Paillamanque Joaquín, Madrid Cristian, Carmona Emerson M, Osses Nelson, Moreno Ricardo D, Oresti Gerardo M, Pino José A, Reyes Juan G
Instituto de Química, Pontificia Universidad Católica de Valparaíso, Valparaíso, Chile.
Departamento de Fisiología, Pontificia Universidad Católica de Chile, Santiago, Chile.
PLoS One. 2016 Jul 18;11(7):e0158518. doi: 10.1371/journal.pone.0158518. eCollection 2016.
The aim of this work was to explore the ability of free arachidonic acid, palmitic acid and the unsaturated fatty acids oleic acid and docosahexaenoic acid to modify calcium homeostasis and mitochondrial function in rat pachytene spermatocytes and round spermatids. In contrast to palmitic acid, unsaturated fatty acids produced significant increases in intracellular calcium concentrations ([Ca2+]i) in both cell types. Increases were fatty acid specific, dose-dependent and different for each cell type. The arachidonic acid effects on [Ca2+]i were higher in spermatids than in spermatocytes and persisted when residual extracellular Ca2+ was chelated by EGTA, indicating that the increase in [Ca2+]i originated from release of intracellular calcium stores. At the concentrations required for these increases, unsaturated fatty acids produced no significant changes in the plasma membrane potential of or non-specific permeability in spermatogenic cells. For the case of arachidonic acid, the [Ca2+]i increases were not caused by its metabolic conversion to eicosanoids or anandamide; thus we attribute this effect to the fatty acid itself. As estimated with fluorescent probes, unsaturated fatty acids did not affect the intracellular pH but were able to induce a progressive decrease in the mitochondrial membrane potential. The association of this decrease with reduced reactive oxygen species (ROS) production strongly suggests that unsaturated fatty acids induced mitochondrial uncoupling. This effect was stronger in spermatids than in spermatocytes. As a late event, arachidonic acid induced caspase 3 activation in a dose-dependent manner both in the absence and presence of external Ca2+. The concurrent but differential effects of unsaturated fatty acids on [Ca2+]i and mitochondrial functions are additional manifestations of the metabolic changes that germ cells undergo during their differentiation.
本研究旨在探讨游离花生四烯酸、棕榈酸以及不饱和脂肪酸油酸和二十二碳六烯酸对大鼠粗线期精母细胞和圆形精子细胞钙稳态及线粒体功能的影响。与棕榈酸不同，不饱和脂肪酸可使两种细胞类型的细胞内钙浓度（[Ca2+]i）显著升高。这种升高具有脂肪酸特异性、剂量依赖性，且在不同细胞类型中有所差异。花生四烯酸对[Ca2+]i的影响在精子细胞中高于精母细胞，并且在EGTA螯合残余细胞外Ca2+时仍然存在，这表明[Ca2+]i的升高源于细胞内钙库的释放。在产生这些升高所需的浓度下，不饱和脂肪酸对生精细胞的质膜电位或非特异性通透性没有显著影响。对于花生四烯酸而言，[Ca2+]i的升高并非由其代谢转化为类花生酸或花生四烯乙醇胺所致；因此，我们将这种效应归因于脂肪酸本身。用荧光探针估计，不饱和脂肪酸不影响细胞内pH，但能够诱导线粒体膜电位逐渐降低。这种降低与活性氧（ROS）产生减少相关，强烈提示不饱和脂肪酸诱导了线粒体解偶联。这种效应在精子细胞中比在精母细胞中更强。作为后期事件，花生四烯酸在有无细胞外Ca2+的情况下均以剂量依赖性方式诱导半胱天冬酶3激活。不饱和脂肪酸对[Ca2+]i和线粒体功能的同时但不同的影响是生殖细胞在分化过程中所经历的代谢变化的额外表现。
