Bone morphogenetic protein (BMP) ligands and receptors in bovine ovarian follicle cells: actions of BMP-4, -6 and -7 on granulosa cells and differential modulation of Smad-1 phosphorylation by follistatin

Abstract

Given the paucity of information on the potential roles of bone morphogenetic proteins (BMPs) in the ruminant ovary we conducted immunolocalization and functional studies on cells isolated from bovine antral follicles. Immunocytochemistry revealed expression of BMP-4 and -7 in isolated theca cells whereas granulosa cells and oocytes selectively expressed BMP-6. All three cell types expressed a range of BMP-responsive type-I (BMPRIB, ActRI) and type-II (BMPRII, ActRII, ActRIIB) receptors supporting autocrine/paracrine roles within the follicle. This was reinforced by functional experiments on granulosa cells which showed that BMP-4, -6 and -7 promoted cellular accumulation of phosphorylated Smad-1 but not Smad-2 and enhanced 'basal' and IGF-stimulated secretion of oestradiol (E2), inhibin-A, activin-A and follistatin (FS). Concomitantly, each BMP suppressed 'basal' and IGF-stimulated progesterone secretion, consistent with an action to prevent or delay atresia and/or luteinization. BMPs also increased viable cell number under 'basal' (BMP-4 and -7) and IGF-stimulated (BMP-4, -6 and -7) conditions. Since FS, a product of bovine granulosa cells, has been shown to bind several BMPs, we used the Biacore technique to compare its binding affinities for activin-A (prototype FS ligand) and BMP-4, -6 and -7. Compared with activin-A (K d 0.28 6 0.02 nM; 100%), the relative affinities of FS for BMP-4, -6 and -7 were 10, 5 and 1% respectively. Moreover, studies on granulosa cells showed that preincubation of ligand with excess FS abolished activin-A-induced phosphorylation of Smad-2 and BMP-4-induced phosphorylation of Smad-1. However, FS only partially reversed BMP-6-induced Smad-1 phosphorylation and had no inhibitory effect on BMP-7-induced Smad-1 phosphorylation. These findings support functional roles for BMP-4, -6 and -7 as paracrine/autocrine modulators of granulosa cell steroidogenesis, peptide secretion and proliferation in bovine antral follicles. The finding that FS can differentially modulate BMP-induced receptor activation and that this correlates with the relative binding affinity of FS for each BMP type implicates FS as a potential modulator of BMP action in the ovary.

Testing the antagonistic effect of follistatin on BMP family members in ovine granulosa cells

“…Particularly, follistatin did not affect BMP-2 and BMP-4 actions on GC, while noggin, a true BMP antagonist, clearly inhibited their actions. In contrast, BMP-6 action was slightly inhibited by follistatin, in accordance with recent results in bovine GC [23].…”

“…This suggests that oocyte-derived BMP could be such anti-luteinizing factors. Among the three oocyte-derived BMP identified yet, follistatin has been shown to bind and reduce the activity of BMP-15 [29] and BMP-6 ( [23], and the present study), reinforcing its role as a luteinizing factor of GC in large antral follicles. In contrast, due to its pattern of expression, follistatin is not expected to interfere with the action of these oocyte-derived BMP factors on the first stages of folliculogenesis.…”

“…This discrepancy might be due to the species difference of the recombinant follistatin used, which was the mouse in the present study and the human in the study of Glister et al [23]. Alternatively, the sensitivity of our biological test might not be sufficient to show an effect of follistatin, the affinity of which being 10-fold lower for BMP-4 than for activin A [23].…”

“…From our experiments, follistatin would not be a modulator of the action of either BMP-2 or BMP-4 on GC. In contrast, recent results of Glister et al [23] indicate that follistatin is able to bind BMP-4 and inhibits BMP-4 dependent Smad-1 phosphorylation in bovine GCs. This discrepancy might be due to the species difference of the recombinant follistatin used, which was the mouse in the present study and the human in the study of Glister et al [23].…”

Expression and localization of bone morphogenetic protein6 (BMP6) in the corpus luteum during different stages of estrous cycle in the buffalo (Bubalus bubalis)

“…The mid CL showed a significantly increased expression of BMP6 transcripts ( Figure. 1), which is consistent with the results of western blot ( Figure. 2A and B) and immunofluorescence ( Figure. 3).Though BMP6 mRNA was highly expressed in the theca cells and the oocytes of rat (Erickson & Shimasaki, 2003) and cow (Glister et al, 2004), it was only expressed in oocytes in the follicle of ewe (Juengel et al, 2006). In bovine GCs, BMP6 upregulated basal and IGF stimulated Estrogen (E2) production (Glister et al, 2004).…”