65 Functional ablation of pregnancy-associated glycoprotein 7 affects attachment and growth of trophectoderm cell lines

Embryonic mortality between Days 8 and 30 of development represents ~20% of pregnancy losses in cattle. During this time, the embryo needs to grow into a conceptus and start the process of placentation. Pregnancy-associated glycoproteins (PAGs) are secreted by the trophectoderm (TE) mononucleated and trophoblast giant cells (TGCs) and have been used to monitor fetal viability and placentation in cattle. Previous research suggests that PAGs 7, 9, and 17 which are highly expressed by the TGCs between Days 20 and 45 of pregnancy, might have a role in extracellular matrix remodeling of the endometrium, which is essential for a successful placentation and therefore, pregnancy. However, the specific function or mechanism of action of PAGs is still unclear. The objective of this study was to elucidate how the functional ablation of PAG7 affects trophectoderm attachment and growth in vitro. To test this, a CRISPR/Cas9 system was used. Single RNA guides were designed to partially cut from exon 2 to 4 of PAG7, for a total edit of 1460 bp. Guides were individually annealed to a universal tracer RNA and then to CAS9mRNA. Abattoir-derived cumulus–oocyte complexes were matured, fertilized, and washed using our group standard protocol. Guide complex and CAS9 mRNA, or just CAS9 mRNA were electroporated into zygotes 15–17 h after insemination. Embryos were cultured until Day 8 of development. Embryos that reached the blastocyst stage were cultured for 2 additional days with 10% fetal bovine serum to promote hatching. On Day 10, hatched blastocysts were individually placed in gelatin-coated plates with an in-house TE culture medium. Daily pictures were taken to measure the area and the day of attachment was recorded for each embryo. On Day 25, cells were collected for endpoint polymerase chain reaction genotyping. A total of 37 PAG7 null TE cell lines and 24 CAS9 control cell lines were produced in three in vitro embryo production rounds. The TE area measurement was done using ImageJ, v1.53 (National Institutes of Health, Bethesda, Maryland, USA). All data were analysed by ANOVA, with TE growth as response variable and replicate, genotype result (PAG7 null or not) and interaction as fixed effects, using the Statistical Analysis System SAS v9.4. The TE cell lines from PAG7 null embryos took 3 days longer (P < 0.05) to attach than CAS9 controls (Day 19 vs 16, respectively). The TE growth area was also affected in PAG7 null embryos compared to controls (P < 0.05). By Day 25 of culture, the TE area was 1.5 mm² for PAG7 null embryos and 4 mm² for the Cas9 controls. These preliminary results indicate that the ablation of PAG7 affects attachment and TE growth, supporting the proposed action of TGC PAGs on matrix remodeling; however, further research is required to elucidate if PAG7 knockout results in an inadequate response of the endometrium, which would not allow a successful placentation, leading to embryonic mortality.

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.