Microinjection

In this study, we microinjected COC, GV, and MII oocytes, zygotes, two-, and four-cell embryos with mRNA. Except for some details, the microinjection process for a different objective is similar. First, the microinjection needle is cleaned several times with the microinjector in the PVP drop, and then some PVP is aspirate drawn to separate the mercury and mRNA samples. Finally, the mRNA samples are aspirate drawn to the needle (Supplementary Video 2). It is worth noting that a bit of air in the microinjection needle will not affect the microinjection, so mercury should not be squeezed into the PVP droplets because it is toxic. In addition, the volume of mRNA in the microinjection needle is an important factor in reducing the intensity of piezoelectric-assisted micromanipulation, so we can use it to control the intensity of piezoelectric-assisted micromanipulation in actual operations. Generally, we can aspirate draw more mRNA for microinjection of COC and GV oocytes because their cell membranes are fragile, while microinjection of MII oocytes and early fertilized eggs need to aspirate draw less mRNA because their cell membrane is more flexible. As shown in Figure 4A and Supplementary Videos 3, 4, for COC and GV oocytes, we first moved the microinjection needle close to the zona pellucida, and then a small piezoelectric pulse was used to penetrate it incompletely. Subsequently, we inserted the microinjection needle with the incomplete zona pellucida and cell membrane into the 1/3 to 2/3 of the oocyte, and then the oil pressure microinjector was turned into a state of vomiting and, at the same time, gave the oocyte a minimum amount of Piezo pulse. When we saw that the injection was completed, we quickly pulled out the microinjection needle. For MII oocytes and embryos of different stages, due to the clear perivitelline space, we used higher piezoelectric pulses to completely penetrate the zona pellucida, and then the microinjection was completed similar to GV oocytes (Figure 4B and Supplementary Videos 5–8).

Schematic diagram of microinjection. (A) Microinjection diagram of GV oocytes [microinjection of cumulus–oocyte complex (COC) is similar]. First, the microinjection pipette should be close to, instead of pressing, the zona pellucida, and then a small piezoelectric pulse was used to incompletely penetrate the zona pellucida. Finally, the microinjection needle with the incomplete zona pellucida and the cell membrane is inserted into 1/3 to 2/3 of the oocyte, and then the oil pressure microinjector is turned into a vomiting state, and a minimum amount of piezoelectric pulse is given at the same time. (B) Microinjection diagram of fertilized eggs (microinjection of MII oocytes, two- and four-cell stage embryos are similar). First, the microinjection pipette presses the zona pellucida, and then an intermediate piezoelectric pulse is used to completely penetrate the zona pellucida. Finally, the microinjection needle is inserted into 1/3 to 2/3 of the cell, and then the oil pressure microinjector enters the vomiting state, and the smallest amount of piezoelectric pulse is given at the same time.