Effect of bone morphogenetic protein 2 (BMP2) on oestradiol and inhibin A production by sheep granulosa cells, and localization of BMP receptors in the ovary by immunohistochemistry.
The bone morphogenetic proteins (BMPs) have been implicated in the paracrine regulation of ovarian follicular development. In this study, we investigated the expression of the BMP receptors (BMPRs) in sheep ovaries by immunohistochemistry and the effect of BMP2, a natural ligand for these receptors, on granulosa cells cultured in vitro. Ovaries from cyclic ewes were fixed, embedded in paraffin wax and cut into sections. The sections were rehydrated, submitted to microwave antigen retrieval and treated with polyclonal antibodies against BMPR1A, BMPR1B and BMPR2. Strong immunostaining for all three receptors was observed in the granulosa cell layer of follicles from the primary to late antral stages of development. Staining was also present in the oocyte, corpus luteum, ovarian surface epithelium and, to a lesser extent, the theca layer of antral follicles. For functional studies, granulosa cells were obtained from immature follicles 1-3 mm in diameter. The cells were cultured for 6 days in serum-free medium containing 1 ng oFSH-20 ml(-1) in the presence of 0, 3, 10 or 30 ng ml(-1) human recombinant BMP2. The medium was replaced every 2 days and oestradiol and inhibin A concentrations were measured in the spent medium. In the absence of BMP2, oestradiol and inhibin A production increased as the granulosa cells differentiated in vitro. The addition of the highest dose of BMP2 enhanced oestradiol production (P < 0.05) without affecting the proliferation of the cells. It is concluded that BMP receptors are present in sheep ovaries and that BMPs may have a role in the differentiation of granulosa cells by enhancing the action of FSH.
Souza
C J H
CJ
Department of Reproductive and Developmental Sciences, Centre for Reproductive Biology, University of Edinburgh, 37 Chalmers Street, Edinburgh EH3 9ET, UK. carlos@obg93.obg.ed.ac.uk
Campbell
B K
BK
McNeilly
A S
AS
Baird
D T
DT
D000818:Animals; D055396:Bone Morphogenetic Protein 2; D052006:Bone Morphogenetic Protein Receptors, Type II; D019485:Bone Morphogenetic Proteins / Q000494:pharmacology*; D002454:Cell Differentiation; D002478:Cells, Cultured; D004958:Estradiol / Q000096:biosynthesis*; D005260:Female; D006107:Granulosa Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D007265:Inhibins / Q000096:biosynthesis*; D010053:Ovary / Q000737:chemistry; D017346:Protein Serine-Threonine Kinases / Q000032:analysis; D011994:Recombinant Proteins / Q000494:pharmacology; D012756:Sheep; D016212:Transforming Growth Factor beta
C525718:BMP2 protein, human; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; C422362:inhibin A; C499469:recombinant human bone morphogenetic protein-2; D004958:Estradiol; D007265:Inhibins; D017346:Protein Serine-Threonine Kinases; C498218:BMPR2 protein, human; D052006:Bone Morphogenetic Protein Receptors, Type II