Effect Of Recombinant Human Calmodulin On Development And Apoptosis Of Mouse Preimplantation Embryo In Vitro

Part1 Effect of recombinant human calmodulin on development of mouse preimplantation embryo in vitroObjective To study the state of embryos cultured in the media in addition of recombinant human calmodulin (rhCaM) and evaluate the influence of rhCaM on the development of mouse preimplantation embryo in vitro.Methods KunMing female mice(8-10 weeks old) were injected with 10IU pregnant mare serum gonadotropin (PMSG) followed by 10IU human chorionic gonadotrophin 48 hours later. Immediately after the second injection, females were paired with mature males of the same strain and checked for mating the next moring. Mated females with vaginal plugs were sacrificed with cervical dislocation at 48 hours post hCG injection. Two-cell mouse embryos were collected and cultured in the CZB media supplemented with rhCaM at the concentrations as the following: 0ug/ml,0.2ug/ml,2.0ug/ml, 20ug/ml and 200ug/ml. Embryo development was recorded at 64h,88h,112h,120h respectively after hCG injection. The expanded blastocytes and hatched blastocytes were dyed by Hoechst 33342 at 120h after human chorionic gonadotrophin injection. The cell numbers in each dyed blastocyst were examined by fluorescene microscope.Results The rates of total blastocyte, expanded blastocyte and hatched blastocyte in the group of supplementation of CZB media with rhCaM at the concentration of 20 ug/ml were markedly higher compared with the group at concentration of 0ug/ml rhCaM, and the rates of 4-8cell embryos at 64th hour after HCG and morulas at 88th hour after HCG in the group of supplementation of CZB media with rhCaM at the concentration of 0.2ug/ml,2.0ug/ml,20 ug/ml were markedly higher compared with the group at concentration of 0ug/ml rhCaM. However, at the concentration of 200 ug/ml the percentages of total blastocyte, expanded blastocyte and hatched blastocyte were significantly lower than those in the other groups. The cell number in each blastocyst between each group did not show statistically significance.Conclusion Within a certain concentration rhCaM can promote the developing of mouse preimplantation embryo in vitro .This mostly by means of promoting cell differentiation in embryo. Nevertheless higher concentration rhCaM restrains embryo development. Part 2 Effect of recombinant human calmodulin on development and apoptosis of mouse preimplantation embryo in vitroObjective To evaluate the effect of recombinant human calmodulin (rhCaM) on development and apoptosis of mouse preimplantation embryos in vitro.Methods KunMing female mice were super-ovulated with PMSG followed by hCG 48hours later and mated. Forty eight hours after hCG, Two-cell mouse embryos were collected and cultured in the IVF-30 media supplemented with rhCaM at the concentrations as the following: 0ug/ml,0.2ug/ml,2.0ug/ml, 20ug/ml and 200ug/ml. Embryo development was recorded every 24 hours after cultured in vitro. Apoptosis in blastocysts and the cell number in each blastocyst were detected by TUNEL (Terminal deoxynucleoside transferase (TdT)-mediated dUTP nick end-labeling) assay. Under a NIKON 80I epi-fluorescene microscope the total number fo nuclei per blastocyst was evaluated using a UV filter, and the DNA fragments were assessed using a fluorescein filter. Images were collected with an OLYMPUS FLUOVIEW FV500 laser scanning spectral confocal microscope. The specimen was excited at 364nm for Hoechst33342, 488nm for TUNEL-FITC.Results The percentage of blastocyst at the 48th hour in vitro was higher in 20ug/ml rhCaM group than that in control group .The rates of blastocyte and hatched blastocyte in the group of supplementation of IVF-30 media with rhCaM at the concentration of 20ug/ml were markedly higher compared with the group at concentration of 0μg/ml rhCaM, also in this group the lower apoptotic indic was observed in blastocysts cultured 72 hours later in vitro compared with control group. However, at the concentration of 200ug/ml the percentages of blastocyte and hatched blastocyte were significantly lower than those in the control group. The higher apoptotic index was observed in blastocysts cultured 72 hours in 200 ug/ml group than that in the control group.Conclusion A certain concentration rhCaM can promote the development of mouse preimplantation embryo in vitro and suppress apoptosis of embryos. Nevertheless higher concentration rhCaM restrains embryo development and increase apoptosis of embryo.