Khan Sana N, Shaeib Faten, Thakur Mili, Jeelani Roohi, Awonuga Awoniyi O, Goud Pravin T, Abu-Soud Husam M
Department of Obstetrics and Gynecology, The C.S. Mott Center for Human Growth and Development, Wayne State University School of Medicine, Detroit, MI, USA.
Department of Obstetrics and gynecology, Division of Reproductive Endocrinology and Infertility, University of California Davis, Sacramento, CA, USA; California IVF Fertility Center, Davis and Sacramento, USA.
Free Radic Biol Med. 2016 Feb;91:275-80. doi: 10.1016/j.freeradbiomed.2015.12.033. Epub 2015 Dec 31.
Previous theoretical studies have suggested that utilization of 3-D imaging to acquire morphologic parameters of meiotic spindles may be useful in infertility related procedures as an assessment of oocyte quality. However, our results show that treatment of oocytes with increasing concentrations of peroxynitrite (ONOO(-)) caused a dramatic alteration in spindle shape in which morphologic parameters are not measurable or are uninformative in terms of oocyte quality. Metaphase II mouse oocytes (n=520) were treated with increasing concentrations of ONOO(-), after which all oocytes were fixed and subjected to indirect immunofluorescence. Oocyte quality was assessed by alterations in the microtubule-organizing center (MTOC), pericentrin location, microtubule morphology, and chromosomal alignment. In untreated oocytes, pericentrin is primarily assembled utilizing the acentrosomal MTOC, which appears as a condensation at both spindle poles. The spindle has a symmetrical pointed barrel shape, assembled around the chromosomal plate at the spindle equator. Oocytes treated with low concentrations of ONOO(-) (<2.5 μM) showed shortening of the spindle apparatus, while pericentrin scatters from a tight condensation to a dispersed cluster around each spindle pole. At higher ONOO(-) concentrations (>2.5μM) the central attachments between microtubules are strained and bend or unevenly break, and the MTOC proteins are further dispersed or undetectable. Peroxynitrite mediated MTOC damage, which deranges the chromosomal scaffold at the time of assembly and separation, caused the deterioration in oocyte quality. These results provide a link between reactive oxygen species and poor reproductive outcomes and elucidate the underlying etiology, which could be used as a superior biomarker for oocyte quality compared to existing assessment tools.
以往的理论研究表明，利用三维成像获取减数分裂纺锤体的形态学参数，可能有助于不孕相关手术中对卵母细胞质量的评估。然而，我们的结果显示，用浓度不断增加的过氧亚硝酸盐（ONOO(-)）处理卵母细胞会导致纺锤体形状发生显著改变，在此情况下，形态学参数无法测量，或者对于卵母细胞质量而言没有参考价值。用浓度不断增加的ONOO(-)处理中期II期小鼠卵母细胞（n = 520），之后将所有卵母细胞固定并进行间接免疫荧光检测。通过微管组织中心（MTOC）、中心粒外周蛋白位置、微管形态和染色体排列的变化来评估卵母细胞质量。在未处理的卵母细胞中，中心粒外周蛋白主要利用无中心体的MTOC进行组装，MTOC在纺锤体两极均表现为凝聚物。纺锤体呈对称的尖桶状，围绕纺锤体赤道处的染色体板组装。用低浓度ONOO(-)（<2.5 μM）处理的卵母细胞显示纺锤体装置缩短，而中心粒外周蛋白从紧密凝聚物分散到每个纺锤体极周围的分散簇。在较高的ONOO(-)浓度（>2.5μM）下，微管之间的中心连接受到拉伸并弯曲或不均匀断裂，并且MTOC蛋白进一步分散或无法检测到。过氧亚硝酸盐介导的MTOC损伤，在组装和分离时扰乱染色体支架，导致卵母细胞质量下降。这些结果提供了活性氧与不良生殖结局之间的联系，并阐明了潜在病因，与现有的评估工具相比，这可作为卵母细胞质量的一种更优生物标志物。
