6 Decelerating embryo development? Characterisation of the uterine environment in European roe deer (Capreolus capreolus) during diapause

The early developing embryo faces a continuously changing microenvironment to supports its growth. In the European roe deer, this environment accompanies embryonic diapause, a period of up to 4 months in which fertilization and subsequent implantation are decoupled. Diapause is characterised by a deceleration of embryonic growth. In most ruminants such as cattle and sheep, interferon tau (IFNt) plays a major role in maternal recognition of pregnancy. Uniquely to ruminants, the roe deer embryo does not secrete IFNt. The roe deer was used as a model species to gain insights into the changing uterine environment devoid of IFNt that supports prolonged decelerated embryo development, resumption of developmental velocity, and subsequent implantation. Uterine fluid samples from 188 female does were collected during regular huntings between September and January, and 4 developmental stages-blastocysts at early, mid, and late diapause and elongated embryos (16, 57, 97, and 18 does per developmental stage, respectively)-were defined. The developmental stages were assigned based on morphological characteristics of the embryo and the embryonic genomic DNA content. For the analysis of amino acids (AA), all 188 uterine fluid samples were subjected to targeted liquid chromatography-tandem mass spectrometry. Almost all AA increased over the course of embryo development. Although most AA showed developmental stage-specific concentration peaks, serine, glycine, alanine, glutamate, and glutamine were most abundantly present irrespective of the developmental progression. For the analysis of the protein abundances in the uterine fluid in a selected subset of samples (n=5 per developmental stage), holistic liquid chromatography-tandem mass spectrometry identified and quantified a total of 819 proteins with a false discovery rate of <1%. Comparison between the developmental stages revealed 106 differentially abundant proteins. Most changes in protein abundance that occurred related to embryo elongation. Interestingly, 713 proteins remained stable during embryo development, indicating that these proteins may contribute to prolonged embryo survival during embryonic diapause. The differentially abundant proteins were clustered with DAVID Bioinformatics Resources 6.8 (https://david.ncifcrf.gov/). The most enriched clusters were cell-cell adhesion, biosynthesis of AA and carbon metabolism, microtubule, structural molecule activity, and chaperone binding. The ongoing detailed identification of stably abundant proteins will advance our basic understanding of the embryos’ needs for sustained survival during prolonged decelerated development. In addition, a comparison with the protein abundances around the time of maternal recognition of pregnancy in other species could advance our knowledge on conserved proteins that support embryo development and establishment of pregnancy in mammals. Our findings may contribute to defining optimal in vitro embryo culture conditions in a species-independent manner and potentially identify factors capable of halting embryo development.