Mammalian Apoptosis in Whole Neonatal Ovaries using Confocal Laser Scanning Microscopy

Abstract

Confocal Laser Scanning Microscopy (CLSM) is being used to visualize folliculogenesis and presumed apoptotic events in whole neonatal mouse/rat ovaries. Our laboratory has previously demonstrated that apoptosis can be visualized in 8-9 day whole mouse embryos, 12 day fetal mouse eyes or 12-15 fetal rat limbs stained with the fluorescent lysosomal stain, LysoTracker Red (LT). The observed LT staining has been correlated with Nile blue staining (lysosomes and phagocytosis), TUNEL staining (DNA breaks) and apoptotic tissue morphology in rat and mouse developmental systems. This increased LT staining intensity appeared to be associated with acidic tissue, lysosomes, phagocytosis and dying cells. Here we describe recent progress using LT to evaluate the apoptotic process in whole ovaries. We are currently optimizing sample preparation and confocal machine operation to improve the image quality. to increase depth of visualization, ovaries derived from 10-45 day old mice/rats are fixed with an aldehyde and then made transparent by MEOH dehydration followed by benzyl alcohol/benzyl aldehyde (BABB) clearing. Such whole ovaries or tissue sections are viewed by CLSM using 568-wavelength laser light to obtain images of oocytes and follicles. Follicles showing gross morphologic changes typical of atresia were also characterized by intense LT staining of granulosa cells. This increased LT staining intensity appeared to be associated with acidic tissue and dying cells contained within the follicle.