Department of Health Sciences, University of L'Aquila, Via Vetoio, 67100 L'Aquila, Italy.
Hum Reprod. 2011 Jul;26(7):1843-59. doi: 10.1093/humrep/der140. Epub 2011 May 9.
Highly reactive carbonyl compounds formed during glycolysis, such as methylglyoxal (MG), can lead to the formation of 'advanced glycation end products' (AGE) and carbonyl stress. Toxic AGEs are suspected to accumulate and play a role in reducing quality and developmental potential of mammalian oocytes of aged females and in PCOS and diabetic patients. Whether and how MG and AGE affect young and aged oocytes at the cellular level is unknown.
The study consists of three parts. In Part A expression of MG-detoxifying enzymes glyoxalases 1 and 2 was analysed by RT-PCR at different stages of maturation in denuded oocytes (DO), cumulus-enclosed oocytes (CEO) and metaphase (M)II oocytes of the CD-1 mouse to obtain information on stage-specific susceptibility to carbonyl stress. DO and CEO from young and aged females and from stimulated cycles were exposed to MG during maturation in vitro to assess also age-related changes in sensitivity to carbonyl stress induced by MG. Induction of apoptosis by MG on in vitro maturing DO was assessed by terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling test. In Part B of the study, DO from large antral follicles of ovaries of adult, young MF-1 mice in late diestrous were exposed to MG to assess direct influences of MG and AGEs formed during continuous exposure to MG on rate and kinetics of maturation to MII, on DNA integrity (by γ-H2AX staining) in the germinal vesicle (GV) stage, and on spindle formation and chromosome alignment (by tubulin and pericentrin immunofluorescence and polarization microscopy), and chromosome segregation (by C-banding) during in vitro maturation. Since MG and AGEs can affect functionality of mitochondria in Part C, mitochondrial distribution and membrane potential was studied using JC-1 probe. Expression of a redox-sensitive mito-Grx1-roGFP2 protein in mitochondria of maturing oocytes by confocal laser scanning microscopy was employed to determine the inner mitochondrial glutathion (GSH)/glutathion disulfide (GSSG)-dependent redox potential.
Part A revealed that mRNA for glyoxalases decreases during meiotic maturation. Importantly, cumulus from aged mice in CEO obtained from stimulated cycles does not protect oocytes efficiently from MG-induced meiotic arrest during in vitro maturation. Part B showed that the MG-induced meiotic delay or arrest is associated with significant rises in spindle aberrations, chromosome congression failure and aberrant telophase I in oocytes. MG exposure of meiotically arrested GV-stage oocytes significantly increases the numbers of γ-H2AX spots in the nucleus suggesting increased DNA damage, while MG exposure during maturation affects chromatin condensation and induces chromosome lagging at anaphase I. Moreover, Part C revealed that carbonyl stress by chronic exposure to MG is associated with delays in changes in mitochondrial distribution and altered inner-mitochondrial GSH/GSSG redox potential, which might be particularly relevant for cytoskeletal dynamics as well as processes after fertilization. Sensitivity to a meiotic block by MG appears dependent on the genetic background.
The sensitivity to carbonyl stress by MG appears to increase with maternal age. Since MG-exposure induces DNA damage, meiotic delay, spindle aberrations, anaphase I lagging and epimutation, aged oocytes are particularly at risk for such disturbances in the absence of efficient protection by cumulus. Furthermore, disturbances in mitochondrial distribution and redox regulation may be especially critical for fertilization and developmental competence of oocytes exposed to MG and carbonyl stress before or during maturation, for instance, in aged females, or in PCOS or diabetic patients, in agreement with recent suggestions of correlations between poor follicular and embryonic development, lower pregnancy rate and presence of toxic AGEs in serum, irrespective of age.
糖酵解过程中形成的高反应性羰基化合物，如甲基乙二醛（MG），可导致“晚期糖基化终产物”（AGE）和羰基应激的形成。有毒的 AGE 被怀疑会积累，并在降低老年雌性哺乳动物卵母细胞的质量和发育潜能以及多囊卵巢综合征和糖尿病患者中发挥作用。MG 和 AGE 是否以及如何在细胞水平上影响年轻和老年的卵母细胞尚不清楚。
该研究由三部分组成。在 A 部分中，通过 RT-PCR 分析了去卵丘卵母细胞（DO）、卵丘包裹卵母细胞（CEO）和 CD-1 小鼠中期（M）II 卵母细胞中 MG 解毒酶糖氧还蛋白 1 和 2 的表达，以了解对羰基应激的阶段特异性敏感性。来自年轻和老年女性以及刺激周期的 DO 和 CEO 被暴露于 MG 中以评估 MG 诱导的羰基应激敏感性的年龄相关变化。通过末端脱氧核苷酸转移酶介导的 dUDP 末端标记试验评估 MG 对体外成熟 DO 诱导的细胞凋亡。在研究的 B 部分中，将来自成年、年轻 MF-1 小鼠发情后期大卵泡的 DO 暴露于 MG 中，以评估 MG 和 AGEs 的直接影响，这些 AGEs 是在连续暴露于 MG 过程中形成的，对向 MII 的成熟率和动力学、GV 期的 DNA 完整性（通过 γ-H2AX 染色）、纺锤体形成和染色体排列（通过微管和中心体免疫荧光和偏振显微镜）以及体外成熟过程中的染色体分离（通过 C-带）。由于 MG 和 AGEs 会影响线粒体的功能，因此在 C 部分中研究了线粒体的分布和膜电位。使用 JC-1 探针研究了成熟卵母细胞中线粒体的线粒体分布和膜电位。通过共聚焦激光扫描显微镜研究了线粒体中氧化还原敏感的 mito-Grx1-roGFP2 蛋白的表达，以确定线粒体中谷胱甘肽（GSH）/谷胱甘肽二硫化物（GSSG）依赖性氧化还原电位。
A 部分显示，在减数分裂成熟过程中，糖氧还蛋白的 mRNA 减少。重要的是，来自刺激周期的老年小鼠的卵丘在体外成熟过程中不能有效地保护卵母细胞免受 MG 诱导的减数分裂阻滞。B 部分显示，MG 诱导的减数分裂延迟或阻滞与纺锤体异常、染色体共聚集失败和异常的第一次减数分裂末期有关。MG 暴露于减数分裂中期的 GV 期卵母细胞会导致核内 γ-H2AX 斑点数量显著增加，表明 DNA 损伤增加，而 MG 在成熟过程中的暴露会影响染色质浓缩，并在第一次减数分裂后期诱导染色体滞后。此外，C 部分表明，慢性暴露于 MG 引起的羰基应激与线粒体分布的变化延迟以及内部线粒体 GSH/GSSG 氧化还原电位改变有关，这对于细胞骨架动力学以及受精后的过程可能特别重要。对 MG 引起的减数分裂阻滞的敏感性似乎取决于遗传背景。
对 MG 引起的羰基应激的敏感性似乎随母体年龄的增加而增加。由于 MG 暴露会导致 DNA 损伤、减数分裂延迟、纺锤体异常、第一次减数分裂后期滞后和表观遗传改变，因此在没有卵丘有效保护的情况下，老年卵母细胞尤其容易受到这种干扰。此外，线粒体分布和氧化还原调节的干扰可能对 MG 暴露的卵母细胞的受精和发育能力特别关键，例如在成熟前或成熟过程中，在老年女性、多囊卵巢综合征或糖尿病患者中，这与最近提出的关于卵泡和胚胎发育不良、妊娠率降低以及血清中存在有毒 AGE 之间存在相关性的建议一致，而与年龄无关。
