Conceptus metabolomic profiling reveals stage-specific phenotypes leading up to pregnancy recognition in cattle†

Abstract

Reproductive efficiency in livestock is a major driver of sustainable food production. The poorly understood process of ruminant conceptus elongation (a) prerequisites maternal pregnancy recognition, (b) is essential to successful pregnancy establishment, and (c) coincides with a period of significant conceptus mortality. Conceptuses at five key developmental stages between Days 8-16 were recovered and cultured in vitro for 6 h prior to conditioned media analysis by untargeted ultrahigh-performance liquid chromatography tandem mass spectroscopy. This global temporal biochemical interrogation of the ex situ bovine conceptus unearths two antithetical stage-specific metabolic phenotypes during tubular (metabolically retentive) vs. filamentous (secretory) development. Moreover, the retentive conceptus phenotype on Day 14 coincides with an established period of elevated metabolic density in the uterine fluid of heifers with high systemic progesterone-a model of accelerated conceptus elongation. These data, combined, suggest a metabolic mechanism underpinning conceptus elongation, thereby enhancing our understanding of the biochemical reciprocity of maternal-conceptus communication, prior to maternal pregnancy recognition.

Keywords: bovine; conceptus; development; elongation; embryo; metabolomics.

Figure 1

Experimental design and conceptus elongation validation. (A) Schematic depiction of the animal synchronization protocol for embryo and conceptus recovery wherein “Σ(ET)” denotes the total number of embryos transferred per cohort and “n” denotes the number of recipient heifers per cohort. Percentages denote the proportion of conceptuses recovered. (B) Diagrammatical representation of embryo and conceptus culture design providing biological (n = 5) replicate values per day in addition to the number of conceptuses pooled (n = 1–14) per replicate. (C) Representative images of recovered embryos/conceptuses (not to scale). Mean (±SEM) total protein concentration per embryo or conceptus per day on (D) logarithmic and (E) linear axes (R² = 0.976). Abbreviations: progesterone releasing intra-vaginal device (PRID), gonadotropin releasing hormone (GnRH), and prostaglandin F2 alpha (PGF2α), embryo transfer (ET).

Figure 2

Qualitative metabolomic analyses of conceptus biochemical turnover. (A-E) Venn diagrams depicting the number of metabolites unique and common to Days 8–16 CCM vs. SOF vs. ULF. Asterisks denote data from [24]. Metabolite distributions by super-pathway are also provided for CCM (A-E), SOF (F), and ULF (G) in terms of proportion (%) of total identified metabolites. (H) Number of metabolites unique and common to CCM from Day 8 vs. 10 vs. 12 vs. 14 vs. 16 conceptuses. (I) List of conceptus-derived metabolites (i.e., neither identified in SOF nor ULF) identified in CCM on Days 8, 10, 12, 14, and 16, wherein green and gray indicate present or not detected, respectively. Metabolite names are provided for the 14 metabolites produced by conceptuses on all days studied. Complete datasets are provided as Supplementary Material. Abbreviations: Day 8-16 (D8-16), conceptus-conditioned medium (CCM), synthetic oviduct fluid (SOF), and uterine luminal fluid (ULF).

Figure 3

Conceptus-normalized semi-quantitative metabolomics. (A) Principal component analysis of biochemical profiles of synthetic oviduct fluid (SOF) and conceptus conditioned medium (CCM) by developmental stage (Days 8–16). Relative mean (±SEM) concentrations (n = 5 per group) of (B) all metabolites, (C) amino acid, (D) carbohydrate, (E) cofactor and vitamin, (F) energy substrate, (G) lipid, (H) nucleotide, (I) peptide, and (J) xenobiotic related metabolites (vertical axes) by day (horizontal axes). Numbers of identified metabolites within each group are provided within the upper frame of each graph. Differences determined by ordinary one-way analysis of variance coupled to a Tukey non-parametric post hoc test (^**** = P ≤ 0.0001, ^*** = P ≤ 0.001, ^** = P ≤ 0.01, and ^* = P ≤ 0.05). (K) Heat-maps of metabolite fold-changes about the mean by sub-pathway across days. Green cells denote a lack of identified metabolites, whereas blue to red (bottom-right) denotes the scaled intensity of the change. Additional abbreviations: s-adenosyl methionine (SAM), tricarboxylic acid cycle (TCA), and branch-chain amino acids (BCAA).

Figure 4

Protein-normalized semi-quantitative metabolomics. (A) Principal component analysis of biochemical profiles of synthetic oviduct fluid (SOF) and conceptus conditioned medium (CCM) by developmental stage (Days 8–16). Relative mean (±SEM) concentrations (n = 5 per group) of (B) all metabolites, (C) amino acid, (D) carbohydrate, (E) cofactor and vitamin, (F) energy substrate, (G) lipid, (H) nucleotide, (I) peptide, and (J) xenobiotic related metabolites (vertical axes) by day (horizontal axes). Numbers of identified metabolites within each group are provided within the upper frame of each graph. Differences determined by ordinary one-way analysis of variance coupled to a Tukey non-parametric post hoc test (^**** = P ≤ 0.0001, ^*** = P ≤ 0.001, ^** = P ≤ 0.01, and ^* = P ≤ 0.05). (K) Heat-maps of metabolite fold-changes about the mean by sub-pathway across days. Green cells denote a lack of identified metabolites, whereas blue to red (bottom-right) denotes the scaled intensity of the change. Additional abbreviations: s-adenosyl methionine (SAM), tricarboxylic acid cycle (TCA), and branch-chain amino acids (BCAA).

Figure 5

Filamentous vs. tubular conceptus biochemical turnover. Network view of metabolites within corresponding super-pathways and sub-pathways in the conceptus-conditioned media on Day 16 vs. 14 when normalized for total protein. Node diameter and color describe metabolite fold-change magnitude and directionality, respectively: Dark red and dark green respectively indicate an increase or decrease (P ≤ 0.05), whereas pink or light green denote a trend (0.05 < P < 0.10) towards a respective increase or decrease. Gray depicts a lack of a statistically significant difference. Asterisks denote predicted metabolites.

Figure 6

Metabolomic signature of maternal-conceptus communication during conceptus elongation. A schematic depiction of our current understanding of the metabolomics surrounding bovine conceptus elongation. Dashed arrows indicate speculative autocrine and paracrine signaling. Abbreviations: uterine luminal fluid (ULF), Days 8–18 conceptus (D8–D18), oxytocin receptor (OTR), estrogen receptor alpha (ESR1), interferon tau (IFNT), progesterone (P4), and conceptus-normalized (CN).