Intracytoplasmic sperm injection（ICSI）

Intracytoplasmic sperm injection（ICSI）

MATERIALS

Mice of the strain B6D2F1 female, C57BL/6 male, CD1 female

Kalium simplex optimized medium (KSOM)：Millipore, cat. no. MR107-D

M2 medium: Sigma, cat. no. M7167 100ml

Embryo-tested bovine testis hyaluronidase : Sigma, cat. no. H-4272

Mineral oil: Sigma, cat. no. M8410

Percoll: Sigma, cat. no. P4937

Hormones for superovulation: human chorionic gonadotropin (hCG): Ningbo Second Hormone Factory, China

pregnant mare serum gonadotropin (PMSG): Ningbo Second Hormone Factory, China

PROCEDURE

Day 1

Select 8-10 weeks old B6D2F1 females, and inject 5 IU PMSG for each intraperitoneally.

Day 3 (45–54 h after PMSG injection)

1. Inject 5 IU hCG to each PMSG treated female for superovulation. 

2. cage 8-12 weeks old CD1 females and vasectomized male mice (2 females : 1 male) to prepare pseudo-pregnant female mice as the recipients of oocytes after ICSI.

Day4

1. Collection of oocytes

1) 12–15 h after hCG injection, sacrifice the female mice were sacrificed and their oviducts were collected in M2 medium. 

2) Transfer the oviducts to a 200 μl drop of M2 containing 0.5mg/ml bovine testis hyaluronidase, tease the oocyte-cumulus complex from the oviduct carefully, and discard the oviductal remnant.

3) Leave the oocyte-cumulus complex in the same dish for 5–30 min to allow the hyaluronidase to digest the intercellular matrix. Collect the oocytes with a transfer pipette and place them in a drop (200 μl) of fresh M2 medium (no hyaluronidase). Wash the oocytes by pipetting them 3 times.

4) Transfer the oocytes to a drop of equilibrated KSOM medium under mineral oil, washing once to remove M2 medium. Place the 3.5-cm dish containing the oocytes in an incubator containing humidified 5% (v/v) CO2/air mix at 37 ℃.

2. collection and sonication of spermatozoa

1) sacrifice the male mice of indicated genotype (WT or SUN5^-/-), collect their epididymides and mince them with fine scissors in a 3.5-cm dish containing 500 μl of M2 medium at room temperature, then place the dish in an incubator containing humidified 5% (v/v) CO2/air mix at 37 ℃ for 15 min to release spermatozoa.

2) filter the sperm suspension with a 70 μm cell strainer to remove tissue debris.

3) transfer the sperm suspension to a 1.5 ml eppendorf tube. WT sperms (intact sperms) were placed in ice-water bath, and sonicated using ultrasonic sonicator with 30% power output for 5-10 sec. 5 sec sonication could yield ~20-30% decapitated sperms, and those sperm heads is enough for the following injection. Long time and higher power sonication should be avoided. 

 SUN5^-/- male mice could produce decapitated sperms, so they should not be sonicated.

4) Isolate the sperm heads by centrifuge the SUN5^-/- sperms or sonicated WT sperms in 60% percoll solution at 1000g, 5min. Wash the sperm heads with M2 solution for once.

3. Microinjection of Sperm Heads Into Oocytes (ICSI)

1) Mix sperm head suspensions with PVP360 solution to final concentration of 10% (w/v) PVP360. 

2) With a transfer pipette, remove MII oocytes from the CO2 incubator and place them into a microinjection droplet (M2 medium containing 5% cytochalasin B) on the microscope stage. The zona of the oocytes was 'drilled' by applying several piezo pulses. And then inject a single sperm head to each oocyte with a minimum amount (~6 pi) of accompanying sperm suspension medium. The sperm-suspening medium was retrieved to the extent possible, and the pipette was withdrawn gently. 

3) the injected oocytes were washed 3 times in KSOM medium and incubated at 37°C for 5-7 h then examined under an inverted microscope, those with two distinct pronuclei and the second polar body were considered fertilized and suitable for embryo transfer. 

4. Embryo transfer 

1) Check the vaginal plug of CD1 females caged with vasectomized male mice, those with obvious vaginal plugs were selected as recipients of the embryos.

2) The recipients were anesthetized with sodium pentobarbital, and their oviducts were exposed through dorso-ventral incisions. Meanwhile, the embryos were transferred to the oviductal ampulla using an embryo transfer pipette (100-150 μm in diameter at its tip).

Full-term pups derived from ICSI embryos were obtained through natural labor.