Follicular Stage-Dependent Tumor Necrosis Factor {alpha}-Induced Hen Granulosa Cell Integrin Production and Survival in the Presence of Transforming Growth Factor {alpha} In Vitro

doi:10.1095/biolreprod65.2.477

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Follicular Stage-Dependent Tumor Necrosis Factor ${alpha}$ -Induced Hen Granulosa Cell Integrin Production and Survival in the Presence of Transforming Growth Factor ${alpha}$ In Vitro¹

J. Soboloff^a^,b^,c, H. Sasaki^a^,c, and B.K. Tsang^a^,b^,c

^a Reproductive Biology Unit and Division of Reproductive Medicine, Departments of Obstetrics & Gynaecology and ^b Cellular & Molecular Medicine, ^c University of Ottawa and Ottawa Health Research Institute, The Ottawa Hospital (Civic Campus), Ottawa, Ontario, Canada K1Y 4E9

ABSTRACT

The link between cell adhesion to extracellular matrix and integrin-mediatedsurvival signals has been established in several physiologicalsystems, and roles for the cytokines tumor necrosis factor alpha(TNF ${alpha}$ ) and transforming growth factor alpha (TGF ${alpha}$ ) have been suggested.TGF ${alpha}$ stimulates fibronectin production in hen granulosa cellsand is an important survival factor during follicular maturation.In contrast, the role of TNF ${alpha}$ and its possible interaction withTGF ${alpha}$ in the regulation of granulosa cell fate (death versus survival)during ovarian follicular development have not been fully elucidated.The object of the current study was to determine if TNF ${alpha}$ andTGF ${alpha}$ interact in the regulation of hen granulosa cell fibronectinand integrin content in the context of cell death and survivalduring follicular development. TGF ${alpha}$ (0.1 or 10 ng/ml), but notTNF ${alpha}$ (0.1 or 10 ng/ml), increased both cellular and secretedfibronectin content in granulosa cell cultures of F5,6 but notF1 follicles. The expression of integrin ß₃ subunitwas also stimulated by TGF ${alpha}$ in a follicular stage-dependent manner,and culture of F5,6 granulosa cells with TNF ${alpha}$ in the presenceof maximal stimulatory concentrations of TGF ${alpha}$ potentiated thisresponse. TGF ${alpha}$ increased both F5,6 and F1 granulosa cell [³H]thymidineincorporation but not 3-(4,5-dimethylthiazol-2-yl)3,5-diphenyltetrazolium bromide (MTT) metabolism. Although TNF ${alpha}$ had no effecton [³H]thymidine incorporation irrespective of the presenceof the growth factor, MTT metabolism was higher in F5,6 granulosacells cultured for 24 h with both TNF ${alpha}$ and TGF ${alpha}$ than with eithercytokine alone. Incubation of F5,6 granulosa cells for 48 and72 h resulted in a TGF ${alpha}$ -inhibited loss of cellular adhesion anddetachment of granulosa cells from the growth surface. AlthoughTNF ${alpha}$ alone had no effect on cell morphology, it facilitated thereorganization of the granulosa cells into multicellular follicle-likestructures in the presence of the growth factor. DNA degradationsignificantly increased between 0 and 72 h of culture in theabsence of the cytokine but was suppressed by the addition ofTGF ${alpha}$ but not of TNF ${alpha}$ . However, fluorometric analysis indicatedthat the primary type of cell death exhibited by F5,6 granulosacells during extended culture and attenuated by the presenceof TNF ${alpha}$ and TGF ${alpha}$ was necrosis and not apoptosis. The current studydemonstrates that TNF ${alpha}$ and TGF ${alpha}$ interact in the regulation ofgranulosa cell integrin content and cell survival in vitro ina follicular stage-dependent manner. These findings suggestthat follicular development is accompanied by a change in theintraovarian role of TNF ${alpha}$ ; it is atretogenic prior to follicularselection but prevents follicular demise during preovulatorygrowth.

FOOTNOTES

First decision: 5 January 2001.

¹ This work was supported by a grant from the Canadian Institutesof Health Research (MOP-10369 to B.K.T.). J.S. is a recipientof a Natural Science and Engineering Research Council studentship.