Serotropin

Porcine ovaries were collected from pre-pubertal cross-bred gilts (Landrace × Large White × Duroc) at a local abattoir and transported to the laboratory in
phosphate buffered saline (PBS; Takara Bio, Otsu, Shiga, Japan) at 35°C within 1 h. Growing oocytes were collected from < 1-mm diameter follicles, whereas
fully-grown oocytes were collected from 2–6-mm follicles. After collection from follicles, cumulus-oocyte complexes were cultured in 500 μl of modified North
Carolina State University (NCSU)-37 medium [11 (link)] according to Kikuchi et al. [12 (link)] in 4-well dishes (Thermo Scientific, Roskilde, Denmark) for 22 h. The IVM medium was modified by adding 10% (v/v) porcine follicular
fluid (pFF), 0.6 mM cysteine (Sigma, St. Louis, MO, USA), 50 mM β-mercaptoethanol (Sigma), 1 mM dibutyryl cAMP (dbcAMP; Sigma), 10 IU/ml eCG (Serotropin; ASKA
Pharmaceutical, Tokyo, Japan), and 10 IU/ml hCG (Puberogen, Novartis Animal Health, Tokyo, Japan). The oocytes were then transferred to IVM medium without
dbcAMP and hormones and cultured for another 22 h. IVM was performed in 5% CO₂, 5% O₂, and 90% N₂ at 39°C.

In the present study, to ensure pregnancy, the embryos obtained in vivo from Duroc gilts were cotransferred.
Donor gilts were prepared according to the method used in previous studies [20 , 21 (link)]. In brief, they were checked for signs of estrus and inseminated artificially with
semen from a 1-year-old Duroc boar. Between 14 and 30 days after artificial insemination, they were injected with 0.526 mg
prostaglandin F2α analogue (cloprostenol-Na, Planate, MSD Animal Health, Tokyo, Japan) to induce miscarriage. Thereafter, the
same amount of cloprostenol-Na plus 1000 IU eCG (Serotropin; ASKA Pharmaceutical, Tokyo, Japan) was injected at 24 h,
followed by 500 IU hCG (Puberogen 1500 U; Novartis Animal Health, Tokyo, Japan) 72 h later. Artificial insemination was
carried out three times, at 24, 31 and 48 h after hCG injection. Blastocysts were collected surgically from the gilts on day
5 or 6 after the initial artificial insemination.

ES cell clones containing the target Wapl gene were aggregated for the
generation of chimeric mice. The cell clones were placed on a MEF feeder layer 2 days
before embryo manipulation. The propagated ES cells were dissociated with 0.05% trypsin
just before the aggregation. Eight-cell-stage-embryos were collected at 2.5 dpc from
female ICR mice that had been superovulated by intraperitoneal (i.p.) injection of 5.0 IU
of equine chorionic gonadotropin (Serotropin; ASKA Pharmaceutical, Tokyo, Japan) at 1600
h, followed by injection of 5.0 IU of human chorionic gonadotropin (Gonadotropin 3000;
ASKA Pharmaceutical) 48 h later and then mated naturally with male ICR mice. The zona
pellucida was removed by treatment with acid Tyrode’s solution (Sigma-Aldrich, St. Louis,
MO). Fifteen to twenty ES cells were aggregated with each zona-free embryo and cultured
overnight in micro-drops of KSOM medium (ARK Resource, Kumamoto, Japan) under mineral oil
at 37°C, 5% CO₂, and 95% humidity. The chimeric embryos were transferred at 2.5
dpc into the uterine horns of pseudopregnant female ICR mice (Sankyo Labo Service
Corporation). Chimeric mice were identified 20 days after birth by their black eyes and
their black coat color. Chimeric males showing germline transmission were crossed with
C57BL/6 females.