Formation of fibrin at sites of conceptus adhesion in the ewe

Abstract

In ruminants, various molecules are involved in regulating conceptus attachment and adhesion; however, molecules that maintain the conceptus adhesion have not been well characterized. We hypothesized that conceptus must produce a molecule(s), yet uncharacterized or overlooked, which maintain conceptus adhesion to the uterine epithelium. In this study, we aimed to identify new candidate(s) in conceptus secretory proteins responsible for maintaining conceptus adhesion in sheep. We performed RNA-sequence analysis with ovine conceptuses, followed by endometria obtained from pregnant animals on day 15 (P15: pre-attachment), 17 (P17: right after attachment), and 21 (P21: post-attachment; adhesion) and iTRAQ analysis of uterine flushing on P15 and P17. To identify the proteins secreted from conceptuses, we cross-referenced the transcriptome and proteome data. These analyses identified 16 and 26 proteins as conceptus secretory proteins on P15 and P17, respectively. Gene ontology analysis revealed that the conceptus secretory proteins were enriched in those categorized to fibrinolysis and coagulation. RT-qPCR analysis verified that the expression levels of transcripts in conceptuses encoding coagulation factors, fibrinogen subunits, and fibrinolysis factors were significantly higher on P21 than on P15 or P17, which were supported by those through in situ hybridization, western blotting and immunohistochemistry. Histology analysis confirmed that fibrin protein was present at the conceptus adhesion region on P21. These results suggest that in addition to the numerous adhesion molecules so far characterized, fibrin is a new candidate molecule for maintaining conceptus adhesion for pregnancy continuation in ruminants.

Epithelial-mesenchymal transition and bi- and multi-nucleated trophoblast cell formation in ovine conceptuses during the peri-implantation period

“…From our previous RNA-sequencing data [ 14 ], EMT-related transcripts were extracted, their expression was evaluated, and heatmap analysis was performed. The heatmap analysis revealed that transcripts encoding EMT-related genes were distinctively upregulated in P21 ovine conceptuses ( Fig.…”

“… ). These analyses indicated that the increase in EMT-related transcripts and conceptus adhesion appeared to occur simultaneously in ovine conceptuses on P21 [ 14 ].…”

“…From our previous RNA-sequencing data [ 14 ], PAG transcripts were extracted, and their expression patterns were analyzed ( Fig. 4

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“… Expression of pregnancy-associated glycoprotein (PAG)-related transcripts in ovine conceptuses. From the RNA-seq analysis done previously [ 14 ], PAG-related transcripts were extracted and their expression values were shown as transcripts per million (TPM). Transcripts encoding various PAG genes, PAG1 , PAG3 , PAG4 , PAG5 , PAG6 , PAG9 , and PAG10 , were found, particularly on P21.…”

“…The original RNA-seq datasets from P15, P17, and P21 ovine conceptuses (n = 4/day each) have been previously reported [ 14 ] and deposited in the Data Bank of Japan (DDBJ, DRA011433 and DRA011655, http://www.ddbj.nig.ac.jp ). The sequences were pseudoaligned, and transcripts were quantified using RaNA-seq (a bioinformatics tool for the analysis of RNA-seq data, https://ranaseq.eu/index.php ) with its default parameters [ 15 ].…”