Díaz-Muñoz Mauricio, de la Rosa Santander Patricia, Juárez-Espinosa Anna Berenice, Arellano Rogelio O, Morales-Tlalpan Verónica
Departamento de Neurobiología Celular y Molecular, Instituto de Neurobiología, Campus UNAM-Juriquilla, Querétaro, QRO, México.
Reprod Biol Endocrinol. 2008 Dec 9;6:60. doi: 10.1186/1477-7827-6-60.
Granulosa cells play an important endocrine role in folliculogenesis. They mobilize Ca2+ from intracellular stores by a coordinated action between 1,4,5 inositol trisphosphate and ryanodine receptors (IP3R and RyR). The aim of this study was to explore the isoforms of IP3Rs expressed in mouse C57BL/6 NHsd granulosa cells, characterizing their intranuclear localization and the relation with other Ca2+-handling proteins.
Ovarian tissue and granulosa cells were analyzed by multiphotonic and confocal microscopy to determine the intracellular presence of IP3R types 1, 2 and 3, RyR, thapsigargin-sensitive Ca2+-ATPase, and endomembranes. Cellular fractionation and Western blot assays were also used to further confirm the nuclear occurrence of the three IP3R isoforms. Free nuclear and cytosolic Ca2+ concentrations were measured using Fluo-4 AM by confocal microscopy.
By using antibodies and specific fluorophores, was shown that granulosa cells endomembranes contain three isoforms of IP3R, the RyR, and the thapsigargin-sensitive Ca2+-ATPase (SERCA). Interestingly, all these proteins were also detected in the nuclear envelope and in well-defined intranuclear structures. Microsomal membranes depicted characteristic bands of the 3 types of IP3R, but also variants of lower molecular weight. Analysis of nuclear membranes and nucleoplasmic fraction confirmed the nuclear localization of the IP3R types 1, 2 and 3. We demonstrated ATP-induced Ca2+ transients in the nuclear and cytoplasmic compartments. Remarkably, the inhibitory effect on ATP-induced Ca2+ mobilization of brefeldin A was more accentuated in the cytoplasm than in the nucleus.
These findings provide evidence that granulosa cells, including nuclei, express the Ca2+-handling proteins that allow Ca2+ mobilization. All three IP3R were also detected in ovarian slices, including the nuclei of granulosa cells, suggesting that these cells use the three IP3R in situ to achieve their physiological responses.
颗粒细胞在卵泡发生过程中发挥重要的内分泌作用。它们通过1,4,5-三磷酸肌醇和兰尼碱受体（IP3R和RyR）之间的协同作用从细胞内储存中动员Ca2+。本研究的目的是探索在小鼠C57BL/6 NHsd颗粒细胞中表达的IP3R亚型，表征其核内定位以及与其他Ca2+处理蛋白的关系。
通过多光子显微镜和共聚焦显微镜分析卵巢组织和颗粒细胞，以确定1型、2型和3型IP3R、RyR、毒胡萝卜素敏感的Ca2+-ATP酶和内膜在细胞内的存在情况。细胞分级分离和蛋白质印迹分析也用于进一步证实三种IP3R亚型在细胞核中的存在。使用Fluo-4 AM通过共聚焦显微镜测量游离的细胞核和细胞质Ca2+浓度。
通过使用抗体和特异性荧光团，显示颗粒细胞内膜含有三种IP3R亚型、RyR和毒胡萝卜素敏感的Ca2+-ATP酶（SERCA）。有趣的是，所有这些蛋白在核膜和明确的核内结构中也被检测到。微粒体膜显示出三种类型IP3R的特征条带，但也有较低分子量的变体。核膜和核质部分的分析证实了1型、2型和3型IP3R的核定位。我们证明了ATP诱导的细胞核和细胞质区室中的Ca2+瞬变。值得注意的是，布雷菲德菌素A对ATP诱导的Ca2+动员的抑制作用在细胞质中比在细胞核中更明显。
这些发现提供了证据，表明颗粒细胞，包括细胞核，表达允许Ca2+动员的Ca2+处理蛋白。在卵巢切片中也检测到所有三种IP3R，包括颗粒细胞的细胞核，这表明这些细胞在原位使用三种IP3R来实现其生理反应。
