141 EXPRESSION OF GENE PRODUCTS ENCODING CNN PROTEINS DURING THE FIRST WEEK OF DEVELOPMENT

Abstract

CCNs (cysteine-rich 61 (Cyr61), connective tissue growth factor (CTGF), and nephroblastoma overexpressed (Nov)) are a newly characterized gene family encoding 30-40 kDa proteins that are cysteine-rich and associated with the extracellular matrix. CCNs bind integrins and activate intracellular signaling, resulting in kinase activation and gene transcription. They are involved in events essential to development, including implantation and placentation. For example, CTGF is increased during decidualization and both CTGF and Cyr61 are produced by trophoblast giant cells. Furthermore, Cyr61-null mice display early embryonic lethality due to placental defects. Although CTGF protein has been reported in mouse 4.5-day embryo, no studies to date have focused on defining the expression of CCN mRNAs during pre-implantation development. The objective of this study was to characterize the expression of CCN mRNAs during the first week of in vivo mouse and in vitro cow development. CD-1 mice were superovulated by i.p. injection of pregnant mare serum gonadotrophin (10 U) and human chorionic gonadotrophin (10 U) 22 hours later. Following mating of the mice with CD-1 males, embryos were flushed from oviducts and uteri at 2-cell, 4-cell, 8-cell, morula, and blastocyst stages. Cow oocytes were obtained from slaughterhouse ovaries. Cow embryos were produced by aspiration of early antral follicles followed by in vitro oocyte maturation, fertilization, and embryo culture methods, and collected at 2-5-cell, 6-8-cell, morula, and blastocyst stages. Groups of five embryos of each stage were pooled for mRNA extraction using a magnetic bead protocol. Reverse transcription-polymerase chain reaction was used to detect CCN mRNAs at each time point, and product identity was verified by sequencing. CCN mRNAs are detectable during the first week of development in both species. In the mouse, Cyr61 mRNAs were present in all embryo stages investigated. CTGF mRNAs were variable at the 2-cell stage, but were consistently present from the 4-cell stage to the blastocyst stage. Interestingly, Nov appeared transiently at the 4- to 8-cell stage only. In the cow, CTGF and Cyr61 mRNAs were also detectable. Nov was undetectable in the cow. This is the first report of CCN mRNA expression in mouse or cow pre-implantation development. The early appearance and continued presence of Cyr61 and CTGF indicate a specific role in early development. The transient nature of Nov expression may indicate a specific role during compaction in the mouse. The expression of CTGF and Cyr61 in the cow indicates that these proteins are conserved between species and supports the hypothesis that they are important in this early phase of development.

This research was funded by NSERC, Canada.