Circannual rhythms of gonadal maturation in female rainbow trout (Oncorhynchus mykiss).

Individually identified, 2-year-old female rainbow trout were maintained for up to 51 months on a constant schedule of 6 hr light and 18 hr darkness (LD 6:18), constant temperature (8.5-9.0 degrees C), and constant feeding rate. The fish exhibited free-running circannual rhythms of gonadal maturation and ovulation, which were self-sustaining for up to three cycles. The periodicity of the rhythm showed variation between fish and in successive cycles for the same fish, ranging from approximately 11 to 15 months.

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.

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.

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.

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.

Thyroxine binding globulin (TBG) and thyroxine binding prealbumin (TBPA) measurement, compared with the conventional T3 uptake in the diagnosis of thyroid disease.

An electroimmunoassay for the determination of thyroxine binding prealbumin is described. The diagnostic efficiency of the assay when used in conjunction with the serum thyroxine as a thyroxine: thyroxine binding prealbumin ratio, is compared with the conventional free thyroxine index and the more recently developed thyroxine: thyroxine binding globulin ratio. The population studied included euthyroid, hypothyroid and hyperthyroid patients and also those who were either pregnant or receiving oral contraceptive therapy. Despite recent evidence establishing the theoretical/practical advantages of using a direct measurement for thyroid binding proteins rather than an indirect method (tri-iodothyronine uptake), results obtained from this study suggest that, for the majority of patients requiring biochemical assessment, the free thyroxine index is still the superior discriminator of thyroid abnormality.

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.

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.