216 IDENTIFICATION OF DIFFERENTIALLY EXPRESSED GENES IN BOVINE EMBRYOS CULTURED IN VIVO OR IN VITRO

Abstract

The post-fertilization culture environment of the bovine embryo is known to influence the quality of the resulting blastocyst, manifested in terms of morphology, cryotolerance, and the relative gene transcript abundance of several candidate genes. This may have consequences for the pregnancy rate following embryo transfer. The objective of the current study was to take a broader approach toward identifying differentially expressed genes in bovine blastocysts derived from either in vivo or in vitro culture. Presumptive zygotes, produced by in vitro maturation and fertilization, were randomly assigned to one of two groups and cultured for 6 days, either in vitro in SOF medium, or in vivo in the ewe oviduct following transfer by mid-ventral laparotomy. Blastocysts were recovered from both systems on Day 7 after insemination, snap frozen in liquid nitrogen, and stored at −80°C. Total RNA was extracted from 50 blastocysts for each culture group from each of four replicates using the PicoPure^TM RNA Isolation Kit (ARCTURUS, Mountain View, CA 94043, USA). The RiboAmp^TM RNA Amplification Kit (ARCTURUS) was used to linearly amplify the mRNA fraction of total RNA using double-stranded cDNA as template in a T7 RNA polymerase-catalyzed amplification. Samples from both culture environments were differentially labelled using N-hydroxysuccinimide (NHS)-activated fluorescent Cy3 or Cy5 dyes (Amersham Pharmacia Ltd., Piscataway, NJ, USA) and were hybridized onto a cDNA microarray. Each microarray contained 3888 total spots, with 932 bovine EST clone inserts developed from a normalized bovine total leukocyte (BOTL) cDNA library and an additional 459 amplicons representing additional genes including cytokines, receptors, signal transduction molecules, transcription and growth factors, enzymes, cell cycle regulators, and cellular components. Data were normalized and an expression ratio calculated between the two groups. This was compared to 1 within each of the 4 replicates by Student's t-test. Microarray analysis identified 15 gene transcripts that were differentially expressed P ≤ 0.05) between blastocysts produced in vivo or in vitro. Among these, four genes involved in transcription (nuclear receptor co-repressor 1, zinc finger protein 22, CCR4-NOT transcription complex, DOT1) and two genes involved in intracellular signalling (proteasome 26S subunit non-ATPase 13 and guanine nucleotide binding protein) had a higher mRNA expression level in blastocysts produced in vivo compared to those produced in vitro. In addition, Connexin 43, a gene involved in gap junction formation, was down regulated following in vitro culture which is consistent with our previous studies. Among the genes up-regulated following in vitro culture were WNT2B wingless-type MMTV integration site, CD103 integrin, and tumor necrosis factor superfamily member 8. In conclusion, we have identified previously uncharacterized, differentially expressed genes involved in cell communication, intracellular signalling, and regulation of transcription in bovine blastocysts cultured in vivo or in vitro.

This work was supported by Science Foundation Ireland under Grant No. 02/IN1/B78.