title: Immunoneutralization of transforming growth factor alpha present in bovine follicular fluid prevents the suppression of the follicle-stimulating hormone-induced production of estradiol by bovine granulosa cells cultured in vitro.
abstract: Growth factors such as transforming growth factors alpha (TGF alpha) and beta (TGFbeta) secreted by theca cells and present in bovine follicular fluid (bFF) have been implicated in granulosa cell growth and differentiation. We investigated these phenomena using two complementary approaches to evaluate the physiological contribution of TGF alpha and TGFbeta in the control of the FSH-induced estradiol production in bovine granulosa cells from large follicles. Granulosa cells (3 x 10(5) viable cells/cm2) harvested from eCG-treated prepubertal calves were cultured (serum free) in wells containing 500 microl/cm2 of defined Ham's F-12 medium supplemented with 0.5 ng/ml FSH for the first 3 days (37 degrees C; 95% air:5% CO2). In the first approach, the effects of individual addition of TGF alpha and TGFbeta at final concentrations of 1 x 10(-4) to 10 ng/ml were determined on Day 4 of culture after stimulation of granulosa cell estradiol production with 6 ng/ml FSH. In a second approach, TGF alpha or TGFbeta was removed specifically from bFF (from large follicles > 10 mm) by immunoneutralization. Thereafter, effects of immunoneutralization of TGF alpha or TGFbeta (0, 0.1, 1, 10, and 100 microg/ml anti-TGF neutralizing antibody) present in bFF (2%) were determined on Day 4 of culture following stimulation of granulosa cell estradiol production with 6 ng/ml FSH. On Day 4, FSH increased (p < 0.001) granulosa cell estradiol production (0 vs. 6 ng/ml FSH). Addition of TGF alpha decreased the granulosa cell estradiol production after 6 ng/ml FSH stimulation in a dose-dependent manner (p < 0.001). In contrast, addition of TGFbeta had no effect on the granulosa cell estradiol production (p > 0.1) after the addition of 6 ng/ml FSH. Addition of bFF (2%) decreased (p < 0.0001) the FSH-induced estradiol production by bovine granulosa cells. After immunoneutralization of TGF alpha in bFF, however, this suppressed FSH-induced estradiol production was restored to levels obtained in the absence of bFF, and this occurred in a dose-dependent manner (p < 0.05). Immunoneutralization of TGFbeta in bFF did not prevent (p > 0.1) the suppressive effect of bFF on FSH-induced estradiol production. These results suggest that TGF alpha produced in vivo by large bovine follicles can act locally (auto/paracrine manner) to suppress granulosa cell estradiol production.
authors.affiliation: Centre de Recherche en Biologie de la Reproduction, Université Laval, Département des Sciences Animales, Ste-Foy, Québec, Canada.
authors.forename: P
authors.initials: P
authors.lastname: Rouillier
authors.forename: M A
authors.initials: MA
authors.lastname: Sirard
authors.forename: P
authors.initials: P
authors.lastname: Matton
authors.forename: L A
authors.initials: LA
authors.lastname: Guilbault
mesh_terms: D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D017209:Apoptosis; D002417:Cattle; D002455:Cell Division; D002478:Cells, Cultured; D004958:Estradiol / Q000096:biosynthesis*; D005260:Female; D005640:Follicle Stimulating Hormone / Q000494:pharmacology*; D015571:Follicular Fluid / Q000378:metabolism*; D006107:Granulosa Cells / Q000378:metabolism*; D006801:Humans; D016211:Transforming Growth Factor alpha / Q000276:immunology / Q000494:pharmacology / Q000502:physiology*; D016212:Transforming Growth Factor beta / Q000494:pharmacology / Q000502:physiology
chemical_list: D000906:Antibodies; D016211:Transforming Growth Factor alpha; D016212:Transforming Growth Factor beta; D004958:Estradiol; D005640:Follicle Stimulating Hormone