This protocol allows for an in vitro in depth examination of the ovarian micro environment. Enabling users to determine follicle growth and development, as well as steroidogenesis and abundance of immune molecules. One advantage of this technique is the ability to directly evaluate changes in the ovarian micro environment and emerging follicles and assess the unique interplay caused by the addition of specific hormones.
This technique can be used to study a variety of reproductive disorders causing follicular arrests. For example, polycystic ovarian syndrome. Since we can directly evaluate the ovarian micro environment in vitro, demonstrating the procedure will be Brooke Bell, an undergraduate researcher from my laboratory.
Using cerated jaw forceps, secure the ovary, slice in half, and begin to remove exterior slices. Ensuring that no more than a one to two millimeter depth of surface is cut away from the ovary so that no medulla is collected. Cut three to four thin strips of the ovarian cortex with a scalpel and place the strips in the third PBS filled Petri dish.
Cut the strips into small square pieces of 0.5 to one cubic millimeter with a scalpel blade. Use a ruler underneath the Petri dishes to ensure the pieces are of similar size and thickness to make consistent ovarian cortex pieces. Wash ovarian cortical pieces in all three PBS and antibiotic field Petri dishes, using curved tip forceps to move the pieces between washes.
Move cortex pieces through the series of LB-15 washes and place them in a final LB-15-filled Petri dish. Label the lid with the animal ID and ovarian side. Collect four ovarian cortex pieces per ovary and fix them for day zero histology and freeze additional pieces for RNA purification.
Use the remaining tissue pieces for culture. Prepare a biological safety cabinet for a final tissue wash and culture preparation. Sanitize supplies with 70%ethanol before placing them in the biological safety cabinet.
Move all ovarian cortex pieces intended for culture to the biological safety cabinet and wash them once more in an LB-15-filled Petri dish. Pipette 350 microliters of Waymouth medium per well into a 24 well tissue culture plate. Place uncoated culture well inserts into each well using forceps making sure that no bubbles are formed under the base of the insert.
Carefully and delicately position four ovarian cortex pieces onto the mesh of each insert without puncturing the mesh. Incubate the tissue at 37 degrees Celsius with 5%carbon dioxide. Change ovarian cortex culture medium daily for seven days making sure to use pre-warmed Waymouth medium.
During the medium changes, use forceps to gently lift the insert out of the well and collect the cultured Waymouth medium in 0.5 milliliter tubes. Set the insert back in the well and add 350 microliters of fresh culture medium by dispensing it between the side of the insert and the well. Store the collected medium from the tissue culture at minus 20 degrees Celsius.
After seven days of culture, image the ovarian cortex pieces using a dissection microscope with an attached camera and a computer imaging software program. Fix two ovarian cortex pieces per well in Bouin's solution for histology and flash freeze to ovarian cortex pieces in liquid nitrogen to obtain RNA for cDNA. Repeat this step for all the wells with tissue then collect the medium from day seven and stored it minus 20 degrees.
Allow the ovarian cortex pieces to remain immersed in Bouin's solution for approximately 1.5 hours, then wash them with 70%ethanol three times, keep the tissue in 70%ethanol and clear it daily until the solution is no longer yellow. Hematoxylin and eosin stain was performed for primordial follicles, early primary follicles, primary follicle, secondary follicle and antral follicle. Follicle staging was conducted on an ovarian cortex fixed prior to and following culture to assess folliculogenesis.
The difference in morphology as determined by collagen deposition can indicate fibrosis in the ovarian cortex from stair-step or control heifers. A comparison of the average area of picrosirius red positive staining per ovarian cortex filled between control and stair-step heifers is shown here. Daily collection of culture medium was pooled over three days to assess varied steroid hormone production, using a radioimmunoassay.
Steroid metabolites and cytokine production were also measured in ovarian cortex culture medium from one well for each animal pooled over four days of culture. The longer the tissue sits, the harder it can be to work with. You may need to practice precise cuts.
This technique is used to understand the effects on signal transduction pathways to rescue excess steroidogenesis to determine the effects of excess steroids on cytokine and chemokine production, and to determine the effects on follicle progression or arrest within the ovarian tissue.
