Melatonin Protects Granulosa Cells for Progesterone Production as an Antioxidant in Human Ovarian Follicles.
Melatonin Protects Granulosa Cells for Progesterone Production as an Antioxidant in Human Ovarian Follicles.
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DOI：
10.1093/biolreprod/81.s1.378
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发表时间：
2009-07
影响因子：
3.6
通讯作者：
K. Taniguchi;T. Taketani;L. Lee;Fumie Kizuka;I. Tamura;N. Sugino
中科院分区：
文献类型：
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作者：
K. Taniguchi;T. Taketani;L. Lee;Fumie Kizuka;I. Tamura;N. Sugino
活性氧（ROS）在卵泡内产生，特别是在排卵过程中。ROS在排卵过程中发挥生理作用，例如卵泡破裂。然而，过量的ROS会引起氧化应激并损伤卵母细胞和黄素化颗粒细胞。另一方面，抗氧化防御系统包括超氧化物歧化酶（SOD）或谷胱甘肽（GSH）存在于卵泡中。卵泡内ROS和抗氧化剂之间的平衡似乎对卵母细胞和黄素化颗粒细胞至关重要。褪黑激素是松果体分泌的一种激素，是一种强大的自由基清除剂和广谱抗氧化剂。我们最近发现，人类排卵前卵泡液中含有高浓度的褪黑激素，褪黑激素可以保护卵母细胞免受氧化应激。本研究旨在探讨褪黑激素是否作为卵泡内的抗氧化剂保护颗粒细胞，以维持孕酮的产生。该项目已获得山口大学IRB的批准。获得所有女性的知情同意。在这项研究中招募了85名接受常规IVF-ET的妇女。分别抽吸每个卵泡，收集含有卵母细胞的卵泡液。取出卵母细胞后，立即离心各卵泡液，获得细胞组分和上清液。在第一项研究中，为了检查卵泡液中褪黑激素、孕酮产生和氧化应激相关分子之间的关系，通过ELISA测量了作为DNA相关氧化应激标志物的8-羟基-2 '-脱氧鸟苷（8-OHdG）、作为脂质相关氧化应激标志物的己酰赖氨酸加合物（HEL）以及作为抗氧化剂的SOD和GSH的水平。用放射免疫法测定褪黑素和孕酮的浓度。卵泡液中褪黑素浓度与孕酮浓度呈显著正相关（p < 0.05），与8-OHdG浓度呈显著负相关（p < 0.05）。然而，SOD和GSH并没有表现出任何相关性的氧化应激标志物，孕酮，或褪黑激素。孕酮与8-OHdG呈负相关（p < 0.05）。在第二项研究中，为了检测褪黑激素对黄体化颗粒细胞产生孕酮的影响，从每个个体的卵泡液的细胞组分中分离细胞。首先，将细胞与单独的褪黑激素（0、1、10、100 μ g/ml）和单独的过氧化氢（30、50、100、200 μ M）孵育12小时。其次，在存在或不存在褪黑激素（10或100 μ g/ml）的情况下，将细胞与过氧化氢（30或50 μ M）孵育12小时。孵育后，通过RIA测量培养基中的孕酮浓度。褪黑激素不直接影响黄体化颗粒细胞孕酮的产生。过氧化氢以剂量依赖性方式显著抑制孕酮的产生。过氧化氢对孕酮生成的抑制作用可被褪黑素显著阻断。总之，褪黑激素似乎在卵泡中起着重要的抗氧化作用。褪黑激素保护颗粒细胞，以维持正常排卵过程中产生的ROS产生孕酮。（海报）
Reactive oxygen species (ROS) are produced within the follicle especially during the ovulatory process. ROS play a physiological role in the process of ovulation, e.g. follicle rapture. However, excessive amount of ROS causes oxidative stress and damages oocytes and luteinized granulosa cells. On the other hand, antioxidant defense systems including superoxide dismutase (SOD) or glutathione (GSH) are present in follicles. The balance between ROS and antioxidants within the follicle seems to be critical for oocytes and luteinized granulosa cells. Melatonin, which is a hormone secreted by pineal gland, is known as a powerful free radical scavenger and a broad-spectrum antioxidant. We recently found that human preovulatory follicular fluids contain high concentrations of melatonin and that melatonin protects oocytes from oxidative stress. The present study was undertaken to examine whether melatonin protects granulosa cells as an antioxidant within follicles to maintain progesterone production. This project was approved by IRB of Yamaguchi University. Informed consent was obtained from all women. Eighty-five women who underwent conventional IVF-ET were recruited in this study. Each follicle was aspirated separately, and the follicular fluid containing an oocyte was collected. Immediately after removal of the oocyte, each follicular fluid was centrifuged, and the cellular component and the supernatant were obtained. In the first study, to examine the relationship between melatonin, progesterone production, and oxidative stress-related molecules in the follicular fluid, levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG) as a DNA-related oxidative stress marker, hexanoyl-lysine adduct (HEL) as a lipid-related oxidative stress marker, and SOD and GSH as antioxidants were measured by ELISA. Melatonin and progesterone concentrations were measured by RIA. Melatonin concentrations in the follicular fluid were significantly and positively correlated with progesterone (p < 0.05) and negatively correlated with 8-OHdG (p < 0.05). However, SOD and GSH did not show any correlation to the oxidative stress markers, progesterone, or melatonin. There was a negative correlation between progesterone and 8-OHdG (p < 0.05). In the second study, to examine the effect of melatonin on progesterone production by luteinized granulosa cells, cells were isolated from the cellular component of follicular fluids in each individual. First, the cells were incubated with melatonin alone (0, 1, 10, 100 micro g/ml) and hydrogen peroxide alone (30, 50, 100, 200 micro M) for 12 h. Secondly, cells were incubated with hydrogen peroxide (30 or 50 micro M) in the presence or absence of melatonin (10 or 100 micro g/ml) for 12 h. After incubation, progesterone concentrations in the medium were measured by RIA. Melatonin directly did not affect progesterone production by luteinized granulosa cells. Progesterone production was significantly inhibited by hydrogen peroxide in a dose-dependent manner. The inhibitory effect of hydrogen peroxide on progesterone production was significantly blocked by melatonin. In conclusions, melatonin seems to play an important role as an anti-oxidant in the follicle. Melatonin protects granulosa cells to maintain progesterone production from ROS which are produced during the normal ovulatory process.(poster)