Gonadotropin regulation of follistatin expression in the cultured ovarian follicle cells of zebrafish, Danio rerio

Follistatin is a single-chain glycoprotein initially identified in the mammalian ovary. As a specific binding protein of activin, it effectively modifies the paracrine/autocrine roles of activin in a variety of tissues including the ovary. In the zebrafish, we have demonstrated that the human chorionic gonadotropin (hCG)-induced oocyte maturation and oocyte maturational competence can be blocked by follistatin, suggesting a role for ovarian activin in the signaling pathway of gonadotropin in the zebrafish ovary. The up-regulation of zebrafish ovarian activin betaA subunit by gonadotropin further supports this hypothesis. Since follistatin has extremely high affinity for activin, its expression level in various tissues is critical in fine-tuning local activin activities. In the present study, we investigated the regulation of follistatin expression by gonadotropin in a primary culture of zebrafish ovarian follicle cells using semi-quantitative RT-PCR. Both hCG and goldfish pituitary extract strongly increased the expression of follistatin in the cultured follicle cells in clear time- and dose-dependant manners. The effect of hCG (15IU/ml) reached the maximal level at 2h of treatment and longer treatment (4-8h) led to decreased response. The up-regulation of follistatin expression by hCG could be mimicked by all the drugs that increase the intracellular cAMP level including 8-Br-cAMP, db-cAMP, forskolin, and 3-isobutyl-1-methylxanthine (IBMX). The hCG (15IU/ml)- and forskolin (10microM)-induced follistatin expression could be blocked by H89 (10microM), a specific protein kinase A (PKA) inhibitor. These results strongly suggest that the regulation of follistatin expression in the zebrafish ovary by gonadotropin is primarily mediated by cAMP-PKA signaling pathway. The up-regulation of follistatin mRNA by gonadotropin in cultured zebrafish ovarian follicle cells, together with our previous studies on gonadotropin regulation of activin beta subunits, suggests that the ovarian activin-follistatin system is tightly controlled by gonadotropin in fish ovary.     

Involvement of cyclic adenosine 3',5'-monophosphate in the differential regulation of activin betaA and betaB expression by gonadotropin in the zebrafish ovarian follicle cells

Activin is a dimeric protein consisting of two similar but distinct beta-subunits, betaA and betaB. In our previous studies, both activin A (betaAbetaA) and activin B (betaBbetaB) have been demonstrated to stimulate oocyte maturation and promote oocyte maturational competence in the zebrafish. Follistatin, a specific activin-binding protein, can block both activin- and gonadotropin-induced final oocyte maturation in vitro, suggesting that activin is likely a downstream mediator of gonadotropin actions in the zebrafish ovary. In the present study, a full-length cDNA encoding zebrafish ovarian activin betaA was cloned and sequenced. The precursor of zebrafish activin betaA consists of 395 amino acids and its mature region exhibits about 78% homology with that of mammals. Using an in vitro primary culture of the ovarian follicle cells and semiquantitative RT-PCR assays, we examined the regulation of activin betaA and betaB expression by human chorionic gonadotropin (hCG) and its intracellular signal transduction mechanisms. hCG (15 IU/ml) increased the mRNA level of activin betaA-subunit; however, it significantly down-regulated the steady-state expression level of activin betaB in a time- and dose-dependent manner. The differential regulation of the two beta-subunits by hCG could be mimicked by 3-isobutyl-1-methylxanthine, forskolin, and dibutyryl-cAMP, suggesting involvement of the intracellular cAMP pathway. Interestingly, H89 (a specific inhibitor of protein kinase A, PKA) could effectively block hCG- and forskolin-stimulated activin betaA expression at 10 micro M, but it was unable to reverse the inhibitory effects of hCG and forskolin on betaB expression. This suggests that the hCG-stimulated activin betaA expression is dependent on the activation of the cAMP-PKA pathway, whereas the inhibitory effect of hCG on activin betaB expression is likely mediated by PKA-independent pathway(s).     

Myelopoiesis in the zebrafish, Danio rerio

Genome-wide chemical mutagenesis screens in the zebrafish (Danio rerio) have led to the identification of novel genes affecting vertebrate erythropoiesis. In determining if this approach could also be used to clarify the molecular genetics of myelopoiesis, it was found that the developmental hierarchy of myeloid precursors in the zebrafish kidney is similar to that in human bone marrow. Zebrafish neutrophils resembled human neutrophils, possessing segmented nuclei and myeloperoxidase-positive cytoplasmic granules. The zebrafish homologue of the human myeloperoxidase (MPO) gene, which is specific to cells of the neutrophil lineage, was cloned and used to synthesize antisense RNA probes for in situ hybridization analyses of zebrafish embryos. Granulocytic cells expressing zebrafish mpo were first evident at 18 hours after fertilization (hpf) in the posterior intermediate cell mass (ICM) and on the anterior yolk sac by 20 hpf. By 24 hpf, mpo-expressing cells were observed along the ICM and within the developing vascular system. Thus, the mpo gene should provide a useful molecular probe for identifying zebrafish mutants with defects in granulopoiesis. The expression of zebrafish homologues was also examined in 2 other mammalian hematopoietic genes, Pu.1, which appears to initiate a commitment step in normal mammalian myeloid development, and L-Plastin, a gene expressed by human monocytes and macrophages. The results demonstrate a high level of conservation of the spatio-temporal expression patterns of these genes between zebrafish and mammals. The morphologic and molecular genetic evidence presented here supports the zebrafish as an informative model system for the study of normal and aberrant human myelopoiesis. (Blood. 2001;98:643-651)     

Gonadotropin regulation of NGFI-B messenger ribonucleic acid expression during ovarian follicle development in the rat.

NGFI-B is an immediate-early gene that encodes an orphan nuclear receptor. The present study was designed to examine the localization and gonadotropin regulation of NGFI-B expression in the rat ovary. Northern blot analysis of ovaries obtained from prepubertal rats revealed the increased expression of NGFI-B during prepubertal development. Treatment of immature rats with PMSG, however, decreased ovarian NGFI-B expression. The major cell types expressing NGFI-B messenger RNA were thecal cells of follicles in different sizes. In contrast, treatment of PMSG-primed rats with human (h) CG resulted in the rapid and transient stimulation of ovarian NGFI-B messenger RNA, reaching a peak within 1 h. In situ hybridization analysis revealed that hCG treatment induced the expression of NGFI-B in granulosa cells of preovulatory follicles. Treatment of cultured preovulatory follicles in vitro with LH further confirmed the time- and dose-dependent stimulation of NGFI-B messenger RNA and protein. LH-stimulated NGFI-B expression in preovulatory follicles was abolished by alpha-amanitin, but was superinduced by cycloheximide. Furthermore, treatment of adult cycling rats with pentobarbital abolished NGFI-B expression on proestrus, and exogenous administration of hCG restored it, indicating the role of the preovulatory surge of LH in the stimulation of NGFI-B expression. These results demonstrate the cell type-specific expression and gonadotropin induction of NGFI-B in granulosa cells of preovulatory follicles and suggest a role for NGFI-B in the ovulatory process.     

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.     

Pharmacological characterization of a nociceptin receptor from zebrafish (Danio rerio)

The nociceptin receptor (NOP) and its endogenous ligand, nociceptin/orphanin FQ (OFQ), are involved in a wide range of biological functions, such as pain, anxiety, learning, and memory. The zebrafish has been proposed as a candidate to study the in vivo effects of several drugs of abuse and to discover new pharmacological targets. We report the cloning, expression, and pharmacological characterization of a NOP receptor from zebrafish (drNOP). The full-length cDNA codes a protein of 363 residues, which shows high sequence similarity to other NOPs. Phylogenetic analysis indicates that NOPs are broadly conserved during vertebrate evolution, and that they stand for the most divergent clade of the opioid/OFQ receptor family. Expression studies have revealed that drNOP mRNA is highly expressed in the central nervous system, and low expression levels are also found in peripheral tissues such as gills, muscle, and liver. Pharmacological analysis indicates that drNOP displays specific and saturable binding for [Leucyl-3,4,5-(3)H]nociceptin, with a K(d)=0.20 ± 0.02 nM and a B(max)=1703 ± 81 fmol/mg protein. [(3)H]Nociceptin binding is displaced by several opioid ligands such as dynorphin A (DYN A), naloxone, bremazocine, or the κ-selective antagonist nor-binaltorphimine. [(35)S]GTPγS stimulation studies showed that drNOP receptor is functional, as nociceptin is able to fully activate the receptor and DYN A behaves as a partial agonist (50% stimulation). Our results indicate that drNOP receptor displays mixed characteristics of both NOP and κ opioid receptors. Hence, drNOP, which has retained more of the likely ancestral features, bridges the gap between nociceptin and opiate pharmacology.     

Identification and characterization of 17 beta-hydroxysteroid dehydrogenases in the zebrafish, Danio rerio

The 17 beta-hydroxysteroid dehydrogenases (17 beta-HSDs) are key enzymes in the final steps of steroid hormone synthesis. 17beta-HSD type 1 (HSD17B1) catalyzes the reduction of estrone to estradiol, while type 3 (HSD17B3) performs the conversion of androstenedione to testosterone. Here we present a functional genomics study of putative candidates of these enzymes in the zebrafish. By an in silico screen of zebrafish EST databases we identified three candidate homologs for both HSD17B1 and HSD17B3. Phylogenetic analysis, unique expression patterns (RT-PCR) during embryogenesis and adulthood, as well as activity measurements revealed that one of the HSD17B1 candidates is the zebrafish homolog, while the other two are paralogous photoreceptor-associated retinol dehydrogenases. All three HSD17B3 candidate genes showed nearly identical, ubiquitous expressions in embryogenesis and adult tissues and were identified to be paralogs of HSD17B12 and a yet uncharacterized putative steroid dehydrogenase. Phylogenetic analysis shows that HSD17B3 and HSD17B12 are descendants from a common ancestor.     

A soluble activin type IIA receptor induces bone formation and improves skeletal integrity

Diseases that affect the regulation of bone turnover can lead to skeletal fragility and increased fracture risk. Members of the TGF-beta superfamily have been shown to be involved in the regulation of bone mass. Activin A, a TGF-beta signaling ligand, is present at high levels in bone and may play a role in the regulation of bone metabolism. Here we demonstrate that pharmacological blockade of ligand signaling through the high affinity receptor for activin, type II activin receptor (ActRIIA), by administration of the soluble extracellular domain of ActRIIA fused to a murine IgG2a-Fc, increases bone formation, bone mass, and bone strength in normal mice and in ovariectomized mice with established bone loss. These observations support the development of this pharmacological strategy for the treatment of diseases with skeletal fragility.     

Cloning of a second form of activin-betaA cDNA and regulation of activin-betaA subunits and activin type II receptor mRNA expression by gonadotropin in the zebrafish ovary

Activins are dimeric proteins consisting of two inhibin beta subunits. Homo- and hetero-dimerizations of two isoforms of beta subunits, betaA and betaB, produce three forms of activins, activin-A, -B, and -AB. Recent studies have suggested that activin-A mediates gonadotropin-induced oocyte maturation in the zebrafish. To further understand the physiological role of activin-A in the zebrafish ovary, we have cloned cDNAs for a second isoform of the activin-betaA subunit and the activin type IIA (ActRIIA) receptor and determined their regulation by gonadotropin. Two sequences were obtained during the cloning of activin-betaA subunit, both of which showed high identity to betaA subunits of other species, and were therefore designated as isoform 1 and 2. Real-time PCR quantification was used to measure mRNA levels of activin-betaA1 and -betaA2, as well as two type II receptors, ActRIIA and ActRIIB, in the zebrafish ovary. Activin-betaA1 mRNA levels in stages III and IV follicles were similar and higher than those in stage II while high activin-betaA2 mRNA levels were only found in stage IV follicles. Highest levels of mRNA expression were detected in small and large stage III follicles for ActRIIA and ActRIIB, respectively. Treatment with human chorionic gonadotropin induced dose- and time-dependent increases in mRNA levels of activin-betaA1 and -betaA, as well as ActRIIA and ActRIIB. These findings further support the involvement of the activin signaling cascade in gonadotropin-regulated gonadal activities.     

Characterization of the heat shock response in mature zebrafish (Danio rerio)

Heat shock proteins (Hsps) are involved in many physiological and pathological processes and are diminished with age in a variety of species. As zebrafish embryos have proven to be excellent models for studying Hsp response during development, we sought to characterize the response in mature zebrafish to demonstrate the utility of the zebrafish model in studying late-life diseases and the biology of aging. Accordingly, mature zebrafish were exposed to a 37 degrees C heat stress and mRNA was isolated from various tissues and subjected to analysis by RT-PCR. We found that Hsp70 was upregulated in brain, liver, and muscle, while Hsp47 was upregulated in brain, but not liver or muscle. Hsp90alpha, Hsp90beta, and heat shock factor 1a (Hsf1a) were expressed in all three tissues, but were not upregulated in response to heat stress. A comparison of Hsp expression in young versus mature zebrafish revealed decreased basal levels of Hsp70 and increased levels of Hsf1a in mature fish. These results indicate that the heat shock response is detectable in mature zebrafish and that there are age differences in their heat shock response, suggesting that mature zebrafish may be a useful model for studying Hsp response during the aging process.     

Determination of lymphatic vascular identity and developmental timecourse in zebrafish (Danio rerio)

Zebrafish lymphatics have been shown to share a number of characteristics with their human counterparts, making the fish a potentially useful model for studying lymphatic development and disease. The utility of the zebrafish lymphatic model would be substantially enhanced by an improved understanding of the spatiotemporal development of the primary lymphatic vasculature. The goal of this project is to identify and map the major zebrafish lymphatic structures throughout embryonic to early juvenile stages of development. Two transgenic lines, kdr-1:RASmCherryxfli1:GFP and stabilin1:YFP, were recently derived to assist in the study of developing lymphatic vasculature, but their specificity has not been rigorously tested. In the course of the present study, we experimentally validate the utility of these two marker lines as potential tools for establishing lymphatic vascular identity and visualizing developmental lymphangiogenesis. We introduced twenty nanometer red florescent microspheres into the blood vasculature of flil:GFP zebrafish and collected tiled optical z-sections at time intervals spanning early development. Three-dimensional reconstructions of the vasculature were used to differentiate between blood and lymphatic vessels. Age-matched injected embryos were compared to the two transgenic lines to further assess their specificity. We created a spatiotemporal map of the major lymphatic vessels in the developing zebrafish including a previously unidentified lymphatic vessel in the gastrointestinal tract. We conclude that the kdr-1:RASmCherryxfli1:GFP line accurately identifies developing lymphatic vessels with the exception of those associated with the gastrointestinal tract. The stabilin1:YFP line, however, is less reliable, as it marks both venous vessels and lymphatic vessels.     

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; 0.57-0.65 mm) and mid-vitellogenic (MV; 0.45-0.56 mm) 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.     

Hormonal regulation of proprotein convertase subtilisin/kexin type 5 expression during ovarian follicle development in the rat

The proprotein convertase subtilisin/kexin (PCSKs), a family of subtilisin-like proteases, is the processing enzymes for the activation of many hormone precursors. The present study was designed to identify the PCSK isoform expressed in the ovary and to examine its expression in gonadotropin-stimulated rat ovary. Northern blot analysis of ovaries obtained from prepubertal rats revealed an increased expression of Pcsk5 messenger RNA (mRNA) during development with the highest levels at 21 days of age. Treatment of immature rats with PMSG further increased ovarian Pcsk5 expression, and in situ hybridization analysis revealed the localization of Pcsk5 mRNA in theca-interstitial cells of follicles in different sizes. Interestingly, treatment of PMSG-primed rats with hCG resulted in a transient stimulation of ovarian Pcsk5 mRNA levels within 3-6 h. In addition to theca-interstitial cells, hCG treatment induced the expression of Pcsk5 in granulosa cells of preovulatory follicles. Pcsk1, 2 and 4 mRNAs were not detected whereas Pcsk7 mRNA was slightly expressed. Injection of a progestin antagonist RU486 or an inhibitor of 3beta-hydroxysteroid dehydrogenase epostane at 1h before hCG treatment inhibited hCG-induced Pcsk5 mRNA levels. Treatment with LH stimulated both Pcsk5 mRNA and protein levels in preovulatory follicles cultured in vitro. In addition, forskolin but not TPA stimulated Pcsk5 mRNA levels. RNase protection assay revealed that the soluble Pcsk5A variant was the predominant form stimulated by gonadotropins in the ovary. Finally, the predicted proprotein substrates cleaved by PCSK5 were analyzed in preovulatory follicles using regular expressions. The present study demonstrates PCSK5A as the gonadotropin-regulated PCSK isoform in the ovary, and its possible contribution to ovulation by processing pro-TGFbeta and matrix metalloproteinase family.     

Zebrafish as a model for vertebrate reproduction: characterization of the first functional zebrafish (Danio rerio) gonadotropin receptor

Vertebrate reproduction is tightly regulated by conserved glycoprotein hormones produced by the pituitary gland. Follicle-stimulating hormone (FSH) in tetrapods and gonadotropic hormone I (GTH-I) in fishes are orthologous glycoprotein hormones that control the timing of egg production and the number of eggs produced. Zebrafish, a well-established genetic model for developmental biology, also offers potential advantages for studies of reproductive toxicology, especially for modeling the impact of pollutants on fish reproductive processes. To facilitate these studies we have identified, expressed, and characterized the zebrafish GTH-I receptor. This receptor (zfGTHR-I)exhibits strong sequence similarity to the tetrapod FSH receptors and to GTHR-I from salmon and catfish. Human 293 cells transfected with zfGTHR-I exhibit increased cAMP levels after treatment with carp pituitary extracts or human FSH, but not when treated with a ligand to a related receptor (human chorionic gonadotropin). Northern blotting and RT-PCR analyses indicate that zfGTHR is expressed in ovaries from sexually mature fish, but not in immature fish. Several alternative splice variants of the receptor affecting putative exons 2-4 that encode dramatically shortened receptor fragments lacking the transmembrane domain as well as regions previously implicated in ligand binding were identified by RT-PCR. The zfGTHR-I sequence opens the way to study effects of genetic mutations or chemicals on ovarian zfGTHR-I expression and function in zebrafish.