An Integrated Bioinformatics Approach to Identify Network-Derived Hub Genes in Starving Zebrafish.

The present study was aimed at identifying causative hub genes within modules formed by co-expression and protein-protein interaction (PPI) networks, followed by Bayesian network (BN) construction in the liver transcriptome of starved zebrafish. To this end, the GSE11107 and GSE112272 datasets from the GEO databases were downloaded and meta-analyzed using the MetaDE package, an add-on R package. Differentially expressed genes (DEGs) were identified based upon expression intensity N(µ = 0.2, σ2 = 0.4). Reconstruction of BNs was performed by the bnlearn R package on genes within modules using STRINGdb and CEMiTool. ndufs5 (shared among PPI, BN and COEX), rps26, rpl10, sdhc (shared between PPI and BN), ndufa6, ndufa10, ndufb8 (shared between PPI and COEX), skp1, atp5h, ndufb10, rpl5b, zgc:193613, zgc:123327, zgc:123178, wu:fc58f10, zgc:111986, wu:fc37b12, taldo1, wu:fb62f08, zgc:64133 and acp5a (shared between COEX and BN) were identified as causative hub genes affecting gene expression in the liver of starving zebrafish. Future work will shed light on using integrative analyses of miRNA and DNA microarrays simultaneously, and performing in silico and experimental validation of these hub-causative (CST) genes affecting starvation in zebrafish.

A chronic exposure to bisphenol A reduces sperm quality in goldfish associated with increases in kiss2, gpr54, and gnrh3 mRNA and circulatory LH levels at environmentally relevant concentrations.

The bisphenol A (BPA)-disrupted reproductive functions have been demonstrated in male animals. In fish, it has been shown that environmentally relevant concentrations of BPA decrease sperm quality associated with inhibition of androgen biosynthesis. However, BPA effects on neuroendocrine regulation of reproduction to affect testicular functions are largely unknown. In the present study, reproductive functions of hypothalamus and pituitary were studied in mature male goldfish exposed to nominal 0.2, 2.0 and 20.0 µg/L BPA. At 90 d of exposure, sperm volume, velocity, and density and motility were decreased in goldfish exposed to 0.2, 2.0, and 20.0 µg/L BPA, respectively (p < 0.05). At 30 d of exposure, there were no significant changes in circulatory LH levels and mRNA transcripts of kiss1, Kiss2, gpr54, and gnrh3. At 90 d of exposure, circulatory LH levels showed trends toward increases in BPA exposed goldfish, which was significant in those exposed to 2.0 µg/L (P < 0.05). At this time, Kiss2, gpr54, and gnrh3 mRNA levels were increased in goldfish exposed to any concentrations of BPA (p < 0.05). This study shows that BPA-diminished sperm quality was accompanied by an increase in circulatory LH levels associated with increases in mRNA transcripts of upstream neuroendocrine regulators of reproduction in goldfish. Further, this is the first study to report circulatory levels of LH in fish exposed to BPA.

A Review on Environmental Contaminants-Related Fertility Threat in Male Fishes: Effects and Possible Mechanisms of Action Learned from Wildlife and Laboratory Studies.

Increasing global rates of diminished fertility in males has been suggested to be associated with exposure to environmental contaminants (ECs). The aquatic environments are the final repository of ECs. As the reproductive system is conserved in vertebrates, studies on the effects of ECs on fertility endpoints in fishes provide us with valuable information to establish biomarkers in risk assessment of ECs, and to understand the ECs-related fertility threat. The aim of the present review was to evaluate associations between ECs and fertility determinants to better understand ECs-related male fertility threat in male fishes. Wildlife studies show that the reproductive system has been affected in fishes sampled from the polluted aquatic environment. The laboratory studies show the potency of ECs including natural and synthetic hormones, alkylphenols, bisphenols, plasticizers, pesticides, pharmaceutical, alkylating, and organotin agents to affect fertility determinants, resulting in diminished fertility at environmentally relevant concentrations. Both wildlife and laboratory studies reveal that ECs adverse effects on male fertility are associated with a decrease in sperm production, damage to sperm morphology, alternations in sperm genome, and decrease in sperm motility kinetics. The efficiency of ECs to affect sperm quality and male fertility highly depends on the concentration of the contaminants and the duration of exposure. Our review highlights that the number of contaminants examined over fertility tests are much lower than the number of contaminants detected in our environment. The ECs effects on fertility are largely unknown when fishes are exposed to the contaminants at early developmental stages. The review suggests the urgent need to examine ECs effects on male fertility when a fish is exposed at different developmental stages in a single or combination protocol. The ECs effects on the sperm genome are largely unknown to understand ECs-related inheritance of reproductive disorders transmitted to the progeny. To elucidate modes of action of ECs on sperm motility, it is needed to study functional morphology of the motility apparatus and to investigate ECs-disrupted motility signaling.

Cytotoxicity Activity and Druggability Studies of Sigmasterol Isolated from Marine Sponge Dysidea avara Against Oral Epithelial Cancer Cell (KB/C152) and T-Lymphocytic Leukemia Cell Line (Jurkat/ E6-1).

BACKGROUND: Marine sponge is a rich natural resource of many pharmacological compounds and various bioactive anticancer agents are derived from marine organisms like sponges. METHODS: studying the anticancer activity and Drug ability of marine sponge Dysidea avara using Cell lines oral epithelial cancer cell (KB/C152) and T-lymphocytic leukemia cell line (Jurkat/ E6-1). Marine sponge was collected from Persian Gulf. Several analytical techniques have been used to obtain and recognize stigmasterol, including column chromatography, thin layer chromatography, and gas chromatography-mass spectrometry. The PASS Prediction Activity was used to investigate the apoptosis-inducing effect of stigmasterol. The cytotoxic activity of stigmasterol was examined using yellow tetrazolium salt XTT (sodium 2, 3,-bis (2methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium) assay. The stigmasterol were docked within the protein tyrosine kinase (PTKs) (PDB code: 1t46) and epidermal growth factor receptor (EGFRK) (PDB code: 1M17). Also, the pharmacological characteristics of stigmasterol were predicted using PerADME, SwissADME, and Molinspi ration tools. Apoptosis-inducing effect of stigmasterol indicate the stigmasterol in terms of the possibility of apoptosis in cells. RESULTS: The apoptosis inducement results of known stigmasterol were determined by PASS on-line prediction. The compound exhibit potent cytotoxic properties against KB/C152 cell compared to Jurkat/ E6-1 cell. The stigmasterol showed the cytotoxicity effects on KB/C152 and HUT78 with IC50 ranges of 81.18 and 103.03 µg/ml, respectively. Molecular docking showed that, stigmasterol bound stably to the active sites of the protein tyrosine kinase (PTKs) (PDB code: 1t46) and epidermal growth factor receptor (EGFRK) (PDB code: 1M17). CONCLUSION: The compound showed desirable pharmacokinetic properties (ADME). This provided direct evidence of how a prospective anti-cancer agent can be stigmasterol. The preclinical studies paved the way for a potential new compound of anti-cancer.

Androgen signaling in male fishes: Examples of anti-androgenic chemicals that cause reproductive disorders.

Similar to other vertebrates, androgens regulate spermatogenesis in fishes. In teleosts, the main androgen is 11-Ketotestosterone (11-KT), which is oxidized testosterone (T) at the C11 position. Compared to T, 11-KT is a nonaromatizable steroid, and does not convert to 17ß-estradiol. However, circulatory levels of both T and 11-KT undergo seasonal variations along with testicular development. Physiological functions of androgens are mediated via androgen receptor (Ar). So far, nuclear Ar and membrane Ar have been identified in fishes. In the present study, we reviewed androgen biosynthesis in fishes, compared molecular structure of nuclear Ar in models of mammals and fishes, and investigated the mechanisms of action of environmental contaminants that differentially disrupt androgen signaling in fish reproduction. In the latter case, the adverse effects of vinclozolin (VZ) and bis(2-ethylhexyl) phthalate (DEHP) are compared. Both VZ and DEHP are capable of decreasing sperm quality in males. Vinclozolin causes an increase in 11-KT production associated with increases in kisspeptin (kiss-1) and salmon gonadotropin-releasing hormone (gnrh3) mRNA levels as well as circulatory levels of luteinizing hormone (Lh). In contrast, DEHP inhibits 11-KT production associated with a decrease in circulatory Lh levels. However, DEHP-inhibited 11-KT production is not associated with changes in kiss-1 and gnrh3 mRNA levels. Studies also show that VZ alters ar mRNA levels, while DEHP is without effect. These suggest that VZ and DEHP act differentially to cause androgen-dependent reproductive disorder in male fishes. Molecular analyses of the nuclear AR show that both DNA and ligand binding domains (DBD and LBD, respectively) are highly conserved within models of mammals and fishes. A phylogeny tree of the AR shows distinct clusters between mammals and fishes. In fishes, subtypes of Arα and Arß are also separated in distinct clusters. Thus, further studies need to generate ar knockout fish model to better elucidate androgen regulation of reproduction in fishes via Ar.

Metabolic stress leads to divergent changes in the ghrelinergic system in goldfish (Carassius auratus) gonads.

Various endocrine factors that regulate energy homeostasis are also implicated in the reproductive physiology of mammals. However, the hormonal link between metabolism and reproduction in fish is poorly understood. Ghrelin is a multifunctional hormone with both metabolic and reproductive roles in vertebrates. Post-translational acylation by ghrelin-O-acyltransferase (GOAT) is critical for its biological actions. The expression of ghrelin, ghrelin or growth hormone secretagogue receptor (GHSR), and GOAT (which forms the ghrelinergic system) in fish under metabolic stress remains unclear. In this research, we used RT-qPCR and Western blot analysis to determine the expression of the ghrelinergic system in goldfish (during the reproductively active phase) hypothalamus and gonads under 7 and 28 days of fasting. We found a significant increase in preproghrelin mRNA expresson in the ovary, and GOAT mRNA expression in the testis of goldfish deprived of food for 7 days. In fish deprived of food for 28 days, preproghrelin, GHSR and GOAT mRNA expression was significantly increased in the hypothalamus of male goldfish. Such differences were not observed in the hypothalamus of female fish, and in the testis of 28 days fasted fish. Meanwhile, preproghrelin, GHSR, and GOAT expression (both mRNA and protein) was significantly increased in the ovary of female fish fasted for 28 days. Ghrelin has been shown to suppress oocyte maturation in fish. The upregulation of a system that has ovarian inbititory roles suggests a role for ghrelin in maintaining reduced reproductive capability during metabolically challenging periods.

Transcripts of genes encoding reproductive neuroendocrine hormones and androgen receptor in the brain and testis of goldfish exposed to vinclozolin, flutamide, testosterone, and their combinations.

Vinclozolin (VZ) is a pesticide that acts as an anti-androgen to impair reproduction in mammals. However, VZ-induced disruption of reproduction is largely unknown in fish. In the present study, we have established a combination exposure in which adult goldfish were exposed to VZ (30 and 100 µg/L), anti-androgen flutamide (Flu, 300 µg/L), and androgen testosterone (T, 1 µg/L) to better understand effects of VZ on reproductive endocrine system. mRNA levels of kisspeptin (kiss-1 and kiss-2) and its receptor (gpr54), salmon gonadotropin-releasing hormone (gnrh3) and androgen receptor (ar) in the mid-brain, and luteinizing hormone receptor (lhr) in the testis were analyzed and compared with those of control following 10 days of exposure. kiss-1 mRNA level was increased in goldfish exposed to 100 µg/L VZ and to Flu, while kiss-2 mRNA level was increased following exposure to Flu and to combinations of 30 µg/L VZ with Flu, 100 µg/L VZ with T, and Flu with T. gpr54 mRNA level was increased in goldfish exposed to Flu and to combination of 30 µg/L VZ with Flu and 100 µg/L VZ with T. gnrh3 mRNA level was increased in goldfish exposed to 100 µg/L VZ, to Flu, and to combinations of 30 µg/L VZ with Flu, 100 µg/L VZ with T, and Flu with T. The mid-brain ar mRNA level was increased in goldfish exposed to Flu and to combinations of 30 µg/L VZ with Flu, 100 µg/L VZ with T, and Flu with T. Testicular lhr mRNA level was increased in goldfish exposed to Flu and to combination of 30 µg/L VZ with Flu. These results suggest that VZ and Flu are capable of interfering with kisspeptin and GnRH systems to alter pituitary and testicular horonal functions in adult goldfish and the brain ar mediates VZ-induced disruption of androgen production.

Di-(2-ethylhexyl)-phthalate disrupts pituitary and testicular hormonal functions to reduce sperm quality in mature goldfish.

Di-(2-ethylhexyl) phthalate (DEHP) interferes with male reproductive endocrine system in mammals, however its effects on fish reproduction are largely unknown. We evaluated sperm quality and investigated reproductive endocrine system in mature goldfish (Carassius auratus) exposed to nominal 1, 10, and 100µg/L DEHP. To examine DEHP estrogenic activity, one group of goldfish was exposed to 17ß-estradiol (5µg/L E2) for comparison. Following 30d of exposure, sperm production was decreased and suppressed in DEHP and E2 treated goldfish, respectively. Sperm motility and velocity were decreased in goldfish exposed to 100 and 10µg/L DEHP at 15s post-sperm activation, respectively. Compared to control, 11-ketotestosterone (11-KT) levels were decreased at 10 and 1µg/L DEHP at day 15 and 30, respectively. In E2 treated goldfish, 11-KT levels were decreased compared to control during the period of exposure. E2 levels were increased in goldfish exposed to E2, but remained unchanged in DEHP treated goldfish during the period of exposure. StAR mRNA levels encoding regulator of cholesterol transfer to steroidogenesis were decreased in DEHP and E2 treated goldfish following 15 and 30d of exposure, respectively. Luteinizing hormone (LH) levels were decreased in DEHP and E2 treated goldfish following 15 and 30d of exposure, respectively. In DEHP treated goldfish, gnrh3, kiss1 and its receptor (gpr54) mRNA levels did not change during the experimental period. In E2 treated goldfish, gnrh3 mRNA levels were decreased at day 7, but kiss1 and gpr54 mRNA levels were increased at day 30 of exposure. The mRNA levels of genes encoding testicular LH and androgen receptors remained unchanged in DEHP and E2 treated goldfish. In contrast to E2 treated goldfish, vitellogenin production was not induced in DEHP treated goldfish and mRNA levels of genes with products mediating estrogenic effects remained unchanged or decreased. In conclusion, DEHP interferes with testis and pituitary hormonal functions to reduce sperm quality in goldfish and does not exhibit estrogenic activity.

Alternations in neuroendocrine and endocrine regulation of reproduction in male goldfish (Carassius auratus) following an acute and chronic exposure to vinclozolin, in vivo.

The fungicide vinclozolin (VZ) is in use globally and known to disrupt reproductive function in male. The present study tested the hypothesis that VZ disrupts testicular function in goldfish (Carassius auratus) by affecting brain-pituitary-testis axis. Goldfish were exposed to 100, 400 and 800 µg/L VZ and 5 µg/L 17ß-estradiol (E2) for comparison. In VZ treated goldfish, 11-ketotesteosterone (11-KT) secretion was changed depending on dose and duration period of treatment. Following 7 days of exposure, 11-KT was decreased in goldfish exposed to 800 µg/L VZ, while it was increased in goldfish exposed to 100 µg/L VZ after 30 days of exposure. Circulating E2 level was unchanged in VZ treated goldfish, however the E2/11-KT ratio was increased in a concentration-related manner. In E2 treated goldfish, circulatory 11-KT and E2 levels were decreased and increased, respectively, which resulted in an increase in the E2/11-KT ratio. Exposure to VZ at 100 µg/L caused a significant increase in the circulatory luteinizing hormone (LH) after 30 days. In E2 treated fish circulatory LH was decreased, significantly. Transcripts of genes encoding gonadotropin-releasing hormone and androgen receptor in the brain, and those of genes encoding LH and follicle-stimulating hormone receptors, StAR, CYP17, and 3ß-HSD in the testis changed in VZ-treated goldfish depending on concentration and period of treatment. mRNA of genes encoding vitellogenin and estrogen receptor in the liver and cytochrome P450 aromatase in the brain were increased in E2-treated goldfish. The results suggest that VZ-induced changes in 11-KT were due to disruption in brain-pituitary-testis axis and provide integrated characterization of VZ-related reproductive disorders in male fish.

Toxicity of environmental contaminants to fish spermatozoa function in vitro--a review.

In vitro techniques for investigating the toxic effects of environmental contaminants (EC) on fish spermatozoa motility kinetics and fertilizing ability are valuable tools to understand toxicity mechanisms and sites of action. In vitro techniques may also be well-suited to studies of endocrine disruption in male fertility in vivo. This review shows ECs to decrease or suppress spermatozoa motility kinetics and fertilizing ability in a dose-dependent manner, with toxic concentrations being much higher than those reported in the aquatic environment. Sites of action depend on EC concentration and duration of exposure. Both instant (immediate) and incubated exposure of spermatozoa to ECs results in damage to the plasma membrane and the axoneme, while disruption of energy metabolism appears only during incubated exposure. Spermatozoa lose fertilizing ability following exposure to ECs in vitro, not only due to inhibition or suppression of the initiation of motility, but also through damage to DNA. This review highlights the significant lack of information about disruption of spermatozoa function associated with exposure to water from polluted areas as well as combined effects of ECs. Specifics of alterations in intracellular signaling cascades involved in the initiation of spermatozoa motility following exposure to sublethal concentrations of ECs remain unknown. Further studies are also needed to elucidate in vitro EC effects during spermatozoa maturation, when spermatozoa acquire the potential for motility.

Anti-androgen vinclozolin impairs sperm quality and steroidogenesis in goldfish.

In mammals, vinclozolin (VZ) is known as anti-androgen, which causes male infertility via androgen receptor (AR) antagonism. In aquatic animals, the VZ effects on reproductive functions are largely unknown and results are somewhat contradictory. To understand VZ adverse effects on male reproduction, mature goldfish (Carassius auratus) were exposed to three nominal VZ concentrations (100, 400, and 800 µg/L) and alternations in gonadosomatic (GSI) and hepatosomatic indices (HSI), 17ß-estradiol (E(2)), 11-ketotestosterone (11-KT) and sperm quality were investigated compared to the solvent control. One group was exposed to E(2) (nominal concentration of 5 µg/L), an estrogenic compound, as a negative control. Following one month exposure, GSI and HSI were unchanged in all VZ treated groups compared to solvent control. Sperm volume, motility and velocity were reduced in fish exposed to 800 µg/L VZ. This was associated with the decrease in 11-KT level, suggesting direct VZ effects on testicular androgenesis and sperm functions. In goldfish exposed to 100 µg/L VZ, 11-KT was increased but E(2) remained unchanged. This is, probably, the main reason for unchanged sperm quality at 100 µg/L VZ. In goldfish exposed to E(2), GSI and 11-KT were decreased, E(2) was increased and no sperm was produced. The present study shows different dose-dependent VZ effects, which lead to impairment in sperm quality via disruption in steroidogenesis. In addition to VZ effects through competitive binding to AR, our data suggests potential effects of VZ by direct inhibition of 11-KT biosynthesis in fish as well as abnormalities in sperm morphology.