Cervix Stromal Cells and the Progesterone Receptor A Isoform Mediate Effects of Progesterone for Prepartum Remodeling

Abstract

The prepartum transition from a soft to ripening cervix is an inflammatory process that occurs well before birth when systemic progesterone is near peak concentration. This 2-part study first determined that stromal fibroblasts but not macrophages in the cervix have progesterone receptors (PRs). Neither the number of PR cells in cervix sections nor the relative abundance or ratio of nuclear PR isoforms (PR-A/PR-B) were diminished in mice between day 15 of pregnancy and term. Second in mice lacking PR-B ( Pgr^tm20mc), the number of cells that expressed the PR-A isoform were maintained during this period of prepartum cervix remodeling. Thus, progesterone effects to sustain pregnancy, as well as soften and ripen the cervix, are mediated by a stable stromal cell population that expresses PR-A and, through interactions with resident macrophages, are likely to mediate inflammatory ripening processes in preparation for birth.

Keywords: mice; cervix; collagen degradation; macrophage; parturition; ripening.

Figure 1.

Photomicrograph of a cervix section from a day 18 postbreeding mouse stained for progesterone receptors (PR-A and PR-B isoforms), as well as macrophages and cell nuclei. Examples of distinct cells stained for PRs (blue arrows points to black stain that obscures methyl green-counterstained cell nuclei) or macrophages (red arrows of brown-stained cellular stain around cell nucleus). Inset is a magnified rare example of a densely stained macrophage with darkened cell nuclei. The methyl green-stained cell nucleus remains evident. Scale bars are 25 and 10 µm in inset.

Figure 2.

Top: Photomicrographs of progesterone receptor (PR) stained cells and counterstained cell nuclei (CN) in cervix sections from nonpregnant (NP), pregnant (days 15 and 18 postbreeding), and day of birth postpartum (PP) mice. Scale bar is 25 µm. Middle: Density of PR cells normalized to CN per area to account for variability in cell nuclei density due to heterogeneity of tissue morphology within and among sections in individuals, as well as within and among groups. Data are the mean ± SE (n = 5-21/group). Bottom: As inversely related to cross-linked collagen in the extracellular matrix, optical density of birefringence of picrosirius red-stained cervix sections cells was normalized to CN per area. Data are mean ± SE (n = 4-10/group; *P < .05 vs NP mice or **vs NP, day 15 and day 16 postbreeding groups by 1-way ANOVA). See Methods for details about staining and analyses. SE indicates standard error; ANOVA, analysis of variance.

Figure 3.

Representative immunoblot of cervix protein extract from pregnant mice on postbreeding days 15 to 18 and the day of birth (PP). Histograms are the optical density of the PR-A-to-PR-B ratio or optical density of the PR-A band normalized to the GAPDH loading control. Data are the mean ± SE (n = 4/group, P > .05; 1-way ANOVA). See Methods for details. PR indicates progesterone receptor; SE, standard error; ANOVA, analysis of variance.

Figure 4.

Photomicrographs of stroma in cervix sections from PRB^−/− mice on days 15, 17, and 18 postbreeding with only PR-A isoform-stained cells and counterstained cell nuclei. Histogram is the numbers of PR-A cell normalized to cell nuclei/area. Data are the mean ± SE (n = 4/group, P > .05; 1-way ANOVA). Scale bar = 50 µm. PR indicates progesterone receptor; SE, standard error; ANOVA, analysis of variance.

Figure 5.

Representative immunoblots from the cervix of a pregnant PRB^−/− mouse on days 15, 16, 17, and 18 postbreeding. With PR-B absent (no band at 116 kDa), only PR-A optical density was normalized to GAPDH (mean ± SE; n = 3/group, P > .05; 1-way ANOVA). PR indicates progesterone receptor; SE, indicates standard error; ANOVA, analysis of variance.