PARENT SESSION
GENE REGULATION AND FUNCTION IN THE REPRODUCTIVE AXIS
Kent
7:30 AM-10:00 AM

(357) SEQUENTIAL EXPRESSION OF SPECIFIC GENES DURING EARLY PREGNANCY OF SOMATIC-CELL CLONE-RECIPIENT COWS.

Kizaki, Keiichiro^1,4, Ishiwata, Hiroko¹, Hirasawa, Akira², Shiojima, Satoshi², Katsuma, Susumu², Ikawa, Hiroshi², Imai, Kei¹, Takahashi, Toru¹, Takahashi, Seiya³, Akagi, Satoshi³, Tsujimoto, Gozo², Hashizume, Kazuyoshi¹, ¹ Department of Developmental Biology, Tsukuba, JP⁴ School of Veterinary Medicine and Sciences, Towada, JP² Department of Pediatric Pharmacology, Tokyo, JP³ National Institute of Livestock and Grassland Sciences, Ibaraki, JP

ABSTRACT- A multitude of abnormalities ranging from placentomegaly to large offspring syndrome has been described in cloned animals. In addition, high rates of peripartum and neonate mortalities have been reported. Aberrations in epigenetic events such as, DNA methylation and demethylation including imprinting of genes have been postulated as a likely cause of the noted anomalies and death. Therefore in this study, gene expression was analyzed using cDNA microarray that was recently developed by this group. The array contained about 2000 identified genes from bovine uterus and placenta. Control pregnancies were derived by artificial insemination (AI) of Japanese black cows (day 0=day of insemination). Diagnosis of pregnancy was made from days 30-60 of gestation (day 0=day of estrus) by transrectal ultrasonography. The viability of fetus was confirmed by a detectable heartbeat 2-3 days prior to slaughter. In vitro fertilized embryos (IVF) were used as controls for microarray analysis on days 25 (n=2 (SNT), n=2 (IVF)). The IVF embryos were non-surgically transferred into synchronized cows on day 7 of estrous cycle (day 0=day of standing estrus). The entire fetal trophoblastic membrane from day 25 of gestation was used for microarray analysis. A number of genes were either overexpressed or underexpressed in cloned cows compared to that of normal pregnancy at day 25 of gestation. Underexpressed genes included various bovine pregnancy-associated glycoprotein, placental lactogen and prolactin-related protein genes. Collagen related genes like type 1 procollagen also showed underexpression. The underexpression profile was maintained in immotile fetus at day 60 of gestation. These results are comparable with histological and RT-PCR data suggesting that the extra-embryonic cell differentiation may be aberrant in cloned embryos. This research was supported by the Organized Research Combination System (ORCS) from the Science and Technology Agency and Technology Corporation, and the Bio-oriented Technology Research Advancement Institution (BRAIN), Japan.

KEY WORDS: cDNA Microarray, Cloned Animal, Bovine, Pregnancy