Effects of embryonic stem cell-conditioned medium on the preimplantation development of mouse embryos.

The production of high-quality embryos in the laboratory and a successful pregnancy are closely related to the condition and contents of oocyte and embryo culture media. In this study, we investigated the effects of embryonic stem cell-conditioned medium (ESCCM) and embryonic stem cells growth medium (ESCGM) compared with potassium-enriched simplex optimized medium (KSOM) on preimplantation embryo development stages during natural or in vitro fertilization (IVF). Birth rate of pups was measured. To obtain mature oocytes, and 2-cell and 8-cell embryos, human chorionic gonadotropin (HCG) was injected 48 h after i.p. injection of 5 units of pregnant mare serum gonadotropin. Mature oocytes were obtained from non-mated female mice 14 h after HCG injection. To obtain 2-cell and 8-cell embryos, mated female mice, 1 day and 3 days, respectively, after HCG injection, were used. Mature oocytes were fertilized in HTF medium. Embryos obtained from natural or in vitro fertilization were cultured in experimental media, ESCCM and ESCGM, or KSOM as the control culture medium. Embryos that developed to the blastocyst stage were transferred to the uteri of pseudopregnant mice and effects of the experimental media on embryo viability were determined. ESCCM and ESCGM could not pass the embryo after the 2-cell stage, but they were suitable for the development of the embryo from the 8-cell stage to the blastocyst. It can be concluded that the embryo has various requirements at different stages of development.

Effect of cyanocobalamin on oocyte maturation, in vitro fertilization, and embryo development in mice.

The aim of this study was to investigate the effect of cyanocobalamin supplementation on in vitro maturation (IVM), in vitro fertilization (IVF), and subsequent embryonic development competence to the blastocyst stage, and in vitro development of mouse 2-cell embryos. Cumulus cells were prepared from mouse cumulus-oocyte complexes (COCs) and incubated for 24 h in an in vitro culture (IVC) medium that contained different concentrations of cyanocobalamin (100, 200, 300 or 500 pM). We collected 2-cell embryos from superovulated NMRI mice and cultured them in the same concentrations of cyanocobalamin (100, 200, 300 or 500 pM). After 42 h of IVM, we observed significantly increased oocyte maturation in the 200 pM cyanocobalamin-treated group compared with the control group (P < 0.0001). Mature oocytes cultured in 200 pM cyanocobalamin were fertilized and cultured in IVC medium with cyanocobalamin (100, 200, 300 or 500 pM) during early embryogenesis. The matured oocytes that were cultured in 200 pM cyanocobalamin had significantly higher 2-cell development rates compared with the control oocytes (P < 0.01). Embryos obtained from in vitro mature oocytes and in vivo fertilized oocytes that were cultured in 200 pM cyanocobalamin had significantly greater frequencies of development to the blastocyst stage and a significant reduction in 2-cell blocked and degenerated embryos compared with the control embryos (P < 0.0001). Embryos derived from oocytes fertilized in vivo with 200 pM cyanocobalamin had a higher percentage of blastocyst embryos compared with those derived from matured oocytes cultured in vitro (P < 0.0001). These finding demonstrated that the effects of cyanocobalamin on oocyte maturation, fertilization, and embryo development in mice depend on the concentration used in IVC medium.