Drosophila mitoferrin is essential for male fertility: evidence for a role of mitochondrial iron metabolism during spermatogenesis

<p>Abstract</p> <p>Background</p> <p>Mammals and <it>Drosophila melanogaster </it>share some striking similarities in spermatogenesis. Mitochondria in spermatids undergo dramatic morphological changes and syncytial spermatids are stripped from their cytoplasm and then individually wrapped by single membranes in an individualization process. In mammalian and fruit fly testis, components of the mitochondrial iron metabolism are expressed, but so far their function during spermatogenesis is unknown. Here we investigate the role of <it>Drosophila </it>mitoferrin (dmfrn), which is a mitochondrial carrier protein with an established role in the mitochondrial iron metabolism, during spermatogenesis.</p> <p>Results</p> <p>We found that P-element insertions into the 5'-untranslated region of the <it>dmfrn </it>gene cause recessive male sterility, which was rescued by a fluorescently tagged transgenic <it>dmfrn </it>genomic construct (<it>dmfrn^venus</it>). Testes of mutant homozygous <it>dmfrn^SH115</it>flies were either small with unorganized content or contained some partially elongated spermatids, or testes were of normal size but lacked mature sperm. Testis squashes indicated that spermatid elongation was defective and electron micrographs showed mitochondrial defects in elongated spermatids and indicated failed individualization. Using a <it>LacZ </it>reporter and the <it>dmfrn^venus</it>transgene, we found that dmfrn expression in testes was highest in spermatids, coinciding with the stages that showed defects in the mutants. Dmfrn-venus protein accumulated in mitochondrial derivatives of spermatids, where it remained until most of it was stripped off during individualization and disposed of in waste bags. Male sterility in flies with the hypomorph alleles <it>dmfrn^BG00456</it>and <it>dmfrn^EY01302</it>over the deletion <it>Df(3R)ED6277 </it>was increased by dietary iron chelation and suppressed by iron supplementation of the food, while male sterility of <it>dmfrn^SH115/Df(3R)ED6277 </it>flies was not affected by food iron levels.</p> <p>Conclusions</p> <p>In this work, we show that mutations in the <it>Drosophila </it>mitoferrin gene result in male sterility caused by developmental defects. From the sensitivity of the hypomorph mutants to low food iron levels we conclude that mitochondrial iron is essential for spermatogenesis. This is the first time that a link between the mitochondrial iron metabolism and spermatogenesis has been shown. Furthermore, due to the similar expression patterns of some mitochondrial iron metabolism genes in <it>Drosophila </it>and mammals, it is likely that our results are applicable for mammals as well.</p

Genome-wide association analyses of physical activity and sedentary behavior provide insights into underlying mechanisms and roles in disease prevention

Although physical activity and sedentary behavior are moderately heritable, little is known about the mechanisms that influence these traits. Combining data for up to 703,901 individuals from 51 studies in a multi-ancestry meta-analysis of genome-wide association studies yields 99 loci that associate with self-reported moderate-to-vigorous intensity physical activity during leisure time (MVPA), leisure screen time (LST) and/or sedentary behavior at work. Loci associated with LST are enriched for genes whose expression in skeletal muscle is altered by resistance training. A missense variant in ACTN3 makes the alpha-actinin-3 filaments more flexible, resulting in lower maximal force in isolated type IIA muscle fibers, and possibly protection from exercise-induced muscle damage. Finally, Mendelian randomization analyses show that beneficial effects of lower LST and higher MVPA on several risk factors and diseases are mediated or confounded by body mass index (BMI). Our results provide insights into physical activity mechanisms and its role in disease prevention.publishedVersionPeer reviewe

Mitochondrial Iron Metabolism : Study of mitoferrin in Drosophila melanogaster

Iron has a dualistic character. On the one hand it is essential for the life of most organisms, on the other hand it is involved in the generation of reactive oxygen species that are implicated in diseases and aging. During evolution efficient mechanisms for uptake, handling and storage of iron in a safe way have developed to keep the balance between iron availability and minimizing the hazards. In eukaryotes, mitochondria are the central organelle for “metabolizing” iron and consequently play an important role in cellular iron homeostasis. Mitoferrins are mitochondrial carrier proteins, which are involved in iron transport into mitochondria. In vertebrates two mitoferrins exist, one (mitoferrin1) of which is essential for heme synthesis during erythropoiesis, while the function of the other (mitoferrin2) is not well defined. In the fruit fly we found only one mitoferrin gene (dmfrn), which codes most likely for a functional homologueof vertebrate mitoferrin2. In Drosophila cell culture, dmfrn overexpression resulted in an overestimation of cell sensed iron levels. The signal responsible for this, is most likely a yet unidentified compound of ISC synthesis. In the cell culture system we also showed that iron chelation blocks the progression of the cell cycle in a reversible and therefore most likely controlled way. Study of different dmfrn mutants indicates a role of dmfrn during spermatogenesis and development to adulthood. As dmfrn deletion mutants are not lethal, it is likely that other lower affinity iron transporters exist. A similar conclusion has been drawn by others from the study of yeast mitoferrin homologuemutants. Rim2p/Mrs12p has recently been implicated in mitochondrial iron transport, and might be an alternative metal carrier. We identified a putative homologuein the fruit fly and found a possible link between mutants in this gene and iron. Our results emphasize the importance of the mitochondrial iron metabolism in cellular iron homeostasis. We also show for the first time, a direct connection between the mitochondrial iron metabolism and spermatogenesis. Mutants characterized and developed by us will help to study these processes in further detail and reveal the underlying mechanisms

Acute loss of the hepatic endolysosomal system in vivo causes compensatory changes in iron homeostasis

Liver cells communicate with the extracellular environment to take up nutrients via endocytosis. Iron uptake is essential for metabolic activities and cell homeostasis. Here, we investigated the role of the endocytic system for maintaining iron homeostasis. We specifically depleted the small GTPase Rab5 in the mouse liver, causing a transient loss of the entire endo-lysosomal system. Strikingly, endosome depletion led to a fast reduction of hepatic iron levels, which was preceded by an increased abundance of the iron exporter ferroportin. Compensatory changes in livers of Rab5-depleted mice include increased expression of transferrin receptor 1 as well as reduced expression of the iron-regulatory hormone hepcidin. Serum iron indices (serum iron, free iron binding capacity and total iron binding capacity) in Rab5-KD mice were increased, consistent with an elevated splenic and hepatic iron export. Our data emphasize the critical importance of the endosomal compartments in hepatocytes to maintain hepatic and systemic iron homeostasis in vivo. The short time period (between day four and five) upon which these changes occur underscore the fast dynamics of the liver iron pool

Modelling Systemic Iron Regulation during Dietary Iron Overload and Acute Inflammation: Role of Hepcidin-Independent Mechanisms

<div><p>Systemic iron levels must be maintained in physiological concentrations to prevent diseases associated with iron deficiency or iron overload. A key role in this process plays ferroportin, the only known mammalian transmembrane iron exporter, which releases iron from duodenal enterocytes, hepatocytes, or iron-recycling macrophages into the blood stream. Ferroportin expression is tightly controlled by transcriptional and post-transcriptional mechanisms in response to hypoxia, iron deficiency, heme iron and inflammatory cues by cell-autonomous and systemic mechanisms. At the systemic level, the iron-regulatory hormone hepcidin is released from the liver in response to these cues, binds to ferroportin and triggers its degradation. The relative importance of individual ferroportin control mechanisms and their interplay at the systemic level is incompletely understood. Here, we built a mathematical model of systemic iron regulation. It incorporates the dynamics of organ iron pools as well as regulation by the hepcidin/ferroportin system. We calibrated and validated the model with time-resolved measurements of iron responses in mice challenged with dietary iron overload and/or inflammation. The model demonstrates that inflammation mainly reduces the amount of iron in the blood stream by reducing intracellular ferroportin transcription, and not by hepcidin-dependent ferroportin protein destabilization. In contrast, ferroportin regulation by hepcidin is the predominant mechanism of iron homeostasis in response to changing iron diets for a big range of dietary iron contents. The model further reveals that additional homeostasis mechanisms must be taken into account at very high dietary iron levels, including the saturation of intestinal uptake of nutritional iron and the uptake of circulating, non-transferrin-bound iron, into liver. Taken together, our model quantitatively describes systemic iron metabolism and generated experimentally testable predictions for additional ferroportin-independent homeostasis mechanisms.</p></div

Hepcidin-mediated Fpn control and inhibition of Fpn transcription contribute to the acute inflammatory response.

<p>A-Experimental data (means with standard deviation) and simulated data (lines) for serum iron content following peritoneal injection of LPS for mice maintained on a standard or iron-enriched diet. Comparison of the full model with models where either hepcidin-mediated ferroportin degradation or inflammation-mediated ferroportin mRNA reduction are removed. The simulations correspond to the best fitting parameter set. B-LPS-induced changes of liver ferroportin mRNA and protein levels relative to the normal diet steady state.</p

Experimental data used for model validation.

<p>The table summarizes the experimental perturbations, time scales and measured quantities for the different datasets used.</p

LPS-induced dynamics of iron-related parameters under normal/enriched iron diet is well reproduced/predicted by the model.

<p>A-Serum iron, B-Liver Iron, C-Liver hepcidin, D-Spleen iron, E-Liver BMP6, F-Liver Fpn mRNA, G-Spleen Fpn protein, H-Liver pSTAT, I-Liver pSMAD, J-Duodenum iron, K-Red blood cells iron, L-Liver Fpn protein. 4–6 weeks old male C57BL/6-mice were administrated a normal diet, containing 200 ppm iron (blue), or a high iron diet, supplemented by 2% carbonyl iron containing about 20000 ppm iron (red). After 4 weeks, mice were injected with 1 <i>μg</i> LPS/g body weight and sacrificed 6/18/48 hours after the injection. Experimental data are given as means with standard deviation of 4–6 replicates and the model simulation for the best fitting parameter set is represented by curves (solid lines: fitted time courses, dashed lines: predicted time courses). Data represented be empty circles were used in fitting as a part of the calibration data set (LPS response for normal diet and the iron parameters after 4 weeks of high iron diet before injection of LPS). The LPS response for high iron diet data (filled circles) was used to test the model predictions. See <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005322#sec014" target="_blank">Materials and Methods</a> for the description of the experiment.</p

Experimental data used for model calibration.

<p>The table summarizes the experimental perturbations, time scales and measured quantities for the different datasets used.</p

Model correctly predicts responses to perturbations in the SMAD4-hepcidin-pathway as well as development of anemia under chronic inflammation.

<p>A-Model quantitatively predicts experimentally measured responses for 2 months old C326S knock-in mice expressing a hepcidin-resistant FPN mutant or or SMAD4-knockout mice. The model simulations are shown as blue bars and the corresponding data from [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005322#pcbi.1005322.ref037" target="_blank">37</a>] and [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005322#pcbi.1005322.ref053" target="_blank">53</a>] as red bars, respectively. Fold changes are referred to the wildtype levels. The model error bars are calculated from the predictions of the 30 best fitting parameter sets (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005322#pcbi.1005322.s001" target="_blank">S1 Text</a>). B-Model prediction for body iron pools when ferroportin regulation by hepcidin is out of action in one of the indicated organs. Shown are model simulations whithout experimental validation. C-Model qualitatively reproduces the development of anemia of inflammation upon chronic elevation of body LPS. Simulation of plasma, RBC and liver iron evolution when the inflammatory Il6/STAT pathway is permanently activated by a persistent LPS stimulus (0.17 <i>μg</i>/g body weight). Shown are model simulations without a quantitative comparison to experiments.</p