Carlos Pendón

Endocrine mediators of seasonal growth in gilthead sea bream (Sparus aurata): the growth hormone and somatolactin paradigm

Regulation of somatolactin (SL) and the somatotropic axis was examined year-around at three different stocking times (spring, summer, and autumn) in a Mediterranean fish, the gilthead sea bream (Sparus aurata). The overall timing of plasma growth hormone (GH) increase was similar among trials (late spring-early summer), but the range of variation year-around was different and followed changes in food intake. Total plasma insulin-like growth factor-I primarily followed changes on growth rates, and a close positive correlation between IGF-I and thermal-unit growth coefficient (TGC) was found irrespective of fish stocking time. Thus, the activation of the somatotropic axis preceded always warm growth spurts, whereas the rise of SL in concurrence with low plasma cortisol levels was found at late autumn. This up-regulation of circulating SL titres preceded the winter inhibition of feeding, and it was more severe in big fish (spring and summer stocking times) than in small fish (autumn stocking time), growing with a relative high efficiency during the cold season despite of a severe hypertriglyceridemia and a high hepatosomatic index. These new insights provide good evidence for a different timing of GH and SL increases, and it is likely that the dominant role of SL in energy homeostasis is to be a mediator of the adaptation to fasting after replenishment of body fat stores, whereas GH and IGF-I are perceived as growth-promoting signals in times of food intake and increasing temperature and day-length.

CENP-C is necessary but not sufficient to induce formation of a functional centromere

CENP‐C is an evolutionarily conserved centromeric protein. We have used the chicken DT40 cell line to test the idea that CENP‐C is sufficient as well as necessary for the formation of a functional centromere. We have compared the effects of disrupting the localization of CENP‐C with those of inducibly overexpressing the protein. Removing CENP‐C from the centromere causes disassembly of the centromere protein complex and blocks cells at the metaphase–anaphase junction. Overexpressed CENP‐C is associated with an increase in errors of chromosome segregation and inhibits the completion of mitosis. However, the excess CENP‐C does not disrupt the native centromeres detectably and does not associate with another conserved centromere protein, ZW10. The distribution of the excess CENP‐C changes during the cell cycle. In metaphase, the excess CENP‐C coats the chromosome arms. At the metaphase–anaphase transition, the excess CENP‐C clusters, and during interphase it is present in large bodies which form around pre‐existing centromeres which are also clustered. These results indicate that CENP‐C is necessary but not sufficient for the formation of a functional centromere and suggest that the structure of CENP‐C may be regulated during the cell cycle.

Somatotropic regulation of fish growth and adiposity: growth hormone (GH) and somatolactin (SL) relationship ☆

Growth hormone (GH) and insulin-like growth factors (IGFs) play a major role in fish development and metabolism, and several studies have allowed discernment of a complex and tissue-specific collection of salmonid IGF-I transcripts (Ea-4, Ea-3, Ea-2, Ea-1), which are the result of the alternative splicing of the E-domain region. However, the pattern of IGF-I expression is different in non-salmonid fish, and only one or two transcripts (Ea-4, Ea-2) have been detected in hepatic and extrahepatic tissues of common carp, barramundi, black sea bream and gilthead sea bream. Despite this, when comparisons are made within Mediterranean fish species (European sea bass, common dentex and gilthead sea bream), plasma IGF-I levels are consistent with fish species differences in growth rates. Changes of growth rates, and plasma IGF-I and GH levels are also found in response to changes in diet composition and ration size, which may serve to assess the suitability of feeding regimes in aquaculture practice. Regulation of plasma somatolactin (SL) levels is also examined in gilthead sea bream, and the resulting plasma SL profile differs from that of GH. Thus, in contrast to GH, plasma SL levels augment with the increase of ration size and fish size (advancement of age). A transient increase in plasma SL levels is also found in short-term fasted fish, and this fish peptide may act as an anti-obesity hormone helping to expedite growth-reproductive processes following replenishment of fat stores, and/or mediate the adaptation to fasting until the lipolytic action of GH and/or other endocrine factors is fully accomplished. This agrees with the known increase of plasma SL levels during acute stress and exhaustive exercise. However, a causal link between SL and energy mobilisation (lipid metabolism) remains to be established, and further research is needed to determine the extent to which SL and GH act in a complementary manner to make available metabolic fuels and to regulate body fat mass and feeding behaviour.

Iodothyronine deiodinases and thyroid hormone receptors regulation during flatfish (Solea senegalensis) metamorphosis

Thyroid hormone-induced metamorphosis seems to represent an ancestral feature of chrordates (urochordates, cephalochordates and vertebrates), but also of nonchordate animals. Although thyroid hormones and thyroid hormone receptor profiles during metamorphosis have been analyzed in different vertebrate taxa, including fish, developmental expression and activity of type 2 (dio2, D2) and type 3 (dio3, D3) iodothyronine deiodinases, two key enzymes in anuran metamorphosis, remain unknown in any fish species. The aim of this work was to investigate the development of thyroid hormone system during the metamorphosis of a flatfish species, the Senegalese sole, focusing on the deiodinases developmental profile. We have cloned sole D2 and D3 and analyzed several parameters of thyroid hormones system in pre-, early-, middle-, and late-metamorphic larvae. Both deiodinases contain in their catalytic centers an UGA triplet encoding for a selenocystein (Sec) residue as expected. Left eye migration and rotation in body position were associated with a significant increase in both thyroid hormones and thyroid hormone receptors at the middle-late metamorphic stages. Although dio2 expression slightly increased during metamorphosis, D2 activity augmentation was much more significant. Sole dio3 expression declined only slightly, whereas the D3 activity clearly decreased at mid-late metamorphic period. This developmental profile of deiodinases sustained the rise of thyroid hormones levels observed during sole metamorphosis. No clear cut daily rhythms were observed in the parameters analyzed although it seemed that thyroid hormone system was more active during daytime, in particular at late metamorphic stages. These developmental changes point out the importance not only of thyroid hormones and their receptors but also of dio2 and dio3 in mediating flatfish metamorphosis, as it has been described in amphibians.

The accuracy of segregation of human mini-chromosomes varies in different vertebrate cell lines, correlates with the extent of centromere formation and provides evidence for a trans-acting centromere maintenance activity

Abstract. We show that the accuracy of mitotic segregation of three engineered, mapped human mini-chromosomes differs between human, mouse and chicken cell lines. We have studied the cause of these differences by analysing the extent of centromere formation on one mini-chromosome immunocytochemically. In human and chicken cell lines the mini-chromosomes segregate accurately and form centromeres but in one mouse cell line centromere formation is undetectable and mitotic segregation is inaccurate. These results indicate that the centromere is maintained by an activity that functions in trans and varies either in amount or specificity between different cells. Structurally defined mini-chromosomes may allow this activity to be studied.

An inducible nitric oxide synthase (NOS) is expressed in hemocytes of the spiny lobster Panulirus argus: cloning, characterization and expression analysis.

Nitric oxide (NO) is a free radical gas involved in a variety of physiological processes in invertebrates, such as neuromodulation, muscle contraction and host defense. Surprisingly, little is known about the involvement of NO synthase (NOS) in the immune system of crustaceans. This work is focused on the study of the NOS gene of the spiny lobster Panulirus argus, a crustacean with commercial interest, and its relationship with the immune response to a microbial elicitor. A NOS full-length DNA was isolated from hemocytes by reverse transcription-polymerase chain reaction (RT-PCR) using degenerated primers. The open reading frame (ORF) encodes a protein of 1200 amino acids, with an estimated molecular mass of 135.9 kDa, which contains the conserved domains and binding motifs of NOS found in a variety of organisms. NOS gene expression in lobster gills, heart, stomach, digestive gland, abdominal muscle, gut and hemocytes was studied by Real Time quantitative PCR (Real Time qPCR). The expression was higher in hemocytes, heart and gills. In addition, when lobster hemocytes were exposed in vitro to Escherichia coli O55:B5 lipopolysaccharide (LPS), an increase in the NOS activity and also in the NOS gene expression evaluated by Real Time qPCR was observed, thus demonstrating the presence of an inducible crustacean NOS by a microbial elicitor of the immune response. The information is relevant in providing basic knowledge for further studies of crustacean defense mechanisms.

The use of recombinant gilthead sea bream (Sparus aurata) growth hormone for radioiodination and standard preparation in radioimmunoassay.

A gilthead sea bream growth hormone (sbGH) obtained by cloning and expression of sbGH cDNA was used to develop a sensitive and specific radioimmunoassay (RIA). Iodination of recombinant sbGH (rsbGH) was performed by the classical Chloramine-T method. Specific antiserum, raised in rabbits, was added in a final dilution of 1/36,000. The minimum detectable dose was 30 pg, and the midrange of the assay (ED50) was 275 pg. Intra- and inter-assay coefficients of variation (CV) were 3.3 and 5.8% at ED50 levels. Human GH (hGH), ovine GH (oGH), carp gonadotropin (cGtH), chinook salmon gonadotropin (sGtH), ovine prolactin (oPRL) and recombinant tilapia prolactin (rtiPRL) did not show cross-reactivity. Serial dilutions of chinook salmon GH (sGH) and recombinant rainbow trout GH (rtGH) showed a low but significant cross-reactivity. A good parallelism between rsbGH standard and serial dilutions of native sbGH, plasma and pituitary extracts was observed. In addition, when plasma and pituitary samples were analyzed for GH quantification, non-significant differences were observed within this and previous RIA for native sbGH. Therefore, it appears conclusive that our rsbGH can be used successfully as a standard and radioiodinated hormone in GH assays for gilthead sea bream, which is extensively cultured in the Mediterranean area.

Cloning of a somatolactin-encoding cDNA from sole (Solea senegalensis)

From a Solea senegalensis cDNA expression library, clones encoding somatolactin (SL), a new pituitary hormone belonging to the growth hormone/prolactin family, were isolated and analyzed. Northern blot analysis showed a unique 1.0-kb mRNA species. The sole SL 778-bp cDNA encoded full-size S. senegalensis SL (ssSL) (230 amino acids), including seven Cys and two potential glycosylation sites. A consensus polyadenylation signal, AATAAA, was found. Protein homology and DNA sequence alignment of SL cDNAs from other evolutionarily distant marine fishes suggest that the SL sequence is highly conserved.

Cloning, tissue expression pattern and daily rhythms of Period1, Period2, and Clock transcripts in the flatfish Senegalese sole, Solea senegalensis

An extensive network of endogenous oscillators governs vertebrate circadian rhythmicity. At the molecular level, they are composed of a set of clock genes that participate in transcriptional–translational feedback loops to control their own expression and that of downstream output genes. These clocks are synchronized with the environment, although entrainment by external periodic cues remains little explored in fish. In this work, partial cDNA sequences of clock genes representing both positive (Clock) and negative (Period1, Period2) elements of the molecular feedback loops were obtained from the nocturnal flatfish Senegalese sole, a relevant species for aquaculture and chronobiology. All of the above genes exhibited high identities with their respective teleost clock genes, and Per–Arnt–Sim or basic helix–loop–helix binding domains were recognized in their primary structure. They showed a widespread distribution through the animal body and some of them displayed daily mRNA rhythms in central (retina, optic tectum, diencephalon, and cerebellum) and peripheral (liver) tissues. These rhythms were most robust in retina and liver, exhibiting marked Period1 and Clock daily oscillations in transcript levels as revealed by ANOVA and cosinor analysis. Interestingly, expression profiles were inverted in retina and optic tectum compared to liver. Such differences suggest the existence of tissue-dependent zeitgebers for clock gene expression in this species (i.e., light for retina and optic tectum and feeding time for liver). This study provides novel insight into the location of the molecular clocks (central vs. peripheral) and their different phasing and synchronization pathways, which contributes to better understand the teleost circadian systems and its plasticity.

The Circadian Clock Machinery During Early Development of Senegalese sole (Solea senegalensis): Effects of Constant Light and Dark Conditions

Circadian rhythms are established very early during vertebrate development. In fish, environmental cues can influence the initiation and synchronization of different rhythmic processes. Previous studies in zebrafish and rainbow trout have shown that circadian oscillation of clock genes represents one of the earliest detectable rhythms in the developing embryo, suggesting their significance in regulating the coordination of developmental processes. In this study, we analyzed the daily expression of the core clock components Per1, Per2, Per3, and Clock during the first several days of Senegalese sole development (0–4 d post fertilization or dpf) under different lighting regimes, with the aim of addressing when the molecular clock first emerges in this species and how it is affected by different photoperiods. Rhythmic expression of the above genes was detected from 0 to 1 dpf, being markedly affected in the next few days by both constant light (LL) and dark (DD) conditions. A gradual entrainment of the clock machinery was observed only under light-dark (LD) cycles, and robust rhythms with increased amplitudes were established by 4 dpf for all clock genes currently studied. Our results show the existence of an embryonic molecular clock from the 1st d of development in Senegalese sole and emphasize the significance of cycling LD conditions when raising embryos and early larvae. (Author correspondence: munoz.cueto@uca.es; carlos.pendon@uca.es)