Melatonin modifies histone acetylation during in vitro maturation of mouse oocytes

Objective: We evaluated the effect of melatonin, as a potent antioxidant agent, on glutathione [GSH] and reactive oxygen species [ROS] levels, as well as histone H3 lysine 9 [H3K9], and H4 lysine 12 [H4K12] acetylation when added to oocytes culture medium

Materials and Methods: In this experimental study, two in vitro and in vivo groups were used. In the in vitro group, cumulus oocyte complexes [COCs] from the ovaries of B6D2F1 mice were cultured in maturation medium containing two doses of melatonin [10-9 and 10-6 M] and without melatonin [control group treated with dimethyl sulfoxide [DMSO]] for 22-24 hour. The cumulus expansion and nuclear status were monitored by an inverted microscope. Next, COCs were isolated from the oviducts of superovulated mice and studied as the in vivo group. In in vitro and in vivo matured oocytes, GSH and ROS levels were assessed by monochlorobimane [MCB] and 2-7-dichlorodihydrofluorescein diacetate [H2DCFDA] staining, respectively. Changes in histone acetylation were examined by immunofluorescent staining with specific antibodies against acetylated H3K9 and H4K12

Results: The H4K12 acetylation and ROS levels were significantly higher in the oocytes matured in the in vitro group compared to the in vivo group [P<0.05]. Furthermore, glutathione levels in the in vitro group were considerably lower than that of the in vivo group [P<0.05]. Melatonin at the concentration of 10-6 M had the most substantial effect on nuclear maturation and histone acetylation as well as glutathione and ROS levels in the in vitro group [P<0.05]

Conclusion: Exogenous melatonin improves the competence of mouse oocytes during in vitro maturation [IVM]

DNA methylation and histone acetylation patterns in cultured bovine adipose tissue-derived stem cells [BADSCs]

Many studies have focused on the epigenetic characteristics of donor cells to improve somatic cell nuclear transfer [SCNT]. We hypothesized that the epigenetic status and chromatin structure of undifferentiated bovine adipose tissue-derived stem cells [BADSCs] would not remain constant during different passages. The objective of this study was to determine the mRNA expression patterns of DNA methyltransferases [DNMT1, DNMT3a, DNMT3b] and histone deacetyltransferses [HDAC1, HDAC2, HDAC3] in BADSCs. In addition, we compared the measured levels of octamer binding protein-4 expression [OCT4] and acetylation of H3K9 [H3K9ac] in BADSCs cultures and different passages in vitro. In this experimental study, subcutaneous fat was obtained from adult cows immediately post-mortem. Relative level of DNMTs and HDACs was examined using quantitative real time polymerase chain reaction [q-PCR], and the level of OCT4 and H3K9ac was analyzed by flow cytometry at passages 3 [P3], 5 [P5] and 7 [P7]. The OCT4 protein level was similar at P3 and P5 but a significant decrease in its level was seen at P7. The highest and lowest levels of H3K9ac were observed at P5 and P7, respectively. At P5, the expression of HDACs and DNMTs was significantly decreased. In contrast, a remarkable increase in the expression of DNMTs was observed at P7. Our data demonstrated that the epigenetic status of BADSCs was variable during culture. The P5 cells showed the highest level of stemness and multipotency and the lowest level of chromatin compaction. Therefore, we suggest that P5 cells may be more efficient for SCNT compared with other passages

effect of melatonin on maturation, glutathione level and expression of HMGB1 gene in brilliant cresyl blue [BCB] stained immature oocyte

Nutrients and antioxidants in the medium of immature oocyte have a profound effect on maturation, fertilization and development of resulting embryos. In this study the effects of melatonin as an antioxidant agent on maturation, glutathione level and expression of high mobility group box-1 [HMGB1] gene were evaluated in immature oocytes of mice stained with brilliant cresyl blue [BCB]. In this experimental study, immature oocytes were harvested from ovaries of Naval Medical Research Institute [NMRI] mice. Oocytes were stained with 26 Micro M BCB for 90 minutes and transferred to in vitro maturation medium containing varying doses of melatonin [10[-12], 10[-9], 10[-6], 10[-3] M] and without melatonin, for 22-24 hours. Maturation was monitored using an inverted microscope. Glutathione was assessed by monochlorobimane [MCB] staining and HMGB1 expression in mature oocyte was analyzed using real-time polymerase chain reaction [PCR]. Melatonin in the concentration of 10[-6] M had the most effect on maturation and HMGB1 expression of BCB+ oocytes [p<0.05]. Meanwhile melatonin had no effects on glutathione levels. Additionally in immature BCB- oocytes, compared to the control group, melatonin did not affect cytoplasm maturation [p>0.05]. In vitro treatment with melatonin increases the maturation and HMGB1 expression in BCB+ immature oocytes and has no significant effect on glutathione levels

effects of pentoxifylline on the wound healing process in a rat experimental pressure sore model

The present study used a histological evaluation method to examine the effects of pentoxifylline [PTX] on healing an experimentally-induced pressure sore in a rat model. There were 36 adult male rats used in this study. Under general anesthesia and sterile conditions, we used forceps to create one pressure sore on each rat. A double layer of folded skin from the dorsal region was held with the highest forceps pressure grade for two hours, followed by 30 minutes of relaxation. This was repeated 12 times over three consecutive working days, and created a pressure sore after seven days. Next, rats were randomly divided into three control and three experimental groups. The experimental groups received intraperitoneal injections of PTX [50 mg/kg] for 14, 21, and 28 days after the pressure sore was created. Control groups received a similar volume of saline solution. Rats were euthanized, after which samples were extracted from the wound area and prepared for light microscopy examination. We calculated the number of neutrophils, macrophages, fibroblasts, blood vessel sections, and thicknesses of the newly formed epidermis and dermis. Although the values of some studied parameters were higher in the experimental group, there were no significant differences noted between the experimental and control groups. In this study PTX did not increase any histological parameters. Thus, the effects of PTX on the pressure sore model seem to result from different mechanisms