Takuya Matsuyama

Bactericidal/Permeability-Increasing Protein Is Associated With the Acrosome Region of Rodent Epididymal Spermatozoa

To elucidate the molecular mechanisms involved in sperm maturation during epididymal transit, we intended to isolate secretory molecules that are region-specifically expressed along the epididymis and secreted into the lumen of epididymal ducts. By using differential display screening and DNA sequence analyses, we isolated a rat bactericidal/permeability-increasing protein (BPI) possessing a signal sequence at its N-terminal, which was expressed in the caput region of epididymis, but not in the caudal region. Reverse transcription polymerase chain reaction analysis and in situ hybridization showed that rat BPI messenger RNA (mRNA) was highly expressed in caput epididymal epithelium and that its expression level was developmentally up-regulated. Confocal laser scanning microscopy with the anti-BPI antibody revealed that in both rats and mice, BPI protein was detected on granulelike structures in the lumen of both caput and cauda epididymal ducts, as well as at the sperm surface covering the acrosome region in spermatozoa freshly isolated from epididymis. Acrosome reaction induced by calcium ionophore A23187 in vitro brought about the disappearance of BPI on mouse spermatozoa. These data suggested that BPI, which is synthesized in caput epididymis and secreted into the lumen, is associated with not only the granulelike structures, but also the sperm surface covering the acrosome region, and that BPI bound to the acrosome region is extinguished by acrosome reaction. Possibly BPI bound to the sperm surface covering the acrosome region in rodent spermatozoa is involved in sperm maturation or fertilization.

Novel Nrf2 activators from microbial transformation products inhibit blood–retinal barrier permeability in rabbits

Nuclear factor erythroid 2‐related factor 2 (Nrf2) is a redox‐sensitive transcription factor that binds to antioxidant response elements located in the promoter region of genes encoding many antioxidant enzymes and phase II detoxifying enzymes. Activation of the Nrf2 pathway seems protective for many organs, and although a well‐known Nrf2 activator, bardoxolone methyl, was evaluated clinically for treating chronic kidney disease, it was found to induce adverse events. Many bardoxolone methyl derivatives, mostly derived by chemical modifications, have already been studied. However, we adopted a biotransformation technique to obtain a novel Nrf2 activator.

Thioacetamide-induced Hepatocellular Necrosis Is Attenuated in Diet-induced Obese Mice

To assess modification of thioacetamide-induced hepatotoxicity in mice fed a high-fat diet, male C57BL/6J mice were fed a normal rodent diet or a high-fat diet for 8 weeks and then treated once intraperitoneally with thioacetamide at 50 mg/kg body weight. At 24 and 48 hours after administration, massive centrilobular hepatocellular necrosis was observed in mice fed the normal rodent diet, while the necrosis was less severe in mice fed the high-fat diet. In contrast, severe swelling of hepatocytes was observed in mice fed the high-fat diet. In addition, mice fed the high-fat diet displayed more than a 4-fold higher number of BrdU-positive hepatocytes compared with mice fed the normal rodent diet at 48 hours after thioacetamide treatment. To clarify the mechanisms by which the hepatic necrosis was attenuated, we investigated exposure to thioacetamide and one of its metabolites, the expression of CYP2E1, which converts thioacetamide to reactive metabolites, and the content of glutathione S-transferases in the liver. However, the reduced hepatocellular necrosis noted in mice fed the high-fat diet could not be explained by the differences in exposure to thioacetamide or thioacetamide sulfoxide or by differences in the expression of drug-metabolizing enzymes. On the other hand, at 8 hours after thioacetamide administration, hepatic total glutathione in mice fed the high-fat diet was significantly lower than that in mice fed the normal diet. Hence, decreased hepatic glutathione amount is a candidate for the mechanism of the attenuated necrosis. In conclusion, this study revealed that thioacetamide-induced hepatic necrosis was attenuated in mice fed the high-fat diet.

Toxicogenomic investigation on rat testicular toxicity elicited by 1,3-dinitrobenzene

Rats were treated with a single oral dose of 10, 25 and 50mg/kg of 1,3-dinitrobenzene (DNB), and the testis was subjected to a GeneChip microarray analysis. A total of 186 and 304 gene probe sets were up- and down-regulated, respectively, by the DNB treatment, where spermatocyte death and Sertoli cell vacuolation in testis and increased debris of spermatogenic cell in epididymis were noted. The expression profile for four sets of genes were investigated, whose expressions are reported to localize in specific cell types in the seminiferous epithelium, namely Sertoli cells, spermatogonia plus early spermtocytes, pachytene spermatocytes and round spermatids. The data demonstrated that pachytene spermatocyte-specific genes elicited explicit down-regulation in parallel with the progression of spermatocyte death, while other gene sets did not show characteristic expression changes. In addition, Gene Ontology analysis indicated that genes associated with cell adhesion-related genes were significantly enriched in the up-regulated genes following DNB treatment. Cell adhesion-related genes, namely Cdh2, Ctnna1, Vcl, Zyx, Itgb1, Testin, Lamc3, Pvrl2 and Gsn, showed an increase in microarray and the up-regulation of Cdh2 and Testin were confirmed by real time RT-PCR. The gene expression changes of pachytene spermatocyte-specific genes and cell adhesion-related genes were thought to reflect a decrease in the number of spermatocytes and dysfunction of Sertoli-germ cells adhesion junction, and therefore these genes would be potential genomic biomarkers for assessing DNB-type testicular toxicity.

Transcriptional profile of ethylene glycol monomethyl ether-induced testicular toxicity in rats

Abstract To clarify the molecular mechanism of ethylene glycol monomethyl ether (EGME)-induced testicular toxicity, the potential for EGME-related changes in transcript levels of genes including spermatocyte-specific genes was evaluated in the testis of rats given single dosing of EGME at 200, 600, or 2000 mg/kg. Furthermore, the contribution of decreased testicular testosterone on EGME-induced spermatocyte toxicity was investigated by comparing to transcriptional profile due to a testosterone synthesis inhibitor, ketoconazole (KET), at 30 or 300 mg/kg. EGME at 600 mg/kg or more dose-dependently caused testicular toxicity characterized by degeneration and necrosis of spermatocytes at stage VII–XIV seminiferous tubules. The spermatocyte injury was well correlated with decreased spermatocyte-specific gene expression. Analysis of upstream regulators by the Ingenuity Pathways Analysis system suggested that up-regulation of oxidative stress, protein kinase activation, and histone acetylation was involved in EGME-induced spermatocyte toxicity. Interestingly, KET decreased testicular testosterone to a similar extent compared to the EGME treatment, but KET at up to 300 mg/kg did not show any histopathological abnormality or change in the expression of spermatocyte-specific genes. These results suggested that the decreased testicular testosterone have little impact on EGME-induced spermatocyte injury. In contrast, KET showed trends toward increases in Hsd3b2 and Hsd17b2 mRNAs, presumably resulting from inhibition of androgen synthesis. Transcriptome analysis clearly demonstrated the differential effects of EGME and KET on androgen synthesis. In conclusion, EGME caused spermatocyte toxicity correlated with decreased expression of spermatocyte-specific genes. Furthermore, oxidative stress, protein kinase activation, and histone acetylation were suggested to be involved in EGME-induced testicular toxicity.