ESR1 inhibits hCG-induced steroidogenesis and proliferation of progenitor Leydig cells in mice.

Oestrogen is an important regulator in reproduction. To understand the role of oestrogen receptor 1 (ESR1) in Leydig cells, we investigated the expression of ESR1 in mouse Leydig cells during postnatal development and the effects of oestrogen on steroidogenesis and proliferation of progenitor Leydig cells (PLCs). In Leydig cells, the ESR1 expression was low at birth, increased until postnatal day 14 at which PLCs were predominant, and then decreased until adulthood. In foetal Leydig cells, ESR1 immunoreactivity increased from birth to postnatal day 14. These suggest that ESR1 is a potential biomarker of Leydig cell development. In PLCs, 17ß-estradiol and the ESR1-selective agonist propylpyrazoletriol suppressed human chorionic gonadotropin (hCG)-induced progesterone production and steroidogenic gene expression. The ESR2-selective agonist diarylpropionitrile did not affect steroidogenesis. In PLCs from Esr1 knockout mice, hCG-stimulated steroidogenesis was not suppressed by 17ß-estradiol, suggesting that oestrogen inhibits PLC steroidogenesis via ESR1. 17ß-estradiol, propylpyrazoletriol, and diarylpropionitrile decreased bromodeoxyuridine uptake in PLCs in the neonatal mice. In cultured PLCs, 17ß-estradiol, propylpyrazoletriol, and diarylpropionitrile reduced hCG-stimulated Ki67 and Pcna mRNA expression and the number of KI67-positive PLCs, suggesting that oestrogen inhibits PLC proliferation via both ESR1 and ESR2. In PLCs, ESR1 mediates the oestrogen-induced negative regulation of steroidogenesis and proliferation.

Changes in aquaporin 5 in the non-ciliated cells of mouse oviduct according to sexual maturation and oestrous cycle.

Aquaporin (AQP) water channels play an important role in fluid homeostasis and the control of epithelial cell volume. To understand the oviductal fluid homeostasis, the expression of aqp5 was examined in mouse oviduct. In the oviduct of cycling females, aqp1, aqp3, aqp4, aqp5, aqp6, aqp7, aqp8, and aqp11 mRNA were detected. Of these, expression of aqp5 mRNA increased significantly from the early prepubertal period to puberty. Epithelial AQP5 immunoreactivity was markedly increased during the same period and was most notable in the infundibulum. In immature female mice (3 weeks old), gonadotropin (pregnant mare's serum gonadotropin (5IU/head) and human chorionic gonadotropin (5IU/head), single intraperitoneal injection) significantly increased oviductal aqp5 mRNA and AQP5 immunoreactivity in oviduct epithelia. In adult mouse oviduct epithelia, AQP5 was primarily found in the apical membrane, subapical cytoplasm and basolateral membrane of secretory non-ciliated cells, whereas weak to negligible immunoreactivity was found in ß-tubulin-positive ciliated cells. Taking into account the fact that non-ciliated cells are well developed with subapical secretory vesicles as well as endosomes, AQP5 may also participate in the secretion and endocytosis in addition to water movement through non-ciliated secretory cells. AQP5 immunoreactivity was also found in the isthmic muscle and lamina propria beneath the epithelia. In cycling females, oviductal aqp5 mRNA levels were the highest at oestrus and the lowest at di-oestrus. AQP5 immunoreactivity in non-ciliated cells was notable in the infundibulum, where AQP5 immunoreactivity was relatively high at oestrus but low at dioestrus and pro-oestrus, indicating synchrony between aqp5 gene activation and the ovarian cycle. Together, the findings of the present study indicate that aqp5 specific to non-ciliated cells is activated during sexual maturation, supporting fluid homeostasis in mouse oviduct.

Effects of bilateral vasectomy on the interleukin 1 system in mouse epididymis.

PROBLEM: Regional difference in the inflammatory response in vasectomized (VAX) epididymis remains unraveled. METHOD OF STUDY: Epididymal expression of interleukin 1α (IL1α), IL1ß, IL1 receptor antagonist (IL1ra), IL1 receptor 1 (IL1R1), IL6, IL10, tumor necrosis factor α (TNF-α), and transforming growth factor-ß1 (TGF-ß1) was examined in bilateral VAX mice by real-time RT-PCR and immunohistochemistry. RESULTS: IL1α, IL1ß, IL1ra, and IL1R1 were expressed in the epididymal epithelia of normal mice. Following VAX, IL1R1 mRNA was not changed, but TNF-α, IL10, IL1α, IL1ß, and IL1ra mRNA were significantly upregulated in whole epididymis, whereas IL6 and TGF-ß1 mRNA were significantly increased in corpus and cauda but not in caput epididymis. Consistently, IL1α, IL1ß, and IL1ra immunoreactivities were visibly increased in epididymis following VAX. CONCLUSION: Following VAX, IL1α, IL1ß, IL1ra, IL10, and TNF-α may mediate immune reaction in whole epididymis, whereas IL6 and TGF-ß1 may mediate regionally different immune response primarily in the lower part of epididymis.

Changes in Inflammatory Cytokines Accompany Deregulation of Claudin-11, Resulting in Inter-Sertoli Tight Junctions in Varicocele Rat Testes.

PURPOSE: To elucidate the changes that occur in the blood-testis barrier during varicocele we examined changes in Cldn11 (claudin-11), an element of the blood-testis barrier, as well as steroidogenesis and proinflammatory cytokines in a model of varicocele rat testes. MATERIALS AND METHODS: Male rats with experimentally induced varicocele were sacrificed 4 weeks after operation. Testicular histology and blood testosterone concentrations were examined. The expression of tight junctions, steroidogenic enzymes, apoptosis and immune cell markers, and proinflammatory cytokines in the testes were evaluated by reverse transcriptase-polymerase chain reaction, Western blot and immunohistochemistry. RESULTS: Weight and Johnsen scores of varicocele testes were lower than those of normal testes. mRNA expression of Bad and Bax increased whereas Bcl-xl and Bcl2 mRNA decreased in varicocele testes compared to controls. Although blood testosterone did not change, Leydig cell 3ßHsd immunoreactivity, testicular 3ßHsd6 and 17ßHsd3 mRNA were significantly decreased in varicocele testes. Cldn11 mRNA and protein levels in varicocele testes were higher than in normal testes together with altered expression of Ocln, Zo1 and N-cadherin mRNA. Cldn11 immunoreactivity appeared as wavy strands at the periphery of the seminiferous epithelium in normal testes but was frequently found in the Sertoli cell cytoplasm in varicocele testes. In varicocele testes Tnfα, Il1α, Il6, Cd45, Cd3g and Cd3d mRNA was increased. CONCLUSIONS: An increase in proinflammatory cytokines might be responsible for deregulation of Cldn11 in the Sertoli cells in varicocele testes, leading to alterations in the permeability of the blood-testis barrier and immunological barriers to normal spermatogenesis.

Coxsackievirus and Adenovirus Receptor, a Tight Junction Protein, in Peri-Implantation Mouse Embryos.

To understand the role of Coxsackievirus and adenovirus receptor (CAR), a tight junction (TJ) protein, in peri-implantation embryos, developmental expression of CAR and its role in paracellular permeability were examined in mouse embryos. Splice variants for transmembrane CAR, Car1, Car2, and Car3 mRNA, were expressed from 2-cell, morula, and blastocyst stages onward, respectively, whereas mRNA for soluble CAR was expressed in MII oocytes and 4-cell stage onward. On Western blot, ∼46 kDa CAR proteins were detected in blastocysts. During the 4-cell embryos to morula stage, CAR was gradually concentrated at the contacts between blastomeres. In blastocysts, CAR was expressed at the cell contacts within the inner cell mass as well as in the trophectoderm (TE) where CAR was found together with ZO1 at the apical contacts, suggesting that CAR builds up apical TJs in TE and mediates cell adhesion in TE and inner cell mass. In blastocysts, CAR-blocking antibodies under Ca(2+) switching increased the dextran permeability and decreased the volume of blastocoel and H19 and Cdx2 mRNA, suggesting the pivotal role of CAR in the blastocyst development and paracellular permeability barrier in TE. CAR was expressed in TE of implanting embryos as well as endometrial epithelium, suggesting the involvement of CAR in the interaction between implanting embryos and endometrium. At 5-6 days postcoitum, CAR was expressed together with ZO1 in the primitive endoderm, visceral endoderm, and epiblasts facing the pro-amniotic cavity, suggesting that CAR TJs contribute to the separation of epiblast from the blastocoel and development of the pro-amniotic cavity within epiblasts.

A new 2-pyrone derivative, 5-bromo-3-(3-hydroxyprop-1-ynyl)-2H-pyran-2-one, suppresses stemness in glioma stem-like cells.

Glioma cells with stem cell properties, termed glioma stem-like cells (GSCs), have been linked to tumor formation, maintenance, and progression and are responsible for the failure of chemotherapy and radiotherapy. Because conventional glioma treatments often fail to eliminate GSCs completely, residual surviving GSCs are able to repopulate the tumor. Compounds that target GSCs might be helpful in overcoming resistance to anticancer treatments in human brain tumors. In this study, we showed that 5-bromo-3-(3-hydroxyprop-1-ynyl)-2H-pyran-2-one (BHP), a new 2-pyrone derivative, suppressed the maintenance of the GSC population in both a glioma cell line and patient-derived glioma cells. Treatment of GSCs with BHP effectively inhibited sphere formation and suppressed the CD133(+) cell population. Treatment with BHP also suppressed expression of the stemness-regulating transcription factors Sox2, Notch2, and ß-catenin in sphere-cultured glioma cells. Treatment of GSCs with BHP significantly suppressed two fundamental characteristics of cancer stem cells: self-renewal and tumorigenicity. BHP treatment dramatically inhibited clone-forming ability at the single-cell level and suppressed in vivo tumor formation. BHP markedly inhibited both phosphoinositide 3-kinase/Akt and Ras/Raf-1/extracellular signal-regulated kinase signaling, which suggests that one or both of these pathways are involved in BHP-induced suppression of GSCs. In addition, treatment with BHP effectively sensitized GSCs to chemotherapy and radiotherapy. Taken together, these results indicate that BHP targets GSCs and enhances their sensitivity to anticancer treatments and suggest that BHP treatment may be useful for treating brain tumors by eliminating GSCs.

PTTG1 oncogene promotes tumor malignancy via epithelial to mesenchymal transition and expansion of cancer stem cell population.

The prognosis of breast cancer patients is related to the degree of metastasis. However, the mechanisms by which epithelial tumor cells escape from the primary tumor and colonize at a distant site are not entirely understood. Here, we analyzed expression levels of pituitary tumor-transforming gene-1 (PTTG1), a relatively uncharacterized oncoprotein, in patient-derived breast cancer tissues with corresponding normal breast tissues. We found that PTTG1 is highly expressed in breast cancer patients, compared with normal tissues. Also, PTTG1 expression levels were correlated with the degree of malignancy in breast cancer cell lines; the more migratory and invasive cancer cell lines MDA-MB-231 and BT549 displayed the higher expression levels of PTTG1 than the less migratory and invasive MCF7 and SK-BR3 and normal MCF10A cell lines. By modulating PTTG1 expression levels, we found that PTTG1 enhances the migratory and invasive properties of breast cancer cells by inducing epithelial to mesenchymal transition, as evidenced by altered morphology and epithelial/mesenchymal cell marker expression patterns and up-regulation of the transcription factor Snail. Notably, down-regulation of PTTG1 also suppressed cancer stem cell population in BT549 cells by decreasing self-renewing ability and tumorigenic capacity, accompanying decreasing CD44(high) CD24(low) cells and Sox2 expression. Up-regulation of PTTG1 had the opposite effects, increasing sphere-forming ability and Sox2 expression. Importantly, PTTG1-mediated malignant tumor properties were due, at least in part, to activation of AKT, known to be a key regulator of both EMT and stemness in cancer cells. Collectively, these results suggest that PTTG1 may represent a new therapeutic target for malignant breast cancer.

Expression of coxsackievirus and adenovirus receptor isoforms in developing mouse bladder uroepithelium.

OBJECTIVES: Tight junctions are important for uroepithelial paracellular permeability barriers. In the present study, we examined the developmental changes in the expression of coxsackievirus and adenovirus receptor (CAR) isoforms in mouse bladder uroepithelium. METHODS: Multiplex reverse transcriptase polymerase chain reaction using CAR isoform-specific primer sets and Western blotting were conducted on gestational day 19 and postnatal days 1, 7, and 55. Subcellular localization of CAR was examined, together with occludin and zonula occludens-1, in neonatal and adult bladder using light microscopy and immunofluorescence microscopy. RESULTS: The total CAR and short CAR isoform mRNA were significantly increased from gestational day 19 to birth. Long CAR isoform mRNA was transiently decreased on postnatal day 7 and had recovered during adulthood. On Western blotting, molecular weight 46-kDa CAR was abundant in the mucosa and increased postnatally. In the neonatal, as well as the adult, bladder uroepithelium, CAR immunoreactivity was observed, together with occludin and zonula occludens-1 at the apical tight junctions and basolateral contacts between the adjacent uroepithelial cells. In adult bladder uroepithelium, CAR was increased at the interface between the basal cells and basal lamina. CONCLUSIONS: The expression patterns of CAR isoforms changed during the late fetal to adult development of the mouse bladder. CAR at the apical tight junctions and cellular adhesions between the uroepithelial cells and the interfaces between the basal cells and basal lamina might support the paracellular permeability barrier and structural integrity of the uroepithelium in the mouse bladder. The expression of CAR in the uroepithelial cells can be integrated as a part of the strategy for virus-mediated gene therapy in the bladder uroepithelium.