Characterization of mouse mediastinal fat-associated lymphoid clusters.

The association between adipose tissue and immunity has been established and fat-associated lymphoid clusters (FALCs) are considered as a source of immune cells. We discovered lymphoid clusters (LCs) in mouse mediastinal fat tissues (MFTs). In Th1-biased C57BL/6N (B6), Th2-biased DBA/2Cr (DBA) and autoimmune-prone MRL/MpJ (MRL) mice strains, LCs without a fibrous capsule and germinal center were observed in white-colored MFTs extending from the diaphragm to the heart. The number and size of the LCs were larger in 12-month-old mice than in 3-month-old mice in all of the examined strains. Moreover, B6 had an especially large number of LCs compared with DBA and MRL. The immune cells in the LCs consisted of mainly T-cells and some B-cells. The majority of T-cells were CD4+ helper T (Th) cells, rather than CD8+ cytotoxic T-cells and no obvious immune cell population difference was present among the strains. Furthermore, high endothelial venules and lymphatic vessels in the LCs were better developed in B6 mice than in the other strains. Interestingly, some CD133+ hematopoietic progenitor cells and some c-Kit+/CD127+ natural helper cells were detected in the LCs. BrdU+ proliferating cells were more abundant in the LCs of B6 mice than in the LCs of the other strains and the number of BrdU+ cells increased with age. This is the first report of LCs in mouse MFTs. We suggest that the mouse genetic background affects LC size and number. We term the LCs "mediastinal fat-associated lymphoid clusters". These clusters can be considered as niches for Th cell production.

Relationship between numerous mast cells and early follicular development in neonatal MRL/MpJ mouse ovaries.

In the neonatal mouse ovary, clusters of oocytes called nests break into smaller cysts and subsequently form individual follicles. During this period, we found numerous mast cells in the ovary of MRL/MpJ mice and investigated their appearance and morphology with follicular development. The ovarian mast cells, which were already present at postnatal day 0, tended to localize adjacent to the surface epithelium. Among 11 different mouse strains, MRL/MpJ mice possessed the greatest number of ovarian mast cells. Ovarian mast cells were also found in DBA/1, BALB/c, NZW, and DBA/2 mice but rarely in C57BL/6, NZB, AKR, C3H/He, CBA, and ICR mice. The ovarian mast cells expressed connective tissue mast cell markers, although mast cells around the surface epithelium also expressed a mucosal mast cell marker in MRL/MpJ mice. Some ovarian mast cells migrated into the oocyte nests and directly contacted the compressed and degenerated oocytes. In MRL/MpJ mice, the number of oocytes in the nest was significantly lower than in the other strains, and the number of oocytes showed a positive correlation with the number of ovarian mast cells. The gene expression of a mast cell marker also correlated with the expression of an oocyte nest marker, suggesting a link between the appearance of ovarian ? 4mast cells and early follicular development. Furthermore, the expression of follicle developmental markers was significantly higher in MRL/MpJ mice than in C57BL/6 mice. These results indicate that the appearance of ovarian mast cells is a unique phenotype of neonatal MRL/MpJ mice, and that ovarian mast cells participate in early follicular development, especially nest breakdown.

Analysis of duodenojejunal flexure formation in mice: implications for understanding the genetic basis for gastrointestinal morphology in mammals.

The mammalian gut undergoes morphological changes during development. We studied the developing mouse duodenojejunal flexure (DJF) to elucidate the mechanism of formation. During embryonic days 10.75-13.75, DJF formation was morphologically classified into three stages: the expansion stage, flexure formation stage, and flexure elongation stage. From the expansion to the flexure formation stages, the DJF wall showed asymmetric morphology and proliferation along the left-right intestinal axis. From the flexure formation to the flexure elongation stage, the DJF started to bend dorsally with counterclockwise rotation along the antero-caudal intestinal axis, indicating that the original right side of the duodenum was rotated towards the dorsal body wall during development of the DJF. The direction of attachment of the dorsal mesentery to the DJF did not correspond to the bending direction of the DJF during flexure formation, and this finding indicated that the dorsal mesentery contributed very little to DJF formation. During DJF formation, Aldh1a2 and hedgehog mRNAs were detected at the DJF, and their expression levels differed along the bending axis. In conclusion, DJF formation might be triggered by asymmetric morphology and proliferation along the left-right intestinal axis under the control of retinoic acid and hedgehog signaling.

Close relations between podocyte injuries and membranous proliferative glomerulonephritis in autoimmune murine models.

BACKGROUND: Membranous proliferative glomerulonephritis (MPGN) is a major primary cause of chronic kidney disease (CKD). Podocyte injury is crucial in the pathogenesis of glomerular disease with proteinuria, leading to CKD. To assess podocyte injuries in MPGN, the pathological features of spontaneous murine models were analyzed. METHODS: The autoimmune-prone mice strains BXSB/MpJ-Yaa and B6.MRL-(D1Mit202-D1Mit403) were used as the MPGN models, and BXSB/MpJ-Yaa(+) and C57BL/6 were used as the respective controls. In addition to clinical parameters and glomerular histopathology, the protein and mRNA levels of podocyte functional markers were evaluated as indices for podocyte injuries. The relation between MPGN pathology and podocyte injuries was analyzed by statistical correlation. RESULTS: Both models developed MPGN with albuminuria and elevated serum anti-double-strand DNA (dsDNA) antibody levels. BXSB/MpJ-Yaa and B6.MRL showed severe proliferative lesions with T and B cell infiltrations and membranous lesions with T cell infiltrations, respectively. Foot process effacement and microvillus-like structure formation were observed ultrastructurally in the podocytes of both MPGN models. Furthermore, both MPGN models showed a decrease in immune-positive areas of nephrin, podocin and synaptopodin in the glomerulus, and in the mRNA expression of Nphs1, Nphs2, Synpo, Actn4, Cd2ap, and Podxl in the isolated glomerulus. Significant negative correlations were detected between serum anti-dsDNA antibody levels and glomerular Nphs1 expression, and between urinary albumin-to-creatinine ratio and glomerular expression of Nphs1, Synpo, Actn4, Cd2ap, or Podxl. CONCLUSION: MPGN models clearly developed podocyte injuries characterized by the decreased expression of podocyte functional markers with altered morphology. These data emphasized the importance of regulation of podocyte injuries in MPGN.

Mice stage-specific claudin 3 expression regulates progression of meiosis in early stage spermatocytes.

Claudin 3 is a protein component of the tight junction strands. Tight junctions between adjacent Sertoli cells form the blood-testis barrier (BTB). During spermatogenesis, seminiferous stage-specific expression of claudin 3 is believed to regulate the migration of preleptotene/leptotene spermatocytes across the BTB. Here, we determined the cell types expressing claudin 3 in adult mouse testis and investigated spermatogenesis after testis-specific in vivo knockdown of claudin 3. The results of in situ hybridization revealed that claudin 3 mRNA was predominantly expressed in germ cells near the basal lamina of seminiferous tubules at stages VI-IX. Furthermore, claudin 3 protein was localized not only to the BTB but also to the cell membrane of STRA8-expressing preleptotene/leptotene spermatocytes in the testis of adult ICR.Cg-Tg(Stra8-EGFP)1Ysa/YsaRbrc mice. Although claudin 3 knockdown did not affect BTB integrity, it did cause a partial delay in spermatocyte migration across the BTB. Moreover, claudin 3 knockdown resulted in a prolonged preleptotene phase during spermatogenesis. These data indicate that the seminiferous stage-specific expression and localization of claudin 3 during spermatogenesis regulate the progression of meiosis by promoting germ cell migration across the BTB.

BXSB/MpJ-Yaa mice develop autoimmune dacryoadenitis with the appearance of inflammatory cell marker messenger RNAs in the lacrimal fluid.

BACKGROUND: Dacryoadenitis is characteristic of an autoimmune exocrinopathy, e.g. Sjögren syndrome. We pathologically examined the lacrimal glands of autoimmune-prone BXSB/MpJ-Yaa mice for the appearance of pathological signs of dacryoadenitis progression in autoimmune dacryoadenitis models, particularly focusing on messenger RNAs in the lacrimal fluid. METHODS: The lacrimal glands of the BXSB/MpJ-Yaa and C57BL/6 mice were histopathologically analyzed in parallel with the evaluation of lacrimation and messenger RNA expression of water channels (Aqp3, Aqp4 and Aqp5). In addition, autoimmune model mice (MRL/MpJ-lpr/lpr and NZB/NZWF1) were used for evaluating cell infiltration and detecting inflammatory cell marker messenger RNAs (Cd68, Ptprc and Cd3e) in the lacrimal fluids by polymerase chain reaction-based methods. RESULTS: B-cell predominant lymphocytic infiltrations and the destruction of acini were observed in the lacrimal glands of BXSB/MpJ-Yaa mice. There was no significant difference in the quantity of lacrimal fluid between the BXSB/MpJ-Yaa and C57BL/6 mice. In the BXSB/MpJ-Yaa mice, Aqp3 expression increased significantly with the cell infiltration score, whereas expression of Aqp4 and Aqp5 tended to decrease. Aqp3 expression increased significantly with the cell infiltration score in BXSB/MpJ-Yaa mice. Among inflammatory cell markers, Cd68 was more frequently detected in the lacrimal fluid of the BXSB/MpJ-Yaa, MRL/MpJ-lpr/lpr and NZB/NZWF1 mice than in that of the C57BL/6 mice. CONCLUSION: BXSB/MpJ-Yaa mice clearly developed autoimmune dacryoadenitis. The altered expression of water channels in lacrimal glands might be associated with the preservation of lacrimal fluid excretion in BXSB/MpJ-Yaa mice. The detection of inflammatory cell markers in lacrimal fluid could be used as a diagnostic marker for autoimmune dacryoadenitis.

Analysis of TdT-mediated dUTP nick end labeling (TUNEL)-positive cells associated with cardiac myogenesis in mouse embryo.

Caspase activation is associated with skeletal muscle differentiation in mouse embryos. We examined the relationship between cardiac myogenesis and cell death using mice hearts at embryonic days (E) 11.5-15.5 and fetal rat heart H9C2 cells. The number of TdT-mediated dUTP nick end labeling (TUNEL)-positive cells increased with fetal age and was much higher than that of single-stranded DNA (ssDNA)- and active caspase-3 (aCasp3)-positive cells. TUNEL and aCasp3 double staining resulted in 3 types of positive cells: TUNEL(+)/aCasp3(+), TUNEL(+)/aCasp3(-) and TUNEL(-)/aCasp3(+). TUNEL(+)/aCasp3(-) cells were the most common but lacked morphological features of apoptosis, such as nuclear condensation or fragmentation. The expression of anti-apoptotic factors increased during E11.5-15.5. Furthermore, TUNEL-positive H9C2 cells without nuclear condensation or fragmentation were observed only in myotubes later in the culture period. In this study, the dynamics of TUNEL-positive cardiomyocyte was inconsistent with the activation of apoptosis cascade, and their morphological feature was clearly different from representative apoptosis. From these findings, we concluded that the increased number of TUNEL-positive cardiomyocyte, having the DNA strand breaks, would be associated with the progression of cardiac myogenesis.

Genomic analysis of the appearance of testicular oocytes in MRL/MpJ mice.

Mammals produce sperm or oocytes depending on their sex; however, newborn MRL/MpJ (MRL) male mice produce oocytes within their testes. We previously reported that one of the genes responsible for this phenotype is present on the MRL-type Y chromosome (Y(MRL)), and that multiple genes, probably autosomal, are also required for the development of this phenotype. In this study we focused on the autosomal genes and examined their relationship with this phenotype by analyzing the progeny from crosses between MRL mice and other strains. We first observed the male F1 progeny from the crosses between female A/J, C57BL/6 (B6), BALB/c, C3H/He, or DBA/2 mice and male MRL mice, and two consomic strains, male B6-Y(MRL) and MRL-Y(B6). Testicular oocytes that were morphologically similar to those of MRL mice were detected in all mouse strains except BALBMRLF1; however, the incidence of testicular oocytes was significantly lower than that in MRL mice. The appearance of testicular oocytes in MRL-Y(B6) mice indicates that this phenotype is strongly affected by genomic factors present on autosomes, and that there is at least one other causative gene on the MRL-type autosomes (MRL testicular oocyte production, mtop) other than that on Y(MRL). Furthermore, a quantitative trait locus (QTL) analysis using N2 backcross progeny from crosses between female MRLB6F1 and male MRL mice revealed the presence of susceptibility loci for the appearance of testicular oocytes at 8-17 cM on Chr 15. These findings demonstrate that the appearance of testicular oocytes is regulated by the genetic factors on Chr 15 and on Y(MRL).

Altered expression of microRNA miR-146a correlates with the development of chronic renal inflammation.

MicroRNAs (miRNAs) are highly conserved small non-coding RNAs that act as post-transcriptional regulators of target mRNA. In this study, we sought to identify the microRNA underlying local inflammation in a murine model of chronic kidney disease (CKD). In microarray analysis of kidneys, the expression of miR-146a/b was elevated in B6.MRLc1 CKD mice that spontaneously develop renal inflammation with age. Primary-microRNA analysis found that elevated miR-146a/b expression in the kidneys of B6.MRLc1 mice was mainly derived from miR-146a rather than miR-146b, and this expression increased with the development of CKD. Histopathological scores for glomerular and interstitial lesions, mRNA expression of inflammatory mediators, and macrophage infiltration were significantly higher in B6.MRLc1 than C57BL/6 mice and were positively correlated with miR-146a expression. In situ hybridization and laser microdissection-RT-PCR showed that miR-146a expression in interstitial lesions containing inflammatory cells was higher than in the glomerulus. The increased expression of the inflammatory-associated genes RELA, IRAK1, IL1B, IL10, and CXCLs was noted in miR-146a/b-silenced human monocytes. The amount of miR-146a was higher in urine sediments of B6.MRLc1 than of C57BL/6 mice. Thus, miR-146a expression in the kidneys and its urinary excretion was specifically associated with the development of interstitial lesions and correlated with inflammatory cell infiltration.

Molecular pathology of murine ureteritis causing obstructive uropathy with hydronephrosis.

Primary causes of urinary tract obstruction that induces urine retention and results in hydronephrosis include uroliths, inflammation, and tumors. In this study, we analyzed the molecular pathology of ureteritis causing hydronephrosis in laboratory rodents.F2 progenies of C57BL/6 and DBA/2 mice were studied histopathologically and by comprehensive gene expression analysis of their ureters. Incidence of hydronephrosis was approximately 5% in F2 progenies. Histopathologically, this hydronephrosis was caused by stenosis of the proximal ureter, which showed fibrosis and papillary malformations of the proliferative epithelium with infiltrations of B-cell-dominated lymphocytes. Additionally, CD16-positive large granular leukocytes and eosinophils infiltrated from the ureteral mucosa to the muscular layer. Eosinophilic crystals were characteristically observed in the lumen of the ureter and the cytoplasm of large granular leukocytes, eosinophils, and transitional epithelial cells. Comprehensive gene profiling revealed remarkably elevated expression of genes associated with hyperimmune responses through activation of B cells in diseased ureters. Furthermore, diseased ureters showed dramatically higher gene expression of chitinase 3-like 3, known as Ym1, which is associated with formation both of adenomas in the transitional epithelium and of eosinophilic crystals in inflammatory conditions. The Ym1 protein was mainly localized to the cytoplasm of the transitional epithelium, infiltrated cells, and eosinophilic crystals in diseased ureters.We determined that the primary cause of hydronephrosis in F2 mice was ureteritis mediated by the local hyperimmune response with malformation of the transitional epithelium. Our data provide a novel molecular pathogenesis for elucidating causes of aseptic inflammation in human upper urinary tracts.

Identifying a new locus that regulates the development of rete ovarian cysts in MRL/MpJ mice.

MRL/MpJ (MRL) is a mouse model for autoimmune disease and develops ovarian cysts with age. The ovarian cysts originate from the rete ovarii, which is considered to be the remnant of fetal mesonephric tubules. In a previous study, we analyzed the genetic background of ovarian cysts by using backcross progenies between MRL and C57BL/6N (B6) mice. By interval mapping, suggestive linkages were detected on several chromosomes (Chrs), and a significant linkage on Chr 14 was designated as MRL Rete Ovarian Cyst (mroc). In the present study, which evaluated 113 F2 intercross progenies, a significant linkage appeared on Chr 6 at the marker position D6Mit188 (likelihood ratio statistic = 18.5). In particular, the peak regions of Chrs 6 and 14, which contain major causative loci by backcross analysis, showed close reverse interaction. From these results, a locus on Chr 6 was identified as mroc2, the second major locus associated with ovarian cyst formation in MRL mice.

Ovarian cysts in MRL / MpJ mice are derived from the extraovarian rete: a developmental study.

MRL/MpJ (MRL) mice, commonly used as a model for autoimmune disease, have a high frequency of ovarian cysts originating from the rete ovarii. In the present study, to clarify how the rete ovarii, which are remnants of mesonephric tubules during embryogenesis, progress to cystic formation with aging, the morphology of MRL rete ovarii was analyzed and compared with that of normal C57BL/6N (B6) mice. In B6 mice, the rete ovarii consisted of a series of tubules, including the extraovarian rete (ER), the connecting rete (CR), and the intraovarian rete (IR), based on their location. Whereas the ER of B6 mice was composed of highly convoluted tubules lined by both ciliated and non-ciliated epithelia, the tubules in the CR and IR had only non-ciliated cells. In MRL mice, dilations of the rete ovarii initiated from the IR rather than the ER or CR. Although the histological types of cells lining the lumen of the rete ovarii were the same as those in B6 mice, the ER in MRL mice showed a variety in morphology. In particular, the connections between the ER and ovary tended to disappear with increasing age and the development of ovarian cysts. Furthermore, the epithelium lining the large ovarian cysts in MRL mice had ciliated cells forming the cluster. On the basis of these findings, it is suggested that cystic changes of the rete ovarii in MRL mice are caused by the dilations of the IR with invasion of the ER and CR into the ovarian medulla. These data provide new pathological mechanisms for ovarian cyst formation.

Quantitative and qualitative urinary cellular patterns correlate with progression of murine glomerulonephritis.

The kidney is a nonregenerative organ composed of numerous functional nephrons and collecting ducts (CDs). Glomerular and tubulointerstitial damages decrease the number of functional nephrons and cause anatomical and physiological alterations resulting in renal dysfunction. It has recently been reported that nephron constituent cells are dropped into the urine in several pathological conditions associated with renal functional deterioration. We investigated the quantitative and qualitative urinary cellular patterns in a murine glomerulonephritis model and elucidated the correlation between cellular patterns and renal pathology.Urinary cytology and renal histopathology were analyzed in BXSB/MpJ (BXSB; glomerulonephritis model) and C57BL/6 (B6; control) mice. Urinary cytology revealed that the number of urinary cells in BXSB mice changed according to the histometric score of glomerulonephritis and urinary albumin; however, no correlation was detected for the levels of blood urea nitrogen and creatinine. The expression of specific markers for podocytes, distal tubules (DTs), and CDs was detected in BXSB urine. Cells immunopositive for Wilms tumor 1 (podocyte marker) and interleukin-1 family, member 6 (damaged DT and CD marker) in the kidney significantly decreased and increased in BXSB versus B6, respectively. In the PCR array analysis of inflammatory cytokines and chemokines, Il10, Cxcl2, C3, and Il1rn showed relatively higher expression in BXSB kidneys than in B6 kidneys. In particular, the highest expression of C3 mRNA was detected in the urine from BXSB mice. Furthermore, C3 protein and mRNA were localized in the epithelia of damaged nephrons.These findings suggest that epithelial cells of the glomerulus, DT, and CD are dropped into the urine, and that these patterns are associated with renal pathology progression. We conclude that evaluation of urinary cellular patterns plays a key role in the early, noninvasive diagnosis of renal disease.

Hepatocyte nuclear factor 4 alpha is associated with mesenchymal-epithelial transition in developing kidneys of C57BL/6 mice.

During kidney development, the metanephric mesenchyme (MM) develops into the nephron through mesenchymal-epithelial transition (MET). We have previously reported that knock-down of the expression of hepatocyte nuclear factor 4 alpha (Hnf4a) gene induces failure of cellular organization in the condensed mesenchyme (CM) of cultured embryonic kidneys. To elucidate the details of MET during nephrogenesis, embryonic mouse kidneys were analyzed by electron microscopy, immunohistochemistry, and molecular biology. The findings showed that the intercellular junction, but not the basal lamina, was present in the CM. Additionally, immediately after Hnf4a gene expression, the expression of epithelial genes (Krt8, Tjp1, and Cdh1) increased, and those of mesenchymal genes (Acta1 and Vim) decreased, in the CM compared to the MM. To clarify the relationship between MET and Hnf4α, the fibroblast cell line with forced expression of Hnf4α protein were analyzed. In this model, it was noted that Hnf4α induced increasing epithelial and decreasing mesenchymal gene expression. In these, up-regulation of Pvrl1, -2, and Mllt4 genes which mediate the formation of apico-basal polarity, were found. These results, and those of previous findings, indicate that Hnf4α protein is associated with the initiation of MET in early nephrogenesis.

Structural characteristics of goat (Capra hircus) parotid salivary glands.

The structural characteristics of the parotid glands in small ruminants (goat, sheep) were observed and compared to those of a major laboratory animal, the mouse. Their parotid glands consist of the purely serous type. Ultrastructurally, the serous acini of goats and sheep were characterized by the presence of well-developed basolateral expansions of folds, which are characteristics of electrolyte- and water-transporting epithelium. Moreover in ruminants, unlike the mouse, the presence of numerous intercellular canaliculi as well as microvilli projecting into both the intercellular canaliculi and the lumina of the serous acini provided a large surface area for osmotic equilibrium and isotonic saliva secretion. Most of the secretory granules in goats and sheep contained peripherally located inclusions that showed dense reaction products for acid phosphatase. This indicates that most of the secretory granules undergo lysosomal degradation rather than secretion. An apocrine mode of secretion of some secretory granules was occasionally observed in some acini of goats and sheep but only exocytotic features were observed in mice. In the goat, the serous acini showed three morphologically different types, which might be an indication of different activity phases. Furthermore, alpha-smooth muscle actin-, and vimentin-positive myoepithelial cells were observed only around the serous acini and the intercalated ducts. From these findings, we consider that the structural characteristics of ruminant parotid glands might reflect their physiological role in the copious isotonic saliva secretion with a low protein concentration.

Cytoarchitectural differences of myoepithelial cells among goat major salivary glands.

Previously, the distribution of myoepithelial cells (mecs) in the salivary glands was studied by both immunohistochemistry, and transmission electron microscopy; however, little was elucidated concerning their morphological features, especially in goats. This study was performed to investigate the correlation between the cytoarchitecture of the mecs in goat major salivary glands (parotid, mandibular, and sublingual glands) and the nature of the saliva secretion. The cytoarchitectural features of the mecs were examined by scanning and transmission electron microscopy as well as immunohistochemically. The secretory endpieces in the parotid gland are of the pure serous type, but in both the mandibular and sublingual glands they are of the mixed type. In all studied glands, the intercalated ducts were covered by mecs which, unlike the large stellate cells that surrounded the secretory endpieces, were spindle-shaped with few cytoplasmic processes. Interestingly, the mecs were found to bulge on the basal surfaces of the serous acini and intercalated ducts in all glands and to be in close contact to the seromucous tubules surface in the mandibular and sublingual glands forming a continuous network around it. In conclusion, the differences in the degree of development of the mecs as well as the number of their cytoplasmic processes may be correlated with the nature of the secretion and the number of the secretory granules. Thus these observations may have some relevance in the diagnosis of atrophy and pathogenic conditions of these glands.

Molecular dynamics of the blood-testis barrier components during murine spermatogenesis.

The blood-testis barrier (BTB) separates the seminiferous epithelium into the adluminal and basal compartments. During murine spermatogenesis, preleptotene/leptotene spermatocytes migrate from the basal to the adluminal compartment through the BTB during stages VIII-IX. In the present study, we focused on the tight junction (TJ) molecules and analyzed their spatiotemporal expression during the murine seminiferous epithelial cycle. Structural analysis revealed that the principal components of the BTB, for example, claudin-3, claudin-11, occludin, and zonula occludens-1 (ZO-1), were localized at the basal and luminal sides of the preleptotene/leptotene spermatocytes during the migration stages (VIII-IX). Although we detected claudin-11, occludin, and ZO-1 throughout spermatogenesis, claudin-3 was only detected during stages VI-IX. Quantitative PCR using dissected seminiferous tubules from three stages (Early: II-VI, Middle: VII-VIII, Late: IX-I) clarified that the mRNA levels of TJ molecules were not correlated with the histoplanimetrical protein levels during spermatogenesis. Additionally, tubulobulbar complexes, considered to be involved in the internalization of TJ, were observed at the BTB site. Furthermore, a significant reduction in the mRNA levels of genes for the degradation of occludin (Itch) and endocytic recycling (Rab13) were observed during the Late and Middle stages, respectively. Therefore, we hypothesized that the lag between mRNA and protein expression of TJ molecules may be due to posttranslational modulation, for example, tubulobulbar complexes and endocytic recycling processes. In conclusion, these findings indicate that the integrity of the BTB is maintained throughout spermatogenesis, and the stage-specific localization of claudin-3 protein plays an important role in regulating BTB permeability.

Hepatocyte nuclear factor 4 alpha is related to survival of the condensed mesenchyme in the developing mouse kidney.

Hepatocyte nuclear factor 4 alpha (Hnf4alpha) is a transcription factor required for embryogenesis and organogenesis. In the adult kidney, Hnf4alpha is expressed at a high level in proximal tubules. Although its expression begins from the embryonic period, its function in the developing kidney has remained almost unknown. In this study, we investigated the role of Hnf4alpha in the cultured embryonic mouse kidney by gene silencing using the RNA interference method. Additionally, we identified the dynamics of Hnf4alpha in the microenvironment of the developing kidney. As a result of gene silencing, the cellular organization in the condensed mesenchyme (CM) fell into disorder and many apoptotic cells appeared. In addition, laser microdissection-reverse transcriptase-polymerase chain reaction provided evidence that Hnf4alpha gene expression began first in the CM. These results suggest the possibility that Hnf4alpha plays an important role in the regulation of the cell survival at the CM stage in nephrogenesis.

Quantitative trait locus analysis of ovarian cysts derived from rete ovarii in MRL/MpJ mice.

MRL/MpJ (MRL) is a model mouse for autoimmune diseases such as dermatitis, vasculitis, arthritis, and glomerulonephritis. In addition to these immune-associated disorders, we found that older MRL mice develop ovarian cysts originating from the rete ovarii, which is lined by ciliated or nonciliated epithelium and considered remnants of mesonephric tubules. Ovarian cysts, which are reported to have several sources, are associated with female infertility, but information regarding the genetic etiology of ovarian cysts originating from the rete ovarii is rare. In this study, to elucidate the genetic background of development of ovarian cysts, we performed quantitative trait locus (QTL) analysis using 120 microsatellite markers, which cover the whole genome of murine chromosomes, and 213 backcross progenies between female MRL and male C57BL/6N mice. The quantitative trait measured was the circumferences of rete ovarii or ovarian cysts. As a result, suggestive linkages were detected on Chrs 3, 4, 6, and 11, but significant linkages were located on Chr 14 by interval mapping. We thereby designated the 27.5-cM region of Chr 14 "MRL Rete Ovarian Cysts (mroc)." The peak regions of Chrs 4 and 14 in particular showed a close additive interaction (p < 0.00001). From these results we concluded that multiple loci on Chrs 3, 4, 6, 11, and 14 interact to result in development of ovarian cysts in MRL mice.

Analysis of factors decreasing testis weight in MRL mice.

MRL/MpJ (MRL) mouse testes have several unique characteristics, including the appearance of oocytes, the occurrence of metaphase-specific apoptosis of meiotic spermatocytes, and the presence of heat-shock-resistant spermatocytes. In the present study we used chromosomal mapping to determine the genomic background associated with small testis size in MRL mice. We prepared and analyzed C57BL/6-based congenic mice carrying MRL mouse loci. Quantitative trait loci (QTL) analysis revealed susceptibility loci for small testis size at 100 cM on chromosome (Chr) 1 and at around 80 cM on Chr 2. Analysis with B6.MRLc1 and B6.MRLc2 congenic mice and double-congenic mice confirmed the QTL data and showed that low testis weight in MRL mice was caused by germ cell apoptosis. Through histological examinations we found that B6.MRLc1 and B6.MRLc2 mice showed stage-specific apoptosis in their testes, the former at metaphase stage XII and the later at pachytene stage IV. Metaphase-specific apoptosis of spermatocytes occurs due to mutation of the exonuclease 1 (Exo1) gene located at 100 cM on Chr 1. Thus, the mutation of the Exo1 gene is also responsible for low testis weight caused by metaphase-specific apoptosis. In conclusion, testis weight is reduced in MRL mice due to apoptosis of germ cells caused by mutations in loci on Chrs 1 and 2.