Disruption of Cytokeratin 18-Containing Intermediate Filaments in Bovine Luteal Cells: Effects of Acrylamide on Progesterone Secretion, Fas Expression, and FasL-Induced Apoptosis.

Abstract

One proposed mechanism of cell death during regression of the bovine corpus luteum (CL) is Fas-Fas Ligand (FasL)-induced apoptosis. Fas is a protein receptor found on the surface of luteal cells that binds with FasL and induces cell death through a series of signaling pathways involving caspase enzymes and cytokeratin-containing intermediate filaments. Others have shown in non-ovarian cell types that cytokeratin 18 (CK18)-containing intermediate filaments impair FasL-induced apoptosis, yet these filaments are also directly cleaved by caspases during apoptotic signaling. The purpose of the present study was to determine whether disruption of CK18-containing intermediate filaments in bovine luteal cells increases the cell surface expression of Fas and enhances FasL-induced apoptosis. Early and late stage CL (Days 5 and 16-18 postovulation, respectively) were collected and prepared for primary cell culture following collagenase dissociation. The cultures were exposed initially to culture medium (control) or acrylamide (5mM) for 4 hours to disrupt CK18-containing intermediate filaments. Subsequently, the culture medium in both groups was exchanged and the cells were then exposed to vehicle (control) or a cytokine cocktail containing FasL (50ng/mL) for 24 hours to induce cell death. Immunocytochemical staining revealed that acrylamide effectively disrupted CK18-containing intermediate filaments, resulting in perinuclear aggregation. In contrast, acrylamide had no effect on microtubule organization, progesterone secretion (177 versus 197 ng/5x105cells in control and acrylamide cultures, respectively), or cell viability (19% versus 14% cell death in control and acrylamide treated cultures, respectively) regardless of the stage of CL. Flow cytometric analysis revealed that Fas expression on the surface of cultured luteal cells from early and late stage CL was relatively high (an average of 84% and 61% of luteal cells from early and late stages, respectively). Pretreatment of the cultures with acrylamide, however, did not substantially enhance Fas expression (86% and 63% of luteal cells from early and late stage CL, respectively). Similarly, acrylamide did not augment FasL-induced cell death (an average of 42% versus 40% cell death for control and acrylamide-treated cultures, respectively), regardless of stage of CL. Consistent with the relatively high expression of Fas on cells from early stage CL, the incidence of FasL-induced death was greater in cultures from early stage CL than late stage CL (52% versus 29% cell death, respectively). The results indicate that while acrylamide effectively disrupts CK18-containing intermediate filaments in bovine luteal cells, its transient actions fail to increase Fas expression on the cell surface or to promote FasL-induced apoptosis. This project was supported by National Research Initiative Competitive Grant no. 2007-35203-18074 from the USDA Cooperative State Research, Education, and Extension Service. (poster)