Acetylcholine/acetylcholine esterase and necroptosis in the ovary

乙酰胆碱(ACh)是在卵巢中产生的，其营养作用是由受体介导的。最近对卵巢胆碱能系统的研究表明，卵巢也产生乙酰胆碱酯酶(AChE)，尤其是颗粒细胞(GCs)，它能抵消ACh的营养作用。在培养的人GC中，药物干预可以阻断AChE。此外，在培养的人GC中发现了一种未知的细胞死亡形式，并与AChE剪接变异体AChE-R的非酶作用有关。在活体内，在人类和非人类灵长类动物的卵巢中，发现了卵泡和黄体(CL)中的坏死性细胞死亡的明显证据。AChE-R多肽强烈促进人GCs的坏死性下垂，另一方面，GCs的坏死性细胞死亡易被Necrostatin-1和NECS阻断。因此，ACh-E阻滞剂和坏死下垂阻滞剂都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。在大鼠身上进行的第一项研究，将AChE阻滞剂局部应用于卵巢，完全支持ACh在促进卵泡生长中的作用。除了正常的卵巢事件外，卵巢病理或卵泡培养的结果也有可能成为靶点。我们现在提出一系列研究来检验这些可能性。我们将使用一种新的标记物(p-MLKL)，通过免疫组织化学方法分析人类和非人类灵长类卵巢中坏死/坏死性事件的位置及其频率。细胞凋亡标记物将使我们能够区分坏死性下垂和细胞凋亡。由于AChE-R可以诱导GCs的坏死性下垂，我们将研究AChE-R在卵巢切片中的表达是否与p-MLKL相关。对人类GC肿瘤的研究也将进行。为了探索AChE-R的调节和作用，我们将使用人类体外受精来源的GCs。我们将研究miRNA132的作用及其激素调节，并试图阐明ARP诱导的坏死性下垂的作用机制。最后，通过国际合作，我们将检查对坏死性下垂的药物干预是否提高了培养的猴子卵泡的存活率，并可能影响大鼠的卵泡命运。我们期待着对一种未知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，这可能会打开新的大门来干预卵巢事件。

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.