'Conceptualizing' the Endometrium: Identification of Conceptus-Derived Proteins During Early Pregnancy in Cattle

Abstract

The aim of this study was to identify conceptus-derived proteins, in addition to IFNT, that may facilitate pregnancy recognition in cattle. Analysis of the protein content of the uterine luminal fluid (ULF) from cyclic heifers on Day 16 by nano liquid chromatography tandem mass spectrometry identified 334 proteins. Comparison of these data with 299 proteins identified in the ULF of pregnant heifers on Day 16 identified 85 proteins only present in the ULF of pregnant heifers. Analysis of Day 16 conceptus-conditioned culture medium revealed the presence of 1005 proteins of which 30 proteins were unique to ULF from Day 16 pregnant heifers. Of these 30 proteins, 12 had mRNA expression values at least 2-fold higher in abundance (P < 0.05) in the conceptus compared to the endometrium (ARPC5L, CAPG, CKMT1, CSTB, HSPA8, HSPE1, LGALS3, MSN, NUTF2, P4HB, PRKAR2A, TKT) as determined by RNA sequencing. In addition, genes that have a significant biological interaction with the proteins (ACO2, CKMT1, CSTB, EEF2, GDI1, GLB1, GPLD1, HNRNPA1, HNRNPA2B1, HNRNPF, HSPA8, HSPE1, IDH2, KRT75, LGALS3, MSN, NUTF2, P4HB, PRKAR2A, PSMA4, PSMB5, PSMC4, SERPINA3, TKT) were differentially expressed in the endometrium of pregnant compared to cyclic heifers during the pregnancy recognition period (Days 16-18). These results indicate that 30 proteins unique to ULF from pregnant heifers and produced by short-term in vitro cultured Day 16 conceptuses could potentially be involved in facilitating the interactions between the conceptus and the endometrium during the pregnancy recognition period.

Keywords: conceptus; endometrium; gene expression; proteomics; uterine luminal fluid.

FIG. 1

A comparison of the number of proteins identified in the ULF of cyclic (n = 4) and confirmed pregnant heifers (n = 4) on Day 16 (Day 0 = estrus). Proteins were determined by total peptide counts following nano liquid chromatography tandem mass spectrometry (nano-LC-MS/MS) and had to be present in at least three out of four heifers in each group. The subsets of proteins listed are on the basis of those described previously as localizing to luminal epithelium/glandular epithelium via in situ hybridization and/or are known in other species to be components of ULF. Full details of all the proteins are listed in Supplemental Table S2, A–C.

FIG. 2

RNA sequencing data for genes whose proteins were detected in ULF from both cyclic and pregnant heifers on Day 16, with largest fold change difference in either the endometrium compared to the conceptus (A) or vice versa (B, C). Gene expression values in the endometrium (n = 5, black bars) and conceptus (n = 5, open bars) are given in average transcripts per million (T.P.M. ± SEM). Significant differences (P < 0.05) in expression values between the endometrium and conceptus are denoted by an asterisk.

FIG. 3

RNA sequencing data for genes whose proteins were detected in ULF only from pregnant heifers on Day 16 of pregnancy and in Day 16 conceptus-cultured medium, with largest fold change differences in the endometrium compared to the conceptus (A) or vice versa (B). Gene expression values in the endometrium (n = 5, black bars) and conceptus (n = 5, open bars) are given in average transcripts per million (T.P.M. ± SEM). Significant differences (P < 0.05) in the expression values between the endometrium and conceptus are denoted by an asterisk.

FIG. 4

Venn diagram comparing the number of proteins identified in ULF from cyclic (n = 4) and confirmed pregnant (n = 4) heifers on Day 16 (Day 0 = estrus) as well as Day 16 conceptus-conditioned cultured medium after culture for 6 h (A) or 24 h (B). Proteins were determined by total peptide counts following nano liquid chromatography tandem mass spectrometry (nano-LC-MS/MS) and had to be present in at least three of four heifers in each group. The 30 proteins listed were those identified only in pregnant ULF (i.e., not cyclic) on Day 16 and in spent culture medium. Full details of all the proteins are listed in Supplemental Tables S3 and S4.