Gonadotropin

Unfertilized eggs of Xenopus laevis were obtained by injection of gonadotropin (ASKA Pharmaceutical). These eggs were artificially fertilized with testis homogenates and dejellied using 4% L-cysteine (adjusted to pH 7.8 with NaOH). Embryos were incubated in 1/10x Steinberg’s solution at 14–17°C and were staged according to Nieuwkoop and Faber, 1967 . Synthesized mRNAs were microinjected into early (2–16 cell) embryos. Amounts of injected mRNAs are described in figure legends.

Eight-week-old ICR female mice (Japan SLC) received 7.5 U of serotropin (ASKA Animal Health) by intraperitoneal injection. Forty-eight hours after of serotropin treatment, mice were injected with 7.5 U of gonadotropin (ASKA Pharmaceutical). These mice were then mated with ICR male mice (Japan SLC). Plug checks were performed on the next morning. Two days later, these female mice were sacrificed by cervical dislocation, and their oviducts were harvested. Two-cell-stage fertilized eggs were collected by perfusion with M2 medium (Sigma) and maintained in KSOM medium. Two days later, the blastocysts were subjected to microinjection. For this purpose, ES cells were treated with trypsin and pipetted up and down 15 times to dissociate them into single cells. The ES cells and MEFs were incubated in a gelatin-coated 10-cm dish with 10 mL ESC medium for 30 min to attach only MEFs onto the dish. 3 mL of supernatant containing ES cells was collected. Three to five ES cells were injected into each ICR blastocyst under on OLYMPUS IX71 microscope. In all, 20–25 injected blastocysts were transplanted into the uterus of each pseudo-pregnant ICR female mouse (Japan SLC).

Eight-week-old ICR female mice (Japan SLC) received 7.5 U of serotropin (ASKA Animal Health) by intraperitoneal injection. Forty-eight hours after serotropin treatment, mice were injected with 7.5 U of gonadotropin (ASKA Pharmaceutical) and then mated with ICR male mice (Japan SLC). Two-cell fertilized eggs were collected by perfusion with mWM medium and maintained in KSOM medium to obtain blastocysts. After injection of six to ten ESCs, the injected blastocysts (22–26 blastocysts/mouse) were transplanted into the uterus of pseudopregnant ICR female mice (Japan SLC). A single ESC line for each genotype was used in this study.

All experiments using Xenopus laevis were approved by The Office for Life Science Research Ethics and Safety, University of Tokyo or The Institutional Animal Care and Use Committee, National Institutes of Natural Sciences. Manipulation of X. laevis embryos and microinjection experiments were carried out according to standard methods^{10 (link), 64} as follows. Briefly, unfertilized eggs were obtained from female frogs injected with gonadotropin (ASKA Pharmaceutical), and artificially fertilized with testis homogenate. Fertilized eggs were dejellied with 2% l-cysteine-HCl solution (pH 7.8), and incubated in 1/10× Steinberg’s solution at 14–20 °C. Embryos were staged according to Nieuwkoop and Faber⁶⁵ . Synthetic mRNAs were transcribed from plasmid DNAs using mMessage mMachine SP6 kit (Ambion) and microinjected into early (2–16 cells) embryos. The amounts of injected mRNAs are described in the Figure legends. For tracing MO-injected cells, 2.5–5.0 ng of FITC-dextran (Molecular Probes, D1820) was coinjected. Heparitinase (25 μU, #100703, Seikagaku Biobusiness, a mixture of Heparitinase I and II at 4:1 ratio) was microinjected into the blastocoel of embryos at stage 6.5 (48 cells)^{66 (link)}. Similarly, blastocoel injection was carried out for PI-PLC (800 μU, Molecular Probes, P6466), anti-HS antibodies or BSA-AF647.