Heparan Sulfate Proteoglycan Sulfation Regulates Uterine Differentiation and Signaling During Embryo Implantation

Abstract

To prepare for embryo implantation, the uterus must undergo a series of reciprocal interactions between the uterine epithelium and the underlying stroma, which are orchestrated by ovarian hormones. During this process, multiple signaling pathways are activated to direct cell proliferation and differentiation, which render the uterus receptive to the implanting blastocysts. One important modulator of these signaling pathways is the cell surface and extracellular matrix macromolecules, heparan sulfate proteoglycans (HSPGs). HSPGs play crucial roles in signal transduction by regulating morphogen transport and ligand binding. In this study, we examine the role of HSPG sulfation in regulating uterine receptivity by conditionally deleting the N-deacetylase/N-sulfotransferase (NDST) 1 gene (Ndst1) in the mouse uterus using the Pgr-Cre driver, on an Ndst2- and Ndst3-null genetic background. Although development of the female reproductive tract and subsequent ovarian function appear normal in Ndst triple-knockout females, they are infertile due to implantation defects. Embryo attachment appears to occur but the uterine epithelium at the site of implantation persists rather than disintegrates in the mutant. Uterine epithelial cells continued to proliferate past day 4 of pregnancy, accompanied by elevated Fgf2 and Fgf9 expression, whereas uterine stroma failed to undergo decidualization, as evidenced by lack of Bmp2 induction. Despite normal Indian hedgehog expression, transcripts of Ptch1 and Gli1, both components as well as targets of the hedgehog (Hh) pathway, were detected only in the subepithelial stroma, indicating altered Hh signaling in the mutant uterus. Taken together, these data implicate an essential role for HSPGs in modulating signal transduction during mouse implantation.

Signaling through retinoic acid receptors is essential for mammalian uterine receptivity and decidualization

“…Despite the absence of luminal closure, which is thought to help immobilize the embryos for implantation, blastocyst attachment appears to successfully occur in the Rara DN Pgr uteri on 4.5 dpc ( Figure 2, B and D , compared with Figure 2, A and C ), raising the possibility that failures in subsequent pregnancy events also contribute to the fertility defects. As we and others have previously reported ( 34 , 35 ), during embryo attachment, strong CDH1 expression is present only in the apical poles of the uterine epithelium and barely detectable on the basal side ( Figure 2C , arrows). Interestingly, this polarized localization of CDH1 is absent in the Rara DN Pgr uteri; strong CDH1 staining was observed on both sides ( Figure 2D , arrowheads).…”

“…In situ hybridization was performed on PFA-fixed, paraffin-embedded, 8 μm tissue sections using RNAscope 2.5 HD Assay-RED kit (Advanced Cell Diagnostics). Gene-specific double-“Z” oligo probes compatible with the kit were ordered from Advanced Cell Diagnostics (probe-Mm- Bmp2 , catalog 406661; probe-Mm- Fst , catalog 454331), and detailed in situ procedure has been previously described ( 35 ).…”

“…Pgr-Cre line was provided by Dr. Francesco DeMayo at the National Institute of Environmental Health Sciences (23) and were mated to RaraDN f/+ mice to generate offspring carrying both alleles (hereinafter referred to as RaraDN Pgr ) and littermate controls (RaraDN f/+ , CTRL). Artificial decidualization and tail vein injection were performed following standard procedures as described previously (35,66,67). All mice used in this study were maintained in a barrier facility at Washington University School of Medicine, Missouri, following the institution's regulations with an approved protocol.…”

Uterine Luminal Epithelium as the Transient Gateway for Embryo Implantation

“…These effects of progesterone are medicated through uterine epithelial PR [12], and most likely stromal PR, in which HAND2 is involved [13]. Studies in genetic mouse models have implicated multiple factors in the autocrine and paracrine regulation of LE proliferation, such as growth factors (e.g., IGF1, FGFs) [9,13,14], cell-cycle related proteins (e.g., MAD2L1, CDKN1A, and CEBPB) [9], and transcription factors (e.g., KLF4 and KLF15) [15][16][17]. Most likely multiple factors are involved because LE proliferation retains in IGF1deficient mice [18].…”

Regulation and Function of Laminin A5 during Mouse and Human Decidualization

“…Previous studies show that embryo implantation triggers a spatiotemporal expression of BMs [ 18 ]. In addition, BMs regulate uterine differentiation during embryo implantation [ 19 , 20 , 21 , 22 ].…”