We hope these in-depth protocols will help to enhance reproducibility in our field while also allowing researchers to make an informed decision when selecting accounting method for their studies. As it uses several sampling parameters, stereology is highly sensitive and accurate. However, direct counts are quicker and easier and can be performed on samples prepared with standard histological techniques.
Stereology is used in studies of ovarian biology for evaluating the impact of toxicants on ovarian reserves. It can also be applied to life sciences for study of the kidney and the brain. Familiarizing yourself with the morphology of the follicles requires experience.
Using previous studies to define the morphology along with establishing standardized criteria of classification can help with consistency. To estimate the primordial follicle number by stereology using an optical fractionator, turn on the computer, multi control unit, camera, and light source within the stereology setup, and set the microscope objective to a low magnification. Open the stereology software and place the first slide securely onto the microscope stage.
Use the joystick to locate the first tissue sample, and bring the sample into focus. In the camera settings panel under exposure, click and drag the sliding scale to adjust the light exposure. In the camera settings panel, click the white balance button and click a blank area of the section to adjust the white balance.
Open the probes menu to select the optical fractionator workflow, then click start new subject, and okay. If an existing sampling configuration has been saved, click yes under sampling parameters, and select the desired sampling configuration. Click next, set the microscope to low magnification, and select the 10x magnification.
Click next and left click to trace the entire ovarian section. Right click to end the selection and select close contour. Click next, set the microscope to high magnification, and select the 100x oil magnification.
After placing a drop of oil on the tissue section on the slide, adjust the light exposure as demonstrated, refocus the sample, and click next. Next, click start counting, focus at the top of the sample, click okay, and begin counting. Use the focusing knob to move through the 10 micron sampling depth, and look within the counting frame for any follicles in which the oocyte nucleus comes into focus.
Then, click next to move to the next site. Classify a follicle as primordial if the oocyte is surrounded by flat squamous granulosa cells but no cuboidal granulosa cells. Count only follicles in which the oocyte nucleus is visible.
The nucleus must appear within the counting frame or be touching the green inclusion lines of the counting frame. If the oocyte nucleus falls outside the counting frame or touches the red exclusion lines of the counting frame, do not count this follicle. When assessing primordial follicle depletion in response to an exogenous chemical, ensure that all of the counted follicles are healthy with a normal morphology.
Count any abnormal or atretic follicles separately. When all of the sites in the section have been counted, click add new section to set up and count the follicles in the next section, or click I finished counting to end the session and exit the software. Then, obtain the sum raw follicle number from each tissue section sampled from the entire ovary, and use the formula as indicated to calculate the final value for each replicate animal.
To estimate primordial follicle numbers by direct follicle counting, obtain multiple stitched high-power photomicrographs of the entire ovarian tissue section. To analyze the images, open them in ImageJ software. To classify the follicles as primordial, oocytes must be clearly visible and surrounded by squamous granulosa cells, but not cuboidal granulosa cells.
Use the counting tool to obtain a raw follicle number of the primordial follicles with each section sampled from the tissue. Once the sum raw follicle number from each tissue section sampled has been obtained, multiply this number by the inverse of the sampling fraction to obtain the final value for each animal as demonstrated. When using stereology, a significant depletion of the mouse primordial follicles can be detected following chemotherapy treatment compared to that detected in control animals.
In contrast, using the contralateral ovaries from the same animals, direct follicle counts failed to detect a significant reduction of the ovarian reserve after chemotherapy compared to controls. Notably, it is clear that the primordial follicle number in young adult wild type animals is variable as the distribution of the saline treated animals is wider compared to the cyclophosphamide groups even when the counts are performed by stereology. It is important to be able to accurately classify primordial follicles to differentiate between healthy and atretic follicles, and to appropriately include or exclude individual follicles from the overall count.
These growing follicle and corpora lutea quantification techniques can be used in conjunction with immunofluorescence and cell viability marker staining to quantify the number of follicles expressing specific markers of interest. Stereology and direct counting are used to evaluate how disease and exogenous insults can alter the ovarian reserve, leading to a more comprehensive evaluations of toxicants and their impacts on female fertility.
