title: Enhanced response of granulosa and theca cells from sheep carriers of the FecB mutation in vitro to gonadotropins and bone morphogenic protein-2, -4, and -6.
abstract: The FecB (Booroola) mutation, which leads to increased ovulation rates and multiple births in sheep, is now known to occur in the signaling domain of the bone morphogenic protein (BMP)-1B receptor. We examined the effect of the mutation on the responsiveness of granulosa (GC) and theca cells (TC) to BMPs and other local regulators using tissue from animals with (Fec(B/B)) and without (Fec(+/+)) the FecB mutation. Experiments examined the effect of BMP-2, -4, and -6 (0.005-50 ng/ml), and their interaction with IGF-I (0.1-10 ng/ml LR3 analog) and gonadotropins, on the proliferation and differentiation of GCs and TCs isolated from small (<2 mm) antral follicles and maintained in serum-free culture for up to 8 d. Dose-finding studies using ovaries from wild-type sheep obtained from the abbattoir showed no difference among the different BMPs in stimulating (P < 0.001) estradiol (E2) production by GCs cultured with FSH (10 ng/ml), but there was a clear interaction (P < 0.001) with IGF-I. BMPs had no effect on GC proliferation or the sensitivity of GCs to FSH. In contrast, higher doses of BMPs (5-50 ng/ml) inhibited LH-stimulated androstenedione production by TCs, whereas lower doses (0.005-0.05 ng/ml) stimulated TC proliferation (P < 0.01). Regardless of dose of IGF-I, at the end of culture (96-192 h) hormone production by GCs (E2, inhibin A) and TCs (androstenedione) was 4- to 5-fold greater (P < 0.001) by cells from Fec(B/B), compared with Fec(+/+) ewes exposed to the same dose of gonadotropin. In the presence of low concentrations of IGF-I (0.1 ng/ml), the maximum increase in the production of E2 and inhibin A by GCs from FF ewes in response to BMPs was observed at doses that were 3- to 10-fold lower (3-10 ng/ml) than ++ (30 ng/ml; P < 0.001). Low doses of BMPs stimulated proliferation of TCs from ++ (P < 0.01) but not FF ewes. Immunohistochemistry confirmed BMP-6 protein expression in the oocyte, granulosa, and thecal layers of antral follicles from both genotypes. These results confirm a major role for BMPs in controlling ovarian somatic cell function in sheep and provide evidence to support the hypothesis that the FecB mutation increases the BMP response of somatic cells when stimulated to differentiate by gonadotropins.
authors.affiliation: Department of Obstetrics and Gynaecology, Queen's Medical Centre, University of Nottingham, UK. bruce.campbell@nottingham.ac.uk
authors.forename: B K
authors.initials: BK
authors.lastname: Campbell
authors.forename: C J H
authors.initials: CJ
authors.lastname: Souza
authors.forename: A J
authors.initials: AJ
authors.lastname: Skinner
authors.forename: R
authors.initials: R
authors.lastname: Webb
authors.forename: D T
authors.initials: DT
authors.lastname: Baird
mesh_terms: D000818:Animals; D055396:Bone Morphogenetic Protein 2; D055415:Bone Morphogenetic Protein 4; D055418:Bone Morphogenetic Protein 6; D052005:Bone Morphogenetic Protein Receptors, Type I / Q000235:genetics*; D019485:Bone Morphogenetic Proteins / Q000032:analysis / Q000494:pharmacology*; D005260:Female; D005640:Follicle Stimulating Hormone / Q000494:pharmacology*; D006107:Granulosa Cells / Q000187:drug effects*; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D007986:Luteinizing Hormone / Q000494:pharmacology*; D009154:Mutation; D010053:Ovary / Q000737:chemistry; D012756:Sheep / Q000235:genetics*; D013799:Theca Cells / Q000187:drug effects*; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
chemical_list: C525718:BMP2 protein, human; C525809:BMP4 protein, human; C525816:BMP6 protein, human; D055396:Bone Morphogenetic Protein 2; D055415:Bone Morphogenetic Protein 4; D055418:Bone Morphogenetic Protein 6; D019485:Bone Morphogenetic Proteins; D016212:Transforming Growth Factor beta; D007334:Insulin-Like Growth Factor I; D007986:Luteinizing Hormone; D005640:Follicle Stimulating Hormone; D052005:Bone Morphogenetic Protein Receptors, Type I