Collagen and elastic fiber remodeling in the pregnant mouse myometrium†

Abstract

The myometrium undergoes progressive tissue remodeling from early to late pregnancy to support fetal growth and transitions to the contractile phase to deliver a baby at term. Much of our effort has been focused on understanding the functional role of myometrial smooth muscle cells, but the role of extracellular matrix is not clear. This study was aimed to demonstrate the expression profile of sub-sets of genes involved in the synthesis, processing, and assembly of collagen and elastic fibers, their structural remodeling during pregnancy, and hormonal regulation. Myometrial tissues were isolated from non-pregnant and pregnant mice to analyze gene expression and protein levels of components of collagen and elastic fibers. Second harmonic generation imaging was used to examine the morphology of collagen and elastic fibers. Gene and protein expressions of collagen and elastin were induced very early in pregnancy. Further, the gene expressions of some of the factors involved in the synthesis, processing, and assembly of collagen and elastic fibers were differentially expressed in the pregnant mouse myometrium. Our imaging analysis demonstrated that the collagen and elastic fibers undergo structural reorganization from early to late pregnancy. Collagen and elastin were differentially induced in response to estrogen and progesterone in the myometrium of ovariectomized mice. Collagen was induced by both estrogen and progesterone. By contrast, estrogen induced elastin, but progesterone suppressed its expression. The current study suggests progressive extracellular matrix remodeling and its potential role in the myometrial tissue mechanical function during pregnancy and parturition.

Keywords: collagen; elastic fibers; extracellular matrix; myometrium; steroid hormones.

Graphical abstract

Figure 1

Structural reorganization of ECM in mouse myometrium during pregnancy. Transmission electron microscopy images of NP, gestation day 12 and day 18 mouse myometrium. Images are taken at 2500× of the circular layer of the myometrium (n = 3/time point). E—extracellular matrix showing collagen fibers, S—smooth muscle bundles.

Figure 2

Gene expression and protein levels of fibrillar collagen I and III in mouse myometrium during pregnancy. (A) mRNA expression of genes encoding collagen subunits, Col1a1 and Col3a1, in mouse myometrium. NP, gestation days 6, 8, 10, 12, 15, 17, 18, IL (in-labor), and PP (n = 6/group, ^*P < 0.05). (B) Western blot analysis of collagen I and III protein levels in PBS and urea soluble fractions of NP, gestation days 6, 8, 10, 12, 15, 18, IL, and PP mouse myometrial tissues. β-Actin was used as a loading control. These are representative images from three independent replicates. Quantitative densitometry analysis of collagen I and III protein levels was determined and is expressed as a ratio to β-actin (mean ± SEM; n = 3). The ratio calculated from both PBS and urea fractions was combined to express total levels.

Figure 3

Gene expression of factors involved in the synthesis, processing, and assembly of fibrillar collagen in the pregnant mouse myometrium. mRNA expression of Pcolce, Pcolce2, Bgn, Dcn, Thbs1 and 2 in mouse myometrium. NP, gestation days 6, 12, 15, 18, and PP (n = 6/group, ^*P < 0.05).

Figure 4

Structural reorganization of fibrillar collagen in the pregnant mouse myometrium. SHG imaging of fibrillar collagen in NP, gestation days 6, 12, 15, and 18 myometrium. The arrows indicate the change in the morphology of collagen fibers from wavy to linear as pregnancy progresses. Representative images from three independent replicates. Imaging settings vary between images for optimal visualization of morphology. (L—longitudinal layer; C—circular layer). Scale bar: 50 μm.

Figure 5

Elastic fibers in the pregnant mouse myometrium—gene expression and protein levels of elastin and structural reorganization of elastic fibers. (A) mRNA expression of Eln in mouse myometrium. NP, gestation days 6, 12, 15, 18, and PP (n = 6/group, ^*P < 0.05). (B) Western blot analysis of elastin levels in PBS and urea soluble fractions of NP, gestation days 6, 8, 10, 12, 15, 18, in-labor (IL) and PP mouse myometrial tissues. β-Actin was used as a loading control. Representative images from three independent replicates. The MW of tropoelastin monomer is 72 kDa and of dimer is 144 kDa. Quantitative densitometry analysis of tropoelastin monomer levels was determined and is expressed as a ratio to β-actin (mean ± SEM; n = 3). The ratio calculated from both PBS and urea fractions was combined to express total levels. (C) Confocal imaging of tropoelastin of NP, gestation days 6, 12, 15, and 18 myometrium. Representative images from three independent replicates. Scale bar: 20 μm.

Figure 6

Gene expression of factors involved in the synthesis, processing, and assembly of elastic fibers in the pregnant mouse myometrium. mRNA expression of Fbn1, 2, and Fbln1-5 in mouse myometrium. NP, gestation days 6, 12, 15, 18, and PP (n = 6/group, ^*P < 0.05).

Figure 7

Steroid hormones differentially induce myometrial collagen and elastin gene expression. (A) Induction of collagen and elastin gene expression in response to 17β-estradiol in ovariectomized mouse myometrium. mRNA expression of Col1a1, Col3a1, Eln, and Fbn1 in response to 17β-estradiol (E) in ovariectomized mouse myometrium. Ovariectomized mice were treated with vehicle (V) or 17β-estradiol (100 ng/mouse, s/c) for 6, 12, 18, or 24 h (n = 6/group, ^*P < 0.05). (B) Induction of collagen and elastin gene expression in response to progesterone and 17β-estradiol in ovariectomized mouse myometrium. mRNA expression of Col1a1, Col3a1, Eln, and Fbn1 in response to progesterone (P) or progesterone and 17β-estradiol (E) in ovariectomized mouse myometrium. Ovariectomized mice were treated with P (1 mg/mouse, s/c), P (1 mg) + E (10 ng), E (100 ng) − 2 days of rest – P (1 mg). The tissues were collected after 24 h (n = 6/group, ^*P < 0.05).