Analysis of Differential Messenger RNA Expression Between Bovine Blastocysts Produced in Different Culture Systems: Implications for Blastocyst Quality1

Abstract

Using reverse transcriptase-amplified fragment length polymorphism (RT-AFLP) analysis of differential mRNA expression and semiquantitative reverse transcriptase-polymerase chain reaction, we compared mRNA expression in bovine blastocysts from 4 sources, known to differ in quality in terms of their ability to withstand cryopreservation: 1) in vitro culture in synthetic oviduct fluid of in vitro-matured (IVM)/in vitro fertilized (IVF) zygotes; 2) in vitro culture in TCM-199 supplemented with granulosa cells (coculture) of IVM/IVF zygotes; 3) in vivo culture in the ewe oviduct of IVM/IVF zygotes; or 4) superovulation, artificial insemination, and nonsurgical embryo recovery. Total mRNA was isolated from pools of blastocysts and reverse transcription was performed. Triplicate reactions from each sample were displayed, and only consistent banding variations were recorded. Using AFLP-differential display assay, we found that cDNA banding patterns are highly conserved between the 4 groups of blastocysts studied; however, there was a difference of 7% in bands either missing or expressed across the groups. Fifty bands were reamplified, and a sequence comparison search revealed similarity of 14 isolated fragments to ribosomal and mitochondrial genes, 16 matched to described cDNA, and 20 corresponded to unknown sequences that may represent novel genes. The study of 7 differentially expressed mRNAs known to be involved in developmental process in the embryo suggests roles for apoptosis, oxidative stress, gap junctions, and differentiation in the determination of embryo quality. The aberrant transcription patterns detected in in vitro-produced bovine embryos compared with those produced in vivo may explain their reduced quality in terms of viability after cryopreservation.

Resveratrol treatment during goat oocytes maturation enhances developmental competence of parthenogenetic and hand-made cloned blastocysts by modulating intracellular glutathione level and embryonic gene expression

“…In case of HMC-derived blastocysts reconstructed from mature oocytes of different resveratrol treatment groups, expression of a set of genes which are having relevance in early embryonic development [13,25,35,42,43] were checked. The genes included in the present study are involved in pre-and postnatal growth (IGF -1 ), metabolism (Glut1 ), pluripotency (Oct4), morphogenesis and biogenesis of the trophoectoderm (E -cad), lineage determination and cavitation (Stat3), gap junction, cavitation and cryosurvival (Cx43), growth arrest (CHOP-10), oxidative stress (MnSOD), and DNA methylation (DNMT).…”

Serum free embryo culture medium improves in vitro survival of bovine blastocysts to vitrification

“…The ability of in vitro produced embryos to survive cryopreservation, an indicator of embryo quality [4,5,8,12,41,42], is affected by culture conditions. Thus, Dinnyés et al [29] reported high survival rates (85% and 90%) after vitrification/warming of Day 8 expanded embryos produced in SOF without and with FCS, respectively; the hatching rates the above authors obtained for embryos produced in SOF + FCS (63%) are comparable to the survival rates in our work in the Vero and BSA groups.…”

“…The freezing sensitivity should be considered as a factor affecting the quality of the in vitro produced bovine blastocysts [10][11][12][13]. Improvement of embryo survival after freezing can be achieved by changing the conditions of in vitro culture, and/or modifying ''standard'' cryopreservation procedures [14].…”

The mRNA expression of brain-derived neurotrophic factor in oocytes and embryos and its effects on the development of early embryos in cattle

“…These early deviations in gene expression patterns seen in NT-derived embryos warrant further investigations in post-implantation and neonatal development to understand the causative mechanism of the abnormalities and elevated mortality after transfer of NT-and activation-derived embryos (Daniels et al, 2001;Wrenzycki et al, 2001;Humpherys et al, 2002). The change of these genes mRNA directly affects the quality of embryos (Rizos et al, 2002;Ruddock et al, 2004). By immunohistochemistry, BDNF protein is localized in trophectoderm cells.…”