233 TEMPOROSPATIAL EXPRESSION PROFILES OF GROWTH AND DIFFERENTIATION FACTOR-9 AND BONE MORPHOGENETIC PROTEIN-15 GENES IN THE BOVINE OVARY

Abstract

Bone morphogenetic protein (BMP) family proteins regulate the folliculogenesis and ovulation rate in mammals. Of the members of BMP family, growth and differentiation factor-9 (GDF-9) and BMP-15 are oocyte-derived proteins that play critical roles in follicular development. In the present study, we characterized the temporospatial expression patterns of GDF-9 and BMP-15 in bovine ovaries. Bovine ovaries were obtained from local abattoir. Cumulus–oocyte complexes (COCs) and mural granulosa cells were collected by aspiration of follicles 2–5 mm in diameter. Follicular aspiration was done the day following slaughter. The COC and mural glanulosa cells were separated and cultured for 20 h according to the protocol for oocyte maturation. Total RNA was isolated from COCs, cumulus cells, denuded oocytes, and mural granulosa cells and used for PCR. For in situ hybridization, collected ovaries were immediately fixed with 4% formaldehyde-PBS and embedded in a paraffin block. In situ hybridization was carried out with digoxigenin-labeled probes. In COCs and cumulus cells, the transcripts encoding GDF-9 and BMP-15 were detected immediately after follicular aspiration and still remained at the end of maturation culture. However, in mural granulosa cells, only GDF-9 expression was detected. Germ cell/oocyte markers, ZAR1 and VASA, were not detected in cumulus and mural granulosa cells. Quantitative real-time RT-PCR revealed extensive expressions of both GDF-9 and BMP-15 in oocytes. In situ hybridization study showed that GDF-9 and BMP-15 were co-localized to oocytes in primary, secondary, and antral follicles. The ovular co-expression of GDF-9 and BMP-15 appeared to commence at fetal life. Expression of GDF-9 and BMP-15 was not clear in cumulus cells possibly because of low expression level. In conclusion, our results show that GDF-9 and BMP-15 are co-localized to oocytes from an early stage of folliculogenesis. Since the transcripts encoding GDF-9 and BMP-15 are detected in cumulus cells, it is suggested that the oocyte is not the sole source of them. Additionally, the cumulus and mural granulosa cells are suggested to be functionally differentiated relative to BMP-15 expression.