Signaling and Signal Transduction in Endocrine Tissues
(M739) EXPRESSION OF TGF-, TGF- RECEPTORS AND Smads IN LUTEINIZING PORCINE FOLLICLES: A ROLE FOR TGF- IN LUTEINIZATION?
Sriperumbudur, Rajagopal1, Gadsby, John1, 1 North Carolina State University, Raleigh, NC
ABSTRACT- It is well known that the LH-surge causes ovulation of follicles and initiates luteinization. There is also evidence from our own studies as well from the literature suggesting that TGF- may also play a role in luteinization. In this study we proposed to examine the expression of TGF- ligands (TGF-1, 2 and 3) and components of its signaling pathway (the TGF- receptors, TBR1 and 2, Smads2, 3, 4, 6 and 7, and the Smad anchor protein, SARA) in porcine follicles induced to luteinize in vivo with hCG treatment and in porcine granulosa cells induced to luteinize in vitro with LH / IGF-I or with TGF-1. For the in vivo experiment, pre-pubertal pigs were injected with PG-600 (to induce follicles) and hCG 3 days later. Ovaries were collected at 0, 1, 24 and 48 h following hCG treatment, and used for RNA extraction. For the in vitro experiment, granulosa cells were obtained from porcine follicles and were placed into culture in M199 + 10% fetal bovine serum on collagen I coated culture dishes. Granulosa cell cultures were treated with either LH (250 ng/ml) / IGF-I (50 ng/ml), or with varying doses of TGF-1 (0.1, 1, 10 and 100 ng/ml). mRNA expression of TGF- signaling components was measured in in vivo and in vitro samples by semi-quantitative RT-PCR using porcine specific primers, and histone 2A to normalize PCR data. In the follicles collected in vivo, hCG up-regulated (compared with 0h) TGF-2 at 48h, TBR1 at 24h and TBR2 at 24 and 48h, Smad3 and Smad4 at 48 h, but down-regulated these genes at other times. Smads 6, 7 and SARA were downregulated by hCG at all times. In the in vitro samples, LH/IGF-I up-regulated most components of the signaling pathway, suggesting that TGF- may mediate (at least in part) the luteinizing action of LH/IGF-I. Furthermore, we showed that there was an increase in the expression of all TGF- signaling components in response to each dose of TGF-1 (max. at 1 ng/ml). Since hCG, LH/IGF and TGF- up-regulate components of the TGF- signaling pathway, and can induce luteinization, we tentatively conclude that TGF- signaling may play a role in mediating LH induced luteinzation in vivo and in vitro. Studies are in progress to explore further the role of TGF- in luteinization. Supported by the State of NC and USDA Grant 2004-35203-14777.
KEY WORDS: luteinization, tgf-, Smads, LH hCG