Confocal laser scanning microscopy of rat follicle development.

Abstract

This study used confocal laser scanning microscopy (CLSM) to study follicular development in millimeter pieces of rat ovary. To use this technology, it is essential to stain the tissue before laser excitation with the confocal microscope. Various fluorescent stains (Yo-Pro, Bo-Pro, LysoTracker Red, hydroethidine, ethidium bromide, and 7-aminoactinomycin-d) were applied either to fresh tissue or to tissue that had been fixed with glutaraldehyde or paraformaldehyde. After fixation and staining, the tissue was dehydrated with MEOH and cleared with benzyl alcohol/benzyl aldehyde. CLSM was then used with the appropriate laser excitation, dichroics, and bandpass filters to acquire images of oocytes contained in follicles. Analysis of the data revealed three principal findings. First, a rapid increase in oocyte size occurred in the preantral stages of follicle development. In the antral stage of follicle development, there was a rapid increase in follicle size without any substantial increase in oocyte size. Second, accompanying these changes in oocyte and follicle growth was a differential staining pattern in which the nucleus stained more than the cytoplasm in a young follicle, but stained less than the cytoplasm as the follicle enlarged into the late antral stage. Lastly, using CLSM, atretic follicles showed increased LysoTracker Red staining in the granulosa region of the antral follicle, suggestive of cell death.