Expression pattern, cellular localization and promoter activity analysis of ovarian aromatase (Cyp19a1a) in protogynous hermaphrodite red-spotted grouper

Aromatase plays a key role in sex differentiation of gonads. In this study, we cloned the full-length cDNA of ovarian aromatase from protogynous hermaphrodite red-spotted grouper (Epinephelus akaara), and prepared the corresponding anti-EaCyp19a1a antiserum. Western blot and immunofluorescence studies revealed ovary-specific expression pattern of EaCyp19a1a in adults and its dynamic expression change during artificial sex reversal. EaCyp19a1a was expressed by follicular cells of follicular layer around oocytes because strong EaCyp19a1a immunofluorescence was observed in the cells of ovaries. During artificial sex reversal, EaCyp19a1a expression dropped significantly from female to male, and almost no any positive EaCyp19a1a signal was observed in testicular tissues. Then, we cloned and sequenced a total of 1967 bp T-flanking sequence of EaCyp19a1a promoter, and showed a number of potential binding sites for some transcriptional factors, such as SOX5, GATA gene family, CREB, AP1, FOXL1, C/EBP, ARE and SF-1. Moreover, we prepared a series of 5' deletion promoter constructs and performed in vitro luciferase assays of EaCyp19a1a promoter activities. The data indicated that the CREB regulation region from -1010 to -898 might be a major cis-acting element to EaCyp19a1a promoter, whereas the elements GATA and SOX5 in the region from -1216 to -1010 might be suppression elements. Significantly, we found a common conserved sequence region in the fish ovary-type aromatase promoters with identities from 93% to 34%. And, the motifs of TATA box, SF-1, SOX5, and CREB existed in the region and were conserved among the most of fish species. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Involvement of Fas/FasL system in apoptotic signaling in testicular germ cells of male Wistar rats injected i.v. with microcystins

Previous studies have shown that gonads were the second target organ of microcystins (MCs), and that MCs exposure exerted obvious toxic effects on male reproductive system of mammals. However, relevant molecular evidences are still lacking. Fas-signaling pathway plays a key role in toxicant-induced germ cell apoptosis. This study was to evaluate the responses of Fas/FasL system related genes and proteins in testes of rats injected intravenously with MCs. Enhanced apoptosis of germ cells in the testes of MCs-treated rats was detected by the terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end labeling (TUNEL) associated with up-regulation of the Fas/FasL system. Both Fas and FasL protein expression were induced evidently from I h post-injection, and this high expression level maintained throughout the experiment. In addition, the activation of caspase-8 and caspase-3 protein was also observed, which were indicators of apoptosis. These results suggested the likely involvement of Fas/FasL system in the MCs-induced germ cell apoptosis. It is also suggested that MCs can cause damage to Sertoli cells directly. (C) 2009 Elsevier Ltd. All rights reserved.

Distribution and depuration of the potentially carcinogenic malachite green in tissues of three freshwater farmed Chinese fish with different food habits

The use of malachite green (MG) in fish farming is prohibited in China due to its potentially toxicological and carcinogenic nature, but it is still illegally used in some places. Uptake, accumulation and deputation of MG in various tissues were studied under laboratory conditions in three common freshwater fish, Parabramis pekinensis (plant-eating fish), Carassius auratus (omnivorous fish) and Ophiocephalus argus (carnivorous fish). The concentrations of MG and its primary metabolite, the reduced and colorless leucomalachite green (LMG), were analyzed by liquid chromatography-mass spectrometry (LC-MS2). Absorption of MG occurred during the waterborne exposure and the MG concentrations in gills of the three fish species all showed a maximum at 0 h after an acute water exposure (6 mg l(-1) MG for 20 min). Afterwards, both MG and LMG declined very rapidly in the blood of the fish. Levels of MG and LMG were still above 0.002 mu g g(-1) in fresh weight muscle at 240 h and may persist for as long as 10 days. Most MG was converted rapidly to LMG in the fish and deputation of LMG was very slow in fat tissue. skin and gonads of the fish. Distribution of LMG was strongly dependent on the fat content in the tissues of the fish, but not related to their different feeding habits. Therefore, it appears that fat tissue, skin and gonads of the fish contaminated by MG and LMG pose the greatest risk for human consumption. (C) 2008 Published by Elsevier B.V.

Chronic effects of water-borne PFOS exposure on growth, survival and hepatotoxicity in zebrafish: A partial life-cycle test

Perfluorooctane sulfonate (PFOS) is widely distributed and persistent in the environment and wildlife. The main aim of this study was to investigate the impact of long-term exposure to low concentrations of PFOS in zebrafish. Zebrafish fry (F-0, 14d post-fertilization, dpf) were exposed via the water for 70d to 0 (control), 10, 50 and 250 mu g L-1 PFOS, followed by a further 30d to assess recovery in clean water. The effects on survival and growth parameters and liver histopathology were assessed. Although growth suppression (weight and length) was observed in fish treated with high concentrations PFOS during the exposure period, no mortality was observed throughout the 70d experiment. Embryos and larvae (F-1) derived from maternal exposure suffered malformation and mortality. Exposure to 50 and 250 mu g L-1 PFOS could inhibit the growth of the gonads (GSI) in the female zebrafish. Histopathological alterations, primary with lipid droplets accumulation, were most prominently seen in the liver of males and the changes were not reversible, even after the fish were allowed to recover for 30d in clean water. The triiodothyronine (T-3)) levels were not significantly changed in any of the exposure groups. Hepatic vitellogenin (VTG) gene expression was significantly up-regulated in both male and female zebrafish, but the sex ratio was not altered. The overall results suggested that lower concentrations of PFOS in maternal exposure could result in offspring deformation and mortality. (c) 2008 Elsevier Ltd. All rights reserved.

Molecular cloning and expression characterization of ApoC-I in the orange-spotted grouper

Endogenous yolk nutrients are crucial for embryo and larval development in fish, but developmental behavior of the genes that control yolk utilization remains unknown. Apolipoproteins have been shown to play important roles in lipid transport and uptake through the circulation system. In this study, EcApoC-I, the first cloned ApoC-I in teleosts, has been screened from pituitary cDNA library of female orange-spotted grouper (Epinephelus coioides), and the deduced amino acid sequence shows 43.5% identity to one zebrafish (Danio rerio) hypothetical protein similar to ApoC-I, and 21.2%, 21.7%, 22.5%, 20%, and 22.5% identities to Apo C-I of human (Homo sapiens), house mouse (Mus musculus), common tree shrew (Tupaia glis), dog (Canis lupus familiaris) and hamadryas baboon (Papio hamadryas), respectively. Although the sequence identity is low, amphipathic alpha-helices with the potential to bind to lipid were predicted to exist in the EcApoC-I. RT-PCR analysis revealed that it was first transcribed in gastrula embryos and maintained a relatively stable expression level during the following embryogenesis. During embryonic and early larval development, a very high level of EcApoC-I expression was in the yolk syncytial layer, indicating that it plays a significant role in yolk degradation and transfers nutrition to the embryo and early larva. By the day 7 after hatching, EcApoC-I transcripts were observed in brain. In adult, EcApoC-I mRNA was detected abundantly in brain and gonad. In transitional gonads, the EcApoC-I expression is restricted to the germ cells. The data suggested that EcApoC-I might play an important role in brain and gonad morphogenesis and growth.

Differential expression and dynamic changes of SOX3 during gametogenesis and sex reversal in protogynous hermaphroditic fish

SOX3 has been suggested to play significant roles in gametogenesis and gonad differentiation of vertebrates, but the exact cellular localization evidence is insufficient and controversial. In this study, a protogynous hermaphrodite fish Epinephelus coioides is selected to analyze EcSox3 differential expression and the expression pattern in both processes of oogenesis and spermatogenesis by utilizing the advantages that gonad development undergoes transition from ovary to intersexual gonad and then to testis, and primordial germ cells and different stage cells during oogenesis and spermatogenesis are synchronously observed in the transitional gonads. The detailed and clear immunofluoresence localization indicates that significantly differential expression and dynamic changes of Sox3 occur in the progresses of gametogenesis and sex reversal, and EcSOX3 protein exists in the differentiating primordial germ cells, oogonia, and different stage oocytes of ovaries, and also in the differentiating primordial germ cells and the Sertoli cells of testis. One important finding is that the EcSox3 expression is a significant time point for enterable gametogenesis of primordial germ cells because EcSOX3 is obviously expressed and localized in primordial germ cells. As EcSox3 continues to express, the EcSOX3-positive primordial germ cells develop toward oogonia and then oocytes, whereas when EcSox3 expression is ceased, the EcSOX3-positive primordial germ cells develop toward spermatogonia. Therefore, the current finding of EcSOX3 in the differentiating primordial germ cells again confirms the potential regulatory role in oogenesis and germ cell differentiation. The data further suggest that SOX3, as a transcription factor, might have more important roles in oogenesis than in spermatogenesis.

Differential and spermatogenic cell-specific expression of DMRT1 during sex reversal in protogynous hermaphroditic groupers

DMRT1 has been suggested to play different roles in sex determination and gonad differentiation, because different expression patterns have been reported among different vertebrates. The groupers, since their gonads first develop as ovary and then reverse into testis, have been thought as good models to study sex differentiation and determination. In this study, we cloned the full-length cDNAs of DMRT] gene from orange-spotted grouper (Epinephelus coioides), and prepared corresponding anti-EcDMRT1] antiserum to study the relationship of DMRT] to sex reversal. One important finding is that the grouper DMRT] is not only differentially expressed in different stage gonads, but also restricted to specific stages and specific cells of spermatogenesis. Grouper DMRT1 protein exists only in spermatogonia, primary spermatocytes and secondary spermatocytes, but not in the supporting Sertoli cells. Moreover, we confirmed that EcSox3 is expressed not only in oogonia and different stage oocytes, but also in Sertoli cells and spermatogonia, and EcSox9 is expressed only in Sertoli cells. The data suggested that grouper DMRT1 might be a more specific sex differentiation gene for spermatogenesis, and play its role at the specific stages from spermatogonia to spermatocytes. In addition, no introns were found in the grouper DMRT1, and no duplicated DMRT1, genes were detected. The finding implicates that the intronless DMRT1 that is able to undergo rapid transcriptional turnover might be a significant gene for stimulating spermatogenesis in the protogynous hermaphroditic gonad. (c) 2006 Published by Elsevier Ireland Ltd.

Differential expression of vasa RNA and protein during spermatogenesis and oogenesis in the gibel carp (Carassius auratus gibelio), a bisexually and gynogenetically reproducing vertebrate

The RNA helicase Vasa is a germ cell marker in animals, and its homolog in vertebrates to date has been limited to bisexual reproduction. We cloned and characterized CagVasa, a Vasa homolog from the gibel carp, a fish that reproduces bisexually or gynogenetically. CagVasa possesses 14 RGG repeats and eight conserved motifs of Vasa proteins. In bisexually reproducing gibel carp, vasa is maternally supplied and its zygotic expression is restricted to gonads. By in situ hybridization on testicular sections, vasa is low in spermatogonia, high in primary spermatocytes, reduced in secondary spermatocytes, but disappears in spermatids and sperm. In contrast, vasa persists throughout oogenesis, displaying low-high-low levels from oogonia over vitellogenic oocytes to maturing oocytes. A rabbit anti-Vasa antibody (alpha Vasa) was raised against the N-terminal CagVasa for fluorescent immunohistochemistry. On testicular sections, Vasa is the highest in spermatogonia, reduced in spermatocytes, low in spermatids, and absent in sperm. In the ovary, Vasa is the highest in oogonia but persists throughout oogenesis. Subcellular localization of vasa and its protein changes dynamically during oogenesis. The aVasa stains putative primordial germ cells in gibel carp fry. It detects gonadal germ cells also in several other teleosts. Therefore, Cagvasa encodes a Vasa ortholog that is differentially expressed in the testis and ovary. Interestingly, the alpha Vasa in combination with a nuclear dye can differentiate critical stages of spermatogenesis and oogenesis in fish. The cross-reactivity and the ability to stain stage-specific germ cells make this antibody a useful tool to identify fish germ cell development and differentiation. (c) 2005 Wiley-Liss, Inc.

Tissue distributions and seasonal dynamics of the hepatotoxic microcystins-LR and -RR in two freshwater shrimps, Palaemon modestus and Macrobrachium nipponensis, from a large shallow, eutrophic lake of the subtropical China

So far no information is available on microcystin (MC) contents in shrimps, prawns or crayfish from natural freshwaters. Tissue distributions and seasonal dynamics of the hepatotoxic MC-LR and -RR in two freshwater shrimps, Palaemon modestus and Macrobrachium nipponensis were studied monthly (during June-November, 2003) in a Chinese lake containing toxic cyanobacterial blooms. The shrimps P. modestus and M. nipponensis accumulated high MCs not only in the hepatopancreas (mean 4.29 and 0.53 mu g g(-1) DW, respectively) but also in the gonad (mean 1.17 and 0.48 mu g g-1 DW, respectively), and the crayfish Procambarus clarkii accumulated as much as 0.93 mu g g(-1) DW in the gonad. This indicates that gonads of these invertebrates are the second important target organ of MCs. P. modestus apparently accumulated more MCs in their organs than M. nipponensis, which might be a reflection of their difference in trophic niche. Eggs of the shrimps accumulated 8.4% (M. nipponensis, 0.27 mu g g(-1) DW) and 29.0% (P. modestus, 2.34 mu g g(-1) DW) of total toxin burden, indicating that MCs had been transferred into offspring from their adults. Among the shrimp muscle samples analyzed, 31% were above the provisional WHO TDI level, suggesting the risk of consuming shrimps in Lake Chaohu. (c) 2005 Elsevier Ltd. All rights reserved.

Germ cell loss induced by C-12(6+) ion irradiation in young female mice

The ovaries of Kun-Ming strain mice (3 weeks) were irradiated with different doses of C-12(6+) ion in the Bragg peak or the plateau region. At 10th day after irradiation, ovarian and uterine weights were measured: normal and atretic (identified with the oocyte to be degenerating or absent) primordial, primary and preantral follicles were identified in the largest cross-section of each ovary. Percentage (%) of normal follicles of each developmental stage of oogenesis was calculated. The data showed that compared to controls, there was a dose-related decrease in percentage of normal follicles in each developmental stage. And the weights of ovary and uterus were also reduced with doses of irradiation. Moreover, these effects were much more significant in the Bragg peak region and the region close to the Bragg peak than in the beam's entrance (the plateau region). Radiosensitivity varied in different follicle maturation stages. Primordial follicles, which are thought to be extremely sensitive to ionizing irradiation, were reduced by 86.6%, while primary and preantral follicles reduced only by 72.5% and 61.8% respectively, by exposure with 6 Gy of C-12(6+) ion in the Bragg peak region and the region close to the Bragg peak. The data suggested that due to their optimal depth-dose distribution in the Bragg peak region, heavy ions are ones of the best particles for radiotherapy of tumors located next of vital organs or/and surrounded by normal tissues, especially radiosensitive tissues such as gonads.

Spring and autumn reproduction of Calanus sinicus in the Yellow Sea

The fecundity, hatching success and naupliar survival of Calanus sinicus were studied in the Yellow Sea during research cruises in April and October 2006, with emphasis on the regulation of reproduction. During both cruises, the egg production rate (EPR) showed large spatial variations (0 to 25.4 eggs female(-1) d(-1)), generally coinciding with the food availability. In April, the abundant phytoplankton and ciliates in the study area supported active reproduction, which would probably initiate the annual population development. In October, females remained immature in the Yellow Sea Cold Water Mass, likely due to the unfavorable environment (poor food and low temperature). However, reproduction and recruitment were high in the neritic region, which may explain the local population recovery in late autumn. Hatching success varied markedly among stations in April (4 to 85%), whereas it was high overall in the neritic region during October (> 90%). Based on the potential recruitment rates, the spring recruitment would be more important for the annual population dynamics, Female gonad maturity, body size and lipid reserve were examined in relation to fecundity. Regression analyses suggested that the reproductive index (defined as the proportion of females with mature gonads) could be an indicator for the EPR of C. sinicus. Among the regulating factors, external food (ciliates and phytoplankton) seems essential for reproduction, whereas inner lipid reserve may mainly serve metabolic needs. Moreover, fecundity is positively related to body size but independent of temperature, which might exert indirect influences on reproduction.

Study on the fatty acid composition of jellyfish Rhopilema esculentum

The fatty acids composition in different parts of full-grown Rhopilema esculentum jellyfish from Yellow Sea was investigated. The lipids, extracted from the umbrella and oral arms and gonads of R. eculentum jellyfish, respectively were analysed by combined capillary gas chromatography/mass spectrometry. The results show that there are more than thirty kinds of fatty acids in jellyfish, and the fatty acid compositions of three parts of R. esculentum are almost the same. In the three parts, percentages of polyunsaturated fatty acids (PUFA) are high, and range from 36.23% to 38.74%. Docosahexaenoic acid (DHA), eicosatetraenoic acid (AA) and eicosapentaenoic acid (EPA) are three major PUFA.

Aspects of the biology of Mya arenaria and Ensis spp. (Mollusca; Bivalvia) in the Irish Sea and adjacent areas

This study was undertaken to investigate the general biology, including the reproductive cycle and health status, of two clam taxa in Irish waters, with particular reference to the Irish Sea area. Monthly samples of the soft shell clam, Mya arenaria, were collected from Bannow Bay, Co. Wexford, Ireland, for sixteen months, and of the razor clam, Ensis spp. from the Skerries region (Irish Sea) between June 2010 and September 2011. In 2010, M. arenaria in Bannow Bay matured over the summer months, with both sexes either ripe or spawning by August. The gonads of both sexes of E. siliqua developed over autumn and winter 2010, with the first spawning individuals being recorded in January 2011. Two unusually cold winters, followed by a warmer than average spring, appear to have affected M. arenaria and E. siliqua gametogenesis at these sites. It was noted that wet weight of E. siliqua dropped significantly in the summer of both 2010 and 2011, after spawning, which may impact on the economic viability of fishing during this period. Additional samples of M. arenaria were collected at Flaxfort (Ireland), and Ensis spp. at Oxwich (Wales), and the pathology of all clams was examined using both histological and molecular methods. No pathogenic conditions were observed in M. arenaria while Prokaryote inclusions, trematode parasites, Nematopsis spp. and inflammatory pathologies were observed at low incidences in razor clams from Ireland but not from Wales; the first time these conditions have been reported in Ensis spp. in northern European waters. Mya arenaria from sites in Europe and eastern and western North America were investigated for genetic variation using both mitochondrial (cytochrome oxidase I (COI) and 16S ribosomal RNA genes) and nuclear markers (10 microsatellite loci). Both mitochondrial CO1 and all nuclear markers showed reduced levels of variation in certain European samples, with significant differences in haplotype and allelic composition between most samples, particularly those from the two different continents, but with the same common haplotypes or alleles throughout the range. The appearance of certain unique rare haplotypes and microsatellite alleles in the European samples suggest a complicated origin involving North American colonization but also possible southern European Pleistocene refugia. Specimens of Ensis spp. were obtained from five coastal areas around Ireland and Wales and species-specific PCR primers were used to amplify the internal transcribed spacer region 1 (ITS1) and the mitochondrial DNA CO1 gene and all but 15 razor clams were identified as Ensis siliqua. Future investigations should focus on continued monitoring of reproductive biology and pathology of the two clam taxa (in particular, to assess the influence of environmental change), and on genetics of southern European M. arenaria and sequencing the CO1 gene in Ensis individuals to clarify species identity

The reproductive biology of the exploited razor clam, Ensis siliqua, in the Irish Sea

Knowledge of the reproductive cycle of a species is a prerequisite for sustainable management of a fishery. The infaunal marine bivalve, Ensis siliqua, is a commercially important species in Europe, and is exploited in many countries, including Ireland, where it is sold by wet weight. Seasonal variations in the reproductive cycle of subtidal razor clams from the Skerries region of the Irish Sea, an important fisheries area, were examined between June 2010 and September 2011 while monitoring weight. Histological examination revealed that the E. siliqua sex-ratio was not different from parity, and no hermaphrodites were observed in the samples collected. In the summer months of 2010 all female clams were either spent or in early development, with just a small percentage of males still spawning. The gonads of both sexes developed over the autumn and winter months of 2010, with the first spawning individuals recorded in January 2011. Spawning peaked in March 2011, but unlike in 2010, spawning continued through June and July with all animals spent in August 2011. The earlier and longer spawning period found in this species in 2011 compared to 2010 may have been due to the colder than normal temperature observed during the winter of 2010 plus the relatively warmer temperatures of Spring 2011, which could have affected the gametogenic development of E. siliqua in the Irish Sea. It was noted that wet weight dropped in the summer months of both years, immediately after the spawning period which may impact on the practicality of fishing for this species during this period. Timing of development and spawning is compared with other sites in the Irish Sea and elsewhere in Europe, including the Iberian Peninsula.

Oestrogen shuts the door on SOX9.

Oestrogen exerts a robust yet imperfectly understood effect on sexual development in vertebrate embryos. New work by Pask and colleagues in BMC Biology indicates that it may interfere with male development by preventing nuclear localization of SOX9, a master regulator of the testis differentiation pathway. See research article http://www.biomedcentral.com/1741-7007/8/113.