Effects of Fatty Acids on Intracellular [Ca2+], Mitochondrial Uncoupling and Apoptosis in Rat Pachytene Spermatocytes and Round Spermatids.

Joaquín Paillamanque,Nelson Osses,Cristian Madrid,Gerardo M. Oresti,José A. Pino,Ricardo D. Moreno,Emerson M. Carmona,Juan G. Reyes,David Holowka

doi:10.1371/journal.pone.0158518

Abstract

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.

Highlights

IntroductionThe functional relationship between germ cells and Sertoli cells (SC) in the mammalian seminiferous tubules occurs either through juxtacrine signalling (adhesion molecules) or paracrine signalling (molecules secreted into the extracellular space of the adluminal compartment) [1]
The functional relationship between germ cells and Sertoli cells (SC) in the mammalian seminiferous tubules occurs either through juxtacrine signalling or paracrine signalling [1]
Our results demonstrated that the changes in [Ca2+]i induced by FA in spermatogenic cells were not associated with entry of Ca2+, the existence of a described voltage-dependence of some GPCRs [46] and voltage-sensing phosphoinositide phosphatases [47] prompted us to look for arachidonic acid (AA)-induced changes in plasma membrane potential

Summary

Introduction

The functional relationship between germ cells and Sertoli cells (SC) in the mammalian seminiferous tubules occurs either through juxtacrine signalling (adhesion molecules) or paracrine signalling (molecules secreted into the extracellular space of the adluminal compartment) [1]. Since the glycolytic activity of SC is stimulated by follicle stimulating hormone (FSH), β-adrenergic agonists, interleukin 1 β (IL1-β) and tumour necrosis factor α (TNF-α) [4,5,6,7], a similar, though inverse, regulation of lactate and glucose secretion toward the adluminal compartment is expected. Accumulated evidence strongly suggests that AA is part of the regulatory signalling networks regulating spermatogenesis. In support of this idea, a diet rich in AA has a large impact on animal fertility [15] and mice lacking the group VI PLA2 isoform show impaired sperm motility and greatly reduced fertility [16]

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