MicroRNAs are involved in ovarian physiology of greater amberjack (Seriola dumerili) under captivity.

Gonad maturation is critical for the reproductive success of any organism, and in fish, captivity can significantly affect their reproductive performance, leading to maturation incompetence and spawning failure. The greater amberjack (Seriola dumerili), a fish species recently introduced to aquaculture fails to undergo oocyte maturation, ovulation, and spawning when reared in aquaculture facilities. Since confinement has been shown to influence gonad maturation and completion of the reproductive cycle, investigations into epigenetic mechanisms may shed light on the reasoning behind the reproductive dysfunctions of fish under captivity. Among the known important epigenetic regulators are small non-coding RNAs (sncRNAs), and in particular microRNAs (miRNAs). In this study, immature, maturing (late vitellogenesis), and spent ovaries of captive greater amberjack were collected, and the differential expression of miRNAs in the three different ovarian development stages was examined. Expression patterns of conserved and novel miRNAs were identified, and potential targets of highly differentially expressed miRNAs were detected. Additionally, read length distribution showed two prominent peaks in the three different ovarian maturation stages, corresponding to miRNAs and putative piwi-interacting RNAs (piRNAs), another type of ncRNAs with a germ-cell specific role. Furthermore, miRNA expression patterns and their putative target mRNAs are discussed, in relevance with the different ovarian maturation stages of captive greater amberjack. Overall, this study provides insights into the role of miRNAs in the reproductive dysfunctions observed in fish under captivity and highlights the importance of epigenetic mechanisms in understanding and managing the reproductive performance of economically important fish species.

Effects of different hormonal treatments on spermatogenesis advancement in hatchery-produced greater amberjack Seriola dumerili (Risso 1810).

In earlier studies, wild-caught greater amberjack Seriola dumerili (Risso, 1810) males reared in sea cages showed gametogenesis impairment and low sperm production and quality. Here, we (a) examined if F1 hatchery-produced males reared in sea cages also exhibit reproductive dysfunctions and (b) evaluated the effects of gonadotropin releasing hormone agonist (GnRHa) administration through injections (GnRHainj) or sustained-release implants (GnRHaimpl), and human chorionic gonadotropin (hGC) injections on spermatogenesis/spermiation enhancement. Fish were given a hormone treatment just prior to the spawning season, and were transferred to land-based tanks, according to an established spawning induction protocol. Blood samples (n = 6) were obtained on Days 0, 7 and 13 after treatment. Testis samples were obtained on Days 0 (n = 4) and 13 (n = 2 per treatment). The fish prior to their transfer from the sea cages to the land-based tanks, exhibited a low gonadosomatic index, altered sex steroid hormone profile and high density of testicular apoptotic cells. After transfer to tanks, there was a general depression of sex steroid plasma levels parallel to an increase in cortisol concentrations. Despite the negative effect on steroidogenesis by the transfer from the sea, the hormonal treatments increased the number of fish from where sperm could be obtained, as well as testis growth, and reduced testicular apoptosis. Treatment with hCG resulted in the most significant changes in spermatogenesis, while GnRHaimpl appeared to induce less intense, but likely longer-lasting effects. The study indicated that F1 hatchery-produced males also exhibited reproductive dysfunctions as wild-caught captive-reared greater amberjack, and that the observed positive effects of the hormone treatments on spermiation/spermatogenesis were likely mediated by factors other than sex steroid hormones.

Thermal manipulation and GnRHa therapy applied to the reproduction of lambari-do-rabo-amarelo, Astyanax altiparanae females (Characiformes: Characidae) during the non-breeding season.

Lambari-do-rabo-amarelo Astyanax altiparanae in the wild reproduce during spring and summer, but females undergo vitellogenesis throughout the year, including the non-spawning winter period when water temperatures are low. The present study investigated the physiological role of temperature modulation on the hypothalamus-pituitary-gonads axis of lambari during winter, as well as the effects of gonadotropin releasing hormone agonist (GnRHa) therapy. Captive females were exposed to two different temperatures (20 °C and 27 °C) and were injected weekly with GnRHa for 21 days during winter (Control, CTR; Low dose; LD and high dose of GnRHa, HD). At the end of the 21-days period gonadosomatic index (GSI), oocyte stage of development and theoretical fecundity were evaluated, together with plasma levels of 17ß-estradiol (E2). Gene expression of the two pituitary gonadotropins follicle-stimulating hormone (fshß) and luteinizing hormone (lhß), as well as hepatic vitellogenin-A (vtgA) expression were also analyzed. At the end of the experimental period, females from the six different experimental conditions were induced to spawn using human chorionic gonadotropin (hCG). Spawning performance parameters and plasma levels of the maturation inducing steroid (MIS) were analyzed. Gene expression of fshß did not change with temperature manipulation, but females exposed to 27 °C and supplemented with a HD of GnRHa exhibited an increased fshß gene expression, associated with higher E2 levels. The higher water temperature alone was able to increase E2 levels. At both water temperatures GnRHa injections induced a decrease in E2 levels. GnRHa injected females had a lower vtgA gene expression levels at 20 °C. Even with differences in the gene expression of gonadotropins among the various temperature/GnRHa treatments, GSI and oocyte diameter did not change, but GnRHa enhanced the number of vitellogenic oocytes at 20 °C. The reproductive performance of lambari induced to spawn with hCG was better after the combined treatment with GnRHa and summer temperature.

The transcriptomic signature of different sexes in two protogynous hermaphrodites: Insights into the molecular network underlying sex phenotype in fish.

Sex differentiation is a puzzling problem in fish due to the variety of reproductive systems and the flexibility of their sex determination mechanisms. The Sparidae, a teleost family, reflects this remarkable diversity of sexual mechanisms found in fish. Our aim was to capture the transcriptomic signature of different sexes in two protogynous hermaphrodite sparids, the common pandora Pagellus erythrinus and the red porgy Pagrus pagrus in order to shed light on the molecular network contributing to either the female or the male phenotype in these organisms. Through RNA sequencing, we investigated sex-specific differences in gene expression in both species' brains and gonads. The analysis revealed common male and female specific genes/pathways between these protogynous fish. Whereas limited sex differences found in the brain indicate a sexually plastic tissue, in contrast, the great amount of sex-biased genes observed in gonads reflects the functional divergence of the transformed tissue to either its male or female character. Α common "crew" of well-known molecular players is acting to preserve either sex identity of the gonad in these fish. Lastly, this study lays the ground for a deeper understanding of the complex process of sex differentiation in two species with an evolutionary significant reproductive system.

Rearing in captivity affects spermatogenesis and sperm quality in greater amberjack, Seriola dumerili (Risso, 1810).

The greater amberjack, (Risso, 1810), is a promising candidate for the diversification of European aquaculture production, but inconsistent reproduction in captivity prevents commercial production. Recent studies showed that greater amberjack confined in sea cages exhibited scarce gonad development and early interruption of gametogenic activity during the reproductive season. The aim of the present study was to improve our understanding of the observed impairment of spermatogenesis. Adult wild and captive-reared males were sampled during 3 different phases of the reproductive cycle: early gametogenesis (EARLY; late April to early May), advanced gametogenesis (ADVANCED; late May to early June), and spawning (SPAWNING; late June to July). Spermatogonial stem cells and proliferating germ cells were identified through the immunohistochemical localization of and proliferating cell nuclear antigen, respectively. Apoptotic germ cells were identified throughout the terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling method. Sperm quality of captive-reared fish was evaluated using computer-assisted sperm analysis. Captive-reared males exhibited seminiferous lobules of a smaller diameter, a precocious and progressive decrease of spermatogonial mitosis, and a high level of apoptosis at the beginning of the reproductive season, concomitant with a many-fold higher 17ß-estradiol plasma concentration. The motile spermatozoa percentage of captive greater amberjack was lower than in other teleosts, and a drastic decrease of spermatozoa motility duration, velocity, and ATP content occurred along the reproductive season. An abnormal increase of sperm concentration as well as an increase of dead spermatozoa occurred during the SPAWNING phase, probably because of lack of sperm hydration and ejaculation and consequent sperm ageing. The present study demonstrates the extreme susceptibility of greater amberjack to rearing stress and underscores the need for improvement of the rearing and handling procedures to ameliorate gametogenesis dysfunctions in commercial aquaculture production.

Skeletal ontogeny of the vertebral column and of the fins in shi drum Umbrina cirrosa (Teleostei: Perciformes: Sciaenidae), a new candidate species for aquaculture.

The osteological development of the vertebral column and fins in shi drum Umbrina cirrosa was studied in order to improve knowledge for its introduction in Mediterranean aquaculture. The osteological development was studied in 171 individuals, of total length (LT ) from 2·7 to 30·2 mm that were reared under the mesocosm technique. Vertebral ontogeny starts at 3·4 and 4·0 mm LT , with the formation of the first cartilaginous neural and haemal arches, and spines, respectively, and is completed with the full attainment of epicentrals (12·5 mm LT ). The formation of vertebral centra occurs between 4·1 and 7·4 mm LT . Pectoral supports are the first fin elements to develop (3·0 mm LT ), followed by those of the caudal fin (3·8 mm LT ), pelvic fin (3·9 mm LT ) and finally by those of the dorsal and anal fins (4·5 mm LT ). The caudal fin is the first to develop fin rays and attain the full count of principal fin rays (4·5-6·8 mm LT ), but the last to be fully completed with the formation of procurrent fin rays (6·9-17·5 mm LT ). The next fins starting to present rays are the dorsal (5·3 mm LT ) and the pectoral fins (5·6 mm LT ), while the anal and pelvic fins are the last (5·7 mm LT ). Following the caudal principal fin rays (6·8 mm LT ), the dorsal, anal (6·9 mm LT ), pelvic (7·4 mm LT ) and pectoral fins (9·8 mm LT ) are the next with fully completed ray counts. Aggregation of qualitative changes, such as the appearance of cartilages, the beginning and the complement of the ossification process and the full complement of elements in U. cirrosa were measured as cumulative frequency counts. These measurements reveal three ontogenetic intervals: one very developmentally active period during early life stages (from 3 to 5·9 mm LT ), a second slower developmental period (from 6·0 to 8·9 mm LT ) and finally a period of ontogeny more focused on structure refinement up to metamorphosis and settlement (>9·0 mm LT ).

Expression of vitellogenin receptor gene in the ovary of wild and captive Atlantic bluefin tuna (Thunnus thynnus).

The cDNA sequences of vitellogenin receptor proteins (VgR(+) and VgR(-)), containing or lacking the O-linked sugar domain, were determined in Atlantic bluefin tuna (Thunnus thynnus L.). VgR(-) gene expression in the ovary was compared in captive-reared and wild Atlantic bluefin tuna during the reproductive cycle. Gonad samples from adult fish were sampled from 2008 to 2010 from stocks reared in captivity at different commercial fattening operations in the Mediterranean Sea and from wild individuals caught either by traditional tuna traps during their migration towards the spawning grounds in the Mediterranean Sea or by the long-line artisanal fishery. In addition, juvenile male and female Atlantic bluefin tuna were sampled from a farming facility, to obtain baseline information and pre-adulthood amounts of VgR(-). The total length of VgR(+) cDNA was 4006 nucleotides (nt) and that of VgR(-) was 3946 nt. Relative amounts of VgR(-) were greater in juvenile females and in those adults having only previtellogenic oocytes (119 ± 55 and 146 ± 26 folds more than juvenile males, respectively). Amounts of VgR(-) were less in individuals with yolked oocytes (ripening stage, May-June) and increased after spawning in July (92 ± 20 and 113 ± 13 folds more than juvenile males in ripening and post-spawning fish, respectively). These data suggest that regulation of VgR(-) is not under oestrogen control. During the ripening period, greater VgR(-) gene expression was observed in wild fish than in fish reared in captivity, possibly because of (a) differences in water temperature exposure and/or energy storage, and/or (b) an inadequate diet in reared Atlantic bluefin tuna.

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 17ß-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 17,20ß-dihydroxy-4-pregnen-3-one (17,20ß-P) or 17,20ß,21-trihydroxy-4-pregnen-3-one (20ß-S), in either the free, conjugated or 5ß-reduced,3α-hydroxylated forms was observed in fish sampled within 6 days after treatment. Interestingly, a significant peak in plasma free 17,20ß-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.

Evidence that severe acute stress and starvation induce rapid atresia of ovarian vitellogenic follicles in Atlantic bluefin tuna, Thunnus thynnus (L.) (Osteichthyes: Scombridae).

The effects of different stressors on the atretic degeneration of ovarian vitellogenic follicles, as well as on the ovarian mass, were examined in female Atlantic bluefin tuna, Thunnus thynnus (L.), from the Mediterranean Sea. The stressors taken into consideration were short-term starvation (up to 14 days), long-term cage rearing (1 year) and crowding-induced severe panic frenzy. Wild-caught individuals were used as a control group. Fish subjected to either severe panic frenzy or starvation exhibited a decrease in gonad mass and had significantly higher intensity of α atresia in the vitellogenic follicles (means: 78% and 58%, respectively; range: 36-100%) than either wild or long-term caged individuals (means: 32% and 30%, respectively; range: 19-44%). The extensive atresia in fish stressed by severe panic frenzy was observed as early as 24 h after the stressing event. The present study represents the first evidence of the extreme susceptibility of Atlantic bluefin tuna to severe acute stress during vitellogenesis; it also shows that starvation is associated with progressive reabsorption of vitellogenic oocytes.

Species specificity in the magnitude and duration of the acute stress response in Mediterranean marine fish in culture.

The aim of the present study was to examine the species-specific stress response for seven Mediterranean fishes in culture. Also, to evaluate the method of measuring free cortisol concentration in the rearing water as a non-invasive and reliable indicator of stress in marine species, of aquaculture importance. Gilthead sea bream, Sparus aurata (Sparidae); common dentex, Dentex dentex (Sparidae); common Pandora, Pagellus erythrinus (Sparidae); sharpsnout sea bream, Diplodus puntazzo (Sparidae); dusky grouper, Epinephelus marginatus (Serranidae); meagre, Argyrosomus regius (Sciaenidae) and European sea bass, Dicentrarchus labrax (Moronidae) were subjected to identical acute stress (5-6 min chasing and 1-1.5 min air exposure) under the same environmental conditions and samples were analyzed by the same procedures. Results indicated that there was a clear species-specificity in the magnitude, timing and duration of the stress response in terms of cortisol, glucose and lactate. European sea bass showed a very high response and dusky grouper and meagre a very low response, except plasma glucose concentrations of dusky grouper which was constantly high, while sharpsnout sea bream presented a protracted stress response, up to 8h. The present study confirmed that free cortisol release rate into the water can be used as a reliable stress indicator.

Comparative study of liver vitellogenin gene expression and oocyte yolk accumulation in wild and captive Atlantic bluefin tuna (Thunnus thynnus L.).

The sequence of vitellogenin A (VgA) and vitellogenin B (VgB) cDNAs in Atlantic bluefin tuna (Thunnus thynnus L.) were determined, and vitellogenin expression levels in the liver and oocyte yolk accumulation were compared in wild and captive-reared individuals. Liver and ovary samples were taken from 31 individuals reared experimentally in three commercial Atlantic bluefin tuna fattening sites in the Mediterranean Sea and from 33 wild individuals caught by commercial traps during the fish's migration towards their Mediterranean spawning grounds. The total length of VgA cDNA was 5585 nucleotides and that of VgB was 5267 nucleotides. The identity and similarity between deduced amino acid sequences of VgA and VgB were 60% and 78%, respectively. The Atlantic bluefin tuna VgA and VgB amino acid sequences have high similarities with those of other teleost fishes. Relative levels of VgA and VgB mRNAs were low in April, increased significantly during the reproductive period in May and June, and declined in July. There was a trend towards higher relative levels of VgA and VgB mRNAs in captive fish compared to wild individuals during the reproductive period. The surface occupied by eosinophilic yolk granules in fully vitellogenic oocytes, as well as the frequency of oocytes in late vitellogenesis, was significantly higher in captive compared to wild individuals. The study suggests that the experimental conditions under which Atlantic bluefin tuna individuals were reared allowed the occurrence of normal vitellogenesis, based on gene expression of VgA and VgB in the liver and yolk accumulation in the oocytes. The higher yolk accumulation and frequency of vitellogenic oocytes observed in the ovaries of captive fish suggest that improvements in feeding practices may result in an improved vitellogenic process.

Proliferation and apoptosis of male germ cells in captive Atlantic bluefin tuna (Thunnus thynnus L.) treated with gonadotropin-releasing hormone agonist (GnRHa).

The effects of administration of gonadotropin-releasing hormone agonist (GnRHa) on proliferation and apoptosis of male germ cells were evaluated on Atlantic bluefin tuna (Thunnus thynnus L.) reared in captivity. Fish (n=19) were treated with a sustained-release delivery system loaded with GnRHa during the natural spawning season of 2004 and 2005 (June-July). Untreated Control fish (n=17) and adult wild spawners were used for comparison. Fish were sacrificed 2-8 d after GnRHa implantation and body weight and gonad weight were recorded, and gonads and blood were taken. Germ cell proliferation and apoptosis were evaluated through the immunohistochemical detection of proliferating cell nuclear antigen (PCNA) and the terminal deoxynucleotidyl transferase-mediated d'UTP nick end labelling (TUNEL) method, respectively. Plasma 11 ketotestosterone (11-KT) levels were measured using an ELISA method. Mean gonado-somatic index and seminiferous lobule diameter did not differ between GnRHa-treated and Control fish, and were significantly lower in captive-reared individuals than in wild spawners. Significant increases in 11-KT plasma levels and spermatogonial mitosis, along with a reduction of germ cell apoptosis were demonstrated in GnRHa-treated fish compared to Controls. The results suggest that GnRHa administration was effective in enhancing germ cell proliferation and reducing apoptosis in captive males through the stimulation of luteinizing hormone (LH) release and testicular 11-KT production.

Ontogeny of the digestive tract in sharpsnout sea bream Diplodus puntazzo (Cetti, 1777).

The ontogeny of the digestive tract was studied histologically and histochemically in sharpsnout sea bream Diplodus puntazzo from hatching (0 DAH, Days After Hatching) until day 57 (57 DAH). At hatching, the digestive tract appeared as a histologically undifferentiated straight tube lying dorsally to the yolk sac. When the mouth opened at 3 DAH, the digestive tract was differentiated into buccopharynx, oesophagus, incipient stomach and intestine. The pancreas, liver and gall bladder were also differentiated at this stage and both the bile and pancreatic duct had opened into the anterior intestine. Active feeding began in 50% of larvae at 4 DAH, although permanence of yolk reserves until 7 DAH suggests a period of both endogenous and exogenous feeding. Nutrient absorption was first visible from 5 DAH, as colourless supra- and infranuclear vacuoles in the anterior intestinal mucosa, suggesting a lipid content, as well as supranuclear, eosinophilic vacuoles, containing protein, in the posterior intestinal mucosa. Early caecal development could be detected from 10 DAH, whereas gastric glands appeared at 30 DAH, indicating the transition from larval to juvenile stage and the acquisition of an adult mode of digestion. Goblet cells appeared in the digestive tract of sharpsnout sea bream larvae shortly after first feeding. The mucus content of goblet cells varied with the digestive region and, in the buccal cavity and oesophagus, also with the developmental phase. This study provides knowledge for better husbandry practices in the aquaculture industry, as well as for the implementation of future nutritional studies.

Temporal profile of brain and pituitary GnRHs, GnRH-R and gonadotropin mRNA expression and content during early development in European sea bass (Dicentrarchus labrax L.).

A likely endocrine control mechanism for sexual differentiation in size-graded populations of European sea bass (Dicentrarchus labrax) is proposed by evaluating the brain expression and pituitary content of two forms of gonadotropin-releasing hormone (GnRH), namely sea bream (sbGnRH) and salmon (sGnRH), the pituitary expression of one subtype of GnRH receptor (dlGnRH-R-2A) and the three gonadotropin (GtH) subunits, namely glycoprotein alpha (GPalpha), follicle-stimulating hormone beta (FSHbeta) and luteinizing hormone beta (LHbeta), as well as the pituitary and plasma LH levels between 50 and 300 days post-hatching (dph). Four gradings were conducted between 2 and 8 months after hatching, resulting in a population of large and small individuals, having 96.5% females (female-dominant population) and 69.2% males (male-dominant population), respectively, after the last grading. The onset of gonadal differentiation was different in the two sexes, and coincided with a peak of expression of sbGnRH or sGnRH. Furthermore, the expression of these GnRHs was correlated with the expression of dlGnRH-R-2A. Sex-related differences in the brain and pituitary content of sbGnRH were also found at the time of sexual differentiation. Moreover, the observed sexual dimorphism at the transcriptional or synthesis level of these GnRH forms suggests that a different neuro-hormonal regulation is operating according to sex. At the onset of sex differentiation, FSHbeta transcriptional activity reached maximal values, which were maintained until the completion of the process. The present study suggests a role for sbGnRH, sGnRH and the dlGnRH-R-2A during gonadal differentiation, possibly through enhancement of FSHbeta gene expression. In males, a different endocrine regulation seems to exist also during spermiogenesis and spermiation, when gene transcription, peptide synthesis and release of LH are of greater importance.

Cloning, characterisation, and expression of three oestrogen receptors (ERalpha, ERbeta1 and ERbeta2) in the European sea bass, Dicentrarchus labrax.

Three oestrogen receptor [ER] subtypes have been described in teleost fish, namely ERalpha, and two ERbeta subtypes, called ERbeta1 and ERbeta2 (or ERbeta and ERgamma in Atlantic croaker). Their expression during embryonic development and gonadal growth has evoked interest in their potential role in sexual differentiation and gonadal development in fish. We cloned three oestrogen receptors from adult liver (sb-ERalpha cDNA) and ovary (partial sb-ERbeta1 and sb-ERbeta2 cDNAs) of the European sea bass, and according to their phylogenetic relatedness to other ERs in teleosts, named them sea bass [sb-] ERalpha, ERbeta1 and ERbeta2. Deduced amino acid numbers for sb-ERalpha, sb-ERbeta1 and sb-ERbeta2 were 639, 517 and 608, respectively, representing in the case of sb-ERbeta1 and sb-ERbeta2 about 90% of the open reading frame. Highest amino acid identities were found for sb-ERalpha with eelpout ERalpha (88.7%), for sb-ERbeta1 with Atlantic croaker ERgamma (85.8%), and for sb-ERbeta2 with Atlantic croaker ERbeta (90.1%). Southern analysis confirmed that all three sea bass oestrogen receptors (sb-ERs) are the products of three distinct genes. In adult sea bass, ERalpha was predominantly expressed in liver and pituitary, while sb-ERbeta1 and sb-ERbeta2 were more ubiquitously expressed, with highest expression levels in pituitary. In a mixed-sex population of juvenile sea bass, sb-ERalpha expression was significantly elevated in gonads at 200 days posthatch (dph), while for sb-ERbeta1 and sb-ERbeta2 highest expression levels were observed in gonads at 250 dph. For sb-ERbeta2, expression was also significantly higher in the brain at 250 dph. The cloning of these three ER subtypes in the European sea bass together with the results obtained on expression levels in adult and juvenile animals has given us the foundation to investigate their possible role in sexual differentiation and development in this species in future studies.

Plasma steroids in mature common dentex (Dentex dentex) stimulated with a gonadotropin-releasing hormone agonist.

The aim of this study was to identify the major C21 steroids produced in vivo during artificially induced final oocyte maturation and spawning in female common dentex (Dentex dentex). During the spawning season, mature females were treated with a gonadotropin-releasing hormone agonist (GnRHa)-loaded delivery system, with or without pimozide (given as a single dose at the beginning of the experiment). Blood samples were collected at various intervals during the experiment and were assayed for GnRHa, 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P), and 17,20beta,21-trihydroxy-4-pregnen-3-one (17,20beta,21-P). A higher percentage of ovulated females was observed in GnRHa-implanted fish, which produced over 10 times more eggs than controls. Relative fecundity was highest in the GnRHa + pimozide group and lowest in controls. The viability of naturally released eggs was low (2 to 15%) in all groups. Plasma concentrations of 17,20beta-P in GnRHa-implanted fish did not increase, but those in control fish decreased, such that there was a significant difference between control and treated fish between 2 and 10 days after treatment. In another experiment, ovulating common dentex were injected intramuscularly with a single dose of 50 microg kg(-1) of GnRHa in saline and were sampled for blood at 0, 3, 6, 12, and 24 h postinjection. A single water sample was taken from the tanks at 9 h postinjection, the tanks having been emptied and refilled at 6 h. Measurements were made of plasma and water concentrations of free and conjugated 17,20beta-P, 17,20beta,21-P, 17beta-oestradiol (E2), and GnRHa (plasma only). The GnRHa injection increased plasma levels of all steroids, with free 17,20beta-P reaching maximal levels within 3 h. GnRHa treatment also increased the amounts of free and conjugated steroids released into the water between 6 and 9 h.

Gonadotrophin-releasing hormone agonist stimulates milt fluidity and plasma concentrations of 17,20beta-dihydroxylated and 5beta-reduced, 3alpha-hydroxylated C21 steroids in male plaice (Pleuronectes platessa).

Spermiating male plaice were caught in the North Sea and acclimatised to laboratory conditions. In two experiments, males were injected intramuscularly with either microspheres or pellets containing gonadotrophin-releasing hormone agonist (GnRHa). Blood was sampled at 2- to 5-day intervals. Individual blood plasma specimens were assayed for testosterone, 5beta-reduced, 3alpha-hydroxy ("5beta,3alpha") steroids and sulphated 17, 20beta-dihydroxy ("17,20beta") steroids. Pooled plasma samples were also assayed for free and sulphated 17, 20beta-dihydroxy-4-pregnen-3-one, free 11-ketotestosterone, and glucuronidated testosterone and 11-ketotestosterone. Plasma concentrations of all steroids were significantly elevated by GnRHa from 2 to 5 days onwards following treatment. The most marked changes occurred in the concentrations of the sulphated 17,20beta steroids, which comprised approximately equal amounts of 5beta-pregnane-3alpha,17,20beta-triol 20-sulphate (3alpha,17, 20beta-P-5beta-S) and 5beta-pregnane-3beta,17,20beta-triol 20-sulphate, rising from ca. 1 to 30-80 ng/ml in the first and from ca. 8 to 80 ng/ml in the second experiment. Concentrations of 5beta, 3alpha steroids matched those of 17,20beta steroids in one experiment. However, in the other experiment, the two RIAs yielded highly disparate results in about 50% of the fish (including males in the control group). The plasma of these fish contained excessive amounts of 5beta,3alpha-immunoreactive material between 10 and 25 days. This material was identified as 3alpha,17, 21-trihydroxy-5beta-pregnan-20-one 21-sulphate (a metabolite of 11-deoxycortisol). All previous studies have indicated that when plasma concentrations of this steroid are high, so are those of 3alpha,17,20beta-P-5beta-S. This is the first indication that these steroids are regulated independently. In a third experiment, milt fluidity and production were assessed at 10, 15, and 25 days following GnRHa implantation. Milt volume and fluidity were significantly enhanced by the GnRHa treatment.

Endocrine profiles of female striped bass (Morone saxatilis) in captivity, during postvitellogenesis and induction of final oocyte maturation via controlled-release GnRHa-delivery systems.

Plasma levels of reproductive and thyroid hormones were measured in captive striped bass females during postvitellogenesis and the spawning period (March-June). Circulating gonadotropin II (GtH II), 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P), and 17,20beta, 21-trihydroxy-4-pregnen-3-one (17,20beta,21-P) remained low and unchanged in nonmaturing females, while 17beta-estradiol (E2) and testosterone (T) declined throughout postvitellogenesis. Plasma thyroxine (T4) declined significantly in mid-April, while triiodothyronine (T3) increased in mid-May. The only female that ovulated spontaneously had markedly different E2, T, and T3 profiles during postvitellogenesis, and had a surge in plasma GtH II during final oocyte maturation (FOM). The lack of a GtH II surge is presumably responsible for the absence of FOM, but earlier, and as of yet unknown, endocrine disruptions during postvitellogenesis may determine the female's ability to undergo FOM. Treatment of females with a gonadotropin-releasing hormone agonist (GnRHa)-delivery system induced FOM and ovulation within 3 and 10 days, respectively, and resulted in the production of fertile eggs. Plasma GtH II increased continually after GnRHa implantation, even in the presence of declining GnRHa plasma levels. Plasma E2 increased first and peaked prior to FOM, whereas T peaked at the peripheral germinal vesicle (GV) stage. Plasma 17,20beta-P and 17,20beta,21-P increased dramatically at the GV breakdown (GVBD) stage. Plasma T4 was unaffected by the GnRHa treatment, whereas T3 decreased after GnRHa implantation and remained low throughout FOM. Based on the observed hormonal profiles, FOM can be separated into an early phase (lipid-droplet coalescence, GV migration) associated with E2 and T elevations, and a late phase (yolk-globule coalescence, GVBD) associated with 17,20beta-P and 17,20beta,21-P elevation.

Modulation of testicular androgen production in adolescent African catfish (Clarias gariepinus).

At 6 months of age the first spermatozoa appear in the testes of the African catfish considered to be adolescent, since the development to adulthood (12 months of age) is accompanied by further morphological and functional differentiation of Leydig cells. There are increasing plasma levels of 11-ketotestosterone (11-KT) and an increasing responsiveness to luteinizing hormone (LH) of testicular androgen secretion in vitro. Whether treatment of adolescent males with key hormones of the brain-pituitary-gonad axis [gonadotropin-releasing hormone (GnRH), LH, and 11-KT] affects the testicular steroidogenic response to a challenge with LH in vitro 7 days later has been investigated. Injection of GnRH (2.5 microg chicken GnRH-II per kilogram of body weight), LH (25 microg/kg), or a high dose of 11-KT (50 microg/kg) down-regulated basal and LH-stimulated testicular androgen secretion to a minimum of 35% of control values. Treatment with LH was, moreover, associated with changes in the ultrastructure of Leydig cell mitochondria which were either swollen and had a less electron-dense matrix or showed an elongated shape. Conversely, a moderate dose of 11-KT (20 microg/kg) enhanced LH-stimulated, but not basal, androgen secretion in vitro to a maximum of 190% of control values. In view of the generally low LH plasma levels and of the steadily increasing 11-KT plasma levels during puberty, 11-KT may be involved in the up-regulation of the testicular steroidogenic capacity observed during development to full maturity.

Plasma gonadotropin II, sex steroids, and thyroid hormones in wild striped bass (Morone saxatilis) during spermiation and final oocyte maturation.

The blood levels of gonadotropin II (GtH II), sex-steroid hormones, and thyroid hormones were determined in wild spermiating male striped bass (Morone saxatilis) in males and in females at various stages of final oocyte maturation (FOM), captured on their spawning grounds. The progression of spermiation was associated with increases in plasma GtH II and decreases in plasma testosterone (T), 11-ketotestosterone, and thyroxine (T4). Plasma triiodothyronine (T3) remained at high and relatively unchanged levels. Plasma levels of 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P) and 17,20beta, 21-trihydroxy-4-pregnen-3-one (17,20beta,21-P), the proposed maturation-inducing steroids (MIS) in striped bass, were low and unchanged during the same period. It was concluded that low progestogen levels are adequate to induce spermiation in striped bass, and that higher levels may be associated with spawning behavior. In the females, based on the profiles of the studied hormones, FOM was separated into two phases. Early FOM, which included germinal vesicle (GV) migration and lipid-droplet coalescence, was associated with elevations in plasma GtH II, T, and estradiol 17beta. Late FOM, which included GV breakdown and yolk-globule coalescence, was associated with a further surge in plasma GtH II, increases in the levels of the two MIS, mainly 17, 20beta-P, and a drop in T4. Plasma T3 levels did not change during FOM. Examination of conjugated steroids demonstrated, in the males, a reduction in conjugated androgens at the peak of the spawning season and, in the females, a small increase in conjugated 17, 20beta-dihydroxylated and 5beta-reduced,3alpha-hydroxylated steroids after spawning. This is the most comprehensive report, to date, on the endocrine regulation of gonadal maturation in wild striped bass, demonstrating that a two-stage process of FOM is regulated by different endocrine signals, providing further evidence for the involvement of 17,20beta-P as a MIS in the females, and indicating that both males and females are in an euthyroid state during the spawning season.