Wojciech Głabowski1. 1. Katedra i Zakład Histologii i Embriologii Pomorskiej Akademii Medycznej al. Powstańiców Wlkp. 72, 70-111 Szczecin.
Abstract
INTRODUCTION: The protective potential of stem cell factor (SCF) on preimplantation embryo development in vitro has not been well investigated. MATERIAL AND METHODS: The aim of the study was to determine the effect of SCF on preimplantation development in vitro of (1) frozen-thawed mouse embryos; (2) mouse embryos exposed to Fas-L. Female mice were stimulated intraperitoneally with 5 IU of pregnant mare serum gonadotropin. An injection of 5 IU of equine chorionic gonadotropin (eCG) was given 48 hours later and the animals were mated. 40 hours after eCG injection, embryos at the stage of 2 blastomeres were flushed out ofthe fallopian tubes. Experiment 1: The two-cell embryos were randomly divided into 3 groups: (A) cultured in standard IVF medium (control); (B) frozen-thawed and cultured without SCF; (C) frozen-thawed and cultured in medium with SCF (100 ng/mL). Experiment 2: two-blastomere embryos were divided into 3 groups: (A) cultured in standard IVF medium (control); (B) cultured in standard IVF medium with Fas-L (500 ng/mL); (C) cultured in standard IVF medium with Fas ligand (500 ng/mL) and SCF (100 ng/mL). Culture was performed for 96 h in groups of 10 embryos in 50 microl droplets under mineral oil at 37 degrees C and 5% CO2. In both experiments the dynamics of embryo development, outgrown blastocyst rates, total blastocyst cell numbers (TBC) and blastocyst cell viability were analyzed. In experiment 2, TUNEL reaction was additionally performed to detect apoptosis of the blastocyst cells. Statistics was based on ANOVA and log-linear analysis with significance taken at p < 0.05. RESULTS: In experiment 1 significantly more hatched and outgrown blastocysts were noted in the control group when compared to embryos exposed or not exposed to SCF after thawing. Control embryos had better cell viability and higher TBC than in the remaining two groups. Blastocyst rates, TBC and cell viability in the frozen-thawed embryos cultured with and without SCF did not differ significantly. Log-linear analysis revealed that the dynamics of embryo growth was highest in control group, followed by thawed embryos cultured with SCF and finally by thawed embryos cultured without SCF. In experiment 2, Fas-L decreased embryo growth, but the effect was not apoptosis-dependent as the TUNEL reaction in embryos exposed to Fas-L was only sporadically positive. The growth of embryos exposed to Fas-L was comparable to that observed in media enriched with both Fas-L and SCF. However, blastocysts grown in Fas-L enriched media had significantly smaller cell numbers (TBC) than when cultured in both Fas-L and SCF. CONCLUSIONS: SCF may improve the dynamics of post-thaw embryo development in vitro but not embryo quality. Fas-L deteriorates preimplantation embryo development in vitro. SCF seems to improve the quality of embryos exposed to Fas-L.
INTRODUCTION: The protective potential of stem cell factor (SCF) on preimplantation embryo development in vitro has not been well investigated. MATERIAL AND METHODS: The aim of the study was to determine the effect of SCF on preimplantation development in vitro of (1) frozen-thawed mouse embryos; (2) mouse embryos exposed to Fas-L. Female mice were stimulated intraperitoneally with 5 IU of pregnant mare serum gonadotropin. An injection of 5 IU of equine chorionic gonadotropin (eCG) was given 48 hours later and the animals were mated. 40 hours after eCG injection, embryos at the stage of 2 blastomeres were flushed out ofthe fallopian tubes. Experiment 1: The two-cell embryos were randomly divided into 3 groups: (A) cultured in standard IVF medium (control); (B) frozen-thawed and cultured without SCF; (C) frozen-thawed and cultured in medium with SCF (100 ng/mL). Experiment 2: two-blastomere embryos were divided into 3 groups: (A) cultured in standard IVF medium (control); (B) cultured in standard IVF medium with Fas-L (500 ng/mL); (C) cultured in standard IVF medium with Fas ligand (500 ng/mL) and SCF (100 ng/mL). Culture was performed for 96 h in groups of 10 embryos in 50 microl droplets under mineral oil at 37 degrees C and 5% CO2. In both experiments the dynamics of embryo development, outgrown blastocyst rates, total blastocyst cell numbers (TBC) and blastocyst cell viability were analyzed. In experiment 2, TUNEL reaction was additionally performed to detect apoptosis of the blastocyst cells. Statistics was based on ANOVA and log-linear analysis with significance taken at p < 0.05. RESULTS: In experiment 1 significantly more hatched and outgrown blastocysts were noted in the control group when compared to embryos exposed or not exposed to SCF after thawing. Control embryos had better cell viability and higher TBC than in the remaining two groups. Blastocyst rates, TBC and cell viability in the frozen-thawed embryos cultured with and without SCF did not differ significantly. Log-linear analysis revealed that the dynamics of embryo growth was highest in control group, followed by thawed embryos cultured with SCF and finally by thawed embryos cultured without SCF. In experiment 2, Fas-L decreased embryo growth, but the effect was not apoptosis-dependent as the TUNEL reaction in embryos exposed to Fas-L was only sporadically positive. The growth of embryos exposed to Fas-L was comparable to that observed in media enriched with both Fas-L and SCF. However, blastocysts grown in Fas-L enriched media had significantly smaller cell numbers (TBC) than when cultured in both Fas-L and SCF. CONCLUSIONS:SCF may improve the dynamics of post-thaw embryo development in vitro but not embryo quality. Fas-L deteriorates preimplantation embryo development in vitro. SCF seems to improve the quality of embryos exposed to Fas-L.