Effects of culture medium and protein supplementation on mRNA expression of in vitro produced bovine embryos

Abstract

Numerous studies have reported aberrant gene expression levels attributed to suboptimal in vitro culture conditions. This study investigated the effects of different culture systems and protein sources on the developmental competence of in vitro production (IVP) embryos measured by cleavage and blastocyst rates, cell number, and relative abundance of POU5F1 (OCT4), nanog, GJA1 (connexin 43), and SLC2A1 (GLUT1) transcripts when compared to in vivo embryos. Experiment 1 compared IVP embryos cultured in either synthetic oviductal fluid (SOFaa) or potassium simplex optimized medium supplemented with amino acids (KSOMaa). Experiment 2 compared the same two culture systems with and without the addition of calf serum (CS). Results from both experiments indicated that despite similar developmental rates, significant differences were observed at the mRNA level. In Experiment 1, OCT4 was the only transcript to have a mean abundance level significantly higher in KSOMaa blastocysts when compared with both SOFaa and in vivo embryos. The same pattern of upregulation of OCT4 mRNA was noted in Experiment 2. There were no significant alterations of the ICM specific transcript nanog in either experiment. In contrast to reports by others, connexin 43 mRNA was not expressed at detectable levels in in vivo embryos analyzed in our studies. Blastocysts cultured in SOFaa with CS or KSOMaa had a significant upregulation of GLUT1 mRNA when compared with other treatments and in vivo embryos. Until differences between IVP and in vivo embryos are minimized, aberrations in IVP will continue to arise.

Effect of different sequential and two‐step culture systems on the development, quality, and RNA expression profile of bovine blastocysts produced in vitro

“…Our results confirmed a significant decrease in the expression of OCT4 , DNMT1 , and SOD2 in embryos cultured in sequential K‐S/FBS, and a decrease in the expression of SOD1 in embryos cultured in two‐step K‐K/BSA relative to embryos cultured in two‐step K‐K/FBS. This is in agreement with previous studies that describe differences at the transcriptional level in embryos cultured in TCM medium compared to embryos cultured in SOF (Wrenzycki et al, 2001) or in embryos cultured in KSOM medium compared to embryos cultured in SOF (Purpera et al, 2009). DNA methyltransferase 1 ( DNMT1 ) is responsible for maintenance of methylation patterns in replicating DNA, and therefore plays a crucial role in the regulation of transcription during the embryonic development (Weissbach et al, 1989).…”

Donor-host mitochondrial compatibility improves efficiency of bovine somatic cell nuclear transfer

“…As summarized in Table 1, after the 8-cell stage, homotype SCNT embryos constructed from enucleated oocytes and donor cells with the same haplotype (A-A or B-B) had significantly higher developmental potential than heterotype SCNT embryos (A-B or B-A), as well as a higher blastocyst rate (42.5% vs. 28.8%) which is similar to rates from IVF embryos (40% based on cleavage number) [10]. Note that the homotype SCNT, heterotype SCNT and IVF embryos were produced from the same OPU session, and the procedures and assays for the three groups were kept as similar as possible.…”

“…Numerous studies have reported the development capability of in vitro produced (IVP) embryos could be influenced by aberrant gene expression levels and epigenetic reprogramming [10-12]. Six to eight days post fertilization or after reconstruction for SCNT embryos is a critical period when various developmentally important events occur, including the first cleavage division which is critical in determining the quality of subsequent embryo development.…”

Comprehensive cross production system assessment of the impact of in vitro microenvironment on the expression of messengers and long non-coding RNAs in the bovine blastocyst

“…Among the bestdescribed negative impacts of IVP are the birth of abnormally large calves or 'large offspring syndrome' (Young et al 1998, McEvoy et al 2000, the skew in the sex ratio in favor of males (Kimura et al 2005(Kimura et al , 2008, and increased sensitivity to cryopreservation (Rizos et al , 2008, which hinders the export potential of embryos obtained in vitro. Due to these problems, a large number of studies are conducted to characterize blastocysts produced in vitro and comparing them to their in vivo counterparts to identify the causes of the deleterious effects of IVP and to improve in vitro conditions for embryo development (Wrenzycki et al 1998, 2001, Niemann & Wrenzycki 2000, Knijn et al 2002, Gutierrez-Adan et al 2004, Mohan et al 2004, McHughes et al 2009, Purpera et al 2009. As is often the case for samples of very small mass such as embryos, most of these studies have focused on gene expression.…”