Effect of antioxidants on BPA-induced stress on sperm function in a mouse model.

In the past few years, bisphenol A, (BPA) an endocrine-disrupting chemical, has received increasing attention because of its detrimental health effects. There is ample evidence to support that BPA interferes with the reproductive health of humans and animals. In spermatozoa, BPA-induced adverse effects are mostly caused by increased oxidative stress. Using an in vitro experimental model, we examined whether antioxidants (glutathione, vitamin C, and vitamin E) have defensive effects against BPA-induced stress in spermatozoa. The results showed that antioxidants inhibit the overproduction of reactive oxygen species (basically cellular peroxides) and increase intracellular ATP levels, thereby preventing motility loss and abnormal acrosome reaction in BPA-exposed spermatozoa. In particular, glutathione and vitamin E reduced the protein kinase A-dependent tyrosine phosphorylation in spermatozoa and, thus, prevented the precocious acrosome reaction from occurring. Furthermore, we found that the compromised fertilisation and early embryo development mediated by BPA-exposed spermatozoa can be improved following their supplementation with glutathione and vitamin E. Based on these findings, we suggest that antioxidants reduce oxidative stress in BPA-exposed spermatozoa, thus preventing detrimental effects on their function and fertility.

Effect of endocrine disruptors on the ratio of X and Y chromosome-bearing live spermatozoa.

Although equal numbers of X and Y spermatozoa are produced during spermatogenesis, the sex chromosome ratio in ejaculated spermatozoa can be altered by exposure to endocrine-disrupting chemicals (EDCs), which can be reflected by altered sex ratios at birth. Here, we hypothesized EDCs affect sperm functions and viability of X and Y chromosome-bearing human spermatozoa. After exposure to genistein (Gen), bisphenol A (BPA), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), dibromochloropropane (DBCP), and diazinon (Diaz), we evaluated motility, viability, capacitation, and differential viability of X and Y spermatozoa. All EDCs tested altered sperm viability, motility, and capacitation. Interestingly, the Y/X ratio of live spermatozoa was significantly lower in sperm treated with TCDD, DBCP, and Diaz than control spermatozoa. Our results suggest that some of EDCs have larger effects on the viability of Y spermatozoa than X spermatozoa, implicating that a reduction in Y sperm viability may result in a female-biased sex ratio of offspring at birth.

Effect of Aminopeptidase N on functions and fertility of mouse spermatozoa in vitro.

Aminopeptidase N (APN) is defined as a multifunctional enzyme, which regulate cellular physiology of a wide variety of cells in human. Earlier studies reported that mammalian semen shares this common enzyme as a major protein of seminal plasma that has correlation with male fertility, while the regulatory mechanisms of APN in spermatozoa are still far from being well understood. Present study was designed to investigate the role of APN in biological and chemical functions of spermatozoa using an in vitro antagonistic approach. Results showed that lower APN activity in sperm culture medium significantly increased sperm motility and the percentage of high speed spermatozoa and decreased the percentage of slow speed spermatozoa after a dose dependent inhibitor treatment (10, 100, and 1000 µM leuhistin) on epididymal mouse spermatozoa in a capacitating media for 90 min. Both 100 µM and 1000 µM decreased APN activity, while only 1000 µM decreased cell viability and increased PKA activity significantly compared to control. Nonetheless capacitation status, acrosome reaction status, and lactate dehydrogenase activity were not affected. Intriguingly, the treatment affected embryonic development through decreasing tyrosine phosphorylation of proteins and increasing reactive oxygen species levels. Further in silico analysis revealed associated regulatory proteins, which have critical functional role for male fertility.

[The Effect of Helicobacter pylori Infection on Recurrence of Gastric Hyperplastic Polyp after Endoscopic Removal].

Background/Aims: Several previous studies suggest that eradication of Helicobacter pylori (H. pylori) leads to the disappearance of gastric hyperplastic polyps. However, little is known about the effect of H. pylori status and eradication on the recurrence of gastric polyps after endoscopic removal. Here, we investigated the recurrence of gastric polyps according to the final H. pylori status in patients who underwent endoscopic removal of gastric hyperplastic polyps. Methods: Between January 2011 and December 2016, patients who underwent endoscopic removal of gastric hyperplastic polyps and were followed-up for more than two months were enrolled. The success of H. pylori eradication was assessed by histology and rapid urease test or urea breath test, at least 4 weeks after the completion of eradication treatment. At follow-up, the recurrence of gastric polyp was evaluated via esophagogastroduodenoscopy. Results: Seventy-nine patients were enrolled. During the mean follow-up period of 16.4 months, the recurrence rate of gastric polyp was 25.3%. Among those who received H. pylori eradication therapy, the H. pylori persistent group showed a higher recurrence of polyp than the H. pylori eradicated group; but there was no statistical significance (42.9% vs. 21.7%, p=0.269). Regarding the final H. pylori infection status, the recurrence rate of gastric polyps was significantly higher in the H. pylori positive group than in the H. pylori negative group (42.9% vs. 18.9%, p=0.031). In multivariate analysis, the final H. pylori infection status was a significant risk factor for gastric polyp recurrence after endoscopic removal. Conclusions: The final positive H. pylori infection status is significantly associated with higher recurrence of gastric hyperplastic polyps after endoscopic removal.

The effect of surgical wound on ovarian carcinoma growth in an animal model.

BACKGROUND/AIMS: We examined the effects according to the extent of surgical wound mimicking laparoscopy or laparotomy on ovarian cancer growth in an orthotopic mouse model. MATERIALS AND METHODS: To mimic surgery effects, we performed laparoscopy or laparotomy on athymic nude mice under isoflurane inhalation at four days after tumor cell injection. For the laparoscopy model, we performed pneumoperitoneum with CO2, by inserting three cannulars. RESULTS: Mice in the laparoscopy-mimicking group had significantly lower tumor weight compared to mice in the laparotomy group (p<0.05). In the immediate postoperative period, serum levels of vascular endothelial growth factor (VEGF) and matrix metalloproteinase-2 (MMP2) were significantly lower in the laparoscopy group. CONCLUSION: These results indicate that a minimal surgical wound such as that on laparoscopy, appears to induce little surgical stress on enhancing tumor growth compared to laparotomy in an ovarian cancer animal model, possibly because it minimally influences the secretion of VEGF and MMP2.

A distal cis-regulatory element, CNS-9, controls NFAT1 and IRF4-mediated IL-10 gene activation in T helper cells.

IL-10 is a multifunctional cytokine that plays a critical role in maintaining the balance between immunity and tolerance. Previously, we identified proximal regulatory elements and alterations of chromatin structure in the IL-10 gene loci of Th1 and Th2 cells. We have now characterized a crucial cis-regulatory element, CNS-9, located 9kb upstream of the transcription start site in IL-10 gene loci. The CNS-9 region is highly conserved in vertebrate genomes, and contains clustered NFAT and IRF binding motifs. In vitro binding of NFAT1 and IRF4 to the CNS-9 region was observed by EMSA. Furthermore, Th2-preferential in vivo binding of NFAT1 and IRF4 to the CNS-9 region was observed by ChIP. Cyclosporine A treatment on wild type Th2 cells or Th2 cells derived from NFAT1 knockout (NFAT1(-/-)) mice showed significantly reduced trans-activity of CNS-9. The Th2 subset-specific enhancer activity of CNS-9 was upregulated synergistically by NFAT1 and its partner IRF4. Mutations in the binding sites for NFAT1 and IRF4 abrogated its enhancer activity of CNS-9. Collectively, our results establish crucial roles for enhancer element CNS-9, and NFAT1 and IRF4 that bind to it, for IL-10 expression in differential T helper subsets.

Differential regulation of the IL-10 gene in Th1 and Th2 T cells.

Interleukin-10 (IL-10), an immunoregulatory cytokine, modulates the function of various immune and nonimmune cells, yet little information is available on the molecular mechanism of transcriptional regulation at the chromatin level. During T cell differentiation from naive T cells into Th1 and Th2 cells, the expression of IL-10 in Th1 cells slowly disappears, whereas Th2 cells produce more IL-10. We examined the chromatin structural changes associated with IL-10 gene transcription by naive and differentiated murine Th1 and Th2 cells. Naive T cells lack DNase I hypersensitivity (HS) sites in the vicinity of the IL-10 gene, whereas differentiated T cells display a strong 3' constitutive HS site as well as several inducible sites. In committed Th1 cells, the mechanism of IL-10 gene silencing is associated with a closed chromatin structure, the lack of an HS site at the promoter region, and the development of repressive histone modification near the IL-10 promoter and introns 3 and 4. We confirm that the majority of HS sites coincide with conserved noncoding sequences (CNSs) identified by comparative genomic sequence alignment between human and mouse genomes. Potential transcription factor binding sites were located by comparing CNSs with the TRANSFAC database. Predicted in vivo binding of specific factors on the CNS locus were confirmed by chromatin immunoprecipitation assays. Our results suggest that the combination of HS site and comparative genomic approaches allows identification of regulatory elements involved in differential IL-10 gene expression between Th1 and Th2 cells during T cell differentiation.

Chromatin-level regulation of the IL10 gene in T cells.

The immunoregulatory cytokine interleukin 10 (IL-10) modulates the function of diverse immune and non-immune cells. Here, we examine the chromatin structural changes associated with IL10 gene transcription by naive and differentiated murine T cells. Naive T cells lack DNase I hypersensitive (HS) sites in the vicinity of the IL10 gene, whereas differentiated T cells display a strong 3' constitutive HS site as well as several inducible sites. The majority of HS sites map to regions that are strongly conserved in sequence between mouse and human genomes. In committed Th1 cells, the mechanism of IL10 gene silencing is associated with the development of repressive histone modifications near the IL10 promoter and also near intronic hypersensitive regions of the IL10 gene. Our results constitute the first report of chromatin structural differences within the IL10 gene in differentiated Th1 and Th2 cells and emphasize the surprising diversity of mechanisms used to regulate cytokine gene expression at the chromatin level.