cDNA microarray analysis of bovine embryo gene expression profiles during the pre-implantation period.
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.
Ushizawa
Koichi
K
Reproductive Biology and Technology Laboratory, Developmental Biology Department, National Institute of Agrobiological Sciences, 2 Ikenodai, Tsukuba, Ibaraki 305-8602, Japan. ushizawa@affrc.go.jp <ushizawa@affrc.go.jp>
Herath
Chandana B
CB
Kaneyama
Kanako
K
Shiojima
Satoshi
S
Hirasawa
Akira
A
Takahashi
Toru
T
Imai
Kei
K
Ochiai
Kazuhiko
K
Tokunaga
Tomoyuki
T
Tsunoda
Yukio
Y
Tsujimoto
Gozoh
G
Hashizume
Kazuyoshi
K
D000818:Animals; D002417:Cattle; D016000:Cluster Analysis; D018076:DNA, Complementary / Q000235:genetics*; D004622:Embryo, Mammalian / Q000737:chemistry* / Q000378:metabolism*; D047108:Embryonic Development / Q000235:genetics*; D005260:Female; D005333:Fetus / Q000737:chemistry / Q000378:metabolism; D020869:Gene Expression Profiling / Q000379:methods* / Q000706:statistics & numerical data / Q000662:veterinary; D018507:Gene Expression Regulation, Developmental / Q000235:genetics*; D005796:Genes / Q000235:genetics; D005865:Gestational Age; D008566:Membranes / Q000737:chemistry / Q000378:metabolism; D046228:Microarray Analysis / Q000379:methods* / Q000706:statistics & numerical data / Q000662:veterinary; D021141:Nucleic Acid Amplification Techniques / Q000379:methods / Q000662:veterinary
D018076:DNA, Complementary