3 X-LINKED GENE EXPRESSION IN BOVINE PRE-IMPLANTATION EMBRYOS OBTAINED IN VIVO AND IN VITRO AS A MEASURE OF IMPACT OF EMBRYO PRODUCTION TECHNOLOGIES
Reproduction, Fertility and Development
The X chromosome provides an ideal system to study the impact of assisted reproduction technologies on gene expression because it holds a wide range of diversely functional genes and also the epigenetic features of X-inactivation are inherently susceptible to in vitro culture-mediated alterations. Using quantitative real-time RT-PCR we studied the expression of a panel of X-linked genes (ANT3, GAB3, RPS4, MECP2, XIAP, XIST) in pooled pre-attachment bovine embryos produced in vivo and in vitro.
... vivo and in vitro. We collected pools of ten embryos in vivo (de la Fuente R. et al. 1999 Biol. Reprod. 60, 769–775) at the morula (n = 3 pools) and blastocyst (n = 3 pools) stages. Pools of ten matured oocytes (n = 10 pools), 2–4 cell (n = 10 pools), 8–16 cell (n = 10 pools), morula (n = 7 pools) and blastocyst (n = 7 pools) stage embryos were produced in LSOF (Robert et al. Biol. Reprod. 67, 1465–1472) and TCM-199/bovine oviductal epithelial cells (BOEC) co-culture (de la Fuente et al. 1999 Biol. Reprod. 60, 769–775). Total RNA was extracted with the Absolutely RNA® Microprep kit (Stratagene, La Jolla, CA, USA) and reverse transcribed using Oligo-dT12–18 primers and Superscript II RT (Invitrogen, Burlington, Ontario, Canada). Specific primers were designed for each gene and PCR products were used to build standard curves for absolute quantification in a Light Cycler™ instrument with the Light Cycler FastStart DNA Master Mix SYBRGreenI kit (Roche Diagnostics, Laval, Quebec, Canada). The data were analyzed with SAS (SAS Institute Inc., Cary, NC, USA) using a factorial ANOVA design and a log transformation. Significance level was set at α = 0.05. There were no significant differences in transcript levels between IVF systems in mature oocytes, morulae, or blastocysts. Significant differences were observed for some of the genes tested at the 2–4-cell (XIAP) and at the 8–16-cell stages (ANT3, GAB3, XIAP) but not others. There were significant differences between IVF (both BOEC and LSOF) and in vivo embryos at the morula and blastocyst stages, with IVF embryos showing an average of 8 (ANT3), 10 (XIAP), 127 (MECP2), and 593 (XIST) times more transcripts than their in vivo counterparts. GAB3 was detected in only a few samples prior to the blastocyst stage where it was consistently detected in IVF but not in vivo embryos. It was concluded that there is an effect of the IVF system on gene transcription just before and during the period of activation of the embryonic genome during the 4th cell cycle, as well as marked differences between IVF and in vivo produced embryos that are evident in the differential expression of X-linked genes. This system provides a good tool to monitor the effects of embryo production conditions and help in their improvement. This project was supported with grants from NSERC, FSBC, and OMAF.