Effect Of In Vitro Fertilization On Imprinted Genes IGF2 And H19 MRNA Expression Of Mouse Early Development

【OBJECTIVES】The study was to evaluate the alteration of expression of the imprinted genes IGF2 and H 19 in embryos and fetuses after in vitro fertilization(IVF), then to reveal whether the IVF can influence the early development of mouse.【METHODS】Use the tip of a pipette to add appropriate amounts(usually about 5μl) of the sperm suspension to the drop of HTF containing the cumulus-enclosed oocytes,such that the final sperm cell concentration was(5-7)×10⁵ cells/μl..Place the fertilization dishes in an incubator(37℃,5%CO₂ in air).6 hours after insemination, wash the oocytes twice using fresh drops of HTF,and then observe the oocytes.Those had female pronucleus and male one and second polar body were fertilized oocytes After 18-22 hours of incubation(37℃,5%CO₂ in air),the two-cell embryos were transferred to pseudo-pregnant recipient females.While natural mating mice were control group.Observing vaginal plug was regarded as the first day of pregnant.The fetus were weighed and frozen with nitrogen quickly after the embryo developed 14 days(E14).Meanwhile,several pregnant mice were maintained until the pups were born and weighed.The newborns were operated,then the heart,liver and brain of newborns were carried out and frozen with nitrogen swiftly.RNA was isolated from the frozen embryos and the heart,liver and brain of newborns according to RNeasy Mini Kit.The concentration of RNA was measured,the RNA was changed DNA by reverse transcription.Last procedure was real-time PCR about IGF2 and H19.【RESULTS】The result found that the mRNA expression ofIGF2 and H19 oflVF group were respectively as 1.57 fold and 1.38 fold as that of control group by real-time reverse transcription-polymerase inspecting El4 fetuses.About the weight of E14 fetuses,the IVF group were 16.5%higher than the control group(P＜0.001, covariance).In the same way,the mRNA expression of IGF2 and H19 of IVF group were respectively as 1.87 fold and 2.06 fold as that of control group by real-time reverse transcription-polymerase inspecting the heart of newborns.That of mouse liver were respectively 1.56 fold and 1.52 fold.That of brain were respectively 1.27 fold and 2.11 fold.The birth weight of IVF group was 13.2%higher than that of control group(p＜0.001,covariance).All the result showed that IVF alters the expression of imprinted genes IGF2 and H19,consequently promote the early development of mouse.【CONCLUTION】Through the present study,the IVF altered the expression of imprinted genes IGF2 and H19,as a result,the weight of El4 fetus and new born increased.