Gonadotropin expression, pituitary and plasma levels in the reproductive cycle of wild and captive-reared greater amberjack (Seriola dumerili).

We compared the endocrine status of the pituitary-gonad axis of wild and captive-reared greater amberjack (Seriola dumerili) during the reproductive cycle (April - July), reporting on the expression and release of the two gonadotropins for the first time in the Mediterranean Sea. Ovaries from wild females were characterized histologically as DEVELOPING in early May and SPAWNING capable in late May-July, the latter having a 3 to 4-fold higher gonadosomatic index (GSI). SPAWNING capable wild females exhibited an increase in pituitary follicle stimulating hormone (Fsh) content, plasma testosterone (T) and 17,20ß-dihydroxy-4-pregnen-3-one (17,20ß-P), while almost a 10-fold increase was observed in pituitary luteinizing hormone (Lh) content. An increasing trend of plasma 17ß-estradiol (E2) was also recorded between the two reproductive stages in wild females. Captive-reared females sampled during the reproductive cycle exhibited two additional reproductive categories, with REGRESSED females having extensive follicular atresia and fish in the REGENERATING stage having only primary oocytes in their ovaries. Pituitary content of Fsh and Lh, fshb and lhb expression and plasma levels of Fsh and Lh remained unchanged among the four reproductive stages in captive females, in contrast with plasma E2 and T that decreased in the REGENERATING stage, and 17,20ß-P which increased after the DEVELOPING stage. In general, no significant hormonal differences were recorded between captive-reared and wild DEVELOPING females, in contrast to SPAWNING capable females, where pituitary Lh content, plasma Fsh and T were found to be lower in females in captivity. Overall, the captive females lagged behind in reproductive development compared to the wild ones and this was perhaps related to the multiple handling of the sea cages where all the sampled fish were maintained. Between wild males in the DEVELOPING and SPAWNING capable stages, pituitary Lh content, plasma T and 17,20ß-P, and GSI exhibited 3 to 4-fold increases, while an increasing trend of pituitary Fsh content, lhb expression levels and plasma 11-ketotestosterone (11-KT) was also observed, and an opposite trend was observed in plasma Lh. Captive males were allocated to one more category, with REGRESSED individuals having no spermatogenic capacity. During the SPAWNING capable phase, almost all measured parameters were lower in captive males compared to wild ones. More importantly, captive males showed significant differences from their wild counterparts throughout the reproductive season, starting already from the DEVELOPING stage. Therefore, it appears that captivity already exerted negative effects in males prior to the onset of the study and the multiple handling of the cage where sampled fish were reared. Overall, the present study demonstrated that female greater amberjack do undergo full vitellogenesis in captivity, albeit with some dysfunctions that may be related to the husbandry of the experiment, while males, on the other hand, may be more seriously affected by captivity even before the onset of the study.

The effect of algal turbidity on larval performance and the ontogeny of digestive enzymes in the grey mullet (Mugil cephalus).

A study comprised of two trials determined the effects of water turbidity produced by live microalgae and inert clay particles on the larval rearing of grey mullet (Mugil cephalus). Trial 1 evaluated the effect of microalgae produced water turbidity on grey mullet larval performance and digestive tract (DT) enzyme ontogeny. Two microalgae (Nannochloropsis oculata and Isochrysis galbana) water turbidity levels (0.76 and 1.20 NTU, respectively) and a non-microalgae control (0.26 NTU) were investigated on 2 to 23 dph grey mullet larvae. The higher turbidity (1.2 NTU) larvae (5 dph) consumed markedly (P < .05) more rotifers than other treatment fish, independently of the microalgae type. There was no clear effect of the turbidity treatments on DT enzyme ontogeny. However, in all treatments lipase and alkaline proteases appeared to be modulated by the diet. Alkaline phosphatase activity was ca. 8 times higher and α-amylase activity increased 5.3 times in 79 dph fish compared to 40 dph individuals. The ratio of alkaline phosphatase and leucine-alanine aminopeptidase indicated gut maturation occurred around 61 dph. Trial 2 compared the most effective N.occulata produced turbidity level (1.2 NTU) with the identical water turbidity produced by inert clay on larval performance. M. cephalus larvae exposed to high algal turbidity demonstrated superior performance (P < .05), in terms of rotifer ingestion, dry weight gain and survival, compared to cohorts reared under the clay treatment and the lower microalgae produced turbidity. These findings suggested that water algal turbidity is not the dominant factor determining improved grey mullet larval performance.

Comparative Study of Reproductive Development in Wild and Captive-Reared Greater Amberjack Seriola dumerili (Risso, 1810).

The greater amberjack Seriola dumerili is a large teleost fish with rapid growth and excellent flesh quality, whose domestication represents an ambitious challenge for aquaculture. The occurrence of reproductive dysfunctions in greater amberjack reared in captivity was investigated by comparing reproductive development of wild and captive-reared individuals. Wild and captive-reared breeders were sampled in the Mediterranean Sea during three different phases of the reproductive cycle: early gametogenesis (EARLY, late April-early May), advanced gametogenesis (ADVANCED, late May-early June) and spawning (SPAWNING, late June-July). Fish reproductive state was evaluated using the gonado-somatic index (GSI), histological analysis of the gonads and determination of sex steroid levels in the plasma, and correlated with leptin expression in the liver and gonad biochemical composition. The GSI and sex steroid levels were lower in captive-reared than in wild fish. During the ADVANCED period, when the wild greater amberjack breeders were already in spawning condition, ovaries of captive-reared breeders showed extensive atresia of late vitellogenic oocytes and spermatogenic activity ceased in the testes of half of the examined males. During the SPAWNING period, all captive-reared fish had regressed gonads, while wild breeders still displayed reproductive activity. Liver leptin expression and gonad proximate composition of wild and captive greater amberjack were similar. However, the gonads of captive-reared fish showed different total polar lipid contents, as well as specific lipid classes and fatty acid profiles with respect to wild individuals. This study underlines the need for an improvement in rearing technology for this species, which should include minimum handling during the reproductive season and the formulation of a specific diet to overcome the observed gonadal decrements of phospholipids, DHA (22:6n-3) and ARA (20:4n-6), compared to wild breeders.

The regulation of thermal stress induced apoptosis in corals reveals high similarities in gene expression and function to higher animals.

Recent studies suggest that controlled apoptotic response provides an essential mechanism, enabling corals to respond to global warming and ocean acidification. However, the molecules involved and their functions are still unclear. To better characterize the apoptotic response in basal metazoans, we studied the expression profiles of selected genes that encode for putative pro- and anti-apoptotic mediators in the coral Stylophora pistillata under thermal stress and bleaching conditions. Upon thermal stress, as attested by the elevation of the heat-shock protein gene HSP70's mRNA levels, the expression of all studied genes, including caspase, Bcl-2, Bax, APAF-1 and BI-1, peaked at 6-24 h of thermal stress (hts) and declined at 72 hts. Adversely, the expression levels of the survivin gene showed a shifted pattern, with elevation at 48-72 hts and a return to basal levels at 168 hts. Overall, we show the quantitative anti-apoptotic traits of the coral Bcl-2 protein, which resemble those of its mammalian counterpart. Altogether, our results highlight the similarities between apoptotic networks operating in simple metazoans and in higher animals and clearly demonstrate the activation of pro-cell survival regulators at early stages of the apoptotic response, contributing to the decline of apoptosis and the acclimation to chronic stress.

Breakdown of coral colonial form under reduced pH conditions is initiated in polyps and mediated through apoptosis.

Certain stony corals can alternate between a calcifying colonial form and noncalcifying solitary polyps, supporting the hypothesis that corals have survived through geologic timescale periods of unfavorable calcification conditions. However, the mechanisms enabling this biological plasticity are yet to be identified. Here we show that incubation of two coral species (Pocillopora damicornis and Oculina patagonica) under reduced pH conditions (pH 7.2) simulating past ocean acidification induce tissue-specific apoptosis that leads to the dissociation of polyps from coenosarcs. This in turn leads to the breakdown of the coenosarc and, as a consequence, to loss of coloniality. Our data show that apoptosis is initiated in the polyps and that once dissociation between polyp and coenosarc terminates, apoptosis subsides. After reexposure of the resulting solitary polyps to normal pH (pH 8.2), both coral species regenerated coenosarc tissues and resumed calcification. These results indicate that regulation of coloniality is under the control of the polyp, the basic modular unit of the colony. A mechanistic explanation for several key evolutionarily important phenomena that occurred throughout coral evolution is proposed, including mechanisms that permitted species to survive the third tier of mass extinctions.

Sexual maturity in western Atlantic bluefin tuna.

We introduce a novel endocrine approach for assessing the unresolved matter of the timing of sexual maturation in western Atlantic bluefin tuna (ABFT), a highly migratory population whose status remains uncertain. Ratios of follicle stimulating hormone to luteinizing hormone, a sexual maturity indicator, in all ABFT ≥ 134 cm curved fork length (CFL) were <0.4, similar to Mediterranean spawners, indicating that western ABFT mature at considerably smaller sizes and at a much younger age than currently assumed (≥ 185 cm CFL).

Urotensin II inhibits arginine vasotocin and stimulates isotocin release from nerve endings in the pituitary of gilthead sea bream (Sparus aurata).

The aim of this in vitro study was to determine whether arginine vasotocin (AVT) and isotocin (IT) release from nerve endings is affected by urotensin II (UII) in gilthead sea bream pituitary. Primary cultures of pituitary cells were exposed to 10(-12), 10(-10), and 10(-8) M UII for 6, 24, and 48 hr. AVT and IT contents were determined in the culture media by high performance liquid chromatography (HPLC). UII at all doses decreased AVT release after 6, 24, and 48 hr of incubation. IT release was increased by UII only after 24 hr of incubation. This study, for the first time, indicates that UII affects AVT and IT release from nerve endings in the pituitary of Sparus aurata. It is presumed that UII together with AVT and IT may control response to different salinities in fishes.

Trioecy, a unique breeding strategy in the sea anemone Aiptasia diaphana and its association with sex steroids.

Reproductive development of anthozoans reveals wide range of breeding strategies. Here, we report the occurrence of trioecy in the sea anemone Aiptasia diaphana (co-occurrence of males, females, and hermaphrodites), which so far was well documented only in plants. Age-homogeneous populations were obtained from pedal lacerates (asexual propagules) and cultured under control conditions. Careful documentation of growth, gamete morphology, and vertebrate-like steroid (i.e., progesterone, testosterone, and estradiol) levels were carried out over a 9-wk period between 4 and 12 wk postlaceration (wpl). First phenotypic signs of gametes development were observed in 6-wk-old anemones, pointing to the differentiation of males and hermaphrodites. While the males exhibited cellular progression of spermatogenesis, the hermaphrodites underwent a process of sex allocation, giving rise to male, female, and hermaphrodite phenotypes. Testosterone levels were relatively high prior to gamete appearance (4 wpl) and later on during gamete maturation (10 wpl). Conversely, estradiol levels steadily increased from 6 to 10 wpl, reaching their peak concomitant with oocyte maturation. Interestingly, increased oocyte atresia incidences were recorded during 9-12 wpl, coinciding with declining levels of steroid hormones. These results point to a strong similarity between the activity of sex steroids in vertebrates and that of vertebrate-like sex steroids on critical stages of A. diaphana's sexual differentiation and gametogenic cycle. The reproductive characteristics of A. diaphana make this anthozoan an important model species for the study of evolutionary drivers and processes underlying sexual development.

Intra-pituitary relationship of follicle stimulating hormone and luteinizing hormone during pubertal development in Atlantic bluefin tuna (Thunnus thynnus).

As part of the endeavor aiming at the domestication of Atlantic bluefin tuna (BFT; Thunnus thynnus), first sexual maturity in captivity was studied by documenting its occurrence and by characterizing the key hormones of the reproductive axis: follicle stimulating hormone (FSH) and luteinizing hormone (LH). The full length sequence encoding for the related hormone ß-subunits, bftFSHß and bftLHß, were determined, revealing two bftFSHß mRNA variants, differing in their 5' untranslated region. A quantitative immuno-dot-blot assay to measure pituitary FSH content in BFT was developed and validated enabling, for the first time in this species, data sets for both LH and FSH to be compared. The expression and accumulation patterns of LH in the pituitary showed a steady increase of this hormone, concomitant with fish age, reaching higher levels in adult females compared to males of the same age class. Conversely, the pituitary FSH levels were elevated only in 2Y and adult fish. The pituitary FSH to LH ratio was consistently higher (>1) in immature than in maturing or pubertal fish, resembling the situation in mammals. Nevertheless, the results suggest that a rise in the LH storage level above a minimum threshold may be an indicator of the onset of puberty in BFT females. The higher pituitary LH levels in adult females over males may further support this notion. In contrast three year-old (3Y) males were pubertal while cognate females were still immature. However, it is not yet clear whether the advanced puberty in the 3Y males was a general feature typifying wild BFT populations or was induced by the culture conditions. Future studies testing the effects of captivity and hormonal treatments on precocious maturity may allow for improved handling of this species in a controlled environment which would lead to more cost-efficient farming.

Reproductive characteristics and steroid levels in the scleractinian coral Oculina patagonica inhabiting contaminated sites along the Israeli Mediterranean coast.

In this study we compared reproductive characteristics and steroid hormone levels in the non-indigenous scleractinian coral, Oculina patagonica, inhabiting contaminated vs. uncontaminated reference sites along the Israeli Mediterranean coast. Our results indicate significantly higher steroid levels in both seawater and coral tissue samples from contaminated sites as compared to reference sites, suggesting that corals tend to accumulate steroids from the surrounding waters. Despite their higher steroid levels, corals from the contaminated sites showed reproductive potential comparable to those of the reference sites. Interestingly, a clear distinction could be seen between corals exposed to pollution for long vs. short durations, with the latter showing a failure to complete gametogenesis. This suggests that reproduction in O. patagonica is susceptible to acute rather than chronic stress. The involvement of adjustment/adaptation processes may explain this species tolerance, and may reflect the ability of O. patagonica to successfully invade new areas in the Mediterranean Sea.

Regulation of apoptotic pathways by Stylophora pistillata (Anthozoa, Pocilloporidae) to survive thermal stress and bleaching.

Elevated seawater temperatures are associated with coral bleaching events and related mortality. Nevertheless, some coral species are able to survive bleaching and recover. The apoptotic responses associated to this ability were studied over 3 years in the coral Stylophora pistillata from the Gulf of Eilat subjected to long term thermal stress. These include caspase activity and the expression profiles of the S. pistillata caspase and Bcl-2 genes (StyCasp and StyBcl-2-like) cloned in this study. In corals exposed to thermal stress (32 or 34°C), caspase activity and the expression levels of the StyBcl-2-like gene increased over time (6-48 h) and declined to basal levels within 72 h of thermal stress. Distinct transcript levels were obtained for the StyCasp gene, with stimulated expression from 6 to 48 h of 34°C thermal stress, coinciding with the onset of bleaching. Increased cell death was detected in situ only between 6 to 48 h of stress and was limited to the gastroderm. The bleached corals survived up to one month at 32°C, and recovered back symbionts when placed at 24°C. These results point to a two-stage response in corals that withstand thermal stress: (i) the onset of apoptosis, accompanied by rapid activation of anti-oxidant/anti-apoptotic mediators that block the progression of apoptosis to other cells and (ii) acclimatization of the coral to the chronic thermal stress alongside the completion of symbiosis breakdown. Accordingly, the coral's ability to rapidly curb apoptosis appears to be the most important trait affecting the coral's thermotolerance and survival.

Apoptosis and the selective survival of host animals following thermal bleaching in zooxanthellate corals.

During the past several decades, numerous reports from disparate geographical areas have documented an increased frequency of "bleaching" in reef-forming corals. The phenomenon, triggered by increased sea surface temperatures, occurs when the cnidarian hosts digest and/or expel their intracellular, photosynthetic dinoflagellate symbionts ("zooxanthellae" in the genus Symbiodinium). Although coral bleaching is often followed by the death of the animal hosts, in some cases, the animal survives and can be repopulated with viable zooxanthellae. The physiological factors determining the ability of the coral to survive bleaching events are poorly understood. In this study, we experimentally established that bleaching and death of the host animal involve a caspase-mediated apoptotic cascade induced by reactive oxygen species produced primarily by the algal symbionts. In addition, we demonstrate that, although some corals naturally suppress caspase activity and significantly reduce caspase concentration under high temperatures as a mechanism to prevent colony death from apoptosis, even sensitive corals can be prevented from dying by application of exogenous inhibitors of caspases. Our results indicate that variability in response to thermal stress in corals is determined by a four-element, combinatorial genetic matrix intrinsic to the specific symbiotic association. Based on our experimental data, we present a working model in which the phenotypic expression of this symbiont/host relationship places a selective pressure on the symbiotic association. The model predicts the survival of the host animals in which the caspase-mediated apoptotic cascade is down-regulated.

Sexual plasticity and self-fertilization in the sea anemone Aiptasia diaphana.

Traits that influence reproductive success and contribute to reproductive isolation in animal and plant populations are a central focus of evolutionary biology. In the present study we used an experimental approach to demonstrate the occurrence of environmental effects on sexual and asexual reproduction, and provide evidence for sexual plasticity and inter-clonal fertilization in laboratory-cultured lines of the sea anemone Aiptasia diaphana. We showed that in A. diaphana, both asexual reproduction by pedal laceration, and sexual reproduction have seasonal components. The rate of pedal laceration was ten-fold higher under summer photoperiod and water temperature conditions than under winter conditions. The onset of gametogenesis coincided with the rising water temperatures occurring in spring, and spawning occurred under parameters that emulated summer photoperiod and temperature conditions. In addition, we showed that under laboratory conditions, asexually produced clones derived from a single founder individual exhibit sexual plasticity, resulting in the development of both male and female individuals. Moreover, a single female founder produced not only males and females but also hermaphrodite individuals. We further demonstrated that A. diaphana can fertilize within and between clone lines, producing swimming planula larvae. These diverse reproductive strategies may explain the species success as invader of artificial marine substrates. We suggest that these diverse reproductive strategies, together with their unique evolutionary position, make Aiptasia diaphana an excellent model for studying the evolution of sex.

Enhancing spawning in the grey mullet (Mugil cephalus) by removal of dopaminergic inhibition.

A dot-blot immunoassay for the detection of vitellogenin (Vtg) in plasma of adult grey mullet (Mugil cephalus) was developed. The assay identified the sex of the tested fish prior to detectable gonadal development, enabling the establishment of broodstock at the desired ratio of 7:4 females to males. This broodstock was maintained under natural photoperiod, and used to study the relative effect of gonadotropin-releasing hormone (GnRH) and dopamine antagonists on oocyte maturation and ovulation, as well as the effect of 17alpha-methyltestosterone (MT) on spermiation. Three groups of females were treated with: (i) a single injection of dopamine antagonist, domperidone (Dom), (ii) GnRH analog (GnRHa) administered via ethylene-vinyl acetate copolymer (EVAc) slow-release implants or (iii) a combination of both Dom and GnRHa. Males were treated with MT, administered via EVAc slow-release implants. An additional group of untreated fish was used as a control. The Dom treatment proved to be more potent than the GnRHa treatment, and did not differ significantly from the combined treatment. The Dom and Dom+GnRHa treatments accelerated oocyte development and increased plasma estradiol levels equally, whereas the GnRHa treatment did not vary significantly from the control. MT was found to be a potent spermiating agent, which enhanced steady milt production in all treated males. In contrast, no spontaneous spermiation occurred in untreated males. Plasma 11-ketotestosterone (11-KT) levels were significantly higher in MT-treated males than in the controls. Interestingly, MT-treated males held with the GnRHa+Dom-treated females showed higher levels of plasma 11-KT than those held with GnRHa-treated females, indicating an additive effect which is probably attributable to female pheromones. Fully mature females were induced to spawn by injecting GnRHa alone or coupled with metaclopramide (a dopamine D2 receptors antagonist). The combined treatment, which included a dopamine antagonist, was found to be more potent in inducing ovulation and spawning as compared to GnRHa alone. In conclusion, our data suggest that dopaminergic inhibition is a major barrier along the reproductive axis that arrests spontaneous spawning in captive mullets.

Regulation of gonadotropin-releasing hormone (GnRH)-receptor gene expression in tilapia: effect of GnRH and dopamine.

The present work was designed to study certain aspects of the endocrine regulation of gonadotropin-releasing hormone receptor (GnRH-R) in the pituitary of the teleost fish tilapia. A GnRH-R was cloned from the pituitary of hybrid tilapia (taGnRH-R) and was identified as a typical seven-transmembrane receptor. Northern blot analysis revealed a single GnRH-R transcript in the pituitary of approximately 2.3 kilobases. The taGnRH-R mRNA levels were significantly higher in females than in males. Injection of the salmon GnRH analog (sGnRHa; 5-50 microg/kg) increased the steady-state levels of taGnRH-R mRNA, with the highest response recorded at 25 microg/kg and at 36 h. At the higher dose of sGnRHa (50 microg/kg), taGnRH-R transcript appeared to be down-regulated. Exposure of tilapia pituitary cells in culture to graded doses (0.1-100 nM) of seabream (sbGnRH = GnRH I), chicken II (cGnRH II), or salmon GnRH (sGnRH = GnRH III) resulted in a significant increase in taGnRH-R mRNA levels. The highest levels of both LH release and taGnRH-R mRNA levels were recorded after exposure to cGnRH II and the lowest after exposure to sbGnRH. The dopamine-agonist quinpirole suppressed LH release and mRNA levels of taGnRH-R, indicating an inhibitory effect on GnRH-R synthesis. Collectively, these data provide evidence that GnRH in tilapia can up- regulate, whereas dopamine down-regulates, taGnRH-R mRNA levels.

Regulation of fish gonadotropins.

Neurohormones similar to those of mammals are carried in fish by hypothalamic nerve fibers to regulate directly follicle-stimulating hormone (FSH) and luteinizing hormone (LH). Gonadotropin-releasing hormone (GnRH) stimulates the secretion of FSH and LH and the expression of the glycoprotein hormone alpha (GPalpha), FSHbeta, and LHbeta, as well as their secretion. Its signal transduction leading to LH release is similar to that in mammals although the involvement of cyclic AMP-protein kinase A (cAMP-PKA) cannot be ruled out. Dopamine (DA) acting through DA D2 type receptors may inhibit LH release, but not that of FSH, at sites distal to activation of protein kinase C (PKC) and PKA. GnRH increases the steady-state levels of GPalpha, LHbeta, and FSHbeta mRNAs. Pituitary adenylate cyclase-activating polypeptide (PACAP) 38 and neuropeptide Y (NPY) potentiate GnRH effect on gonadotropic cells, and also act directly on the pituitary cells. Whereas PACAP increases all three subunit mRNAs, NPY has no effect on that of FSHbeta. The effect of these peptides on the expression of the gonadotropin subunit genes is transduced differentially; GnRH regulates GPalpha and LHbeta via PKC-ERK and PKA-ERK cascades, while affecting the FSHbeta transcript through a PKA-dependent but ERK-independent cascade. The signals of both NPY and PACAP are transduced via PKC and PKA, each converging at the ERK level. NPY regulates only GPalpha- and LHbeta-subunit genes whereas PACAP regulates the FSHbeta subunit as well. Like those of the mammalian counterparts, the coho salmon LHbeta gene promoter is driven by a strong proximal tripartite element to which three different transcription factors bind. These include Sf-1 and Pitx-1 as in mammals, but the function of the Egr-1 appears to have been replaced by the estrogen receptor (ER). The GnRH responsive region in tilapia FSHbeta 5' flanking region spans the canonical AP1 and CRE motifs implicating both elements in conferring GnRH responsiveness. Generally, high levels of gonadal steroids are associated with high LHbeta transcript levels whereas those of FSHbeta are reduced when pituitary cells are exposed to high steroid levels. Gonadal or hypophyseal activin also participate in the regulation of FSHbeta and LHbeta mRNA levels. However, gonadal effects are dependent on the gender and stage of maturity of the fish.