4 Aryl hydrocarbon receptor targets are upregulated in porcine blastocyst-stage embryos that were cultured in vitro: A transcriptional analysis

Abstract

In vitro-produced (IVP) porcine embryos are developmentally delayed compared with those derived in vivo. Thus, efforts have been made to modify the current medium for IVP of porcine embryos in an attempt to shift the abundance of target transcripts towards the in vivo level. The objective of the current study was to identify differences in mRNA abundance that may account for reduced developmental competence in IVP embryos and to determine whether alterations to maturation and culture media directed the transcriptional profiles of IVP embryos towards an in vivo state. Following AI of gilts, an oviduct and tip of the uterine horn were flushed on Day 2 to recover 4-cell stage embryos; these were cultured for 4 days in MU3, generating IVM and in vitro-cultured (IVC) blastocyst-stage embryos. On Day 6, the gilts were killed, and the contralateral horns were flushed to obtain in vivo-derived (IVV) blastocyst-stage embryos. The third group of blastocyst-stage embryos, referred to as in vitro-matured and cultured (IVMC), were created by aspirating cumulus–oocyte complexes from slaughterhouse-derived ovaries, maturing and fertilizing in vitro, and culturing for 6 days in MU3. Total RNA was extracted from pools of 10 blastocyst-stage embryos with 3 replicates per group. First- and second-strand cDNA was synthesised and sequenced by using the Illumina platform (Illumina Inc.). After removal of adapters and reads that mapped to porcine rRNA genes and the PhiX genome, reads were mapped to the Sus scrofa genome by using STAR (version 2.7.1a) with default options. Pairwise comparisons were performed to test for differential expression of genes by using the Bioconductor package DESEqn 2. Transcripts were differentially abundant (false discovery rate <0.05) between IVV and IVC embryos (2,450), between IVV and IVMC embryos (3,045), and between IVC and IVMC embryos (262). Pathways related to cell cycle were downregulated in IVC and IVMC compared with IVV embryos, and pathways related to amino acid transport in metabolism were upregulated in IVC and IVMC compared with IVV embryos. Of particular interest, message for cytochrome P450 family 1 subfamily A member 1 (CYP1A1) was only present in the IVC (956 reads) and IVMC (381 reads) embryos but not in the IVV embryos (0 reads). The aryl hydrocarbon receptor (AHR) promotes transcription of CYP1A1, which encodes a monooxygenase involved in xenobiotic metabolism. Moreover, abundance of 12 other AHR targets was increased in IVC and IVMC embryos (false discovery rate <0.05) compared with IVV embryos. Thus, production of porcine embryos in vitro may activate AHR, resulting in altered transcriptional profiles and reduced competence.

This research was funded by USDA-NIFA (2019-67011-29543).