Elongation and gene expression in bovine cloned embryos transferred to temporary recipients

Abstract

SummaryElongated embryos provide a unique source of information about trophoblastic differentiation, gene expression and maternal-embryonic interactions; however they are difficult and costly to obtain, especially elongated cloned embryos. One alternative is their production in heterologous temporary recipients such as sheep and goats. We aimed to produce elongated bovine cloned embryos using heterologous transfer to temporary recipients. Day-7 cloned cattle blastocysts were transferred to the uteri of ewes and goats and recovered as elongated structures at day 17. We evaluated elongation, length, presence of embryonic disc and expression of several important genes for embryonic development. We also produced homologous (cloned cattle embryos transferred into cattle uteri). Cloned bovine blastocysts were able to proceed with preimplantation development through elongation with high efficiency despite the species to which they were transferred. In qualitative and quantitative RT-PCR experiments we found differences in the pattern of gene expression among embryos recovered from different species. Sox2, Nanog and FGF-4 were markedly deregulated. No previous reports about the expression pattern of the studied genes had been published for elongated bovine cloned embryos produced in intermediate recipients, furthermore, the pattern of expression of Nanog, Oct4, Eomes, Cdx2, IFN-tau, Dicer, FGF-4 and Sox2 shown here are novel for elongated cloned bovine embryos created by hand-made cloning. Our data confirmed that sheep and goats can be used as temporary recipients. This model could serve as a basis for further research on gene expression and cellular changes during bovine peri-implantation development.

Changes in the expression of pluripotency-associated genes during preimplantation and peri-implantation stages in bovine cloned and in vitro produced embryos

“…We found OCT4 gene and protein expression in the adult cell line used for nucleus transfer (Rodríguez-Alvarez et al ., 2009 and this paper). Expression of OCT4 has been reported in adult stem cells and in differentiated cells (Pochampally et al ., 2004; Tai et al ., 2005; Tondreau et al ., 2005; Zangrossi et al ., 2007; Misuno and Kosaka, 2008; Rodríguez-Alvarez et al ., 2009).…”

“…We found OCT4 gene and protein expression in the adult cell line used for nucleus transfer (Rodríguez-Alvarez et al ., 2009 and this paper). Expression of OCT4 has been reported in adult stem cells and in differentiated cells (Pochampally et al ., 2004; Tai et al ., 2005; Tondreau et al ., 2005; Zangrossi et al ., 2007; Misuno and Kosaka, 2008; Rodríguez-Alvarez et al ., 2009). However, controversy has arisen about this issue, due to the existence of alternative splice variants of OCT4 , (Takeda et al ., 1992) and of several pseudogenes in the human and mouse genome (Liedtke et al ., 2007), whose expression must be ruled out if true OCT4 expression is investigated.…”

“…The expression level of specific genes changes from blastogenesis to implantation Ushizawa et al ., (2004) reported in bovine embryos more than 500 genes are up-regulated between day 7 versus day 14, whereas only 26 genes were down-regulated in the same time period. Likewise, differential gene expression was shown in bovine embryos during elongation (Degrelle et al ., 2005; Rodríguez-Alvarez et al ., 2009). Recent research using somatic cell nucleus transfer (SCNT) technology suggested that aberrant expression of genes during the earliest stages of development could be the cause of embryonic losses (Arnold et al ., 2008).…”

“…This scarcity is probably related to difficulties and costs associated with the production of elongated embryos in vitro or via SCNT. Recently we and others have developed systems for elongation of ruminant cloned embryos using heterologous recipients as intermediate hosts (Rodríguez-Alvarez et al ., 2009; Rodríguez-Alvarez et al ., submitted). This approach can be an attractive model to study changes in gene expression during elongation, however the contribution of a heterologous uterine environment to correct embryonic gene expression requires still further research.…”

“…Incomplete reprogramming of the donor cell could be the cause of aberrant patterns of gene expression first manifested in bovine blastocyst, but lasting throughout embryonic development (Latham & Sapienza, 2004;Piedrahita et al, 2004;Arnold et al, 2008). Aberrant gene reprogramming after nucleus transfer has been documented also for miRNA (Castro et al, 2009). This finding implies that de-regulated gene expression is a common phenomenon in cattle cloning.…”