Accurate Follicle Enumeration in Adult Mouse Ovaries

Ovaries were collected from female C57BL6J mice. All animal procedures and experiments were performed in accordance with the NHMRC Australian Code of Practice for the Care and Use of Animals and approved by the Monash Animal Research Platform Animal Ethics Committee.

NOTE: A chemotherapy agent shown to deplete primordial follicle oocytes, as determined using stereology¹¹ and direct counts^12,13 was used in this report to compare the two counting methods in the same animal. Female, 8-week-old (young adult) mice were weighed prior to a single intraperitoneal injection of 75 mg/kg/bodyweight of cyclophosphamide, or saline vehicle control (n=5/group). This dose has been shown to cause an approximate 50% depletion of primordial follicles, but not reported to cause morbidity or mortality in mice¹⁴. Ovaries were harvested 48 hours after treatment. One ovary from each animal was fixed in 10% (v/v) neutral buffered formalin solution for 24 hours, and the other fixed in Bouin’s solution for 24 hours. Tissue was then embedded in either glycolmethacrylate resin and serially sectioned at 20 µm, or in paraffin and serially sectioned at 5 µm. All tissues were stained with periodic acid Schiff and haematoxylin.

1. Histological preparation: fixation, processing, embedding and sectioning mouse ovaries

1. Dissect mouse ovaries by trimming the oviduct and all surrounding fat, without damaging or cutting the ovary itself. If necessary, use a dissecting microscope and fine blade for this step (Figure 1A).
2. Fix tissues immediately by placing into a small labelled tube containing either Bouin’s fixative (stereology), or formalin fixative (direct counts) for 24 hours (Figure 1B), before transferring tissues into 70% ethanol.
   NOTE: Follicle morphology is conserved best within Bouin’s fixed ovarian tissue, embedded into glycolmethacrylate resin (Figure 2).
3. Process whole fixed ovaries and then embed in glycolmethacrylate resin for stereology (Supplementary File 1), or paraffin wax for direct counts using a standard histological protocol.
   CAUTION: Resin is toxic, so ensure all tissue processing steps are performed in a fume hood and gloves are worn at all times.
4. Use a specialized resin methacrylate microtome (Figure 1C) fitted with a glass knife (Figure 1D) to exhaustively cut thick glycolmethacrylate resin sections (e.g., 20 µm). Collect the sections at a regular interval (e.g., every 3^rd section) onto glass microscope slides for stereology.
5. Use a standard microtome to cut thin paraffin sections (e.g., 4-6 µm). Collect tissue sections at a regular interval (e.g., every 9^th section) onto a glass microscope slide for direct follicle counts.
6. Stain the slides with periodic acid Schiff and haematoxylin (Supplementary File 2).
7. Coverslip with standard DPX for paraffin sections, or thick DPX for glycolmethacrylate resin sections (Figure 1E).
   CAUTION: Glycolmethacrylate resin DPX is hazardous, so perform this step in to fume hood.
   NOTE: Glycolmethacrylate resin DPX is extremely viscous. The glass coverslip must be adhered firmly by pressing it down with a spatula to ensure the DPX is evenly and thinly dispersed, and there are no air bubbles present under the coverslip (Figure 1F).

2. Stereological estimation of primordial follicle number using the optical fractionator

1. Turn on the computer, the multi-control unit, the camera and the light source within the stereology setup and set the microscope objective to a low magnification (e.g., 10x).
2. Open the stereology software.
3. Put the first slide securely on the microscope stage.
4. Adjust the light exposure by checking Automatic under Exposure in the Camera Settings panel (Supplementary Figure 1A). Alternatively, manually adjust the light exposure.
5. Use the joystick to locate the first tissue sample and bring the sample into focus.
6. Adjust the white balance, either by clicking on More Settings (located bottom right of the Camera Settings panel), and then click White Balance and click on Automatic (Supplementary Figure 1B). Alternatively, click the White Balance button adjacent to the More Settings button (or Select Area in More Settings), to set the white balance manually by selecting a white area on the section.
7. Go to the Probes drop down menu and click on Optical Fractionator Workflow. Then click Start New Project and click OK.
   1. If an existing sampling configuration has been saved, under Sampling Parameters click Yes | … and select the desired sampling configuration.
   2. If not, click No and manually enter the serial section information (Supplementary Figure 1C) and define the probe configuration at step 2.13.
8. Click on Next, set the microscope to Low Magnification and choose 10x magnification from the dropdown menu.
9. Click on Next, and then trace around the entire ovarian section – start by left clicking around the section and at the end, right click and choose Close Contour.
10. Click on Next, set the microscope to High Magnification and choose 100x Oil magnification from the dropdown menu.
11. Place a drop of oil on the tissue section on the slide and move the microscope objective to 100x magnification.
12. Adjust the light exposure (as in step 2.4) and click Next.
13. Set up the Sampling Parameters to define the probe configuration. Here, the counting frame was set to 47.5 µm x 47.5 µm (2,256.25 µm²) and the step length was set to 100 µm x 100 µm (10,000 µm²) (Supplementary Figure 2). Once the sampling parameters are established, save the template and re-open during subsequent analysis sessions at step 2.7.
14. Click on Start Counting (Supplementary Figure 1D). Focus to the top of the sample, click OK and begin counting.
15. Use the focusing knob to move through the 10 µm sampling depth and count any primordial follicles whose oocyte nucleus comes into focus. Click Next to move to the next area.
    1. Classify follicles as a primordial if the oocyte is surrounded by squamous (flattened) granulosa cells, but no cuboidal granulosa cells (Figure 2A).
       NOTE: Primordial follicles are distinct from intermediate/transitional follicles, which comprise a combination of cuboidal and squamous granulosa cells (Figure 2B), and primary follicles, which are surrounded predominantly by cuboidal granulosa cells (Figure 2C). These follicle classes should be quantified separately.
    2. Only count follicles in which the oocyte nucleus is visible. The oocyte nucleus must appear within the counting frame or be touching the green inclusion lines of the counting frame (Supplementary Figure 1E,F).
    3. If the oocyte nucleus falls outside the counting frame (Supplementary Figure 1G) or touches the red exclusion lines of the counting frame, do not count this follicle.
    4. When assessing primordial follicle depletion in response to an exogenous chemical (e.g., chemotherapy), ensure all follicles counted are healthy and thus have normal morphology (Figure 2). Count any abnormal or atretic follicles separately. Often, follicle death is induced by insults such as chemotherapy, and quantification of the atretic follicles should be obtained separately in order to distinguish between healthy and atretic follicles, as only healthy follicles comprise the ovarian reserve¹⁵.
16. Once counting is complete on that section, do one of the following:
    1. Click Add New Section, and then return to step 2.3 to set up the next section for counting.
    2. Click I’ve Finished Counting to end the session. Return the objective to 10x, exit the stereology software and turn off the light source, camera, multi-control unit and computer.
17. Obtain the sum raw follicle number (Q) from each tissue section sampled from the entire ovary, then using a spreadsheet, use the equation below to obtain the final value from each replicate animal (N)⁴.
    , where:
    N = Total estimated number of follicles within the ovary.
    Q = Raw primordial follicle count.
    f₁ = Sampling interval. Every 3^rd section was sampled thus .
    f₂ = Relationship between the counting frame (sample area) and stepper, calculated as . Since the sample area was 2256 µm² (47.5 µm x 47.5 µm) and the stepper area was 10000 µm² (100 µm x 100 µm), .
    f₃ = Fraction of ovarian section sampled. Since 10 µm of the 20 µm section was sampled, .
    Therefore, .
    NOTE: This protocol describes how to apply these principles of stereological analyses using a widely cited stereology software (Table of Materials); however, other stereological software is available. The principles applied during stereological analyses of ovarian follicles are the same, regardless of the software used to set up the parameters. Stereology is most accurate when 100 or more objects are counted in an adult mouse ovary⁴, as this gives a coefficient of error for the estimate below 10%¹⁶. The sampling parameters outlined here have been optimized to ensure a minimum of approximately 100 objects (i.e., primordial, transitional and primary follicles) can be counted in control adult wild-type C57BL6J ovaries. A pilot study can be conducted including a small sample size to establish the optimal sampling parameters, such as the interval and number of sections to be analyzed and the number of optical dissectors within the sampled sections¹⁷.

3. Estimation of primordial follicle number by direct ovarian follicle counts

1. Place the microscope slide securely under a standard light microscope and perform direct counts to obtain raw primordial follicle number.
   1. Classify follicles as a primordial if the oocyte is clearly visible and is surrounded by squamous (flattened) granulosa cells, but no cuboidal granulosa cells (Figure 2D).
      NOTE: Primordial follicles are distinct from intermediate/transitional follicles, which comprise a combination of cuboidal and squamous granulosa cells (Figure 2E), and primary follicles, which are surrounded predominantly by cuboidal granulosa cells (Figure 2F). These follicle classes should be quantified separately.
   2. Ensure all follicles counted are healthy and thus have normal morphology (Figure 2). Count any abnormal or atretic follicles separately, as only healthy follicles comprise the ovarian reserve.
2. Alternatively, take multiple, or stitched high-power (e.g., 20x) photomicrographs of the entire ovarian tissue section to perform counts by opening the image file(s). This can be done manually, or using an automated slide scanner.
3. Obtain the sum raw follicle number (Q) from each tissue section sampled from the entire ovary at the predetermined interval. Multiply this number by the inverse of the sampling fraction to obtain the final value for each replicate animal (N), using the equation below.
   , where:
   N = Total estimated number of follicles within the ovary.
   Q = Raw follicle count (of each individual type, calculated separately).
   f₁ = Sampling interval. Every 9^th section was sampled thus .
   Therefore, .