Female Medaka Research Articles

Characterization of Immune Aging in the Japanese Medaka (Oryzias latipes)

The prevalence of chronic inflammation increases with age and may be aggravated by environmental exposures. Similarly, during immune aging, inflammatory disease incidence increases as protective immunity decreases. To better understand disease and exposure risks, an immune aging model outlining key changes in immune function is crucial. Utilizing the lowest possible vertebrate class, we propose the Japanese medaka (Oryzias latipes) as a model to investigate sex-specific immune aging including changes in immune gene expression, leukocyte profiles, and organismal level immune response. Evaluating the expression of immune initiators (CRP, TLR5-s, TLR5-m, TCRb, and MHCII), immune mediators (MYD88, Nf-kß, C3, and IL1b), and immune effectors (LYZ and C8) in concomitance with alterations in leukocyte populations and host resistance to pathogens will inform about immune competence across ages. The data presented here demonstrate a critical decrease in the expression of immune initiators (CRP, TLR5-soluble, TCRb, and MHCII), mediators (MYD88, Nf-kß, C3, and IL1b), and effector (LYZ) in both females and males after 11 months post hatching (mph). Interestingly, both sexes displayed an upregulation for the immune effector, C8, during this older life stage (11–13 mph). Gene expression profiles for both sexes at the most elderly age (20 or 23 mph) appear to revert to a younger profile of expression indicating a second change in immune function during aging rather than a steady decline. Significant changes in leukocyte populations were observed in both male and female medaka after peaking sexual maturation at 3 mph. Organismal level immune competence data revealed male medaka at the elderly age to be more vulnerable than their female and younger male counterparts while no differences were observed in females based on age. Together, these data provide a holistic profile for immune aging in medaka, a useful tool for future immunological studies considering age as a factor influencing disease susceptibility.

Exposure effects of benzalkonium chloride (BAC) on gonadal physiology and fertility suppression in medaka (Oryzias latipes)

Benzalkonium chloride (BAC), a commonly used quaternary ammonium compound in various products like antiseptics, cosmetics, and disinfectants, has raised concerns due to its potential to contaminate aquatic environments and subsequently affect the reproductive performance of the organisms within those ecosystems. The article underscores a critical concern regarding the impact of BAC on aquatic ecosystems, particularly its effect on fish reproductive quality, using medaka (Oryzias latipes) as a model organism. Firstly, while measuring lethal dose of BAC in adult medaka, we observed a dose dependent mortality in BAC treated fish (100 and 200 ppm: 100%; 60 ppm: 51.7%; 30 ppm or less: no mortality at 24 h post treatment (hpt)) and calculated the LD50 at 96 hpt as 39.291 ppm (95% confidence interval: 28.817–53.570 ppm). Further, we assessed the molecular, cellular and histological changes through long-term exposure. Enlarged sperm pockets and reduced spermatocyte were seen in BAC exposed testis while no significant structural changes were observed in the ovaries. Following BAC exposure, drastic alterations in the gene expression and cellular localization related to sex, estrogen signaling, and autophagy were also noted from gonads and liver. Subsequently, using a short-term exposure analysis, we confirmed the sex and time responsive transcriptional kinetics and found that BAC sequentially affected the gonadal somatic cells followed by germ cell differentiation. Finally, using reproductively competent male and female medaka, we conducted progeny production and performance analysis and depicted a drastic reduction in fecundity, and fertilization and hatching rate, indicating adverse effects of BAC on reproductive success. Cumulatively, these findings emphasize the consequences of widespread use of BAC on reproductive security of aquatic animals and highlights the need for further research to comprehend the potential harm posed by such compounds to aquatic animal health and ecosystem integrity.

Medaka liver developed Human NAFLD-NASH transcriptional signatures in response to ancestral bisphenol A exposure.

The progression of fatty liver disease to non-alcoholic steatohepatitis (NASH) is a leading cause of death in humans. Lifestyles and environmental chemical exposures can increase the susceptibility of humans to NASH. In humans, the presence of bisphenol A (BPA) in urine is associated with fatty liver disease, but whether ancestral BPA exposure leads to the activation of human NAFLD-NASH-associated genes in the unexposed descendants is unclear. In this study, using medaka fish as an animal model for human NAFLD, we investigated the transcriptional signatures of human NAFLD-NASH and their associated roles in the pathogenesis of the liver of fish that were not directly exposed, but their ancestors were exposed to BPA during embryonic and perinatal development three generations prior. Comparison of bulk RNA-Seq data of the liver in BPA lineage male and female medaka with publicly available human NAFLD-NASH patient data revealed transgenerational alterations in the transcriptional signature of human NAFLD-NASH in medaka liver. Twenty percent of differentially expressed genes (DEGs) were upregulated in both human NAFLD patients and medaka. Specifically in females, among the total shared DEGs in the liver of BPA lineage fish and NAFLD patient groups, 27.69% were downregulated, and 20% were upregulated. Of all DEGs, 52.31% of DEGs were found in ancestral BPA-lineage females, suggesting that NAFLD in females shared the majority of human NAFLD gene networks. Pathway analysis revealed beta-oxidation, lipoprotein metabolism, and HDL/LDL-mediated transport processes linked to downregulated DEGs in BPA lineage males and females. In contrast, the expression of genes encoding lipogenesis-related proteins was significantly elevated in the liver of BPA lineage females only. BPA lineage females exhibiting activation of myc, atf4, xbp1, stat4, and cancerous pathways, as well as inactivation of igf1, suggest their possible association with an advanced NAFLD phenotype. The present results suggest that gene networks involved in the progression of human NAFLD and the transgenerational NAFLD in medaka are conserved and that medaka can be an excellent animal model to understand the development and progression of liver disease and environmental influences in the liver.

Sex-specific expression of the human NAFLD-NASH transcriptional signatures in the liver of medaka with a history of ancestral bisphenol A exposure.

The progression of fatty liver disease to non-alcoholic steatohepatitis (NASH) is a leading cause of death in humans. Lifestyles and environmental chemical exposures can increase the susceptibility of humans to NASH. In humans, the presence of bisphenol A (BPA) in urine is associated with fatty liver disease, but whether ancestral BPA exposure leads to the activation of human NAFLD-NASH-associated genes in the unexposed descendants is unclear. In this study, using medaka fish as an animal model for human NAFLD, we investigated the transcriptional signatures of human NAFLD-NASH and their associated roles in the pathogenesis of the liver of fish who were not directly exposed but their ancestors were exposed to BPA during embryonic and perinatal development three generations prior. Comparison of bulk RNA-Seq data of the liver in BPA lineage male and female medaka with publicly available human NAFLD-NASH patient data revealed transgenerational alterations in the transcriptional signature of human NAFLD-NASH in medaka liver. Twenty percent of differentially expressed genes (DEGs) were upregulated in both human NAFLD patients and medaka. Specifically in females, among the total shared DEGs in the liver of BPA lineage fish and NAFLD patient groups, 27.69% DEGs were downregulated and 20% DEGs were upregulated. Off all DEGs, 52.31% DEGs were found in ancestral BPA-lineage females, suggesting that NAFLD in females shared majority of human NAFLD gene networks. Pathway analysis revealed beta-oxidation, lipoprotein metabolism, and HDL/LDL-mediated transport processes linked to downregulated DEGs in BPA lineage males and females. In contrast, the expression of genes encoding lipogenesis-related proteins was significantly elevated in the liver of BPA lineage females only. BPA lineage females exhibiting activation of myc, atf4, xbp1, stat4, and cancerous pathways, as well as inactivation of igf1, suggest their possible association with an advanced NAFLD phenotype. The present results suggest that gene networks involved in the progression of human NAFLD and the transgenerational NAFLD in medaka are conserved and that medaka can be an excellent animal model to understand the development and progression of liver disease and environmental influences in the liver.

Effects of salinity on behavior and reproductive toxicity of BPA in adult marine medaka

Salinity is an important environmental factor influencing the toxicity of chemicals. Bisphenol A (BPA) is an environmental endocrine disruptor with adverse effects on aquatic organisms, such as fish. However, the influence of salinity on the biotoxicity of BPA and the underlying mechanism are unclear. In this study, we exposed marine medaka (Oryzias melastigma) to BPA at different salinities (0 psμ, 15 psμ, and 30 psμ) for 70days to investigate the toxic effects. At 0 psμ salinity, BPA had an inhibitory effect on the swimming behavior of female medaka. At 15 psμ salinity, exposure to BPA resulted in necrotic cells in the ovaries but not on the spermatozoa. In addition, BPA exposure changed the transcript levels of genes related to the nervous system (gap43, elavl3, gfap, mbpa, and α-tubulin) and the hypothalamic-pituitary-gonadal (HPG) axis (fshr, lhr, star, arα, cyp11a, cyp17a1, cyp19a, and erα); the expression changes differed among salinity levels. These results suggest that salinity influences the adverse effects of BPA on the nervous system and reproductive system of medaka. These results emphasize the importance of considering the impact of environmental factors when carrying out ecological risk assessment of pollutants.

Unregistered Hexaphenoxycyclotriphosphazene and Its Metabolite Antagonize Retinoic Acid and Retinoic X Receptors and Cause Early Developmental Damage.

Hexaphenoxycyclotriphosphazene (HPCTP), an unregistered chemical, has been used as a substitute for triphenyl phosphate in flame retardants and plasticizers. Here, we identified its metabolite, pentaphenoxycyclotriphosphazene (PPCTP) in the liver of Japanese medaka exposed to HPCTP. When sexually mature female medaka were exposed to HPCTP at 37.0, 90.4, and 465.4 ng/L for 35 days, the HPCTP concentration (642.1-2531.9 ng/g lipid weight [lw]) in the embryos considerably exceeded that (34.7-298.1 ng/g lw) in the maternal muscle, indicating remarkable maternal transfer. During 0-9 days postfertilization, the HPCTP concentration in the embryos decreased continuously, while the PPCTP concentration increased. HPCTP and PPCTP antagonized the retinoic X receptor with 50% inhibitory concentrations (IC50) of 34.8 and 21.2 μM, respectively, and PPCTP also antagonized the retinoic acid receptor with IC50 of 2.79 μM. Such antagonistic activities may contribute to eye deformity (4.7% at 465.4 ng/L), body malformation (2.1% at 90.4 ng/L and 6.8% at 465.4 ng/L), and early developmental mortality (11.6-21.7% in all exposure groups) of the embryos. HPCTP was detected in a main tributary of the Yangtze River Basin. Thus, HPCTP poses a risk to wild fish populations, given the developmental toxicities associated with this chemical and its metabolite.

Dietary exposure to sulfamethazine alters fish intestinal homeostasis and promotes resistance gene transfer

The present study was undertaken to explore the effects of sulfamethazine (SMZ) dietary exposure on the enrichment of the intestine microbial structure, and antibiotic resistance gene (ARGs) transmission in marine medaka, with respect to antibiotic dose, duration, and sex. In male fish, a dietary exposure of 10 μg/L SMZ led to a heightened SMZ enrichment in the intestine, whereas metabolite (N-SMZ) levels were elevated at a higher exposure concentration (100 μg/L). Conversely, female fish exhibited stable levels of accumulation and metabolic rates across the exposure period. The composition of intestinal microorganisms revealed that exposure duration exerted a greater impact on the abundance and diversity of gut microbes, and microbial responses to SMZ varied across exposure time points. The expansion of Bacteroidetes and Ruegeria likely stimulated SMZ metabolism and contributed to the more balanced level of SMZ and N-SMZ observed in females. In males, short-term SMZ stress resulted in a disruption of intestinal homeostasis, while the rise in the abundance of the Fusobacteria and Propionigeniuma suggested a potential enhancement in intestinal anti-inflammatory capacity over time. Overall, female medaka exhibited greater adaptability to SMZ, and males appear to experience prolonged effects due to SMZ. A total of 11 ARGs and 5 mobile genetic elements (MGEs) were identified. Ruegeria is the main carrier of two types of MGEs (IS1247, ISSm2-Xanthob), and may serve as an indicator of ARG transmission. Therefore, it is rational to consider some fish breeding areas in natural waters as potential "reservoirs" of antibiotic resistance. This research will provide a valuable reference for the transmission of drug resistance along the food chain.

The PCOS-NAFLD Multidisease Phenotype Occurred in Medaka Fish Four Generations after the Removal of Bisphenol A Exposure.

As a heterogeneous reproductive disorder, polycystic ovary syndrome (PCOS) can be caused by genetic, diet, and environmental factors. Bisphenol A (BPA) can induce PCOS and nonalcoholic fatty liver disease (NAFLD) due to direct exposure; however, whether these phenotypes persist in future unexposed generations is not currently understood. In a previous study, we observed that transgenerational NAFLD persisted in female medaka for five generations (F4) after exposure to an environmentally relevant concentration (10 μg/L) of BPA. Here, we demonstrate PCOS in the same F4 generation female medaka that developed NAFLD. The ovaries contained immature follicles, restricted follicular progression, and degenerated follicles, which are characteristics of PCOS. Untargeted metabolomic analysis revealed 17 biomarkers in the ovary of BPA lineage fish, whereas transcriptomic analysis revealed 292 genes abnormally expressed, which were similar to human patients with PCOS. Metabolomic-transcriptomic joint pathway analysis revealed activation of the cancerous pathway, arginine-proline metabolism, insulin signaling, AMPK, and HOTAIR regulatory pathways, as well as upstream regulators esr1 and tgf signaling in the ovary. The present results suggest that ancestral BPA exposure can lead to PCOS phenotypes in the subsequent unexposed generations and warrant further investigations into potential health risks in future generations caused by initial exposure to EDCs.

Variation in responses to photoperiods and temperatures in Japanese medaka from different latitudes

Seasonal changes are more robust and dynamic at higher latitudes than at lower latitudes, and animals sense seasonal changes in the environment and alter their physiology and behavior to better adapt to harsh winter conditions. However, the genetic basis for sensing seasonal changes, including the photoperiod and temperature, remains unclear. Medaka (Oryzias latipes species complex), widely distributed from subtropical to cool-temperate regions throughout the Japanese archipelago, provides an excellent model to tackle this subject. In this study, we examined the critical photoperiods and critical temperatures required for seasonal gonadal development in female medaka from local populations at various latitudes. Intraspecific differences in critical photoperiods and temperatures were detected, demonstrating that these differences were genetically controlled. Most medaka populations could perceive the difference between photoperiods for at least 1 h. Populations in the Northern Japanese group required 14 h of light in a 24 h photoperiod to develop their ovaries, whereas ovaries from the Southern Japanese group developed under 13 h of light. Additionally, Miyazaki and Ginoza populations from lower latitudes were able to spawn under short-day conditions of 11 and 10 h of light, respectively. Investigation of the critical temperature demonstrated that the Higashidori population, the population from the northernmost region of medaka habitats, had a critical temperature of over 18 °C, which was the highest critical temperature among the populations examined. The Miyazaki and the Ginoza populations, in contrast, were found to have critical temperatures under 14 °C. When we conducted a transplant experiment in a high-latitudinal environment using medaka populations with different seasonal responses, the population from higher latitudes, which had a longer critical photoperiod and a higher critical temperature, showed a slower reproductive onset but quickly reached a peak of ovarian size. The current findings show that low latitudinal populations are less responsive to photoperiodic and temperature changes, implying that variations in this responsiveness can alter seasonal timing of reproduction and change fitness to natural environments with varying harshnesses of seasonal changes. Local medaka populations will contribute to elucidating the genetic basis of seasonal time perception and adaptation to environmental changes.

Toxicity of perfluorobutanesulfonate on gill functions of marine medaka (Oryzias melastigma): A time course and hypoxia co-exposure study

Perfluorobutanesulfonate (PFBS) is found in hypoxia regions. Results of previous studies have shown that hypoxia was capable of altering the inherent toxicity of PFBS. However, regarding gill functions, hypoxic influences and time course progression of toxic effects of PFBS remain unclear. In this study, with the aim to reveal the interaction behavior between PFBS and hypoxia, adult marine medaka Oryzias melastigma were exposed for 7 days to 0 or 10 μg PFBS/L under normoxic or hypoxic conditions. Subsequently, to explore the time-course transition in gill toxicity, medaka were exposed to PFBS for 21 days. The results showed that hypoxia dramatically increased the respiratory rate of medaka gill, which was further enhanced by exposure to PFBS; although exposure to PFBS under normoxic conditions for 7 days did not alter respiration, exposure to PFBS for 21 days significantly accelerated the respiration rate of female medaka. Concurrently, both hypoxia and PFBS were potent to interrupt the gene transcriptions and Na+, K+-ATPase enzymatic activity that play pivotal roles in the osmoregulation in gills of marine medaka, consequently disrupting homeostasis of major ions in blood, such as Na+, Cl−, and Ca2+. In addition, composition and diversity of the microbiome residing on surfaces of the gill were profiled by using amplicon sequencing. Acute exposure to hypoxia for only 7 days caused a significant decrease in diversity of the bacterial community of gill whatever the presence of PFBS, while PFBS exposure for 21 days increased the diversity of gill microbial community. Principal component analysis revealed that, compared with PFBS, hypoxia was the predominant driver of gill microbiome dysbiosis. Depending on duration of exposure, a divergence was caused in the microbial community of gill. Overall, the current findings underline the interaction between hypoxia and PFBS on gill function and demonstrate the temporal variation in PFBS toxicity.

Cholesterol accumulation in ovarian follicles causes ovulation defects in Abca1a−/− Japanese medaka (Oryzias latipes)

ATP-binding cassette A1 (ABCA1) is a membrane protein, which exports excess cellular cholesterol to generate HDL to reduce the risk of the onset of cardiovascular diseases (CVD). In addition, ABCA1 exerts pleiotropic effects on such as inflammation, tissue repair, and cell proliferation and migration. In this study, we explored the novel physiological roles of ABCA1 using Japanese medaka (Oryzias latipes), a small teleost fish. Three Abca1 genes were found in the medaka genome. ABCA1A and ABCA1C exported cholesterol to generate nascent HDL as human ABCA1 when expressed in HEK293 cells. To investigate their physiological roles, each Abca1-deficient fish was generated using the CRISPR-Cas9 system. Abca1a−/− female medaka was found to be infertile, while Abca1b−/− and Abca1c−/− female medaka were fertile. In vitro ovarian follicle culture suggested that Abca1a deficiency causes ovulation defects. In the ovary, ABCA1A was expressed in theca cells, an outermost layer of the ovarian follicle. Total cholesterol content of Abca1a−/− ovary was significantly higher than that of the wild-type, while estrogen and progestin contents were compatible with those of the wild-type. Furthermore, cholesterol loading to the wild-type follicles caused ovulation defects. These results suggest that ABCA1A in theca cells regulates cholesterol content in the ovarian follicles and its deficiency inhibits successful ovulation through cholesterol accumulation in the ovarian follicle.

Prostaglandin E2 receptor Ptger4b regulates female-specific peptidergic neurons and female sexual receptivity in medaka

In vertebrates, female receptivity to male courtship is highly dependent on ovarian secretion of estrogens and prostaglandins. We recently identified female-specific neurons in the medaka (Oryzias latipes) preoptic area that express Npba, a neuropeptide mediating female sexual receptivity, in response to ovarian estrogens. Here we show by transcriptomic analysis that these neurons express a multitude of neuropeptides, in addition to Npba, in an ovarian-dependent manner, and we thus termed them female-specific, sex steroid-responsive peptidergic (FeSP) neurons. Our results further revealed that FeSP neurons express a prostaglandin E2 receptor gene, ptger4b, in an ovarian estrogen-dependent manner. Behavioral and physiological examination of ptger4b-deficient female medaka found that they exhibit increased sexual receptivity while retaining normal ovarian function and that their FeSP neurons have reduced firing activity and impaired neuropeptide release. Collectively, this work provides evidence that prostaglandin E2/Ptger4b signaling mediates the estrogenic regulation of FeSP neuron activity and female sexual receptivity.

Liver comparative transcriptome analysis reveals the mechanism of the Hainan medaka, Oryzias curvinotus, to adapt to salinity

AbstractTo explore the adaptation mechanism of Hainan medaka, Oryzias curvinotus, to salinity, this study performed a histological and RNA‐Seq comparative analysis of female medaka liver with different salinities (0, 15 and 30 practical salinity unit [psu]). In the 30 psu group, the volume of hepatocytes was increased and cytoplasmic fat accumulated. A total of 83,758 unigenes were assembled by RNA‐Seq, of which 31,147 were annotated. In total, 922 differentially expressed genes (DEGs) were obtained, and the detection results of 24 DEGs by quantitative polymerase chain reaction (qPCR) were consistent with the RNA‐Seq data. Gene ontology (GO) enrichment showed that DEGs were mainly involved in biological processes, such as substance synthesis and transport, energy metabolism, signal transduction, and had catalytic activity and binding functions. Kyoto Encyclopedia of Genes and Genomes enrichment analysis showed that DEGs were significantly enriched in steroid biosynthesis, glycerophospholipid metabolism, peroxisome proliferator‐activated receptor signaling pathway, and insulin signaling pathway that regulate lipid metabolism. The results of this study highlight the response of the liver transcriptome to long‐term environmental salinity changes and provide a basis for the application of the medaka genetic resources.

Oxidative Stress Causes Masculinization of Genetically Female Medaka Without Elevating Cortisol.

Medaka (Oryzias latipes) is a teleost fish with an XX/XY sex determination system. Sex reversal from female-to-male (masculinization of XX fish) can be induced through cortisol elevation from exposure to environmental stress such as high temperature during sexual differentiation. However, the effects of oxidative stress, generated via metabolic reactions and biological defense mechanisms, on the sexual differentiation of medaka are unclear. Here, we investigated the effect of oxidative stress on medaka sexual differentiation using hydrogen peroxide (H2O2), which induces oxidative stress in vertebrates. H2O2 treatment from 0 to 5 days post-hatching induced masculinization of wild-type XX medaka, but not of gonadal soma-derived growth factor (gsdf) or peroxisome proliferator-activated receptor alpha-a (pparaa) knockout XX fish. Co-treatment with an oxidative stress inhibitor caused masculinization recovery but co-treatment with a cortisol synthesis inhibitor did not. H2O2 treatment significantly upregulated gsdf and pparaa expression in XX medaka. However, H2O2 did not elevate cortisol levels in medaka larvae during sexual differentiation. These results strongly indicate that oxidative stress induces masculinization of XX medaka without causing elevation of cortisol.

The light-dependent daily cycle of ovulation in the oviparous medaka fish, Oryzias latipes (Atherinomorpha: Beloniformes: Adrianichthyidae) artificially pregnant with developing embryos.

In the oviparous medaka fish, Oryzias latipes, mature spermatozoa that were artificially introduced into the ovarian cavity retaining ovulated eggs could internally fertilize these eggs. This enabled us to examine the effect of ovarian gestation on the ovulation cycle. Most freshly ovulated eggs could be internally fertilized in the ovarian cavity. Yet eggs ovulated 24 h after single insemination remained unfertilized in the ovarian cavity. Artificially pregnant females persisted in a daily cycle of ovulation, which occurred shortly before the onset of light under the present reproductive conditions. Females continuously ovulated a certain number of eggs despite ovarian gestation, that is, the presence of embryos within the ovarian cavity. Repeated cycles of ovulation led to crowding in the ovarian cavity because the group of fertilized eggs, with their hardened egg envelope (chorion or zona radiata), plugged the genital orifice. The development of fertilized eggs was retarded and ceased around the initiation stage of blood circulation, but when they were transferred from the ovarian cavity into regular saline, they regained their ability to develop normally up to hatching. These results show that in oviparous female medaka, ovarian gestation exerted little effect on the time of ovulation and the number of ovulated eggs.

DIFFERENCES IN THE VULNERABILITY OF MEDAKA FISH (Oryzias celebensisis) MALE AND FEMALE TO CLORPYRIPHOS INSETICIDE

Chlorpyrifos is one of the most widely used organophosphate insecticides used to control pests on plants. The use of insecticides will produce residues in the soil and on plants and can be carried by rain flows to water bodies. This can pollute aquatic ecosystems and could negatively affected the growth of aquatic biota like Oryzias celebensis. This study aimed to determine the susceptibility of male and female medaka celebes (Oryzias celebensis) to chlorpyrifos insecticide. In this study, five fish medaka celebes were put in each jar in one jar for male medaka fish and female medaka fish with five replications. The parameters measured were survival rate, oxygen consumption rate between male and female of O. celebensis, temperature and pH. Statistically the survival rate of male and female medaka fish showed no statistically significant difference (P>0.05). Likewise, there was no difference between the oxygen consumption levels of male and female medaka fish before and after exposure to chlorpyrifos insecticide (P>0.05). This study concluded that there was no difference in susceptibility to chlorpyrifos insecticides between male and female medaka fish.

Estrogen upregulates the firing activity of hypothalamic gonadotropin-releasing hormone (GnRH1) neurons in the evening in female medaka.

The reproductive function of vertebrates is regulated by the hypothalamic-pituitary-gonadal axis. In sexually mature females, gonadotropin-releasing hormone (GnRH) neurons in the preoptic area (POA) are assumed to be responsible for a cyclic large increase in GnRH release, the GnRH surge, triggering a luteinizing hormone (LH) surge, which leads to ovulation. Precise temporal regulation of the preovulatory GnRH/LH surge is important for successful reproduction because ovulation should occur after follicular development. The time course of the circulating level of estrogen is correlated with the ovulatory cycle throughout vertebrates. However, the neural mechanisms underlying estrogen-induced preovulatory GnRH surge after folliculogenesis still remain unclear, especially in non-mammals. Here, we used a versatile non-mammalian model medaka for the analysis of the involvement of estrogen in the regulation of POA-GnRH (GnRH1) neurons. Electrophysiological analysis using a whole brain-pituitary in vitro preparation, which maintains the hypophysiotropic function of GnRH1 neurons intact, revealed that 17β-estradiol (E2 ) administration recovers the ovariectomy-induced lowered GnRH1 neuronal activity in the evening, indicating the importance of E2 for upregulation of GnRH1 neuronal activity. The importance of E2 was also confirmed by the fact that GnRH1 neuronal activity was low in short-day photoperiod-conditioned females (low E2 model). However, E2 failed to upregulate the firing activity of GnRH1 neurons in the morning, suggesting the involvement of additional time-of-day signal(s) for triggering GnRH/LH surges at an appropriate timing. We also provide morphological evidence for the localization of estrogen receptor subtypes in GnRH1 neurons. In conclusion, we propose a working hypothesis in which both estrogenic and time-of-day signals act in concert to timely upregulate the firing activity of GnRH1 neurons that trigger the GnRH surge at an appropriate timing in a female-specific manner. This neuroendocrinological mechanism is suggested to be responsible for the generation of ovulatory cycles in female teleosts in general.

Roles of Gonadotropin Receptors in Sexual Development of Medaka.

The gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), are secreted from the pituitary and bind to the FSH receptor (FSHR) and LH receptor (LHR) to regulate gonadal development in vertebrates. Previously, using fshr-knockout (KO) medaka (Oryzias latipes), we demonstrated that FSH regulates ovarian development by elevating estrogen levels. However, the lhr-KO phenotype in medaka is poorly characterized. Here, we generated lhr-KO medaka using the transcription activator-like effector nuclease (TALEN) technique. We analyzed its phenotype and that of fshr-KO, lhr;fshr double-heterozygotes (double-hetero), and double-KO fish. All genetically male medaka displayed normal testes and were fertile, whereas fshr-KO and double-KO genetically female fish displayed small ovaries containing many early pre-vitellogenic oocytes and were infertile. Although lhr-KO genetically female fish had normal ovaries with full-grown oocytes, ovulation did not occur. Levels of 17α,20β-dihydroxy-4-pregnen-3-one, which is required for meiotic maturation of oocytes and sperm maturation in teleost fish, were significantly decreased in all KO female medaka ovaries except for double-heteros. Further, 17β-estradiol levels in fshr-KO and double-KO ovaries were significantly lower than those in double-heteros. These findings indicate that LH is necessary for oocyte maturation and FSH is necessary for follicle development, but that neither are essential for spermatogenesis in medaka.

Long photoperiod impairs learning in male but not female medaka

SummaryDay length in conjunction with seasonal cycles affects many aspects of animal biology. We have studied photoperiod-dependent alterations of complex behavior in the teleost, medaka (Oryzias latipes), a photoperiodic breeder, in a learning paradigm whereby fish have to activate a sensor to obtain a food reward. Medaka were tested under a long (14:10 LD) and short (10:14 LD) photoperiod in three different groups: mixed-sex, all-males, and all-females. Under long photoperiod, medaka mixed-sex groups learned rapidly with a stable response. Unexpectedly, males-only groups showed a strong learning deficit, whereas females-only groups performed efficiently. In mixed-sex groups, female individuals drove group learning, whereas males apparently prioritized mating over feeding behavior resulting in strongly reduced learning performance. Under short photoperiod, where medaka do not mate, male performance improved to a level similar to that of females. Thus, photoperiod has sex-specific effects on the learning performance of a seasonal vertebrate.

Effects of opioids on reproduction in Japanese medaka, Oryzias latipes.

To study the effects of exposure of fish to opioid drugs, we exposed Japanese medaka (Oryzias latipes) over a full life cycle to codeine spiked into river water at nominal concentrations of 100, 1,000 and 25,000ng/L and to fentanyl spiked into river water at nominal concentrations of 5, 25 and 1,000ng/L. The measured concentrations during medaka exposures were consistent with the nominal concentrations. Treatments with codeine at all test concentrations reduced the number of eggs produced by female medaka, as well as the number of mature oocytes observed histologically in the ovaries. Exposures to codeine also resulted in altered concentrations of hormones within the hypothalamic-pituitary-gonadal axis, including reduced levels of 17β-estradiol in female medaka. Fentanyl did not affect reproduction or the levels of hormones in medaka at the concentrations tested. Monitoring of surface waters in southern Ontario, Canada downstream of wastewater treatment plants showed that the test concentrations of fentanyl and codeine were environmentally relevant. The results of this work contribute to the literature on the impacts of opioids and other drugs of abuse released into surface waters.