Zebrafish Gonads Research Articles

Kisspeptin stimulates oocyte maturation, and food deprivation modulates the abundance of kisspeptin system in zebrafish gonads

Kisspeptin is a hormone involved in the neuroendocrine control of reproduction in fish. We hypothesized that kisspeptin stimulates oocyte maturation and modulates other reproductive hormones in zebrafish; and the gonadal kisspeptin system in zebrafish is affected during energy unavailability. The main goals of this research were to test in vitro effects of kisspeptin on oocyte maturation and mRNA abundance in zebrafish ovarian follicles and determine how short-term feed restriction affects kisspeptin and its receptors in zebrafish testis and ovary. This study demonstrates the presence of kisspeptin and its receptors in zebrafish (Danio rerio) gonads and its direct action on ovarian follicles in vitro. Kisspeptin (10 ng/mL) induced oocyte maturation, as indicated by germinal vesicle break down at 18- and 24- hours post-treatment. Kisspeptin significantly increased the abundance of mRNAs encoding reproductive hormones and its receptors in zebrafish oocytes. This suggests that kisspeptin-10 affects ovarian functions by modulating other hormones. Reproduction is a process that requires energy. Therefore, whether energy availability affects the kisspeptin system in zebrafish gonads was determined. Food deprivation modulated kisspeptin expression differently in zebrafish testis and ovary. Kiss2 and kiss1ra were upregulated while kiss1rb was downregulated in the testis post-food deprivation. Meanwhile, no changes in kiss in the ovary were found after food deprivation. However, kiss1rb was downregulated in unfed fish at 3- and 7-days post-food deprivation. Overall, our results suggest sex- and tissue-specific changes in the gonadal abundance of the kisspeptin system in zebrafish. The fine tuning of reproduction during energy fluctuations in fish is likely mediated via changes in hormones, including kisspeptin as shown in this research.

Loss of Nobox prevents ovarian differentiation from juvenile ovaries in zebrafish.

As a species without master sex-determining genes, zebrafish displays high plasticity in sex differentiation, making it an excellent model for studying the regulatory mechanisms underlying gonadal differentiation and gametogenesis. Despite being a gonochorist, zebrafish is a juvenile hermaphrodite that undergoes a special phase of juvenile ovary before further differentiation into functional testis and ovary. The mechanisms underlying juvenile ovary formation and subsequent gonadal differentiation remain largely unknown. In this study, we explored the role of Nobox/nobox (new born ovary homeobox protein), another oocyte-specific transcription factor in females, in early zebrafish gonadogenesis using CRISPR/Cas9 technology. As in mammals, nobox is specifically expressed in zebrafish gonads with a dimorphic pattern at juvenile stage. In contrast to the mutant of figla (factor in the germline alpha, another oocyte-specific transcription factor), the nobox mutants showed formation of typical perinucleolar (PN) follicles at primary growth (PG) stage in juvenile gonads, suggesting occurrence of follicle assembly from cystic oocytes (chromatin nucleolar stage, CN). These follicles, however, failed to develop further to form functional ovaries, resulting in all-male phenotype. Despite its expression in adult testis, the loss of nobox did not seem to affect testis development, spermatogenesis and male spawning. In summary, our results indicate an important role for Nobox in zebrafish ovarian differentiation and early folliculogenesis.

Effects of Diethylstilbestrol on Zebrafish Gonad Development and Endocrine Disruption Mechanism.

Environmental estrogen is a substance that functions as an endocrine hormone in organisms and can cause endocrine system disruption. A typical environmental estrogen, diethylstilbestrol (DES), can affect normal sexual function and organism development. However, even though the effects of different exposure stages of DES on the endocrine system and gonadal development of zebrafish juveniles are unknown, sex determination is strongly influenced by endocrine-disrupting chemicals (EDCs). From 10–90 days post fertilization (dpf), juvenile zebrafish were exposed to DES (100 and 1000 ng/L) in three different stages (initial development stage (IDS), 10–25 dpf; gonadal differentiation stage (GDS), 25–45 dpf and gonadal maturity stage (GMS), 45–60 dpf). Compared with that of IDS and GMS, the growth indicators (body length, body weight, and others) decreased significantly at GDS, and the proportion of zebrafish females exposed to 100 ng/L DES was significantly higher (by 59.65%) than that of the control; in addition, the zebrafish were biased towards female differentiation. The GDS is a critical period for sex differentiation. Our results show that exposure to environmental estrogen during the critical gonadal differentiation period not only affects the development of zebrafish, but also affects the population development.

Zebrafish male differentiation: Do all testes go through a “juvenile ovary” stage?

Zebrafish (Danio rerio) studies describe before the onset of mature gonads differentiation all individuals go through a “juvenile ovary” stage. However, the sequential events of the early zebrafish gonad differentiation are still not described in full detail and recent works indicate that some individuals never form a “juvenile ovary” structure. Therefore, the present study aimed to confirm the existence of two processes of zebrafish male differentiation. For this purpose, every two days between 20 and 30 days post-fertilization (dpf) zebrafish were collected for a stereological analysis of the differentiating gonads.The histological evaluation showed that prior to 22 dpf, zebrafish gonads were still undifferentiated. At 24 dpf, some individuals started to present a “juvenile ovary” and from 26 to 30 dpf, it was possible to discern two processes of gonad development. The majority of the individuals (80 %) developed a “juvenile ovary”, while in the remaining (20 %) it was not possible to detect this structure.The results of the present study show the existence of two distinct processes of zebrafish male gonad development, indicating that not all individuals go through the “juvenile ovary” stage.

Histopathological and apoptotic examination of zebrafish (Danio rerio) gonads exposed to triclosan

Triclosan, produced as a broad-spectrum antibiotic in the early 1960s, is generally used as a preservative in personal care products, fabrics, plastic products such as kitchenware and toys. As a result of the high demand for triclosan, this chemical threatens the aquatic ecosystem by contaminating wastewater sources. Environmental pollutants affect the reproductive potential of fish, one of the most critical aquatic organisms. This study aimed to investigate the histopathological and apoptotic effects of triclosan in zebrafish gonads. Fish were exposed to sublethal concentrations of triclosan for 5 days, and general histological methods were applied. Histological sections were examined under a light microscope after staining with hematoxylin and eosin and toluidine blue. Triclosan exposure caused deterioration in ovarian tissue, such as shrinkage in the ooplasm, accumulation of proteinaceous fluid in the interstitial tissue, morphological changes of oocyte and the zona radiata. In testicular tissue, triclosan exposure caused fusion in seminiferous tubules, hypertrophy in spermatogenic and Leydig cells, edema in seminiferous tubules, and karyorrhexis in spermatogenic cells. The TUNEL assay was used for the determination of apoptotic cells. Brown-colored apoptotic cells were visualized under the light microscope. TUNEL positive cells were observed in all exposure groups. Triclosan administration was found to cause apoptosis in zebrafish gonads. These findings indicate that triclosan potentially affects fish reproduction, and that its judicious disposal is essential for protecting the environment and maintaining the reproductive potential of fish.

Histological Analysis of Gonads in Zebrafish.

Zebrafish is an excellent system for the study of gonad development due to available genetic tools and its utilization as a human disease model. The zebrafish serves as an experimental system to model human disorders affecting the reproductive system, toxicological effects on fertility and sexual development, and hormonal regulation of fertility. Forward genetic screens have been used to uncover genetic causes of infertility and reverse genetic approaches have demonstrated that genes involved in germ cell development have similar functions in zebrafish and mammals. The most comprehensive picture of the gonad can be visualized by histology. There are a variety of methods that give excellent histology of zebrafish gonads. Below are methods for two staining approaches for the histology of paraffin-embedded zebrafish gonads.

Acute Exposure to the Neonicotinoid Insecticide Imidacloprid of Zebrafish (Danio rerio) Gonads: A Histopathological Approach

Neonicotinoids are the new class of insecticides that are high target specificity to insects. Imidacloprid is a neonicotinoid that is the most widely used insecticide in the world. As a result of its widespread use in agriculture, imidacloprid interferes with the aquatic system and threatens the aquatic environment. In this study, an investigation of the histopathological effects of imidacloprid on zebrafish gonads was aimed. Zebrafish were exposed to 9.5 mg/L, 19 mg/L, and 38 mg/L of imidacloprid for 5 days, considering the 96 h LC50 value. After dissecting the gonadal tissues, routine histological techniques were applied, and the tissues were stained with Periodic Acid-Schiff (PAS), Toluidine Blue (TB), and Haematoxylin and Eosin (H&E). Sections were examined under a light microscope. While normal gonad histology was observed in the control group, histopathological alternations such as degeneration and union in the seminiferous tubules, hypertrophy in spermatogenic and Leydig cells, and interstitial fibrosis were detected in testicular tissue of the experimental groups. In the ovarian tissues of the experimental groups, structural deterioration in oocytes, autolysis, increase in the number of atretic oocytes, vacuolization in cortical alveoli, thickening and curling in the zona radiata, and opening in the perifollicular layers were detected.

In Vivo and Ex Vivo CT Imaging of Zebrafish Gonads.

3D imaging of the gonads in adult zebrafish in vivo is of great interest, as it allows to follow up on their development and/or the egg development in the same individual over time. Optical-based imaging methods can hardly be applied on the adult zebrafish, due to their limited transparency. In this chapter, we will demonstrate the application of microcomputer tomography (CT) imaging for in vivo 3D imaging of the gonads in adult zebrafish. We explain how the limited soft-tissue contrast in CT can be overcome and which X-ray dose levels can be expected using this technique. Moreover, we will use high-resolution microCT to perform ex vivo 3D virtual histology of the adult zebrafish, which allows a simple quantitative analysis of the gonad regions, malformation or alterations in the development of the follicles.

Parental exposure to polystyrene microplastics at environmentally relevant concentrations has negligible transgenerational effects on zebrafish (Danio rerio)

Waterborne exposure to highly persistent microplastic pollutants is a major concern for aquatic species worldwide. There are still gaps in knowledge on microplastics’ potential transgenerational effects on offspring generation. Using zebrafish, this study investigated the survival and early development of offspring through a 21-day continuous parental treatment with polystyrene microplastics. The results showed that continuous waterborne exposure to high concentrations (>100 μg/L) of polystyrene microplastics (1 μm) for 21 days resulted in notable microplastic accumulation in adult fish intestines. Exposure at microplastic concentrations greater than 100 μg/L also induced significant changes in steroidogenic mRNA expression in zebrafish gonads. However, no significant changes in the cumulative number of eggs spawned and fertilization rate were observed at any parental exposure concentrations when compared to the control. Early development of derived offspring, in terms of hatching rate, body length, malformation rate and mortality rate, did not significantly differ from that of the control. This study showed that transgenerational effects of parental exposure to polystyrene microplastics in zebrafish might be negligible or recoverable. This study provided new results and insights on the transgenerational effects of microplastics on a freshwater fish species and can help to understand impacts of microplastics on freshwater ecosystems.

Effects of BPA on zebrafish gonads: Focus on the endocannabinoid system

Bisphenol A (BPA), a monomer used for polycarbonate manufacture, has been widely reported as an endocrine-disrupting chemical (EDC). Among other alterations, BPA induces reproductive dysfunctionalities. Changes in the endocannabinoid system (ECS) have been recently shown to be associated with reproductive disorders. The ECS is a lipid-based signaling system (cannabinoid receptors, endocannabinoids and enzymatic machinery) involved in several physiological functions. The main goal of the present study was to assess the effects of two environmental concentrations of BPA (10 and 20μg/L) on the ECS in 1-year old zebrafish gonads. In males, BPA increased the gonadosomatic index (GSI) and altered testicular levels of endocannabinoids as well as reduced the testicular area occupied by spermatogonia. In male liver, exposure to 20μg/L BPA significantly increased vitellogenin (vtg) transcript levels. In female zebrafish, BPA altered ovarian endocannabinoid levels, elevated hepatic vtg mRNA levels as well as increased the percentage of vitellogenic oocytes in the ovaries. In conclusion, exposure to two environmentally relevant concentrations of BPA altered the ECS and consequently, gonadal function in both male and female zebrafish.

Phoenixin-20 Stimulates mRNAs Encoding Hypothalamo-Pituitary-Gonadal Hormones, is Pro-Vitellogenic, and Promotes Oocyte Maturation in Zebrafish

Phoenixin-20 (PNX-20) is a bioactive peptide with hormone-like actions in vertebrates. In mammals, PNX stimulates hypothalamo-pituitary-gonadal hormones and regulate reproductive processes. Our immunohisto/cytochemical studies show PNX-like and the putative PNX receptor, SREB3-like immunoreactivity in the gonads of zebrafish, and in zebrafish liver (ZFL) cells. Intraperitoneal injection of zebrafish PNX-20 upregulates mRNAs encoding both salmon gonadotropin-releasing hormone (GnRH), and chicken GnRH-II and kisspeptin and its receptor in zebrafish hypothalamus. Similarly, luteinizing hormone receptor mRNA expression in the testis, follicle-stimulating hormone receptor in the ovary, and the kisspeptin system were upregulated in the gonads of PNX-20 injected fish. We also observed the upregulation of genes involved in the sex steroidogenic pathway (cyp11a1, cyp17a1, 17βhsd, cyp19a1a) in the gonads of PNX-20 administered fish. PNX-20 upregulates the expression of vitellogenin isoforms and estrogen receptor (esr2a and 2b) mRNAs in ZFL cells in vitro. Meanwhile, siRNA-mediated knockdown of PNX-20 resulted in the downregulation of all vitellogenin transcripts, further suggesting its possible role in vitellogenesis. PNX-20 treatment resulted in a significant increase in germinal vesicle breakdown in zebrafish follicles in vitro. Collectively, these results provide strong evidence for PNX-20 effects on the HPG axis and liver to promote reproduction in zebrafish.

The Dietary Effect of Vitex agnus-castus Hydroalcoholic Extract on Growth Performance, Blood Biochemical Parameters, Carcass Quality, Sex Ratio and Gonad Histology in Zebrafish (Danio rerio)

The main purpose of this study was to consider the impact of Vitex agnus-castus hydroalcoholic extract, containing phytoestrogenic compounds on growth indices, sex ratio and histology of gonads of female Zebrafish. Fish larvae (4-day-old after hatching) were nourished with investigational diets (0 (control group T0), 5 g (T1), 10 g (T2) and 15 g (T3) kg−1 food) from first active feeding for 90 days. The results showed that Condition Factor (CF), Food Conversion Rate (FCR), Body Weight Gain (BWG), and Specific Growth Rate (SGR) were affected meaningfully by hydroalcoholic extract of Vitex agnus-castus (p < 0.05). These factors were significantly higher in compare to T3 treatment (p < 0.05). With increasing concentration of the extract in the diet, the ratio of female to male increased Treatment of T3 resulted in 87.23% feminization. Overall, this study suggests the use of T3 treatment achieves the best reproduction performance and a higher percentage of females in zebrafish as a model for aquaculture species. According to the results, the lowest levels of glucose and cholesterol were observed in T3 treatment and showed a significant changes with the control and treatments (p < 0.05).

Toxicity of Deltamethrin to Zebrafish Gonads Revealed by Cellular Biomarkers

Deltamethrin is responsible for health and reproduction problems both in mammals and aquatic organisms. In this study, zebrafish adults were exposed for 15 days to 0.25, 0.5, 1, and 2 μg L−1 non-lethal concentrations of deltamethrin, knowing that is used worldwide on agricultural crops. We investigated the chronic effects of deltamethrin on gonads by histopathological examination, immunohistochemistry, and immunofluorescence using biomarkers for apoptosis (anti-p53, anti-H2A.XS139ph antibodies, and TUNEL assay), oxidative stress (anti-Cox4i1 antibody) and proliferation (anti-PCNA antibody). Among the histopathological changes, the apoptotic response was elevated in ovary and testis of deltamethrin exposed groups as it was seen in the IHC and IF for p53, H2A.XS139ph, and confirmed by TUNEL assay. These were observed in the case of all studied concentrations compared with the control group. Thereby, the gonadal tissue exhibited an up-regulated activity of this cell-death signaling markers, while the proliferation marker (PCNA) increased in the ovary due to its presence not only in primary growth and cortical-alveolar stage follicles but also in atretic follicles, meanwhile decreased notably in the testis. Cox4i1, a mitochondrial marker, decreased both in ovary and testis during deltamethrin treatment, probably inhibited by the overproduction of the free radicals after pesticide exposure.

TCDD-induced multi- and transgenerational changes in the methylome of male zebrafish gonads.

The legacy endocrine disrupting chemical and aryl hydrocarbon receptor agonist, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), is produced as a byproduct of industrial processes and causes adverse health effects ranging from skin irritation to cancer. TCDD endpoints are also observed in subsequent, unexposed generations; however, the mechanisms of these multi- and transgenerational effects are unknown. We hypothesized an epigenetic mechanism, specifically DNA methylation for the transgenerational, male-mediated reproductive effects of developmental TCDD exposure. Using whole genome bisulfite sequencing, we evaluated DNA methylation changes in three generations of zebrafish, the first of which was exposed to TCDD during sexual development at 50 ppt for 1 h at both 3- and 7-week post-fertilization. We discovered that TCDD induces multi- and transgenerational methylomic changes in testicular tissue from zebrafish with decreased reproductive capacity, but most significantly in the indirectly exposed F1 generation. In comparing differentially methylated genes to concurrent transcriptomic changes, we identified several genes and pathways through which transgenerational effects of low level TCDD exposure are likely inherited. These include significant differential methylation of genes involved in reproduction, endocrine function, xenobiotic metabolism, and epigenetic processing. Notably, a number of histone modification genes were both differentially methylated and expressed in all generations, and many differentially methylated genes overlapped between multiple generations. Collectively, our results suggest that DNA methylation is a promising mechanism to explain male-mediated transgenerational reproductive effects of TCDD exposure in zebrafish, and these effects are likely inherited through integration of multiple epigenetic pathways.

Immune genes, IL1β and Casp9, show sexual dimorphic methylation patterns in zebrafish gonads

There is crosstalk between the immune and reproductive systems in which sexual dimorphism is a common pattern in vertebrates. In recent years, epigenetics has emerged as a way to study the molecular mechanisms involved in gonadal development, those responsible for integrating environmental information that contribute to assigning a specific sexual phenotype (either an ovary or a testis). The knowledge of epigenetic mechanisms in certain molecular processes allows the development of epigenetic markers. In fish gonads, the existence of reproduction-immune system interactions is known, although the epigenetic mechanisms involved are far from clear. Here, we used the zebrafish (Danio rerio) as a model to study the DNA methylation patterns in gonads of two well-known innate immune genes: IL1β and Casp9. DNA methylation levels were studied by a candidate gene approach at single nucleotide resolution and gene expression analyses were also carried out. Results showed that there was clear sexual dimorphism in the DNA methylation levels of the two immune genes studied, being significantly higher in the testes when compared to the ovaries. In summary, and although further research is needed, this paper presents sexual dimorphic methylation patterns of two immune-related genes, thus sex-biased differences in methylation profiles should considered when analyzing immune responses in fish. Data showed here can help to develop epimarkers with forthcoming applications in livestock and fish farming production, for example, in immune fish diseases or sexual control programs as epigenetic molecular tools to predict environmental pressure in the gonads.

Chronic exposure to venlafaxine and increased water temperature reversibly alters microRNA in zebrafish gonads (Danio rerio)

MicroRNA (miRNA) are short, non-coding RNA that act by downregulating targeted mRNA transcripts. Only recently have they been used as endpoints in studies of aquatic toxicology. The purpose of this study was to determine the effect of an antidepressant contaminant, venlafaxine (VFX), and increased temperature on specific microRNA levels in zebrafish (Danio rerio) reproductive tissue. Adult zebrafish were exposed to one of four conditions; control, 1 μg/L VFX (VFX), 32 °C (Temp), or 1 μg/L VFX + 32 °C (VFX & Temp) for 21 days. Half of the fish were returned to control conditions for a 21-day recovery period. RT-qPCR was performed to measure relative abundances of several miRNAs known to respond to antidepressant exposure: dre-miR-22b-3p, dre-miR-301a, dre-miR-140-5p, dre-let-7d-5p, dre-miR-210-5p, and dre-miR-457b-5p. After the exposure period, dre-miR-22b-3p and dre-miR-301a showed a significant downregulation in response to all treatments. In contrast, after the recovery period, there were no significant differences in microRNA abundance. These altered microRNA are predicted to target several genes, including phosphofructokinase, and are associated with ovarian pathologies. Combined, we have shown that VFX and increased water temperature alter miRNA abundances in zebrafish reproductive tissue, an effect correlated with a functional stress response and cell cycle dysregulation.

A Hormone That Lost Its Receptor: Anti-Müllerian Hormone (AMH) in Zebrafish Gonad Development and Sex Determination.

Fetal mammalian testes secrete Anti-Müllerian hormone (Amh), which inhibits female reproductive tract (Müllerian duct) development. Amh also derives from mature mammalian ovarian follicles, which marks oocyte reserve and characterizes polycystic ovarian syndrome. Zebrafish (Danio rerio) lacks Müllerian ducts and the Amh receptor gene amhr2 but, curiously, retains amh To discover the roles of Amh in the absence of Müllerian ducts and the ancestral receptor gene, we made amh null alleles in zebrafish. Results showed that normal amh prevents female-biased sex ratios. Adult male amh mutants had enormous testes, half of which contained immature oocytes, demonstrating that Amh regulates male germ cell accumulation and inhibits oocyte development or survival. Mutant males formed sperm ducts and some produced a few offspring. Young female mutants laid a few fertile eggs, so they also had functional sex ducts. Older amh mutants accumulated nonvitellogenic follicles in exceedingly large but sterile ovaries, showing that Amh helps control ovarian follicle maturation and proliferation. RNA-sequencing data partitioned juveniles at 21 days postfertilization (dpf) into two groups that each contained mutant and wild-type fish. Group21-1 upregulated ovary genes compared to Group21-2, which were likely developing as males. By 35 dpf, transcriptomes distinguished males from females and, within each sex, mutants from wild types. In adult mutants, ovaries greatly underexpressed granulosa and theca genes, and testes underexpressed Leydig cell genes. These results show that ancestral Amh functions included development of the gonadal soma in ovaries and testes and regulation of gamete proliferation and maturation. A major gap in our understanding is the identity of the gene encoding a zebrafish Amh receptor; we show here that the loss of amhr2 is associated with the breakpoint of a chromosome rearrangement shared among cyprinid fishes.

Male-biased zebrafish sex differentiation and metabolomics profile changes caused by dydrogesterone

Some progestins, including the widely used dydrogesterone (DDG), have been shown to cause male-biased sex ratio in teleost. However, there is a gap to fully understand the mechanisms of the sex differentiation disturbance by progestins, particularly from the metabolic aspect. We thus aimed to examine the sex changes by exposing zebrafish embryos to 4.4 (L), 44 (M) and 440 (H) ng/L DDG for up to 140 days, and investigated metabolomic profile changes during the critical period of sex differentiation at fry stage (35 dpf). DDG increased the percentage of male zebrafish in a dose-dependent manner, with 98% male fish in the high concentration group. In zebrafish fry, DDG increased the levels of some free fatty acids, monoglycerides, acylcarnitines, organic acids, free amino acids, while decreased lysophospholipids, uric acid and bile acids. DDG exposure also decreased the nucleoside monophosphates and UDP-sugars while increased nucleosides and their bases. These metabolite changes, namely increase in n-3 PUFAs (polyunsaturated fatty acids), myo-inositol, taurine, palmitoleic acid, oleic acid, lactic acid, fumaric acid, and uracil, and decrease in uric acid and bile acids, might account for the male-biased sex ratio in zebrafish. It appears that many of these metabolites could inhibit several pathways that regulate zebrafish gonad differentiation, including NF-κB/COX-2 and Wnt/β-catenin pathways, and activate p53 pathway. Thus we proposed a hypothesis that DDG might induce oocytes apoptosis through the above pathways and finally lead to female-to-male sex reversal. The results from this study suggest that DDG at environmentally relevant concentrations could affect zebrafish metabolomic profiles and finally disturb fish sex differentiation.

Ovarian transcriptomic signatures of zebrafish females resistant to different environmental perturbations

Sex is remarkably plastic in fish and can be easily influenced by environmental cues, in which temperature has been the most studied abiotic factor. However, it has been shown that elevated population densities can increase the number of males in several species but little is known about the underlying molecular mechanisms and whether general patterns exist. Here, we studied the long-term effects of population density on the gene expression program in zebrafish gonads. The ovarian transcriptome of females exposed to high versus low population densities contained 4,634 differentially expressed genes. Among them, a set of promale genes (amh, sypc3, spata6, and sox3) were upregulated in the high-population density group. Next, we compared the transcriptomes of ovaries of female zebrafish resistant to the masculinizing effects of either high density or elevated temperature. Results showed a set of 131 and 242 common upregulated and downregulated genes, respectively, including the upregulation of known male-related genes (e.g., amh and sycp3) but also genes involved in other functions (e.g., faima, ccm21, and ankrd6b) and a downregulation of cyp19a1a together with other genes (e.g., lgals9l1 and ubxn2a). We identified the common Gene Ontology terms involved in the reproduction and sexual development that were consistently affected in both environmental factors. These results show that regardless of the environmental perturbation there are common genes and cellular functions involved in the resistance to masculinization. These altered gene-expression profiles can be used as markers indicative of previous exposure to environmental stress independent of conspicuous alterations in sex ratios or gonadal morphology.

Developmental Dioxin Exposure Alters the Methylome of Adult Male Zebrafish Gonads.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental toxicant and endocrine disrupting compound with reproductive and developmental effects in humans and model organisms, including zebrafish. Our previous microarray and histological studies found defects in spermatogenesis and fertility of zebrafish in response to acute developmental TCDD exposure. These effects are apparent following exposure during reproductive development, modeling fetal basis of adult-onset disease. Some outcomes of these previous studies (reduced fertility, changes in sex ratio, transcriptomic alterations) are also transgenerational – persisting to unexposed generations – through the male germline. We hypothesized that DNA methylation could be a possible mechanism for these reproductive effects and performed whole genome bisulfite sequencing (WGBS), which identifies whole genome DNA methylation status at the base pair level, on testes of adult zebrafish exposed to TCDD (two separate hour-long exposures to 50 pg/mL TCDD at 3 and 7 weeks post fertilization). In response to TCDD exposure, multiple genes were differentially methylated; many of which are involved in reproductive processes or epigenetic modifications, suggesting a role of DNA methylation in later-life health outcomes. Additionally, several differentially methylated genes corresponded with gene expression changes identified in TCDD-exposed zebrafish testes, indicating a potential link between DNA methylation and gene expression. Ingenuity pathway analysis of WGBS and microarray data revealed genes involved in reproductive processes and development, RNA regulation, the cell cycle, and cellular morphology and development. We conclude that site-specific changes in DNA methylation of adult zebrafish testes occur in response to acute developmental TCDD exposure.