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.

Confocal laser scanning microscopy of whole mouse ovaries: Excellent morphology, apoptosis detection, and spectroscopy

“…We have previously demonstrated that apoptosis (cell death) can be visualized in 8-9 days old mouse embryos, rat fetal limbs, and other tissues using a fluorescent dye (LysoTracker Red, LT), which stains lysosomes and acidic regions of cells/tissues (16)(17)(18)(19)(20)(21)(22)(23). The current study extends our previous work to visualize apoptosis within follicles of a whole ovary (17).…”

“…The current study extends our previous work to visualize apoptosis within follicles of a whole ovary (17). To study the morphology of oocytes and granulosa cells located within the follicles in whole ovaries, we had to improve the tissue preparation protoThe information in this document has been funded wholly (or in part) by the U.S. Environmental Protection Agency.…”

“…As previously described for rat limbs, rat ovaries, and mouse embryos (16)(17)(18)(19), the ovaries were stained with LT, fixed with aldehyde, dehydrated with MEOH, and cleared with BABB (1:2 by volume; Sigma, St. Louis, MO).…”

“…After fixation for 3-4 h at 37°C, the ovaries were stored overnight at 4°C. This fixation procedure was developed after subjective observations from preliminary trials that varied the temperature (16,17) between 4, 25, and 37°C with different aldehyde cocktails that varied the percentage of Glut between 0, 0.5, and 1% and the percentage of PF between 2% with 4%.…”

“…Ovaries were cleared by adding sequentially 50% BABB in MEOH for 0.5-2 h, followed by 70% BABB, and finally in 100% BABB (17,(24)(25)(26)(27). The refractive index of BABB (RI 5 1.54) was similar to that of the cleared ovaries.…”