Functional Assessment of Kinesin-7 CENP-E in Spermatocytes Using In Vivo Inhibition, Immunofluorescence and Flow Cytometry

Meiosis is a evolutionary conserved event in eukaryotes, which is essential for gametogenesis and sexual reproduction. Our protocol provides an effective method for studies of meiotic division and spermatogenesis. We have described a series of protocols for the analysis of spermatocytes.

This technique can be applied for observation of meiotic spindle, homologous chromosomes, and the subcellular organelles in spermatocytes. This method can be used for the analysis of meiotic division, the generation of gene editing animal models and gene transfection in tissues or organs. Experimenters should maintain a sterile environment during abdominal surgery, and pay attention to the temperature, time, and key experimental conditions of this protocol.

Demonstrating the procedure will be Miss Meng-Fei Xu, a post-graduate from my laboratory. Begin the construction of the GSK923295 mediated centromere protein E or CENP-E inhibition mouse model by tying up the anesthetized mouse limbs and fixing them on the wax tray. After disinfecting the surgical area, use a sterile scalpel to make an opening of five millimeters in the abdominal cavity of the mouse followed by placing sterile surgical clamps on the skin.

Then pull the epididymal fat pad with sterile dissecting forceps to locate the testes with the help of sterile tweezers. After fixing the testes with sterile forceps under a stereoscope, slowly inject 10 microliters of GSK923295 into seminiferous tubules at a final concentration of 10 micromolar using 10 microliters of Riodine. When done, push the testes back into the abdominal cavity and suture the surgical site as described in the text manuscript.

For the gradient dehydration of the collected mouse testes, sequentially incubate the sample in various media as described in the manuscript. After the serial incubation, place the tissue at the bottom of the embedding box and add the melted paraffin into the box. For the complete solidification of the paraffin, cool the samples at four degrees Celsius for six hours.

Later, fix the sample on the holder of the ultra microtome while keeping the angle between the sample and the knife surface at 5 to 10 degrees. Adjust the slice thickness to five micrometers to prepare the five micrometer thick sections using an ultra microtome. Upon preparation, spread the sample slide in a water bath at 40 degrees Celsius before drying the sections in the slide dryer for 12 hours at 37 degrees Celsius.

After 12 hours, perform sequential incubation of the slides as explained earlier. Next, rinse the slides in distilled water for five minutes followed by staining with Mayer's Hematoxylin Solution for six minutes at room temperature. Then rinse the stained slides in running water for five minutes and incubate with distilled water for two minutes.

Incubate the slides in 1%ethanol hydrochloride for three seconds and then rinse and running water for two minutes. Stain the sample with 1%eosin for 15 seconds, followed by serial incubation. When done, seal the slides using 15 microliters of neutral gum and a 24 by 50 millimeter coverslip.

For immunofluorescence, place the slide in the antigen retrieval solution to boil under high pressure in a pressure cooker for four minutes for antigen retrieval. Then cool the slide naturally to room temperature before rinsing twice with distilled water for five minutes and with PBS for five minutes. To permeabilize the cells, incubate the slide in 500 microliters of 0.25%Triton X-100 in PBS for 10 minutes and then rinse the slides with PBS for five minutes three times.

For antigen blocking, incubate the sample with 300 microliters of 3%bovine serum albumin or BSA for one hour and with the primary antibodies in 3%BSA in PBST for 16 hours at four degrees Celsius. After incubation, put the slide in a humidified box to prevent the tissues drying out. Rewarm the slides naturally to room temperature for 30 minutes.

Discard the primary antibody, followed by rinsing the slide in PBST for five minutes thrice. Dilute secondary antibodies with 3%BSA and PBST. Then incubate the sample with diluted secondary antibodies for one to two hours at 37 degrees Celsius.

After rinsing the sample in PBST for five minutes five times, stain the nuclei with 50 microliters DAPI for five minutes at room temperature. Mount the cover-slip with the anti-fade mounting medium and seal the coverslip with nail polish. For flow cytometry, collect mouse testes in six centimeter Petri dishes and cut the testes into one cubic millimeter pieces using surgical scissors.

Then digest the testes in one milliliter of 1%collagenase in a 1.5 milliliter centrifuge tube for 10 minutes at 37 degrees Celsius. To precipitate spermatogenic cells, centrifuge the sample at 1, 000 G for five minutes and discard the supernatant before adding one milliliter of 0.25%trypsin EDTA solution in the sample for 20 minutes at 37 degrees Celsius. Later, centrifuge the sample at 1, 000 G for five minutes.

Discard the supernatant to incubate the precipitated cells with one milliliter of 70%cold ethanol for more than eight hours at four degrees Celsius. After centrifugation at 1, 000 G for five minutes, collect cell sediments and stain the spermatogenic cells with 500 microliters propidium iodide or PI staining solution at 37 degrees Celsius for 30 minutes. Following incubation, filter the sample using a 300 mesh screen to eliminate cell debris and collect the cells in a flow tube to store the cells at four degrees Celsius.

Detect fluorescence signals and light scattering at the excitation wavelength at 488 nanometers using a flow cytometer. Analyze the DNA content in the sample and light scattering using the ModFit MF LT 32 software. After testicular injection of GSK923295, the spermatogenic wave was altered in the seminiferous tubules due to CENP-E inhibition.

However, the spermatogenic wave in the seminiferous tubules was regular and organized in the control group. It was observed that the several homologous chromosomes were not aligned at the equatorial plate after CENP-E inhibition. Furthermore, CENP-E inhibition led to the increase of metaphase I spermatocytes in seminiferous tubules.

The immunofluorescence analysis demonstrated the decreased number of anti-synaptonemal complex protein three or SYCP3 positive cells per seminiferous tubule after CENP-E inhibition. On the other hand, the SYP3 dots per metaphase cell and the SYP3 stretches per cell were not affected in the GSK92395 groups. The distances of spindle poles in metaphase I spermatocytes were increased due to CENP-E inhibition.

The representative analysis demonstrates that CENP-E inhibition resulted in the decrease of the haploid cells in the GSK 923295 group when compared to control. The ratios of the diploid cells and aneuploidy cells were not significantly influenced after CENP-E inhibition. Conversely, the ratios of the tetraploid cells increased in the GSK923295 group than the control group.

The transmission electron microscopy analysis of the spermatogenic cells displayed the disrupted organization of spermatogenic cells in the the GSK923295 group. The experimenter should pay attention to the injection position, drug dose, injection method, and suture method during abdominal surgery and testicular injection. This technique together with in vivo electroporation focused on the targeted proteins and the gene editing tools can be used in the field of miotic division and spermatogenesis.