The overall goal of this standardized procedure is to isolate distinct male germ cell types from different mammalian species using a single protocol. This method can help facilitate the molecular study of spermatogenesis by the high-throughput analysis of different germ cell types. The main advantage of this technique is that a single set of reagents can be applied to various mammalian species for the simultaneous isolation of four different male germ cell populations.
We first had the idea for this method when we decided to investigate the evolution of specific patterns of gene expression regulation in different mammals. Chris Holley, a medical technologist from the Siteman Flow Cytometry Core, will be demonstrating how to operate the cell sorter and set the gates. Begin by decapsulating a single murine testis and cutting the tissue into small pieces.
Then use forceps to transfer small tissue pieces onto a pre-wetted 50 micrometer tissue disaggregation cartridge. Add one milliliter of DMEM medium to the cartridge, and load the tissue disaggregation cartridge onto the tissue disaggregation system. Turn the knob from standby to run, and process the tissue for five minutes.
Then remove the cartridge, and use a disposable, three milliliter syringe to aspirate the cell suspension from the syringe port. When all of the liquid has been recovered, filter the cells through two individual, disposal, pre-wetted 40 micrometer filters to remove any cell aggregates, and return the suspension to the disaggregation cartridge. Then process the cells for another five minutes.
To stain the cells for fluorescence-activated cell sorting, collect the dissociated cell suspension in a 1.5 milliliter tube. Then transfer 150 microliters of cells into each of three new 1.5 milliliter tubes, adding the remaining volume to a fourth 1.5 milliliter tube. Setting aside the first tube as an unstained control, add 2.5 microliters of Hoechst to the second tube, one microliter of propidium iodide to the third tube, and both Hoechst and propidium iodide to the fourth tube.
After 30 minutes at room temperature in the dark with gentle rotation, filter the cell suspensions through individual, pre-wetted 40 micron strainers into five milliliter round-bottom tubes. Next place four collection tubes containing DMEM supplemented with FBS under the sorting spouts of the flow cytometer, and load the unstained control tube for analysis. Use a forward versus side scatter plot to exclude the cell debris, and adjust the forward scatter threshold and pulse width to allow gating of the single cells.
Stringent gating is crucial to optimize the quality of the collected sample. Ensure selection of single cells by excluding cellular debris and multicellular aggregates. Use the single-stained cells to establish the threshold for Hoechst and propidium iodide fluorescence intensity in relation to the unstained sample.
Then load the double-stained experimental sample, and set the three parent gates as just demonstrated. When the three parent gates have been set, select live cells based on their lack of propidium iodide staining, and set the DNA content by plotting a histogram of cell counts based on the Hoechst blue fluorescence. Set a gate for each of the three peaks of increasing Hoechst blue fluorescence concentration, representing the haploid, diploid, and tetraploid cells, and observe at least 500, 000 events on the forward versus side scatter plot.
Plot the propidium iodide-negative cells according to their Hoechst blue and red fluorescence intensities. The spermatogonia will appear as a side population. Hoechst blue fluorescence intensity correlates with DNA content.
Therefore, setting this gate prior to generating Hoechst blue and red plots ensures selection of the cells with desired ploidy. For spermatid isolation, gate the haploid peak and select the population that appears when Hoechst blue and red fluorescence are plotted. For spermatocytes II isolation, gate the diploid peak and select the population that appears when Hoechst blue and red fluorescence are plotted.
For spermatocytes I isolation, gate the tetraploid peak and select the population that appears when Hoechst blue and red fluorescence are plotted. Then sort the selected subpopulations into the individual collection tubes to collect approximately 0.5 to six times 10 to the six cells for each cell subset. Cell suspensions obtained by mechanical dissociation of fresh tissue unstained or stained with Hoechst demonstrate the presence of single cells in various stages of differentiation.
Importantly, the cellular structures appear to be preserved, including the flagella of the spermatozoa. After cellular debris exclusion, 95 to 98%of cells are singlets, 86 to 93%of which are alive. Plotting the function of blue red Hoechst fluorescence during FACS facilitates the identification of specific testicular germ cell populations.
As expected, the haploid spermatid population is the most abundant, followed by diploid spermatocyte II cells and tetraploid primary spermatocytes, a pattern that is preserved across species. Microscopic analysis of the sorted cells reveals that spermatogonia display distinct, small, round, pericentric heterochromatin that stains brightly for Hoechst. Spermatocytes are larger, granulated cells with easily identifiable nuclei that exhibit chromatin variations characteristic of meiotic cells in the spermatocyte I subpopulation and by nucleated or diakinetic nuclei in the spermatocyte II subpopulations.
Spermatids are small haploid cells with a round or elongated shape that can be distinguished from spermatogonia by the presence of localized chromocenters. One mastered, this technique can be completed in two hours if it is performed properly. While attempting this procedure, it is important to remember that Hoechst is both highly sensitive and genotoxic and that the resolution of the cell populations can be improved by longer incubation periods as needed.
Following this procedure, high-throughput omics methods can be performed to answer questions regarding epigenetic modifications or transcript and protein abundance and diversity within mammalian species in an evolutionary context. After its development, this technique paved the way for researchers in the field of male reproductive biology to explore the regulatory mechanisms driving spermatogenesis progression in several mammalian species. After watching this video, you should have a good understanding of how to process testicular samples for Hoechst FACS and how to identify and isolate different male germ cell type based on their Hoechst signal.
DNA-binding dyes such as Hoechst and propidium iodide can be extremely hazardous. Always wear personal protective equipment when handling these reagents.
