Effects of macrophage depletion on characteristics of cervix remodeling and pregnancy in CD11b-dtr mice

Abstract

To test the hypothesis that macrophages are essential for remodeling the cervix in preparation for birth, pregnant homozygous CD11b-dtr mice were injected with diphtheria toxin (DT) on days 14 and 16 postbreeding. On day 15 postbreeding, macrophages (F4/80+) were depleted in cervix and kidney, but not in liver, ovary, or other non-reproductive tissues in DT-compared to saline-treated dtr mice or wild-type controls given DT or saline. Within 24 h of DT-treatment, the density of cell nuclei and macrophages declined in cervix stroma in dtr mice versus controls, but birefringence of collagen, as an indication of extracellular cross-linked structure, remained unchanged. Only in the cervix of DT-treated dtr mice was an apoptotic morphology evident in macrophages. DT-treatment did not alter the sparse presence or morphology of neutrophils. By day 18 postbreeding, macrophages repopulated the cervix in DT-treated dtr mice so that the numbers were comparable to that in controls. However, at term, evidence of fetal mortality without cervix ripening occurred in most dtr mice given DT-a possible consequence of treatment effects on placental function. These findings suggest that CD11b+ F4/80+ macrophages are important to sustain pregnancy and are required for processes that remodel the cervix in preparation for parturition.

Keywords: collagen; diphtheria toxin; monocytes; parturition; preterm birth; ripening.

Figure 1.

A Timeline of injection of saline (Sal) or diphtheria toxin (DT) administered intraperitoneal on days 14 and 16 postbreeding into homozygous dtr mice. X indicated day of euthanasia when blood and tissues were collected for study (n = 4–10 mice/group). B. Serum progesterone concentrations on days 15 and 18 postbreeding in Saline- or DT-treated dtr mice. Treatments described in Methods. P > 0.05 two-way ANOVA (n = 4–5 mice/group).

Figure 2.

A Representative photographs of the reproductive tract from dtr mice on day 15 (D15) postbreeding that had been injected 24 h earlier with Saline (7 of 7) or DT (3 of 11). Distinct uterine compartments, each with a single fetal sac, are indicated by white arrows in saline-treated mice compared with compact compartments demarcated by a sinuous vascularized boundary in DT-treated dtr mice. B Photographs of dtr mice on day 18 postbreeding treated with saline (5 of 5; 1 day before expected delivery) or DT (5 of 7) given 4 and 2 days earlier on days 14 and 16 postbreeding. Note the 9 fetal compartments in the saline-injected mouse compared to the estimated 11 compact segments with resorbing fetuses in the DT-treated dtr mouse. C Histogram of the percentage of viable pregnancy in DT-treated dtr mice based upon morphology and firmness of uterus, as well as assessment of fetal viability with respect to presence of dark deoxygenated blood, compactness, resorption, and diminished segment size. On day 19 postbreeding, all saline dtr controls gave birth in the morning (<9a), while 7 of 10 DT-treated CD11b-dtr mice had not delivered by 4 pm in the afternoon when the study concluded.

Figure 3.

A Photomicrographs of cervix sections on day 15 (D15) from wild-type (WT) or dtr mice that were stained for F4/80 macrophages (Mϕ) and counterstained with methyl green to identify cell nuclei (CN) as described in Methods. Scale bar = 50 μm or 6.5 μm (inset). B Histograms of the density of cell nuclei/volume and macrophages/cell nuclei/volume of WT (left) or dtr mice (right) injected 24 h earlier with saline (Sal) or DT.*P < 0.05 D15 WT vs dtr mice Mϕ/CN, ^aP < 0.05 vs day 15 saline dtr mice (Student t-test, n = 4–9 mice/group).

Figure 4.

Photograph of a picrosirius red-stained section of cervix from a dtr mice on day 15 postbreeding, 24 h after injection of Saline (Sal) or DT. The 9 non-overlapping boxes represent the area analyzed for optical density (9 photomicrographs in each of 3 sections/cervix). Scale bar = 50 μm. The histogram is the optical density assessment of polarized light birefringence (OD/CN), an indication of collagen content and structure degradation. Details provided in Methods and previous studies [19, 41] P > 0.05 for all comparisons (two-way ANOVA, n = 4–9 mice/group).

Figure 5.

A Photomicrographs of cervix sections stained for cell nuclei and macrophages from a day 18 (D18) postbreeding saline (Sal)- or DT-treated dtr mouse. Scale bar = 50 μm. B Histograms of the density of cell nuclei or macrophages/cell nuclei/area in the cervix stroma of dtr mice on day 18 postbreeding that had been injected 4 and 2 days earlier with saline or DT (n = 5–7 mice/group). Note scale change for macrophages/CN compared to Figure 4, an indication of increased abundance as pregnancy neared term. P > 0.05 vs Sal group (Student t-test).

Figure 6.

Photomicrographs of other tissues from saline- or DT-treated dtr mice on day 15 postbreeding stained for F4/80 macrophages and cell nuclei. Scale bar is 50 μm. Note the diminished density of macrophages in kidney, but not liver or ovary.

Figure 7.

Photomicrographs of placenta sections from CD11b-dtr mice on D 15 postbreeding treated 24 h earlier with DT without or with evidence of fetal morbidity (described in Figure 2 legend). Sub-regions are demarcated by brackets, i.e., Troph = Trophoblast layer, Spongiotroph = Spongiotrophoblast layer. Boxes are magnified at right. Scale bar is 500 μm or 25 μm in right 4 panels.