194 EXPRESSION OF STANNIOCALCIN FAMILY GENES DURING PREIMPLANTATION STAGE BOVINE EMBRYO DEVELOPMENT

Abstract

Stanniocalcins (STC) are a small family of secreted homodimeric glycoprotein hormones consisting of STC1 and STC2. A previous study in Drosophila (Tolias and Stroumbakis 1998 Dev. Genes Evol. 208, 274–282) indicated that maternally derived STC is required during embryogenesis. However, little information is available for mammalian embryos. The aim of this study was to examine the expression of STC and assess their roles during the preimplantation stage of bovine embryo development. Immature cumulus–oocyte complexes were aspirated from follicles of bovine ovaries collected at a local abattoir and matured in vitro for 24 h at 39°C under an atmosphere of 5% CO₂ in air with maximum humidity in TCM-199 supplemented with 10% (vol/vol) fetal calf serum and 10 ng mL^–1 of epidermal growth factor. Matured cumulus–oocyte complexes were inseminated with fertile bull semen (Day 0). Embryos were cultured in vitro, and subsequently, 4 pools of 10 embryos each at the zygote, 2-cell, 4-cell, 8-cell, 16-cell, morula, and blastocyst stages were collected from 4 different replicate cultures and stored at –80°C until analysis. Total RNA was isolated using an RNeasy Micro Kit and a random primer was used during cDNA synthesis. The expression of STC1, STC2, and reference genes (YWHAZ, PPIA, SDHA) was examined. Quantitative real-time PCR was used to compare transcript abundance, and data were normalized to the geometric averages of the reference genes. The expression levels were analysed using the relative standard curve method, and means were compared using Student’s t-test. Despite being members of the same family and having large sequence similarity, the expression of each gene was unique and stage dependent during embryo development. Expression of STC1 was detected in all the stages examined. Expression was transiently reduced at the 2-cell stage, with no significant change until the 8-cell stage but with a slight increase at the 16-cell stage. In contrast, STC2 was barely detectable before the 8-cell stage. Expression at the 8- and 16-cell stages was significantly (P < 0.0001) higher compared with all other stages, with a peak at the 16-cell stage. This significantly higher expression pattern of STC2 during the critical stages of maternal to zygotic control of development may suggest an important role during this critical period of embryo development.