Hanna Rosenfeld

Regulation of Gonadotropin-Releasing Hormone (GnRH)-Receptor Gene Expression in Tilapia: Effect of GnRH and Dopamine

Abstract 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 μg/kg) increased the steady-state levels of taGnRH-R mRNA, with the highest response recorded at 25 μg/kg and at 36 h. At the higher dose of sGnRHa (50 μg/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 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 corals ability to rapidly curb apoptosis appears to be the most important trait affecting the corals thermotolerance and survival.

Reproductive development of male and female tilapia hybrids (Oreochromis niloticus × O. aureus) and changes in mRNA levels of gonadotropin (GtH) Iβ and IIβ subunits.

A study was carried out in tilapia in order to see whether the gonadotropin (GtH) β subunits show distinct patterns of expression at different stages of their reproductive development. Male and female tilapia hybrids (Oreochromis niloticus × O. aureus) were collected at various times of the year, and a number of parameters were measured in order to establish the reproductive state of the fish. Circulating testosterone (T), estradiol (E2) and 11 ketotestosterone (11KT) levels were assayed, gonads were removed for calculation of gonadosomatic index (GSI) values and histological studies, and RNA was extracted from the pituitaries for measurement of GtH Iβ and IIβ mRNA levels. In maturing fish of both sexes, the circulating steroid levels were positively correlated with each other (r2 = 0.66–0.91) and in males, also with the GSI values (r2 = 0.68). A positive correlation was also seen in these fish between GSI values and the prevalence of spermatocytes and spermatids (r2 = 0.54). In maturing females, the maximal oocyte diameter was positively correlated with circulating E2 levels (r2 = 0.63), while GSI values showed no correlation; this presumably relates to the cycling nature of this asynchronous spawner. In regressing fish of both sexes, no clear correlation between these reproductive parameters was seen. In all fish, the GtH Iβ mRNA levels were highest in fish with steroids ranging 1–10 ng T or E2/ml for males or females, respectively, and were lower in fish with steroids at higher or lower levels. In fish with high steroid levels, the IIβ mRNA levels were also high, and in regressed males the increases were positively correlated. Exposure of cultured pituitary cells to either steroid (T at >10 nM, or E2 at >1 nM) was followed by a decrease in the steady-state levels of the Iβ transcript, while those of IIβ were left unaltered. In situ hybridization studies revealed that in pituitaries of both sexes, the cells producing each of these mRNAs are located in a distinct location. These results suggest that gonadal steroids may exert differential feedback mechanisms at the level of the pituitary to control transcription of each GtH β subunit in distinct cell types specific for each hormone. J. Exp. Zool. 286:64–75, 2000.

Characterization of tilapia FSHβ gene and analysis of its 5′ flanking region

Abstract The objective of the current study was to unveil molecular mechanisms underlying transcriptional regulation of the FSHβ gene expression in the pituitary of tilapia (Oreochromis mossambicus). The full-length sequence of tilapia FSHβ (tFSHβ) gene was determined. Its transcriptional unit (2.7 kb) exhibits the conserved genomic organization, i.e. three exons and two introns. Primer extension and RT-PCR analysis revealed heterogeneity of the tFSHβ transcripts, due to alternate mRNA splicing and multiple initiation sites for transcription. Examination of the 5′ flanking region (5′FR) of the tFSHβ gene identified potential CAAT and TATA promoter proximal elements as well as several sequences of cis-acting motifs known to dictate inducible and tissue-specific transcriptional regulation in other gonadotropin genes. Chimeric constructs containing 1.7 kb of the tFSHβ 5′FR fused to a luciferase (LUC) reporter gene were transiently transfected into primary culture of tilapia pituitary cells. The tFSHβ-LUC construct was efficiently expressed under basal conditions and was rapidly induced by GnRH stimulation. Our data indicate that the 5′FR contains a functional promoter, which is responsive to GnRH treatment. In addition, 5′ deletion analysis showed that the 1.7 kb, DNA sequence of the FSHβ 5′FR encompasses both positive and negative regulatory elements.

GnRHa-mediated stimulation of the reproductive endocrine axis in captive Atlantic bluefin tuna, Thunnus thynnus.

A controlled-release implant loaded with GnRH agonist (GnRHa) was used to induce spawning in Atlantic bluefin tuna (Thunnus thynnus) during two consecutive reproductive seasons. The fish were implanted underwater and sampled between days 2 and 8 after treatment. At the time of GnRHa treatment, females were in full vitellogenesis and males in spermiation. There was a rapid burst of pituitary luteinizing hormone (LH) release at day 2 after treatment in GnRHa-treated fish, and circulating LH remained elevated up to day 8 after treatment. In contrast, control fish had significantly lower levels in the plasma, but higher LH content in the pituitary, as observed in many other cultured fishes that fail to undergo oocyte maturation, ovulation and spawning unless induced by an exogenous GnRHa. Plasma testosterone (T) and 17β-estradiol (E(2)) were elevated in response to the GnRHa treatment in females, while 11-ketotestosterone (11-KT) but not T was elevated in males. Even though oocyte maturation and ovulation did occur in GnRHa-induced fish, no significant elevations in 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) or 17,20β,21-trihydroxy-4-pregnen-3-one (20β-S), in either the free, conjugated or 5β-reduced,3α-hydroxylated forms was observed in fish sampled within 6 days after treatment. Interestingly, a significant peak in plasma free 17,20β-P levels occurred in both males and females at day 8 after treatment. Histological sections of the ovaries in these females contained oocytes at the migrating germinal vesicle stage, suggesting the role of this hormone as a maturation-inducing steroid in Atlantic bluefin tuna. In conclusion, the GnRHa implants activated effectively the reproductive endocrine axis in captive Atlantic bluefin tuna broodstocks, through stimulation of sustained elevations in plasma LH, which in turn evoked the synthesis and secretion of the relevant sex steroids leading to gamete maturation and release.

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

Significance Stony corals have survived through geologic timescale periods of unfavorable calcification conditions, including low-pH regimes, possibly by alternating between a calcifying colonial form and noncalcifying soft body solitary polyps. Although experimentally supported, the mechanisms enabling this biological plasticity are unidentified. We show that incubating two coral species under reduced pH conditions induced tissue-specific apoptosis that leads to loss of coloniality because of the dissociation of polyps (the basic modular units of the colony) from their connective tissue (coenosarc). Apoptosis was initiated in the polyps and subsided once the dissociation between polyps and coenosarc terminated. When conditions returned to normal, these solitary polyps initiated calcification and regenerated coenosarcs. These results indicate that regulation of coloniality is under the control of the polyp. 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 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.

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.

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).

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.