Near-anoxia induces immobilization and sustains viability of sperm stored in ant queens.

After copulation, insect females store sperm in a spermatheca for some duration until fertilization. At the beginning of their adult lives, ant queens can preserve numerous viable sperm cells from copulation for over ten years. However, the key factors influencing long-term sperm storage have not been identified. Here we show that the spermathecal environment is nearly anoxic, which induces sperm immobilization. Furthermore, mitochondrial respiratory inhibitors suppress sperm motility, suggesting that sperm immobilization may be caused by a shortage of ATP generated from only glycolysis under near-anoxic conditions. Sperm immobilization is not induced by acidification via glycolytic metabolism because the spermathecal fluid is not acidic. Finally, we show that artificial anoxic conditions rather than aerobic conditions sustain viable sperm cells. Therefore, near-anoxia is a key factor influencing long-term sperm storage in ant queens. The viability of sperm cells under artificial anoxia, however, is lower than that of those dissected immediately from queens. Moreover, the immotile sperm cells under more than 4 h of anoxia do not begin swimming after aerobic exposure, unlike those under anoxic conditions for less than 2 h. This finding indicates that factors other than anoxia are also necessary for long-term sperm preservation.

Characterization and functional analysis of BmSR-B1 for phytosterol uptake.

Insects cannot synthesize sterols, such as cholesterol, and require sterols in their diet. Phytophagous insects use dietary phytosterols as a source of cholesterol. Sterols are transported from the midgut by the insect lipoprotein, lipophorin (Lp), although mechanisms for uptake of phytosterols into tissues are unclear. This study characterizes Scavenger Receptor class B type1 (SR-B1) from Bombyx mori (BmSR-B1) as molecules related to phytosterol uptake. According to sterol quantification using LC-MS/MS analysis, the midgut and fat body were phytosterol-rich relative to cholesterol-rich brain and prothoracic glands. Gene expression analysis of Bmsr-b1 in silkworm tissues showed that the genes Bmsr-b1_2, 3, 4, 6, and 10 were expressed in the midgut and fat body. To characterize the function of BmSR-B1, 11 BmSR-B1 homologs expressed in Bombyx ovary-derived BmN cells and Drosophila melanogaster embryo-derived Schneider 2 (S2) cells were incubated with purified Lp. Our analysis showed that BmSR-B1_3 induced the accumulation of campesterol and BmSR-B1_4 induced the accumulation of ß-sitosterol and campesterol in culture cells. BmSR-B1 incorporated specific phytosterols into insect cells by selective uptake across the cell membrane where BmSR-B1 was localized. In conclusion, our study demonstrated that one function of BmSR-B1 is the uptake of phytosterols into silkworm tissues.

Twenty-hydroxyecdysone produced by dephosphorylation and ecdysteroidogenesis regulates early embryonic development in the silkmoth, Bombyx mori.

Ecdysteroids are key regulators of embryonic development as well as molting and metamorphosis in insects. Although an active form of ecdysteroids, 20-hydroxyecdysone (20E) is known to be produced through ecdysteroidogenesis from cholesterol and dephosphorylation of 20E-phosphate during embryogenesis in Lepidoptera, the importance of these production mechanisms in embryonic development has been unclear. Here, we investigated the activation timing of ecdysteroidogenesis from cholesterol and 20E-phosphate dephosphorylation during early embryogenesis in non-diapause eggs of the silkmoth Bombyx mori by observing morphological development, quantifying 20E and 20E-phosphate, measuring transcripts of enzymes involved in 20E production, and detecting activity of these enzymes using egg extracts. Stage-dependent 20E fluctuation and changes in mRNA amounts of enzymes suggest that the two 20E-producing mechanisms are activated at different stages during embryogenesis. Furthermore, knockdown of a dephosphorylation enzyme delayed development at early embryogenesis, whereas knockdown of an ecdysteroidogenic enzyme delayed development at early-middle embryogenesis. These results suggest that 20E is primarily produced initially by dephosphorylation of 20E-phosphate, and then by ecdysteroidogenesis from cholesterol to induce progression of embryonic development in B. mori.

Functional analysis of ecdysteroid biosynthetic enzymes of the rice planthopper, Nilaparvata lugens.

Ecdysteroids, insect steroid hormones, play key roles in regulating insect development and reproduction. Hemipteran insects require ecdysteroids for egg production; however, ecdysteroid synthesis (ecdysteroidogenesis) details have not been elucidated. We identified all known genes encoding ecdysteroidogenic enzymes in Nilaparvata lugens and clarified their necessity during nymphal and ovarian development. We confirmed that N. lugens utilized 20-hydroxyecdysone as an active hormone. Assays using heterologous expression of enzymes in Drosophila S2 cells showed conserved functions of enzymes Neverland, CYP306A2, CYP314A1 and CYP315A1, but not CYP302A1. RNA interference and rescue analysis using 20-hydroxyecdysone demonstrated that most of the genes were necessary for nymphal development. The identified N. lugens enzymes showed conserved functions and pathways for ecdysteroidogenesis. Knockdown of ecdysteroidogenic enzyme genes in newly molted females caused failure of egg production: less vitellogenic and mature eggs in ovaries, fewer laid eggs and embryonic development deficiency of laid eggs. Considering the high expressions of ecdysteroidogenic enzyme genes in adults and ovaries, ecdysteroidogenesis in ovaries was critical for N. lugens ovarian development. Our study presents initial evidence that hemipteran insects require ecdysteroidogenesis for ovarian development.

Effects of maternal bisphenol A diglycidyl ether exposure during gestation and lactation on behavior and brain development of the offspring.

Bisphenol A diglycidyl ether (BADGE) is an epoxy resin used for the inner coating of canned food and beverages. BADGE can easily migrate from the containers and become a contaminant. In this study, we examined the effects of BADGE exposure to the dams on the behavioral, structural, and developmental abnormalities in the offspring. Female pregnant mice were fed with a diet containing BADGE (0.15 or 1.5 mg/kg/day) during gestation and lactation periods. In an open field test, the time spent in the corner area significantly increases in male mice of high-dose BADGE group at 5 weeks old. The histological analysis using offspring brain at postnatal day 1 delivered from BADGE (1.5 mg/kg/day)-treated dams demonstrates that positive signals of Forkhead box P2- and COUP-TF interacting protein 2 are restricted in each cortical layer, but not in the control brain. In addition, the maternal BADGE exposure reduces nestin-positive fibers of the radial glia and T-box transcription factor 2-positive intermediate progenitors in the inner subventricular zone. Furthermore, a direct BADGE exposure promotes neurite outgrowth and neuronal connection in the primary cultured cortical neurons. These data suggest that maternal BADGE exposure can accelerate neuronal differentiation in fetuses and induce anxiety-like behavior in juvenile mice.

Sterol Characteristics in Silkworm Brain and Various Tissues Characterized by Precise Sterol Profiling Using LC-MS/MS.

Sterols, especially cholesterol (Chl), are fundamental for animal survival. Insects lacking the ability to synthesize Chl are sterol auxotrophic animals and utilize dietary Chl and phytosterols to survive. The sterols obtained from a diet are distributed to the tissues; however, sterol homeostasis in insect tissues remains to be elucidated. This study sought to understand the sterol characteristics of insect tissues through detailed sterol quantification and statistics. The combination of sterol quantification using liquid chromatography tandem mass spectrometry (LC-MS/MS) and principal component analysis (PCA) revealed tissue-specific sterol characteristics in the silkworm, Bombyx mori, a phytophagous insect. We found that insect tissues have tissue-intrinsic sterol profiles. The brain has a unique sterol composition as compared to other tissues-high concentration of Chl and less accumulation of phytosterols. Other tissues also have intrinsic sterol characteristics, which when defined by dietary sterols or Chl metabolites, indicate preference for a sterol and consistently manage their own sterol homeostasis. Though most tissues never change sterol profiles during development, the brain drastically changes its sterol profile at the wandering stage, indicating that it could alter sterol composition in preparation for metamorphosis. These results suggest the existence of tissue- and sterol-specific systems for sterol homeostasis in insects.

Deep sequencing of the prothoracic gland transcriptome reveals new players in insect ecdysteroidogenesis.

Ecdysteroids are steroid hormones that induce molting and determine developmental timing in arthropods. In insect larva, the prothoracic gland (PG) is a major organ for ecdysone synthesis and release. Released ecdysone is converted into the active form, 20-hydroxyecdysone (20E) in the peripheral tissues. All processes from ecdysone synthesis and release from the PG to its conversion to 20E are called ecdysteroidogenesis and are under the regulation of numerous factors expressed in the PG and peripheral tissues. Classical genetic approaches and recent transcriptomic screening in the PG identified several genes responsible for ecdysone synthesis and release, whereas the regulatory mechanism remains largely unknown. We analyzed RNA-seq data of the silkworm Bombyx mori PG and employed the fruit fly Drosophila melanogaster GAL4/UAS binary RNAi system to comprehensively screen for genes involved in ecdysone synthesis and/or release. We found that the genes encoding δ-aminolevulinic acid synthase (CG3017/alas) and putative NAD kinase (CG33156) were highly expressed in the PG of both B. mori and D. melanogaster. Neither alas nor CG33156 RNAi-induced larvae could enter into the pupal stage, and they had a lower abundance of the active form ecdysteroids in their prolonged larval stage. These results demonstrated that alas and CG33156 are indispensable for ecdysteroidogenesis.

l-Theanine protects against excess dopamine-induced neurotoxicity in the presence of astrocytes.

l-Theanine (γ-glutamylethylamide), a component of green tea, is considered to have regulatory and neuroprotective roles in the brain. The present study was designed to determine the effect of l-theanine on excess dopamine-induced neurotoxicity in both cell culture and animal experiments. The primary cultured mesencephalic neurons or co-cultures of mesencephalic neurons and striatal astrocytes were pretreated with l-theanine for 72 h, and then treated with excess dopamine for further 24 h. The cell viability of dopamine neurons and levels of glutathione were evaluated. Excess dopamine-induced neurotoxicity was significantly attenuated by 72 h preincubation with l-theanine in neuron-astrocyte co-cultures but not in neuron-rich cultures. Exposure to l-theanine increased the levels of glutathione in both astrocytes and glial conditioned medium. The glial conditioned medium from l-theanine-pretreated striatal astrocytes attenuated dopamine-induced neurotoxicity and quinoprotein formation in mesencephalic neurons. In addition, replacement of l-glutamate with l-theanine in an in vitro cell-free glutathione-synthesis system produced glutathione-like thiol compounds. Furthermore, l-theanine administration (4 mg/kg, p.o.) for 14 days significantly increased glutathione levels in the striatum of mice. The results suggest that l-theanine provides neuroprotection against oxidative stress-induced neuronal damage by humoral molecules released from astrocytes, probably including glutathione.

Lack of dopaminergic inputs elongates the primary cilia of striatal neurons.

In the rodent brain, certain G protein-coupled receptors and adenylyl cyclase type 3 are known to localize to the neuronal primary cilium, a primitive sensory organelle protruding singly from almost all neurons. A recent chemical screening study demonstrated that many compounds targeting dopamine receptors regulate the assembly of Chlamydomonas reinhardtii flagella, structures which are analogous to vertebrate cilia. Here we investigated the effects of dopaminergic inputs loss on the architecture of neuronal primary cilia in the rodent striatum, a brain region that receives major dopaminergic projections from the midbrain. We first analyzed the lengths of neuronal cilia in the dorsolateral striatum of hemi-parkinsonian rats with unilateral lesions of the nigrostriatal dopamine pathway. In these rats, the striatal neuronal cilia were significantly longer on the lesioned side than on the non-lesioned side. In mice, the repeated injection of reserpine, a dopamine-depleting agent, elongated neuronal cilia in the striatum. The combined administration of agonists for dopamine receptor type 2 (D2) with reserpine attenuated the elongation of striatal neuronal cilia. Repeated treatment with an antagonist of D2, but not of dopamine receptor type 1 (D1), elongated the striatal neuronal cilia. In addition, D2-null mice displayed longer neuronal cilia in the striatum compared to wild-type controls. Reserpine treatment elongated the striatal neuronal cilia in D1-null mice but not in D2-null mice. Repeated treatment with a D2 agonist suppressed the elongation of striatal neuronal cilia on the lesioned side of hemi-parkinsonian rats. These results suggest that the elongation of striatal neuronal cilia following the lack of dopaminergic inputs is attributable to the absence of dopaminergic transmission via D2 receptors. Our results provide the first evidence that the length of neuronal cilia can be modified by the lack of a neurotransmitter's input.

Protective effects of phytochemical antioxidants against neurotoxin-induced degeneration of dopaminergic neurons.

The specific toxicity to dopaminergic neurons of psychostimulants and neurotoxins has been extensively studied in vivo and in vitro, and findings have been used to establish animal models of amphetamine psychosis or Parkinson's disease. The multiple mechanisms of neurotoxicity operating in these disorders are known to involve oxidative stress or neuroinflammation, producing the characteristic behavioral and neuropathlogical changes arising from injured dopaminergic neurons and glial cells. A number of studies have shown that glia-targeting antioxidants play important roles in protecting against the neurotoxicity caused by psychostimulants or neurotoxins. Phytochemicals, which are non-nutritive plant chemicals, protect dopaminergic neurons and glial cells from damage caused by psychostimulants or neurotoxins. The objective of this review was to evaluate the involvement of glial cells in dopaminergic neuron-specific toxicity and to explore the neuroprotective activity of phytochemicals in terms of anti-inflammatory and antioxidant action.

Targeting 5-HT(1A) receptors in astrocytes to protect dopaminergic neurons in Parkinsonian models.

Astrocytes are abundant neuron-supporting glial cells that harbor a powerful arsenal of neuroprotective antioxidative molecules and neurotrophic factors. Here we examined whether enrichment with healthy striatal astrocytes can provide neuroprotection against progressive dopaminergic neurodegeneration. Serotonin 1A (5-HT1A) agonist 8-OH-DPAT induced astrocyte proliferation and increased metallothionein-1/-2 (MT-1/-2), antioxidative molecules, in cultured astrocytes and the striatum of mice. Primary cultured mesencephalic dopamine neurons were protected against oxidative stress by preincubation with conditioned media from 8-OH-DPAT-treated astrocytes. These protective effects were canceled by 5-HT1A antagonist or MT-1/-2-specific antibody. Furthermore, reduction of nigrostriatal dopaminergic neurons in 6-hydroxydopamine-lesioned parkinsonian model mice was significantly abrogated by repeated injections of 8-OH-DPAT. Treatment with 8-OH-DPAT markedly increased the expression of MT in striatal astrocytes in the hemi-parkinsonian mice. Our study provides a promising therapeutic strategy of neuroprotection against oxidative stress and progressive dopaminergic neurodegeneration by demonstrating the efficacy of targeting 5-HT1A receptors in astrocytes.

Cyclooxygenase-independent neuroprotective effects of aspirin against dopamine quinone-induced neurotoxicity.

Prostaglandin H synthase exerts not only cyclooxygenase activity but also peroxidase activity. The latter activity of the enzyme is thought to couple with oxidation of dopamine to dopamine quinone. Therefore, it has been proposed that cyclooxygenase inhibitors could suppress dopamine quinone formation. In the present study, we examined effects of various cyclooxygenase inhibitors against excess methyl L-3,4-dihydroxyphenylalanine (L-DOPA)-induced quinoprotein (protein-bound quinone) formation and neurotoxicity using dopaminergic CATH.a cells. The treatment with aspirin inhibited excess methyl L-DOPA-induced quinoprotein formation and cell death. However, acetaminophen did not show protective effects, and indomethacin and meloxicam rather aggravated these methyl L-DOPA-induced changes. Aspirin and indomethacin did not affect the level of glutathione that exerts quenching dopamine quinone in dopaminergic cells. In contrast with inhibiting effects of higher dose in the previous reports, relatively lower dose of aspirin that affected methyl L-DOPA-induced quinoprotein formation and cell death failed to prevent cyclooxygenase-induced dopamine chrome generation in cell-free system. Furthermore, aspirin but not acetaminophen or meloxicam showed direct dopamine quinone-scavenging effects in dopamine-semiquinone generating systems. The present results suggest that cyclooxygenase shows little contribution to dopamine oxidation in dopaminergic cells and that protective effects of aspirin against methyl L-DOPA-induced dopamine quinone neurotoxicity are based on its cyclooxygenase-independent property.

Protective effects of baicalein against excess L-DOPA-induced dopamine quinone neurotoxicity.

OBJECTIVES: Baicalein, a flavonoid derived from the root of Scutelaria baicalensis Georgi, possesses anti-oxidative properties including reactive oxygen species scavenging and lipid peroxidation inhibiting activities. The present study was undertaken to investigate the neuroprotective effect of baicalein against dopamine (DA) neurotoxicity induced by exposure to a synthetic DA precursor, L-3,4-dihydroxyphenylalanine (L-DOPA), in cultured dopaminergic CATH.a cells. METHODS AND RESULTS: Exposure to L-DOPA for 24 hours reduced the number of viable cells and enhanced protein-bound quinone (quinoprotein) formation in the cell. Both effects were prevented by simultaneous treatment with baicalein. In addition, baicalein prevented the formation of DA semiquinone radicals from DA in an in vitro cell-free system. Long-term baicalein treatment for 96 hours also protected against excess L-DOPA-induced cell death, and also increased glutathione (GSH) levels in CATH.a cells. DISCUSSION: Our results indicate that baicalein has neuroprotective properties against excess L-DOPA-induced DA neurotoxicity through the suppression of DA quinone formation. Furthermore, the long-term treatment of baicalein upregulates intracellular GSH contents, which may also exert neuroprotective effects against oxidative stress-induced neuronal damage.

Astrocyte-derived metallothionein protects dopaminergic neurons from dopamine quinone toxicity.

Our previous studies demonstrated the involvement of quinone formation in dopaminergic neuron dysfunction in the L-DOPA-treated parkinsonian model and in methamphetamine (METH) neurotoxicity. We further reported that the cysteine-rich metal-binding metallothionein (MT) family of proteins protects dopaminergic neurons against dopamine (DA) quinone neurotoxicity by its quinone-quenching property. The aim of this study was to examine MT induction in astrocytes in response to excess DA and the potential neuroprotective effects of astrocyte-derived MTs against DA quinone toxicity. DA exposure significantly upregulated MT-1/-2 in cultured striatal astrocytes, but not in mesencephalic neurons. This DA-induced MT upregulation in astrocytes was blocked by treatment with a DA-transporter (DAT) inhibitor, but not by DA-receptor antagonists. Expression of nuclear factor erythroid 2-related factor (Nrf2) and its binding activity to antioxidant response element of MT-1 gene were significantly increased in the astrocytes after DA exposure. Nuclear translocation of Nrf2 was suppressed by the DAT inhibitor. Quinone formation and reduction of mesencephalic DA neurons after DA exposure were ameliorated by preincubation with conditioned media from DA-treated astrocytes. These protective effects were abrogated by MT-1/-2-specific antibody. Adding exogenous MT-1 to glial conditioned media also showed similar neuroprotective effects. Furthermore, MT-1/-2 expression was markedly elevated specifically in reactive astrocytes in the striatum of L-DOPA-treated hemi-parkinsonian mice or METH-injected mice. These results suggested that excess DA taken up by astrocytes via DAT upregulates MT-1/-2 expression specifically in astrocytes, and that MTs or related molecules secreted specifically by astrocytes protect dopaminergic neurons from damage through quinone quenching and/or scavenging of free radicals.

Neuroprotective effects of zonisamide target astrocyte.

OBJECTIVE: Recent double-blind, controlled trials in Japan showed that the antiepileptic agent zonisamide (ZNS) improves the cardinal symptoms of Parkinson's disease. Glutathione (GSH) exerts antioxidative activity through quenching reactive oxygen species and dopamine quinone. GSH depletion within dopaminergic neurons impairs mitochondrial complex I activity, followed by age-dependent nigrostriatal neurodegeneration. This study examined changes in GSH and GSH synthesis-related molecules, and the neuroprotective effects of ZNS on dopaminergic neurodegeneration using 6-hydroxydopamine-injected hemiparkinsonian mice brain and cultured neurons or astrocytes. METHODS AND RESULTS: ZNS increased both the cell number and GSH levels in astroglial C6 cells, but not in dopaminergic neuronal CATH.a cells. Repeated injections of ZNS (30mg/kg intraperitoneally) for 14 days also significantly increased GSH levels and S100beta-positive astrocytes in mouse basal ganglia. Repeated ZNS injections (30mg/kg) for 7 days in the hemiparkinsonian mice increased the expression of cystine/glutamate exchange transporter xCT in activated astrocytes, which supply cysteine to neurons for GSH synthesis. Treatment of these mice with ZNS also increased GSH levels and completely suppressed striatal levodopa-induced quinone formation. Reduction of nigrostriatal dopamine neurons in the lesioned side of hemiparkinsonian mice was significantly abrogated by repeated injections of ZNS with or without adjunctive levodopa starting 3 weeks after 6-hydroxydopamine lesioning. INTERPRETATION: These results provide new pharmacological evidence for the effects of ZNS. ZNS markedly increased GSH levels by enhancing the astroglial cystine transport system and/or astroglial proliferation via S100beta production or secretion. ZNS acts as a neuroprotectant against oxidative stress and progressive dopaminergic neurodegeneration.

Dopamine-induced behavioral changes and oxidative stress in methamphetamine-induced neurotoxicity.

High-dose administration of amphetamine-like compounds is associated with acute behavioral toxicity (including stereotypic and self-injurious behavior and schizophrenic-like psychoses) as well as long-lasting damage to dopaminergic neurons. Several mechanisms are thought to be responsible for methamphetamine-induced neurotoxicity including the formation of reactive oxygen species, dopamine quinones, glutamatergic activity, apoptosis, etc. Recently, new factors regarding glial cell line-derived neurotorophic factor, tumor necrosis factor-alpha, and interferon-gamma have also been associated with methamphetamine-induced neurotoxicity. The objective of this review is to link the behavioral and neurotoxic responses of the amphetamines, emphasizing their common underlying mechanism of monoaminergic release together with inhibition of monoamine oxidase activity. The amphetamine-induced release of dopamine and inhibition of monoamine oxidase increases both cytosolic and synaptic levels of dopamine leading to the acute manifestation of stereotypic and self-injurious behavior. In turn, the enhanced extravesicular levels of dopamine lead to oxidative stress through the generation of reactive oxygen species and dopamine quinones, and cause the long-lasting neuronal damage. Thus, we propose that acute behavioral observation of subjects immediately following methamphetamine administration may provide insight into the long-lasting toxicity to dopaminergic neurons.

[New perspectives on the mechanism of methamphetamine-induced neurotoxicity].

Various hypotheses have been proposed concerning the mechanisms responsible for methamphetamine (METH)-induced neurotoxicity including reactive oxygen species (ROS), dopamine quinones, glutamatergic activity, apoptosis, etc. Recently, new factors regarding glial cell line-derived neurotorophic factor, tumor necrosis factor-alpha, and alpha-synuclein contained in striatal interneural inclusions have also been associated with METH-induced neurotoxicity. In addition, METH-induced self-injurious behavior (SIB) has been proposed to be an acute or immediate behavioral marker predicting the long-lasting neurotoxicity induced by METH. Specifically, it has been proposed that the SIB response may accurately reflect the underlying mechanistic changes occurring in the neuron that eventually result in the long-lasting damage. Several studies have demonstrated that endogenous dopamine (DA) plays an important role in mediating METH-induced neuronal damage. DA release and redistribution from synaptic vesicles to cytoplasmic compartments is thought to involve METH-induced changes in both the vesicular monoamine transporter-2 and DA transporter function. In turn, the consequent elevation of cytosolic auto-oxidizable DA concentrations is thought generate ROS such as superoxide and hydroxyl radicals and cause the DA terminal injury. Finally, the inflammatory response of microglia and glutamatergic toxicity in astrocytes have been related to the METH-induced neurotoxicity. The objective of the present review will be to consolidate the new perspectives in an attempt to formulate a more cohesive explanation of the underlying mechanism responsible for METH-induced DA damage and its early biological markers.