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CONFOCAL LASER SCANNING MICROSCOPY OF APOPTOSIS IN WHOLE MOUSE AND RAT OVARIES
Citation:
Zucker, R M., S C. Jeffay, AND S P. Darney. CONFOCAL LASER SCANNING MICROSCOPY OF APOPTOSIS IN WHOLE MOUSE AND RAT OVARIES. Presented at Triangle Consortium for Reproductive Biology, Research Triangle Park, NC, January 27, 2001.
Description:
Confocal Laser Scanning Microscopy of Apoptosis in Whole Mouse and Rat Ovaries. Robert M. Zucker Susan C. Jeffay and Sally D. Perreault Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina, 27711.
Confocal Laser Scanning Microscopy (CLSM) is being used to visualize folliculogenesis and apoptotic events in whole neonatal mouse and rat ovaries. Our laboratory has previously demonstrated that apoptosis can be visualized in 8-9 day whole embryos or millimeter sections of adult ovaries stained with the fluorescent lysosomal stain, LysoTracker Red (LT). Recently progress has been made in using LT to evaluate apoptosis in whole ovaries. Sample preparation and confocal machine operation are being optimized to improve the image quality. To increase depth of visualization, ovaries are fixed with an aldehyde and then made transparent by MEOH dehydration followed by benzyl alcohol/benzyl aldehyde clearing. Whole ovaries or tissues sections are viewed by CLSM using 568-wavelength laser light to obtain images of oocytes contained within follicles of different sizes and developmental stages. In ovaries from 19-45 day old mice, follicles showing gross morphologic changes typical of atresia are also characterized by intense LT staining derived from the granulosa cells. This increased LT staining intensity appears to be associated with acidic tissue, dying cells and abnormal follicular morphology. Oocytes contained in such brightly stained, atretric follicles may exhibit minimal LT staining but often show nuclear fragmentation typical of apoptotic cells. These methods for visualizing apoptosis in developing ovaries will be used to establish baseline data for our future studies examining the impact of potential reproductive toxicants on neonatal and prepubertal ovaries.
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