Igf2 Gene imprinting in preimplantation mouse embryos

Abstract

Background and Significance: Epigenetic effects on embryos produced from in vitro fertilization are unknown. Recent epidemiological studies show that individuals conceived through in vitro fertilization (IVF) are at twice the risk of birth defects which are associated with a loss of imprinting or altered imprinting of Igf2,and other genes, as compared with babies conceived naturally. These birth defects include diseases associated with aberrant imprinting, such as, Beckwith-Wiedemann Syndrome, Prader-Willi and Angelman’s Syndrome.Objective: In this study, we use a mouse model system to investigate Igf2 imprinting in embryos conceived in vitro.Materials and Methods: M. Musculus female mice (C57 Black, Jackson Laboratories, Maine), were superovulated with pregnant mare serum gonadotropin (10 IU of PMSG, Sigma, St. Louis MO). In vitro fertilization was performed using oocytes recovered from the superovulated mice, 36–40 hours after an injection of 7.5 IU of human chorionic gonadotropin (hCG). The oocytes were fertilized with sperm removed from the epididymus of Mus Spretus male mice. Embryos were placed in Quinn’s Advantage Cleavage Medium (Sage Biopharma, Inc.). The embryos were recovered and analyzed at the 2-cell, 4-cell, morula and blastocyst stages of development.Igf2 expression was evaluated by RT PCR (1Hu J.F. Vu T.H. Hoffman A.R. Promoter-specific modulation of insulin-like growth factor II genomic imprinting by inhibitors of DNA methylation.JBC. 1996; 271: 18253-18262Crossref PubMed Scopus (62) Google Scholar). Maternal and paternal alleles were differentiated by restriction endonuclease digestion specific for polymorphic sites between M. Musculus and Mus Spretus.Results: Our results show that Igf2 gene expression is detectable in morula and blastocyst of our mouse model system. Igf2 expression is biallelic in the morula stage, whereas, some blastocyst have only monallelic expression. Further studies will compare Igf2 imprinting in embryos conceived in vitro vs. in vivo, as well as, evaluate the imprinting patterns of other genes.Conclusion: We have developed a mouse model system for evaluating Igf2 imprinting for in vitro studies. This model enables us to discriminate maternal vs. paternal gene expression in morula and blastocyst. Background and Significance: Epigenetic effects on embryos produced from in vitro fertilization are unknown. Recent epidemiological studies show that individuals conceived through in vitro fertilization (IVF) are at twice the risk of birth defects which are associated with a loss of imprinting or altered imprinting of Igf2,and other genes, as compared with babies conceived naturally. These birth defects include diseases associated with aberrant imprinting, such as, Beckwith-Wiedemann Syndrome, Prader-Willi and Angelman’s Syndrome. Objective: In this study, we use a mouse model system to investigate Igf2 imprinting in embryos conceived in vitro. Materials and Methods: M. Musculus female mice (C57 Black, Jackson Laboratories, Maine), were superovulated with pregnant mare serum gonadotropin (10 IU of PMSG, Sigma, St. Louis MO). In vitro fertilization was performed using oocytes recovered from the superovulated mice, 36–40 hours after an injection of 7.5 IU of human chorionic gonadotropin (hCG). The oocytes were fertilized with sperm removed from the epididymus of Mus Spretus male mice. Embryos were placed in Quinn’s Advantage Cleavage Medium (Sage Biopharma, Inc.). The embryos were recovered and analyzed at the 2-cell, 4-cell, morula and blastocyst stages of development. Igf2 expression was evaluated by RT PCR (1Hu J.F. Vu T.H. Hoffman A.R. Promoter-specific modulation of insulin-like growth factor II genomic imprinting by inhibitors of DNA methylation.JBC. 1996; 271: 18253-18262Crossref PubMed Scopus (62) Google Scholar). Maternal and paternal alleles were differentiated by restriction endonuclease digestion specific for polymorphic sites between M. Musculus and Mus Spretus. Results: Our results show that Igf2 gene expression is detectable in morula and blastocyst of our mouse model system. Igf2 expression is biallelic in the morula stage, whereas, some blastocyst have only monallelic expression. Further studies will compare Igf2 imprinting in embryos conceived in vitro vs. in vivo, as well as, evaluate the imprinting patterns of other genes. Conclusion: We have developed a mouse model system for evaluating Igf2 imprinting for in vitro studies. This model enables us to discriminate maternal vs. paternal gene expression in morula and blastocyst.