Probiotics as Potential Tool to Mitigate Nucleotide Metabolism Alterations Induced by DiNP Dietary Exposure in Danio rerio.

Diisononyl phthalate, classified as endocrine disruptor, has been investigate to trigger lipid biosynthesis in both mammalian and teleostean animal models. Despite this, little is known about the effects of DiNP exposure at tolerable daily intake level and the possible mechanisms of its toxicity. Probiotics, on the other hand, were demonstrated to have beneficial effects on the organism's metabolism and recently emerged as a possible tool to mitigate the EDC toxicity. In the present study, using a metabolomic approach, the potential hepatic sex-related toxicity of DiNP was investigated in adult zebrafish together with the mitigating action of the probiotic formulation SLAB51, which has already demonstrated its ability to ameliorate gastrointestinal pathologies in animals including humans. Zebrafish were exposed for 28 days to 50 µg/kg body weight (bw)/day of DiNP (DiNP) through their diet and treated with 109 CFU/g bw of SLAB51 (P) and the combination of DiNP and SLAB51 (DiNP + P), and the results were compared to those of an untreated control group (C). DiNP reduced AMP, IMP, and GMP in the purine metabolism, while such alterations were not observed in the DiNP + P group, for which the phenotype overlapped that of C fish. In addition, in male, DiNP reduced UMP and CMP levels in the pyrimidine metabolism, while the co-administration of probiotic shifted the DiNP + P metabolic phenotype toward that of P male and closed to C male, suggesting the beneficial effects of probiotics also in male fish. Overall, these results provide the first evidence of the disruptive actions of DiNP on hepatic nucleotide metabolism and mitigating action of the probiotic to reduce a DiNP-induced response in a sex-related manner.

Arginine Vasotocin Directly Regulates Spermatogenesis in Adult Zebrafish (Danio rerio) Testes.

The neuropeptide vasopressin is known for its regulation of osmotic balance in mammals. Arginine vasotocin (AVT) is a non-mammalian homolog of this neuropeptide that is present in fish. Limited information suggested that vasopressin and its homologs may also influence reproductive function. In the present study, we investigated the direct effect of AVT on spermatogenesis, using zebrafish as a model organism. Results demonstrate that AVT and its receptors (avpr1aa, avpr2aa, avpr1ab, avpr2ab, and avpr2l) are expressed in the zebrafish brain and testes. The direct action of AVT on spermatogenesis was investigated using an ex vivo culture of mature zebrafish testes for 7 days. Using histological, morphometric, and biochemical approaches, we observed direct actions of AVT on zebrafish testicular function. AVT treatment directly increased the number of spermatozoa in an androgen-dependent manner, while reducing mitotic cells and the proliferation activity of type B spermatogonia. The observed stimulatory action of AVT on spermiogenesis was blocked by flutamide, an androgen receptor antagonist. The present results support the novel hypothesis that AVT stimulates short-term androgen-dependent spermiogenesis. However, its prolonged presence may lead to diminished spermatogenesis by reducing the proliferation of spermatogonia B, resulting in a diminished turnover of spermatogonia, spermatids, and spermatozoa. The overall findings offer an insight into the physiological significance of vasopressin and its homologs in vertebrates as a contributing factor in the multifactorial regulation of male reproduction.

The probiotic SLAB51 as agent to counteract BPA toxicity on zebrafish gut microbiota -liver-brain axis.

A plethora of studies have so far described the toxic effects of bisphenol A (BPA) on organism health, highlighting the urgent need to find new strategies not only to reduce the presence of this toxicant but also to counteract its adverse effects. In this context, probiotics emerged as a potential tool since they promote organism welfare. Using a multidisciplinary approach, this study explores the effects of SLAB51 dietary administration to counteract BPA toxicity using zebrafish as a model. Adult males and females were maintained under standard conditions (control group; C), exposed for 28 days via the water to an environmental relevant dose of BPA (10 µg/L; BPA), dietary treated with SLAB51 (109 CFU/g of body weight; P) and co-treated with BPA plus SLAB51 (BPA + P). In the gut, exposure to BPA resulted in altered architecture in both males and females, with females also experiencing an increase of pathogenic bacterial species. Co-administration of BPA + P led to the restoration of normal gut architecture, favored beneficial bacteria colonization, and decreased the abundance of pathogenic species. In the liver, male BPA exposure led to steatosis and glycogen depletion, which was partially mitigated by SLAB51 co-administration. In contrast, in females exposed to BPA, the lack of steatosis along with the greater glycogen depletion, suggested an increase in energy demand as supported by the metabolomic phenotype. The analysis of liver metabolites in BPA + P males revealed increased levels of anserine and reduced levels of glutamine, which could lie behind the counteraction of the brain histopathological damage caused by BPA. In BPA + P females, a reduction of retinoic acid was found in the liver, suggesting an increase in retinoids responsible for BPA detoxification. Overall, these results demonstrate that SLAB51 exerts its beneficial effects on the gut microbiota-brain-liver axis through distinct molecular pathways, effectively mitigating the pleiotropic toxicity of BPA.

Prenatal exposure to a low dose of BPS causes sex-dependent alterations to vascular endothelial function in adult offspring.

Background: Bisphenol S (BPS) is among the most commonly used substitutes for Bisphenol A (BPA), an endocrine disrupting chemical used as a plasticizer in the manufacture of polycarbonate plastics and epoxy resins. Bisphenols interfere with estrogen receptor (ER) signaling, which modulates vascular function through stimulation of nitric oxide (NO) production via endothelial nitric oxide synthase (eNOS). BPS can cross into the placenta and accumulates in the fetal compartment to a greater extent than BPA, potentially interfering with key developmental events. Little is known regarding the developmental impact of exposure to BPA substitutes, particularly with respect to the vasculature. Objective: To determine if prenatal BPS exposure influences vascular health in adulthood. Methods: At the time of mating, female C57BL/6 dams were administered BPS (250 nM) or vehicle control in the drinking water, and exposure continued during lactation. At 12-week of age, mesenteric arteries were excised from male and female offspring and assessed for responses to an endothelium-dependent (acetylcholine, ACh) and endothelium-independent (sodium nitroprusside, SNP) vasodilator. Endothelium-dependent dilation was measured in the presence or absence of L-NAME, an eNOS inhibitor. To further explore the role of NO and ER signaling, wire myography was used to assess ACh responses in aortic rings after acute exposure to BPS in the presence or absence of L-NAME or an ER antagonist. Results: Increased ACh dilation and increased sensitivity to Phe were observed in microvessels from BPS-exposed females, while no changes were observed in male offspring. Differences in ACh-induced dilation between control or BPS-exposed females were eliminated with L-NAME. Increased dilatory responses to ACh after acute BPS exposure were observed in aortic rings from female mice only, and differences were eliminated with inhibition of eNOS or inhibition of ER. Conclusion: Prenatal BPS exposure leads to persistent changes in endothelium-dependent vascular function in a sex-specific manner that appears to be modulated by interaction of BPS with ER signaling.

Cylindrospermopsin induces oocyte maturation and disrupts gene expression in zebrafish ovarian follicles.

There is evidence that cylindrospermopsin (CYN) exerts reproductive toxicity in mice. However, little information is available concerning the toxicity of CYN in nonmammalian vertebrates. Here, we investigated the direct action of CYN on female reproduction by studying germinal vesicle breakdown, transcript abundance, caspase-3 activity, and testosterone production using cultured follicle-enclosed zebrafish oocytes as a model system. Treatment of follicles with 1,000 µg/L CYN significantly increased GVBD, Caspase-3 activity, and hCG-induced testosterone secretion. Exposure to CYN also reduced the abundance of 3ßhsd as well as hCG-induced fshr and era transcripts and increased cyp19a1 mRNA levels. In summary, this study provides a framework for a better understanding of the adverse action of CYN on female reproduction in zebrafish and other vertebrate species. The findings are also relevant to developing valid biomarkers for CYN by measuring zebrafish oocyte maturation and gene expression.

Thyroid Hormones Deficiency Impairs Male Germ Cell Development: A Cross Talk Between Hypothalamic-Pituitary-Thyroid, and-Gonadal Axes in Zebrafish.

In vertebrates, thyroid hormones are critical players in controlling different physiological processes such as development, growth, metabolism among others. There is evidence in mammals that thyroid hormones are also an important component of the hormonal system that controls reproduction, although studies in fish remain poorly investigated. Here, we tested this hypothesis by investigating the effects of methimazole-induced hypothyroidism on the testicular function in adult zebrafish. Treatment of fish with methimazole, in vivo, significantly altered zebrafish spermatogenesis by inhibiting cell differentiation and meiosis, as well as decreasing the relative number of spermatozoa. The observed impairment of spermatogenesis by methimazole was correlated with significant changes in transcript levels for several genes implicated in the control of reproduction. Using an in vitro approach, we also demonstrated that in addition to affecting the components of the brain-pituitary-peripheral axis, T3 (triiodothyronine) also exerts direct action on the testis. These results reinforce the hypothesis that thyroid hormones are an essential element of multifactorial control of reproduction and testicular function in zebrafish and possibly other vertebrate species.

NMR-based metabolomic profiling can differentiate follicular lymphoma from benign lymph node tissues and may be predictive of outcome.

Follicular lymphoma (FL) is a cancer of B-cells, representing the second most common type of non-Hodgkin lymphoma and typically diagnosed at advanced stage in older adults. In contrast to the wide range of available molecular genetic data, limited data relating the metabolomic features of follicular lymphoma are known. Metabolomics is a promising analytical approach employing metabolites (molecules < 1 kDa in size) as potential biomarkers in cancer research. In this pilot study, we performed proton nuclear magnetic resonance spectroscopy (1H-NMR) on 29 cases of FL and 11 control patient specimens. The resulting spectra were assessed by both unsupervised and supervised statistical methods. We report significantly discriminant metabolomic models of common metabolites distinguishing FL from control tissues. Within our FL case series, we also report discriminant metabolomic signatures predictive of progression-free survival.

TBBPA downregulates thyroid receptor and estrogen receptor mRNA levels in goldfish gonadal tissue.

Tetrabromobisphenol A (TBBPA) is a brominated flame retardant that has been shown to be a potential thyroid disrupting chemical. Currently, TBBPA is not included in the UN's list of endocrine disruptors and adverse effects of TBBPA in mammals has not been fully investigated. However, there is clear evidence that TBBPA exerts adverse health effects on reproduction of aquatic species. Therefore, it is important to provide more information on potential endocrine disruptive effects of TBBPA in vertebrate species. In this study we investigated the effect of TBBPA on transcript levels of estrogen receptors (ERs) and thyroid receptors (TRs) in the gonadal tissue of goldfish in vivo and in vitro. ERß mRNA levels were significantly lower in testis and ovary following exposure to TBBPA. TRα mRNA levels were also downregulated in testis tissue. Importantly, these phenotypic effects occurred at lower, environmentally relevant, concentrations in vivo. Furthermore, exposure to TBBPA also reduced ERß and TRα mRNA abundance in goldfish testes and ovaries in vivo, which is similar to previously observed T3 responses in these tissues. These findings suggest that TBBPA may be a thyroid hormone mimic, capable of disrupting reproduction by affecting steroid hormone receptors. Our findings suggest that it is important to study TBBPA as an endocrine disruptor in aquatic organisms as it may have implications for both conservation and aquaculture.

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.

Metabolic Changes During Growth and Reproductive Phases in the Liver of Female Goldfish (Carassius auratus).

Hormones of the brain-pituitary-peripheral axis regulate metabolism, gonadal maturation, and growth in vertebrates. In fish, reproduction requires a significant energy investment to metabolically support the production of hundreds of eggs and billions of sperms in females and males, respectively. This study used an LC-MS-based metabolomics approach to investigate seasonally-related changes in metabolic profile and energy allocation patterns in female goldfish liver. We measured basal metabolic profile in female goldfish at three phases of the reproductive cycle, including 1) Maximum growth period in postovulatory regressed phase, 2) mid recrudescence in fish with developing follicles, and 3) late recrudescence when the ovary contains mature ovulatory follicles. We also investigated changes in the liver metabolism following acute treatments with GnRH and GnIH, known to be involved in controlling reproduction and growth in goldfish. Chemometrics combined with pathway-driven bioinformatics revealed significant changes in the basal and GnRH/GnIH-induced hepatic metabolic profile, indicating that metabolic energy allocation is regulated to support gonadal development and growth at different reproductive cycles. Overall, the findings support the hypothesis that hormonal control of reproduction involves accompanying metabolic changes to energetically support gonadotropic and somatotropic activities in goldfish and other oviparous vertebrates.

Metabolomic and Transcript Analysis Revealed a Sex-Specific Effect of Glyphosate in Zebrafish Liver.

Glyphosate is a component of commonly used herbicides for controlling weeds in crops, gardens and municipal parks. There is increasing awareness that glyphosate-based herbicides, in addition to acting on plants, may also exert toxicity in wildlife and humans. In this study, male and female adult zebrafish were exposed to 700 µg/L of glyphosate (GLY), for 28 days. We used the metabolomic approach and UHPLC-ESI-MS to analyze liver samples to investigate the adverse effects of glyphosate on hepatic metabolism. The impact of GLY was found to be sex-specific. In female, GLY exposure affected purine metabolism by decreasing the levels of AMP, GMP and inosinic acid, consequently increasing uric acid levels with respect to the control (CTRL). Exposure to GLY also caused a decrease of UMP levels in the pyrimidine metabolism pathway. In male, GLY exposure decreased the aminoadipic acid within the lysine degradation pathway. Transcript analysis of genes involved in stress response, oxidative stress and the immune system were also performed. Results demonstrated an increased stress response in both sexes, as suggested by higher nr3c1 expression. However, the hsp70.2 transcript level was increased in female but decreased in male. The results demonstrated reduced sod1, sod2, and gpx1a in male following exposure to GLY, indicating an impaired oxidative stress response. At the same time, an increase in the cat transcript level in female was observed. mRNA levels of the pro-inflammatory interleukins litaf and cxcl8b.1 were increased in female. Taken together, the results provide evidence of disrupted nucleotide hepatic metabolism, increased stress inflammatory response in female and disruption of oxidative stress response in male.

Probiotic Administration Mitigates Bisphenol A Reproductive Toxicity in Zebrafish.

Although the use of bisphenol A (BPA) has been banned in a number of countries, its presence in the environment still creates health issues both for humans and wildlife. So far, BPA toxicity has been largely investigated on different biological processes, from reproduction to development, immune system, and metabolism. In zebrafish, Danio rerio, previous studies revealed the ability of environmentally relevant concentrations of this contaminant to significantly impair fertility via epigenetic modification. In addition, several studies demonstrated the ability of different probiotic strains to improve organism health. This study provides information on the role of the probiotic mixture SLAb51 to counteract adverse BPA effects on reproduction. A 28-day trial was set up with different experimental groups: BPA, exposed to 10 µg/L BPA; P, receiving a dietary supplementation of SLAb51 at a final concentration of 109 CFU/g; BPA+P exposed to 10 µg/L BPA and receiving SLAb51 at a final concentration of 109 CFU/g and a C group. Since oocyte growth and maturation represent key aspects for fertility in females, studies were performed on isolated class III (vitellogenic) and IV (in maturation) follicles and liver, with emphasis on the modulation of the different vitellogenin isoforms. In males, key signals regulating spermatogenesis were investigated. Results demonstrated that in fish exposed to the combination of BPA and probiotic, most of the transcripts were closer to C or P levels, supporting the hypothesis of SLAb51 to antagonize BPA toxicity. This study represents the first evidence related to the use of SLAb51 to improve reproduction and open new fields of investigation regarding its use to reduce endocrine disrupting compound impacts on health.

Effects of Di-Isononyl Phthalate (DiNP) on Follicular Atresia in Zebrafish Ovary.

Di-isononyl phthalate (DiNP) is a plasticizer reported to elicit hormone-like activity and disrupt metabolism and reproduction in fish and other vertebrates. In general, phthalates have been used at high concentrations beyond reported environmental levels to assess their adverse effects on fish gonadal physiology. The present study exposed adult female zebrafish to a wide range of DiNP concentrations [0.42 µg L-1 (10-9 M), 4.2 µg L-1 (10-8 M), and 42 µg L-1 (10-7 M)] for 21 days. We evaluated gene expression profiles related to apoptosis, autophagy, and oxidative stress; DNA fragmentation (TUNEL assay: terminal deoxynucleotidyl transferase dUTP nick end labeling) and caspase activity (CAS3) were also examined. Exposure to 0.42 and 4.2 µg L-1 upregulated the genes coding for tnfa and baxa, sod1, prkaa1, respectively. CAS3 immunohistochemistry revealed a higher number of positive vitellogenic oocytes in ovaries exposed to 0.42 µg L-1. Subsequently, we examined the relationship between CAS3 signaling and DNA fragmentation. Accordingly, DNA fragmentation was observed in vitellogenic follicles of fish exposed to 0.42 and 4.2 µg L-1. Our results demonstrate that follicular atresia can occur after exposure to environmental levels of DiNP for 21 days, which may adversely affect the reproductive performance of female zebrafish in a non-monotonic manner.

Interaction between thyroid hormones and gonadotropin inhibitory hormone in ex vivo culture of zebrafish testis: An approach to study multifactorial control of spermatogenesis.

Reproduction is under multifactorial control of neurohormones, pituitary gonadotropins, as well as of local gonadal signaling systems including sex steroids, growth factors and non-coding RNAs. Among the factors, gonadotropin-inhibitory hormone (Gnih) is a novel RFamide neuropeptide which directly modulates gonadotropin synthesis and release from pituitary, and in the gonads, Gnih mediated inhibitory actions on gonadotropin response of zebrafish spermatogenesis. Thyroid hormones are peripheral hormones which are also known to interact with reproductive axis, in particular, regulating testicular development and function. This study investigated the interaction between Gnih and thyroid hormones in zebrafish spermatogenesis using in vivo and ex vivo approaches. Three experimental groups were established: "control" (non-treated fish), "methimazole" and "methimazole + T4". Fish were exposed to goitrogen methimazole for 3 weeks; T4 (100 µg/L) was added in the water from the second week only in the "reversal treatment" group. After exposure, testes were dissected out and immediately incubated in Leibovitz's L-15 culture medium containing hCG, Gnih or hCG + Gnih for 7 days. Germ cell cysts and haploid cell population were evaluated by histomorphometry and flow cytometry, respectively. Our results showed that hypothyroidism affected germ cell development in basal and gonadotropin-induced spermatogenesis, in particular, meiosis and spermiogenesis. Hypothyroid testes showed lower amount of spermatozoa, and decreased potency of hCG. We also showed that goitrogen treatment nullified the inhibitory actions of Gnih on the gonadotropin-induced spermatogenesis. This study provided evidences that thyroid hormones are important regulatory factors for hCG- and Gnih-mediated functions in zebrafish spermatogenesis.

Seasonally related metabolic changes and energy allocation associated with growth and reproductive phases in the liver of male goldfish (Carassius auratus).

Reproduction and growth follow a seasonal pattern in many fish species involving changes in gonadal development, growth, and metabolism. Significant metabolic energy is needed during gametogenesis in both female and male to produce hundreds of eggs and billions of sperms. Seasonal variations are controlled by the hormones of brain-pituitary-peripheral axis and are accompanied by significant metabolic changes. There is evidence that GnRH and GnIH are among the key neurohormones that regulate the reciprocal control of growth and reproduction. The objective of this study was to investigate changes in metabolic profile and energy allocation patterns at different stages of reproduction, using goldfish as a model organism and LC-MS as analytical platform for metabolic analysis. Goldfish undergoes a clear seasonal cycle of growth and reproduction. In vivo experiments were conducted at three different time point of the annual cycle: regressed gonadal phase (peak growth phase), mid gametogenesis and late gametogenesis. Emphasis is placed on changes in liver metabolic pathways to energetically sustain the physiological processes related to growth and reproduction. Moreover, we tested the hypothesis that GnRH and GnIH may play a role in the regulation of metabolism by investigating acute effects of these peptides at different stages of reproductive cycle. SIGNIFICANCE: The findings in this paper provide novel information on the seasonal changes in basal metabolism during different stages of reproductive cycle, and evidence for differential allocation of energy during reciprocal control of reproduction and growth in goldfish. Chemometrics combined with pathway-driven bioinformatics elucidated a shift in the metabolic profile, indicating distinct patterns of energy allocation in the reproductive and growth seasons. Furthermore, to our knowledge this is the first study to provide evidence for a possible regulatory role of GnRH and GnIH in liver metabolism and energy allocation patterns associated with growth and reproductive processes. Together our findings present a framework for better understanding of the hormonally induced changes in metabolism to energetically sustain growth and reproduction in fish and other oviparous species undergoing seasonal cycle.

Seasonally Related Disruption of Metabolism by Environmental Contaminants in Male Goldfish (Carassius auratus).

Endocrine disrupting chemicals mimic or disrupt action of the natural hormones, adversely impacting hormonal function as well as cardiovascular, reproductive, and metabolic health. Goldfish are seasonal breeders with an annual reproductive cycle regulated by neuroendocrine signaling which involves allocation of metabolic energy to sustain growth and reproduction. We hypothesize that seasonal changes in physiology alter overall vulnerability of goldfish to metabolic perturbation induced by environmental contaminants. In this study, we assess effects of endogenous hormones, individual contaminants and their mixture on metabolism of goldfish at different reproductive stages. Exposure effects were assessed using 1H-NMR metabolomics profiling of male goldfish midbrain, gonad and liver harvested during early recrudescence (October), mid-recrudescence (February) and late recrudescence (June). Compounds assessed include bisphenol A, nonylphenol, bis(2-ethylhexyl) phthalate, fucosterol and a tertiary mixture (DEHP + NP + FS). Metabolome-level responses induced by contaminant exposure across tissues and seasons were benchmarked against responses induced by 17ß-estradiol, testosterone and thyroid hormone (T3). We observe a clear seasonal dependence to metabolome-level alteration induced by hormone or contaminant exposures, with February (mid-recrudescence) the stage at which male goldfish are most vulnerable to metabolic perturbation. Responses induced by contaminant exposures differed from those induced by the natural hormones in a season-specific manner. Exposure to the tertiary mixture induced a functional gain at the level of biochemical pathways modeling over responses induced by individual components in select tissues and seasons. We demonstrate the importance of seasonally driven changes in physiology altering overall vulnerability of goldfish to metabolic perturbation induced by environmental contaminants, the relevance of which likely extends to other seasonally-breeding species.

Effects of gonadotropin-inhibitory hormone on early and late stages of spermatogenesis in ex-vivo culture of zebrafish testis.

Gonadotropin-inhibitory hormone (Gnih) is known to play a role in the regulation of reproduction in vertebrates by influencing gonadotropin release and synthesis. While the endocrine actions of Gnih have been identified in several species, its paracrine/autocrine effects in the control of spermatogenesis are less defined. We have used ex vivo culture of zebrafish testis to investigate the role of gonadal zebrafish Gnih (zGnih) in the regulation of the spermatogenic process. We used FACScan cell cycle analysis, morphometric quantifications, BrdU incorporation and caspase-3 activity assays as well as measuring 11-Ketotestosterone (11-KT) level in the culture media. FACScan analysis and morphometric quantification results demonstrated direct action of zGnih on basal and gonadotropin (Lh and Fsh)-induced spermatogenesis. Treatment with zGnih (10 nM) significantly decreased the number of G0/G1 cells after 7-days of culture while no significant changes were found in the proportion area of spermatogonia cell types. Investigation of DNA synthesis using BrdU (5-Bromo-2'-Deoxyuridine) labeling showed that treatment with zGnih (10 nM) significantly decreased proliferative activity of type A spermatogonia, while increased the mitotic activity of type B spermatogonia. We also showed that treatment with zGnih (100 nM) completely eliminated 11-KT release induced by 100 ng/ml Fsh. Treatment with zGnih (10 and 100 nM) also inhibited both hCG and Fsh-induced spermatogenesis. These results, plus our previous findings, demonstrate that zGnih produced locally in the testis is a component of a complex multifactorial system that regulates testicular function in zebrafish.

Role of GnRH and GnIH in paracrine/autocrine control of final oocyte maturation.

The control of oocyte growth and its final maturation is multifactorial and involves a number of hypothalamic, hypophyseal, and peripheral hormones. In this study, we investigated the direct actions of the gonadotropin-releasing hormone (GnRH) and the gonadotropin-inhibitory hormone (GnIH), which are expressed in the ovarian follicles, on final oocyte maturation in zebrafish, in vitro. Our study demonstrates the expression of GnRH and GnIH in the ovarian follicles of zebrafish (Danio rerio) at different stages of development and provides information on the direct action of these hormones on final oocyte maturation. Treatment with both GnRH and GnIH peptides stimulated the germinal vesicle breakdown (GVBD) of the late-vitellogenic oocyte. Both the GnRH and GnIH treatments showed no significant change in the caspase-3 activity of pre-vitellogenic and mid-vitellogenic oocytes, while they displayed different responses in the late-vitellogenic follicles. The GnRH treatment increased caspase-3 activity, whereas the GnIH reduced caspase-3 activity in the late-vitellogenic follicles. We also investigated the effects of GnRH and GnIH on the hCG-induced resumption of meiosis and caspase activity in vitro. GnRH and GnIH were found to have a similar effect on the hCG-induced resumption of meiosis, while they showed the opposite effect on caspase-3 activity. Furthermore, we investigated the effects of concomitant treatment of GnRH and GnIH peptides with hCG. The results demonstrated that the presence of both GnRH3 and GnIH are necessary for the normal induction of final oocyte maturation by gonadotropins. The findings support the hypothesis that GnIH and GnRH peptides produced in the ovary are part of a complex multifactorial regulatory system that controls zebrafish final oocyte maturation in paracrine/autocrine manner working in concert with gonadotropin hormones.

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 20 µg/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 20 µg/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.

Seasonal Related Multifactorial Control of Pituitary Gonadotropin and Growth Hormone in Female Goldfish: Influences of Neuropeptides and Thyroid Hormone.

Female reproduction is under multifactorial control of brain-pituitary-peripheral origin. The present study provides information on seasonal changes in circulating LH and GH concentrations, as well as transcript levels for a number of genes involved in the regulation of reproduction and growth in female goldfish. We also provide information on the effects of treatments with GnRH and/or GnIH, and their interaction with T3, at three stages of gonadal recrudescence. Maximum basal concentration of LH was observed at late recrudescence (Spring) while no seasonal changes in basal serum GH levels was detected. Serum LH and GH levels were stimulated by GnRH as expected, depending on the season. GnIH stimulated basal GH concentrations in gonadally regressed fish. GnIH inhibitory action on GnRH-induced LH response was observed in late, but not in mid recrudescence. T3 actions on basal and GnRH- or GnIH-induced GH secretion were generally inhibitory, depending on season. Administration of T3 attenuated GnRH-induced LH responses in mid and late stages of gonadal recrudescence, and the presence of GnIH abolished inhibitory actions of T3 in fish at mid recrudescence. Our results also demonstrated seasonal patterns in basal and GnRH- and/or GnIH-induced transcript levels for ERα, ERßI, FSHR, aromatase, TRαI, TRß, IGF-I, and Vtg in the liver and ovary. However, there were no clear correlations between changes in transcript levels and circulating levels of LH and GH. The results support the hypothesis that GnRH, GnIH, and T3 are contributing factors in complex reciprocal control of reproduction and growth in goldfish.