Maternal Recognition of Pregnancy in the Horse: Are MicroRNAs the Secret Messengers?

Abstract

The signal for maternal recognition of pregnancy (MRP) has still not been identified in the horse. High-throughput molecular biology at the embryo–maternal interface has substantially contributed to the knowledge on pathways affected during MRP, but an integrated study in which proteomics, transcriptomics and miRNA expression can be linked directly is currently lacking. The aim of this study was to provide such analysis. Endometrial biopsies, uterine fluid, embryonic tissues, and yolk sac fluid were collected 13 days after ovulation during pregnant and control cycles from the same mares. Micro-RNA-Sequencing was performed on all collected samples, mRNA-Sequencing on the same tissue samples and mass spectrometry was conducted previously on the same fluid samples. Differential expression of miRNA, mRNA and proteins showed high conformity with literature and confirmed involvement in pregnancy establishment, embryo quality, steroid synthesis and prostaglandin regulation, but the link between differential miRNAs and their targets was limited and did not indicate the identity of an unequivocal signal for MRP in the horse. Differential expression at the embryo–maternal interface was prominent, highlighting a potential role of miRNAs in embryo–maternal communication during early pregnancy in the horse. These data provide a strong basis for future targeted studies.

Spatiotemporal endometrial transcriptome analysis revealed the luminal epithelium as key player during initial maternal recognition of pregnancy in the mare

“…The fact that some classical downstream regulators of E2 and other factors were not found as upregulated or even downregulated or not detectable at mRNA level, suggested that several signaling molecules are involved affecting each other’s downstream regulatory network. For example, ERRFI1, strongly upregulated in LE in this and in a previous study 16 , has been described as critical for implantation in mice by suppressing ESR1 activity in the uterine epithelium and mediating P4′s suppression of E2 signaling during embryo implantation 51 . Furthermore, ERRFI1 is acting as a negative feedback regulator of EGF receptor signaling and is stimulated by epidermal growth factor 52 which mRNA was highly upregulated in LE in P.…”

“…With the advent of new techniques for comprehensive transcriptome analysis, several studies have been performed to analyze endometrial gene expression changes in response to the presence of a conceptus during the period of MRP in the mare, mainly focusing on days 8, 12, 13, 13.5 or 16 11 – 16 . Two studies analyzed endometrial miRNA expression during MRP in addition to mRNAs 13 , 16 , and Smits et al 17 performed also a comparison to gene expression in the embryo and protein expression in the uterine fluid. At day 8 after ovulation, Merkl et al 11 did not identify changes in endometrial gene expression of pregnant versus non-pregnant mares, but changes were evident on day 12.…”

Maternal recognition of pregnancy in the mare: does it exist and why do we care?

“…Many of the proteins identified have putative or reported roles in pregnancy or related processes and could serve as a nidus for countless new ideas, hypotheses and directions of scientific investigation. Meanwhile, integration and co-analysis of these proteomic results with the transcriptomic analyses of the pregnant endometrium ( Gebhardt et al 2012 , Klein et al 2010 , Klein & Troedsson 2011 , Klein 2015 b , Smits et al 2020 ) promise to yield valuable new insights by highlighting specific networks and pathways that appear to become more active in the pregnant state.…”

“…Overlapping genes pointed to conserved mechanisms in early pregnancy but unique features and some surprising findings were revealed for each species. Most recently, Smits et al integrated comprehensive novel miRNA sequencing data of pregnant and control mare endometrium, uterine fluid, embryonic tissue and yolk sac fluid with corresponding transcriptome (mRNA) and proteome datasets ( Smits et al 2020 ) on day 13 post-ovulation. This approach was particularly interesting as it encompassed multiple tissues/fluids, and three levels of the transcription/translation cascade, yielding important information on the origins and regulation of protein expression.…”

Embryo–Maternal Interactions Underlying Reproduction in Mammals

“…Furthermore, two original articles provide new clues about the roles of miRNAs in embryo–maternal communication in the horse. Smits and co-workers [ 21 ] performed an integrated analysis of differential miRNA, mRNA, and protein expression analysis at the embryo–maternal interface. In agreement with previous studies [ 22 , 23 ], the results derived from their study did not point at of an unequivocal signal for MRP in the horse but highlighted a potential role of miRNAs in embryo–maternal communication during pregnancy establishment in the horse.…”