Nobuyuki Shirasawa

Peroxiredoxin 4 knockout results in elevated spermatogenic cell death via oxidative stress

Prx (peroxiredoxin) is a multifunctional redox protein with thioredoxin-dependent peroxidase activity. Prx4 is present as a secretory protein in most tissues, whereas in sexually mature testes it is anchored in the ER (endoplasmic reticulum) membrane of spermatogenic cells via an uncleaved N-terminal hydrophobic peptide. We generated a Prx4 knockout mouse to investigate the function of Prx4 in vivo. Prx4(-/y) mice lacking Prx4 expression in all cells were obtained by mating Prx4(flox/+) female mice with Cre-transgenic male mice that ubiquitously expressed Cre recombinase. The resulting Prx4(-/y) male mice were fertile, and most organs were nearly normal in size, except for testicular atrophy. The number of deoxynucleotidyl transferase-mediated dUTP nick end labelling-positive spermatogenic cells was higher in Prx4(-/y) mice than in Prx4(+/y) mice and increased remarkably in response to warming the lower abdomen at 43 degrees C for 15 min. Cells reactive to antibodies against 4-hydroxynonenal and 8-hydroxyguanine were high in the Prx4(-/y) mice and concomitant with elevated oxidation of lipid and protein thiols. The cauda epididymis of Prx4(-/y) mice contained round spermatocytes, which were not found in Prx4(+/y) mice, and displayed oligozoospermia. However, mature spermatozoa from the epididymis of Prx4(-/y) mice exhibited normal fertilization In vitro. Taken together, these results indicate that spermatogenic cells lacking Prx4 are more susceptible to cell death via oxidative damage than their wild-type counterparts. Our results suggest that the presence of Prx4, most likely the membrane-bound form, is important for spermatogenesis, but not an absolute requisite.

Novel clonal strains from adult rat anterior pituitary producing S-100 protein.

S-100 protein, one of the unique proteins found in the nervous system1,2, has recently been discovered unexpectedly in the rat anterior pituitary3,4. Immunocytochemistry reveals that stellate, follicular and folliculostellate cells5, and marginal cells4 of the rat anterior pituitary contain this protein; however, as far as we know, there are no reports on the physiological role of this protein in the anterior pituitary. In the study reported here, three S-100 protein-producing clonal strains (JH-S3, JH-S8 and JH-S12) from adult rat anterior pituitaries were established by using the single cell-plating feeder layer method6. These new clonal strains reveal that the S-100 protein-producing cell is an independent cell type of the anterior pituitary. Both cultures and grafts of the JH-S3 cells stain immunocytochemically with anti-S100 protein IgG fraction. Moreover, the S-100 protein and conditioned medium of JH-S3 clonal cells both stimulate release of prolactin from prolactin-secreting clonal cells (1G4)7 in vitro.

Nano-scaled particles of titanium dioxide convert benign mouse fibrosarcoma cells into aggressive tumor cells.

Nanoparticles are prevalent in both commercial and medicinal products; however, the contribution of nanomaterials to carcinogenesis remains unclear. We therefore examined the effects of nano-sized titanium dioxide (TiO(2)) on poorly tumorigenic and nonmetastatic QR-32 fibrosarcoma cells. We found that mice that were cotransplanted subcutaneously with QR-32 cells and nano-sized TiO(2), either uncoated (TiO(2)-1, hydrophilic) or coated with stearic acid (TiO(2)-2, hydrophobic), did not form tumors. However, QR-32 cells became tumorigenic after injection into sites previously implanted with TiO(2)-1, but not TiO(2)-2, and these developing tumors acquired metastatic phenotypes. No differences were observed either histologically or in inflammatory cytokine mRNA expression between TiO(2)-1 and TiO(2)-2 treatments. However, TiO(2)-2, but not TiO(2)-1, generated high levels of reactive oxygen species (ROS) in cell-free conditions. Although both TiO(2)-1 and TiO(2)-2 resulted in intracellular ROS formation, TiO(2)-2 elicited a stronger response, resulting in cytotoxicity to the QR-32 cells. Moreover, TiO(2)-2, but not TiO(2)-1, led to the development of nuclear interstices and multinucleate cells. Cells that survived the TiO(2) toxicity acquired a tumorigenic phenotype. TiO(2)-induced ROS formation and its related cell injury were inhibited by the addition of antioxidant N-acetyl-l-cysteine. These results indicate that nano-sized TiO(2) has the potential to convert benign tumor cells into malignant ones through the generation of ROS in the target cells.

Interaction between spontaneous and neurally mediated regulation of smooth muscle tone in the rabbit corpus cavernosum

Interaction between spontaneous and neurally mediated regulation of tone in the corpus cavernosum smooth muscle (CCSM) of the rabbit was investigated. Changes in isometric muscle tension, intracellular Ca2+ concentration ([Ca2+]i) and membrane potential were recorded. CCSM developed spontaneous contractions, transient increases in [Ca2+]i (Ca2+ transients) and depolarizations. This spontaneous activity was abolished by blocking L‐type Ca2+ channels (nicardipine, 1 μm), sarcoplasmic reticulum Ca2+ pump activity (cyclopiazonic acid, 10 μm), Ca2+‐activated Cl− channels (niflumic acid, 10 μm) or cyclooxygenase‐2 (COX‐2; NS‐398, 1 μm). Transmural nerve stimulation initiated either α‐adrenergic contractions or nitrergic relaxations of CCSM depending on the level of muscle tone. NS‐398 suppressed nerve‐evoked contractions by about 70% but caused only a 40% reduction in the corresponding Ca2+ transient. Blocking nitric oxide synthase with Nω‐nitro‐l‐arginine (LNA, 100 μm) reinforced nerve‐evoked Ca2+ transients by about 150%, whilst increasing the corresponding Ca2+ transients by only 20%. In CCSM preparations that had been pre‐contracted with either noradrenaline (0.3 μm) or prostaglandin F2α (0.1 μm), nerve stimulation inhibited about 70% of the contraction and caused only a 20% decrease in [Ca2+]i. Fluorescent immunohistochemistry with COX‐2 antibodies and the reverse transcriptase‐polymerase chain reaction (RT‐PCR) method showed that the enzyme and its mRNA were highly expressed in the CCSM. These results suggest that spontaneously produced prostaglandins (PGs) not only contribute to the generation of spontaneous contractions but also facilitate nerve‐evoked contractions. Conversely, spontaneously released nitric oxide (NO) suppresses excitation. Thus, interaction between spontaneous and neurally mediated regulation of CCSM tone may be fundamental to maintaining the muscle contractility. In addition, both PGs and NO appear to alter CCSM tone with only small changes in [Ca2+]i.

Intercellular Communication Within the Rat Anterior Pituitary: XIV Electron Microscopic and Immunohistochemical Study on the Relationship Between the Agranular Cells and GnRH Neurons in the Dorsal Pars Tuberalis of the Pituitary Gland

Although numerous investigators in 1970s to 1980s have reported the distribution of LH‐RH nerve fibers in the median eminence, a few LH‐RH fibers have been shown to be present in the pars tuberalis. The significance of the finding remains to be elucidated, and there are few studies on the distribution of LH‐RH neurons in the pars tuberalis, especially in the dorsal pars tuberalis (DPT). Adult male Wistar‐Imamichi rats were separated into two groups: one for electron microscopy and the other for immunohistochemistry to observe LH‐RH and neurofilaments. Pituitary glands attached to the brain were fixed by perfusion, and the sections were prepared parallel to the sagittal plane. The typical glandular structure of the pars tuberalis was evident beneath the bottom floor of the third ventricle, and the thick glandular structure was present in the foremost region. Closer to the anterior lobe, the glandular structure changed to be a thin layer, and it was again observed at the posterior portion. Then the pituitary stalk was surrounded with the dorsal, lateral, and ventral pars tuberalis. LH‐RH and neurofilaments fibers were noted in the bottom floor, and some of them vertically descended to the gland. Adjacent to the glandular folliculostellate cells in the pars tuberalis, Herring bodies with numerous dense granules invading into the gland were present between the pituitary stalk and DPT. It was postulated that the “message” carried by LH‐RH might have been transmitted to the cells in the DPT to aid in the modulation of LH release. Anat Rec, 290:1388–1398, 2007.

Gastric estradiol-17β (E2) and liver ERα correlate with serum E2 in the cholestatic male rat

Cholestasis is associated with changes in hepatic cholesterol metabolism and serum estrogen levels. Ueyama and colleagues reported that the gastric estradiol-17β (E2) level in the portal vein is several times higher than that in the artery. This study aimed to clarify the relationships between gastric E2, hepatic estrogen receptor (ER) α and cholesterol metabolism in cholestatic male rats induced by bile duct ligation (BDL). After BDL, serum E2 levels in the portal vein and artery were measured by ELISA. The gene expression of gastric estrogen-synthesizing enzymes and various hepatic enzymes for cholesterol metabolism were measured by real-time RT-PCR, and gastric aromatase and hepatic ERα proteins were determined by immunohistochemistry and western blotting. Portal E2 levels increased by 4.9, 5.0, and 3.6 times that of controls at 2 days after BDL (BDL2d), BDL4d, and BDL7d respectively. The change in arterial E2 levels was positively correlated with that in the portal vein. Under these conditions, the expression of hepatic Ers1 (ERα) mRNA and protein was significantly reduced in a negative correlation with serum E2 levels in the portal vein after BDL. The expression of hepatic male-specific cytochrome P450 (CYP) genes Cyp2c55 and Cyp3a2 decreased and female-specific Cyp2c12 increased after BDL. It is postulated that the increase in gastric E2 levels, which occurs after BDL, results in the reduction of hepatic ERα, the elevation of arterial E2 level and leads to cholesterol metabolism becoming sex steroid dependent.

Immunoreactive luteinizing hormone (ir-LH) cells in the lung and stomach of chick embryos

Abstract.Luteinizing hormone (LH) immunoreactivity was detected in the lung and stomach of chick embryos by the immunoperoxidase staining technique using specific antiserum to chicken LH. Immunoreactive LH (ir-LH) cells first appeared in the primordial cells of the epithelial layer of lung bud and foregut as well as of Rathke’s pouch in the 3-day-old embryo, Hamburger and Hamilton stage 21. Ir-LH cells increased in number with advancing age of embryos in the lung, stomach, and pituitary gland. In the lung of 7-day-old embryos, stage 31, the ir-LH cells were distributed in the epithelium of primary, secondary, and tertiary bronchi, and their shapes were pseudostratified columnar, simple columnar, and simple cuboidal, depending on their sites in the intrapulmonary airway. Ir-LH cells were more numerous in the median part than in the lateral part of the lung, and the population in the epithelial layer of entobronchi of the secondary bronchi was 4 times higher than that in ectobronchi and laterobronchi of the secondary bronchi and in the primary bronchi. The immunoreactive products were found, either in the entire cell or in the apical part, facing the lumina of bronchi. In the stomach, ir-LH cells were found in the epithelial layer of gastric glands. No ir-LH cells were observed in interstitial regions, which consisted of mesenchymal cells and blood vessels, in the lung and stomach tissues. With advancing age, ir-LH cells changed their shapes to flat or squamous, coincident with the formation of parabronchi. Other pituitary hormones were not observed immunohistochemically in either the lung or stomach before hatching. Preabsorption of the antiserum against avian LH with the purified chicken LH or the extract of pituitaries from 10-day-old embryos completely destroyed the immunoreactivity to the cells in the lung and the pituitary. A single band of the immunoreaction products, whose molecular weight was around 25 K daltons, was shown by the immunostaining of nitrocellulose membrane transblotted after sodium dodecylsulphate-polyacrylamide gel electrophoresis of the purified pituitary LH, extracts of pituitaries from 10-day-old embryos, and the extracts of lungs from 7-, 10-, and 14-day-old chick embryos. These results demonstrated that ir-LH cells are present in extrapituitary tissues, and may play an important role during the development of chick embryonic lung and stomach.

Postnatal development of gastric aromatase and portal venous estradiol-17β levels in male rats

Gastric parietal cells synthesize and secrete estradiol-17β (E₂) into gastric veins joining the portal vein, and a large amount of gastric E₂ first binds to its receptors in the liver. However, the role of the gastric E₂ is not entirely clear during postnatal development. The objective of this study was to reveal the onset of aromatase and other steroid-synthesizing enzymes in the gastric mucosa; to determine the period of rising E₂ levels in the portal vein; and to further understand the relationship between gastric E₂ and liver estrogen receptor α (ERα). The immunoblot bands and the immunohistochemistry of gastric mucosa revealed that aromatase protein began to express itself at 20 days and then increased in the levels of aromatase protein from 20 days onward. Expression of mRNAs for gastric aromatase (Cyp19a1) and other steroid-synthesizing enzymes, 17α-Hydroxylase (Cyp17a1) and 17β-hydroxysteroid dehydrogenase (HSD17b3), also increased similar to the increment of aromatase protein. Portal venous E₂ levels were elevated after 20 days and increased remarkably between 23 and 30 days, similar to gastric aromatase mRNA levels. The E₂ level was approximately three times higher at 40 days than that at 20 days. The liver weight and Esr1 levels began to increase after 20 days and the increment was positively correlated with the change of portal venous E₂ levels. These findings suggest that some changes may occur around 20 days to regulate the gastric E₂ synthesis and secretion.

Intercellular communication within the rat anterior pituitary gland. XV. Properties of spontaneous and LHRH-induced Ca2+ transients in the transitional zone of the rat anterior pituitary in situ.

In the transitional zone of the rat anterior pituitary, spontaneous and LHRH-induced Ca(2+) dynamics were visualized using fluo-4 fluorescence Ca(2+) imaging. A majority of cells exhibited spontaneous Ca(2+) transients, while small populations of cells remained quiescent. Approximately 70% of spontaneously active cells generated fast, oscillatory Ca(2+) transients that were inhibited by cyclopiazonic acid (10 μm) but not nicardipine (1 μm), suggesting that Ca(2+) handling by endoplasmic reticulum, but not Ca(2+) influx through voltage-dependent L-type Ca(2+) channels, plays a fundamental role in their generation. In the adult rat anterior pituitary, LHRH (100 μg/ml) caused a transient increase in the Ca(2+) level in a majority of preparations taken from the morning group rats killed between 0930 h and 1030 h. However, the second application of LHRH invariably failed to elevate Ca(2+) levels, suggesting that the long-lasting refractoriness to LHRH stimulation was developed upon the first challenge of LHRH. In contrast, LHRH had no effect in most preparations taken from the afternoon group rats euthanized between 1200 h and 1400 h. In the neonatal rat anterior pituitary, LHRH caused a suppression of spontaneous Ca(2+) transients. Strikingly, the second application of LHRH was capable of reproducing the suppression of Ca(2+) signals, indicating that the refractoriness to LHRH had not been established in neonatal rats. These results suggest that responsiveness to LHRH has a long-term refractoriness in adult rats, and that the physiological LHRH surge may be clocked in the morning. Moreover, LHRH-induced excitation and associated refractoriness appear to be incomplete in neonatal rats and may be acquired during development.

Expression of microRNA-1, microRNA-133a and Hand2 protein in cultured embryonic rat cardiomyocytes

In this study, we investigated the expression of the pathway, SRF–microRNA-1/microRNA-133a–Hand2, in the Wistar rat embryonic ventricular cardiomyocytes under conventional monolayer culture. The morphological observation of the cultured cardiomyocytes and the mRNA expression levels of three vital constituent proteins, MLC-2v, N-cadherin, and connexin43, demonstrated the immaturity of these cultured cells, which was featured by less myofibril density, immature sarcomeric structure, and significantly lower mRNA expression of the three constituent proteins than those in neonatal ventricular samples. More importantly, results in this study suggest that the change of SRF–microRNA-1/microRNA-133a–Hand2 pathway results into the attenuation of the Hand2 repression in cultured cardiomyocytes. These outcomes are valuable to understand the cellular state as embryonic cardiomyocytes to be in vitro model and might be useful for the assessment of engineered cardiac tissue and cardiac differentiation of stem cells.