Mechanisms underlying the effect of infection and inflammation on oocyte health and embryo development

Development of a vigorous mammalian embryo depends on ovulation and fertilisation of a competent oocyte from a healthy ovarian follicle. However, uterine bacterial infections or inflammation during follicle growth, oocyte maturation and early embryo development are associated with infertility. The scientific question this project addresses is: How do bacterial infections or inflammation affect oocytes and early embryos? We study cattle because bacterial infection of the uterus is ubiquitous after parturition in this species, and 40% of animals have uterine disease. We identified that cattle with uterine disease have perturbed ovarian function and low conception rates - even after 'successful' treatment. Indeed, dairy cattle have the lowest conception rate of all domestic animals and our CASE partner needs to develop new therapeutics to solve this problem. Host immunity and inflammatory responses are dependent on receptors on host cells recognising pathogen associated molecular patterns (PAMPs). We found that follicular fluid contains PAMPs, and follicle cells express the necessary immune receptors. We believe that the release of PAMPs or the inflammation associated with uterine infection, impairs oocyte maturation and embryo development. The project tests the hypothesis that ovarian follicle, oocyte and early embryo health is perturbed by PAMPs either directly or via inflammatory mediators. We will pursue 4 objectives: 1. Measure the effect of PAMPs and inflammatory mediators on oocytes and embryos in vitro Ovarian follicles, follicle cells, oocytes and IVF blastocysts will be challenged with PAMPs (LPS, LTA, flagellin) or inflammatory mediators in long-term culture, and we will evaluate: - Markers of follicle and oocyte health: steroid secretion (oestradiol, androstenedione, and progesterone), hormone receptor expression (LHR, FSHR, EGFR), and developmental genes (Mater, Zar-1, Mos, BMP15, GDF9, JY-1) - Gene expression of inflammatory mediators: TNF, IL-1, IL6, iNOS, prostaglandins - Embryo cleavage and blastocyst rates following IVF - Cytoskeleton structure of the oocyte and embryo by confocal imaging of trans-zonal projections, nuclear configuration and mitochondria 2. Determine the vulnerability of oocytes and embryos to infection and inflammation in vivo Oocytes collected for IVF and their blastocysts will be treated with a PAMP or a pro-inflammatory cytokine (identified in objective 1) before embryo transfer into normal animals, and the establishment of pregnancy will be monitored. In addition, blastocysts and early embryos will be collected from animals that had a normal postpartum period or uterine disease, and embryo development compared using IHC and gene expression. 3. Identify the cellular pathways activated in response to PAMPs and inflammation Follicle cells, oocytes and embryos will be treated with PAMPs or cytokines. The expression of immune (TLRs, NOD) and endocrine (LHR, FSHR, EGFR) receptors will be measured by qPCR or Western blotting. There appears to be considerable cross talk between the receptor signalling pathways used for reproduction and immunity. So, immune (nuclear factor-kappa B, Caspase-1 and MAPK) and endocrine (cAMP, PKA, MAPK) signalling pathways, and receptor regulators (ARF6, arrestins) will be explored using Western blotting, IP, ELISA and commercial kits. 4. Mechanistic studies to limit the effect of PAMPs and inflammation on fertility At Pfizer and the ILS, the student will test which agonists and inhibitors of cellular pathways limit the detrimental effect of PAMPs or cytokines on the vulnerable stages of oocyte and embryo development. By the end of the studentship we will have answered fundamental questions about how infection, immunity and inflammation affect reproduction. Knowledge of these mechanisms will be exploited by the Pfizer bovine reproduction international research network to select drug targets of clinical relevance for future in-vivo studies.