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.

Expression of cubilin in mouse testes and Leydig cells.

Cubilin (cubn) is a receptor for vitamins and various protein ligands. Cubn lacks a transmembrane domain but anchors to apical membranes by forming complexes with Amnionless or Megalin. In an effort to better understand the uptake of nutrients in testis, we analysed cubn expression in the developing mice testes. In testes, cubn mRNA increased from birth to adulthood. In the inter-stitium and isolated seminiferous tubules, neonatal increase in cubn mRNA until 14 days post-partum (pp) was followed by a marked increase at puberty (28 days pp). Cubn was found in the gonocytes, spermatogonia, spermatocytes and spermatids in the developing testes. In adult testes, strong Cubn immunoreactivity was found in the elongating spermatids, suggesting the role of Cubn in endocytosis during early spermiogenesis. In Sertoli cells and peritubular cells, Cubn immunoreactivity was weak throughout the testis development. In the inter-stitium, Cubn immunoreactivity was found in foetal Leydig cells, was weak to negligible in the stem cells and progenitor Leydig cells and was strong in immature and adult Leydig cells, demonstrating a positive association between Cubn and steroidogenic activity of Leydig cells. Collectively, these results suggest that Cubn may participate in the endocytotic uptake of nutrients in germ cells and somatic cells, supporting the spermatogenesis and steroidogenesis in mouse testes.

Claudin-1 induces epithelial-mesenchymal transition through activation of the c-Abl-ERK signaling pathway in human liver cells.

Claudins (CLDNs) are a family of integral membrane proteins central to the formation of tight junctions, structures that are involved in paracellular transport and cellular growth and differentiation, and are critical for the maintenance of cellular polarity. Recent studies have provided evidence that CLDNs are aberrantly expressed in diverse types of human cancers, including hepatocellular carcinomas (HCCs). However, little is known about how CLDN expression is involved in cancer progression. In this study, we show that CLDN1 has a causal role in the epithelial-mesenchymal transition (EMT) in human liver cells, and that the c-Abl-Ras-Raf-1-ERK1/2 signaling axis is critical for the induction of malignant progression by CLDN1. Overexpression of CLDN1 induced expression of the EMT-regulating transcription factors Slug and Zeb1, and thereby led to repression of E-cadherin, ß-catenin expression, enhanced expression of N-cadherin and Vimentin, a loss of cell adhesion, and increased cell motility in normal liver cells and HCC cells. In line with these findings, inhibition of either c-Abl or ERK clearly attenuated CLDN1-induced EMT, as evidenced by a reversal of N-cadherin and E-cadherin expression patterns, and restored normal motility. Collectively, these results indicate that CLDN1 is necessary for the induction of EMT in human liver cells, and that activation of the c-Abl-Ras-Raf-1-ERK1/2 signaling pathway is required for CLDN1-induced acquisition of the malignant phenotype. The present observations suggest that CLDN1 could be exploited as a biomarker for liver cancer metastasis and might provide a pivotal point for therapeutic intervention in HCC.

Fucosyl neoglycoprotein binds to mouse epididymal spermatozoa and inhibits sperm binding to the egg zona pellucida.

Glycan epitopes of cellular glycoconjugates act as versatile biochemical signals, and this sugar coding plays an important role in cell-to-cell recognition processes. In this study, our aims were to determine the distribution of sperm receptors with activity for fucosyl- and galactosyl glycans and to address whether monosugar neoglycoproteins functionally mimic the binding between zona pellucida (ZP) glycoproteins and spermatozoa. In mouse epididymal spermatozoa with intact acrosomes, fucopyranosyl bovine serum albumin (BSA-Fuc) bound to the segment of the acrosome, the equatorial segment, and the postacrosome region of the sperm head. Galactosyl BSA (BSA-Gal) binding activity was similar to that of BSA-Fuc, but was weaker. In acrosome-reacted spermatozoa treated with the Ca(2+) ionophore A23187, BSA-zuc binding was lost in the apical segment of the acrosome but remained in the equatorial segment and postacrosome regions. BSA-Gal binding to the equatorial region was increased. In the presence of 2.5 µg ml(-1) BSA-Fuc, in vitro sperm-ZP binding was significantly decreased, indicating that fucosyl BSA functionally mimics ZP glycoproteins during sperm-egg ZP interactions. At the same concentration, BSA-Gal was not effective. Fucosyl BSA that efficiently inhibited the sperm-ZP binding can mimic the ZP glycoconjugate and has potential for use as a sperm fertility control agent in mouse.

Butyl paraben-induced changes in DNA methylation in rat epididymal spermatozoa.

Parabens have been shown to affect male rodent reproductive parameters, including testosterone levels and sperm production. In this study, we examined the effect of long-term exposure to butyl paraben (BP) on rat epididymal sperm DNA methylation. Adult male rats were exposed to BP (0, 10, 100 and 1000 mg kg(-1) per day) according to OECD TG407 for a repeated 28-day oral toxicity study. Sperm DNA methylation was examined by differential display random amplification of polymorphic DNA (RAPD) following methylation-specific restriction digestion of DNA. Among the 57 RAPD amplicons, six were methylation specific. Of these, five amplicons increased by 1.4- to 3.8-fold in epididymal sperm DNA at testing dose of BP. This indicates that BP can cause DNA hypermethylation in germ cells from the mitotic through post-meiotic stage in adult rat testes. To our knowledge, this is the first report on the epigenetic modification of sperm DNA by parabens.

Expression of amnionless in mouse testes and Leydig cells.

Vitamin B(12) (cobalamin) deficiency results in atrophy of seminiferous tubules and aplasia of spermatozoa and spermatid. The transmembrane protein amnionless (AMN) directs endocytosis of cubilin with its ligand, contributing to intrinsic factor-vitamin B(12) absorption. To understand vitamin B(12) transport in testis, we analysed AMN expression in developing mouse testes and in Leydig cells and speculated the possible role of AMN in testis. In testes, Amn mRNA levels were low until 14 days post partum (pp) and markedly increased from puberty onwards. In the interstitium, Amn mRNA levels were low at 14 days pp and increased at puberty (28 days pp) together with 3-beta-hydroxysteroid dehydrogenase type 6 mRNA. Strong AMN immunoreactivity was observed in early spermatocytes from 7 days pp, suggesting that AMN participates in meiosis. In Leydig cells, AMN was not observed until 14 days pp but was strongly expressed after 28 days pp, suggesting a positive relationship between AMN expression and functional differentiation of adult Leydig cells. Together, AMN may participate in meiosis in early spermatocytes and in functional differentiation of adult Leydig cells through the mediation of vitamin B(12) transport in the mouse testes. This is the first report on AMN expression in the germ cells and soma of mammalian testes.

Expression of claudin-1 and -11 in immature and mature pheasant (Phasianus colchicus) testes.

The expression of claudin-1 and -11, tight junctions (TJs) proteins was examined in immature and adult pheasant (Phasianus colchicus) testes. Claudin-1 and -11 cDNA were highly similar to those of human, mice, and chicken. Claudin-1 mRNA and protein (21 kDa) levels in immature testes were higher than those of adult testis. In immature testes until 6 weeks of age, Claudin-1 was found at contacts between adjacent Sertoli cells and between Sertoli cells and germ cells. In adult testis, Claudin-1 was found in early spermatocytes migrating the blood testis barrier (BTB). Blood vessels were positive for claudin-1. Claudin-11 mRNA and protein (21 kDa) increased during adulthood development of testis. In immature testis, Claudin-11 was found in apicolateral contacts between adjacent Sertoli cells, indicating its involvement in cell adhesion in immature testis. In adult testis, strong wavy Claudin-11 immunoreactivity was parallel to basal lamina at the basal part of seminiferous epithelium, indicating that Claudin-11 at the inter-Sertoli TJs may act as a structural element of the BTB. Weak Claudin-1 and -11 immunoreactivity at contacts between Sertoli cells to elongating/elongated spermatids, meiotic germ cells, and basal lamina suggests that they also participate in the cell-cell and cell-extracellular matrix adhesion in pheasant testis. Testosterone increased claudin-11 mRNA in testis organ culture and Sertoli cell primary culture, suggesting positive regulation of claudin-11 gene by androgen in Sertoli cells of pheasant testis. This is the first report on the claudins expression at BTB in avian testis.

Effects of molinate on survival and development of Bombina orientalis (Boulenger) embryos.

Molinate, a thiocarbamate chemical is a slightly to moderately toxic herbicide in EPA (Environmental Protection Agency) toxicity class III, and is a registered as a General Use Pesticide (GUP). Bombina orientalis is one of the most common amphibians in the world and comprise a large proportion of their total number in Korea. B. orientalis spawns in the rice fields at spring when the massive application of agricultural chemicals occurs. In the present study, we examined the effects of molinate on embryonic survival and developmental abnormality in B. orientalis embryos. The difference in survival rate between vehicle control and molinate treated embryos was not observed until the blastula stage. The first statistically significant decrease in embryonic survival was observed at mouth open stage following exposure to 100 microM molinate (46.8% vs. 81.1% in control). When the embryos develop to tadpole stage survival was significantly decreased at 50 microM molinate (35.9% vs. 68.9% in control), suggesting that the lowest observed effective dose (LOED) for systemic toxicity in B. orientalis embryos is 50 microM. In survived embryos molinate exposure produced several types of severe developmental abnormalities in order of frequency with bent trunk, neurula with yolk plug, bent tail, tail dysplasia, ventral blister, eye dysplasia, thick-set body and cephalic dysplasia. This suggests that molinate targets multiple events in embryonic and larval development in this frog species. Together this suggested that molinate was detrimental for survival and development following zygotic transcription after midblastula transition in B. orientalis embryos.

Expression of occludin in testis and epididymis of wild rabbits, Lepus sinensis coreanus.

Tight junctions (TJs) in inter-Sertoli junctional areas and epididymal epithelia build up the blood-testis barrier (BTB) and the blood-epididymal barrier (BEB), respectively. In this study, the expression of occludin, an integral member of the TJs, was examined in testis and different regions of epididymis of Lepus sinensis coreanus, an Korean wild rabbit species. In testis, intense occludin immunoreactivity was found in the basally located inter-Sertoli junctional area together with diffused immunoreactivity of occludin in the cytoplasm of Sertoli cells. It can be suggested that occludin is one of the robust elements of BTB in seminiferous tubules of rabbit testis. In proximal and distal caput epididymis, occludin immunoreactivity was found in the lateral as well as apical contacts of epithelial cells. In corpus epididymis, intense occludin immunoreactivity was found in the basolateral as well as apical contacts of epithelial cells together with cytoplasmic signal. In cauda epididymis, occludin immunoreactivity in luminal epithelia was relatively strong but largely found in the cytoplasm. This suggests that intriguing regulatory mechanisms differentially recruit occludin to the TJ in the different regions of epididymal epithelia. The differences in the subcellular localization as well as expression levels of occludin among the epididymal segments may reflect differential paracellular permeability of epithelia along the epididymal tubules and be correlated with sperm maturation in rabbit. In Western blot, a major form of occludin was MW 62 kDa together with small fragments of MW 34-39 kDa in testis and epididymis, suggesting the peptide cleavage of occludin. This is the first report on the molecular nature of TJs in a wild rabbit testis and epididymis.

Expression of p57kip2 in germ cells and Leydig cells in human testis.

p57kip2, a KIP family cyclin-dependent kinase (Cdk) inhibitor, blocks the cell cycle by acting on multiple cyclin-Cdk complexes. To investigate the role of p57kip2 in human fertility, the expression of p57kip2 was investigated in testes from normal and obstructive azoospermic male patients who were positive for p57kip2 mRNA. In the seminiferous tubule, strong immunoreactivity of p57kip2 was found in nuclei of early spermatocytes, but not in the spermatogonia. The p57kip2 immunoreactivity in spermatocytes was markedly heterogeneous. Preleptotene spermatocytes showed strong p57kip2 immunoreactivity, but no visible signal was found in late pachytene spermatocytes. Nuclei of the elongating spermatids was also positive for p57kip2 immunoreactivity. Taken together, this suggests that p57kip2 may play a role in the regulation of meiotic progression of early spermatocytes and cell cycle arrest and differentiation of spermatids. p57kip2 immunoreactivity was found in the perinuclear region of the peritubular cells, but not in the Sertoli cells. In Leydig cells, moderate immunoreactivity of p57kip2 was largely found in the cytoplasm, suggesting the noble function of p57kip2 in the differentiation of adult Leydig cells.

Effects of calcium channel blockers on the spermatogenesis and gene expression in peripubertal mouse testis.

Treatment of Ca(2+) channel blockers (CCB) to relieve hypertension causes reversible male infertility, suggesting deregulation of Ca(2+) homeostasis in testis is closely related with male infertility. To investigate the possible toxicity of therapeutic application of CCB in childhood, the effect of nifedipine and ethosuximide, an L-type and T-type CCB, respectively, on the spermatogenesis and testicular gene expression was examined. Following the intraperitoneal injection of either drug for 7 days to 18 days on old mice, the paired testes weights were significantly lower in mice treated with nifedipine (> or = 10 mg/kg/day) or ethosuximide (100 mg/kg/day) than vehicle controls. In mice given high drug dosing (100 mg/kg), seminiferous tubules showed immaturity with spermatogenic arrest at elongating spermatid stage and poorly developed lumen. Unexpectedly, the expression of activator isoform of transcription factor cAMP-responsive element modulator (CREM) mRNA increased together with transition protein 2 and protamine 2 mRNA in drug-treated mice testes, suggesting that CCB may deregulate expression of activator isoform of CREM in male germ cells and that spermatogenic defect following CCB treatment may attribute to ectopic expression of CREM-dependent gene battery in testis. Therapeutic application of CCB in childhood should be cautious because of their potential to cause spermatogenic defect and altered gene expression in testis.

Expression of beta-catenin in human testes with spermatogenic defects.

beta-catenin is a multifunctional molecule that functions in intercellular adhesion and signal transduction during assembly of AJs between Sertoli cells as well as between Sertoli cells and germ cells. To assess changes in the testicular beta-catenin in male infertility conditions, testicular tissues from obstructive azoospermia with normal spermatogenesis, spermatogenic arrest (SA) and Sertoli cell-only syndrome (SCO) patients were examined for immunohistochemical localization of beta-catenin. In normal spermatogenic tissue, expression of beta-catenin was largely found in the Sertoli cell-germ cell (primarily spermatocytes) contact areas. Interestingly, perinuclear localization of beta-catenin was found in spermatocytes and spermatids. In spermatogenic arrest, beta-catenin in cell contact areas between Sertoli cells and germ cells was greatly decreased, but perinuclear beta-catenin in spermatocytes was not. In SCO, weak or negligible immunoreactivity of beta-catenin was found in cell contacts between Sertoli cells. Nuclear localization of beta-catenin was found in myotubular cells in all samples. Taken together, altered expression of beta-catenin in cell contacts within the seminiferous epithelia in spermatogenic arrest and SCO suggests that interactions between Sertoli cells and germ cell are crucial for expression of beta-catenin, and thus functional development of AJs in seminiferous epithelia in human testis. It should be also emphasized that perinuclear beta-catenin in germ cells may play a specific role in spermatogenesis.

Expression of cannabinoid receptor 1 in mouse testes.

Marijuana smoke and cannabinoids adversely affect male reproductive function in human and rodent through the cannabinoid receptors. To understand the possible function of cannabinoid receptor 1 (CB1) in spermatogenesis, expression of CB1 in testis during the postnatal development was examined in mice. Semiquantitative RT-PCR analysis revealed that testicular CB1 mRNA level was relatively high at 1 week post partum (p.p.). Following decrease during prepubertal development (2 weeks p.p.) and CB1 mRNA level re-increased during puberty (4 weeks p.p.) and reached the peak in adult testis. At 1 week p.p., some spermatogonia and Leydig cells showed strong immunoreactivity of CB1. At 2 weeks p.p., CB1 immunoreactivity was largely found in the primary spermatocytes as well as spermatogonia, and Leydig cells showed a weak signal. In adult testis, strong immunoreactivity was found in Leydig cells and luminal epithelia of seminiferous tubule. Germ cells including spermatozoa were positive for CB1 immunoreactivity. On Western blot, multiple forms of CB1 proteins were detected in testes, suggesting oligomerization of CB1. Ubiquitous, but spatiotemporal difference in expression of CB1 in soma and germ line during postnatal development of testis suggests functional involvement of CB1 signaling in steroidogenesis, spermatogenesis and fertilization.

The expression of Cdk inhibitors p27kip1 and p57kip2 in mouse placenta and human choriocarcinoma JEG-3 cells.

This study was to investigate the expression of Cdk inhibitors p27kip1 and p57kip2 during the development of mouse placenta and during the steroid-treated culture of human choriocarcinoma JEG-3 cells. The p27kip1 mRNA in mouse placenta was highly expressed in 18 days p.c. than that in other groups. But, p57kip2 mRNA expression was high in 12, 14, and 16 days p.c., then decreased in 18 days p.c. The p27kip1 protein expression pattern was similar to mRNA. But, p57kip2 expression was higher in 14 days p.c. than that in other groups. The p27kip1 protein in mouse placenta was gradually increased in labyrinth zone from 12 days to 18 days p.c. However, p57kip2 protein was slightly decreased in labyrinth zone from 12 days to 18 days p.c. These reverse patterns of p27kip1 and p57kip2 expression were also shown in decidua and spongiotrophoblast. The p27kip1 mRNA expression was very low in human choriocarcinoma JEG-3 cells with estradiol concentration-independent manner. In 5 and 50 ng DEX-treated groups, p27kip1 mRNA was dramatically increased in comparison with control groups. The p57kip2 mRNA was not detected in JEG-3 cells. This result shows that p27kip1 may play a role in late period of mouse placental development and p57kip2 may play a role in middle period of mouse placental development, and that p27kip1 may play a role in growth inhibition of human choriocarcinoma cells and could be up-regulated by DEX in human choriocarcinoma.

Expression of p50 C-terminal Src kinase (Csk) in mouse testis.

C-terminal Src Kinase (Csk) is a cytoplasmic tyrosine kinase that phosphorylates a critical tyrosine residue in each of the Src family kinases to inhibit their activities. To investigate the possible regulation of spermatogenesis by Src-Csk loop, the postnatal changes in the expression of Csk were examined in mouse testes. Semiquantitative RT-PCR analysis revealed that Csk mRNA increased during neonatal development and peaked at 2 weeks of age. Following the decrease during pubertal development, Csk expression re-increased in adult testes. In Western blot, immature testes showed higher expression of Csk protein than the pubertal or adult testes. In immature testis, Csk immunoreactivity was largely found in the Sertoli cell and there was no visible difference in the Csk immunoreactivity among the seminiferous tubules. In adult testis, however, a differential Csk immunoreactivity was found among the seminiferous tubules. Intense signal was found in the adluminal cytoplasm of the Sertoli cells bearing the post-meiotic differentiating germ cells, suggesting that Csk may participate in the remodeling of seminiferous tubule during late phase of spermatogenesis. Csk immunoreactivity was also found in the Leydig cells, suggesting the possible regulation of Leydig cell function. Src-Csk loop may participate in the differentiation of the seminiferous epithelia and Leydig cells in mouse testis.