Spatial Characterization of Bioenergetics and Metabolism of Primordial to Preovulatory Follicles in Whole Ex Vivo Murine Ovary.
Spatial Characterization of Bioenergetics and Metabolism of Primordial to Preovulatory Follicles in Whole Ex Vivo Murine Ovary.
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DOI：
10.1095/biolreprod.116.142141
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发表时间：
2016-12
影响因子：
3.6
通讯作者：
Luderer U
中科院分区：
文献类型：
--
作者：
Cinco R;Digman MA;Gratton E;Luderer U
以前的工作特征卵巢生物能量学定义卵泡代谢通过测量代谢副产物在培养基中。然而，培养条件干扰卵泡的天然状态，并且这些方法不能区分卵母细胞或颗粒细胞内发生的代谢。我们应用相量的方法，荧光寿命成像显微镜（相量FLIM）在740 nm的双光子激发检查游离和蛋白结合的烟酰胺腺嘌呤二核苷酸氢化物（NADH）的空间分布在原始通过排卵前阶段的卵泡发育在新鲜的离体小鼠新生儿和促性腺激素刺激青春期前卵巢。我们获得了原始卵泡至初级卵泡的亚细胞分辨率相量FLIM图像，并分别定量了卵母细胞核和卵母细胞胞质的游离/结合NADH比率（相对NADH/NAD+）。我们发现，卵母细胞核游离/结合型NADH的动态变化与原始卵泡到初级卵泡的发育成熟有关。NAD+依赖性去乙酰化酶SIRTUIN 1（SIRT 1）在新生儿卵巢的免疫组化显示，增加SIRT 1在卵母细胞核中的表达呈负相关，减少游离/结合的NADH在原始初级卵泡过渡。我们将这些早期阶段的卵母细胞代谢表征为产生NADH（糖酵解/Krebs）。我们扩展了先前的研究结果，表明卵丘和壁颗粒细胞在次级通过排卵前卵泡的代谢主要是产生NADH（糖酵解/Krebs循环），而卵母细胞代谢主要是消耗NADH（氧化磷酸化）。两者合计，我们的数据特征的动态变化，游离/结合的NADH和SIRT 1表达在早期卵泡发育，并确认从以前的研究结果定义窦和排卵前卵泡代谢的文化。
Previous work characterizing ovarian bioenergetics has defined follicular metabolism by measuring metabolic by-products in culture media. However, culture conditions perturb the native state of the follicle, and these methods do not distinguish between metabolism occurring within oocytes or granulosa cells. We applied the phasor approach to fluorescence lifetime imaging microscopy (phasor FLIM) at 740-nm two-photon excitation to examine the spatial distribution of free and protein-bound nicotinamide adenine dinucleotide hydride (NADH) during primordial through preovulatory stages of follicular development in fresh ex vivo murine neonatal and gonadotropin stimulated prepubertal ovaries. We obtained subcellular resolution phasor FLIM images of primordial through primary follicles and quantified the free/bound NADH ratio (relative NADH/NAD+) separately for oocyte nucleus and oocyte cytoplasm. We found that dynamic changes in oocyte nucleus free/bound NADH paralleled the developmental maturation of primordial to primary follicles. Immunohistochemistry of NAD+-dependent deacetylase SIRTUIN 1 (SIRT1) in neonatal ovary revealed that increasing SIRT1 expression in oocyte nuclei was inversely related to decreasing free/bound NADH during the primordial to primary follicle transition. We characterized oocyte metabolism at these early stages to be NADH producing (glycolysis/Krebs). We extended the results of prior studies to show that cumulus and mural granulosa cell metabolism in secondary through preovulatory follicles is mainly NADH producing (glycolysis/Krebs cycle), while oocyte metabolism is mainly NADH consuming (oxidative phosphorylation). Taken together, our data characterize dynamic changes in free/bound NADH and SIRT1 expression during early follicular development and confirm results from previous studies defining antral and preovulatory follicle metabolism in culture.