Autophagy of germ-granule components, PGL-1 and PGL-3, contributes to DNA damage-induced germ cell apoptosis in C. elegans.

Germ granules, termed P granules in nematode C. elegans, are the germline-specific cytoplasmic structures widely observed from worms to humans. P granules are known to have critical functions for postembryonic germline development likely through regulating RNA metabolism. They are localized at the perinuclear region of germ cells during most of the developmental stages. However, the biological significance of this specific localization remains elusive. PGL-1 and PGL-3, the defining components of P granules, were shown to be lost from the perinuclear region prior to germ cell apoptosis. Furthermore, this loss was shown to be significantly enhanced upon DNA damage. Here, we show that the removal of PGL-1 and PGL-3 from the perinuclear region following UV-induced DNA damage is significantly reduced in autophagy mutants. Autophagy was previously shown to be required for DNA damage-induced germ cell apoptosis. We show that the apoptosis defect of autophagy mutants is bypassed by depletion of pgl-1 or pgl-3. These findings are consistent with time-lapse observations of LGG-1 foci formation, showing that autophagy is activated following UV irradiation and that maximal accumulation of LGG-1 foci occurs before PGL-1 removal. We also show that some of the autophagy genes are transcriptionally activated following UV irradiation by CEP-1, the worm p53-like protein. Taken together, our results indicate that autophagy is required to remove the major P granule components, PGL-1 and PGL-3, and that their removal is required for the full induction of DNA damage-induced germ cell apoptosis. Our study contributes to a better understanding of germ cell apoptosis, a process that leads to the elimination of the vast majority of germ cells in various animals from worms to mammals.

Depletion of gipc-1 and gipc-2 causes infertility in Caenorhabditis elegans by reducing sperm motility.

The G-protein signaling pathway plays a key role in multiple cellular processes and is well conserved in eukaryotes. Although GIPC (G-protein α subunit interacting protein (GAIP)-interacting protein, C terminus) has been studied in several model organisms, little is known about its role in Caenorhabditis elegans. In the present study, we investigated the roles of gipc-1 and gipc-2 in C. elegans. We observed that they were exclusively expressed in sperm throughout the development and that gipc-1; gipc-2 double mutants were infertile. Further examination of sperm development in gipc-1; gipc-2 mutants revealed defective sperm activation and abnormal pseudopod extension that resulted in reduced sperm motility. Moreover, major sperm protein (MSP) was abnormally segregated between spermatids and residual bodies in gipc-1; gipc-2 mutants. Our findings indicate that gipc-1 and gipc-2 are required for the proper pseudopod extension of sperm during the terminal differentiation of spermatids. During this process, the segregation of MSP into spermatids is important for ensuring normal sperm motility during fertilization.

The B-type cyclin CYB-1 maintains the proper position and number of centrosomes during spermatogenesis in Caenorhabditis elegans.

Depletion of cyb-1, a major B-type cyclin expressed during Caenorhabditis elegans spermatogenesis, causes a meiotic division arrest in diakinesis-stage spermatocytes with multiple and mispositioned centrosomes. Association of the two nuclear membrane proteins SUN-1 and ZYG-12 is essential for centrosome-nuclear envelope attachment. We found that depletion of sun-1 causes centrosome defects similar to those caused by cyb-1 depletion in diakinesis-stage spermatocytes. In addition, Ser8 and Ser43 residues in SUN-1 are dephosphorylated in cyb-1-depleted diakinesis-stage spermatocytes. Nevertheless, dephosphorylation of these residues was not sufficient to reproduce the cyb-1-related centrosome defects. We then found that the ZYG-12::GFP signal in the nuclear envelope was significantly reduced in the cyb-1-depleted diakinesis-stage spermatocytes. However, only mispositioned but not multiplied centrosomes were observed in zyg-12 mutant diakinesis-stage spermatocytes, suggesting that zyg-12 is not involved in the centrosome duplication at this stage. Our results suggest that CYB-1 functions to maintain proper positioning of centrosomes during spermatogenesis by regulating phosphorylation of SUN-1, which is possibly crucial for the association between SUN-1 and ZYG-12. This phosphorylation of SUN-1 may also regulate centrosome duplication independently of ZYG-12.

Loss of PGL-1 and PGL-3, members of a family of constitutive germ-granule components, promotes germline apoptosis in C. elegans.

In Caenorhabditis elegans, the mechanisms regulating germline apoptosis remain largely unknown, except for the core machinery. Here, we found that mutants of pgl-1 and pgl-3, encoding members of a family of constitutive protein components of germline-specific P granules, showed increased germline apoptosis under both physiological and DNA-damaged conditions. We also found that the number of germ cells that lost PGL proteins increased significantly following UV irradiation, and that only those PGL-absent germ cells were selectively engulfed by gonadal sheath cells in adult hermaphrodite gonads. We further revealed that CEP-1, the p53 homolog, and the caspase CED-3 promoted elimination of PGL-1 from germ cells following UV irradiation. Furthermore, protein levels of CED-4, the Apaf-1 homolog, and cytoplasmic translocation of SIR-2.1, a Sirtuin homolog, significantly increased in pgl mutants and increased even more following UV irradiation. CED-4 and SIR-2.1 were essential for high levels of germline apoptosis in pgl mutants. We conclude that PGL proteins suppress excessive germline apoptosis by repressing both the protein levels of CED-4 and the cytoplasmic translocation of SIR-2.1. Our study has revealed new roles for PGL-1 and PGL-3 in the control of germline apoptosis.

Gliadin intake induces oxidative-stress responses in Caenorhabditis elegans.

Clinical attention to gluten-related disorders, such as celiac disease and nonceliac gluten sensitivity, is on the rise. However, identifying the pathophysiological mechanisms of gluten-related disorders remains elusive. Gliadin, a component of gluten, is known to play a major role in gluten toxicity. Caenorhabditis elegans has been widely used as the predominant experimental animal model to study toxicity and stress response in biomedical research. We investigated the stress response induced by gliadin intake in C. elegans to evaluate its toxicity and found brood size, body bending, and pumping rates to be significantly altered in response to gliadin. Notably, reactive oxygen species (ROS) production and Pgst-4::GFP transgene expression, an indicator of the oxidative-stress response, were significantly increased after gliadin intake. Reduced pumping rates were most likely caused by gliadin-induced oxidative stress, since pumping rates in oxidative stress-sensitive mev-1 mutants were more severely reduced than in oxidative stress-resistant daf-2 mutants following gliadin intake. Our results indicated that gluten/gliadin intake in C. elegans triggered ROS production and induced an oxidative stress response that reduced pumping rates and decreased brood size. We suggest C. elegans to be a useful model system for studying gluten/gliadin toxicity.

cdc-25.2, a Caenorhabditis elegans ortholog of cdc25, is required for male tail morphogenesis.

Cell division cycle 25 (Cdc25) is an evolutionarily conserved phosphatase that promotes cell cycle progression by activating cyclin-dependent kinases (Cdks) which are inactivated by Wee1/Myt1 kinases. It was previously reported that cdc-25.2 promotes oocyte maturation and intestinal cell divisions in Caenorhabditis elegans hermaphrodites. Here, we report a novel function of cdc-25.2 in male tail development which was significantly deformed by cdc-25.2 RNAi depletion and in cdc-25.2 mutant males. The deformation was also observed after RNAi depletion of other cell cycle regulators, cdk-1, cyb-3, cyd-1, and cyl-1. Furthermore, wee-1.3 counteracted cdc-25.2 in male tail development as observed in oocyte maturation and intestine development. The number of cells in ray precursor cell lineages was significantly reduced in cdc-25.2 depleted males. These results indicate that CDC-25.2 is essential for cell divisions in ray precursor cell lineages for proper male tail development.

Transgenerational effects of proton beam irradiation on Caenorhabditis elegans germline apoptosis.

When treating cancer using radiation therapy, it is critical to increase patient survival rates and to reduce side effects. In this respect, proton beam radiation treatment performs better than other radiation treatments because of its high target specificity. However, complications still remain after proton beam radiation treatment. Among them, the risk to progeny after irradiation of their parents is a major concern. In this study, we analyzed the transgenerational effects of proton beam irradiation using the model organism Caenorhabditis. elegans. We found that germline apoptosis increased after proton beam irradiation and its effects were sustained transgenerationally. Moreover, we identified that a germline-specific histone methyltransferase component, SET-2, has a critical role in transmitting the transgenerational effect on germline apoptosis to the next generation after proton beam irradiation.

Methionine Supplementation Alleviates the Germ Cell Apoptosis Increased by Maternal Caffeine Intake in a C. elegans Model.

Caffeine (1,3,7-trimethylxanthine) is a widely consumed bioactive substance worldwide. Our recent study showed that a reduction in both reproduction and yolk protein production (vitellogenesis) caused by caffeine intake were improved by vitamin B12 supplementation, which is an essential co-factor in methionine metabolism. In the current study, we investigated the role of methionine in the reproduction of caffeine-ingested animals (CIAs). We assessed the effect of methionine metabolism on CIAs and found that caffeine intake decreased both methionine levels and essential enzymes related to the methionine cycle. Furthermore, we found that the caffeine-induced impairment of methionine metabolism decreased vitellogenesis and increased germ cell apoptosis in an LIN-35/RB-dependent manner. Interestingly, the increased germ cell apoptosis was restored to normal levels by methionine supplementation in CIAs. These results indicate that methionine supplementation plays a beneficial role in germ cell health and offspring development by regulating vitellogenesis.

Vitamin B12 Supplementation Improves Oocyte Development by Modulating Mitochondria and Yolk Protein in a Caffeine-Ingested Caenorhabditis elegans Model.

Vitamin B12 is an essential cofactor involved in the function of two enzymes: cytosolic methionine synthase and mitochondrial methylmalonic-CoA mutase. In our previous studies, caffeine (1,3,7-trimethylxanthine), the most popular bioactivator, was shown to reduce yolk protein (vitellogenin) and fertility in a Caenorhabditis elegans model. Based on the previous finding that methionine supplementation increases vitellogenesis in C. elegans, we investigated the role of vitamin B12 in methionine-mediated vitellogenesis during oogenesis in caffeine-ingested animals (CIA). Vitamin B12 supplementation improved vitellogenesis and reduced oxidative stress by decreasing mitochondrial function in CIA. Furthermore, the decreased number of developing oocytes and high levels of reactive oxygen species in oocytes from CIA were recovered with vitamin B12 supplementation through a reduction in mitochondrial stress, which increased vitellogenesis. Taken together, vitamin B12 supplementation can reverse the negative effects of caffeine intake by enhancing methionine-mediated vitellogenesis and oocyte development by reducing mitochondrial stress.

The HUPO initiative on Model Organism Proteomes, iMOP.

The community working on model organisms is growing steadily and the number of model organisms for which proteome data are being generated is continuously increasing. To standardize efforts and to make optimal use of proteomics data acquired from model organisms, a new Human Proteome Organisation (HUPO) initiative on model organism proteomes (iMOP) was approved at the HUPO Ninth Annual World Congress in Sydney, 2010. iMOP will seek to stimulate scientific exchange and disseminate HUPO best practices. The needs of model organism researchers for central databases will be better represented, catalyzing the integration of proteomics and organism-specific databases. Full details of iMOP activities, members, tools and resources can be found at our website http://www.imop.uzh.ch/ and new members are invited to join us.

cdc-25.2, a C. elegans ortholog of cdc25, is required to promote oocyte maturation.

Cdc25 is an evolutionarily conserved protein phosphatase that promotes progression through the cell cycle. Some metazoans have multiple isoforms of Cdc25, which have distinct functions and different expression patterns during development. C. elegans has four cdc-25 genes. cdc-25.1 is required for germline mitotic proliferation. To determine if the other members of the cdc-25 family also contribute to regulation of cell division in the germ line, we examined phenotypes of loss-of-function mutants of the other cdc-25 family genes. We found that cdc-25.2 is also essential for germline development. cdc-25.2 homozygous mutant hermaphrodites exhibited sterility as a result of defects in oogenesis: mutant oocytes were arrested as endomitotic oocytes that were not fertilized successfully. Spermatogenesis and male germline development were not affected. Through genetic interaction studies, we found that CDC-25.2 functions upstream of maturation-promoting factor containing CDK-1 and CYB-3 to promote oocyte maturation by counteracting function of WEE-1.3. We propose that cdc-25 family members function as distinct but related cell cycle regulators to control diverse cell cycles in C. elegans germline development.

3,3'-Diindolylmethane Supplementation Maintains Oocyte Quality by Reducing Oxidative Stress and CEP-1/p53-Mediated Regulation of Germ Cells in a Reproductively Aged Caenorhabditis elegans Model.

In recent decades, maternal age at first birth has increased, as has the risk of infertility due to rapidly declining oocyte quality with age. Therefore, an understanding of female reproductive aging and the development of potential modulators to control oocyte quality are required. In this study, we investigated the effects of 3,3'-diindolylmethane (DIM), a natural metabolite of indole-3-cabinol found in cruciferous vegetables, on fertility in a Caenorhabditis elegans model. C. elegans fed DIM showed decreased mitochondrial dysfunction, oxidative stress, and chromosomal aberrations in aged oocytes, and thus reduced embryonic lethality, suggesting that DIM, a dietary natural antioxidant, improves oocyte quality. Furthermore, DIM supplementation maintained germ cell apoptosis (GCA) and germ cell proliferation (GCP) in a CEP-1/p53-dependent manner in a reproductively aged C. elegans germ line. DIM-induced GCA was mediated by the CEP-1-EGL-1 pathway without HUS-1 activation, suggesting that DIM-induced GCA is different from DNA damage-induced GCA in the C. elegans germ line. Taken together, we propose that DIM supplementation delays the onset of reproductive aging by maintaining the levels of GCP and GCA and oocyte quality in a reproductively aged C. elegans.

Maternal Gliadin Intake Reduces Oocyte Quality with Chromosomal Aberrations and Increases Embryonic Lethality through Oxidative Stress in a Caenorhabditis elegans Model.

Oocyte quality is essential for reproductive capacity, but it rapidly declines with age. In addition to aging, maternal nutrition is a major concern in maintaining oocyte quality. Gliadin, a major component of gluten, causes gluten toxicity, which has been reported in a variety of gluten-related disorders. The basis of gluten toxicity in reproduction is being understood using simple animal models such as Caenorhabditis elegans. In this study, we examined the effects of gliadin peptide (GP; amino acids 151-170) intake on oocyte quality control in C. elegans. We found that GP intake impaired oocyte quality through chromosomal aberrations and mitochondrial oxidative stress, which was suppressed by antioxidant treatment. The reduced oocyte quality by GP intake consequently increased embryonic lethality. Furthermore, the expression of oxidative stress-responding genes prdx-3 and gst-4 was significantly increased by GP intake. The increased DAF-16 activity by GP intake suggests that DAF-16 is a possible transactivator of these antioxidant genes. Taken together, GP intake reduced reproductive capacity in C. elegans by decreasing oocyte quality and increasing embryonic lethality through mitochondrial oxidative stress.

Peripubertal requirement of Tsg101 in maintaining the integrity of membranous structures in mouse oocytes.

OBJECTIVE: As a component of Endosomal Sorting Complex Required for Transport (ESCRT) complex I, the tumor susceptibility gene 101 (Tsg101) carries out multiple functions. In this work, we report that oocyte-specific deletion of tumor susceptibility gene 101 (Tsg101) leads to age-dependent oocyte demise in mice. MATERIALS AND METHOD: Tsg101 floxed mice (Tsg101f/f ) were bred with Zp3cre transgenic mice to examine oocyte-specific roles of Tsg101. Multiple cellular and molecular biological approaches were taken to examine what leads to oocyte demise in the absence of Tsg101. RESULTS: The death of oocytes from Zp3cre /Tsg101f/f (Tsg101d/d thereafter) mice showed a strong correlation with sexual maturation, as gonadotropin-releasing hormone antagonist injections improved the survival rate of oocytes from 5-week-old Tsg101d/d mice. Maturation of oocytes from prepubertal Tsg101d/d mice proceeded normally, but was largely abnormal in oocytes from peripubertal Tsg101d/d mice, showing shrinkage or rupture. Endolysosomal structures in oocytes from peripubertal Tsg101d/d mice showed abnormalities, with aberrant patterns of early and late endosomal markers and a high accumulation of lysosomes. Dying oocytes showed plasma membrane blebs and leakage. Blockage of endocytosis in oocytes at 4°C prevented cytoplasmic shrinkage of oocytes from Tsg101d/d mice until 9 h. The depletion of tsg-101 in Caenorhabditis elegans increased the permeability of oocytes and embryos, suggesting a conserved role of Tsg101 in maintaining membrane integrity. CONCLUSIONS: Collectively, Tsg101 plays a dual role in maintaining the integrity of membranous structures, which is influenced by age in mouse oocytes.

A circulatory transcriptional regulation among daf-9, daf-12, and daf-16 mediates larval development upon cholesterol starvation in Caenorhabditis elegans.

C. elegans shows dauer-like larvae formation upon cholesterol starvation (CS), but the genetic epistasis among abnormal dauer formation (daf) genes during the process remains unclear. To clarify the genetic interactions among daf-9, daf-12, and daf-16 in this process, mRNA levels of these genes upon CS were measured. CS increased the mRNA levels of daf-9, daf-12, and daf-16. CS also induced DAF-16 nuclear localization, which was positively and negatively regulated by DAF-12 and DAF-9 activities, respectively. Activated DAF-16, a FOXO transcription factor, enhanced daf-12 but suppressed daf-9 expression, whereas DAF-9 inhibited daf-12 expression. Concomitantly, CS-induced larval arrest was regulated positively by DAF-12 and DAF-16, but negatively by DAF-9. The larval arrest in daf-9 mutant was suppressed by daf-12 RNAi, placing DAF-12 downstream of DAF-9. These results altogether suggest that circulatory mutual regulation among daf-9, daf-12, and daf-16 at the expression level mediates cholesterol signal to control larval development upon CS.

Long-Term Caffeine Intake Exerts Protective Effects on Intestinal Aging by Regulating Vitellogenesis and Mitochondrial Function in an Aged Caenorhabditis Elegans Model.

Caffeine, a methylxanthine derived from plants, is the most widely consumed ingredient in daily life. Therefore, it is necessary to investigate the effects of caffeine intake on essential biological activities. In this study, we attempted to determine the possible anti-aging effects of long-term caffeine intake in the intestine of an aged Caenorhabditis elegans model. We examined changes in intestinal integrity, production of vitellogenin (VIT), and mitochondrial function after caffeine intake. To evaluate intestinal aging, actin-5 (ACT-5) mislocalization, lumenal expansion, and intestinal colonization were examined after caffeine intake, and the levels of vitellogenesis as well as the mitochondrial activity were measured. We found that the long-term caffeine intake (10 mM) in the L4-stage worms at 25 °C for 3 days suppressed ACT-5 mislocalization. Furthermore, the level of autophagy, which is normally increased in aging animals, was significantly reduced in these animals, and their mitochondrial functions improved after caffeine intake. In addition, the caffeine-ingesting aging animals showed high resistance to oxidative stress and increased the expression of antioxidant proteins. Taken together, these findings reveal that caffeine may be a potential anti-aging agent that can suppress intestinal atrophy during the progression of intestinal aging.

Nicotinamide Supplementation Improves Oocyte Quality and Offspring Development by Modulating Mitochondrial Function in an Aged Caenorhabditis elegans Model.

Aging is associated with a decline in the quality of biological functions. Among the aging processes, reproductive aging is a critical process because of its intergenerational effects. However, the mechanisms underlying reproductive aging remain largely unknown. Female reproductive aging is the primary reason for limited fertility in mammals. Therefore, we attempted to investigate a modulator that can control female reproductive aging using a Caenorhabditis elegans model. In the present study, we examined the role of nicotinamide (NAM) in oocyte quality and offspring development. The levels of reactive oxygen species (ROS) and oxidative stress responses in aged oocytes, embryonic lethality, and developmental growth of the offspring were examined with maternal NAM supplementation. Supplementation with NAM improved oocyte quality, decreased embryonic lethality, and promoted germ cell apoptosis. Furthermore, NAM supplementation in aged mothers reduced ROS accumulation and improved mitochondrial function in oocytes. Consequently, the developmental growth and motility of offspring were improved. These findings suggest that NAM supplementation improves the health of the offspring produced by aged mothers through improved mitochondrial function. Taken together, our results imply that NAM supplementation in the aged mother improves oocyte quality and protects offspring by modulating mitochondrial function.

Caenorhabditis elegans proteomics comes of age.

Caenorhabditis elegans, a free-living soil nematode, is an ideal model system for studying various physiological problems relevant to human diseases. Despite its short history, C. elegans proteomics is receiving great attention in multiple research areas, including the genome annotation, major signaling pathways (e.g. TGF-beta and insulin/IGF-1 signaling), verification of RNA interference-mediated gene targeting, aging, disease models, as well as peptidomic analysis of neuropeptides involved in behavior and locomotion. For example, a proteome-wide profiling of developmental and aging processes not only provides basic information necessary for constructing a molecular network, but also identifies important target proteins for chemical modulation. Although C. elegans has a simple body system and neural circuitry, it exhibits very complicated functions ranging from feeding to locomotion. Investigation of these functions through proteomic analysis of various C. elegans neuropeptides, some of which are not found in the predicted genome sequence, would open a new field of peptidomics. Given the importance of nematode infection in plants and mammalian pathogenesis pathways, proteomics could be applied to investigate the molecular mechanisms underlying plant- or animal-nematode pathogenesis and to identify novel antinematodal drugs. Thus, C. elegans proteomics, in combination of other molecular, biological and genetic techniques, would provide a versatile new tool box for the systematic analysis of gene functions throughout the entire life cycle of this nematode.

Maternal Caffeine Intake Disrupts Eggshell Integrity and Retards Larval Development by Reducing Yolk Production in a Caenorhabditis elegans Model.

During pregnancy, most women are exposed to caffeine, which is a widely consumed psychoactive substance. However, the consequences of maternal caffeine intake on the child remain largely unknown. Here, we investigated the intergenerational effects of maternal caffeine intake on offspring in a Caenorhabditis elegans model. We treated a young mother (P0) with 10 mM of caffeine equivalent to 2-5 cans of commercial energy drinks and examined its reproduction and growth rate from P0 to F2 generation. The fertility decreased and embryonic lethality increased by defective oocytes and eggshell integrity in caffeine-ingested mothers, and F1 larval development severely retarded. These results were due to decreased production of vitellogenin protein (yolk) in caffeine-ingested mothers. Furthermore, effects of RNA interference of vitellogenin (vit) genes, vit-1 to vit-6, in P0 mothers can mimic those by caffeine-ingested mothers. In addition, RNA interference (RNAi) depletion of unc-62 (human Meis homeobox), a transcriptional activator for vit genes, also showed similar effects induced by caffeine intake. Taken together, maternal caffeine intake reduced yolk production mediated by the UNC-62 transcription factor, thereby disrupting oocyte and eggshell integrity and retarding larval development. Our study suggests the clinical significance of caffeine intake for prospective mothers.

Effects of Phosphoethanolamine Supplementation on Mitochondrial Activity and Lipogenesis in a Caffeine Ingestion Caenorhabditis elegans Model.

Caffeine intake is strongly linked to lipid metabolism. We previously reported the age-dependent physiological effects of caffeine intake in a Caenorhabditis elegans model. Since nutritional status can actively influence metabolism and overall health, in this study, we evaluated the effect of caffeine intake on lipid metabolism in adult-stage C. elegans. We found that, in C. elegans, fat storage and the level of phosphoethanolamine (PE) were significantly reduced with caffeine intake. In addition, mitochondrial activity decreased and mitochondrial morphology was disrupted, and the expression of oxidative stress response genes, hsp-6, gst-4, and daf-16, was induced by caffeine intake. Furthermore, the level of an energy metabolism sensor, phospho-AMP-activated protein kinase, was increased, whereas the expression of the sterol regulatory element binding protein gene and its target stearoyl-CoA desaturase genes, fat-5, -6, and -7, was decreased with caffeine intake. These findings suggest that caffeine intake causes mitochondrial dysfunction and reduces lipogenesis. Interestingly, these changes induced by caffeine intake were partially alleviated by PE supplementation, suggesting that the reduction in mitochondrial activity and lipogenesis is in part because of the low PE level, and proper dietary supplementation can improve organelle integrity.