The majority of embryonic mortality in cattle occurs before maternal recognition of pregnancy at Day 16 postconception. In vitro-derived embryos exhibit a greater incidence of loss than their in vivo-derived counterparts. To better understand the causes of such embryonic loss, the aim of the current study was to compare transcript profiles of Days 7 and 13 bovine embryos derived in vitro or in vivo using the bovine Affymetrix microarray. We wanted to answer 3 questions: (1) what genes differ on Day 7 between blastocysts derived in vivo or in vitro, (2) what genes differ between Day 13 embryos derived from in vitro or in vivo embryos, and (3) what genes change between the blastocyst stage (Day 7) and the initiation of elongation (Day 13) and how are these temporal changes affected by the origin of the embryo. Day 7 bovine blastocysts were produced either in vitro by maturation, fertilization, and culture or in vivo by superovulation, AI, and nonsurgical embryo recovery. Half of the Day 7 blastocysts were snap frozen in liquid nitrogen in pools of 25 (microarray) or 10 (quantitative RT-PCR), and the other half were transferred in groups of 10 to synchronized heifers (10 recipients per group) ipsilateral to the corpus luteum and recovered on Day 13 by flushing the uterus after slaughter. Day 13 conceptuses were snap frozen individually. Three replicate pools of 25 Day 7 blastocysts and 5 Day 13 conceptuses were used for microarray analysis. Of the 24 128 probe-sets on the array, approximately 9500 genes were actively expressed in Days 7 and 13 embryos, irrespective of source. In Day 7 blastocysts, 50 genes were found to be differentially expressed (≥ 1.5-fold; P ≤ 0.05), of which 19 were up-regulated and 31 down-regulated in the in vivo compared with in vitro embryos. In Day 13 conceptuses, 288 genes were found to be differentially expressed (≥1.5-fold; P ≤ 0.05), of which 133 were up-regulated and 155 down-regulated in the in vivo compared with in vitro embryos. The comparison between Days 7 and 13 embryos revealed significant temporal changes in transcript profile, with 1806 and 909 transcripts differentially expressed in in vitro and in vivo-derived embryos, respectively. Across the 3 array comparisons between Days 7 and 13 embryos, 444 genes were consistently exclusively present in in vivo embryos, whereas 1341 were exclusively present in in vitro embryos. Array validation was done by quantitative RT-PCR analysis of fatty acid desaturase 1 (FADS1), cytochrome P450, family 51, subfamily A, polypeptide I (CYP51), and hyaluronan binding protein 2 (HABP2) genes. In conclusion, these results indicate that the origin of the blastocyst can have a significant effect on the transcript profile of the conceptus at the initiation of elongation and might be associated with the likelihood of embryo survival/loss subsequently. Further hierarchical clustering analysis and quantitative RT-PCR data will address the functional roles for certain known genes and novel candidate genes related to embryonic mortality.
This work was supported by a grant (AGL2006-05616) from the Spanish Ministry of Science and Technology.
Quantitative RT-PCR analysis of differentially expressed genes in Quercus suber in response to Phytophthora cinnamomi infection
