👤 Alfonso Gutierrez-Adan

The majority of embryonic loss in cattle occurs before maternal recognition of pregnancy, at around Day 16 postconception. The origin of the embryo can have a significant impact on the dynamics of embryo mortality. The aim of this study was to examine the temporal changes in transcriptional profile as the embryo develops from a spherical blastocyst on Day 7 to an ovoid conceptus at the initiation of elongation on Day 13 and to highlight differences in these temporal gene expression dynamics between in vivo- and in vitro-derived blastocysts that may be associated with embryonic survival/mortality using the bovine Affymetrix microarray. All embryos were produced either in vitro by in vitro fertilization or in vivo by superovulation. A proportion of Day 7 blastocysts were snap frozen, and the remainder were transferred (n = 10 per recipient) to synchronized heifers, recovered on Day 13, and snap frozen individually. Three pools of Day 7 blastocysts (n = 25 per pool) and Day 13 conceptuses (n = 5 per pool) were used for microarray analysis. In Day 7 blastocysts, 50 genes were found to be differentially expressed (P < 0.05), of which 19 were up-regulated and 31 down-regulated in the in vivo compared to in vitro embryos. In Day 13 conceptuses, 288 genes were found to be differentially expressed (P < 0.05), of which 133 were up-regulated and 155 down-regulated in the in vivo compared to in vitro embryos. The comparison between Day 7 and Day 13 embryos revealed significant temporal changes in transcript profile with 1806 and 909 transcripts differentially expressed in the in vitro- and in vivo-derived embryos, respectively. Across the three array comparisons between Day 7 and Day 13 embryos, 444 genes were consistently exclusively present in the in vivo embryos, whereas 1341 were exclusively present in the in vitro embryos. Regardless of the origin of the embryo, 465 differentially expressed genes between Day 7 and 13 were common to both in vivo- and in vitro-derived embryos; these genes are likely critical for the transition between the blastocyst (Day 7) and ovoid conceptus (Day 13) stages of embryo development. In order to validate the microarray findings, differences in the expression of six genes (CYP51A1, FADS1, TDGF1, HABP2, APOA2, and SLC12A2) were confirmed by quantitative real-time PCR on in vivo- and in vitro-derived embryos on Day 7 and Day 13 using independent samples from those used for the microarray. Subsequent mapping of these differentially expressed genes into relevant functional groups and pathways identified important pathways involved in conceptus elongation in cattle. In conclusion, this analysis has identified genes and pathways crucial for the transition from a spherical blastocyst to an ovoid conceptus as well as those uniquely associated with a greater likelihood of embryonic survival (those unique to in vivo embryos) or loss (those unique to in vitro embryos). Show less

For epigenetic phenotypes to be passed on from one generation to the next, it is required that epigenetic marks between generations are not cleared during the two stages of epigenetic reprogramming: mammalian gametogenesis and preimplantation development. The molecular nature of the chromatin marks involved in these events is unknown. Using the epigenetically inherited allele Axin1(Fu) (the result of a retrotransposon insertion upstream of the Axin1 gene) we sought to establish the heritable mark during early embryonic development that determines transgenerational epigenetic inheritance and to examine a possible shift in the expression of this epiallele in future progeny induced by in vitro culture (IVC). To identify the heritable mark we analyzed 1) the level of DNA methylation shown by the Axin1(Fu) allele in sperm and embryos at blastocysts stage and 2) the histone marks (H3K4 me2, H3K9 me3, H3K9 ac, and H4K20 me3) present at the blastocyst stage at the specific Axin1(Fu) locus. According to our data, histone H3K4 me2 and H3K9 ac mark the differences between the Axin1(Fu) penetrant and the silent locus during the first period of demethylation of the preimplantation development. Moreover, suboptimal IVC (reported to produce epigenetic alterations in embryos) and the histone deacetylase inhibitor trichostatin A affect the postnatal expression of this epigenetically sensitive allele through histone modifications during early development. This finding indicates that altered histone modifications during preimplantation can drive altered gene expression later on in development. Show less