Relationships between environmental changes, maturity, growth rate and plasma insulin-like growth factor-I (IGF-I) in female rainbow trout.

Size reflecting growth rate, energy balance or nutritional status is regarded as an important determinant of the ability of trout to undergo puberty. The relationship of a change in photoperiod, either natural (SNP) or advancing (ADV), with growth, IGF-I and reproduction was investigated in virgin female rainbow trout. Under SNP 63% of the population attained maturity while only 29% spawned 6 months in advance in the ADV regime. Under SNP both size and growth rate in late spring-early summer appeared to determine whether an individual may initiate reproduction while condition factor appeared to be a better predictor in the ADV regime. A complete seasonal relationship between plasma IGF-I, daylength and temperature was demonstrated under natural conditions, and provides direct evidence for the relationship between reproduction and IGF-I. Conversely, trout maintained under ADV exhibited a significantly different plasma IGF-I profile relative to those under a natural photoperiod. Furthermore, IGF-I levels accurately reflected growth rate prior to elevations in sex steroids, suggesting that IGF-I may provide an endocrine signal between the somatotropic and reproductive axes that growth rate and/or size is sufficient to initiate gonad development. In addition, maturing individuals under SNP typically expressed higher circulating IGF-I levels than those that remained immature and may reflect a greater opportunity for IGF-I to act on the pituitary to stimulate gonadotropin production. We observed elevated levels in maturing fish for 3 months under SNP compared to only 1 month under ADV were observed. This may reflect a reduction in the window of opportunity to initiate reproduction under advancing photoperiods and hence explain the reduction in fish successfully recruited.

A comparative ex vivo and in vivo study of day and night perception in teleosts species using the melatonin rhythm.

The purpose of this study was to determine and compare the light sensitivity of two commercially important, phylogenetically different teleost species in terms of melatonin production. Three series of experiments were performed on both Atlantic salmon and European sea bass. First, a range of light intensities were tested ex vivo on pineal melatonin production in culture during the dark phase. Then, light transmission through the skull was investigated, and finally short-term in vivo light sensitivity trials were performed. Results showed that sea bass pineal gland ex vivo are at least 10 times more sensitive to light than that of the salmon. Light intensity threshold in sea bass appeared to be between 3.8 x 10(-5) and 3.8 x 10(-6) W/m2 in contrast to 3.8 x 10(-4) and 3.8 x 10(-5) W/m2 in salmon. These highlighted species-specific light sensitivities of pineal melatonin production that are likely to be the result of adaptation to particular photic niches. Light transmission results showed that a significantly higher percentage of light penetrates the sea bass pineal window relative to salmon, and confirmed that penetration is directly related to wavelength with higher penetration towards the red end of the visible spectrum. Although results obtained in vivo were comparable, large differences between ex vivo and in vivo were observed in both species. The pineal gland in isolation thus appeared to have different sensitivities as the whole animal, suggesting that retinal and/or deep brain photoreception may contribute, in vivo, to the control of melatonin production.

Photoperiod influences growth rate and plasma insulin-like growth factor-I levels in juvenile rainbow trout, Oncorhynchus mykiss.

The effect of different photoperiod regimes and the subsequent influence of melatonin on growth and insulin-like growth factor-I (IGF-I) were assessed in juvenile rainbow trout. In Experiment 1, triplicate groups of all female underyearling rainbow trout were exposed to one of three photoperiods; simulated natural photoperiod (SNP), constant short-days (LD 8:16), or constant long-days (LD 18:6) from June to December 2000 under ambient water temperatures. Fish exposed to LD 18:6 grew to a significantly heavier mean weight than the other treatments. Regression analysis showed a strong correlation between circulating plasma IGF-I, growth rate and temperature. Furthermore, it was apparent that fish exposed to LD 18:6 expressed significantly higher circulating levels of IGF-I. In a second experiment, duplicate groups of all female yearling trout were exposed to one of three photoperiods; SNP, LD 8:16, or constant light (LL), with sub groups receiving either a slow-release melatonin implant (18 mg), sham implant or left intact (control). LL increased growth rate in controls, reaching a significantly greater weight than SNP or LD 8:16 photoperiods but did not affect circulating IGF-I levels. Melatonin implants reduced growth rate in all photoperiod treatments below that of their respective controls but again did not affect circulating IGF-I levels. No differences in growth rate were found in implanted fish between photoperiods suggesting that a diel cycle of melatonin is necessary for the perception of daylength. These results would indicate that extended photoperiods (LD 18:6) may cause direct photostimulation of growth through up-regulation of IGF-I production. In contrast, in the absence of a changing diel melatonin signal, growth appeared to be maintained by a possible underlying endogenous rhythm, which was phase advanced under LL, as such plasma IGF-I levels simply reflected growth rate rather than photostimulation of the somatotropic axis. Overall, these findings indicate that measuring plasma IGF-I may be a useful tool for studying environmental influences on growth in rainbow trout.

Analysis of repetitive DNA sequences in the sex chromosomes of Oreochromis niloticus.

In the Nile tilapia, Oreochromis niloticus, sex determination is primarily genetic, with XX females and XY males. While the X and Y chromosomes (the largest pair) cannot be distinguished in mitotic chromosome spreads, analysis of comparative hybridization of X and Y chromosome derived probes (produced, by microdissection and DOP-PCR, from XX and YY genotypes, respectively) to different genotypes (XX, XY and YY) has demonstrated that sequence differences exist between the sex chromosomes. Here we report the characterization of these probes, showing that a significant proportion of the amplified sequences represent various transposable elements. We further demonstrate that concentrations of a number of these individual elements are found on the sex chromosomes and that the distribution of two such elements differs between the X and Y chromosomes. These findings are discussed in relation to sex chromosome differentiation in O. niloticus and to the changes expected during the early stages of sex chromosome evolution.

Molecular-cytogenetic analysis reveals sequence differences between the sex chromosomes of Oreochromis niloticus: evidence for an early stage of sex-chromosome differentiation.

Sex determination in the Nile tilapia, Oreochromis niloticus, is primarily genetic, with XX females and XY males. A candidate sex-determining region in the terminal region of the largest chromosome pair has been identified by analysis of meiotic chromosomes. This region shows an inhibition of pairing and synapsis in the XY genotype, but not in XX or YY genotypes, suggesting that recombination is inhibited. Here we show that chromosome microdissection and subsequent amplification by degenerate oligonucleotide-primed PCR (DOP-PCR) can be used to produce in situ hybridization probes to this largest pair of O. niloticus chromosomes. Furthermore, analysis of the comparative hybridization of X and Y chromosome-derived probes to different genotypes provides the first demonstration that sequence differences exist between the sex chromosomes of O. niloticus. This provides further support for the theory that this chromosome pair is related to sex determination and further suggests that the sex chromosomes are at a very early stage of divergence.

Karyotype evolution in Tilapia: mitotic and meiotic chromosome analysis of Oreochromis karongae and O. niloticus x O. karongae hybrids.

The karyotype of Oreochromis species is considered to be highly conserved, with a diploid chromosome complement of 2n = 44. Here we show, by analysis of mitotic and meiotic chromosomes, that the karyotype of O. karongae, one of the Lake Malawi 'chambo' species, is 2n = 38. This difference in chromosome number does not prevent the production of inter-specific hybrids between O. niloticus (2n = 44) and O. karongae (2n = 38). Analysis of the meiotic chromosomes of the O. niloticus x O. karongae hybrids indicates that three separate chromosome fusion events have occurred in O. karongae. Comparison of the O. karongae and O. niloticus karyotypes suggests that these consist of one Robertsonian fusion and two fusions of a more complex nature.

Early origins of the X and Y chromosomes: lessons from tilapia.

Differentiated sex chromosome pairs in diverse species display certain common characteristics, normally comprising one largely heterochromatic genetically inactive chromosome and one euchromatic genetically active chromosome (e.g. the mammalian Y and X respectively). It is widely accepted that dimorphic sex chromosomes evolved from homologous pairs of autosomes. Although the exact mechanisms through which the pair diverged are not fully understood, an initial suppression of recombination in the sex-determining region is required by all of the major theories. Here we address the question of the mechanism by which this initial suppression of recombination occurs. Our model postulates that the stochastic, de novo accumulation of heterochromatin in the sex determining region can delay pairing of the sex chromosomes in meiosis, resulting in a decrease in recombination. Data to support this model is presented from the cichlid fish, Oreochromis niloticus. Although such a decrease would in most circumstances be evolutionarily disadvantageous, if the region concerned included the major sex determining gene and other gene(s) with sex-specific functions, then this would be selectively advantageous and could trigger the process(es) which, ultimately, lead to the differentiation of the sex chromosomes.

Identification of putative sex chromosomes in the blue tilapia, Oreochromis aureus, through synaptonemal complex and FISH analysis.

Sex determination in the blue tilapia, Oreochromis aureus, is primarily a ZW female-ZZ male system. Here, by analysis of the pachytene meiotic chromosomes of O. aureus, we demonstrate the presence of two distinct regions of restricted pairing present only in heterogametic fish. The first, a subterminal region of the largest bivalent is located near to the region of unpairing found in the closely related species O. niloticus, while the second is in a small bivalent, most of which was unpaired. These results suggest that O. aureus has two separate pairs of sex chromosomes.

Effects of melatonin on liver estrogen receptor and vitellogenin expression in rainbow trout: an in vitro and in vivo study.

Although melatonin is believed to mediate many seasonal and circadian effects of photoperiod on reproduction in salmonids, the precise mechanisms underlying such effects are still largely unknown. Recent data of the literature indicate a relationship between melatonin and expression of estrogen receptors (ER) in various tissues. In this study, the effects of melatonin on estrogen receptor and/or vitellogenin expression were studied by a combination of in vivo and in vitro experiments. In yeast stably expressing ER and transfected with an estrogen-responsive element-beta-galactosidase reporter gene, melatonin had no effect on basal or E2-stimulated ER expression. Incubation of hepatocyte aggregates with melatonin (10(-8) to 10(-4)) for 16 or 48 h did not modify the E2-stimulated ER and vitellogenin mRNA, as measured by dot blots. Finally, neither pinealectomy nor melatonin implants caused any effect on basal or E2-stimulated ER and vitellogenin mRNA contents in the liver. Altogether, these results suggest that, although we cannot exclude potential effects at the brain or pituitary levels, melatonin has no or little effects on estrogen receptor in the liver.

Expression of clock gene in the brain of rainbow trout: comparison with the distribution of melatonin receptors.

To identify brain structures potentially acting as biological clocks in rainbow trout (Oncorhynchus mykiss), the expression sites of a trout homolog of the mouse clock gene were studied and compared with that of melatonin receptors (Mel-R). For this purpose, a partial sequence of the trout clock gene, including a PAS domain, was obtained by reverse transcription-polymerase chain reaction and used to perform in situ hybridization. The highest density of clock transcripts was observed in the periventricular layer (SPV) of the optic tectum, but a weaker expression was detected in some pretectal nuclei, such as the posterior pretectal nucleus (PO) and the periventricular regions of the diencephalon. Comparison of the hybridization signal in fish sacrificed at 08:00 and 17:00 did not indicate major changes in clock expression levels. Comparison of adjacent sections alternatively treated with clock and Mel-R probes suggests that both messengers are probably expressed in the same cells in the SPV and PO. In addition, in situ hybridization with a glutamate decarboxylase 65 probe, demonstrates that cells expressing clock and Mel-R in the optic tectum are gamma-aminobutyric acid neurons. The tight overlapping between the expression of Mel-R and clock transcripts in cells of the PO and SPV suggests a functional link between these two factors. These results indicate that the optic tectum and the pretectal area of the rainbow trout are major sites of integration of the melatonin signal, express the clock gene, and may act as biological clocks to influence behavioral and endocrine responses in trout.

The effects of oral administration with 17 alpha-methyltestosterone on chromosomal synapsis in Oreochromis niloticus (Pisces, Cichlidae).

The pattern of chromosomal synapsis after treatment with 17 alpha-methyltestosterone (MT), a testosterone analogue routinely used for the reversal of phenotypic sex in aquaculture, was investigated using the Nile tilapia (Oreochromis niloticus) as a model teleost species. Progeny-tested, monosex diploid (2n = 44) individuals were orally administered with diets containing 50 mg/kg MT for 30 days after first feeding (XX(MT) neomales and XY(MT) males) and compared to controls (XY males). The formation and structure of the synaptonemal complex (SC) and the nature of chromosomal synapsis were investigated in control and treated groups by computer-assisted image analysis of transmission electron microscope (TEM) microphotographs taken from SC spreads. Nuclei at the pachytene stage were first observed in XX(MT) neomales, indicating an earlier commitment of genetically female spermatocytes to enter the first meiotic prophase. Administration of MT did not result in obvious SC lesions, breakage, asynapsis or formation of multivalents in genotypic females (XX(MT) neomales). Administration of MT resulted in a significant increase in the SC lengths in XY(MT) males, although it did not significantly alter the pattern of synapsis (SC structure and number and morphology of bivalents) in comparison to XY controls. The significance of the effects and the putative mode(s) of action of MT on chromosomal synapsis in teleosts is discussed.

Central melatonin receptors in the rainbow trout: comparative distribution of ligand binding and gene expression.

To better define the role of melatonin in fish, we have compared in detail the distribution of 2-[125I]iodomelatonin binding sites with gene expression for melatonin receptor subtypes in a widely studied seasonal species, the rainbow trout. Three distinct partial sequences of the melatonin receptor gene were cloned from trout genomic DNA. Two of the sequences corresponded to the Mella receptor subtype, and one corresponded to the Mellb receptor subtype. Analysis of numerous clones failed to find a sequence equivalent to the Mel1c receptor subtype. Comparison of receptor gene expression with 2-[125I]iodomelatonin binding distribution indicated dendritic transport of the receptor. Melatonin receptors were associated predominantly with visually related areas of the trout brain, such as the thalamic region, the pretectal area, and the optic tectum. The pituitary was devoid of 2-[125I]iodomelatonin binding, and melatonin receptor gene expression was not detectable. It would appear from the results of the present study that melatonin in this species is involved primarily in the processing of visual signals. How melatonin interacts with circannual rhythms of growth and reproduction is unclear, although a direct interaction between melatonin and the hypothalamo-pituitary axis is not clearly indicated.

Natural copepods are superior to enriched artemia nauplii as feed for halibut larvae (Hippoglossus hippoglossus) in terms of survival, pigmentation and retinal morphology: relation to dietary essential fatty acids.

Replicate groups of halibut larvae were fed to d 71 post-first feeding (PFF) either the marine copepod, Eurytemora velox, or Artemia nauplii doubly enriched with the marine chromist or golden algae, Schizochytrium sp., (Algamac 2000) and a commercial oil emulsion (SuperSelco). The fatty acid compositions of eyes, brains and livers from larvae fed the two diets were measured, and indices of growth, eye migration and skin pigmentation were recorded along with histological examinations of eye and liver. The docosahexaenoic acid [22:6(n-3); DHA]/eicosapentaenoic acid [20:5(n-3); EPA] ratios in Artemia nauplii enriched with the SuperSelco and Algamac 2000 were 0.4 and 1.0, respectively. The E. velox copepods were divided into two size ranges (125-250 and 250-400 microm) with the smaller size range containing the highest level of (n-3) highly unsaturated fatty acids (HUFA). The DHA/EPA ratios for the two size ranges of copepods were 2.0 and 0.9, respectively. The total lipids of eyes, brains and livers of larvae fed copepods had higher levels of DHA and lower levels of EPA than those of larvae fed enriched Artemia. The percentage of survival of the halibut larvae was significantly higher when copepods rather than enriched Artemia nauplii were fed, but larval specific growth rates did not differ. The indices of eye migration were high and not significantly different in larvae fed the two diets, but the percentage of larvae undergoing successful metamorphosis (complete eye migration and dorsal pigmentation) was higher in larvae fed copepods (40%) than in larvae fed enriched Artemia (4%). The rod/cone ratios in histological sections of the retina were 2.5 +/- 0.7 in larvae fed copepods and 1.3 +/- 0.6 in larvae fed enriched Artemia (P < 0.01). Histological examination of the livers and intestines of the larvae were consistent with better assimilation of lipid from copepods than lipid from Artemia nauplii up to 46 d post-first feeding. Thus, marine copepods are superior to enriched Artemia as food for halibut larvae in terms of survival, eye development and pigmentation, and this superiority can be related to the level of DHA in the feed.

The brain-pituitary-gonadal axis of female rainbow trout Oncorhynchus mykiss: effects of photoperiod manipulation1.

Two groups of post-spawned female rainbow trout were exposed to two different photoperiods, an ambient photoperiod (56 degrees N) and a combination of long and short photoperiods (a constant 18L:6D from February 1 until May 10, then a constant 6L:18D), which acted to advance maturation and spawning. The stimulatory long-short photoperiod advanced spawning by 3-4 months and correspondingly advanced peaks in serum levels of 17beta-estradiol, testosterone, calcium (an index of vitellogenin), and GTH II. Earlier events in gonadal recrudescence appeared to be less affected by the photoperiod. The initiation of exogenous vitellogenesis coincided with high levels of both pituitary salmon gonadotropin-releasing hormone (sGnRH) content and serum follicle-stimulating hormone (FSH, GTH I) levels. High levels of serum FSH were associated with rapid gonadal growth in the fish exposed to the stimulatory long-short photoperiod. In contrast, the fish exposed to the ambient photoperiod showed gonadal steroid production, formation of vitellogenin, and secondary oocyte growth without any detectable increase in serum FSH levels. The possible roles and interactions of sGnRH, gonadotropins, and steroids with respect to normal and artificially stimulated ovarian maturation are discussed.

Yolk formation and degradation during oocyte maturation in seabream Sparus aurata: involvement of two lysosomal proteinases.

Oocyte growth within the follicle is preponderantly due to the accumulation of hepatically derived yolk protein (vitellogenin, VTG) by receptor-mediated endocytosis; once in the oocyte, VTG is partially processed and stored in yolk globules. In some pelagic egg-laying marine teleosts, additional cleavages of yolk proteins followed by a pronounced water uptake occur concomitantly with final oocyte maturation. The aim of this study was to establish the lysosomal enzymes involved in these two proteolytic processes that characterize oocyte maturation of seabream Sparus aurata. The enzymatic activities of several cathepsins were assessed in the various classes of oocytes. Changes in cathepsin B, D, and L activity were found depending on the oocyte maturation stage; cathepsin B and D were found to be at maximum level in early-vitellogenesis oocytes, and cathepsin L in mid-vitellogenesis ones. Cathepsin D and L were purified from seabream ovary, and their roles in VTG and lipovitellin (LV) proteolysis, respectively, were analyzed. Here we demonstrate directly that one of the catalysts for the intraoocytic processing of VTG in yolk proteins is cathepsin D; however, we cannot exclude also a role of cathepsin B in the same process. On the other hand, cathepsin L is responsible for the second proteolytic cleavage of the LV components. We postulate that the acquisition of buoyancy by eggs through the hydration process may be regulated by enzymatic activation at the appropriate time of oocyte maturation, this process probably being the key event in the reproduction of this marine pelagic egg spawner.

Long-term, quantitative analysis of gametogenesis in autotriploid rainbow trout, Oncorhynchus mykiss.

A long-term, quantitative analysis was conducted on the gametogenesis of autotriploid rainbow trout (Oncorhynchus mykiss) to quantify their degree of germline development and reproductive potential. Triploid and diploid (control) trout siblings were raised separately under identical conditions and sampled randomly for histological analysis. Triploid males underwent testicular development and proliferation of germ cells by mitosis and meiosis, progressing through initial phases of spermatogenesis at a similar pace to diploid controls. The effects of triploidy on males were most evident during the final stages of spermatogenesis, when all diploid males contained free spermatozoa in the lumen of most tubules (average relative frequency, ARF = 68.5%), whereas triploid males contained predominantly spermatocytes (ARF = 36.3%) and morphologically abnormal spermatozoa (ARF = 31.8%). In contrast, the gonadal development of triploid females was affected during its early stages; the major patterns observed were the arrest of the oogonia within oogonial clusters (ARF = 30.4-71.1%), the appearance of small numbers (ARF = 1.5-6.0%) of previtellogenic and early vitellogenic follicles, and the proliferation of non-follicular elements (vascular lacunae, fibrosis and tubular adenomas). In agreement with previous reports on the ovarian development of chromosomally female (3A:ZZW) triploid chickens, male-differentiating areas (ARF = 0.2-12.2%) were observed in most triploid females examined, which by the end of the sampling period appeared as gonadal hermaphrodites. It is hypothesized that the lack of proper somatic-to-germ cell interactions prevents the segregation of the oocytes from the gonial clusters and may explain the early blockage observed during the gonadal morphogenesis of autotriploid female rainbow trout.

Photoperiod-induced phase-shifts of the endogenous clock controlling reproduction in the rainbow trout: a circannual phase-response curve.

Different groups of winter-spawning female rainbow trout that had been maintained under seasonally changing daylength and temperature were exposed to 2 months of continuous light at different times of the year. The same photoperiod produced advances in the time of spawning of up to 232 days and delays of up to 80 days, depending upon the timing of exposure in relation to the phase of the reproductive cycle. The proportion of fish spawning in each group varied from 18% to 100%, again dependent on the timing of exposure to continuous light. The photoperiod-induced changes in spawning time can be interpreted as phase-dependent phase-shifts of an endogenous circannual clock controlling maturation. It is proposed that long days, occurring earlier or later than they would under a natural photoperiod, were perceived as indications that the clock was running slow or fast, thus initiating corrective forward adjustments (advance phase-shifts) or backward adjustments (delay phase-shifts), respectively. Collectively, these responses can be described in the form of a circannual phase-response curve.

Melatonin rhythms in Atlantic salmon (Salmo salar) maintained under natural and out-of-phase photoperiods.

Diel changes in circulating melatonin were measured in juvenile Atlantic salmon, Salmo salar, maintained under natural and out-of-phase seasonal photocycles. Under natural daylengths of autumn, winter, spring, and summer circulating melatonin levels were inversely related to light intensity, with levels low during the day and high at night. The duration of the nocturnal increase in circulating melatonin was related to the duration of darkness, i.e., longer in winter than in summer. Under simulated seasonal photocycles circulating melatonin concentrations measured in August, October, and December were also elevated for the duration of darkness, irrespective of whether the photoperiods were synchronized or 6 months out-of-phase with the natural light and temperature cycles. Circulating melatonin also provided an accurate representation of the prevailing photoperiod in fish initially maintained on simulated natural photocycles, either synchronized or 6 months out-of-phase with the natural light cycle, and then held for 3 months on daylengths approximating the summer and winter solstices. Well-defined melatonin rhythms were always present, irrespective of time of year, photoperiod, and temperature. The amplitude of the nocturnal increase in circulating melatonin was similar in groups of fish maintained under simulated seasonal photoperiods 6 months out-of-phase with each other, but otherwise identical conditions, indicating that daylength per se did not influence the amplitude of the melatonin rhythm. The amplitude of the melatonin rhythm was slightly higher during the summer months, suggesting that temperature may modify circulating melatonin levels. These results demonstrate that circulating melatonin profiles always reflect the prevailing daylength and hence have the potential to provide the Atlantic salmon with accurate information on daily and calendar time, which could be utilised to time daily and seasonal events.