Embryo implantation triggers dynamic spatiotemporal expression of the basement membrane toolkit during uterine reprogramming

Abstract

Basement membranes (BMs) are specialized extracellular scaffolds that influence behaviors of cells in epithelial, endothelial, muscle, nervous, and fat tissues. Throughout development and in response to injury or disease, BMs are fine-tuned with specific protein compositions, ultrastructure, and localization. These features are modulated through implements of the BM toolkit that is comprised of collagen IV, laminin, perlecan, and nidogen. Two additional proteins, peroxidasin and Goodpasture antigen-binding protein (GPBP), have recently emerged as potential members of the toolkit. In the present study, we sought to determine whether peroxidasin and GPBP undergo dynamic regulation in the assembly of uterine tissue BMs in early pregnancy as a tractable model for dynamic adult BMs. We explored these proteins in the context of collagen IV and laminin that are known to extensively change for decidualization. Electron microscopic analyses revealed: 1) a smooth continuous layer of BM in between the epithelial and stromal layers of the preimplantation endometrium; and 2) interrupted, uneven, and progressively thickened BM within the pericellular space of the postimplantation decidua. Quantification of mRNA levels by qPCR showed changes in expression levels that were complemented by immunofluorescence localization of peroxidasin, GPBP, collagen IV, and laminin. Novel BM-associated and subcellular spatiotemporal localization patterns of the four components suggest both collective pericellular functions and distinct functions in the uterus during reprogramming for embryo implantation.

Basement membrane ultrastructure and component localization data from uterine tissues during early mouse pregnancy

“…The following primary antibodies were applied to the sections: rat anti-collagen IV NC1 (1:500 dilution, JK2, were from Y. Sado, Shigei Medical Research Institute, Okayama, Japan [5], [6]), rabbit anti-laminin (1:50 dilution for tissues not treated with dissociation buffer and 1:500 dilution for those that were pre-treated with dissociation buffer, ab11575, Abcam, Cambridge, MA), rabbit anti-peroxidasin (1:250 dilution, were from G. Bhave, Vanderbilt University, Nashville, TN [7]), Alexa546 conjugated mouse anti-GPBP (1:50 dilution, mAb N26 to the N-terminal serine-rich domain conserved across all GPBP isoforms in several species [8], from Fibrostatin, SL, Valencia, Spain), and Alexa546 conjugated mouse anti-GPBP-1 (1:50 dilution, mAb e11-2 to the 26-amino acid residue of exon 11 not present in GPBP-2, also called CERT [9], from Fibrostatin, SL, Valencia, Spain). A previously described dissociation buffer was used on sections co-stained with collagen IV antibody (JK2) [1], [5] followed by several washes with PBS and PBS/0.2% Tween. Detection and co-detection with GPBP antibodies (mAbs N26 and e11-2) were performed without dissociation treatment.…”

“…The sections were stained with uranyl acetate and Reynold lead citrate before they were imaged at 30,000x on an electron microscope (Philips T-12 equipped with an AMT CCD camera system, FEI, Hillsboro, OR). Further details of immunofluorescence and electron microscopy methods are available in the accompanying research article [1].…”

“…A schematic (Fig. 2) (included in part in [1]) is presented for anatomical spatial orientation of dynamic basement membranes (BMs) that is beneficial in cross-examination of BM ultrastructure (Fig. 3) and localization of individual components: peroxidasin (Fig.…”

Unicellular ancestry and mechanisms of diversification of Goodpasture antigen–binding protein

“…These proteins include collagen IV, laminin, nidogen, and agrin/perlecan (17)(18)(19). Recently, additional proteins were identified: peroxidasin (20), lysyl oxidase II (21), and Goodpasture antigen-binding protein (GPBP) (22)(23)(24). Recent studies in nonbilaterian animals have shown that collagen IV and laminin (25) are the earliest components of basement membrane, as evidenced in ctenophores and sponges, the two oldest animal lineages, and were likely essential for animal multicellularity (2,3).…”

“…Among BM proteins, GPBP was uniquely poised to also play a role in the appearance of BMs and the transition to multicellularity, owing to its multiple isoforms (23,26,27) with varied functions both inside (28,29) and outside of cells (22)(23)(24)30).…”