Assessment of environmental exposure to betamethasone on the reproductive function of female Japanese medaka (Oryzias latipes).

Betamethasone has been extensively used in medicine in recent years and poses potential hazards to aquatic organisms. This study investigated the reproductive toxic effects of betamethasone exposure in fish, employing female Japanese medaka (Oryzias latipes) as a model. Betamethasone exposure at environmentally relevant concentrations (0, 20, 200, and 2000 ng/L) for a period of 15 weeks resulted in its high accumulation in the ovary, leading to abnormal oogenesis in female Japanese medaka. The production of gonadotropins (LH and FSH) in the pituitary gland was inhibited, and sex steroid biosynthesis in the ovary was significantly influenced at the transcriptional level. The imbalance of androgens and estrogens resulted in a decrease in the E2/T ratio and hepatic VTG synthesis, and the suppression of estrogen receptor signaling was also induced. Furthermore, betamethasone exposure delayed spawning and reduced fertility in the F0 generation, and had detrimental effects on the fertilization rate and hatchability of the F1 generation. Our results showed that environmental betamethasone had the potential to adversely affect female fertility and steroid hormone dynamics in fish.

Reproductive endocrine disruption and gonadal intersex induction in male Japanese medaka chronically exposed to betamethasone at environmentally relevant levels.

The broad utilization of betamethasone in medical treatments may pose a significant ecotoxicological risk to aquatic organisms, yet its potential reproductive toxicity remains unclear. The present study examined the impacts of environmental exposure on male reproduction using Japanese medaka (Oryzias latipes). After 110 days of betamethasone exposure at environmentally relevant concentrations (0, 20 and 200 ng/L), LH/FSH synthesis and release in the pituitary was inhibited, and the production of sex hormones and their signaling pathways in the gonads of male medaka were greatly influenced. This synthetic glucocorticoid restrained testosterone (T) synthesis and gave rise to a significant increase in E2/T and E2/11-KT ratios. Furthermore, chronic betamethasone exposure (20 and 200 ng/L) led to the suppression of androgen receptor (AR) signaling and enhancement of estrogen receptors (ERs) signaling. An increase in hepatic vitellogenin contents was also detected, and testicular oocytes were observed in both 20 and 200 ng/L betamethasone-treated groups. It showed that 20 and 200 ng/L betamethasone could induce male feminization and even intersex, triggering abnormal spermatogenesis in medaka males. With its adverse effects on male fertility, betamethasone could potentially influence the fishery productivity and population dynamics in aquatic ecosystems.

Osmoregulatory strategies of estuarine fish Scatophagus argus in response to environmental salinity changes.

BACKGROUND: Scatophagus argus, an estuarine inhabitant, can rapidly adapt to different salinity environments. However, the knowledge of the molecular mechanisms underlying its strong salinity tolerance remains unclear. The gill, as the main osmoregulatory organ, plays a vital role in the salinity adaptation of the fish, and thus relative studies are constructive to reveal unique osmoregulatory mechanisms in S. argus. RESULTS: In the present study, iTRAQ coupled with nanoLC-MS/MS techniques were employed to explore branchial osmoregulatory mechanisms in S. argus acclimated to different salinities. Among 1,604 identified proteins, 796 differentially expressed proteins (DEPs) were detected. To further assess osmoregulatory strategies in the gills under different salinities, DEPs related to osmoregulatory (22), non-directional (18), hypo- (52), and hypersaline (40) stress responses were selected. Functional annotation analysis of these selected DEPs indicated that the cellular ion regulation (e.g. Na+-K+-ATPase [NKA] and Na+-K+-2Cl- cotransporter 1 [NKCC1]) and ATP synthesis were deeply involved in the osmoregulatory process. As an osmoregulatory protein, NKCC1 expression was inhibited under hyposaline stress but showed the opposite trend in hypersaline conditions. The expression levels of NKA α1 and ß1 were only increased under hypersaline challenge. However, hyposaline treatments could enhance branchial NKA activity, which was inhibited under hypersaline environments, and correspondingly, reduced ATP content was observed in gill tissues exposed to hyposaline conditions, while its contents were increased in hypersaline groups. In vitro experiments indicated that Na+, K+, and Cl- ions were pumped out of branchial cells under hypoosmotic stress, whereas they were absorbed into cells under hyperosmotic conditions. Based on our results, we speculated that NKCC1-mediated Na+ influx was inhibited, and proper Na+ efflux was maintained by improving NKA activity under hyposaline stress, promoting the rapid adaptation of branchial cells to the hyposaline condition. Meanwhile, branchial cells prevented excessive loss of ions by increasing NKA internalization and reducing ATP synthesis. In contrast, excess ions in cells exposed to the hyperosmotic medium were excreted with sufficient energy supply, and reduced NKA activity and enhanced NKCC1-mediated Na+ influx were considered a compensatory regulation. CONCLUSIONS: S. argus exhibited divergent osmoregulatory strategies in the gills when encountering hypoosmotic and hyperosmotic stresses, facilitating effective adaptabilities to a wide range of environmental salinity fluctuation.

Effects of environmental salinity on the immune response of the coastal fish Scatophagus argus during bacterial infection.

The coastal aquaculture is characterized with environmental salinity fluctuation, and the effects of salinity stress on the immunity of cultured fish are needed to be further explored. Scatophagus argus is an important species in the wild fisheries and aquaculture industry, it would be of great value to reveal the impact of salinity change on the immune response in this species. Understanding the effects of salinity stress on immune response can provide valuable insights into salinity management in the aquacultural process. The head kidney, which is an organ unique for teleost fish, functions not only as a central immune organ but also as a crucial role in the stress response during which the secretion of immunoregulatory molecules i.e. cytokines is facilitated. In the present study, Individuals of S. argus acclimated to 3 different salinities [0‰ (FW), 10‰ (BW), and 25‰ (SW)] were injected intraperitoneally with A. hydrophila, and then monitored throughout one week. The effects of environmental salinity on the immune response in S. argus stimulated by A. hydrophila infection were investigated. mRNA expression profiles of cytokine genes IL-1ß, IL-6, IL-10 and TNF-α in different salinity groups was quite different. mRNA expression of cytokine genes in BW group and SW group rose more quickly and significantly higher than FW group (p < 0.05) at early stages (6-24 hpi) after bacterial injection, and before 96 hpi, the highest value of cytokine expression at each time point was recorded in SW group. Immune parameters such as lysozyme level, complement C3 activity and IgM content in BW and FW groups were lower than SW group at each time point from 24 to 144 hpi after bacterial injection. In addition, leukocyte profiles in the head kidney and blood were also investigated. Although hypoosmotic acclimation could temporarily stimulate monocyte and neutrophil proliferation, it was observed that the number of monocytes, neutrophils and lymphocytes of the head kidney and blood in SW group increased more quickly than BW and FW groups after bacterial infection. Our results indicate that hypoosmotic stress due to the decrease of environmental salinity has suppressive immunoregulatory effects on the immune response of S. argus.

Inflammatory responses associated with hyposaline stress in gill epithelial cells of the spotted scat Scatophagus argus.

The molecular processes of immune responses in mucosal tissues such as fish gills under environmental stress are poorly understood. In the present study, pro-inflammatory response under hyposaline stress and its regulation by cortisol/corticosteroid receptors (CRs) in gill epithelial cells of the spotted scat Scatophagus argus were analyzed. The fish were transferred to freshwater for 6 days (144 h) of acclimation. Following freshwater exposure, the cortisol concentration increased transiently before returning to the control level over time. mRNA expression of pro-inflammatory cytokines (TNF-a, IL-1b and IL-6) was stimulated by cortisol through CR signals at early stages of acclimation, but hyposaline stress inhibited their levels by the end of the experimental period. The transcriptional profile of anti-inflammatory cytokine IL-10 was quite different from these pro-inflammatory cytokines, and its value fluctuated within a narrow range during the experimental period. Full-length cDNAs of mineralocorticoid receptor (MR) and glucocorticoid receptor 1 (GR1) (different kinds of CRs) were cloned from the gills. Our results showed that MR and GR displayed mutually antagonistic effects during hyposaline stress. MR responded quickly at early stages, and its expression decreased with the drop of cortisol concentration. By contrast, GR expression was maintained at high levels after the acclimation of freshwater exposure. The tight coordination of GR and MR helps to shape the effects of stress on the immune system, which in turn, regulates the stress response. Our results confirm the interaction between endocrine and cytokine messengers and a clear difference in the sensitivity of GR and MR during the hyposaline challenge in gill epithelial cells of the spotted scat Scatophagus argus.

Modulation of inflammatory response by cortisol in the kidney of spotted scat (Scatophagus argus) in vitro under different osmotic stresses.

Salinity changes on renal osmoregulation have often been investigated while the immune response of the kidney under osmotic stress is poorly understood in teleosts. Acute stress is generally associated with enhancement of circulating cortisol. The effects of osmotic stress on renal immune response and its regulation by cortisol deserve more attention. In the present study, the effects of exogenous cortisol treatment on the lipopolysaccharide (LPS)-induced immune response were analyzed in renal masses of Scatophagus argus under different osmotic stresses in vitro. mRNA expression of pro-inflammatory cytokines (TNF-α, IL1-ß and IL-6) and immune-regulatory related genes (GR and SOCS1) was measured over a short course (15 h). Comprehensive analysis reveals that transcript abundances of pro-inflammatory cytokine genes such as TNF-α, IL-1ß, and IL-6 induced by LPS, alone or in the combination of cortisol, are tightly associated with osmoregulation under acute osmotic stress. Our results showed that osmotic challenge could significantly enhance mRNA expression levels of pro-inflammatory cytokines in renal masses in vitro. Based on our analysis, it can be inferred that cortisol suppresses the magnitude of renal inflammatory response and attenuates LPS-induced immune response through GR signaling in the face of challenging environmental conditions.

Transcriptional analysis of renal dopamine-mediated Na+ homeostasis response to environmental salinity stress in Scatophagus argus.

BACKGROUND: To control the osmotic pressure in the body, physiological adjustments to salinity fluctuations require the fish to regulate body fluid homeostasis in relation to environmental change via osmoregulation. Previous studies related to osmoregulation were focused primarily on the gill; however, little is known about another organ involved in osmoregulation, the kidney. The salinity adaptation of marine fish involves complex physiological traits, metabolic pathways and molecular and gene networks in osmoregulatory organs. To further explore of the salinity adaptation of marine fish with regard to the role of the kidney, the euryhaline fish Scatophagus argus was employed in the present study. Renal expression profiles of S. argus at different salinity levels were characterized using RNA-sequencing, and an integrated approach of combining molecular tools with physiological and biochemical techniques was utilized to reveal renal osmoregulatory mechanisms in vivo and in vitro. RESULTS: S. argus renal transcriptomes from the hyposaline stress (0‰, freshwater [FW]), hypersaline stress (50‰, hypersaline water [HW]) and control groups (25‰) were compared to elucidate potential osmoregulatory mechanisms. In total, 19,012 and 36,253 differentially expressed genes (DEGs) were obtained from the FW and HW groups, respectively. Based on the functional classification of DEGs, the renal dopamine system-induced Na+ transport was demonstrated to play a fundamental role in osmoregulation. In addition, for the first time in fish, many candidate genes associated with the dopamine system were identified. Furthermore, changes in environmental salinity affected renal dopamine release/reuptake by regulating the expression of genes related to dopamine reuptake (dat and nkaα1), vesicular traffic-mediated dopamine release (pink1, lrrk2, ace and apn), DAT phosphorylation (CaMKIIα and pkcß) and internalization (akt1). The associated transcriptional regulation ensured appropriate extracellular dopamine abundance in the S. argus kidney, and fluctuations in extracellular dopamine produced a direct influence on Na+/K+-ATPase (NKA) expression and activity, which is associated with Na+ homeostasis. CONCLUSIONS: These transcriptomic data provided insight into the molecular basis of renal osmoregulation in S. argus. Significantly, the results of this study revealed the mechanism of renal dopamine system-induced Na+ transport is essential in fish osmoregulation.

Dopamine regulates renal osmoregulation during hyposaline stress via DRD1 in the spotted scat (Scatophagus argus).

Dopamine is an important regulator of renal natriuresis and is critical for the adaptation of many animals to changing environmental salinity. However, the molecular mechanisms through which dopamine promotes this adaptation remain poorly understood. We studied the effects of dopamine on renal hypo-osmoregulation in the euryhaline fish Scatophagus argus (S. argus) during abrupt transfer from seawater (SW) to freshwater (FW). Following the transfer, serum dopamine concentration was decreased, and dopamine activated expression of the dopamine receptor 1 (designated SaDRD1) in the kidney, triggering the osmoregulatory signaling cascade. SaDRD1 protein is expressed in the renal proximal tubule cells in vivo, and is localized to the cell membrane of renal primary cells in vitro. Knockdown of SaDRD1 mRNA by siRNA significantly increased Na+/K+-ATPase (NKA) activity in cultured renal primary cells in vitro, suggesting that expression of SaDRD1 may oppose the activity of NKA. We demonstrate that exogenous dopamine enhances the response of NKA to hyposaline stress after transferring primary renal cells from isosmotic medium to hypoosmotic medium. Our results indicate that dopamine regulation via SaDRD1 ignited the renal dopaminergic system to balance the osmotic pressure through inhibiting NKA activity, providing a new perspective on the hyposaline adaptation of fish.

Adaptive responses to osmotic stress in kidney-derived cell lines from Scatophagus argus, a euryhaline fish.

The euryhaline fish, the spotted scat (Scatophagus argus), is exceptional for its ability to tolerate rapid fluctuations in salinity. To better understand fish osmoregulation and enable more precise analyses of specific features of adaptive responses to the osmotic stress in fish, a S. argus kidney-derived cell line (SK) was developed and subcultured for more than 70 passages. The cells were mostly fibroblast-like, with a normal diploid karyotype (2n=48). A low-osmolarity-adapted SK cell line (SK-la) was induced by growth in a hypotonic solution (150 mOsm). Effects of different osmotic stresses (150, 300 and 450 mOsm) on cell growth, cell morphology, cell volume changes and cell damage in SK, SK-la and CIK (a kidney-derived cell line from freshwater grass carp) cells were studied. These were compared by use of microscopic observation, flow cytometry and a Na-K-ATPase (NKA) assay. SK cells became smaller and grew rapidly in response to hypotonic stress (150 mOsm), and exhibited no visible morphological changes in response to hypertonic stress (450 mOsm). SK-la grew well by moderate hypertonicity (300 mOsm) but depressed in severe hypertonicity (450 mOsm), the number of unhealthy SK-la cells rose as osmolarity increased. In contrast, CIK cells became unhealthy with anisotonic challenge. The NKA activities of SK and CIK cells were assayed after exposure to anisotonic conditions, and rapid decreases were detected immediately except SK cells which were not affected in hypotonicity. Unlike in SK and CIK, an increase following a down-regulation of NKA activity was observed in SK-la cells upon moderate hypertonic stress. These results suggested that SK and SK-la cells had stronger osmoregulatory capacity than CIK cells, and provided new insights on the osmosensing and osmotic adaption in euryhaline fish kidney.

Characterization and gonadal expression of FOXL2 relative to Cyp19a genes in spotted scat Scatophagus argus.

In the present study, we cloned the full-length cDNAs of FOXL2, Cyp19a1a and Cyp19a1b and analyzed their expression patterns during gonadal development in spotted scat, Scatophagus argus. All three genes were expressed in ovaries and testes but showed sexual dimorphism. At early stages of gonadal development, the expression of FOXL2 in ovaries was higher than testes. FOXL2 expression deceased gradually as gonadal development continued, and reached the lowest level at the mature stage. Cyp19a1a and Cyp19a1b were expressed coordinately with FOXL2, except at the early vitellogenic stage in the ovary. The expression of FOXL2, Cyp19a1a and Cyp19a1b was mainly localized in granulosa cells of ovaries. In S. argus testes, strong expression of FOXL2 gene was observed in the interstitial cells including tubules and Leydig cells, while Cyp19a1a and Cyp19a1b were mainly expressed in Sertoli cells throughout gametogenesis. These results show that FOXL2 plays an essential role in sexual development, and imply that it may regulate Cyp19a1a and Cyp19a1b expression in S. argus.

Comparative renal gene expression in response to abrupt hypoosmotic shock in spotted scat (Scatophagus argus).

Scatophagus argus, a euryhaline fish, is notable for its ability to tolerate a wide range of environmental salinities and especially for its tolerance to a rapid, marked reduction in salinity. Therefore, S. argus is a good model for studying the molecular mechanisms mediating abrupt hyperosmoregulation. The serum osmotic pressure decreased steeply within one hour after transferring S. argus from seawater (SW) to freshwater (FW) and remained at new balance throughout the duration of one week. To explain this phenomenon and understand the molecular responses to an abrupt hypoosmotic shock, hypoosmotic stress responsive genes were identified by constructing two suppression subtractive hybridization (SSH) cDNA libraries from the kidneys of S. argus that had been transferred from SW to FW. After trimming and blasting, 52 ESTs were picked out from the subtractive library. Among them, 11 genes were significantly up-regulated (p < 0.05). The kinetics studies of gene expression levels were conducted for 1 week after the transfer using quantitative real-time PCR. A significant variation in the expression of these genes occurred within 12h after the hypoosmotic shock, except for growth hormone (GH) and polyadenylate binding protein 1 (PBP1), which were significantly up-regulated 2 days post-transfer. Our results suggest different functional roles for these genes in response to hypoosmotic stress during the stress response phase (1 hpt-12 hpt) and stable phase (12 hpt-7 dpt). Furthermore, the plasma growth hormone level was detected to be significantly elevated at 1 hpt and 24 hpt following abrupt hypoosmotic shock. Meanwhile, several hematological parameters, hemoglobin (HGB), red blood cell (RBC) and mean cellular hemoglobin concentration (MCHC), were observed to be significantly increased at 12 hpt and 2 dpt compared with that of control group. Our results provide a solid basis from which to conduct future studies on the osmoregulatory mechanisms in the euryhaline fish.