PARENT SESSION
BIOLOGY OF PREGNANCY AND IMMUNE FUNCTION
Harborside C
7:30 AM-10:00 AM

(329) EXPRESSION OF DNA METHYLTRANSFERASE IN SOMATIC-CELL CLONED BOVINE PREGNANCIES.

Imai, Kei¹, Ishiwata, Hiroko¹, Akagi, Satoshi², Takahashi, Seiya², Yamada, Osamu¹, Patel, Osman¹, Todoroki, Junichi³, Kizaki, Keiichiro^1,4, Izaike, Yoshiaki¹, Hashizume, Kazuyoshi¹, ¹ Laboratory of Reproductive Biology and Tecnology, Ibaraki, Japan² National Institute of Livestock and Grassland Sciences, Ibaraki, Japan³ Kagoshima Prefectural Cattle Breeding and Development Institute, Kagoshima, Japan⁴ University of Kitazato, Aomori, Japan

ABSTRACT- Cloning has been accomplished in bovine by somatic cell nuclear transfer (SNT). However, the net yield of cloned calves remains quite low due to a high rate of embryonic/fetal mortalities. A possible cause of the low success rate of cloning is the incomplete reprogramming following SNT. DNA methylation in mammals is thought to be essential in the regulation of transcription in embryonic development, cell differentiation and imprinting. The objective of the present study was to determine the relative expression of DNA methyltransferase (Dnmt1) in SNT and AI cows. The concepti and placenti were recovered on days 14, 30 and 60 of gestation from SNT and AI cows. Total RNA was extracted from embryos (Days 14), cotyledonary, intercotyledonary, caruncular and intercaruncular tissues using ISOGEN (Nippon Gene, Toyama, Japan) and then total RNA was reverse transcribed with Super Script II (Life Technologies Inc., Rockville, MD). The PCR primers were designed on the catalytic domain region of human Dnmt1 using the OLIGO program and GAPDH was used as an internal standard. The intensity of each band was assessed by densitometry (ATTO Co., Tokyo, Japan). The Dnmt1 was detected in all AI embryos by day 14, however, it has not expressed in some of the SNT embryos at that stage. By days 30 and 60 of gestation there was no significant difference in Dnmt 1 expression in the cotyledonary and caruncular tissue of the two groups. Dnmt 1 was expressed in the intercotyledonary and intercaruncular tissues of all AI cows at days 30 of gestation; however, it was not detected in some of the SNT cows at parallel stage. No significant difference was observed in the intercotyledonary and intercaruncular tissues of the two groups at Day 60 of gestation. These data indicates that abnormal Dnmt1 expression could be linked to early embryonic death in the SNT embryos. This study was supported by Grants-in-aid from ORCS, BRAIN and MAFF, Japan.

KEY WORDS: cloned animal, DNA methyltransferase, pregnancy, bovine