Research

cDNA microarray analysis of bovine embryo gene expression profiles during the pre-implantation period

Koichi Ushizawa¹ , Chandana B Herath¹ , Kanako Kaneyama¹ , Satoshi Shiojima² , Akira Hirasawa² , Toru Takahashi¹ , Kei Imai^1,6 , Kazuhiko Ochiai⁵ , Tomoyuki Tokunaga³ , Yukio Tsunoda⁴ , Gozoh Tsujimoto² and Kazuyoshi Hashizume^1,5

¹Reproductive Biology and Technology Laboratory, Developmental Biology Department, National Institute of Agrobiological Sciences, 2 Ikenodai, Tsukuba, Ibaraki 305-8602, Japan

²Department of Genomic Drug Discovery Science, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida Shimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan

³Development and Differentiation Laboratory, Developmental Biology Department, National Institute of Agrobiological Sciences, 2 Ikenodai, Tsukuba, Ibaraki 305-8602, Japan

⁴Laboratory of Animal Reproduction, College of Agriculture, Kinki University, 3327-204 Nakamachi, Nara 631-8505, Japan

⁵Department of Veterinary Medicine, Faculty of Agriculture, Iwate University, 3-18-8 Ueda, Morioka, Iwate 020-8550, Japan

⁶Department of Technology, National Livestock Breeding Center, 1 Odakurahara, Odakura, Nishigo, Fukushima 961-8511, Japan

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Reproductive Biology and Endocrinology 2004, 2:77doi:10.1186/1477-7827-2-77

Published:	24 November 2004

Abstract

Background

After fertilization, embryo development involves differentiation, as well as development of the fetal body and extra-embryonic tissues until the moment of implantation. During this period various cellular and molecular changes take place with a genetic origin, e.g. the elongation of embryonic tissues, cell-cell contact between the mother and the embryo and placentation. To identify genetic profiles and search for new candidate molecules involved during this period, embryonic gene expression was analyzed with a custom designed utero-placental complementary DNA (cDNA) microarray.

Methods

Bovine embryos on days 7, 14 and 21, extra-embryonic membranes on day 28 and fetuses on days 28 were collected to represent early embryo, elongating embryo, pre-implantation embryo, post-implantation extra-embryonic membrane and fetus, respectively. Gene expression at these different time points was analyzed using our cDNA microarray. Two clustering algorithms such as k-means and hierarchical clustering methods identified the expression patterns of differentially expressed genes across pre-implantation period. Novel candidate genes were confirmed by real-time RT-PCR.

Results

In total, 1,773 individual genes were analyzed by complete k-means clustering. Comparison of day 7 and day 14 revealed most genes increased during this period, and a small number of genes exhibiting altered expression decreased as gestation progressed. Clustering analysis demonstrated that trophoblast-cell-specific molecules such as placental lactogens (PLs), prolactin-related proteins (PRPs), interferon-tau, and adhesion molecules apparently all play pivotal roles in the preparation needed for implantation, since their expression was remarkably enhanced during the pre-implantation period. The hierarchical clustering analysis and RT-PCR data revealed new functional roles for certain known genes (dickkopf-1, NPM, etc) as well as novel candidate genes (AW464053, AW465434, AW462349, AW485575) related to already established trophoblast-specific genes such as PLs and PRPs.

Conclusions

A large number of genes in extra-embryonic membrane increased up to implantation and these profiles provide information fundamental to an understanding of extra-embryonic membrane differentiation and development. Genes in significant expression suggest novel molecules in trophoblast differentiation.