This protocol allows for the efficient and repeatable isolation of early stage pre-antral follicles from a single bovine ovary and it creates new opportunities for the study of ovarian folliculogenesis in a non-bovine species. By using a greater number of follicles from a single ovary, this technique enables in vivo treatments before ovary harvest, or obtention of multiple samples over time from the same animal via ovarian biopsy. Isolation and cryo-preservation of early stage pre-antral follicles can be a valuable option for fertility preservation in young women undergoing gonadotoxic treatment such as chemotherapy, as well as germplasm preservation from endangered species.
Demonstrating the procedure will be Stephanie McDonnell, Amanda Morton, and Juliana Candelaria, PhD students from my laboratory. To begin, transfer ovaries in the laboratory to warm, sterile PBS containing Pen-Strep and select ovaries with many small antral follicles and no prominent corpus lutetium. Remove any excess connective tissue and fat from the ovaries using scissors.
Transfer one ovary to the cutting board on the bench paper. Using a scalpel, cut the ovary in half longitudinally from one ligament attachment site to the opposite attachment site. Keep one half of the ovary on the cutting board to be processed and place the other half of the ovary back into warm PBS containing Pen-Strep.
To remove the medulla, slice along the curvature of the ovary approximately 2 millimeter away from the surface without cutting through the cortex with the exposed medulla facing upward. Slice down through the cut to remove the majority of the medulla. Flip the ovary half over so that the epithelium faces upward and use the scalpel to finish cutting the medulla away from the cortex and trim away any remaining white connective tissue around the edge of the ovary piece that was connected to the ligaments.
Once the majority of the medulla is removed, use the scalpel to cut the cortex to approximately 1-millimeter thickness while manipulating the scalpel with small back-and-forth motions to shave away the remainder of the medulla, then cut each ovary cortex half into two pieces. Keep the cortex pieces in warm PBS containing Pen-Strep until ready to chop. Transfer a single piece of the cortex to the Petri dish on the tissue chopper and wet the tissue with three or four drops of warm PBS containing Pen-Strep.
While holding the piece of tissue steady with a pair of forceps, press the Reset button once to start the tissue chopper. After the entire piece of the cortex has been cut into strips, use the blade holder knob to lift the blade off the Petri dish and the forceps to remove any tissue from the blade. After rotating the plate holder by 90 degrees, again, press the Reset button and pass the blade entirely through the tissue strips.
Use the blade holder knob to lift the blade off the Petri dish and the forceps to remove any tissue from the blade as demonstrated previously. Using a transfer pipette and warm PBS containing Pen-Strep, wash the chopped tissue into a pre-warmed 50-milliliter conical tube. Return the conical tube to the water or bead bath to keep the chopped tissue warm while repeating the chopping procedure with the other three halves of the ovary.
For best cutting results, use the nut driver to remove the nut from the chopping arm and remove the washer and blade clasp. Using forceps, remove the blade from the chopping arm. Flip it over so that the unused edge is facing the Petri dish and place it back onto the chopping arm.
After ensuring that the homogenizer unit is plugged in and the speed is set to the second bar, insert the 10-millimeter generator probe into the unit according to the manufacturer's specifications. Next, set a timer for 1 minute and insert the probe into the 50-milliliter conical tube containing the chopped cortex tissue from one ovary and enough PBS containing Pen-Strep to fill the tube to the 25 milliliter line. The depth to which the probe is inserted must be 1/3 of the liquid's height measured from the bottom of the chamber.
Once the timer starts, turn on the homogenizer, ensuring that the bottom of the probe does not touch the tube and hold the tube still while the homogenizer is turned on. After 1 minute of homogenization, remove the probe from the tube, then remove any connective tissue clogging the venting holes in the space between the rotor knife and rotor tube or cortex pieces stuck in the probe using forceps and place them back into the tube. Pour the dispersed tissue into the cheesecloth-covered funnel, inserted into the Erlenmeyer flask and rinse the contents of the tube into the funnel using warm PBS.
Finally, rinse the cheesecloth with warm PBS. Force the liquid in small fragments to pass through the holes of the cloth by twisting the cheesecloth around the tissue fragments down and squeezing until all excess fluid and tissue are removed from the cheesecloth. After reopening the cheesecloth over the funnel, rinse the cheesecloth with PBS containing Pen-Strep using a transfer pipette, and again, squeeze any residual tissue fragments through the cloth.
Using a hemostat, hold the 300-micrometer cell strainer over a 200-milliliter beaker and pour the filtrate in the Erlenmeyer flask through the cell strainer. If the cell strainer becomes clogged with tissue, gently tap it against the beaker, then turn the strainer upside down and tap out the large tissue debris onto the bench paper. Rinse the contents of the flask using warm PBS containing Pen-Strep until no tissue fragments remain and pour it into the cell strainer.
Next, while holding the 40-micrometer cell strainer with a hemostat over a second 200-milliliter beaker, pour all the initial filtrate present in the first 200-milliliter beaker through the strainer, then rinse the contents of the beaker using warm PBS containing Pen-Strep until no tissue fragments remain and pour it into the cell strainer. After fitting the 18 gauge needle to the 20-milliliter syringe, fill the syringe with follicle wash medium. With a hemostat, hold the 40-micrometer cell strainer upside down over a square Petri dish and use the syringe to wash out the contents of the cell strainer into the dish.
Transfer the square Petri dish to a stereoscope with a warm stage set to 38.5 degrees Celsius with a magnification set between 1.25 and 3.2x. After identifying follicles from the square Petri dish, transfer the follicle to wash medium drops using the micropipette. The total number of isolated pre-antral follicles per replicate using 6-minute tissue homogenization shows an average of 41 follicles per replicate, ranging from 11 to 135 follicles.
Assessment of the developmental stage of 476 isolated follicles showed that the majority were in the primary stage of development, measuring 40 to 79 micrometers in containing one layer of cuboidal granulosa cells. Homogenization of the ovarian cortex for 6 minutes yielded a greater number of pre-antral follicles compared to 5 minutes of homogenization at the same speed. Transcript expression of the granulosa cell marker FSH receptor and germ cell marker DAZL in the follicles was evaluated using reverse transcription quantitative PCR demonstrating that both primary and early secondary follicles expressed FSH receptor and DAZL transcripts.
Immunofluorescent localization of CX37 in isolated pre-antral follicles at both the primary and early secondary stages shows that CX37 was localized to the O plasm, being absent from the nucleus of the O site and to the membrane of the granulosa cells. In our experience, proper dissection of a thin layer of cortical tissue, chopping the tissue to the right size, and proper homogenization are critical steps to ensure release of viable follicles from the tissue. Isolated pre-antral follicles can be used to answer questions such as regulation of pre-antral follicle growth and the nature of interactions between granulosa cells and the oocyte, thus facilitating the development of more efficient culture conditions.
