SPATIAL AND TEMPORAL REGULATION OF BMP-2, -6, -15 AND GDF-9 mRNA EXPRESSION IN DEVELOPING PORCINE OVARIAN FOLLICLES

Abstract

Members of the transforming growth factor beta (TGF-beta) superfamily, namely the bone morphogenetic proteins (BMPs) and growth differentiation factor 9 (GDF-9), have been shown to play important roles during follicular development and ovulation. However, despite the wealth of information available in rodent and ruminant species, very little is known about the role of these genes in the porcine ovary. Therefore, the aims of the present study were to investigate the expression of BMP-2, -6, -15 and GDF-9 mRNA in the oocyte, granulosa cells and theca cells of porcine follicles and to determine if changes occur in the levels of expression of these genes during follicular growth. Oocyte, granulosa cells and theca cells were recovered from antral follicles (2-10 mm in diameter) of first parity sows at day 1, 2 and 4 after weaning or at day 14, 16 and 20 of their subsequent estrous cycle. These time points correspond to specific phases of follicular growth; the recruitment of a growing cohort of follicles, the mid-selection phase and the final selection of the pre-ovulatory follicle population. Plasma estrogen and progesterone analysis was used to confirm the expected physiological status of each animal. Follicular fluid estradiol concentration in day 4 and day 20 animals was also used to determine whether the follicles had been exposed to the LH surge. Total RNA was extracted, reverse transcribed and the levels of expression for these genes were determined using real-time PCR. The delta Ct values were calculated using cyclophilin as the internal control and the data were analysed using the general linear model procedure of SAS (SAS Institute Inc., Cary, NC). It is particularly interesting to notice that both BMP-2 and BMP-15 were expressed in all three cell types but were expressed at considerably higher levels in the oocyte (p < 0.001). BMP-2 mRNA levels increased in granulosa cells (P = 0.018) and theca cells (P < 0.001) from the recruitment phase to the mid-selection phase, remained constant until the final selection phase and decreased after the LH surge. BMP-15 mRNA levels also increased in both cell types from the recruitment phase to the mid-selection phase, however the levels decreased in the granulosa cells of the final pre-ovulatory follicle population while remaining constant during this phase in the theca cells (P < 0.05). As opposed to BMP-2, the level of BMP-15 mRNA did not change in response to the LH surge. Finally, BMP-15 mRNA levels showed a strong tendency to decrease in the oocytes from recruitment to the final selection phase (P = 0.054). These results confirm that the oocyte is the main source of BMP-2 and BMP-15 but contrary to other studies, their expression was also found in granulosa cells and theca cells. These differences suggest that follicular growth and development may be regulated differently in swine. (poster)