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CONFOCAL LASER SCANNING MICROSCOPY OF APOPTOSIS IN WHOLE MOUSE OVARIES
Citation:
Zucker, R M., S C. Jeffay, AND S P. Darney. CONFOCAL LASER SCANNING MICROSCOPY OF APOPTOSIS IN WHOLE MOUSE OVARIES. Presented at Society of Toxicology, San Francisco, CA, March 25-29, 2001.
Description:
Confocal Laser Scanning Microscopy of Apoptosis in Whole Mouse 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 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). Here we describe recent progress in using LT to evaluate apoptosis in whole ovaries. We are currently optimizing sample preparation and confocal machine operation 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. Such 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. We have observed, in ovaries from 19-45 day old mice, that 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. However, the oocytes in such brightly stained, atretric follicles exhibit minimal LT staining and contain morphologically normal nuclei with no evidence of the nuclear fragmentation typical of apoptotic cells. These methods for visualizing apoptosis in developing ovaries will be used to establish baseline data for our studies on the impact of potential reproductive toxicants on neonatal and prepubertal ovaries.
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