FISH analysis of ovarian cells from females with X chromosomal aberrations is a unique technique to gain insight into the X chromosomal content of ovarian cells in this specific group. These techniques could be helpful to learn more about reproductive potential in females with X chromosomal aberrations. It is important to mention that both methods are intended to facilitate future research, and are not designed to be used as a diagnostic tool in clinical practice.
Demonstrating the procedure will be Ronald Peek, a senior researcher from our fertility laboratory, and Milad Intezar, an analyst from our pathology department. To begin, cut the cryopreserved, or thawed ovarian cortex tissue into small pieces of one by one by one millimeter using a scalpel. Enzymatically digest the tissue fragments in four liters of pre-warmed L15 for 75 minutes at 37 degrees Celsius.
Pipette the digestion mixture up and down every 15 minutes. Stop the enzymatic digestion by adding four milliliters of cold L15 supplemented with 10%Fetal Bovine Serum, or FBS. Wash the dissociated tissue once with eight milliliters of cold L15 medium by centrifugation at 500 g before re-suspending in 500 microliters of L15 medium.
Transfer the cell suspension containing largely individual stromal cells and small follicles to a plastic Petri dish, and examine the suspension under a stereo microscope. Pick the follicles less than 50 micrometers in size manually using a 75 micrometer plastic pipette, and transfer them to the droplet of L15 medium supplemented with 10%FBS at four degrees Celsius. Then transfer the purified follicles to a solution of 0.06%trypsin, one milligram per milliliter EDTA, and one milligram per milliliter glucose, and incubate for 20 minutes at 37 degrees Celsius.
After incubation, obtain ovarian stromal cells from the cortex cell suspension using a 75 micrometer plastic pipette. For Fluorescence In Situ Hybridization, or FISH analysis of individual ovarian cells, transfer the treated ovarian follicles with trypsin/EDTA/glucose, or stromal cells to droplets of five microliters of 0.15 millimolar potassium chloride, and 15 microliters of DPBS on a slide, and incubate for 20 minutes at 37 degrees Celsius. Dry and pre-fixate the slide in 300 microliters of 0.05 millimolar potassium chloride, 7.5%acetic acid, and 22.5%methanol for two minutes.
Cover the slide with three-to-one methanol acetic acid for two minutes to finalize fixation. After fixation, wash the sample twice in freshly prepared SSC at 73 degrees Celsius. Cover it with 100 microliters of 2%Proteinase K and seal it with a cover slip.
Incubate the slide for 10 minutes at 37 degrees Celsius in the hybridization station. After incubation, remove the cover slip, and wash the slide for five minutes in DPBS at room temperature under shaking. Fix the sample for five minutes with 1%formaldehyde.
Then wash the slide in DPBS, and serially dehydrate it in increasing ethanol concentrations for two minutes each. Air dry the sample before hybridizing it with fluorescently labeled probes. Next, add one microliter of CEPX, one microliter of CEP18, and 18 microliters of the hybridization buffer to the sample and seal with a cover slip that is glued to the slide.
Transfer the slide to the hybridization station for denaturation at 73 degrees Celsius for three minutes, followed by hybridization during an overnight incubation at 37 degrees Celsius. After hybridization, remove the cover slip and any remaining glue from the slide. Wash the slide in 0.4X SSC, 0.3%Tween-20 at 72 degrees Celsius for two minutes, followed by incubation for one minute in 2X SSC and 0.1%Tween-20.
Dehydrate the slide as demonstrated, and air dry it in the dark before covering it with a mounting medium containing DAPI. Place the slide at minus 20 degrees Celsius for 10 minutes before analysis by fluorescence microscopy. To hybridize paraffin sections, select new sections before, or after the section containing follicles from the paraffin ribbon.
Mount one section on a glass slide, and dry it for 45 minutes on a stove at 56 degrees Celsius. Deparaffinize the section in xylene for 10 minutes, then immerse the slide in 99.5%ethanol, and rinse it for five minutes in tap water. Pre-treat the slide with the target retrieval solution for 10 minutes at 96 degrees Celsius.
After cooling down, rinse the slide in distilled water. Treat the slide for five minutes with 0.01 molar hydrochloric acid, followed by pepsin digestion for 15 minutes at 37 degrees Celsius. Rinse the slide again in hydrochloric acid and subsequently in PBS.
Fix the slide in 1%formaldehyde PBS for five minutes. Then briefly rinse the slide in PBS, and again in demineralized water before dehydrating it in 99.5%ethanol. Apply five microliters of probe CEP18 and CEPX on the pre-treated slide.
Then apply a cover slip and seal the area with photo glue. Place the slide in a hybridizer for denaturation at 80 degrees Celsius for 10 minutes and hybridization overnight at 37 degrees Celsius. The next day, rinse the slide twice for five minutes each in SSC at 42 degrees Celsius, followed by a two minute and a one minute rinse in 0.3%Nonidet P-40 at 73 degrees Celsius.
Again, rinse the slide in 2X SSC followed by rinsing in distilled water before dehydrating it with 99.5%ethanol. Air dry the slide, and mount it in DAPI-containing mounting medium. Cryopreserved ovarian cortex tissue in Patient A showed 50%of the lymphocytes, had a 45, X karyotype, and 50%had 46, XX.In Patient B, 38%of the lymphocytes were 45, X, 28%were 46, XX, and 34%were 47, XXX.
Surrounding stromal cells were also analyzed for X chromosomal content. Enzymatic digestion of ovarian cortex tissue resulted in a suspension of largely dissociated cells, but leaving the primordial follicles, consisting of an intact oocyte surrounded by a single layer of granulosis cells. Small follicles provided difficulties in interpreting signals after FISH, due to the clumping of the granulosis cells.
Additional digestion of the follicles with trypsin before FISH resulted in individual granulosis cells contracting into a spherical morphology on the surface of the follicles. Hematoxylin eosin staining showed morphologically normal secondary and antral follicles in ovarian cortex tissue after five months of xenografting. The X chromosomal content of granulosis cells of these follicles was determined by FISH analysis.
Fixating grafted ovarian cortex tissue in Bouin solution resulted in blurred and largely obscured fluorescent signals in follicular cells due to a green haze. In contrast, ovarian cortex tissue fixed in formaldehyde provided excellent fluorescent signals. The main challenge of this protocol lies in the isolation of ovarian cells and the enzymatic digestion of the tissue.
Deviating from this protocol can cause a substantial loss of ovarian cells during the process, and this should especially be avoided in females who already have a low ovarian reserve. Additional gene expression profiling could be helpful to learn more about the impact of aneuploid ovarian cells on folliculogenesis, and hence the process of premature ovarian failure in females with X chromosomal aberrations.
