Type-dependent differences in Fas expression and phagocytes distribution in rat corpora lutea during natural regression: an immunohistochemical evidence

Abstract

Though Fas/Fas ligand (FasL) system-dependent apoptosis is considered to be the primary form of cell death in regressing corpus luteum (CL), the cellular identity and regulation of expression of the ligand and receptor molecules are not fully understood. Here, we focused on immunohistochemical determination of Fas expression during natural regression with comparison of three different types of rat CLs. Detected Fas was in good spatial association with cleaved caspase-3 and FasL proteins and with macrophages and neutrophils. In CLs of the cycle and pseudopregnancy, Fas-positive cell types included large and small luteal (steroidogenic) cells and capillary endothelial cells mainly, and blood-derived immune cells occasionally. Fas signals were abundant at multiple focal inflammatory-like sites. In contrast, Fas signals in CL of pregnancy did not localize in steroidogenic cells, but almost exclusively in endothelial cells and granulocytes. The signals scattered evenly throughout the CL tissue as phagocytes also did. In all CLs types, the numbers of Fas-expressing cells increased transiently after functional inactivation and at the early phase of structural regression. This observation revealed spatio-temporally regulated expression of Fas that was highly associated with apoptotic and phagocytotic systems and type-dependent differences in Fas expression and phagocytes dynamics in naturally regressing CL of rats.

Fig. 1.

Immunolocalization of Fas and related factors in CyCL of the estrous rat. (A) Fas immunostaining in CyCL. (B) Pre-absorption of the antibody with the blocking peptide completely abolished the immunoreaction. (C) Immunoreactive Fas-positive cell types include large (black arrowheads) and small (white arrowheads) luteal cells, capillary endothelial cells (blue arrowheads) and blood derived granulocytes and macrophages (green arrowheads). Positive immunoreactions for FasL (D), Fas (E) and cleaved caspase-3 (F), the most of which were densely scattered in inflammatory-like and fibrotic sites (closed dashed lines in E) in the CyCL. Larger magnification of portions of central zone of D and F was available in Supplementary Fig. 1A and 1B, respectively, to identify the immunopositive cell types. Associated immunostaining for Fas (G), MPO (H) and CD68 (I) was also abundant to inflammatory-like and fibrotic sites (surrounded by the dashed line in G). Magnification of rectangles with dashed lines in H and I was available in Supplementary Fig. 2A and 2B, respectively. Scale bars, 50 µm (A, B), 25 µm (C), 100 µm (D–I).

Fig. 2.

Fas and associated phagocytes distribution in PsCL and PrCL. Immunoreactive Fas signals on PSP15 located frequently at focal sites of inflammatory-like reactions (marked by the dashed lines) in regressed PsCL (A). The signals were present on steroidogenic cells (black arrowhead), endothelial cells (blue arrowhead) and granulocytes (green arrowheads) (B). Fas signals (highlighted with black arrowheads) (C) as well as MPO-positive cells (D) and CD68-positive cells (E) were fairly evenly scattered throughout the PrCL tissue sampled on PP0. Fas signals were present on endothelial cells (blue arrowhead) and migratory cells (green arrowheads), but not steroidogenic cells with intact nuclear structure (black arrowheads) (F). Signals of cleaved caspase-3 in a portion of PrCL were predominant in endothelial cells (blue arrowheads) and absent in steroidogenic cells (black arrowheads) (G). Scale bars, 100 µm (A, C, D, E), 12.5 µm (B, F), 20 µm (G).