal day 0. In addition, developed follicles were more frequently observed in MRL/MpJ mice than in C57BL/6 mice between postnatal days 0 and 4. In conclusion, the oocyte development during nest breakdown and folliculogenesis was accelerated in MRL/MpJ mice when compared to that observed in C57BL/6 mice.

Asymmetric morphology of the cells comprising the inner and outer bending sides of the murine duodenojejunal flexure.

The asymmetric shape of component cells determines the asymmetric features of developing organs. Here, we focused on the murine duodenojejunal flexure (DJF), which bends without affecting the mesentery, and analyzed the morphological asymmetries of the mucosal epithelium and gut wall cells between the inner and outer bending sides at embryonic days 10.75-11.75. In the mucosal epithelium, the cell shape and the expression of epithelial markers (Cdx2, E-cadherin) showed no differences between the two DJF sides. In contrast, the gut wall cells comprising the inner and outer sides of the DJF were elongated along the inner-outer axis and perpendicular to this axis, respectively. Furthermore, the gut wall cells in the outer side possessed cytoplasmic processes connecting cells via adherens junctions, but those in the inner side were attached via adherens junctions of juxtaposed cell bodies and were relatively more crowded. In immunohistochemistry experiments, there was no remarkable difference in the positive reactions of markers for mesenchyme (vimentin), smooth muscle cells (αSMA), endothelial cells (LYVE-1, CD34), and undifferentiated neurons (Sox10) between the DJF sides. Interestingly, Tuj1-positive cells, indicating differentiated neurons, were observed in the middle layer of the gut wall, and these cells were significantly more abundant and tended to be larger in the inner side than in the outer side of the DJF. In conclusion, we clarified the asymmetries of gut wall cell morphology and neural differentiation between the inner and outer sides of the DJF. These characteristics of the developing murine DJF indicate its asymmetric formation.

Overexpression of Toll-like receptor 8 correlates with the progression of podocyte injury in murine autoimmune glomerulonephritis.

Members of the Toll-like receptor (TLR) family serve as pathogen sensors and participate in local autoimmune responses. This study found a correlation between glomerular injury and TLR expression by analysing BXSB/MpJ-Yaa (BXSB-Yaa) lupus model mice. In isolated glomeruli, the mRNA expression of several TLRs was higher in BXSB-Yaa mice than in healthy control BXSB mice. In particular, the expression of Tlr8 and its downstream cytokines was markedly increased. In mouse kidneys, TLR8 protein and mRNA localized to podocytes, and TLR8 protein expression in the glomerulus was higher in BXSB-Yaa mice than in BXSB mice. In BXSB-Yaa mice, the glomerular levels of Tlr8 mRNA negatively correlated with the glomerular levels of podocyte functional markers (Nphs1, Nphs2, and Synpo) and positively correlated with urinary albumin levels. Furthermore, the glomerular and serum levels of miR-21, a putative microRNA ligand of TLR8, were higher in BXSB-Yaa mice than in BXSB mice. The urinary levels of Tlr8 mRNA were also higher in BXSB-Yaa mice than in BXSB mice. In conclusion, the overexpression of TLR8 correlates with the progression of podocyte injury in glomerulonephritis. Thus, altered levels of urinary Tlr8 mRNA might reflect podocyte injury.

Decreased miR-26a expression correlates with the progression of podocyte injury in autoimmune glomerulonephritis.

MicroRNAs contribute to the pathogenesis of certain diseases and may serve as biomarkers. We analyzed glomerular microRNA expression in B6.MRLc1, which serve as a mouse model of autoimmune glomerulonephritis. We found that miR-26a was the most abundantly expressed microRNA in the glomerulus of normal C57BL/6 and that its glomerular expression in B6.MRLc1 was significantly lower than that in C57BL/6. In mouse kidneys, podocytes mainly expressed miR-26a, and glomerular miR-26a expression in B6.MRLc1 mice correlated negatively with the urinary albumin levels and podocyte-specific gene expression. Puromycin-induced injury of immortalized mouse podocytes decreased miR-26a expression, perturbed the actin cytoskeleton, and increased the release of exosomes containing miR-26a. Although miR-26a expression increased with differentiation of immortalized mouse podocytes, silencing miR-26a decreased the expression of genes associated with the podocyte differentiation and formation of the cytoskeleton. In particular, the levels of vimentin and actin significantly decreased. In patients with lupus nephritis and IgA nephropathy, glomerular miR-26a levels were significantly lower than those of healthy controls. In B6.MRLc1 and patients with lupus nephritis, miR-26a levels in urinary exosomes were significantly higher compared with those for the respective healthy control. These data indicate that miR-26a regulates podocyte differentiation and cytoskeletal integrity, and its altered levels in glomerulus and urine may serve as a marker of injured podocytes in autoimmune glomerulonephritis.

Podocyte injury caused by indoxyl sulfate, a uremic toxin and aryl-hydrocarbon receptor ligand.

Indoxyl sulfate is a uremic toxin and a ligand of the aryl-hydrocarbon receptor (AhR), a transcriptional regulator. Elevated serum indoxyl sulfate levels may contribute to progressive kidney disease and associated vascular disease. We asked whether indoxyl sulfate injures podocytes in vivo and in vitro. Mice exposed to indoxyl sulfate for 8 w exhibited prominent tubulointerstitial lesions with vascular damage. Indoxyl sulfate-exposed mice with microalbuminuria showed ischemic changes, while more severely affected mice showed increased mesangial matrix, segmental solidification, and mesangiolysis. In normal mouse kidneys, AhR was predominantly localized to the podocyte nuclei. In mice exposed to indoxyl sulfate for 2 h, isolated glomeruli manifested increased Cyp1a1 expression, indicating AhR activation. After 8 w of indoxyl sulfate, podocytes showed foot process effacement, cytoplasmic vacuoles, and a focal granular and wrinkled pattern of podocin and synaptopodin expression. Furthermore, vimentin and AhR expression in the glomerulus was increased in the indoxyl sulfate-exposed glomeruli compared to controls. Glomerular expression of characteristic podocyte mRNAs was decreased, including Actn4, Cd2ap, Myh9, Nphs1, Nphs2, Podxl, Synpo, and Wt1. In vitro, immortalized-mouse podocytes exhibited AhR nuclear translocation beginning 30 min after 1 mM indoxyl sulfate exposure, and there was increased phospho-Rac1/Cdc42 at 2 h. After exposure to indoxyl sulfate for 24 h, mouse podocytes exhibited a pro-inflammatory phenotype, perturbed actin cytoskeleton, decreased expression of podocyte-specific genes, and decreased cell viability. In immortalized human podocytes, indoxyl sulfate treatment caused cell injury, decreased mRNA expression of podocyte-specific proteins, as well as integrins, collagens, cytoskeletal proteins, and bone morphogenetic proteins, and increased cytokine and chemokine expression. We propose that basal levels of AhR activity regulate podocyte function under normal conditions, and that increased activation of podocyte AhR by indoxyl sulfate contributes to progressive glomerular injury.

Genomic analysis of the appearance of ovarian mast cells in neonatal MRL/MpJ mice.

In MRL/MpJ mice, ovarian mast cells (OMCs) are more abundant than in other mouse strains, and tend to distribute beneath the ovarian surface epithelium at birth. This study investigated the factors regulating the appearance of neonatal OMCs in progeny of the cross between MRL/MpJ and C57BL/6N strains. F1 neonates had less than half the number of OMCs than MRL/MpJ. Interestingly, MRLB6F1 had more neonatal OMCs than B6MRLF1, although they were distributed over comparable areas. Furthermore, in MRL/MpJ fetuses for which parturition was delayed until embryonic day 21.5, the number of OMCs was significantly higher than in age-matched controls at postnatal day 2. These results suggest that the number of OMCs was influenced by the environmental factors during pregnancy. Quantitative trait locus analysis using N2 backcross progeny revealed two significant loci on chromosome 8: D8Mit343-D8Mit312 for the number of OMCs and D8Mit86-D8Mit89 for their distribution, designated as mast cell in the ovary of MRL/MpJ 1 (mcom1) and mcom2, respectively. Among MC migration-associated genes, ovarian expression of chemokine (C-C motif) ligand 17 at mcom1 locus was significantly higher in MRL/MpJ than in C57BL/6N, and positively correlated with the expression of OMC marker genes. These results indicate that the appearance of neonatal OMCs in MRL/MpJ is controlled by environmental factors and filial genetic factors, and that the abundance and distribution of OMCs are regulated by independent filial genetic elements.