Ovine conceptuses express phospholipase inhibitory genes on days 14-15 of pregnancy, interacting with IFNT pathways

Abstract

In brief: Ovine conceptuses highly express phospholipase inhibitory genes just before the conceptus attachment period. Phospholipase inhibitors could synergistically work with the interferon pathway on the endometrium.

Abstract: In mammals, various molecules are involved in the biochemical interaction between the conceptus and endometrium for pregnancy recognition and establishment. In ruminants, interferon tau (IFNT) is the pregnancy recognition factor; however, IFNT alone does not explain corpus luteum maintenance. Although data on factors expressed during implantation have been accumulated, we hypothesized that the conceptus produces additional uncharacterized molecules during the period of conceptus attachment. This study aimed to identify new conceptus secretory proteins involved in the biochemical interaction between the conceptus and endometrium in sheep. We analyzed RNA-sequence data of ovine conceptuses from pregnant animals on days 12, 14, 15, 16, 17, 19, 20 and 21. To identify novel secretory proteins, we focused on highly expressed but uncharacterized genes and performed in silico protein function analysis, identifying genes encoding phospholipase inhibitory proteins expressed on days 14 and 15. Recombinant proteins from these genes were produced, and the effects on cultured bovine endometrial epithelial cells (EECs) and stromal cells (STRs) were analyzed by RNA-sequence analysis. Differentially expressed gene (DEG) analysis demonstrated that the recombinant protein treatment upregulated 31 genes and downregulated 4 genes in EECs; it also upregulated 398 genes and downregulated 66 genes in STRs, including implantation-related genes, such as ISG15, OAS1X, OAS1Y, PARP9, PARP14, MX1 and PTGS2. Gene set enrichment analysis revealed that DEGs were enriched in several implantation-related pathways, including ISG15 antivirus mechanisms. These results suggest that, in addition to numerous characterized molecules, phospholipase inhibitory protein is a new candidate molecule in enabling biochemical communication between the conceptus and endometrium.

Keywords: conceptus; phospholipase inhibitor; sheep; uncharacterized genes.

Figure 1

Study design to identify the highly expressed uncharacterized genes in ovine conceptuses during peri-implantation period. The RNA-sequencing data of ovine conceptuses at pregnant days 12, 14, 15, 16, 17, 19, 20 and 21 (data sets from PRJDB11121 and GSE87017) were used in this study. The top 100 expression genes based on transcripts per million value were extracted at each pregnant day. Of these genes, the genes that were not annotated with gene-symbol were subjected to in silico protein function analysis.

Figure 2

Expression of the phospholipase inhibitory genes in ovine conceptus during the peri-implantation period. (A) The expression values were shown as transcripts per million (TPM). (B) RT-qPCR analysis of transcripts encoding LOC101108413 and LOC101111695 in ovine conceptuses at pregnant days 15, 17, 19 and 21. RNA was extracted from frozen conceptuses (n = 3/day). Data relative to the value of day 15 are shown. The values represent each sample. The values with different letters (a and b) are significantly different (P < 0.05).

Figure 3

Schematic illustration of the phospholipase inhibitory gene structures. The predicted protein domains are shown in blue (Phospholipase A2 inhibitor, N-terminal domain, InterPro ID: IPR004126) and red (Ly-6 antigen/uPA receptor-like, InterPro ID: IPR016054), respectively.

Figure 4

Transcript expression profiles of cultured bovine endometrial epithelial cells (EEC) and stromal cells (STRs) treated with or without the phospholipase inhibitory recombinant protein. (A) Principal component analysis of the transcripts of the cultured bovine EECs and STRs treated with or without the phospholipase inhibitory recombinant protein. (B) Volcano plot analysis of the differentially expressed transcripts. Adjusted P-value (padj) <0.05 was considered as significantly different. The blue and red dots represent significantly decreased and increased transcripts, respectively.

Figure 5

Gene set enrichment analysis of the differentially expressed genes in the cultured bovine endometrial epithelial cells (EECs) and stromal cells (STRs) treated with or without the phospholipase inhibitory recombinant protein. Enriched pathways are shown. The larger circles indicate a higher percentage of annotated genes. The color of the circles represents the number of significantly differentially expressed genes in each pathway, with blue indicating a higher number and black indicating a lower number. Details are shown in Supplemental Table 4.