Whole Ovary Immunofluorescence, Clearing, and Multiphoton Microscopy for Quantitative 3D Analysis of the Developing Ovarian Reserve in Mouse

This method can help answer one of the key questions in the reproductive field, namely how and when genetically regulated processes influence the size of the ovarian reserve which we know impact females, reproductive longevity This protocol can be adopted for large scale studies using the same techniques for intact ovaries from different developmental stages. It generates produceable data by providing an efficient quantification of germ cells. This method applied to genetically diverse animals of the same species can provide insight into the genetic factors directly germ cell and ovarian development and other development organs.

Demonstrating the procedure will be Nathaniel Boechat, a research intern from my laboratory To start with the profusion step place and secure the anesthetized pubertal female mouse on its back by gently pinning all legs through the paw pads in a relaxed position onto a board. To open the thoracic cavity of the mouse, carefully cut the ribs on both sides of the sternum just below the scapula, avoiding puncturing the lungs grab the skin or rib flaps with the forceps and pin the flaps to expose the internal organs use dissection scissors to snip the right atrium of the heart and release blood from the system. Then by holding the heart with forceps insert a perfusion needle into the left ventricle and pump 10 milliliters of PBS with a gentle and constant pressure.

When the tissues such as the liver kidneys and reproductive tract turned from pink to white replace the PBS with freshly made 1%paraformaldehyde or PFA and continue perfusion with approximately 10 milliliters of PFA. Next locate the fat pad with the ovaries below the kidney and gently dissect the entire reproductive tract including the fat pad ovaries and uterine horns to place it in a vial with 4%PFA. Fix the ovaries at room temperature for 16 to 24 hours before replacing the 4%PFA with 70%ethanol.

On the day of the staining trim the ovaries before proceeding with the immuno staining. On day one of the immuno staining protocol pipette one milliliter of PBS per well into the desired number of wells in a clean 24 well plate. Cut off the tip of a 1000 microliter pipette tip to make a wide opening and use the wide tip to gently transfer the pubertal ovaries from vials with 70%ethanol into the Wells, then replace PBS in the well with fresh 0.8 milliliter of PBS The next day aspirate PBS from the wells to replace it with 0.8 milliliters of the permeation buffer per well.

After incubating the plates on the shaker at room temperature for four hours replace the permeation buffer with blocking buffer On day three, prepare the primary antibodies by dilution in the blocking buffer. To visualize the oocytes incubate the prenatal and prepubertal ovaries with the oocyte markers. On day eight wash the samples with a fresh washing buffer for two hours then replace the washing buffer with 0.5 milliliters of the secondary antibody mixes diluted in a blocking buffer.

Seal the plate with a parafilm to prevent evaporation and incubate the samples at room temperature in the dark for three days. On day 11 after the immuno staining wash, aspirate the washing buffer and incubate the samples with 0.8 milliliters of the scale cubic one solution at 37 degrees Celsius for three days in the dark. Changing the scale cubic one solution daily.

On day 15, wash the samples three times for 10 minutes and 0.8 milliliters of PBS at room temperature with gentle shaking. Then replace the PBS with 0.8 milliliters of the freshly prepared degassed 20%sucrose with PBS followed by gentle shaking at room temperature. After one hour replace the 20%sucrose solution to treat the samples with 0.8 milliliters of the scale cubic two solution for four to five days, as explained earlier.

To prepare a sample set up, use a 200 microliter pipette tip with the tip cutoff to transfer the pubertal ovaries with 15 to 20 microliters of scale cubic two solution into the middle of the adhesive well. Once the ovaries are transferred into the individual well with a drop of clearing solution gently place a cover glass on the adhesive well and press to create a seal sandwiching the samples and clearing solution between two cover slips. For imaging place, the cover slip sandwich in a 3D printed microscope adapter slide.

Set up the parameters for image acquisition according to the microscopy cores or the manufacturer's specifications. If available use the bidirectional image acquisition to reduce the scanning time and improve efficiency. Adjust the line average, frame average and frame accumulation in the software.

Set up the Z stack by identifying the beginning and the end position, then select a Z step size of two micrometers for prepubertal ovaries and five micrometers for pubertal ovaries. Next activate the Z compensation feature and the excitation gain. Set the Z compensation for the bottom and top of the sample by selecting a laser intensity for both.

Use the tiling mode for larger samples that cannot be captured in a single field of view. Activate the image navigator and indicate the number of tiles needed to capture the entire sample with the rectangular marking tool. Once the setting is done, start with the image acquisition for the images with multiple tiles, begin image acquisition with the navigator to capture all tiles.

After the image acquisition, save all the images. And if multiple tiles were acquired, run the mosaic merged tool to merge the tiles into a single image. To determine the size of the oocytes, open the image and select the slice option to open the Zs stack image.

Select the line option and the measure panel and measure the distance by drawing a line from one edge of an oocytes or marker to the other edge at the widest point to determine the X Y diameter of the oocyte. Move through the stack and obtain the range of diameters for multiple oocytes of the same type. Use the shortest length as the size selection criterion for oocytes counts.

Next, in the 3D view option, select the spots feature. In the scene panel activate the add new spots function to open the algorithm panel. Then deselect all the algorithm settings as the size selection filter with the oocytes size obtained earlier.

Click on the channel forward arrow to move to the source channel. Then select the channel with the preferred marker and type the acquired diameter in the estimated X, Y diameter. After the size selection activate the model PSF elongation and background subtraction which are automatically determined.

Then, choose the single forward arrow to move to the filter spots panel and add the quality filter to determine a threshold. To ensure the accurate estimation of oocytes numbers enlarge the image. Later, click on the double arrow to finish automated counts.

Use the edit tab to manually select missed oocytes or deselect nonoocyte particles selected by the automatic threshold. When done, record the data in the statistics tab under the overall tab for further analysis. To estimate the total oocyte numbers and the damaged ovaries, open images of the intact ovaries with MRS and select the frame feature.

Under the frame settings, select the box grid tick marks and access labels. In the position XYZ tab select 200 microme to generate tick marks with 200 micrometer spaces in all XYZ positions. Using the 200 micrometer tick marks, select oocytes that fall within 50%of the volume of each ovary.

Click on the spots feature and select the edit labels to classify the oocytes that fall within the 50%region by color. In the representative grayscale, multi photon images of the non perfused and perfused ovaries, the small oocytes in primordial follicles with the thin layer of cytoplasmic DDX4 staining could be seen. Additionally, the larger oocytes within the growing follicles were spotted.

The 3D renderings from the multi photon images of the prenatal and the postnatal ovaries were studied. At the postnatal day 28, the ovary from the control non irradiated female contained a large population of small oocytes in the primordial follicles. In contrast, the ovaries from the irradiated female with 0.5 gray of the gamma radiation was completely devoid of small oocytes in the primary follicles.

But larger oocytes were still present. For the quantification of the oocytes, The 3D multi photon images were processed in the MRS software using the goucian filter and the oocytes of small and large sizes were identified and quantified using the spot feature. Next, the average percentage of the oocytes was calculated at each stage compared to the average number present at the earliest stage E 15.5 oocytes numbers sharply declined between E 15.5 and E 18.5 the E 15.5 and E 18.5 fetal oocytes expressed line one orf one P as seen in the multi photon images.

The intensity analysis showed higher expressions of line one orf one P per oocytes at E 18.5 than E 15.5. The ovaries with small damage can be used for analysis. Total oocytes numbers are determined using computational correction.

The representative model shows G CNA positive oocytes in the E 15.5 ovary with a five 10%regions highlighted in an intact ovary. Similarly, a simulated ovary with 10%incremental regions missing up to 50%of the ovary was generated. The total oocytes numbers and the simulated ovaries were compared to the original intact ovaries.

And the difference was presented as the percent deviation Good perfusion results in good image quality. So ensure all tissues are cleared with PBS before switching to PFA. Ensure that the ovaries is completely exposed by trimming away the extra tissue.

The protocol allows efficient analysis of a large number of samples, quantitative data obtained from genetic reference populations such as collaborative cross or diversity output will help identify genetic modifiers of oocytes development and oocytes numbers.