Differential release of cell-signaling metabolites by male and female bovine embryos cultured in vitro

Highlights

• Male and female blastocysts release different metabolites with potential signaling effects.

• Genes previously identified in endometrial cells can be responsive to such metabolites.

• Metabolic differences between male and female embryos may help design culture media adapted to the embryonic sex.

Abstract

Male and female early bovine embryos show dimorphic transcription that impacts metabolism. Individual release of metabolites was examined in a 24h single culture medium from Day-6 male and female morulae that developed to Day-7 expanded blastocysts. Embryos were produced in vitro, fertilized with a single bull and cultured in SOFaaci+6  g/L BSA. The embryonic sex was identified (amelogenin gene amplification). Embryos (N = 10 males and N = 10 females) and N = 6 blank samples (i.e. SOFaaci+6  g/L BSA incubated with no embryos) were collected from 3 replicates. Metabolome was analyzed by UHPLC-TOF-MS in spent culture medium. After tentative identification, N = 13 metabolites significantly (P < 0.05; ANOVA) differed in their concentrations between male and female embryos, although N = 10 of these metabolites showed heterogeneity (Levene's test; P > 0.05). LysoPC(15:0) was the only metabolite found at higher concentration in females (fold change [FC] male to female = 0.766). FC of metabolites more abundant in male culture medium (N = 12) varied from 1.069 to 1.604. Chemical taxonomy grouped metabolites as amino-acids and related compounds (DL-2 aminooctanoic acid, arginine, 5-hydroxy-l-tryptophan, and palmitoylglycine); lipids (2-hexenoylcarnitine; Lauroyl diethanolamide; 5,6 dihydroxyprostaglandin F1a; LysoPC(15:0); DG(14:0/14:1(9Z)/0:0) and triterpenoid); endogenous amine ((S)-N-Methylsalsolinol/(R)-N-Methylsalsolinol); n-acyl-alpha-hexosamine (N-acetyl-alpha-d-galactosamine 1-phosphate); and dUMP, a product of pyrimidine metabolism. Among the compounds originally contained in CM, female embryos significantly depleted more arginine than males and blank controls (P < 0.001). Male and female embryos induce different concentrations of metabolites with potential signaling effects. The increased abundance of metabolites released from males is consistent with the higher metabolic activity attributed to such blastocysts.

Introduction

The bovine endometrium partially responds quantitatively to the presence of embryos [1] and soon becomes sensitive to embryonic sex in vivo [2] and in vitro [3], sugestting that differential release of molecules depending on embryonic sex may occur as shown with miRNAs [3]. Male and female early bovine embryos differ not only in sex chromosomes, but also in the somatic complement, which shows dimorphic transcription [[4], [5], [6]]. Sexual dimorphism affects metabolisms of carbohydrates, amino-acid, mitochondria and the overall metabolomics fingerprint [[7], [8], [9], [10], [11], [12]]. Biological metabolites are generally small molecules (SMs) (<900 Da) that may use passive and transport-mediated mechanisms to cross cell membranes [13] and reach intracellular sites of action [14,15]. SMs are included in the repertory of compounds released by embryos [16] and they can inhibit specific functions of multifunctional proteins or disrupt interactions between proteins [17] and modify gene expression [18]. Their involvement in cell signaling, together with the rapid responses they can elicit, make SMs good candidates for exerting a role within early embryo maternal interactions. Therefore, the study of the metabolome released by the embryo is of maximal interest within maternal interactions.

In this study, we analyzed whether or not embryonic sex determines differential release of metabolites to culture medium (CM) by single embryos in vitro using UHPLC-MS. The information collected may shed light on mechanisms of endometrial sex recognition at early stages.