Cinco Rachel, Digman Michelle A, Gratton Enrico, Luderer Ulrike
Department of Developmental and Cell Biology, University of California Irvine, Irvine, California.
Laboratory for Fluorescence Dynamics, University of California Irvine, Irvine, California.
Biol Reprod. 2016 Dec;95(6):129. doi: 10.1095/biolreprod.116.142141. Epub 2016 Sep 28.
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.
以往对卵巢生物能量学的研究通过测量培养基中的代谢副产物来定义卵泡代谢。然而，培养条件会干扰卵泡的天然状态，而且这些方法无法区分卵母细胞或颗粒细胞内发生的代谢。我们应用相量法进行740纳米双光子激发的荧光寿命成像显微镜（相量FLIM），以检查新鲜离体小鼠新生卵巢和促性腺激素刺激的青春期前卵巢中卵泡从原始阶段到排卵前阶段发育过程中游离和蛋白结合的烟酰胺腺嘌呤二核苷酸（NADH）的空间分布。我们获得了从原始卵泡到初级卵泡的亚细胞分辨率相量FLIM图像，并分别对卵母细胞核和卵母细胞质中的游离/结合NADH比率（相对NADH/NAD+）进行了量化。我们发现，卵母细胞核中游离/结合NADH的动态变化与原始卵泡到初级卵泡的发育成熟过程平行。新生卵巢中NAD+依赖性脱乙酰酶SIRTUIN 1（SIRT1）的免疫组织化学显示，在原始卵泡到初级卵泡的转变过程中，卵母细胞核中SIRT1表达的增加与游离/结合NADH的减少呈负相关。我们将这些早期阶段的卵母细胞代谢特征描述为产生NADH（糖酵解/三羧酸循环）。我们扩展了先前研究的结果，表明在次级卵泡到排卵前卵泡中，卵丘和壁层颗粒细胞的代谢主要是产生NADH（糖酵解/三羧酸循环），而卵母细胞的代谢主要是消耗NADH（氧化磷酸化）。综上所述，我们的数据描述了卵泡早期发育过程中游离/结合NADH和SIRT1表达的动态变化，并证实了先前关于培养中窦状卵泡和排卵前卵泡代谢的研究结果。
