Effect of leukemia inhibitory factor (LIF) on the quality of in vitro produced mouse blastocysts and subsequent derivation of embryonic stem (ES) cells (original) (raw)
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Journal of Assisted Reproduction and Genetics, 2008
The aim of this study was to investigate the developmental potential of isolated blastomeres in the presence and absence of leukemia inhibitory factor (LIF) and granulocyte-macrophage colony stimulating factor (GM-CSF). Methods The blastomeres of two (1/2) and eight cells (1/8) embryos were isolated and cultured in T6 medium in the presence and absence of LIF (1,000 IU/ml) and or GM-CSF (2 ng/ml) up to 120 h. The diameter and cell number of blastocysts were measured. Results The developmental rates of 1/2 isolated blastomeres developed to blastocysts stages in the presence and absence of LIF and GM-CSF were 45.80, 35.10 and 48.66, 41.66, respectively. The diameter of blastocysts was higher in GM-CSF group and total cell number of blastocyst in both treated groups was higher than control (P<0.05). No 1/8 blastomeres developed to morula and blastocyst stages. Conclusions LIF and GM-CSF could improve the development of 1/2 isolated blastomeres.
Relevance of LIF and EGF on mouse preimplantation embryo development
Yakhteh Medical Journal, 2008
, a member of interleukin-6 family, has biological actions on preimplantation embryo development. Also it is established that Epidermal Growth Factor (EGF), a strong mitosis-promoting agent, improves the preimplantation embryo development by increasing the cell metabolism and proliferation. The purpose of the present study is to investigate the effects of these factors, alone and in combination together, on preimplantation and development of the embryo. Materials and Methods: Six to eight weeks old NMRI mice were super ovulated by injection of 10IU PMSG and 10IU hCG, then the mated mice were killed 46 hours later. Their oviducts were flushed, two-cell embryos collected and divided randomly to the four groups as following: Control, treatment 1 (LIF), treatment 2 (EGF), treatment 3 (LIF+EGF). In each group, the embryos were cultured in an incubator at 37°C with 5% CO 2 and 90% humidity for 72hrs. The state of embryo development was evaluated in 24,36,48,60 and 72hrs following the embryos cultures. By the end of the cultures, cell apoptosis was studied by the terminal deoxynucleotidyl transferas-mediated dUTP nick end-labeling (TUNEL) technique. Results: Significant difference was detected in the rate of hatching in the LIF and LIF+EGF groups. This difference was also seen in the rate of blastocyst formation after 36hrs (p<0.05) and in the average of the total cell number (p<0.05) after 72hrs. In comparison to the apoptotic index, there was no significant difference between the control and treatment groups. Conclusion: The findings in this study show a beneficial effect of LIF and EGF on the blastocyst formation, hatching and its total cell numbers in vitro.
The stimulating effect of recombinant cytokine LIF on the mouse blastocysts during implantation
Biochemistry (Moscow) Supplement Series B: Biomedical Chemistry, 2007
The effect of recombinant LIF cytokine (Leukemia inhibitory factor) on the isolated mouse embryos at the stages of middle and late blastocyst has been investigated. We have demonstrated here that this agent is necessary in vitro at the stage of normal trophoblast formation after the blastocysts hatch from zona pellucida. This cytokine (10 ng/ml) caused intensification of adhesion and proliferative activity of the trophoblast cells. This is important for intercellular interactions with endometrium and for invasion of embryos into the uterus. The recombinant LIF insignificantly influenced cells of the inner cell mass.
Theriogenology, 1994
Three experiments were conducted to evaluate the effect of addition of human leukemia inhibitory factor (hLIF1 to synthetic oviduct fluid medium (SOFM) supplemented with human serum (HSI. bovine serum albumin (BSAI or polyvinyl alcohol (PVA) on the development of bovine embryos matured and fertilized in vitro. In vitro matured and fertilized bovine oocytes were cultured in SOFM supplemented with 10% HS to obtain embryos at 1-cell, 4-or 8-cell, and morula or early blastocyst stages. In Experiment 1, embryos at the different developmental stages were cultured In SOFM supplemented with 10% HS and 1 of 6 different dosages (0. 500, 1000. 2000, 4000, 6000 U/ml) of hLIF. In Experiments 2 and 3, the embryos were cultured in SOFM + BSA and SOFM + PVA, respectively with or without hLIF 15000 U/ml). In, Experiment 1, the addition of any hLIF dosages did not improve development to the expanding blastocysts as compared with the control (without hLIF) in each embryonic stage. Embryonic stages at the tune of hLIF addition affected the development: early blastocysts resulted in significantly (PcO.01) better development than the other stages. The addition of hLIF at 1-, 4-and 8-cell stages in Experiment 2 and 3 had no effect on development to the expanding blastocyst stages significantly (P~O.011 unproved the development. The results indicate that the effect of hLIF addition is critical to embryonic stages and the advantage of hLIF addition is only observed when SOFM is supplemented with BSA or PVA. A stimulating effect of hLIF was not observed when SOFM was supplemented with HS.
Fertility and Sterility, 2008
Objective: To examine the role of leukemia inhibitory factor (LIF) during in vitro maturation (IVM) on human and mice cumulus expansion and mice oocyte competence by in vitro fertilization (IVF), culture, and embryo transfer (ET). Design: Prospective animal and human study. Setting: Serono laboratories and IVF clinic. Patient(s): Healthy women volunteers and 8-week-old female mice. Intervention(s): Cumulus compacted human and mice oocytes were matured in IVM media with and without recombinant follicle-stimulating hormone (FSH) and with and without LIF. Mice IVM oocytes with and without 0.2 IU/mL of recombinant FSH; or with and without recombinant FSH þ LIF (0.1, 1.0, 1000.0 ng/mL) and ovulated oocytes were in vitro fertilized and cultured. We transferred 395 blastocysts to the uterine horn of 2.5-day pseudopregnant female mice. Main Outcome Measure(s): Cumulus expansion in human and mice oocytes, and two-cell rate, blastocyst rate, and delivered rate of live pups in mice. Result(s): In human and mouse oocytes, LIF induced cumulus expansion. When 1000 ng/mL of LIF was added in combination with recombinant FSH, a statistically significant increase in cleavage rate, embryo development rate, and birth rate was observed when compared with oocytes matured with FSH alone. Conclusion(s): Leukemia inhibitory factor induced cumulus expansion similarly in human and mouse cumulusoocyte complexes, and recombinant FSH plus LIF supplementation during mouse IVM significantly improved oocyte competence as measured by cleavage rate, blastocyst development, and birth rate.
The effects of LIF and EGF on mouse oocyte maturation, fertilization and development in vitro
Iranian Journal of …, 2009
Background: Mammalian oocytes are exposed to a mixture of many different growth factors and cytokines which provides an optimized microenvironment for oocyte maturation. In the lack of this natural microenvironment in vitro, the quality of oocyte and embryos appears to be suboptimal. Objective: This study was undertaken to investigate the effects of EGF and LIF on in vitro maturation, fertilization and cleavage rates in mouse oocytes. Materials and Methods: The GV oocytes were collected from female NMRI mice and randomly divided into control and 3 treatment groups. Oocytes in treatment groups were cultured in the maturation medium supplemented with 50 ng/ml rhLIF (Treatment 1), 10ng/ml EGF (Treatment 2) and 50 ng/ml LIF+ 10ng/ml EGF (Treatment 3) for 24 hours at 37°C in humidified 5% CO 2 in air. The matured oocytes were fertilized in vitro and cultured for 96 hours. Finally, the developmental rates were assessed and embryos were stained using Hoechst 33258. Results: There was a higher maturation rate in treatment groups compared to the control group. There was not any significant difference in the rate of fertilization among the groups. The rates of cleavage (79.1%) and blastocyst formation (62.2%) were significantly higher in LIF + EGF group comparing to the other groups. The rates of hatching in groups treatment 1 (35.2%) and 3(41%) was significantly higher comparing to the other groups. Also the mean of total cell number in treatment groups significantly was higher than control (p< 0.05). Conclusion: The findings of this study suggest a beneficial effect of LIF and EGF on mouse oocyte maturation and cleavage rates.
Journal of Applied Animal Research, 2011
Feeder layer and leukaemia inhibitory factor (LIF) play an important role in maintaining the embryonic stem cells in undifferentiated state and help in their unlimited proliferation. The aim of the present investigation was to see the effect of LIF and different types of feeder layers on growth and pluripotent nature of goat embryonic stem cells. Goat blastocysts were produced in vitro by standard methods of in vitro maturation (IVM), in vitro fertilisation (IVF) and in vitro culture (IVC) techniques. Inner cell mass (ICM) cells were isolated mechanically and cultured in embryonic stem (ES) cell medium on inactivated feeder layer supplemented with or without LIF. When ICM cells were cultured on goat fibroblast feeder layer in ES cell culture medium supplemented with LIF they maintained undifferentiated state up to 15 passages. The undifferentiated ES cells showed stem cell specific morphological features, normal karyotype and expressed stem cell specific surface markers like alkaline phosphatase,TRI-1-61, TRI-1-81 and intracellular markers Oct4, Sox2 and Nanog. It could be concluded that goat embryonic stem cells were successfully cultured on feeder layers which were not of goat origin but to maintain the undifferentiated state for long time was found the best on goat fetal fibroblast with LIF.
Effects of culture milieus on the development of mouse blastocysts in vitro
In Vitro, 1977
Various culture milieus were examined for their support of mouse blastocyst development. Two important variables were the time at which human cord serum was added to the medium and the concentration of amino acids. In the best medium, Eagle's Minimum Essential Medium (fortified with six times the usual amino-acid concentration plus 20% fetal bovine serum, replaced after 48 hr with human cord serum), 83% of the blastocysts shed the zona pellucida, 58% developed to the early egg cylinder stage, 42% to the advanced egg cylinder stage and 22% attained the primitive streak stage after 6 to 8 days of culture. their generous help in obtaining the human cord serum. We are also very grateful to Dr. Y. I-Isu for giving advice and teaching us the methodology of culturing mouse blastocysts.