Cervical softening during pregnancy: regulated changes in collagen cross-linking and composition of matricellular proteins in the mouse

Abstract

A greater understanding of the parturition process is essential in the prevention of preterm birth, which occurs in 12.7% of infants born in the United States annually. Cervical remodeling is a critical component of this process. Beginning early in pregnancy, remodeling requires cumulative, progressive changes in the cervical extracellular matrix (ECM) that result in reorganization of collagen fibril structure with a gradual loss of tensile strength. In the current study, we undertook a detailed biochemical analysis of factors in the cervix that modulate collagen structure during early mouse pregnancy, including expression of proteins involved in processing of procollagen, assembly of collagen fibrils, cross-link formation, and deposition of collagen in the ECM. Changes in these factors correlated with changes in the types of collagen cross-links formed and packing of collagen fibrils as measured by electron microscopy. Early in pregnancy there is a decline in expression of two matricellular proteins, thrombospondin 2 and tenascin C, as well as a decline in expression of lysyl hydroxylase, which is involved in cross-link formation. These changes are accompanied by a decline in both HP and LP cross-links by gestation Days 12 and 14, respectively, as well as a progressive increase in collagen fibril diameter. In contrast, collagen abundance remains constant over the course of pregnancy. We conclude that early changes in tensile strength during cervical softening result in part from changes in the number and type of collagen cross-links and are associated with a decline in expression of two matricellular proteins thrombospondin 2 and tenascin C.

FIG. 1.

Cervical collagen Iα1 and IIIα1 levels remain constant during pregnancy. A) Collagen Iα1 (Col1a1) and (B) collagen IIIα1 (Col3a1) mRNA levels were measured via QPCR using cervices at NP metestrus and Day 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18, and 2 h postpartum and 24 h postpartum and normalized to Day 18. For NP, n = 15 cervices; for pregnant and postpartum, n = 4–6 cervices per time point. *, Significance at P < 0.05. C) The amount of collagen in the cervix was measured through hydroxyproline content. Hydroxyproline assay was carried out in NP estrus, gestation Day 5, 8, 10, 12, 14, 16, and 18, 2-h postpartum, 10-h postpartum, and 24-h postpartum cervices and normalized to dry weight; n = 5 cervices per time point. D) Dot blot analysis of collagen I and collagen III shows soluble protein levels at NP estrus and gestation Days 6, 8, 10, 12, 15, and 18. E and F) Optical density analysis is shown of (E) collagen I and (F) collagen III dot blot; n = 3 cervices per time point. G and H) Immunofluorescence of (G) collagen I and (H) collagen III is shown in gestation Day 6, 12, 15, and 18 cervical sections.

FIG. 2.

mRNA expression of collagen processing proteins is shown. A and B) mRNA expression of C-propeptide collagen processing enzymes Bmp1 (A) and Tll1 (B) were evaluated at NP, gestation Day 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18, and 2 h postpartum and 24-h postpartum and normalized to Day 18. For NP, n = 15 cervices; for pregnant and postpartum, n = 4–6 cervices per time point. C) C-propeptide was evaluated via Western blotting in NP, gestation Day 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18, and 4-h postpartum, 12-h postpartum, 24-h postpartum, and 48-h postpartum cervices. A 30-kDa band corresponding to the collagen C-propeptide is present. D) The N-propeptide collagen processing enzyme Adamts2 mRNA expression was evaluated at NP, and gestation Day 10, 12, 15, and 18, and 24-h postpartum and normalized to Day 18. For NP, n = 15 cervices; for pregnant and postpartum, n = 4–5 cervices per time point. E) The of N-propeptide collagen processing enzyme Adamts14 mRNA expression was evaluated at the same time points as described in the legend to panel D. For NP, n = 15 cervices; for pregnant and postpartum, n = 4–5 cervices per time point. *, Significance at P < 0.05.

FIG. 3.

Decline in Plod2 mRNA expression correlates to a reduction in pyridinoline cross-links during pregnancy and an increase in soluble collagen. A) mRNA levels of the Plod family were evaluated in NP, gestation Days 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18 and 2 h postpartum and 24 h postpartum and normalized to Day 18. For NP, n = 15 cervices; for pregnant and postpartum, n = 4–6 cervices per time point. B) HPLC analysis of HP cross-links in NP estrus, gestation Day 5, 8, 10, 12, 14, 16, and 18 and 2-h postpartum, 10-h postpartum, and 24-h postpartum cervices; n = 5 cervices per time point. C) LP cross-links were evaluated in the same time-points shown in panel B; n = 5 cervices per time-point. D) Extractable collagen was assessed in NP, gestation Day 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18 and 4-h postpartum, 12-h postpartum, 24-h postpartum, and 48-h postpartum cervices. Blots were probed with rabbit anti-collagen I. Bands are present at ∼250 kDa and 140 kDa (indicated as Kd in figure), representing procollagen and mature collagen, respectively. An unidentified 50-kDa band is also present in NP and postpartum tissues. *, Significance at P < 0.05.

FIG. 4.

Cervical proteoglycan mRNA expression is shown during pregnancy and parturition. mRNA levels of (A) decorin, (B) biglycan, (C) osteomodulin, (D) asporin, and (E) fibromodulin were determined via QPCR on gestation Days 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18 and 2 h postpartum and 24 h postpartum and normalized to Day 18; n = 3–6 cervices per time point. *Significance at P < 0.05.

FIG. 5.

Cervical matricellular protein expression is shown during pregnancy and parturition. mRNA levels of (A) thrombospondin 2, (B) Sparc, and (C) tenascin C were analyzed via QPCR at NP, gestation Days 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18 and 2-h postpartum and 24-h postpartum and normalized to Day 18. For NP, n = 11 cervices for pregnant, and postpartum n = 5–7 cervices per time point. *Significance at P < 0.05.

FIG. 6.

Cervical ECM becomes dispersed throughout pregnancy. Electron micrographs of cervical ECM taken at a magnification of ×4200 on (A) Day 6 and (B) Day 18 of pregnancy. Bar = 1000 nm.

FIG. 7.

Collagen fibrils increase in size during pregnancy. Electron micrographs were taken at a magnification of ×20 500 of (A) NP metestrus, (B) Day 6, (C) Day 12, (D) Day 15, and (E) Day 18. Bar = 1000 nm. Analysis of frequency of fibril diameter of (F) NP metestrus, (G) Day 6, (H) Day 12, (I) Day 15, and (J) Day 18 is shown; n = 8260–13 030 fibrils.