Zebrafish Ovary Research Articles

Nanos3 maintains germline stem cells and expression of the conserved germline stem cell gene nanos2 in the zebrafish ovary

Female zebrafish have a prolific reproductive capacity, suggesting that a germline stem cell (GSC) population drives oocyte production. However, a zebrafish female GSC population has yet to be identified. Adult stem cells are defined by their ability to both self-renew and differentiate, and by their localization to a stem cell niche. We show here that mitotic and early meiotic germ cells are present in the adult ovary and that the zebrafish homolog of the conserved vertebrate GSC marker, nanos2, is expressed in a subset of pre-meiotic oogonia in the adult gonad. We propose that these nanos2+ cells are GSCs. Importantly, we find that mitotic, nanos2+, and early meiotic germ cells localize to the germinal zone, thus identifying this region as the probable ovarian GSC niche in zebrafish. nanos3, which encodes a conserved RNA-binding protein, is known to be required for the continued production of oocytes in the zebrafish. Although mammalian homologs of nanos3 are expressed in early spermatogonia, no study has defined the role of nanos3 in the regulation of vertebrate GSCs. Here we demonstrate that nanos3 function is required for the maintenance of GSCs, but not for their specification, and propose that nanos2 and nanos3 are partially redundant in this role.

Endocrine-active compound evaluation: Qualitative and quantitative histomorphological assessment of zebrafish gonads after bisphenol-A exposure

There is great social concern about the risk involved from exposure to BPA as an endocrine disrupter in humans, as well as the possible repercussion of this chemical on the environment. In this study, the short-term effects of BPA at a gonadal level were assessed by means of different biomarkers in a model animal organism in vogue, the zebrafish (Danio rerio). For this purpose, 60 female zebrafish aged 16 weeks were used. These were exposed for 14 days in aquariums (following OECD Directive no.204) to BPA concentrations of 1, 10, 100 and 1000μg/L, in addition to a control batch. After the exposure period, the zebrafish were sacrificed and samples taken for a histopathological study by light and electron microscopy and morphometric analysis. During the fourteen days of exposure, water samples were taken from the aquariums to analyze the BPA levels. The BPA concentration in the fish and the water was determined by LC–MS/MS.The gonads of the zebrafish exposed to the BPA had a normal external appearance and there were no variations in their size or body weight. An accumulation of BPA was produced in the zebrafish tissues, and this increased as the BPA concentration to which the fish were exposed did. In the histopathological and morphometric studies, multiple alterations were observed in the zebrafish ovaries, particularly highlighting the vacuolization of the follicular cytoplasm, a great degeneration of all the cell components, and an important increase in the percentage of atretic follicles as from concentrations of 100 and 1000μg/L of BPA, verified by morphometry.These data indicate that morphological endpoints are sufficiently sensitive to individuate early effects of environmental concentration of BPA on gonads after two weeks of exposure.

Interplay between autophagy and apoptosis in the development of Danio rerio follicles and the effects of a probiotic

The present study investigated autophagic processes in Danio rerio preovulatory follicles (Stage III and IV). There were more autophagosomes, as revealed by electron microscopy, in follicles from females fed the probiotic Lactobacillus rhamnosus IMC 501. This was confirmed by increased expression of genes involved in the autophagic process, namely ambra1, becn1, lc3 and uvrag. In addition, preovulatory follicles from females fed the probiotic contained more microtubule-associated protein 1 light chain 3 isoform II (LC3-II) and less p62 protein. The increased autophagy in preovulatory follicles from females fed the probiotic was concomitant with a decrease in the apoptotic process in the ovary, as evidenced by terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling analysis and confirmed by lower expression of genes involved in apoptosis (i.e., p53, bax, apaf and cas3) and higher expression as igfII and igf1r. The results of the present study provide preliminary evidence of the involvement of autophagy during follicle development in the zebrafish ovary. In addition, we have demonstrated for the first time that a functional food, such as L. rhamnosus IMC 501, can modulate the balance between apoptosis and autophagy that regulates ovary physiology in zebrafish by inhibiting follicular apoptosis and improving follicular survival.

Differential regulation of gonadotropin receptors (fshr and lhcgr) by epidermal growth factor (EGF) in the zebrafish ovary

Epidermal growth factor (egf) is expressed in the zebrafish oocyte whereas its receptor EGF receptor (egfr) is expressed in the somatic follicle layer, strongly suggesting a role for Egf in the intrafollicular paracrine communication that mediates an oocyte-to-follicle cell signaling pathway. However, the exact function of Egf in the follicle remains largely unknown. The present study aimed to explore the possible role of Egf in regulating gonadotropin receptors (fshr and lhcgr) in cultured zebrafish follicle cells. EGF down-regulated lhcgr expression dose-dependently in a biphasic manner with significant effect observed at 1.5 and 24h. The effect was mediated via Egfr on the follicle cells. On the contrary, EGF also tended to decrease fshr expression at 1.5h but it appeared to up-regulate fshr at 24h. The EGF suppression of lhcgr expression was functionally relevant as pre-exposure to EGF reduced the follicle cell responsiveness to LH/hCG. We have recently reported that estradiol (E2) strongly stimulated lhcgr expression in the zebrafish ovary. In the current study, we further demonstrated that EGF and other EGF family members, heparin-binding EGF-like growth factor (HBEGF), transforming growth factor α (TGFα) and betacellulin (BTC), all reduced basal and E2-induced lhcgr expression. This study provides evidence for a potential paracrine role of Egf and its related peptides in the zebrafish follicle. The oocyte-derived EGF family ligands may actively control the process of follicle growth and maturation by differentially controlling the expression of fshr and lhcgr in the follicle cells in a paracrine manner.

Histological analysis of acute toxicity of 2,4-diclorophenoxy acetic acid in ovary of zebrafish

Plant growth regulators are the chemicals that are found in plants and produced synthetically. In agricultural applications, plant hormones are used in minor quantities for fixing the problems. In our research, we studied the effects of 2,4 diclorophenoxyacetic acid (2,4D), which is an auxin used in agricultural applications. Auxins are the group that are used most popularly in plant growth regulators. In our study, different doses of 2,4 diclorophenoxyacetic acid are given to zebrafish, and ovarium tissues are observed histomorphologically. We generated one control and three experiment groups from the stock solution. The experiment was carried out in 20 liter capacity complete glass aquarium at 24±1°C water temperature. After five days of application, fishes were dissected. Histomorphological changes of the ovarium were investigated under a light microscope. A decrease in the number of oocytes in zebrafish ovarium was observed when compared with the control group. Many deformed and underdeveloped oocytes were detected. An increase in the number of atretic oocytes was observed. It was deduced that acute doses of 2,4 diclorophenoxyacetic acid decelerates oogenesis in fishes.

Epidermal growth factor differentially regulates activin subunits in the zebrafish ovarian follicle cells via diverse signaling pathways

Epidermal growth factor (EGF) promotes oocyte maturation in the zebrafish and its effect is mediated via the activin system. However, the mechanisms by which EGF regulates activin subunits in the follicle cells remain unknown. The present study demonstrated that EGF controlled expression of three activin subunits (inhbaa, inhbab and inhbb) in the follicle cells via diverse signaling pathways. The expression of inhbaa and inhbb was often co-regulated via similar pathways. Suppression of MAPK3/1, p38 MAPK, PKC and PKA each blocked or partially reduced the stimulatory effects of EGF on the expression of inhbaa and inhbb while up-regulated that of inhbab. Conversely, inhibition of PI3K did not have any effect on the expression of inhbaa and inhbb but significantly suppressed the stimulatory effect of EGF on inhbab. In summary, EGF action in the zebrafish ovary involves activin system and its regulation of activin subunits is mediated by diverse signaling pathways downstream of EGFR.

Regulation and actions of insulin-like growth factors in the ovary of zebrafish (Danio rerio)

Insulin-like growth factors (Igf) are known paracrine/autocrine regulators of ovarian development in teleosts. Initial studies investigated the hormonal and intracellular signalling cascades involved in regulating the expression of ovarian-derived Igfs in zebrafish (Danio rerio). Quantitative real-time PCR was used to quantify the expression of igf3, igf2a, and igf2b in full grown immature (FG; 0.57–0.65mm) and mid-vitellogenic (MV; 0.45–0.56mm) follicles. Addition of the gonadotropin analogue human chorionic gonadotropin (hCG) and the adenylate cyclase activator forskolin increased igf3 expression in FG and MV follicles, but had no effect on igf2a or igf2b expression. The effects of hCG on igf3 expression were blocked by the addition of the protein kinase A inhibitor H-89. Pituitary adenylate cyclase activating peptide also stimulated a small increase in igf3 expression in FG follicles, while growth hormone and salmon gonadotropin releasing hormone had no effect on igf3, igf2a, or igf2b expression. Secondary studies investigated the involvement of ovarian-derived Igfs in mediating the ovarian actions of gonadotropins on cell survival and steroidogenesis. Treatment of FG follicles with recombinant human IGF1, hCG, or forskolin inhibited the induction of caspase-3/7 activity, which was used as a measure of apoptosis. The effects of hCG and forskolin on caspase-3/7 were attenuated by co-treatment with NVP-AEW54, an IGF1 receptor antagonist. In other studies, hCG was shown to increase the production of the maturation-inducing steroid 17,20β-dihydroxy-4-pregnen-3-one, but this action was not affected by co-treatment with NVP-AEW54. These results suggest there is a high degree of hormonal specificity in regulating Igfs in the zebrafish ovary and the ovarian-derived Igfs, presumably Igf3, are downstream mediators of gonadotropin-dependent cell survival, but are not involved in gonadotropin-induced steroidogenesis.

Ghrelinergic system in fish ovaries and ghrelin inhibition of germinal vesicle breakdown in zebrafish oocytes

Ghrelin, the only known orexigenic gut hormone has been proposed to integrate energy balance and reproduction in mammals. There is a large set of data available on the orexigenic and LH stimulatory roles of ghrelin in fish. Ghrelin and ghrelin receptor mRNAs are expressed in the gonads of several fishes. However, the direct roles of ghrelin on fish gonads remain unclear. Our objective was to identify the ghrelinergic system in fish ovaries, at the protein level in the cross sections of paraffin fixed ovaries, and test the direct effects of ghrelin on oocyte maturation. Both ghrelin and ghrelin receptor like immunoreactivity was detected in the follicle cells in the cross section of goldfish and zebrafish ovaries. This agrees with the mRNA expression data and further confirms the presence of the ghrelinergic system in fish ovaries. We found that native ghrelin at 10, 50 and 100ng/mL concentrations inhibited both basal and maturation-inducing hormone stimulated stage IV germinal vesicle breakdown (GVBD) of zebrafish oocyte maturation in vitro. This result indicates that ghrelin acts directly on zebrafish follicles. When zebrafish follicles were co-incubated with ghrelin and a well-characterized ghrelin receptor antagonist, d-lys3-GHRP-6, the inhibitory effects of ghrelin on stage IV GVBD was abolished. This result indicates that ghrelin inhibits stage IV GVBD via its receptor(s). Collectively, our results for the first time indicate a direct role for ghrelin in the ovarian physiology of fish.

Differential regulation of gonadotropin receptors by bone morphogenetic proteins in the zebrafish ovary

Follicle-stimulating hormone receptor (fshr) and luteinizing hormone/choriogonadotropin receptor (lhcgr) exhibit differential temporal expression patterns during zebrafish folliculogenesis with fshr being dominant during vitellogenic growth and lhcgr increasing its expression dramatically before maturation. The dynamic and distinct expression patterns of fshr and lhcgr suggest that they are under tight regulatory control. However, the underlying mechanisms for the differential expression of the two receptors remain unknown. We have recently demonstrated that members of bone morphogenetic protein (BMP) family are largely expressed in the oocyte, while their receptors are exclusively localized on the follicle cells, suggesting a potential paracrine signaling from the oocyte to the follicle cells by BMPs. In this study, we investigated the effects of zebrafish BMP2b (zfBmp2b) and BMP4 (zfBmp4) on the expression of fshr and lhcgr using a novel co-culture approach. The recombinant zfBmp2b or zfBmp4-producing CHO cells were co-cultured with the zebrafish follicle cells followed by real-time qPCR analysis of fshr and lhcgr expression. Our results showed that zfBmp2b and zfBmp4 both down-regulated fshr, while up-regulated lhcgr expression at 24h of co-culturing. This finding, together with the high expression level of BMP receptors in the follicle cells prior to oocyte maturation, strongly suggests a potential role for BMPs in the differential expression of fshr and lhcgr, especially in the full-grown follicles before maturation. As BMPs are largely expressed in the oocyte, this also implies an important role for the oocyte in orchestrating the differentiation and function of the follicle cells.

Immunohistochemical Demonstration of Lymphatic Vessels in Adult Zebrafish

Morphological profiles of lymphatic vessels in adult zebrafish trunk and ovary were studied by immunohistochemistry and electron microscopy. The present immunohistochemistry for Prox1 was successful in demonstrating lymphatic vessels in zebrafish. The zebrafish trunk revealed two types of bilateral longitudinal lymphatic trunks draining lymph centripetally along dorsal aorta and posterior cardinal veins. Large honeycomb lymphatic sinus was further shown around common cardinal veins. In the zebrafish ovary, the lymphatic vessels, comprising endothelial cells only, encompassed arterioles in their lumen. This peculiar structure appeared to be conserved in vertebrates including mammals and might serve for control of blood temperature and tissue homeostasis. The present study is first to delineate lymphatic vessels in adult zebrafish by immunohistochemistry. Our immunohistochemical results showed usefulness of immunostaining for Prox1 not only for demonstration of lymphatic vessels in zebrafish, but also for examination of their function and dynamics in pathophysiological condition.

Effect of lambda-cyhalothrin and Neemgold on some biochemical parameters in the gill, liver, and ovary of zebrafish, Danio rerio (Cyprinidae)

The aim of the present work was to study the effect of lambda-cyhalothrin and Neemgold on the total protein, total free amino acid, and nucleic acid contents in tissues (gill, liver, and ovary) of zebrafish, Danio rerio (Hamilton), after exposure to 96 h LC10 ,L C 20, and LC40 of lambda-cyhalothrin and the neem-based pesticide Neemgold. It was found that the total protein content was reduced to 38, 46, and 45% in gill, liver, and ovary, respectively, after lambda-cyhalothrin exposure for 21 days at the LC40 dose. The total free amino acid content in the liver was enhanced to 172 and 154% of control (100%) after exposure to LC40 of lambda-cyhalothrin and Neemgold, respectively. However, the total free amino acid content in the gill and ovary of treated fish was significantly reduced. In addition, DNA content was reduced to 45, 41, and 41% of controls (100%) in gill, liver, and ovary, respectively, after 21 days of exposure to LC40 of lambda-cyhalothrin. The reduction in DNA content from the control was 51, 53, and 55% in gill, liver, and ovary, respectively, from Neemgold exposure at the same concentration (LC40) and period as lambda-cyhalothrin. It was observed that all the changes were dependent on concentration as well as time. Similarly, significant reductions in RNA content were also observed in gill, liver, and ovary after lambda-cyhalothrin and Neemgold exposure. The probable causes are discussed.

Differential Regulation of Gonadotropin Receptors (fshr and lhcgr) by Estradiol in the Zebrafish Ovary Involves Nuclear Estrogen Receptors That Are Likely Located on the Plasma Membrane

FSH and LH are gonadotropins (GTH) that control all major events of gonadal function. FSH and LH signal through their cognate receptors, FSH receptor and LH/choriogonadotropin receptor, respectively, across vertebrates. Compared with the information in mammals, very little is known about these receptors in fish, especially the regulation of their expression. In female zebrafish, fshr and lhcgr exhibit significant temporal difference in expression, with fshr increasing first when the follicles are activated to enter the vitellogenic growth phase and lhcgr lagging behind. This raises an interesting question on the differential regulation of these two GTH receptors (GTHR) during folliculogenesis. Using a primary follicle cell culture, the present study demonstrated that 17β-estradiol (E2), but not testosterone, was a potent endocrine hormone that differentially regulated the expression of fshr and lhcgr. Although E2 stimulated both receptors, its effect on the steady-state level of lhcgr mRNA was much higher (>8-fold up-regulation) than that of fshr (∼0.5-fold increase). E2 likely acted at the transcription level via its nuclear estrogen receptors (ERα and ERβ), because ICI 182,780 could abolish its effects. However, our evidence suggested that these receptors might be localized on the plasma membrane, because β-estradiol 6-(O-carboxy methyl)oxime:BSA could fully mimic the effects of E2. Demonstrating that E2 is likely one of the differentiating factors for the distinct expression of the two GTHR in the zebrafish ovary, this study sheds important light on the functions of the two GTH and their receptors in fish as well as the conservation and diverse aspects of GTHR regulation across vertebrates.

Identification of Z-OTU protein during zebrafish oogenesis and early embryogenesis.

Zebrafish (Danio rerio) Z-OTU, containing OTU and TUDOR domains, was predicted to be a member of OTU-related protease, a family of the deubiquitylating enzymes (DUBs). A previous report from our laboratory clearly describes the expression patterns of z-otu mRNA. Here, we characterized the Z-OTU protein during zebrafish oogenesis and early embryogenesis. After prokaryotic expression, the recombinant protein of the OTU domain and GST was purified and injected into rabbits to obtain the polyclonal antibody-anti-Z-OTU, which was used for immunohistochemistry in zebrafish ovaries and embryos. Interestingly, obvious differences existed between the expression patterns of z-otu mRNA and its protein during oogenesis and early embryogenesis. In stage I oocytes, z-otu mRNA was detected in cytoplasm while its protein existed in the germinal vesicle. In addition, its protein was distributed during entire oogenesis, while mRNA was not detected in oocytes at stage IV or mature oocytes. The z-otu mRNA disappeared after midblastula transition (MBT) and its protein gradually decreased after this stage. We inferred that Z-OTU protein, like other OTU-related protease with DUB activity, was required for germinal vesicle breakdown of oocytes during meiosis, germinal vesicle migration, and embryo cleavage maintenance.

Differential Regulation of Gonadotropin Receptors ( fshr and lhcgr ) by Estradiol in the Zebrafish Ovary Involves Nuclear Receptors that are Likely Located on the Plasma Membrane

Differential Regulation of Gonadotropin Receptors ( fshr and lhcgr ) by Estradiol in the Zebrafish Ovary Involves Nuclear Receptors that are Likely Located on the Plasma Membrane

A transgenic zebrafish model of targeted oocyte ablation and de novo oogenesis

We describe here a novel transgenic zebrafish, Tg(zpc:G4VP16/UAS:nfsB-mCherry) that effectively demonstrates the targeted oocyte ablation in the adult zebrafish ovary. This transgenic line expresses bacterial nitroreductase enzyme (nfsB) under the control of the oocyte-specific zona pellucida C (zpc) gene promoter. Adult transgenic females exposed to the prodrug metronidazole demonstrated near-complete ablation of growing oocytes, resulting in ovarian degeneration and complete cessation of reproductive function. Within 4 weeks of prodrug removal, treated fish demonstrated complete anatomical regeneration of the ovary and, within 7 weeks, ovarian function (fertility) was fully restored. Together, these results demonstrate functional renewal of the oocyte pool in the adult zebrafish ovary. Accordingly, this transgenic zebrafish model system provides a novel means to investigate ovarian growth dynamics in a genetically tractable vertebrate, and may be useful for evaluating signaling interactions that regulate gonadal development processes such as de novo oogenesis.

Spatiotemporal Expression of Bone Morphogenetic Protein Family Ligands and Receptors in the Zebrafish Ovary: A Potential Paracrine-Signaling Mechanism for Oocyte-Follicle Cell Communication1

The bone morphogenetic proteins (BMPs), originally identified by their abilities to induce bone and/or cartilage formation, have been reported to be involved in various growth and differentiation processes, including reproduction. Although mammalian models are more frequently used to study the BMP system in reproduction, we have extended the study to the zebrafish, an excellent model for studying female reproduction in teleosts. Reverse transcription-PCR analysis revealed the expression of the Bmp ligands (bmp2a, bmp2b, bmp4, bmp6, and bmp7a) and the type II Bmp receptors (bmpr2a and bmpr2b) in various tissues, including the ovary. Spatiotemporal distribution of these Bmp ligands and receptors in the ovary was then investigated in this study. Reverse transcription-PCR on isolated follicle layers and denuded oocytes demonstrated that all Bmp ligands examined were exclusively or abundantly expressed in the oocyte, whereas the two receptors were expressed exclusively in the follicle layers, strongly suggesting a potential paracrine signaling from the oocyte towards the follicle layer by various Bmp ligands. This supports the current view that instead of being passively controlled and nurtured by the follicle layer for its growth and development, the oocyte may play an active role by releasing various growth differentiation factors to regulate follicle layer function. Quantitative analysis of temporal expression profiles during folliculogenesis revealed an increased expression of bmp2a, bmp2b, bmp4, and bmp6 from primary growth (stage I) to previtellogenic (stage II) stages, followed by steady declines toward the end of folliculogenesis when the follicles became fully grown. On the contrast, the BMP receptors (bmpr2a and bmpr2b) consistently showed an increase in expression during folliculogenesis, with the peak levels reached at the full-grown stage prior to final oocyte maturation. The spatiotemporal expression patterns of the Bmp family in the zebrafish follicles provide important insights into potential roles for Bmps during follicle development as oocyte-derived factors. Further experiments using recombinant zebrafish Bmp4 showed that Bmp4 had an inhibitory effect on spontaneous oocyte maturation in vitro, but not 17alpha,20beta-dihydroxy-4-pregnen-3-one (DHP)-induced oocyte maturation in vitro.

Cadmium-induced ovarian pathophysiology is mediated by change in gene expression pattern of zinc transporters in zebrafish (Danio rerio)

This study explored the potential for expression pattern of genes encoding zinc (Zn) transporters to be involved in the cadmium (Cd)-induced reproductive toxicity in female of zebrafish. For this purpose, oocytes maturity and ovarian histology as well as Cd, Zn and metallothioneins (MTs) accumulation and expression of genes encoding Zrt-,Irt-related protein 10 (ZIP10), Zn transporter 1 (ZnT1) and zebrafish metallothionein (zMT) were examined in ovaries of adult zebrafish exposed to 0.4mg/L Cd in water and supplemented with Zn (5mgkg−1) in their diet for 21days. Cd-exposure decreased the expression of ZnT1 and caused up-regulation of ZIP10 and zMT gene expression. These changes were accompanied by increased Cd and MTs accumulation, decreased Zn contents as well as by histopathological damages in ovarian tissues. The co-exposure of fish to Cd and Zn abolished ZnT1 down-regulation and rendered a persistently increased ZIP10 mRNA level. This treatment also decreased Cd and MTs accumulation, reversed Cd-induced Zn depletion and partially restored Cd-induced histological changes in ovarian tissues. These results imply that the downregulation of ZnT1 as well as the overexpression of ZIP10, in responses to the ovarian Zn depletion induced by Cd, play a major role in Cd accumulation and consequently in its toxicity. The protective effect of dietary Zn supplementation against Cd-induced toxicity is mediated, at least in part, by the increase of Zn availability and subsequently the induction of ZnT1 gene expression.

The toxicity potential of pharmaceuticals found in the Douro River estuary (Portugal): Assessing impacts on gonadal maturation with a histopathological and stereological study of zebrafish ovary and testis after sub-acute exposures

Concerns associated with pharmaceuticals in aquatic systems demand the establishment of links between xenobiotics and their respective concentrations and impacts on aquatic organisms. Herein, effects of non-steroidal pharmaceuticals in the gonadal maturation of zebrafish ( Danio rerio) were evaluated by histopathological and stereological analyses after 21 days of exposure. Carbamazepine, fenofibric acid, propranolol, sulfamethoxazole and trimethoprim were selected, considering their detection in the Douro estuary (Portugal). Exposures were performed with single compounds and mixtures, the exposure concentrations including environmental levels. Overall, quantitative analyses showed a decreasing trend for late maturation stages in male and female gametogenesis with parallel increases in immature gametes. In females, and at the highest concentration mixture, a significant switch between the volume densities of late/mature oocytes versus primary oocytes was observed. On the verge of statistical significance, oocyte atresia was higher in both mixtures (5.75 ± 4.02% for MXA and 5.65 ± 5.27% for MXB) versus control (2.21 ± 1.88%), in accordance with the histological identification of large atretic areas in some fish. Unlike females, males showed significant effects with single exposures. Spermatozoa in controls totalled 53.25 ± 7.13% of the testis volume, decreasing with carbamazepine (47.19 ± 5.30%), fenofibric acid (46.36 ± 4.30%), propranolol (37.22 ± 2.38%) and sulfametoxazole (39.37 ± 5.15%). An increase in spermatocyte percentage was noted with propranolol (40.13 ± 7.36%) and sulfametoxazole (40.84 ± 1.66%) versus control (30.93 ± 6.53%). The changes in maturation dynamics did not impact the gonadosomatic index. The results show that pharmaceuticals from various therapeutic classes can disrupt the maturation dynamics of fish ovaries and testes. Further studies are justified to tackle the underlying mechanisms and to gauge the full extent of effects/risks.

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) and Its Receptors in the Zebrafish Ovary: Evidence for Potentially Dual Roles of PACAP in Controlling Final Oocyte Maturation

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide originally purified from ovine hypothalamus for its potent activity to stimulate cAMP production. However, its presence and action have also been demonstrated in various peripheral tissues including the ovary. In the zebrafish, two forms of PACAP (PACAP(38)-1, adcyap1a; and PACAP(38)-2, adcyap1b) and three PACAP receptors (PAC(1)-R, adcyap1r1; VPAC(1)-R, vipr1; and VPAC(2)-R, vipr2) were all expressed in the ovary. Interestingly, although both follicle cells and oocytes express adcyap1b, the expression of adcyap1a was restricted to the oocytes only. Among the three receptors, adcyap1r1 and vipr2 were expressed in the oocytes, whereas the expression of vipr1 was exclusively located in the follicle cells. Temporal expression analysis of PACAP ligands and receptors during folliculogenesis suggested that PACAP might play differential roles in regulating follicle growth and maturation through different receptors. The two receptors that are expressed in the oocyte (adcyap1r1 and vipr2) showed a significant increase in expression at the transition from the primary growth (PG) stage to previtellogenic (PV) stage and their levels maintained high during follicle growth. However, when the follicle development approached full-grown (FG) stage, these two receptors both decreased significantly in expression. In contrast, vipr1, the receptor expressed in the follicle cells, showed little change in expression at the PG-PV transition and afterwards during follicle growth; however, its expression surged dramatically at the FG stage prior to oocyte maturation. Based on these results, we hypothesized that PACAP might play dual roles in regulating follicle growth and maturation through different receptors located in different compartments. PACAP may stimulate oocyte growth but block its maturation in early follicles by acting directly on the oocyte via PAC1-R and VPAC2-R, whose expression is dominant in growth phase; however, PACAP may promote oocyte maturation in the maturation phase via VPAC1-R on the follicle cells, whose expression surges in FG follicles prior to maturation and is consistently high in the follicles undergoing final maturation. This hypothesis was further supported by the observation that PACAP promoted maturation of follicle-enclosed oocytes but suppressed spontaneous maturation of denuded oocytes in vitro. This study provides strong evidence for a PACAP-mediated signaling network in the zebrafish ovarian follicle, which may play roles in orchestrating follicle growth and maturation via different types of receptors located in different compartments of the follicle.

Mechanisms underlying the protective effect of zinc and selenium against cadmium-induced oxidative stress in zebrafish Danio rerio

The present study was designed to elucidate the protective effect mechanism of Zinc (Zn) and Selenium (Se) on cadmium (Cd)-induced oxidative stress in zebrafish. For this purpose we investigate the response of oxidative stress markers, metallothionein accumulation and gene expression in liver and ovary of female zebrafish exposed to 0,4mg/l Cd in water and supplemented with Zn (5mg kg(-1)) and/or Se (2mg kg(-1)) for 21days in their diet. Liver and ovary Cd uptake was evaluated after the exposure period. Cd exposure significantly inhibited the antioxidant enzyme activities termed as catalase (CAT), superoxide dismutase (SOD) and glutathione peroxydase (GPx) and caused a pronounced malondialdehyde (MDA) accumulation in both organs. Co-administration of Zn and Se reversed the Cd-induced toxicity in liver and ovary measured as MDA accumulation. Interestingly, gene expression patterns of Cat, CuZnSod and Gpx were up-regulated when related enzymatic activities were altered. Zebrafish metallothionein transcripts (zMt) significantly decreased in tissues of fish supplemented with Zn and/or Se when compared to Cd-exposed fish. Our data would suggest that Zn and Se protective mechanism against Cd-induced oxidative stress is more depending on the correction of the proteins biological activities rather than on the transcriptional level of related genes.