Early life adversity promotes resilience to opioid addiction-related phenotypes in male rats and sex-specific transcriptional changes.

Experiencing some early life adversity can have an "inoculating" effect that promotes resilience in adulthood. However, the mechanisms underlying stress inoculation are unknown, and animal models are lacking. Here we used the limited bedding and nesting (LBN) model of adversity to evaluate stress inoculation of addiction-related phenotypes. In LBN, pups from postnatal days 2 to 9 and their dams were exposed to a low-resource environment. In adulthood, they were tested for addiction-like phenotypes and compared to rats raised in standard housing conditions. High levels of impulsivity are associated with substance abuse, but in males, LBN reduced impulsive choice compared to controls. LBN males also self-administered less morphine and had a lower breakpoint on a progressive ratio reinforcement schedule than controls. These effects of LBN on addiction-related behaviors were not found in females. Because the nucleus accumbens (NAc) mediates these behaviors, we tested whether LBN altered NAc physiology in drug-naïve and morphine-exposed rats. LBN reduced the frequency of spontaneous excitatory postsynaptic currents in males, but a similar effect was not observed in females. Only in males did LBN prevent a morphine-induced increase in the AMPA/NMDA ratio. RNA sequencing was performed to delineate the molecular signature in the NAc associated with LBN-derived phenotypes. LBN produced sex-specific changes in transcription, including in genes related to glutamate transmission. Collectively, these studies reveal that LBN causes a male-specific stress inoculation effect against addiction-related phenotypes. Identifying factors that promote resilience to addiction may reveal novel treatment options for patients.

Germ plasm-related structures in marine medaka gametogenesis; novel sites of Vasa localization and the unique mechanism of germ plasm granule arising.

Germ plasm, a cytoplasmic factor of germline cell differentiation, is suggested to be a perspective tool for in vitro meiotic differentiation. To discriminate between the: (1) germ plasm-related structures (GPRS) involved in meiosis triggering; and (2) GPRS involved in the germ plasm storage phase, we investigated gametogenesis in the marine medaka Oryzias melastigma. The GPRS of the mitosis-to-meiosis period are similar in males and females. In both sexes, five events typically occur: (1) turning of the primary Vasa-positive germ plasm granules into the Vasa-positive intermitochondrial cement (IMC); (2) aggregation of some mitochondria by IMC followed by arising of mitochondrial clusters; (3) intramitochondrial localization of IMC-originated Vasa; followed by (4) mitochondrial cluster degradation; and (5) intranuclear localization of Vasa followed by this protein entering the nuclei (gonial cells) and synaptonemal complexes (zygotene-pachytene meiotic cells). In post-zygotene/pachytene gametogenesis, the GPRS are sex specific; the Vasa-positive chromatoid bodies are found during spermatogenesis, but oogenesis is characterized by secondary arising of Vasa-positive germ plasm granules followed by secondary formation and degradation of mitochondrial clusters. A complex type of germ plasm generation, 'the follicle cell assigned germ plasm formation', was found in late oogenesis. The mechanisms discovered are recommended to be taken into account for possible reconstruction of those under in vitro conditions.

Multigenerational Disruption of the Thyroid Endocrine System in Marine Medaka after a Life-Cycle Exposure to Perfluorobutanesulfonate.

Accumulation of perfluorobutanesulfonate (PFBS) is frequently detected in biota, raising concerns about its ecological safety. However, hazardous effects of PFBS remain largely unexplored, especially for endocrine disrupting potency. In the present study, the multigenerational endocrine disrupting potential of PFBS was investigated by exposing F0 marine medaka eggs to PFBS at different concentrations (0, 1.0, 2.9, and 9.5 µg/L) until sexual maturity. The F1 and F2 generations were reared without continued exposure. Thyroidal disturbances were examined in all three generations. PFBS exposure decreased the levels of 3,5,3'-triiodothyronine (T3) in F0 female blood; however, it increased T3 or thyroxine (T4) levels in F0 brains, in which hyperthyroidism suppressed the local transcription of 5'-deiodinase 2 ( Dio2). Obviously decreased T3 was transferred to F1 eggs, although the parental influences were reversed in F1 larvae. Delayed hatching was coupled with elevated T3 levels in F1 larvae. F1 adults showed comparable symptoms of thyroidal disruption with F0 adults. A slight recovery was noted in the F2 generation, although F2 larvae still exhibited thyroid disruption and synthesized excessive T4. Our results suggested that the offspring suffered more severe dysfunction of the thyroidal axis albeit without direct exposure. This study provided the first molecular insight about PFBS toxicology on the thyroid, beneficial to both human and environmental risk assessment.

Modification of the plasma complement protein profile by exogenous estrogens is indicative of a compromised immune competence in marine medaka (Oryzias melastigma).

Growing evidence suggests that the immune system of teleost is vulnerable to xenoestrogens, which are ubiquitous in the marine environment. This study detected and identified the major circulatory immune proteins deregulated by 17α-ethinylestradiol (EE2), which may be linked to fish susceptibility to pathogens in the marine medaka, Oryzias melastigma. Fish immune competence was determined using a host resistance assay to pathogenic bacteria Edwardsiella tarda. Females were consistently more susceptible to infection-induced mortality than males. Exposure to EE2 could narrow the sex gap of mortality by increasing infection-induced death in male fish. Proteomic analysis revealed that the major plasma immune proteins of adult fish were highly sexually dimorphic. EE2 induced pronounced sex-specific changes in the plasma proteome, with the male plasma composition clearly becoming "feminised". Male plasma was found to contain a higher level of fibrinogens, WAP63 and ependymin-2-like protein, which are involved in coagulation, inflammation and regeneration. For the first time, we demonstrated that expression of C1q subunit B (C1Q), an initiating factor of the classical complement pathway, was higher in males and was suppressed in both sexes in response to EE2 and bacterial challenge. Moreover, cleavage and post-translational modification of C3, the central component of the complement system, could be altered by EE2 treatment in males (C3dg down; C3g up). Multiple regression analysis indicated that C1Q is possibly an indicator of fish survival, which warrants further confirmation. The findings support the potential application of plasma immune proteins for prognosis/diagnosis of fish immune competence. Moreover, this study provides the first biochemical basis of the sex-differences in fish immunity and how these differences might be modified by xenoestrogens.

Identification of Molecular Targets for 4,5-Dichloro-2-n-octyl-4-isothiazolin-3-one (DCOIT) in Teleosts: New Insight into Mechanism of Toxicity.

Environmental pollutants are capable of concomitantly inducing diverse toxic effects. However, it is largely unknown which effects are directly induced and which effects are secondary, thus calling for definitive identification of the initiating molecular event for a pollutant to elucidate the mechanism of toxicity. In the present study, affinity pull-down assays were used to identify target proteins for 4,5-dichloro-2-n-octyl-4-isothiazolin-3-one (DCOIT), a costal pollutant of emerging concern, in various tissues (e.g., brain, liver, plasma, and gonad) from marine medaka (Oryzias melastigma) and zebrafish (Danio rerio). Pull-down results showed that, in male and female brains from medaka and zebrafish, DCOIT had a consistently high affinity for G protein alpha subunits (Gα), suggesting the targeted effects of DCOIT on signaling transduction from G protein-coupled receptors (GPCRs) and an extrapolatable mode of action in teleost brains. Validation using recombinant proteins and molecular docking analysis confirmed that binding of DCOIT to Gα protein competitively inhibited its activation by substrate. Considering the involvement of GPCRs in the regulation of myriad biological processes, including the hypothalamus-pituitary-gonadal-liver axis, binding of DCOIT to upstream Gα proteins in the brain may provide a plausible explanation for the diversity of toxic effects resulting from DCOIT challenge, especially abnormal hormonal production through the mitogen-activated protein kinase pathway. A new mechanism of action based on GPCR signaling is thus hypothesized for endocrine disrupting chemicals and warrants further research to clearly elucidate the link between GPCR signaling and endocrine disruption.

Endocrine Disruption throughout the Hypothalamus-Pituitary-Gonadal-Liver (HPGL) Axis in Marine Medaka (Oryzias melastigma) Chronically Exposed to the Antifouling and Chemopreventive Agent, 3,3'-Diindolylmethane (DIM).

Despite being proposed as a promising antifouling and chemopreventive agent, the environmental risks of 3,3'-diindolylmethane (DIM) are scarcely investigated. Therefore, this study used adult marine medaka (Oryzias melastigma) as a model organism to examine the toxicological effects and underlying mechanism of DIM throughout the hypothalamus-pituitary-gonadal-liver (HPGL) axis following 28 days of exposure to low DIM concentrations (0 and 8.46 µg/L). The results showed that altered gene transcription in the hypothalamus, pituitary, and gonads contributed to the great imbalance in hormone homeostasis. The lowered estradiol (E2)/testosterone (T) and E2/11-keto-testosterone (11-KT) ratios in female plasma resulted in decreased synthesis and levels of vitellogenin (VTG) and choriogenin in the liver and plasma, and vice versa in males. Subsequently, VTG and choriogenin deficiency blocked the reproductive function of the ovary as indicated by decreased fecundity and offspring viability, whereas in male medaka, DIM mainly targeted the liver and induced severe vacuolization. Proteomic profiling of plasma revealed that the sex-specific susceptibility to DIM could be attributed to the increased detoxification and oxidative defense in males. Overall, this study identified the endocrine disruption and reproductive impairment potency of DIM and first elucidated its mechanisms of action in medaka. The differential responses to DIM (estrogenic activities in the male but antiestrogenic activities in the female) provided sensitive biomarkers characteristic of each sex. Considering the chemical stability and potent endocrine disturbance at low concentration, the application of DIM either as an antifouling or chemopreventive agent should be approached with caution in marine environments.

Multigenerational impacts of EE2 on reproductive fitness and immune competence of marine medaka.

Estrogenic endocrine disrupting chemicals (EEDC) have been suspected to impact offspring in a transgenerational manner via modifications of the germline epigenome in the directly exposed generations. A holistic assessment of the concentration/ exposure duration-response, threshold level, and critical exposure windows (parental gametogenesis and embryogenesis) for the transgenerational evaluation of reproduction and immune compromise concomitantly will inform the overall EEDC exposure risk. We conducted a multigenerational study using the environmental estrogen, 17α-ethinylestradiol (EE2), and the marine laboratory model fish Oryzias melastigma (adult, F0) and their offspring (F1-F4) to identify transgenerationally altered offspring generations and phenotype persistence. Three exposure scenarios were used: short parental exposure, long parental exposure, and a combined parental and embryonic exposure using two concentrations of EE2 (33ng/L, 113ng/L). The reproductive fitness of fish was evaluated by assessing fecundity, fertilization rate, hatching success, and sex ratio. Immune competence was assessed in adults via a host-resistance assay. EE2 exposure during both parental gametogenesis and embryogenesis was found to induce concentration/ exposure duration-dependent transgenerational reproductive effects in the unexposed F4 offspring. Furthermore, embryonic exposure to 113 ng/L EE2 induced feminization of the directly exposed F1 generation, followed by subsequent masculinization of the F2 and F3 generations. A sex difference was found in the transgenerationally impaired reproductive output with F4 females being sensitive to the lowest concentration of EE2 (33 ng/L) upon long-term ancestral parent exposure (21 days). Conversely, F4 males were affected by ancestral embryonic EE2 exposure. No definitive transgenerational impacts on immune competence were identified in male or female offspring. In combination, these results indicate that EEDCs can be transgenerational toxicants that may negatively impact the reproductive success and population sustainability of fish populations.

Paternal morphine exposure enhances morphine self-administration and induces region-specific neural adaptations in reward-related brain regions of male offspring.

Background: A growing body of preclinical studies report that preconceptional experiences can have a profound and long-lasting impact on adult offspring behavior and physiology. However, less is known about paternal drug exposure and its effects on reward sensitivity in the next generation. Methods: Adult male rats self-administered morphine for 65 days; controls received saline. Sires were bred to drug-naïve dams to produce first-generation (F1) offspring. Morphine, cocaine, and nicotine self-administration were measured in adult F1 progeny. Molecular correlates of addiction-like behaviors were measured in reward-related brain regions of drug naïve F1 offspring. Results: Male, but not female offspring produced by morphine-exposed sires exhibited dose-dependent increased morphine self-administration and increased motivation to earn morphine infusions under a progressive ratio schedule of reinforcement. This phenotype was drug-specific as self-administration of cocaine, nicotine, and sucrose were not altered by paternal morphine history. The male offspring of morphine-exposed sires also had increased expression of mu-opioid receptors in the ventral tegmental area but not in the nucleus accumbens. Conclusions: Paternal morphine exposure increased morphine addiction-like behavioral vulnerability in male but not female progeny. This phenotype is likely driven by long-lasting neural adaptations within the reward neural brain pathways.

Transcriptomics in the nucleus accumbens shell reveal sex- and reinforcer-specific signatures associated with morphine and sucrose craving.

Incubation of craving is a well-documented phenomenon referring to the intensification of drug craving over extended abstinence. The neural adaptations that occur during forced abstinence following chronic drug taking have been a topic of intense study. However, little is known about the transcriptomic changes occurring throughout this window of time. To define gene expression changes associated with morphine consumption and extended abstinence, male and female rats underwent 10 days of morphine self-administration. Separate drug-naive rats self-administered sucrose in order to compare opioid-induced changes from those associated with natural, non-drug rewards. After one or 30 days of forced abstinence, rats were tested for craving, or nucleus accumbens shell tissue was dissected for RNA sequencing. Morphine consumption was predictive of drug seeking after extended (30 days) but not brief (1 day) abstinence in both sexes. Extended abstinence was also associated with robust sex- and reinforcer-specific changes in gene expression, suggesting sex differences underlying incubation of morphine and sucrose seeking respectively. Importantly, these changes in gene expression occurred without re-exposure to drug-paired cues, indicating that chronic morphine causes long-lasting changes in gene expression that prime the system for increased craving. These findings lay the groundwork for identifying specific therapeutic targets for curbing opioid craving without impacting the natural reward system in males and females.

Sex-specific immunomodulatory action of the environmentalestrogen 17α-ethynylestradiol alongside with reproductive impairment in fish.

Estrogenic endocrine disrupting chemicals (EEDCs) are present ubiquitously in sediments and aquatic ecosystems worldwide. The detrimental impact of EEDCs on the reproduction of wildlife is widely recognized. Increasing evidence shows the immunosuppressive effects of EEDCs in vertebrates. Yet, no studies have considered concomitantly EEDC-induced impacts on reproductive impairment and immune suppression in vivo, which are deemed essential for risk assessment and environmental monitoring. In this study, EE2 was used as a representative EEDC, for parallel evaluation of EEDC-induced immune suppression (immune marker gene expression, leukocyte numbers, host resistance assay, and immune competence index) and reproductive impairment (estrogen responsive gene expression, fecundity, fertilization success, hatching success, and reproductive competence index) in an established fish model (marine medaka Oryzias melastigma), considering sex-specific induction and adaptation and recovery responses under different EE2 exposure scenarios. The findings in marine medaka reveal distinct sex differences in the EE2-mediated biological responses. For female fish, low concentration of exogenous EE2 (33 ng/L) could induce hormesis (immune enhancement), enable adaptation (restored reproduction) and even boost fish resistance to bacterial challenge after abatement of EE2. However, a prolonged exposure to high levels of EE2 (113 ng/L) not only impaired F0 immune function, but also perturbed females recovering from reproductive impairment, resulting in a persistent impact on the F1 generation output. Thus, for female fish, the exposure concentration of EE2 is more critical than the dose of EE2 in determining the impacts of EE2 on immune function and reproduction. Conversely, male fish are far more sensitive than females to the presence of low levels of exogenous EE2 in water and the EE2-mediated biological impacts are clearly dose-dependent. It is also evident in male fish that direct contact of EE2 is essential to sustain impairments of immune competence and reproductive output as well as deregulation of immune function genes in vivo. The immunomodulatory pathways altered by EE2 were deciphered for male and female fish, separately. Downregulation of hepatic tlr3 and c3 (in female) and tlr3, tlr5 and c3 (in male) may be indicative of impaired fish immune competence. Taken together, impaired immune competence in the EE2-exposed fish poses an immediate thread on the survival of F0 population. Impaired reproduction in the EE2-exposed fish can directly affect F1 output. Parallel evaluation of immune competence and reproduction are important considerations when assessing the risk of sublethal levels of EE2/EEDCs in aquatic environments.

Sex-dependent telomere shortening, telomerase activity and oxidative damage in marine medaka Oryzias melastigma during aging.

Marine medaka Oryzias melastigma at 4months (young), 8months (middle-aged) and 12months old (senior) were employed to determine age-associated change of sex ratios, sex hormones, telomere length (TL), telomerase activity (TA), telomerase transcription (omTERT) and oxidative damage in the liver. Overall, O. melastigma exhibited gradual senescence, sex differences in longevity (F>M), TL (F>M) and oxidative damage (F5kb), TA and omTERT expression (p≤0.01), and negatively correlated with liver DNA oxidation (p≤0.05). The results suggest high levels of E2 in female O. melastigma may retard TL shortening by enhancing TA via TERT transcription and/or reducing oxidative DNA damage. The findings support TL shortening as a biomarker of aging and further development of accelerated TL shortening, abnormal suppression of TA and excessive oxidative DNA damage as early molecular endpoints, indicative of advanced/premature aging in marine medaka/fish.

Immune competence assessment in marine medaka (Orzyias melastigma)-a holistic approach for immunotoxicology.

Many anthropogenic pollutants in coastal marine environments can induce immune impairments in wild fish and reduce their survival fitness. There is a pressing need to establish sensitive and high throughput in vivo tools to systematically evaluate the immunosuppressive effects of contaminants in marine teleosts. This study reviewed a battery of in vivo immune function detection technologies established for different biological hierarchies at molecular (immune function pathways and genes by next generation sequencing (NGS)), cellular (leukocytes profiles by flow cytometry), tissues/organ system (whole adult histo-array), and organism (host resistance assays (HRAs)) levels, to assess the immune competence of marine medaka Oryzias melastigma. This approach enables a holistic assessment of fish immune competence under different chemical exposure or environmental scenarios. The data obtained will also be useful to unravel the underlying immunotoxic mechanisms. Intriguingly, NGS analysis of hepatic immune gene expression profiles (male > female) are in support of the bacterial HRA findings, in which infection-induced mortality was consistently higher in females than in males. As such, reproductive stages and gender-specific responses must be taken into consideration when assessing the risk of immunotoxicants in the aquatic environment. The distinct phenotypic sexual dimorphism and short generation time (3 months) of marine medaka offer additional advantages for sex-related immunotoxicological investigation.

Insight into the transgenerational effect of benzo[a]pyrene on bone formation in a teleost fish (Oryzias latipes).

Recent cross-generational studies in teleost fish have raised the awareness that high levels of benzo[a]pyrene (BaP) could affect skeletal integrity in the directly exposed F0 and their F1-F2. However, no further details were provided about the causes for abnormalities on the molecular and cellular level and the persistence of such sub-organismal impairments at the transgenerational scale (beyond F2). Adult Oryzias latipes were exposed to 1µg/L BaP for 21days. The F1-F3 were examined for skeletal deformities, histopathological alterations of vertebral bodies and differential expression of key genes of bone metabolism. Significant increase of dorsal-ventral vertebral compression was evident in ancestrally exposed larvae. Histopathological analysis revealed abnormal loss of notochord sheath, a lack of notochord epithelial integrity, reduced bone tissue and decreased osteoblast abundance. A significant downregulation of ATF4 and/or osterix and a high biological variability of COL10, coupled with a significant deregulation of SOX9a/b in the F1-F3 suggest that ancestral BaP exposure most likely perturbed chordoblasts, chondroblast and osteoblast differentiation, resulting in defective notochord sheath repair and rendering the vertebral column more vulnerable to compression. The present findings provide novel molecular and cellular insights into BaP-induced transgenerational bone impairment in the unexposed F3. From the ecological risk assessment perspective, BaP needs to be regarded as a transgenerational skeletal toxicant, which exerts a far-reaching impact on fish survival and fitness. Given that basic mechanisms of cartilage/bone formation are conserved between medaka and mammals, the results may also shed light on the potential transgenerational effect of BaP on the genesis of skeletal diseases in humans.