Pretreatment with pancaspase inhibitor (Z-VAD-FMK) delays but does not prevent intraperitoneal heat-killed group B Streptococcus-induced preterm delivery in a pregnant mouse model

Abstract

Caspases and apoptosis are thought to play a role in infection-associated preterm-delivery. We have shown that in vitro treatment with pancaspase inhibitor Z-VAD-FMK protects trophoblasts from microbial antigen-induced apoptosis. Objective. To examine whether in vivo administration of Z-VAD-FMK would prevent infection-induced preterm-delivery. Methods. We injected 14.5 day-pregnant-mice with heat-killed group B streptococcus (HK-GBS). Apoptosis within placentas and membranes was assessed by TUNEL staining. Calpain expression and caspase-3 activation were assessed by immunohistochemistry. Preterm-delivery was defined as expulsion of a fetus within 48 hours after injection. Results. Intrauterine (i.u.) or intraperitoneal (i.p.) HK-GBS injection led to preterm-delivery and induced apoptosis in placentas and membranes at 14 hours. The expression of calpain, a caspase-independent inducer of apoptosis, was increased in placenta. Treatment with the specific caspase inhibitor Z-VAD-FMK (i.p.) prior to HK-GBS (i.p.) delayed but did not prevent preterm-delivery. Conclusion. Caspase-dependent apoptosis appears to play a role in the timing but not the occurrence of GBS-induced preterm delivery in the mouse.

Figure 1

Intrauterine HK-GBS injection leads to TUNEL positive apoptosis in the membranes. Day 14.5 timed pregnant mice were injected with either HK-GBS (panel d) or PBS (a) and euthanized at 5 or 14 hours to isolate the placenta and membranes. The sections shown were obtained after 14 hours of stimulation. TUNEL positive apoptotic cells are stained black-brown. Slides treated with endonuclease served as a positive control (b); animals injected with media indicate baseline apoptosis levels (c). Data shown are representative of 3 separate experiments.

Figure 2

Intrauterine HK-GBS injection leads to TUNEL-positive apoptosis in the placenta. In the mouse placenta, there was TUNEL positive apoptosis after 14 hours of exposure to HK-GBS (d). The slides were treated with PBS for the negative control (a) and endonuclease for the positive control (b); animals were injected with media to assess baseline apoptosis levels (c). Data shown are the representative of 3 separate experiments.

Figure 3

Intrauterine HK-GBS injection leads to caspase 3 activation in the membranes and placenta. Day 14.5 timed pregnant mice were injected with either PBS or HK-GBS and euthanized at 5 or 14 hours to isolate fetal membranes (a–c) and placentas (d–f). Caspase 3 activation was assessed by performing immunohistochemistry analysis using an antibody against active-cleaved caspase 3. Representative data from three separate experiments are shown. HK-GBS exposure led to an increase in caspase 3 positive cells in the membranes (c) and above the spongiform trophoblast layer at 14 hours in the placenta (f).

Figure 4

Apoptosis in the ovarian follicle. Mouse atretic follicles are known to undergo caspase mediated apoptosis. As anticipated, caspase 3 was cleaved and activated in the atretic follicle, which were used as positive control for the caspase 3 antibody specificity. Representative data from three separate experiments are shown.

Figure 5

Intrauterine HK-GBS injection induces placental m-calpain expression in a time-dependent manner. Day 14.5 pregnant mice were euthanized 14 hours after exposure to PBS (i) or HK-GBS (ii). Placentas were removed and stained for m-calpain. Data from a representative of three separate experiments is shown.