Analysis of Gene Expression in Mouse 2-Cell Embryos Using Fluorescein Differential Display: Comparison of Culture Environments1

Abstract

The effect of the oviductal environment on gene expression in 2-cell mouse embryos was examined with mRNA differential display. Embryos used for experiments were cultured in modified Whitten medium with or without oviductal tissue until late 2-cell stage. The results of sequencing indicated that the genes for ATP synthase (ATPase 6), S:-adenosylmethionine decarboxylase (S:-AMDC) and nuclear autoantigenic sperm protein (NASP) were differentially expressed in embryos cultured in the oviductal environment (nonblocking culture condition). The ATPase 6 gene is encoded by mitochondrial DNA and is essential for the production of ATP. This indicates that the expression of ATP synthesis-related genes at the 2-cell stage may be required to maintain normal development in vitro. S:-Adenosylmethionine decarboxylase decarboxylates adenosylmethionine, which is a substrate of DNA methylation. The expression of S:-AMDC may be responsible for the low level of methylation of preimplantation development. As NASP is a histone-binding protein that is thought to be testis and sperm specific, its function in embryos remains unclear. On the other hand, the Tcl1 gene and a novel gene, the c-1 gene, were strongly expressed in embryos cultured without oviductal tissue (blocking culture condition). The expression patterns of these genes are quite similar. However, the detailed functions of these genes in embryos remain to be determined.

TCL1 participates in early embryonic development and is overexpressed in human seminomas

“…Our observation that Tcl1 expression is higher under in vitro versus in vivo conditions is concordant with the findings of Minami et al (22) who have described enhanced Tcl1 gene expression in embryos cultured without oviductal tissues. This higher expression of Tcl1 might account also for the more dramatic in vitro phenotype observed for Tcl1 Ϫ/Ϫ embryos compared with the in vivo phenotype, wherein the absence of Tcl1 in females reduces their litter size and significantly shortens their reproductive life.…”

“…Semiquantitative reverse transcription-PCR and immunofluorescence analysis indicated that Tcl1 mRNA and protein are present in unfertilized eggs, 1-cell embryos and 2-cell embryos, and the Tcl1 levels increase significantly under in vitro culture conditions (Fig. 3A) (22). At the late preimplantation stages, however, the embryonic Tcl1 content progressively declines irrespectively of the culture conditions.…”

“…Tcl1 may thus act as a structural amplification loop in the phosphatidylinositol 3-kinase (PI3-kinase) Akt pathway (25). Although the mechanism of Tcl1 action in early embryo development is not yet fully elucidated, the Tcl1 cofactor role in the PI3-kinase Akt pathway may be essential under conditions where growth factors are minimal, as for the in vitro conditions used in this study or in the absence of oviduct factors (22).…”

Molecular and subcellular characterisation of oocytes screened for their developmental competence based on glucose-6-phosphate dehydrogenase activity

“…NASP is an H1 histone-binding protein that is cell cycle regulated and occurs in two major forms: tNASP, found in gametes, embryonic cells and transformed cells; and sNASP, found in all rapidly dividing somatic cells (Richardson et al 2000). Moreover, it was strongly expressed in mouse embryos developed under non-blocking culture conditions in which embryos do not exhibit developmental arrest at the two-cell stage; however, the function of this transcript in early embryonic development remains unknown (Minami et al 2001). NASP was one of the genes with increased expression in very fast moving bovine oocytes, which showed higher blastocyst rate compared with the slow groups after dielectrophoretic separation (Dessie et al 2007).…”

Evaluation of gene expression in pigs selected for enhanced reproduction using differential display PCR: II. Anterior pituitary1

“…The ddPCR method has been used for transcriptional analysis to understand reproductive processes in mouse (Lee et al, 2001;Minami et al, 2001), cow (Robert et al, 2001), human (Xu et al, 1999), Rhesus monkey (Ace and Okulicz, 1999), and pig (Li et al, 1996). Gene expression analysis is also useful for understanding the biological basis that underlies polygenic traits and the response to long-term genetic selection.…”