Peroxynitrite deteriorates oocyte quality through disassembly of microtubule organizing centers

• Spindle morphologic changes may be secondary to movement of microtubule organizing center proteins, pericentrin, or disruption of the spindle force balance.
• Previous dimensional analysis of spindle morphology may be lacking as assessment systems for oocyte quality.
• Analysis of spindle morphologic patterns may be used as superior biomarkers of oocyte quality.

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 (>2.5 μM) 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.

With increasing concentrations of peroxynitrite (ONOO-) spindle morphologies change in a reversible series from a normal well organized symmetrical barrel shape with aligned chromosomes to, initially, a shorter wider spindle, then to a elongated spindle with a “C” or “V” shape as the forces regulating spindle shape are deregulated. At the highest concentrations of treatment, the spindle fibers are deteriorated and only a disorganized cluster of chromosomal material remains. These morphologies are also seen in other inflammatory conditions.Figure optionsDownload high-quality image (198 K)Download as PowerPoint slide