Fig 1.
Dynamic changes in the histoarchitecture and ECM deposition accompany PS remodelling.
(A-F) Giemsa staining of PS and IpL transverse sections. (A) NP PS consists of a narrow fibrocartilaginous disc (FC) situated between two high metachromatic hyaline cartilaginous pads (HC) on the surface of the subchondral pubic bones, which are caudally and ventrally connected by non-metachromatic dense connective tissue. (B-E) The absence of highly metachromatic hyaline cartilage (HC) and the presence of IpL attached to the pubic bone via an osteoligamentous junction (OJ). (F) The return of similar NP PS histoarchitecture and highly metachromatic hyaline cartilage (HC) tissue at 10dpp in the PS. (G) Morphometric measurement of hyaline cartilage metachromatic tissues volume in the NP and 10dpp mice PS (U = 3; p = 0.7). Data from a Mann-Whitney test are presented with the means and SEM. (A, F) Scale bars = 50 μm. (B-E) Scale bars = 100 μm.
Fig 2.
Morphological characterization of distinct regions of the IpL osteoligamentous junction at D19.
(A-C) There are two distinct regions in the IpL osteoligamentous junction (dotted line): the BPR is composed of elongated cells (arrows) with poorly birefringent collagen fibrils at the ECM, while the BDR contains angular chondrocyte-like cells (arrowheads) organized as isogenous groups surrounded by dense bundles of collagen. (A) Giemsa staining, scale bar = 30 μm. (B, C) Sirius Red staining and polarized microscopy, scale bar = 20 μm.
Fig 3.
IpL osteoligamentous junction remodelling during late pregnancy (D19) and early postpartum (1-3dpp).
(A-C) Identification of chondroitin sulphate at BDR and BPR regions (dotted lines). (D-I) Typical cell phenotypes present in BPR (arrow) and BDR (arrowhead). (J-L) Isogenous groups of chondrocytes seen as dark areas (arrowhead) distributed in BDR and BPR regions that exhibit differently birefringent collagenous ECM. Scale bar = 20 μm.
Fig 4.
Spatiotemporal expression of Sox9 in the PS cartilage pads and IpL osteoligamentous junction during pregnancy and the postpartum period.
(A, F, G and L) Both Sox9 mRNA and protein were localized at the hyaline cartilage (HC) and fibrocartilage (FC) in angular and elliptical chondrocyte-like cells and at round cells near the subchondral bone (SB) in NP and 10dpp PS (arrowheads). (B and H) At the IpL osteoligamentous junction, Sox9 mRNA and protein were observed in elongated cells at the BPR and mainly in BDR angular chondrocyte-like cells at D19 (arrowheads). (E and K) At 5dpp, Sox9 mRNA and protein were localized in round cells at the BPR region of the IpL osteoligamentous junction, but only cells testing positive for Sox9 mRNA were observed at the BDR (arrowheads). (C, D, I and J) From 1dpp to 3dpp, no cells positive for either Sox9 mRNA or protein were observed at the IpL osteoligamentous junction (1:2000 anti-DIG pod/H-O). In situ hybridization (ISH) and Immunohistochemistry (IHC) experiments. Scale bars = 20 μm.
Fig 5.
Spatiotemporal expression of Col2a1 in the PS cartilage pads and IpL osteoligamentous junction during pregnancy and the postpartum period.
(A- F) Distribution of Col2a1 mRNA expression in hyaline cartilage (HC), near the subchondral bone (SB), in fibrocartilage (FC) at NP and 1dpp PS, and in BDR and BPR regions of the osteoligamentous junction from D19 to 5dpp. Col2a1-positive cells exhibit variable phenotypes: round cells at SB, elongated cells at the BPR, angular chondrocyte-like cells (HC/BDR) and elliptical chondrocyte-like cells (FC) from NP to 10dpp PS (arrowheads). (G-L) Areas temporally immunostaining to procollagen encoded by Col2a1. Positive cells presented a coincident phenotype and localization with Col2a1 mRNA-positive cells (arrowheads) (1:2000 anti-DIG pod/H-O). In situ hybridization (ISH) and Immunohistochemistry (IHC) experiments. Scale bars = 20 μm.
Fig 6.
Spatiotemporal expression of Dcx in the PS cartilage pads and IpL osteoligamentous junction during pregnancy and the postpartum period.
(A and G) Both Dcx mRNA and protein were observed in round cells near the NP PS subchondral bone (SB) and in hyaline cartilage (HC) angular chondrocyte-like cells (arrowheads). (B, E, H and K). At the osteoligamentous junction, Dcx mRNA and protein were observed in elongated cells and round cells of the BPR region at D19 and 5dpp, respectively (arrowheads). (C, D, F, I, J and L) Only DCX protein-positive cells were localized in both the BPR and BDR regions of the IpL osteoligamentous junction at 1dpp and 3dpp, and this occurred mostly in round cells near the PS subchondral bone (SB) at 10dpp PS (arrowheads). (1:2000 anti-DIG pod/H-O In situ hybridization (ISH) and Immunohistochemistry (IHC) experiments. Scale bars = 20 μm.
Fig 7.
Spatiotemporal expression of Runx2 in the PS cartilage pads and IpL osteoligamentous junction during pregnancy and the postpartum period.
(A, F, G and L) Runx2 mRNA and protein were localized in round cells at the subchondral bone (SB) and hyaline cartilage (HC) cells with faded staining in NP and 10dpp PS (arrowheads). (B, C, E, H, I and K) At the IpL osteoligamentous junction, Runx2 mRNA expression and protein were observed primarily in elongated or round cells at the BPR region at D19, 1dpp and 5dpp (arrowheads), (D and J) while at 3dpp, faded staining for mRNA and protein were observed in elongated cells of both the BPR and BDR (arrowheads) regions (1:2000 anti-DIG pod/H-O). In situ hybridization (ISH) and Immunohistochemistry (IHC) experiments. (A-F) Scale bars = 20 μm. (G-L) Scale bars = 30 μm.
Table 1.
ISH probes: Primers, amplicon sizes and temperatures used in the hybridization assays.
Gene-specific forward (F) and reverse (R) primers were used to generate antisense RNA probes for use in the in situ hybridization (ISH) assays. All R primers also contained the T7 RNA promoter sequence (T7PS) at the 5’-end (5´-TAATACGACTCACTATAGGGAGA-3´). The amplicon sizes and temperatures used in the ISH assays are also shown.