220 AN EXPRESSION PROFILE OF GENES CRUCIAL FOR PLACENTAL DEVELOPMENT IN SINGLE IN VIVO, IN VITRO AND CLONED BOVINE BLASTOCYSTS

Abstract

Abnormalities of the placenta are a major factor contributing to early death in cloned bovine conceptuses. This is primarily due to incomplete chromatin remodeling and reprogramming of the donor nucleus. It is unknown whether genetic aberrations of genes crucial for placental development can be detected in pre-implantation cloned bovine embryos. This study looked at the expression profile of four genes in single bovine blastocysts derived from in vivo, in vitro produced (IVP), or cloning techniques, including handmade cloning (HMC) and serial HMC (SHMC). The genes studied included acrogranin, caudal type homeobox 2 (cdx2), estrogen-receptor-related receptor beta (essrb), and the mammalian relative of DnaJ (MRJ). These genes play a role in trophoblast regulation and placental development. Messenger RNA expression was analyzed by using PCR following cDNA amplification by means of SMART cDNA synthesis (Clontech, Palo alto, CA, USA). Primers were designed from homologous human and mouse sequence. PCR products were sequenced for verification. Five single blastocysts were analyzed from each of the following treatments: in vivo, IVP, HMC, and SHMC. Pooled (n = 10) IVP blastocyst cDNA produced by standard RT was used as a positive control. Grade 1 Day 7 blastocysts were selected for all treatments. Amplified cDNA was tested using control genes polyA, IFN-τ and GDF9. In vitro-produced embryos were matured, fertilized and cultured as published by Ruddock et al. (2004 Biol. Reprod. 70, 1131). Cloned HMC embryos were produced as described by Tecirlioglu et al. (2003 Reprod. Fertil. Dev. 15, 361). Serial HMC embryos were produced as per the HMC embryos, followed by a second round of nuclear transfer at the pronuclear stage. The pooled IVP, in vivo, and IVP blastocysts expressed all four genes of interest. In the HMC-cloned embryos, all four genes were expressed. However, in the SHMC cloned embryos, although MRJ was found to be expressed in all blastocysts, three of the five blastocysts did not express acrogranin. Similarly, two SHMC embryos did not express cdx2, and essrb was weakly expressed in three of the five embryos analyzed. Initial pregnancy rates of HMC and SHMC embryo transfers are similar. Further pregnancy results are pending. These results indicate that aberrations of genes crucial for placental development can be detected in single cloned blastocysts. It also suggests that failed implantation and/or placental defects may stem from patterned genetic defects in the pre-implantation embryo. An increase in the number of embryos analyzed would further strengthen results. These genes could act as markers to identify cloning techniques that produce more embryos with normal genetic profiles. The benefits of developing a screening tool to assess abnormalities in single pre-implantation embryos would be significant.