Acetylcholine/acetylcholine esterase and necroptosis in the ovary

乙酰胆碱（ACh）在卵巢中产生并发挥营养作用，这是由受体介导的。最近对卵巢胆碱能系统的研究表明，乙酰胆碱酯酶（AChE）也与卵巢一起产生，特别是由颗粒细胞（GCs）产生，并抵消乙酰胆碱酯酶的营养作用。乙酰胆碱酯酶可通过药物干预阻断培养的人GCs。此外，程序性细胞死亡（坏死性死亡）在培养的人类GCs中被鉴定为一种未知的细胞死亡形式，与乙酰氨基乙酰酯剪接变体AChE- r的非酶作用有关。在人类和非人灵长类动物的卵巢中，发现了这种卵泡和黄体（CL）中坏死细胞死亡的变异和明确证据。AChE-R肽可增强人GCs的坏死坏死，另一方面，GCs的坏死细胞死亡可被坏死他汀-1和坏死磺酰胺阻断。因此，乙酰胆碱受体阻滞剂和坏死因子阻滞剂可能是干扰重要卵巢过程（卵泡生长、卵泡闭锁和/或黄体溶解）的新工具。首先在大鼠身上进行的研究中，乙酰胆碱酯受体阻滞剂局部应用于卵巢，充分支持乙酰胆碱在促进卵泡生长中的作用。除了卵巢正常事件外，卵巢病变或卵泡培养的结果也可能是目标。我们现在提出一系列的研究来检验这些可能性。我们将使用一种新的标记（p-MLKL），通过免疫组织化学分析人类和非人类灵长类动物卵巢中坏死/坏死事件的部位及其频率。细胞凋亡标志物将使我们能够区分坏死下垂和细胞凋亡。由于AChE-R可诱导GCs坏死下垂，我们将研究AChE-R表达是否与卵巢切片p-MLKL相关。人类gc肿瘤的研究也将进行。为了探索AChE-R的调控和作用，我们将使用人类体外受精衍生的gc。我们将研究miRNA132及其激素调节的作用，并试图阐明arp诱导的坏死性坏死的作用机制。最后，通过国际合作，我们将研究药物干预坏死性下垂是否能提高培养猴卵泡的存活率，并可能影响大鼠卵泡的命运。我们期待对一种未被探索的细胞死亡形式及其在卵巢生理和病理中的作用的重要翻译见解，这种见解可能为干扰卵巢事件打开新的大门。

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.