The expression profiles of cyp19a1, sf-1, esrs and gths in the brain-pituitary during gonadal sex differentiation in juvenile Japanese eels.

Eels are gonochoristic species whose gonadal differentiation initiates at the yellow eel stage and is influenced by environmental factors. We revealed some sex-related genes were sex dimorphically expressed in gonads during gonadal sex differentiation of Japanese eel (Anguilla japonica); however, the expression of sex-related genes in the brain-pituitary during gonadal sex differentiation in eels is still unclear. This study aimed to investigate the sex-related gene expressions in the brain-pituitary and tried to clarify their roles in the brain and gonads during gonadal sex differentiation. Based on our previous histological study, the control eels developed as males, and estradiol-17ß (E2) was used for feminization. Our results showed that during testicular differentiation, the brain cyp19a1 transcripts and aromatase proteins were increased significantly; moreover, the cyp19a1, sf-1, foxl2s, and esrs (except gperb) transcripts in the midbrain/pituitary also were increased significantly. Forebrain gnrh1 transcripts increased slightly during gonadal differentiation of both sexes, but the gnrhr1b and gnrhr2 transcripts in the midbrain/pituitary were stable during gonadal differentiation. The expression levels of gths and gh in the midbrain/pituitary were significantly increased during testicular differentiation and were much higher in males than in E2-feminized females. These results implied that endogenous estrogens might play essential roles in the brain/pituitary during testicular differentiation, sf-1, foxl2s, and esrs may have roles in cyp19a1 regulation in the midbrain/pituitary of Japanese eels. For the GnRH-GTH axis, gths, especially fshb, may be regulated by esrs and involved in regulating testicular differentiation and development in Japanese eels.

Involvement of Transforming Growth Factor Beta Family Genes in Gonadal Differentiation in Japanese Eel, Anguilla japonica, According to Sex-Related Gene Expressions.

The gonochoristic feature with environmental sex determination that occurs during the yellow stage in the eel provides an interesting model to investigate the mechanisms of gonadal development. We previously studied various sex-related genes during gonadal sex differentiation in Japanese eels. In the present study, the members of transforming growth factor beta (TGF-ß) superfamily were investigated. Transcript levels of anti-Müllerian hormone, its receptor, gonadal soma-derived factor (amh, amhr2, and gsdf, respectively) measured by real-time polymerase chain reaction (qPCR) showed a strong sexual dimorphism. Transcripts were dominantly expressed in the testis, and their levels significantly increased with testicular differentiation. In contrast, the expressions of amh, amhr2, and gsdf transcripts were low in the ovary of E2-feminized female eels. In situ hybridization detected gsdf (but not amh) transcript signals in undifferentiated gonads. amh and gsdf signals were localized to Sertoli cells and had increased significantly with testicular differentiation. Weak gsdf and no amh signals were detected in early ovaries of E2-feminized female eels. Transcript levels of amh and gsdf (not amhr2) decreased during human chorionic gonadotropin (HCG)-induced spermatogenesis in males. This study suggests that amh, amhr2, and especially gsdf might be involved in the gene pathway regulating testicular differentiation of Japanese eels.

Elevated estradiol-17ß levels inhibit final oocyte maturation via G protein-coupled estrogen receptor (Gper) in yellowfin porgy, Acanthopagrus latus.

Yellowfin porgy a protandrous teleost, exhibits asynchronous oocyte development and multiple spawning. Seasonal profiles of plasma estradiol-17ß (E2) levels showed a peak in three-year-old females during the spawning season, when batches of fully-grown oocytes undergo final oocyte maturation (FOM). Because E2 has been shown to inhibit FOM via the G protein-coupled estrogen receptor (Gper) in several teleost species, we investigated the role of this "paradoxical" increase in E2 during FOM in yellowfin porgy. In vivo treatment with a GnRH-agonist stimulated germinal vesicle breakdown (GVBD) and increased E2 plasma levels, and ovarian cyp19a1a transcripts, confirming the increase in E2 production at the time of FOM. Ovarian transcripts of gper peaked at the time of FOM, indicating an increase in ovarian responsiveness to Gper-mediated E2 effects. In vitro, E2 and the Gper agonist, G-1, inhibited the stimulatory effect of maturation-inducing steroids (MIS) on GVBD, while an aromatase inhibitor enhanced the MIS effect, in agreement with a physiological inhibitory role of E2 on FOM via Gper. Immunohistological studies showed that the Gper protein was specifically located on the oocyte plasma membrane. Ovarian membranes displayed high-affinity and limited-capacity specific [3H]-E2 receptor binding which was displaced by G-1, characteristic of Gper. Expression of gper increased at the time of FOM in mid-vitellogenic oocytes, but not in larger oocytes undergoing GVBD. These results suggest increases in both E2 production and E2 responsiveness via Gper upregulation in mid-vitellogenic oocytes, may maintain meiotic arrest in this oocyte stage class during the period when full-grown oocytes are undergoing FOM. This study indicates a critical involvement of E2 in the control of asynchronous oocyte maturation and the multiple spawning pattern in Sparidae.

Basal teleosts provide new insights into the evolutionary history of teleost-duplicated aromatase.

Duplicated cyp19a1 genes (cyp19a1a encoding aromatase a and cyp19a1b encoding aromatase b) have been identified in an increasing number of teleost species. Cyp19a1a is mainly expressed in the gonads, while cyp19a1b is mainly expressed in the brain, specifically in radial glial cells, as largely investigated by Kah and collaborators. The third round of whole-genome duplication that specifically occurred in the teleost lineage (TWGD or 3R) is likely at the origin of the duplicated cyp19a1 paralogs. In contrast to the situation in other teleosts, our previous studies identified a single cyp19a1 in eels (Anguilla), which are representative species of a basal group of teleosts, Elopomorpha. In the present study, using genome data mining and phylogenetic and synteny analyses, we confirmed that the whole aromatase genomic region was duplicated in eels, with most aromatase-neighboring genes being conserved in duplicate in eels, as in other teleosts. These findings suggest that specific gene loss of one of the 3R-duplicated cyp19a1 paralogs occurred in Elopomorpha after TWGD. Similarly, a single cyp19a1 gene was found in the arowana, which is a representative species of another basal group of teleosts, Osteoglossomorpha. In eels, the single cyp19a1 is expressed in both the brain and the gonads, as observed for the single CYP19A1 gene present in other vertebrates. The results of phylogenetic, synteny, closest neighboring gene, and promoter structure analyses showed that the single cyp19a1 of the basal teleosts shared conserved properties with both teleost cyp19a1a and cyp19a1b paralogs, which did not allow us to conclude which of the 3R-duplicated paralogs (cyp19a1a or cyp19a1b) was lost in Elopomorpha. Elopomorpha and Osteoglossomorpha cyp19a1 genes exhibited preserved ancestral functions, including expression in both the gonad and brain. We propose that the subfunctionalization of the 3R-duplicated cyp19a1 paralogs expressed specifically in the gonad or brain occurred in Clupeocephala, after the split of Clupeocephala from Elopomorpha and Osteoglossomorpha, which represented a driving force for the conservation of both 3R-duplicated paralogs in all extant Clupeocephala. In contrast, the functional redundancy of the undifferentiated 3R-duplicated cyp19a1 paralogs in elopomorphs and osteoglossomorphs would have favored the loss of one 3R paralog in basal teleosts.

Dmrt1 (doublesex and mab-3-related transcription factor 1) expression during gonadal development and spermatogenesis in the Japanese eel.

Dmrt1, doublesex- and mab-3-related transcription factor-1, has been suggested to play critical roles in male gonadogenesis, testicular differentiation and development, including spermatogenesis, among different vertebrates. Vasa is a putative molecular marker of germ cells in vertebrates. In this study, we cloned the full-length dmrt1 cDNA from Japanese eel, and the protein comprised 290 amino acids and presented an extremely conserved Doublesex and Mab-3 (DM) domain. Vasa proteins were expressed in gonadal germ cells in a stage-specific manner, and were expressed at high levels in PGC and spermatogonia, low levels in spermatocytes, and were absent in spermatids and spermatozoa of Japanese eels. Dmrt1 proteins were abundantly expressed in spermatogonia B cells, spermatocytes, spermatids, but not in spermatozoa, spermatogonia A and Sertoli cells. To our knowledge, this study is the first to show a restricted expression pattern for the Dmrt1 protein in spermatogonia B cells, but not spermatogonia A cells, of teleosts. Therefore, Dmrt1 might play vital roles at the specific stages during spermatogenesis from spermatogonia B cells to spermatids in the Japanese eel. Moreover, the Dmrt1 protein exhibited a restricted localization in differentiating oogonia in the early differentiating gonad (ovary-like structure) of male Japanese eels and in E2-induced feminized Japanese eels. We proposed that dmrt1 may be not only required for spermatogenesis but might also play a role in oogenesis in the Japanese eel.

The germline-specific expression of Foxl3a and its paralogous Foxl3b are associated with male gonadal differentiation in the Japanese eel, Anguilla japonica.

Unlike its paralog Foxl2, which is well known for its role in ovarian development in vertebrates, the function of Foxl3 is still unclear. Foxl3 is an ancient duplicated copy of Foxl2. It is present as a single copy in ray-finned fish. But, due to repeated losses, it is absent in most tetrapods. Our transcriptomic data, however, show that two Foxl3s (Foxl3a and its paralog Foxl3b) are present in Japanese eel. Foxl3a is predominantly expressed in the pituitary, and Foxl3b is predominantly expressed in the gills. Both Foxl3s show a sex-dimorphic expression, being higher expression in testes than in ovaries. Moreover, Foxl3a and Foxl3b were exclusively expressed during gonadal differentiation in control eels (100% male). Conversely, Foxl3a and Foxl3b significantly decreased after gonadal differentiation in E2-treated eels (100% female). Furthermore, in accordance the difference in adhesive ability between somatic cells and germline cells in testes, Foxl3s showed a high expression in suspension cells (putative germline cells) and low expression in adhesive cells (putative somatic cells). In situ hybridization further showed that Foxl3a and Foxl3b were expressed in the testicular germline cells. In addition, Foxl3s expression was not changed by sex steroids in in vitro testes culture. Taken together, our results suggest that the teleost-specific Foxl3 paralog was repeatedly lost in most fish after the third round of whole genome duplication. The two germline-expressed Foxl3s had higher expression levels in males than in females during gonadal differentiation in Japanese eel. These results demonstrated that Foxl3s might play an important role in germline sexual fate determination from ancient fish to modern fish.

Gonadal development and expression of sex-specific genes during sex differentiation in the Japanese eel.

The process of gonadal development and mechanism involved in sex differentiation in eels are still unclear. The objectives were to investigate the gonadal development and expression pattern of sex-related genes during sex differentiation in the Japanese eel, Anguilla japonica. For control group, the elvers of 8-10cm were reared for 8months; and for feminization, estradiol-17ß (E2) was orally administered to the elvers of 8-10cm for 6months. Only males were found in the control group, suggesting a possible role of environmental factors in eel sex determination. In contrast, all differentiated eels in E2-treated group were female. Gonad histology revealed that control male eels seem to differentiate through an intersexual stage, while female eels (E2-treated) would differentiate directly from an undifferentiated gonad. Tissue distribution and sex-related genes expression during gonadal development were analyzed by qPCR. The vasa, figla and sox3 transcripts in gonads were significantly increased during sex differentiation. High vasa expression occurred in males; figla and sox3 were related to ovarian differentiation. The transcripts of dmrt1 and sox9a were significantly increased in males during testicular differentiation and development. The cyp19a1 transcripts were significantly increased in differentiating and differentiated gonads, but did not show a differential expression between the control and E2-treated eels. This suggests that cyp19a1 is involved both in testicular differentiation and development in control males, and in the early stage of ovarian differentiation in E2-treated eels. Importantly, these results also reveal that cyp19a1 is not a direct target for E2 during gonad differentiation in the eel.

Neuroendocrine gene expression reveals a decrease in dopamine D2B receptor with no changes in GnRH system during prepubertal metamorphosis of silvering in wild Japanese eel.

Silvering is a prepubertal metamorphosis preparing the eel to the oceanic reproductive migration. A moderate gonad development occurs during this metamorphosis from the sedentary yellow stage to the migratory silver stage. The aim of this study was to elucidate the molecular aspects of various endocrine parameters of BPG axis at different ovarian developmental stages in wild yellow and silver female Japanese eels. The GSI of the sampled female eels ranged between 0.18 and 2.3%, corresponding to yellow, pre-silver and silver stages. Gonad histology showed changes from previtellogenic oocytes in yellow eels to early vitellogenic oocytes in silver eels. Both serum E2 and T concentrations significantly increased with ovarian development indicating a significant activation of steroidogenesis during silvering. In agreement with previous studies, significant increases in pituitary gonadotropin beta subunits FSH-ß and LH-ß transcripts were also measured by qPCR, supporting that the activation of pituitary gonadotropin expression is likely responsible for the significant ovarian development observed during silvering. We investigated for the first time the possible brain neuroendocrine mechanisms involved in the activation of the pituitary gonadotropic function during silvering. By analyzing the expression of genes representative of the stimulatory GnRH control and the inhibitory dopaminergic control. The transcript levels of mGnRH and the three GnRH receptors did not change in the brain and pituitary between yellow and silver stages, suggesting that gene expression of the GnRH system is not significantly activated during silvering. The brain transcript levels of tyrosine hydroxylase, limiting enzyme of DA synthesis did not change during silvering, indicating that the DA synthesis activity was maintained. In contrast, a significant decrease in DA-D2B receptor expression in the forebrain and pituitary was observed, with no changes in DA-D2A receptor. The decrease in the pituitary expression of DA-D2BR during silvering would allow a reduced inhibitory effect of DA. We may raise the hypothesis that this regulation of D2BR gene expression is one of the neuroendocrine mechanisms involved in the slight activation of the pituitary gonadotropin and gonadal activity that occur at silvering.

Multiple kisspeptin receptors in early osteichthyans provide new insights into the evolution of this receptor family.

Deorphanization of GPR54 receptor a decade ago led to the characterization of the kisspeptin receptor (Kissr) in mammals and the discovery of its major role in the brain control of reproduction. While a single gene encodes for Kissr in eutherian mammals including human, other vertebrates present a variable number of Kissr genes, from none in birds, one or two in teleosts, to three in an amphibian, xenopus. In order to get more insight into the evolution of Kissr gene family, we investigated the presence of Kissr in osteichthyans of key-phylogenetical positions: the coelacanth, a representative of early sarcopterygians, the spotted gar, a non-teleost actinopterygian, and the European eel, a member of an early group of teleosts (elopomorphs). We report the occurrence of three Kissr for the first time in a teleost, the eel. As measured by quantitative RT-PCR, the three eel Kissr were differentially expressed in the brain-pituitary-gonadal axis, and differentially regulated in experimentally matured eels, as compared to prepubertal controls. Subfunctionalisation, as shown by these differences in tissue distribution and regulation, may have represented significant evolutionary constraints for the conservation of multiple Kissr paralogs in this species. Furthermore, we identified four Kissr in both coelacanth and spotted gar genomes, providing the first evidence for the presence of four Kissr in vertebrates. Phylogenetic and syntenic analyses supported the existence of four Kissr paralogs in osteichthyans and allowed to propose a clarified nomenclature of Kissr (Kissr-1 to -4) based on these paralogs. Syntenic analysis suggested that the four Kissr paralogs arose through the two rounds of whole genome duplication (1R and 2R) in early vertebrates, followed by multiple gene loss events in the actinopterygian and sarcopterygian lineages. Due to gene loss there was no impact of the teleost-specific whole genome duplication (3R) on the number of Kissr paralogs in current teleosts.

Expression of aromatase in radial glial cells in the brain of the Japanese eel provides insight into the evolution of the cyp191a gene in Actinopterygians.

The cyp19a1 gene that encodes aromatase, the only enzyme permitting conversion of C19 aromatizable androgens into estrogens, is present as a single copy in the genome of most vertebrate species, except in teleosts in which it has been duplicated. This study aimed at investigating the brain expression of a cyp19a1 gene expressed in both gonad and brain of Japanese eel, a basal teleost. By means of immunohistochemistry and in situ hybridization, we show that cyp19a1 is expressed only in radial glial cells of the brain and in pituitary cells. Treatments with salmon pituitary homogenates (female) or human chorionic gonadotrophin (male), known to turn on steroid production in immature eels, strongly stimulated cyp19a1 messenger and protein expression in radial glial cells and pituitary cells. Using double staining studies, we also showed that aromatase-expressing radial glial cells exhibit proliferative activity in both the brain and the pituitary. Altogether, these data indicate that brain and pituitary expression of Japanese eel cyp19a1 exhibits characteristics similar to those reported for the brain specific cyp19a1b gene in teleosts having duplicated cyp19a1 genes. This supports the hypothesis that, despite the fact that eels also underwent the teleost specific genome duplication, they have a single cyp19a1 expressed in both brain and gonad. Such data also suggest that the intriguing features of brain aromatase expression in teleost fishes were not gained after the whole genome duplication and may reflect properties of the cyp19a1 gene of ancestral Actinopterygians.

17,20ß,21-Trihydroxy-4-pregnen-3-one biosynthesis and 20ß-hydroxysteroid dehydrogenase expression during final oocyte maturation in the protandrous yellowfin porgy, Acanthopagrus latus.

The purpose of this study was to investigate the physiological maturation-inducing steroid (MIS) in the marine protandrous yellowfin porgy (Acanthopagrus latus). Female fish were injected with 2 doses of LHRH analog (10 and 40 µg per kg). Ovarian tissue was obtained at 6 h intervals for in vitro analysis of oocyte maturation. The most effective steroids for inducing in vitro maturation (germinal vesicle breakdown and GVBD) in cultured oocytes were 17,20ß-dihydroxy-4-pregnen-3-one (17,20ßP) and 17,20ß,21-trihydroxy-4-pregnen-3-one (20ß-S). 17,20ßP was less potent than 20ßS in inducing oocyte maturation. At higher concentrations, 11-deoxycortisol, 17α-hydroxy-progesterone, and 20ß-21-dihydroxy-4-pregnen-3-one also significantly induced oocyte maturation. A tritiated precursor [(3)H]-pregnenolone, was cultured in vitro together with the maturing ovarian tissue. The tritiated metabolites were purified and identified by solvent extraction, HPLC, TLC, acetylation reaction and recrystallization. HPLC, TLC and recrystallization analysis showed that significant levels of tritiated 11-deoxycortisol (a precursor of 20ß-S) and 20ß-S, but not 17,20ßP, were biosynthesized from [(3)H]-pregnenolone. Similar TLC profiles were obtained from the tritiated products that were isolated from the HPLC/TLC 20ß-S fraction and standard 20ß-S after the acetylation reaction. Constant specific radioactivity of tritiated 11-deoxycortisol and 20ß-S but not 17,20ßP by recrystallization was obtained in the tritiated metabolites isolated from HPLC and TLC fractions. The expression of 20ß-hydroxysteroid dehydrogenase (20ß-HSD) mRNA (a key enzyme that converts 11-deoxycortisol to 20ß-S) was significantly increased in maturing ovarian tissue. This study provides the first evidence that 20ß-S is converted from 11-deoxycortisol and is the possible MIS in yellowfin porgy.

Differential regulation of the expression of cytochrome P450 aromatase, estrogen and androgen receptor subtypes in the brain-pituitary-ovarian axis of the Japanese eel (Anguilla japonica) reveals steroid dependent and independent mechanisms.

This study aimed at investigating the role of sexual steroids in the regulation of the expression of the single aromatase gene and steroid receptor subtypes in the brain-pituitary-ovarian axis of the Japanese eel. Unlike other teleosts, which possess duplicated genes for aromatase, cyp19a1a and cyp19a1b, expressed in the gonads and in the brain, respectively, eel species possess a single cyp19a1. Phylogenetic analysis indicated that eel brain/gonadal cyp19a1 branches at the basis of both teleost gonadal cyp19a1a and brain cyp19a1b clades. Female eels treated with catfish pituitary homogenate (CPH) to induce sexual maturation showed an increase in the expression of cyp19a1 and aromatase enzymatic activity in the brain and in the ovaries. Treatments with sex steroids (estradiol-17ß, E(2) or testosterone, T) revealed that the increase in cyp19a1 expression in the brain may result from E(2)-specific induction. In contrast, the increase in cyp19a1 expression in the ovaries of CPH-treated eels is a result of steroid-independent control, probably from a direct effect of gonadotropins contained in the pituitary extract. Analysis of the expression of estrogen and androgen receptor subtypes, esr-α, esr-ß, ar-α and ar-ß, in eels treated with CPH or sex steroids revealed differential regulations. In CPH-treated eels, the expression of esr-α and ar-α was significantly increased in the brain, while the expression of ar-α and ar-ß was increased in the ovaries. No change was observed in esr-ß in any organ. Steroid treatments induced an upregulation by E(2) of esr-α, but not esr-ß expression, in the brain, pituitary and ovaries, while no autoregulation by T of its own receptors could be observed. These results reveal both steroid-dependent and -independent mechanisms in the regulation of cyp19a1 and steroid receptor subtype expression in the eel.

Pituitary gonadotropins FSH and LH are oppositely regulated by the activin/follistatin system in a basal teleost, the eel.

European eels are blocked at a prepubertal silver stage due to a deficient production of pituitary gonadotropins. We investigated the potential role of activin/follistatin system in the control of eel gonadotropins. Through the development of qPCR assays for European eel activin ß(B) and follistatin, we first analyzed the tissue distribution of the expression of these two genes. Both activin ß(B) and follistatin are expressed in the brain, pituitary and gonads. In addition, a striking expression of both transcripts was also found in the retina and in adipose tissue. The effects of recombinant human activins and follistatin on eel gonadotropin gene expression were studied using primary cultures of eel pituitary cells. Activins A and B strongly stimulated FSHß subunit expression in a time- and dose-dependent manner. In contrast, activin reduced LHß expression, an inhibitory effect which was highlighted in the presence of testosterone, a known activator of eel LHß expression. No effect of activin was observed on other pituitary hormones. Follistatin antagonized both the stimulatory and inhibitory effects of activin on FSHß and LHß expression, respectively. Activin is the first major stimulator of FSH expression evidenced in the eel. These results in a basal teleost further support the ancient origin and strong conservation of the activin/follistatin system in the control of FSH in vertebrates. In contrast, the opposite regulation of FSH and LH may have emerged in the teleost lineage.

Differential expression and regulation of gonadotropins and their receptors in the Japanese eel, Anguilla japonica.

Eel species have a striking life cycle with a blockade of puberty until the oceanic migration. We report the first molecular data on eel gonadotropin receptors. The partial sequences cloned covered two-third of the open reading frame and included most of the extracellular and transmembrane domains. Phylogenetic analysis partitioned the two eel gonadotropin receptors into the two teleost FSHR and LHR clusters, respectively. Real-time quantitative RT-PCR was used to quantify the expression of eel gonadotropins and their receptors. Similar levels of pituitary FSH-beta and LH-beta transcripts were found in the immature previtellogenic female eels. In contrast, ovarian FSHR mRNA level was at 100- to 185-fold higher than that of LHR. This revealed that FSHR rather LHR would mediate gonadotropin stimulation of the early stages of ovarian growth. Chronic treatment with fish pituitary homogenates, applied to induce eel sexual maturation, stimulated pituitary LH-beta but suppressed FSH-beta transcripts. In the ovaries, both FSHR and LHR mRNA were significantly increased in experimentally matured eels. Treatments with sexual steroids showed a stimulatory effect of estradiol-17beta (E(2)) on pituitary LH-beta mRNA levels, while FSH-beta transcripts were suppressed by E(2) or testosterone (T). In contrast, neither E(2) nor T-treatment had any significant effect on ovarian FSHR nor LHR transcripts. This suggests that steroid feedbacks may be responsible for the opposite regulation of pituitary gonadotropins in experimentally matured eels, but are not involved in the regulation of gonadotropin receptors. In conclusion, these are the first data on the sequence, expression and regulation of gonadotropin receptors in the eel. They provide new foundation for basic and applied research on eel reproduction.

An endothelial-cell-enriched primary culture system to study vascular endothelial growth factor (VEGF A) expression in a teleost, the Japanese eel (Anguilla japonica).

A partial gene for eel (Anguilla japonica) vascular endothelial growth factor (VEGF) has been cloned and an endothelial-cell-enriched primary culture derived from rete mirabile established to study regulation of the expression of the eel VEGF gene. Cells were cultured in M199 medium containing 0.1% fetal calf serum (FCS) and serum-free M199 medium for long-and short-term experiments, respectively. Cells were separately treated with cobalt ions (Co2+), basic fibroblast growth factor (bFGF), and estradiol (E2), which have been demonstrated to stimulate mammalian VEGF A expression, followed by quantification of the VEGF mRNA levels by real-time reverse transcription polymerase chain reaction. Our results show that: (1) the deduced eel VEGF protein encoded by the cloned gene is about 130 amino acids in length, and is closely related to a zebrafish (Danio rerio) VEGF A; (2) the endothelial-cell-enriched rete mirabile primary culture containing mainly (over 70%) the capillary endothelial cells; (3) the expression levels of the eel VEGF transcript were increased by Co2+, bFGF, and E2 treatments in a dose-and time-dependent manner. Our data demonstrate that an eel partial VEGF gene has been cloned and its regulation of expression in endothelial-cell-enriched rete mirabile cell culture is similar to that in higher vertebrates.

The presence and ancestral role of gonadotropin-releasing hormone in the reproduction of scleractinian coral, Euphyllia ancora.

The objectives of this study were to investigate the presence of immunoreactive GnRH (irGnRH) in scleractinian coral, Euphyllia ancora, study its seasonal variation, and evaluate its biological activity. irGnRH was detected and quantified in coral polyps. The biological activity of coral irGnRH was tested on pituitary cells from black porgy by evaluating its ability to stimulate LH release. Coral extracts (10(-9)-10(-5) M irGnRH) as well as mammalian (m) GnRH agonist (10(-10)-10(-6) M) had a similar dose-dependent effect on LH release. Furthermore, GnRH receptor antagonist dose-dependently inhibited the stimulation of LH release in response to coral extracts (10(-5) M irGnRH) and mGnRH agonist (10(-6) M). Peak levels of irGnRH (10-fold increase) were observed during the spawning period in a 3-yr investigation. Significantly higher aromatase activity and estradiol (E2) levels were also detected during the period of spawning compared with the nonreproductive season. In in vivo experiments, mGnRH agonist time- and dose-dependently stimulated aromatase activity as well as the concentrations of testosterone and E2 in free and glucuronided forms in coral. In conclusion, our data indicate that irGnRH does exist in coral, with its ability to stimulate LH release in fish. Seasonal variations of coral irGnRH, with a dramatic increase during the spawning period, concomitant to that in aromatase and E2, as well as the ability of mGnRH agonist to stimulate coral aromatase, steroidogenesis, and steroid glucuronization suggest that irGnRH plays an important role in the control of oocyte growth and mass spawning in corals.

Differential expression of neural and gonadal aromatase enzymatic activities in relation to gonadal development in Japanese eel, Anguilla japonica.

The objectives of the present study were to investigate the enzymatic characteristics of brain aromatase in Japanese eel, Anguilla japonica, as well as the correlations between aromatase activities in various tissues (brain regions, pituitary, and gonads) and ovarian development. Eel brain aromatase exhibited a K(m) of 75 nM and a V(max) of 1.14 fmol/min mg protein (91.5 fmol/h mg protein), indicating an enzymatic activity much lower than in other teleosts but similar to that in mammals. This supports the hypothesis of the occurrence of a single aromatase gene in eels (representative of an ancient group of teleosts, Elopomorphs), as in mammals, but unlike what is observed in more recent teleosts. Aromatase inhibitors could significantly suppress brain and pituitary aromatase activity. There was no significant sex difference in aromatase activity in the forebrain, midbrain, hindbrain, or pituitary, but there was in the gonads, where aromatase could be detected in the ovaries but not in the testes, in accordance with the role of this enzyme in ovarian differentiation. Comparison with another teleost, black porgy (Percomorph), under the same experimental conditions, further confirmed the low activity of aromatase in the eel. We investigated variations in brain, pituitary, and gonad aromatase activity in relation to ovarian development in control female eels (gonadosomatic index, GSI, 0.1-1.6%) as well as in eels treated with pituitary extract (experimental ovarian maturation; GSI up to 25%). Differential expression of neural and gonadal aromatase was observed in relation to the course of gonadal development. Pituitary aromatase activity increased with GSI at all stages. Brain (specially forebrain) aromatase activity significantly increased in early vitellogenic control eels (GSI>0.8%) and in treated eels. The low activity of eel aromatase may be related to the characteristic life cycle of the eel, in which there is a long delay of the onset of puberty before oceanic reproductive migration.

In vivo and in vitro sex steroids stimulate seabream gonadotropin-releasing hormone content and release in the protandrous black porgy, Acanthopagrus schlegeli.

The objective of the present study was to investigate the regulation of seabream gonadotropin-releasing hormone (sbGnRH) release using in vivo and in vitro approaches in the protandrous black porgy, Acanthopagrus schlegeli. Estradiol-17beta (E2), testosterone (T), and 11-ketotestosterone (11-KT) were found to significantly stimulate the increase of sbGnRH levels in pituitary of black porgy after 5-96 h of injection. An in vitro culture system using dispersed brain neurons was also developed to investigate the effects of various steroids on sbGnRH release. Different doses (10(-6) - 10(-12) M) of E2, T, 11-KT, and cortisol were applied during 6 h experiment. KCl stimulated sbGnRH release at a dose- and time-dependent manner. The concentration of sbGnRH increased 2-fold in the highest dose of KCl treatment compared to the control. Treatments with E2, T, 11-KT and cortisol significantly stimulated the release of sbGnRH from the cultured brain neurons. The concentration of sbGnRH in medium was increased by 2-, 1.9-, 2.1-, and 4.9-fold when treated with E2, T, 11-KT, and cortisol, respectively, as compared to the respective control. Cholesterol did not have any stimulatory effects in the release of sbGnRH. The results showed that sex steroids and cortisol had direct effect on brain neuronal cells stimulating the release of sbGnRH.