Prostaglandin F2alpha- and FAS-activating antibody-induced regression of the corpus luteum involves caspase-8 and is defective in caspase-3 deficient mice

Abstract

We recently demonstrated that caspase-3 is important for apoptosis during spontaneous involution of the corpus luteum (CL). These studies tested if prostaglandin F2alpha (PGF2alpha) or FAS regulated luteal regression, utilize a caspase-3 dependent pathway to execute luteal cell apoptosis, and if the two receptors work via independent or potentially shared intracellular signaling components/pathways to activate caspase-3. Wild-type (WT) or caspase-3 deficient female mice, 25-26 days old, were given 10 IU equine chorionic gonadotropin (eCG) intraperitoneally (IP) followed by 10 IU human chorionic gonadotropin (hCG) IP 46 h later to synchronize ovulation. The animals were then injected with IgG (2 micrograms, i.v.), the FAS-activating antibody Jo2 (2 micrograms, i.v.), or PGF2alpha (10 micrograms, i.p.) at 24 or 48 h post-ovulation. Ovaries from each group were collected 8 h later for assessment of active caspase-3 enzyme and apoptosis (measured by the TUNEL assay) in the CL. Regardless of genotype or treatment, CL in ovaries collected from mice injected 24 h after ovulation showed no evidence of active caspase-3 or apoptosis. However, PGF2alpha or Jo2 at 48 h post-ovulation and collected 8 h later induced caspase-3 activation in 13.2 +/- 1.8% and 13.7 +/- 2.2 % of the cells, respectively and resulted in 16.35 +/- 0.7% (PGF2alpha) and 14.3 PlusMinus; 2.5% TUNEL-positive cells when compared to 1.48 +/- 0.8% of cells CL in IgG treated controls. In contrast, CL in ovaries collected from caspase-3 deficient mice whether treated with PGF2alpha, Jo2, or control IgG at 48 h post-ovulation showed little evidence of active caspase-3 or apoptosis. CL of WT mice treated with Jo2 at 48 h post-ovulation had an 8-fold increase in the activity of caspase-8, an activator of caspase-3 that is coupled to the FAS death receptor. Somewhat unexpectedly, however, treatment of WT mice with PGF2alpha at 48 h post-ovulation resulted in a 22-fold increase in caspase-8 activity in the CL, despite the fact that the receptor for PGF2alpha has not been shown to be directly coupled to caspase-8 recruitment and activation. We hypothesize that PGF2alpha initiates luteolysis in vivo, at least in part, by increasing the bioactivity or bioavailability of cytokines, such as FasL and that multiple endocrine factors work in concert to activate caspase-3-driven apoptosis during luteolysis.

Figure 1

Histochemical assessment of apoptosis in CL derived from WT and caspase-3-null mice following treatment with IgG, Jo2 or PGF_2α, each administered at 48 h post-ovulation. The ovaries were harvested 8 h post-injection. The data shown depict the incidence of apoptotic (TUNEL-positive, brown staining) cells in CL of ovaries derived from WT (A, C, and E) and caspase-3 deficient mice (B, D, and F) following injection with IgG (A, B), Jo2 (C, D) or PGF_2α (E, F). Original magnifications, × 600. The insets represent a higher magnification (× 1000), demonstrating the presence or absence of TUNEL positive cells. The photomicrographs shown are representative of similar results obtained in at least three independent experiments.

Figure 2

Detection of active caspase-3 in CL derived from WT and caspase-3-deficient mice following treatment with IgG, Jo2 or PGF_2α. The presence of active caspase-3 was evaluated by immunohistochemistry using CM-1 antibody in sections of CL derived from WT or caspase-3-deficient mice 8 h following injection at 48 h post ovulation with IgG (A and B), Jo2 (C and D) or PGF_2α (E and F). Original magnification: × 600. The insets represent a high magnification (original magnification × 1000) demonstrating the presence or absence of CM-1 positive cells (dark brown reaction). Medial sections were obtained from CL from three independent mice for analysis. Photomicrographs shown are representative of identical results in at least three separate experiments.

Figure 3

Histochemical assessment of FAS in CL derived from WT and caspase-3-deficient mice following treatment with IgG, Jo2 or PGF_2α. administered 24 h post ovulation and ovaries harvested 8 h post injection. Panel represents a photomicrograph of a CL section derived from WT mouse used as a negative control (no primary antibody). Panel B and C are photomicrographs of CL sections derived from WT and caspase-3-deficient mice (respectively) probed with anti-FAS antibody. Original magnification for panels A, B and C × 600.

Figure 4

Quantitative analysis of Caspase-8 activity in CL in response to IgG, Jo2 or PGF_2α in vivo. The level of caspase-8 activity was determined in CL isolated from ovaries collected from WT mice 8 h following injection IgG (2 μg/IV), Jo2 (2 μg/IV) or PGF_2α (10 μg/IP). The asterisk (*) represent differences from the control P < 0.05. The experiment was replicated three separate times (n = 3) with different groups of mice.