The current study is the first to directly measure relative changes in the expression of Fas on the surface of bovine luteal cells across the estrous cycle. The observation of quantifiably higher Fas receptor expression on luteal cells from early stage compared to late stage CL was unexpected and somewhat contrary to what was anticipated based upon earlier published studies. In these studies, the investigators examined the gross expression of Fas mRNA [5, 7] and protein [34, 35] in ovarian tissues, without reference to cell-specificity and they found that Fas increased only in CL undergoing regression. In the current study, Fas protein was quantified for individual cells obtained from CL following tissue dissociation and cell culture, and then analyzed using flow cytometry. Similarly, Fas mRNA expression for the two stages of CL was measured by Q-RTPCR. The current methods are arguably more quantitative than the mRNA detection, immunoblot analysis, and immunohistochemistry methods described in the cited studies, but fall short of identifying specific cell type(s) known to exist within the CL. Nevertheless, dissociation of the luteal tissue and establishing serum-free culture conditions, as described, removes many of the various cell types, while enriching the population of luteal steroidogenic cells. Thus, we suggest the pattern of Fas expression observed in the current study is essentially representative of the luteal steroidogenic cell population within the bovine CL at the two extremes of the estrous cycle. Moreover, our observation of no measureable difference in relative steady-state amounts of mRNA for Fas in early versus late stage CL, as evaluated by Q-RTPCR, is consistent with a previously published study .
Overall, a 72% decline in the number of bovine luteal cells expressing Fas at their cell surface, and a 59% decline in the density of Fas expressed at the cell surface across the estrous cycle was observed. Total Fas expression (surface and intracellular) for freshly isolated cells was higher for early stage CL than late stage CL. A similar difference in Fas surface expression was observed for cultured luteal cells, but was further enhanced by culture alone. Exposure of the cultured cells to the cytokine cocktail of IFN + TNF + FasL, however, resulted in similar estimates of cell death for both stages of CL. This indicates cultured luteal cells from both stages of CL are equally vulnerable to cytokine-mediated cell death despite clear differences in the cell surface expression of Fas.
The observation that Fas expression is elevated on luteal cells of early stage CL without further enhancing their susceptibility to cytokine-induced death indicates mechanisms exist to protect the cells against Fas-induced apoptosis. For instance, a soluble secreted isoform of Fas has been identified in other tissues that sequesters FasL prior to binding at the target cell surface, thus preventing cell death [36–38]. This isoform of Fas lacks the transmembrane domain of wild-type Fas, causing it to be secreted rather than expressed on the surface of cells . The murine ovary expresses a soluble form of Fas, which has protective effects . Thus, it is possible a soluble form of Fas exists within the bovine CL to modulate the effect of elevated Fas expression in early stage CL as seen in the current study. Certainly this possibility merits additional exploration.
Another intrinsic “protective” mechanism of cells of early stage bovine CL might include the expression of membrane-bound splice variants of the Fas receptor. The cytokine TRAIL (TNF-related apoptosis-inducing ligand) for example, which is structurally similar to FasL, binds to receptors, DR4 and DR5, yet membrane-bound decoy receptors also exist for TRAIL. These receptors, named DcR1 and DcR2, have a cytoplasmic domain structurally similar to DR4 and DR5, respectively, but lack the intracellular death domain necessary for transmitting an apoptotic signal [39–41]. Recently, Sugimoto and coworkers identified a putative Fas decoy receptor, DcR3, in granulosa cells of porcine ovaries . Similar to DcR1 and DcR2, DcR3 contains an extracellular and cytoplasmic domain similar to wild-type Fas, but lacks the intracellular death domain. Unlike soluble Fas, the decoy receptor is expressed on the plasma membrane and retains its ability to bind FasL, but does not induce cell death . It is tempting to speculate that a decoy receptor of Fas may exist on bovine luteal cells, explaining the high prevalence of Fas expression observed for cells of early stage CL, but not late stage CL, in the current study. Further research is needed to determine whether or not a Fas decoy receptor exists within the bovine ovary, and to explore its possible role in ovarian function.
Alternatively, enhanced expression of Fas on cells of early stage CL can be explained by a non-apoptotic or even proliferative role of Fas in the early stage CL. In recent years, diverse non-apoptotic functions of Fas have been documented , such as the acceleration of liver regeneration after partial hepatectomy , the induction of cell migration and invasiveness of apoptotic-resistant tumor cells , and the stimulation of cardiomyocyte hypertrophy . The ability of Fas to control the fate of the cell likely hinges on the regulation of Fas-induced downstream signaling events, such as activation/inhibition of the ERK, JNK, p38, and NF-κB pathways. These same pathways have suggested roles in luteal cell function and fate [5, 48–50], but their influence on the developing early CL, especially in the context of elevated Fas expression, is unknown. Overall, the concept that Fas might facilitate development of the CL is consistent with the premise suggested by Pate and Keyes , in which immune-response mechanisms exist within the ovary to abate damaging inflammatory responses caused by dead or dying cells. In the current scenario, these cells would arise from postovulation trauma during the initial development of the CL.
In the present study, acrylamide selectively disrupted the K8/K18 filaments in the luteal cells, but did not enhance Fas expression or otherwise influence Fas-mediated cell death. In effect, this result did not support the concept that K8/K18 filaments influence Fas trafficking at the cell surface. However, acrylamide causes intermediate filaments to only partially disassemble and undergo acute dephosphorylation . Dephosphorylation provokes a 50% loss of phosphate from the keratin protein which corresponds with the morphological changes observed for intermediate filament expression . At best, the dephosphorylation event is transient, and the striking changes in intermediate filament organization are reversible. In fact, the filaments re-establish their ‘net-like’ organization generally within 12 h after acrylamide removal , and complete rephosphorylation occurs within 18 h . In the current investigation, the K8/K18 filaments of bovine luteal cells were exposed to acrylamide for only 4 h. This was sufficient time to noticeably disrupt the filaments, but perhaps insufficient to sustain a change in Fas trafficking or in downstream signaling that would otherwise enhance cell death. It is noteworthy, however, that these same conditions increased Fas expression on HepG2 cells in the current study. For the time-being, we cannot reject the possibility that K8/K18 filaments influence events downstream from Fas binding; however, it seems unlikely that the filaments directly impair Fas expression on the cell surface as has been suggested in other studies [20–22].