Keshavarzi Somayeh, Salehi Mohammad, Farifteh-Nobijari Fattaneh, Hosseini Taher, Hosseini Sara, Ghazifard Alaleh, Ghaffari Novin Marefat, Fallah-Omrani Vahid, Nourozian Mohsen, Hosseini Ahmad
Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Department of Biology and Anatomical Sciences, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Cell J. 2018 Jul;20(2):244-249. doi: 10.22074/cellj.2018.4860. Epub 2018 Mar 18.
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.
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 and 10 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.
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 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).
Exogenous melatonin improves the competence of mouse oocytes during in vitro maturation (IVM).
我们评估了褪黑素作为一种强效抗氧化剂，添加到卵母细胞培养基中时对谷胱甘肽（GSH）、活性氧（ROS）水平以及组蛋白H3赖氨酸9（H3K9）和H4赖氨酸12（H4K12）乙酰化的影响。
在本实验研究中，使用了两个体外和体内组。在体外组中，将B6D2F1小鼠卵巢中的卵丘卵母细胞复合体（COCs）在含有两种剂量褪黑素（10和10μM）和不含褪黑素[用二甲基亚砜（DMSO）处理的对照组]的成熟培养基中培养22 - 24小时。通过倒置显微镜监测卵丘扩展和核状态。接下来，从超排卵小鼠的输卵管中分离出COCs并作为体内组进行研究。在体外和体内成熟的卵母细胞中，分别通过单氯双氢乙锭（MCB）和2,7 - 二氯二氢荧光素二乙酸酯（H2DCFDA）染色评估GSH和ROS水平。通过用针对乙酰化H3K9和H4K12的特异性抗体进行免疫荧光染色来检查组蛋白乙酰化的变化。
与体内组相比，体外组成熟卵母细胞中的H4K12乙酰化和ROS水平显著更高（P<0.05）。此外，体外组中的谷胱甘肽水平明显低于体内组（P<0.05）。浓度为10μM的褪黑素对体外组的核成熟、组蛋白乙酰化以及谷胱甘肽和ROS水平影响最大（P<0.05）。
外源性褪黑素可提高小鼠卵母细胞在体外成熟（IVM）过程中的能力。
