Expression and Analysis of the Imprinted Genes, IGF2R and AIRN, During Development of In Vivo and In Vitro Produced Bovine Pregnancies

The objectives of this study were to examine the expression of IGF2R and AIRN in cattle; and determine the effects of in vitro embryo production on expression of these imprinted genes during bovine fetal and placental development. Study 1 was conducted to determine if AIRN was expressed in bovine fetal tissues and whether or not bovine AIRN exhibited timespecific expression during development, indicative of its role in regulating imprinted expression of IGF2R. The existence of bovine AIRN was validated using RNA extracted from gestational Day 70 bovine fetal liver. The presence of AIRN in early gestation bovine fetal tissues was examined. No AIRN was detected in any blastocyst pools (n = 11). The number of conceptuses expressing AIRN increased from 1 of 9 at Day 15 of gestation to 8 of 10 at Day 18 of gestation. AIRN was expressed in all fetal livers recovered at Day 35 to 55 (n =5) and Day 70 (n = 7). In contrast, IGF2R was expressed in all blastocyst pools, Day 18 conceptuses, Day 35 to 55 fetal livers, and Day 70 fetal livers. Only 1 conceptus out of 9 at Day 15 gestation did not express IGF2R. The relative level of AIRN expression was decreased (P < 0.05) in in vitro produced fetal livers at Day 70 of gestation compared to in vivo produced controls. Our results indicate that, in cattle, expression of AIRN is not expressed in blastocyst-stage embryos, is expressed in an increasing proportion of embryos around the time of maternal recognition of pregnancy, and is expressed following implantation. Additionally, the relative expression level of AIRN in bovine fetal liver can be altered by the method of embryo production. The objective of study 2 was to characterize the novel bovine AIRN non-coding RNA. Total RNA was extracted from gestational day 150 bovine fetal liver and DNase-treated prior to cDNA synthesis and PCR amplification. PCR primer sets (n=16) were designed based on genomic sequences to "walk" down the predicted AIRN ncRNA sequence. All PCR amplicons were sequence verified. A putative AIRN promoter was located 441 base-pairs upstream of differentially methylated region 2 (DMR2) within intron 2 of IGF2R. A strong polyadenylation signal was found 117 kb downstream of the promoter. Primer sets designed upstream of the promoter and downstream of the polyadenylation signal yielded no PCR amplicons. In conclusion, bovine AIRN is a long non-coding RNA approximately 117kb in length with transcriptional overlap of IGF2R. These results provide further evidence that AIRN most likely plays a role in regulating imprinted expression of IGF2R. In the third and final study, the objectives were to examine the expression of IGF2R mRNA and AIRN non-coding RNA in in vivo (IVO) and in vitro produced (IVP) bovine conceptuses, and in fetal and placental tissues at early and late gestation; and to analyze the level of DNA methylation of cytosine-guanine (CpGs) repeats within differentially methylated region 2 (DMR2) of IGF2R in these tissues. Expression of IGF2R mRNA did not vary between treatments regardless of the method used for embryo production, tissue, or stage of development. The level of expression for AIRN non-coding RNA was decreased in Day 70 IVP fetal cotyledons and Day 222 IVP-AOS fetal livers, and increased in Day 222 IVP-AOS fetal cotyledons compared to IVO controls. DNA hypomethylation was observed in Day 15 IVP conceptuses, Day 70 IVP and Day 222 IVPAOS fetal liver and cotyledons. Therefore, a mechanism proposed to regulate and maintain imprinted expression of IGF2R, expression of AIRN , was affected by the methods used for in vitro embryo production. However, a direct correlation between the level of AIRN expression and the level of imprinted expression of IGF2R was not identified. To summarize, AIRN in cattle is a 117kb long non-coding RNA whose expression was correlated with the onset of imprinted expression of IGF2R in bovine conceptuses. Furthermore, expression of AIRN varies in a time and tissue-specific manner. DNA methylation at DMR2 of IGF2R also appears to be involved in maintaining imprinted expression of AIRN and IGF2R; and expression of these imprinted genes can be affected by in vitro embryo production.