Round spermatid injection can solve the problem of reproduction in some non-obstructive azoospermia patients, but its efficiency is very low. Our study used mouses as models to search for methods to improve the embryonic development efficiency of ROSI in mouse. Incomplete statistic indicates that fewer than 200 human ROSI-conceived fetuses have been delivered globally.
A turning point in understanding the potential of ROSI technology occurred in 2015, when Tanaka and colleagues reported on the successful births of 14 fetuses through ROSI technology, instilling renewed confidence in its clinical application and feasibility. The current focus of ROSI research is on abnormalities in epigenetic modifications and abnormal oocyte assisted activation, while accurately identifying round sperm data is also a challenge. The development efficiency of ROSI embryos in mouse is already very high in our laboratory, which is similar to other laboratories.
However, the development efficiency of non-rodents, like humans, is still very new. In the future, we will focus on the improvement of development efficiency of non-rodents. To begin, inject a six to eight-week-old female mouse intraperitoneally with 7.5 international units each of pregnant mare serum gonadotropin at 5:00 PM and human chorionic gonadotropin 48 hours later.
Ensure no contact with male mice occurs after the injection. After euthanizing the mouse, open its abdominal cavity. Using scissors, sever the fallopian tubes near both the ovaries and place them in M2 buffer preheated at 37 degrees Celsius.
Under the 10X objective of an upright microscope, locate the transparent, enlarged part of the fallopian tube. Then, using a one milliliter syringe, secure the fallopian tube, cut the enlarged part, and collect the oocyte coronal cumulus complexes. To remove granulosa cells, place the oocyte complex in a preheated M2 operating solution containing 0.1%hyaluronidase, and gently blow through an oral pipette.
Transfer the oocyte serially to KSOMaa droplets to rinse three times, and place in a 37 degree Celsius incubator with 5%carbon dioxide. To begin, obtain a euthanized eight to 10-week-old male C57BL/6 mouse. Open its abdominal cavity and gently excise the epididymis near the testis with scissors.
Place the dish containing epididymis in M2 medium under 10X objective of an upright microscope. And using a one milliliter syringe, gently excise the epididymis to let the spermatozoa flow out. Siphon the flowing spermatozoa in suspension into the bottom of a tube containing 0.5 milliliter of M2 buffer.
For round spermatid isolation, transfer the dissected testicle to the M2 buffer. Using a one milliliter syringe, gently cut the white membrane under the microscope, and then squeeze and excise the convoluted seminiferous tubules out. After extracting the suspension, filter with a 400 mesh sieve and centrifuge the filtered cells.
Discard the supernatant and incubate the cells with 200 microliters of Hoechst 33342 at 37 degrees Celsius for 10 minutes. Place the cytometry tube containing stained cells in the flow cytometer. After adjusting the voltage, select the cell population based on forward and side scatter.
Then, remove the cell adhesions according to forward scatter and trigger pulse width. Using two detection channels under 355 nanometer wavelength laser, sort the round spermatids and store them at four degrees Celsius. Under the microscope, mouse round spermatids were approximately 10 micrometers in diameter and displayed a protrusion-like nucleolus structure in the middle.
To begin, obtain oocytes and round spermatids from mice. Then, prepare the holding and injection needles with appropriate diameters. Place the oocytes into 10 microliter M2 droplets with or without cytochalasin B for softening and storing the gametes.
Then, place the round spermatids'M2 droplets and mount the operating dish on the microscopic operating table. Adjust the position of the injection and fix the needle. Using polyvinylpyrrolidone, or PVP, moisten and cleanse the injection needle.
Next, extract the round spermatid into the injection needle. Rotate the oocyte with the holding needle to orient the oocytes'polar body at 12 o'clock position. Then, place the injection needle carefully at the three o'clock position on the oocyte.
Using a PiezoXpert device, create a hole in the oocytes'zona pellucida. Then, gently advance the injection needle into the oocyte horizontally and rupture the oocyte membrane. Gradually inject the round spermatid into the oocyte cytoplasm.
Withdraw the injection needle and aspirate a small amount of the oocytes'membrane near the opening to seal it. For intracytoplasmic sperm injection, place the spermatozoa on a PVP runway. Aspirate the spermatozoa from the tail and position its neck at the mouth of the injection needle.
Apply a piezo to separate the sperm head from the tail. Then, inject the sperm into the oocyte as demonstrated earlier, with an injection needle having nine to 10 micrometer diameter. For assisted oocyte activation, place the oocytes in 20 microliter droplets of calcium and magnesium-free CZB medium containing strontium chloride hexahydrate.
Then, transfer them to KSOMaa culture medium for cultivation. Simultaneously, establish a group for parthenogenesis activation without injecting round spermatids or spermatozoa for embryological studies. After activation, transfer to KSOMaa culture medium for cultivation.
Round spermatid-injected embryos displayed reduced developmental efficiency compared to the intracytoplasmic sperm-injected ones.
