Adult exposure of atrazine alone or in combination with carbohydrate diet hastens the onset/progression of type 2 diabetes in Drosophila.

AIM: The combined impact of traditional and non-traditional risk factors of type 2 diabetes (T2D) on the development and progression of insulin resistance and associated complications is poorly understood. Therefore, we assessed the effect of moderately rich sugar diet coupled with environmental chemical exposure on the development and progression of T2D using Drosophila as a model organism. MAIN METHODS: We reared newly eclosed Drosophila males on a diet containing atrazine (20 µg/ml; non-traditional risk factor) and/or moderately high sucrose (0.5 M/1 M; to mimic binge eating, Traditional risk factor) for 20-30 days. Subsequently, we assessed diabetic parameters, oxidative stress parameters and also the abundance of advanced glycation end products (AGEs) along with their receptor (RAGE) in these flies. For diabetic cardiomyopathy, we examined the pericardin (tissue fibrosis marker) level in Drosophila heart. KEY FINDINGS: Flies reared on 20 µg/ml atrazine alone showed T2D hallmarks at 30 days. In contrast, flies reared on 0.5 M sucrose+ 20 µg/ml atrazine showed insulin resistance characterized by hyperglycemia and increased Drosophila insulin-like peptides along with reduced insulin signaling at 20 days, similar to those reared on high sucrose diet. In addition, both groups had high levels of oxidative stress and showed starvation response (converting triglycerides into fatty acids). Alarmingly, flies fed with sucrose+atrazine for 20 and 30 days had elevated pericardin in heart tissues, indicating early onset of diabetic complications such as cardiomyopathy. SIGNIFICANCE: Lifestyle-chemical exposure synergistically impairs glucose metabolism, affects organisms' redox state and leads to the early onset of T2D and associated complications like cardiomyopathy.

Altered sperm fate in the reproductive tract milieu due to oxidative stress leads to sub-fertility in type 1 diabetes females: A Drosophila-based study.

AIMS: Female sub-fertility, a prominent complication due to Type 1 diabetes (T1D), is generally attributed to disturbances in menstrual cycles and/or ovarian defects/disorders. T1D women, however, are high in oxidative stress, although the impact of the same on their reproduction and associated events remains unknown. Therefore, we assessed the repercussions of elevated oxidative stress on the sperm fate (storage/utilization) in the reproductive tract milieu of T1D females and their fertility using the Drosophila T1D model (Df[dilp1-5]), which lacks insulin-like peptides and displays reduced female fertility. MAIN METHODS: We mated Df[dilp1-5] females to normal males and thereafter examined sperm storage and/or utilization in conjunction with oxidative stress parameters in mated Df[dilp1-5] females at different time points. Also, the impact of antioxidant (Amla or Vitamin C) supplementation on the above oxidative stress parameters in Df[dilp1-5] females and the consequences on their sperm and fertility levels were examined. KEY FINDINGS: Df[dilp1-5] females showed elevated oxidative stress parameters and a few of their reproductive tract proteins are oxidatively modified. Also, these females stored significantly fewer sperm and also did not utilize sperm as efficiently as their controls. Surprisingly, amelioration of the oxidative stress in Df[dilp1-5] females' milieu through antioxidant (Amla or vitamin C) supplementation enhanced sperm storage and improved fertility. SIGNIFICANCE: Hyperglycemia coupled with elevated oxidative stress within the female reproductive tract environment affects the sperm fate, thereby reducing female fertility in T1D. In addition, these findings suggest that antioxidant supplementation may substantially aid in the mitigation of sub-fertility in T1D females.

Estrogen-related receptor is critical for testicular mitochondrial homeostasis and sperm motility: a Drosophila-based study.

OBJECTIVE: To study the role of estrogen-related receptors (ERRs) in testicular function, with particular emphasis on mitochondrial homeostasis, testicular steroidogenesis, and sperm motility using Drosophila as a model. DESIGN: Experimental study. SETTING: Academic research laboratory. ANIMAL(S): Wild-type and transgenic strains of Drosophila melanogaster. INTERVENTION(S): Using a ribonucleic acid interference-based approach, ERR was knocked down specifically in the testes to generate Drosophila males with reduced ERR levels in their testes. Genetically matched sibling males without the knockdown formed the controls. MAIN OUTCOME MEASURE(S): Analysis of the testicular mitochondrial structure and function in relation to energy production, steroidogenesis, and sperm motility in Drosophila. RESULT(S): Depletion of ERR affects mitochondrial homeostasis (biogenesis, fission, fusion, mitophagy, and transport) and oxidative respiration in the testes. Consequently, ERR knockdown testes have significantly reduced mitochondrial size, mass, and depleted adenosine triphosphate levels resulting in testicular oxidative stress. Further, Halloween genes, associated with steroidogenesis in Drosophila, are misregulated in ERR knockdown testes, and knockdown of some of the steroidogenic genes in a testis-specific manner results in significantly reduced fertility. In addition, sperm from ERR knockdown testes have significantly reduced levels of glucose transporter, Na+K+ ATPase, Dynein heavy chain, and adenosine triphosphate-5α synthase essential for sperm function. Corroborating this, sperm from ERR knockdown males are significantly less motile compared with control. CONCLUSION(S): The ERR is crucial for meeting the cellular energy requirements of the testes and the generation of normal motile sperm and hormone synthesis/secretion in the testes. To our knowledge, this is the first report implicating ERR in these ultimate functions of the testes. These findings can potentially contribute to the etiologic understanding of asthenozoospermia or infertility at large in men.

Drosophila ecdysone receptor activity-based ex vivo assay to assess the endocrine disruption potential of environmental chemicals.

Insect pollinators, critical for both agricultural output and the ecosystem, are declining at an alarming levels partly due to human-made chemicals. Majority of environmental chemicals hamper the endocrine function and studies on the same in insects remain neglected. Here, we report a Drosophila-based ex vivo assay system that employs a reproductive tissue from transgenic males carrying a reporter gene (lacZ) downstream of ecdysone receptor response element (EcRE) and permits the evaluation of chemical-mediated activity modulation of all three isoforms of ecdysone receptor, which are critical for male fertility. We show agonistic [plasticizers, cypermethrin, atrazine, methyl parathion, imidacloprid, cadmium chloride, mercuric chloride or 3-(4-methylbenzylidene) camphor] or antagonistic (apigenin, tributyltin chloride) effects or lack of effect thereof (rutin hydrate, dichlorvos, lead acetate, parabens) for seven different classes of environmental chemicals on ecdysone receptor activity reflecting the specificity and sensitivity of the developed ex vivo assay. Exposure to a few of these chemicals in vivo hampers the fertility of Drosophila males, thus linking the observed endocrine disruption to a quantifiable reproductive phenotype. The developed ex vivo assay offers a quick Drosophila-based screening tool for throughput monitoring of environmental chemicals for their ability to hamper the endocrine function of insect pollinators and other invertebrates.

Xenobiotic mediated diabetogenesis: Developmental exposure to dichlorvos or atrazine leads to type 1 or type 2 diabetes in Drosophila.

The increased incidence of diabetes to the magnitude of a global epidemic is attributed to non-traditional risk factors, including exposure to environmental chemicals. However, the contribution of xenobiotic exposure during the development of an organism to the etiology of diabetes is not fully addressed. Developing stages are more susceptible to chemical insult, but knowledge on the consequence of the same to the onset of diabetes is residual. In this context, by using Drosophila melanogaster having conserved Insulin/Insulin growth factor-like signaling (IIS) as well as glucose homeostasis as a model, we evaluated the potential of developmental exposure to dichlorvos (DDVP, an organophosphorus pesticide) or atrazine (herbicide) to cause diabetes in exposed organisms. Flies exposed to DDVP during their development display insulin deficiency or type 1 diabetes (T1D) while those exposed to atrazine show insulin resistance or type 2 diabetes (T2D), suggesting that exposure to these xenobiotics during organismal development can result in diabetes and that different mechanisms underlie pesticide mediated diabetes. We show that oxidative stress-mediated c-Jun N-terminal kinase (JNK) signaling activation underlies insulin resistance in flies exposed to atrazine during their development while DDVP-mediated T1D involves activation of caspase-mediated cell death pathway. Mitigation of oxidative stress through over-expression of SOD2 in atrazine (20µg/ml) exposed flies, revealed significantly decreased oxidative stress levels and reduced phosphorylation of JNK. Moreover, glucose and Akt phosphorylation levels in SOD2 over-expression flies exposed to atrazine were comparable to those in controls, suggesting restoration in insulin sensitivity. Therefore, exposure to xenobiotics during development is a common risk factor for the development of type 1 or type 2 diabetes. Accordingly, the present study cautions against the use of such diabetogenic pesticides. Also, mitigation of oxidative stress or anti-oxidant supplementation could be a potential therapy for xenobiotic mediated type 2 diabetes.

Correction: Functional male accessory glands and fertility in Drosophila require novel ecdysone receptor.

[This corrects the article DOI: 10.1371/journal.pgen.1006788.].

Mlh1 is required for female fertility in Drosophila melanogaster: An outcome of effects on meiotic crossing over, ovarian follicles and egg activation.

Mismatch repair (MMR) system, a conserved DNA repair pathway, plays crucial role in DNA recombination and is involved in gametogenesis. The impact of alterations in MMR family of proteins (bacterial MutS and MutL homologues) on mammalian fertility is well documented. However, an insight to the role of MMR in reproduction of non-mammalian organisms is limited. Hence, in the present study, we analysed the impact of mlh1 (a MutL homologue) on meiotic crossing over/recombination and fertility in a genetically tractable model, Drosophila melanogaster. Using mlh1e00130 hypomorphic allele, we report female specific adverse reproductive outcome for reduced mlh1 in Drosophila: mlh1e00130 homozygous females had severely reduced fertility while males were fertile. Further, mlh1e00130 females contained small ovaries with large number of early stages as well as significantly reduced mature oocytes, and laid fewer eggs, indicating discrepancies in egg production and ovulation. These observations contrast the sex independent and/or male specific sterility and normal follicular development as well as ovulation reported so far for MMR family proteins in mammals. However, analogous to the role(s) of mlh1 in meiotic crossing over and DNA repair processes underlying mammalian fertility, ovarian follicles from mlh1e00130 females contained significantly increased DNA double strand breaks (DSBs) and reduced synaptonemal complex foci. In addition, large proportion of fertilized eggs display discrepancies in egg activation and fail to proceed beyond stage 5 of embryogenesis. Hence, reduction of the Mlh1 protein level leads to defective oocytes that fail to complete embryogenesis after fertilization thereby reducing female fertility.

Correction: Functional male accessory glands and fertility in Drosophila require novel ecdysone receptor.

[This corrects the article DOI: 10.1371/journal.pgen.1006788.].

Functional male accessory glands and fertility in Drosophila require novel ecdysone receptor.

In many insects, the accessory gland, a secretory tissue of the male reproductive system, is essential for male fertility. Male accessory gland is the major source of proteinaceous secretions, collectively called as seminal proteins (or accessory gland proteins), which upon transfer, manipulate the physiology and behavior of mated females. Insect hormones such as ecdysteroids and juvenoids play a key role in accessory gland development and protein synthesis but little is known about underlying molecular players and their mechanism of action. Therefore, in the present study, we examined the roles of hormone-dependent transcription factors (Nuclear Receptors), in accessory gland development, function and male fertility of a genetically tractable insect model, Drosophila melanogaster. First, we carried out an RNAi screen involving 19 hormone receptors, individually and specifically, in a male reproductive tissue (accessory gland) for their requirement in Drosophila male fertility. Subsequently, by using independent RNAi/ dominant negative forms, we show that Ecdysone Receptor (EcR) is essential for male fertility due to its requirement in the normal development of accessory glands in Drosophila: EcR depleted glands fail to make seminal proteins and have dying cells. Further, our data point to a novel ecdysone receptor that does not include Ultraspiracle but is probably comprised of EcR isoforms in Drosophila male accessory glands. Our data suggest that this novel ecdysone receptor might act downstream of homeodomain transcription factor paired (prd) in the male accessory gland. Overall, the study suggests novel ecdysone receptor as an important player in the hormonal regulation of seminal protein production and insect male fertility.

Estrogen related receptor is required for the testicular development and for the normal sperm axoneme/mitochondrial derivatives in Drosophila males.

Estrogen related receptors (ERRs), categorized as orphan nuclear receptors, are critical for energy homeostasis and somatic development. However, significance of ERRs in the development of reproductive organs/organelles/cells remain poorly understood, albeit their homology to estrogen receptors. In this context, here, we show that knockdown of ERR in the testes leads to improperly developed testes with mis-regulation of genes (aly, mia, bruce, bam, bgcn, fzo and eya) involved in spermatogenesis, resulting in reduced male fertility. The observed testicular deformity is consistent with the down-regulation of SOX-E group of gene (SOX100B) in Drosophila. We also show dispersion/disintegration of fusomes (microtubule based structures associated with endoplasmic reticulum derived vesicle, interconnecting spermatocytes) in ERR knockdown testes. A few ERR knockdown testes go through spermatogenesis but have significantly fewer sperm. Moreover, flagella of these sperm are defective with abnormal axoneme and severely reduced mitochondrial derivatives, suggesting a possible role for ERR in mitochondrial biogenesis, analogous to mammalian ERRα. Interestingly, similar knockdown of remaining seventeen nuclear receptors did not yield a detectable reproductive or developmental defect in Drosophila. These findings add newer dimensions to the functions envisaged for ERR and provide the foundation for deciphering the relevance of orphan nuclear receptors in ciliopathies and testicular dysgenesis.

Exposure to endosulfan influences sperm competition in Drosophila melanogaster.

Dwindling male fertility due to xenobiotics is of global concern. Accordingly, male reproductive toxicity assessment of xenobiotics through semen quality analysis in exposed males, and examining progeny production of their mates is critical. These assays, in part, are biased towards monogamy. Females soliciting multiple male partners (polyandry) is the norm in many species. Polyandry incites sperm competition and allows females to bias sperm use. However, consequences of xenobiotic exposure to the sperm in the light of sperm competition remain to be understood. Therefore, we exposed Drosophila melanogaster males to endosulfan, and evaluated their progeny production as well as the ability of their sperm to counter rival control sperm in the storage organs of females sequentially mated to control/exposed males. Endosulfan (2 µg/ml) had no significant effect on progeny production and on the expression of certain genes associated with reproduction. However, exposed males performed worse in sperm competition, both as 1(st) and 2(nd) male competitors. These findings indicate that simple non-competitive measures of reproductive ability may fail to demonstrate the harmful effects of low-level exposure to xenobiotics on reproduction and advocate consideration of sperm competition, as a parameter, in the reproductive toxicity assessment of xenobiotics to mimic situations prevailing in the nature.

Targeted gene deletion and phenotypic analysis of the Drosophila melanogaster seminal fluid protease inhibitor Acp62F.

Internally fertilizing organisms transfer a complex assortment of seminal fluid proteins, a substantial fraction of which are proteolysis regulators. In mammals, some seminal protease inhibitors have been implicated in male infertility and these same molecular classes of protease inhibitors are also found in Drosophila seminal fluid. Here, we tested the reproductive functions of the Drosophila melanogaster seminal fluid protease inhibitor Acp62F by generating a precise deletion of the Acp62F gene. We did not detect a nonredundant function for Acp62F in modulating the egg laying, fertility, remating frequency, or life span of mated females. However, loss of Acp62F did alter a male's defensive sperm competitive ability, consistent with the localization of Acp62F to sperm storage organs. In addition, the processing of at least one seminal protein, the ovulation hormone ovulin, is slower in the absence of Acp62F.

Evolution in the fast lane: rapidly evolving sex-related genes in Drosophila.

A large portion of the annotated genes in Drosophila melanogaster show sex-biased expression, indicating that sex and reproduction-related genes (SRR genes) represent an appreciable component of the genome. Previous studies, in which subsets of genes were compared among few Drosophila species, have found that SRR genes exhibit unusual evolutionary patterns. Here, we have used the newly released genome sequences from 12 Drosophila species, coupled to a larger set of SRR genes, to comprehensively test the generality of these patterns. Among 2505 SRR genes examined, including ESTs with biased expression in reproductive tissues and genes characterized as involved in gametogenesis, we find that a relatively high proportion of SRR genes have experienced accelerated divergence throughout the genus Drosophila. Several testis-specific genes, male seminal fluid proteins (SFPs), and spermatogenesis genes show lineage-specific bursts of accelerated evolution and positive selection. SFP genes also show evidence of lineage-specific gene loss and/or gain. These results bring us closer to understanding the details of the evolutionary dynamics of SRR genes with respect to species divergence.

Predicted seminal astacin-like protease is required for processing of reproductive proteins in Drosophila melanogaster.

During mating, males provide females with seminal fluids that include proteins affecting female physiology and, in some cases, reproductive behavior. In several species these male-derived modulators of reproduction are processed upon transfer to the female, suggesting molecular interaction between the sexes. Males could increase their reproductive success by contributing to regulation of this processing; consistent with this hypothesis, seminal fluids are rich in proteolysis regulators. However, whether these molecules carry out processing of male-derived reproductive modulators is unknown. We tested for this role using RNAi to knock down individually 11 Drosophila seminal fluid proteases and protease inhibitors. We found that CG11864, a predicted astacin-type metalloprotease in seminal fluid, is necessary to process two other seminal proteins: the ovulation hormone ovulin (Acp26Aa) and the sperm storage protein Acp36DE. This processing occurs only after all three proteins have entered the female. Moreover, CG11864 itself is processed inside males while en route to the female and before its action in processing ovulin and Acp36DE. Thus, processing of seminal proteins is stepwise in Drosophila, beginning in the male after the proteins leave their site of synthesis and continuing within another organism, the mated female, and the male-donated protease CG11864 is an agent of this latter processing.