FSH and TGF-beta superfamily members regulate granulosa cell connective tissue growth factor gene expression in vitro and in vivo.
Connective tissue growth factor (CTGF) is a heparin-binding growth factor implicated in diverse epithelial cell types as a paracrine regulator of mitosis, angiogenesis, cellular taxis, and remodeling of the extracellular matrix. To understand the possible roles of CTGF in the ovarian paracrine system, we studied CTGF gene expression by granulosa cells in relation to their stage of cellular differentiation using both in vitro and in vivo methodologies. Untreated monolayer granulosa cell cultures from immature rats abundantly expressed the approximately 2.5-kb CTGF mRNA transcript (determined by Northern analysis), but had low levels of aromatase activity (an index of granulosa cell differentiation). Treatment for 48 h with FSH (0.1-10 ng/ml) dose-dependently inhibited (>or=50%) CTGF mRNA expression, but enhanced aromatase enzyme activity. This in vitro observation of CTGF mRNA down-regulation coinciding with FSH-induced granulosa cell maturation is substantiated by studies of in vivo ovarian CTGF expression in FSHbeta knockout mice. Northern blot and in situ hybridization analyses demonstrate high levels of CTGF expression in the granulosa cells of preantral follicles blocked from further development by the absence of FSH. The action of FSH (10 ng/ml) was mimicked in vitro by 8-bromo-cAMP (1.0 mM) and was augmented by the additional presence of androgen (1 micro M 5alpha-dihydrotestosterone), consistent with mediation by intracellular cAMP. Conversely, treatment of granulosa cell cultures with TGFbeta1 (0.1-10 ng/ml) dose-dependently increased CTGF mRNA levels up to 12-fold at a dose of 10 ng/ml, without affecting aromatase activity. Cotreatment with FSH (0.1-10 ng/ml) dose-dependently suppressed the stimulatory action of TGFbeta1 (10 ng/ml) on CTGF mRNA, but substantially enhanced aromatase activity beyond levels induced by FSH alone. Importantly, other TGFbeta superfamily members known to be produced in the ovary (growth/differentiation factor-9 and activin A; 10 ng/ml) stimulated granulosa cell CTGF mRNA in a similar fashion as TGFbeta1 (10 ng/ml), and this was also inhibited by FSH (10 ng/ml). These data show that granulosa cell CTGF gene expression is inversely related to the stage of granulosa cell differentiation, being directly inhibited by FSH via cAMP-mediated signaling. CTGF mRNA abundance in nondifferentiated granulosa cells is up-regulated in vitro by TGFbeta1, growth/differentiation factor-9, and activin, suggesting paracrine roles for these growth/differentiation factors in the regulation of CTGF synthesis in mammalian ovaries.
Department of Reproductive and Developmental Sciences, University of Edinburgh, Center for Reproductive Biology, Edinburgh, United Kingdom EH3 9ET. c.harlow@ed.ac.uk
Christopher R
CR
Harlow
Lindsay
L
Davidson
Kathleen H
KH
Burns
Changning
C
Yan
Martin M
MM
Matzuk
Stephen G
SG
Hillier
D028341:Activins / Q000494:pharmacology; D000818:Animals; D015152:Blotting, Northern; D055430:Bone Morphogenetic Protein 15; D002454:Cell Differentiation; D002478:Cells, Cultured; D055513:Connective Tissue Growth Factor; D000242:Cyclic AMP / Q000502:physiology; D005260:Female; D005640:Follicle Stimulating Hormone / Q000172:deficiency / Q000494:pharmacology*; D037201:Follicle Stimulating Hormone, beta Subunit; D005786:Gene Expression Regulation / Q000187:drug effects*; D006107:Granulosa Cells / Q000378:metabolism*; D055429:Growth Differentiation Factor 9; D006133:Growth Substances / Q000235:genetics* / Q000494:pharmacology; D017874:Immediate-Early Proteins / Q000235:genetics*; D017403:In Situ Hybridization; D028322:Inhibin-beta Subunits / Q000494:pharmacology; D036341:Intercellular Signaling Peptides and Proteins; D051379:Mice; D018345:Mice, Knockout; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017208:Rats, Wistar; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D053773:Transforming Growth Factor beta1
C525902:Bmp15 protein, mouse; C525904:Bmp15 protein, rat; D055430:Bone Morphogenetic Protein 15; C526318:CCN2 protein, mouse; C526316:CCN2 protein, rat; D037201:Follicle Stimulating Hormone, beta Subunit; C525898:Gdf9 protein, mouse; C525899:Gdf9 protein, rat; D055429:Growth Differentiation Factor 9; D006133:Growth Substances; D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; D012333:RNA, Messenger; C508961:Tgfb1 protein, mouse; C508962:Tgfb1 protein, rat; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; C422405:activin A; D028341:Activins; D055513:Connective Tissue Growth Factor; D005640:Follicle Stimulating Hormone; D028322:Inhibin-beta Subunits; D000242:Cyclic AMP