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Collection, in vitro culture, and in vivo development of 2-cell stage embryos

 

1.       In this experiment, two-cell stage embryos were used.

2.       Female mice received 5 IU of equine chorionic gonadotropin (Serotropin; ASUKA Pharmaceutical Co., Ltd., Tokyo, Japan) and 5 IU of human chorionic gonadotropin (Gonatropin; ASUKA Pharmaceutical Co., Ltd.) intra-abdominally with an interval of 50 h (17:00 – 19:00) to induce excessive ovulation.

3.       Two-cell stage embryos of BALB/cA, BALB/cByJ, CBA/N, and ICR mice were collected by oviduct flushing after natural mating. Those of other strains were conducted by in vitro fertilization (IVF).

4.       We used modified Whitten's (mW) medium for oviduct flushing and in vitro culture (IVC) of embryos [12,25].

5.       In IVF, we used a modified human tubal fluid in which the calcium concentration of human tubal fluid was doubled [30].

6.       All reagents used for making the media were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). The media were added to Petri dishes (351008; Becton Dickinson & Co., Franklin Lakes, NJ) in a drop-wise manner (modified human tubal fluid, 200 μL; mW medium, 50 μL) and then covered with mineral oil (26117-45; Nacalai Tesque, Kyoto, Japan).

7.       IVF and IVC were performed in an atmosphere of 5% CO2 and 95% air at 37°C.

8.       Oviduct flushing was conducted according to the method of Horgan et al. [13].

9.       Immediately after the administration of Gonatropin to female mice, they were mated with males of the same strain. On day 1.5 after mating, the female mice were euthanized for collection of two-cell stage embryos.

10.     IVF was conducted using a modified method of Toyoda et al. [35,36].

11.     Approximately 16 h after Gonatropin administration to female mice, the culture medium containing oocytes (300 μL) was inseminated with precultured sperm (100 – 150 sperm/μL).

12.     About 6 h after insemination, fertilized ova were washed with mW medium and IVC was performed with embryos of the two-cell stage or less.

13.     Embryo transfer for examination of in vivo development was conducted in the oviduct of pseudopregnant female mice on day 0.5 after mating [14].

14.     On day 17.5 18.5 after embryo transfer, the pseudopregnant mice were euthanized and a laparotomy was performed to observe embryo development.