Developement of Diagnostic Tools to Decipher Para- and Endocrine Effects of IFN Tau

Abstract

Reproductive success is indispensable for efficient animal husbandry of modern dairy farms. Nevertheless, embryonic losses occur in every second cow during the first three weeks of gestation. The comprehension of key factors involved in the reproduction events is thus a prerequisite to reveal dysregulations. The protein Interferon tau (IFN tau) is the major pregnancy recognition signal in ruminants. It is not only responsible to extend the luteal life span but also involved in establishment of uterine receptivity, embryo elongation, nutrient supply and antiviral protection. While IFN tau mainly acts in a paracrine manner via interferon receptor signalling on endometrial cells, endocrine actions in peripheral blood cells, corpus luteum and liver have also been described. As IFN tau is solely produced in trophoblast tissue during the critical pregnancy recognition phase and as its production is influenced by embryo characteristics and culture conditions, it may be an interesting molecular marker for pregnancy and embryo monitoring. Deeper understanding of paracrine and endocrine actions of IFN tau may therefore not only help to understand its influence in embryo-maternal crosstalk but also its potential as a biomarker. Therefore, the objective of this thesis was to provide the prerequisite for an in-depth investigation of IFN tau and its effects. For this purpose, a highly sensitive enzyme-linked Immunosorbent Assay (ELISA) for IFN tau quantification was developed and validated as well as a system to sense minute IFN activity. To monitor endocrine IFN tau or pregnancy induced effects, miRNA expression in milk was analysed and evaluated regarding its use for a bovine pregnancy detection system. The ELISA was established with polyclonal and monoclonal antibodies. They were generated in rabbits and rodents, respectively. Recombinantly produced IFN tau and IFN tau specific peptides were used as antigens. For IFN tau activity determination endometrial stroma cells (SC) were isolated, and cellular response to IFN tau exposure was determined via mRNA expression changes of ISG15, MX2 and OAS1. Both assays were biologically validated in a SC-blastocyst co-culture by determination of sex-dependent IFN tau production. The miRNA expression profiles of milk cells, skim milk and whole milk of pregnant and non-pregnant cows were determined using high-throughput sequencing and validated by RT-qPCR. The limit of detection of the IFN tau-ELISA was as low as 7 pg/ml in uterine flushings and embryo culture supernatants, which is lower than in any reported assay. The SC reacted to actions of only 0.4 pg/ml IFN tau. We thereby evidenced five times higher IFN tau production in female than in male blastocysts. Furthermore, the IFN tau activity, mRNA and protein expression were significantly correlated. The indirect analysis of endocrine IFN tau action, using miRNA expression changes in milk, on the other hand only displayed minor miRNA regulations with high variances that could not be validated in biological replicates. In conclusion, the miRNA analysis in milk lagged behind expectations, while the ELISA and SC culture form an excellent fundament for in-depth investigation of IFN tau and embryo development.