Global gene expression analysis comparing bovine blastocysts flushed on day 7 or produced in vitro

Abstract

In vitro produced (IVP) bovine embryos have darker cytoplasm, reduced buoyant density, fragile zonae pellucidae, chromosomal abnormalities, higher pregnancy failure rates, and altered gene expression compared to embryos produced in vivo. Characterization of early deviations in gene expression would enable us to better understand the biology of early embryo development and improve in vitro culture systems. Here we compared gene expression between Day 7 blastocysts generated in TCM199 with 5% FBS and Day 7 in vivo derived blastocysts and using suppression-subtractive hybridization (SSH). Pools of 25 embryos for both driver and tester were used in the RNA extraction process. The subtracted products were cloned and subjected to differential hybridization screening analysis. cDNAs were isolated, single-pass sequenced, and subjected to BLAST search. Of 32 in vivo ESTs (expressed sequence tags) that provided sequence information, 30 matched homologous sequences in GenBank. Of 32 in vitro ESTs, 22 provided specific matches while the remaining ten represented novel transcripts. Two in vivo ESTs, galectin-1 and fibronectin, and one in vitro EST, filamin A, were further characterized using real-time quantitative PCR. To further examine the reproducibility of the SSH data, three different pools of embryos with each pool containing ten embryos produced from each of the following production systems, namely, in vivo, IVP in TCM199 with 5% FBS and CR1aa with 5% FBS were used for real-time reverse transcription-polymerase chain reaction (RT-PCR) confirmation studies. Significant increases in the expression level of galectin-1 and fibronectin were observed in the in vivo derived blastocysts compared to blastocysts produced in TCM199 with 5% FBS and CR1aa cultures. No significant difference in filamin A expression was found between blastocysts produced in vivo and those derived from either of the in vitro production systems. We conclude that these techniques are useful to characterize the transcriptome of the early preattachment embryo and observed deviations in mRNA expression may partially explain the differences in quality between in vivo and IVP embryos.

The Human Endometrium Expresses the Glycoprotein Mucin-1 and Shows Positive Correlation for Thomsen-Friedenreich Epitope Expression and Galectin-1 Binding

“…Galectin-1 expression by the bovine blastocyst was described previously by Mohan et al (2004). Here we show that human oocytes and irregularly fertilized human oocytes also express galectin-1, TF, and, in addition, bind galectin-1 on their surfaces.…”

Endometrial expression of progesterone-induced blocking factor and galectins-1, -3, -9, and -3 binding protein in the luteal phase and early pregnancy in cattle

“…It also regulates homoeostasis (43) and inhibits the LGALS3BP cyclic 0.78 Ϯ 0.13 0.83 Ϯ 0. activity of CD4ϩ and CD25ϩ regulatory T cells (19). Expression of LGALS1 has been reported to be higher in in vivoderived day 7 bovine embryos compared with their less viable in vitro-derived counterparts and may contribute to lower pregnancy rates following embryo transfer as LGALS1 promotes maternal fetal tolerance (37). Extensive studies in mice showed control of Lgals1 to be under the influence of estrogen and progesterone (9).…”

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.…”