This protocol describes an efficient method to isolate ovarian follicles from the surrounding stroma cells to assess gene expression analyses on these germ cells. This technique, using controlled enzymatic and mechanical isolations, allows the retrieval of follicles while maintaining their integrity. Following this protocol, the researcher can assess specific gene expressions from these cells.
The technique could be used to understand further the deficit of gene expression in the follicles related to diseases affecting ovarian function or after exposure to toxic agents. Begin preparing a six-well plate by adding five milliliters of cryoprotectant solution to the first well and five milliliters of Leibovitz 15 medium with decreasing concentrations of cryoprotectant to the next four wells. Remove a vial containing an ovarian cortical fragment from liquid nitrogen in compliance with safety rules.
After 30 seconds at room temperature, or RT, soak the vial in double distilled water for two minutes. Inside a vertical hood, gently agitate and open the ovarian cortical fragment vial before transferring the content into the first well of the six-well plate placed on ice. Then, transfer the ovarian cortical fragment in the first well successively into each well of the six-well plate containing decreasing concentrations of cryoprotectant in five milliliters of Leibovitz 15 medium.
Gently agitate the tissue in each medium for five minutes. For follicle isolation, transfer the thawed ovarian tissue into a two-millimeter gridded Petri dish filled with 10 milliliters of dissection medium, 30 micrograms per milliliter penicillin G, and 50 micrograms per milliliter streptomycin. Adjust the tissue size with the scalpel, if necessary.
Pile up the three pieces of the tissue slicer and place the ovarian fragment between the two blocks. Cut the fragment in half using a blade, sliding through the blocks to obtain two fragments of 0.5-millimeter thickness. Cut the tissue using the tissue chopper to obtain the smaller pieces.
If necessary, cut the remaining pieces manually with the scalpel until the tissue is smashed. Once the tissue is smashed, transfer the fragmented tissue into a gridded Petri dish filled with seven milliliters of digestion medium before placing the dish in the incubator at 5%carbon dioxide and 37 degrees Celsius. Take the Petri dish from the incubator every 10 minutes.
Flush the tissue by pipetting the digestion medium up and down with a one-milliliter pipette. After 45 minutes of incubation in the digestion medium, place the dish under a stereo microscope with the magnification range of five to 6.3 times and retrieve the follicles using a micro capillary pipette. Select the follicles of interest and isolate them by sucking them up with a mouth pipette.
If the follicles remain stuck in a piece of cortex, isolate them mechanically with two 27-gauge syringes by ripping the cortex off with the tip of the syringes and releasing follicles from the stroma. Using the micro capillary, transfer the isolated follicles to a four-well plate containing 15 microliters of the calibrated culture medium covered by 500 microliters of oil culture. After transferring one to 10 follicles, rinse them two times in two drops of 15 microliters of PBS covered with 500 microliters of oil culture for five seconds each.
Using the mouth pipette, collect 20 follicles with minimal PBS in an empty tube and keep the tube on ice. After 90 minutes of incubation in the digestion medium, stop the enzymatic reaction by adding a cold-blocking solution. Under the chemical hood, suspend the isolated follicles in 100 microliters of the lysis solution provided with the RNA Extraction Kit.
Vortex the isolated follicles at 2500 RPM per minute to break their structure, then add 50 microliters of 100%ethanol to the tube. And briefly vortex the tube at high speed. After vortexing, transfer the total volume of the tube to a micro filter cartridge assembly, comprising a column and a collection tube.
And centrifuge the assembly for 10 seconds at 16, 363g at four degrees Celsius. Wash the column with 180 microliters of Wash Solution 1 provided with the kit before centrifuging the tube for 10 seconds. After giving two washes of 180 microliters of Wash Solution 2, 3 to the column, dry the filter by centrifuging the tube one last time and place a new collection tube under the cartridge.
To perform the elution of the nucleic acids, first add eight microliters of 75 degrees Celsius Elution Solution to the filter assembly. After one minute, centrifuge the assembly for 30 seconds. Repeat this step with seven microliters of Elution Solution.
Once done, add two international unit DNase and DNase buffer to the tube and incubate the tube for 20 minutes at 37 degrees Celsius. Block the enzymatic activity with one by 10 DNase inactivation reagent for two minutes at room temperature. Next, centrifuge the tube for 1.5 minutes at 16, 363g at four degrees Celsius and transfer the suspension containing the RNA to a new tube.
Using a spectrophotometer, assess the quantity of RNA in the sample. Use one microliter Elution Solution as a blank and one microliter solution of RNA extracted from isolated follicles. Check if the 260/280 ratio is around 2.0 for RNA purity.
And store the sample at minus 80 degrees Celsius or directly retrotranscribe it to perform RT-qPCR. Using this protocol, the two homogenized ovarian fragments of 4x2x1 millimeter from the same patient were processed to isolate the follicles. RNA quantification revealed that 4.8 nanograms per microliter of total RNA was extracted from follicles less than 75 micrometers in size with a purity ratio of 1.89.
From the less than 200 micrometers follicles, 10.5 nanograms per microliter of total RNA was extracted with a purity ratio of 1.74. The RNA integrity number, or RIN, values were 7.1 and 7.9 in tubes one and two respectively validating the quality of the RNA from our samples. After running a standard cycle on a real-time PCR system, the cycle thresholds, or CT, obtained were 30.87 and 29.56 for HPRT, 33.5 and 31.77 for kit ligand, and 30.71 and 30.57 for GDF9.
The enzymatic digestion of the tissue is a critical point. The experimenter has to continuously evaluate and ensure follicle integrity during the reaction by stopping it when necessary. Besides gene expression analyses, isolated follicles are used to develop experimental systems to preserve cancer patients'fertility, like follicle in vitro culture or artificial ovary.
The isolation technique required a learning curve for retrieving a high number of intact follicles. The researchers are advised to ensure the tissue is well-shattered before digestion.
