Peroxynitrite deteriorates oocyte quality through disassembly of microtubule organizing centers

Abstract

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− (<2.5μM) 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.