155 Effect of PAG7 ablation on matrix remodeling markers in the bovine endometrium

Pregnancy-associated glycoproteins (PAGs) have been widely used as a biomarker of fetal viability and placentation in cattle. Several hypotheses have been proposed regarding the roles of PAGs, such as extracellular matrix (ECM) remodeling in tissues, immunomodulation, and luteotropic effects. During placental development and parturition, metalloproteinases (MMPs) play an important role in tissue remodeling and endometrial ECM degradation. This process is essential for increasing surface area and promoting changes in cell-cell interaction for successful placentation. Previous studies indicated that binucleated cell-specific PAGs upregulate matrix MMPs in the endometrium. Recent work from our group showed that PAG7 ablation reduces trophectoderm (TE) attachment and growth, which could be due to a lack of proteolytic activity in the absence of PAG7, delaying attachment of TE cells. The aim of this study was to elucidate if PAG7 in particular has a role in endometrial ECM remodeling. Functional ablation of PAG7 was done using a CRISPR/CAS9 system. A single guide-RNA targeting Exon 2 of PAG7 was annealed to a Universal Tracer sequence and CAS9-mRNA. Bovine embryos were electroporated 14 h post-insemination, either with the guide-CAS9 complex (treated) or CAS9-mRNA (control). After transfection, embryos were cultured until Day 8 post-insemination. Hatched blastocysts were individually cultured in gelatin-coated plates for 45 days. Supernatant (150 μL) was collected and replenished every 2 days; the collected medium was snap-frozen for later analysis. To confirm the effective knockout of PAG7, Day 45 TE cells were genotyped by topo-cloning and Sanger sequencing. A successful gene editing was determined by frame-disrupting indels that introduced a stop codon. A 70% biallelic editing efficiency was achieved. Individual 8-mm endometrial explants were cultured for 24 h with TE cell culture medium, supplemented with 20% supernatant obtained from TE cells derived from either PAG7 ablated (n = 7) or CAS9 control (n = 7) embryos. To evaluate the effect of PAG7 on the endometrial explants, the expression of matrix metalloproteinase 1 (MMP1), matrix metalloproteinase 3 (MMP3), and metallopeptidase inhibitor 2 (TIMP2) was quantified by qPCR. All data were analyzed by ANOVA using the GLM procedure of SAS v9.4. Treatment was included as a fixed effect, and replicates (n = 3) were considered random. While the expression of MMP1 and TIMP2 showed no significant changes, MMP3 exhibited a significant downregulation (P < 0.05) in the endometrial explants treated with PAG7 ablated (0.6-fold change) compared with those treated with CAS9 control supernatant. Our findings suggest that PAG7 might be necessary to activate MMP3 in the endometrium and contribute to ECM remodeling to promote conceptus attachment. However, further studies are necessary to assess the expression of additional genes associated with endometrial remodeling.

This work was supported by Agriculture and Food Research Initiative Competitive Grant no. 2021-67015-33675 from the USDA National Institute of Food and Agriculture.