Acetylcholine/acetylcholine esterase and necroptosis in the ovary

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Acetylcholine (ACh) is produced in the ovary and exerts trophic actions, which are mediated by receptors. Recent studies on the cholinergic system of the ovary revealed that ACh-esterase (AChE) is also produced with the ovary, specifically by granulosa cell (GCs) and that it counteracts the trophic actions of ACh. AChE can be blocked by pharmacological intervention in cultured human GCs. Furthermore, programmed cell death (necroptosis) was identified in cultured human GCs as an unknown form of cell death, and was linked to a non-enzymatic action of an AChE splice variant, AChE-R. This variant and clear evidence for necroptotic cell death in follicles and the corpus luteum (CL) were found in vivo, in the human and non-human primate ovary. An AChE-R peptide strongly enhanced necroptosis of human GCs, and on the other side, necroptotic cell death of GCs is amenable to pharmacological blockage by necrostatin-1 and necrosulfonamide. Thus, both ACh-E-blockers and necroptosis-blockers may be novel tools to interfere with important ovarian processes (follicular growth, follicular atresia and/or luteolysis). First studies in rats, in which an AChE blocker was applied locally to the ovary, are in full support for a role of ACh in the promotion of follicular growth. It is possible that besides normal ovarian events, ovarian pathologies, or the outcome of follicular cultures could be targeted. We now propose a series of studies to examine these possibilities. We will analyze sites of necrotic/necroptotic events and their frequency in human and non-human primate ovary by immunohistochemistry, using a novel marker (p-MLKL). Apoptosis markers will allow us to distinguish between necroptosis and apoptosis. Since AChE-R can induce necroptosis in GCs, we will study, whether AChE-R expression correlates with p-MLKL in ovarian sections. Studies in human GC-tumors will be performed as well. To explore regulation and actions of AChE-R, we will use human IVF-derived GCs. We will among others examine a role of miRNA132 and its hormonal regulation and will attempt to elucidate the mechanisms of action of ARP-induced necroptosis. Finally, through international collaboration we will examine, whether pharmacological intervention with necroptosis improves the survival of cultured monkey follicles and may influence follicular fate in rat. We expect important translational insights into an unexplored form of cell death and its role in ovarian physiology and pathology, insighty which may open novel doors to interfere with ovarian events.