Fig. S7.

Development of mouse oocytes after ICSI using ACRBP-deficient sperm. (A) Development of the oocytes following microinjection of sperm. The head of a single epididymal sperm from wild-type mice (WT), type 1/type 2, or type 3 Acrbp^−/− mice was separated from the tail by applying a few Piezo pulses and injected into the cumulus-free oocyte. The injected oocytes were cultured at 37 °C for 24 h under 5% (vol/vol) CO₂ in air. Embryos that reached the two-cell stage were transferred into the oviducts of pseudopregnant mice on the day after sterile mating with a vasectomized male (day 0.5). On day 19.5, the uteri of the recipient mice were examined for the presence of live term fetuses. Note that type 1 Acrbp^−/− sperm were indistinguishable from the type 2 sperm based on morphology, without staining of the acrosome and nucleus, and that the type 4 sperm, whose head and tail could not be separated by Piezo pulses, were injected as the whole cell. (B) Genotyping of ICSI-derived fetuses. PCR was carried out using fetal genomic DNAs as templates. The tail DNA of Acrbp^+/− mouse (Het) was also analyzed by PCR as a control. Two DNA fragments with the sizes of 1.3 and 0.5 kbp, originated from the wild-type (WT) and null-mutated (KO) alleles, respectively, were detected by agarose gel electrophoresis.