Genomic organization and sequence of the Gus-s alpha allele of the murine beta-glucuronidase gene.

The Gus-s alpha allele of the mouse beta-glucuronidase gene exhibits a high degree of inducibility by androgens due to its linkage with the Gus-r alpha regulatory locus. We isolated Gus-s alpha on a 28-kilobase pair fragment of mouse chromosome 5 and found that it contains 12 exons and 11 intervening sequences spanning 14 kilobase pairs of this genomic segment. The mRNA cap site was identified by ribonuclease protection and primer extension analyses which revealed an unusually short 5' noncoding sequence of 12 nucleotides. Proximal regulatory sequences in the 5'-flanking DNA and the complete sequence of the Gus-s alpha mRNA transcript were also determined. Comparison of the amino acid sequence determined from the Gus-s alpha nucleotide sequence with that of human beta-glucuronidase indicated that the two human mRNA species differ due to alternate splicing of an exon homologous to exon 6 of the mouse gene.

Quantitative RT-PCR analysis and immunohistochemical localization of HSP70 in sea bass Dicentrarchus labrax exposed to transport stress.

In aquaculture, fish are exposed to stressful conditions, which cause an increased synthesis of heat shock proteins (HSPs) at the cellular level. In this work we considered the expression of the constitutive and inducible forms of HSP70 as an indicator of stress caused by transport, during development of the sea bass (Dicentrarchus labrax), a teleost fish of high value for aquaculture. Qualitative RT-PCR analysis revealed expression of inducible HSP70 gene in larvae and fry (25, 40 and 80 days) as well as in adult tissues (liver, brain, muscle, gills, kidney, gonads, heart, spleen and skin) of both control and stressed animals. Expression of inducible HSP70 mRNA examined in different adult tissues by Real-Time PCR, was significantly higher in skin and skeletal muscle of stressed animals than in controls. Immunolocalization of inducible and constitutive forms of heat shock protein 70 (HSP70 and HSC70), reported here for the first time, demonstrated an ubiquitous distribution of HSC70 protein in several tissues of both stressed and control animals (at all stages), while inducible HSP70 protein was found only in skeletal muscle of stressed animals. In all stressed animals, regardless of their developmental stage, cortisol levels were higher than in control animals.

Long-term culture of muscle explants from Sparus aurata.

Although there are mammalian myoblast cell lines, no fish myoblast cell line has been developed so far. The aim of this study was to develop a culture system of muscle explants for fish, as explants provide an approximation of the in vivo conditions for cell proliferation and differentiation, and enable a close comparison with events in muscle regenerating in vivo. Here we describe the main features of a long-term in vitro culture system for muscle explants from Sparus aurata fry. At the time of sampling, the original fibres were damaged and subsequently degenerated as shown by the loss of parvalbumin (PV) and presence of apoptotic nuclei. This mechanical damage provoked a myogenic response by activation of myogenic precursor cells. After a few days, new mononucleate cells aligned with the original fibres were seen in the explants, some with proliferating cell nuclear antigen (PCNA-) and Myf-5-positive nuclei, indicating proliferation and their myogenic fate. By 1 week, multinucleate cells with desmin immunoreactivity but PCNA- and Myf5-negative nuclei were present, equivalent to differentiated, postmitotic myotubes. Some of these myotubes were also immunoreactive for PV and insulin-like growth factors (IGFs). By 11 days, many of the myotubes were also immunoreactive for myostatin (MSTN). By 23 days, many of the myotubes had increased in diameter, were packed with myofibrils, and were strongly PV-positive and immunoreactive for MSTN, IGF-I and IGF-I receptor. This study shows that a proliferative process occurs in the explants despite the death of the original muscle fibres, and new muscle fibres expressing growth regulators are formed by regeneration from myogenic precursors present in the explants at the time of sampling.

Hormonal control of the IGF system in the sea bream ovary.

Numerous studies have described the presence of an intragonadal IGF system involved in regulation of gametogenesis in teleost fish. In the present study, the in vivo effects of estradiol-17beta (E2) and growth hormone (GH) exposure on IGF-I, IGF-II, IGF1R, and IGFBP2 gene expression in sea bream ovary were monitored by RT-PCR during prereproductive and reproductive periods. The evidence demonstrates that both hormones investigated here affect the ovarian IGF system, showing that it is not only under GH control, but also can be regulated by sexual hormones; this hormonal modulation is related to reproductive phase.

Molecular cloning of fish alcohol dehydrogenase cDNA.

A cDNA encoding a putative alcohol dehydrogenase class III (ADH) was cloned from a cDNA library constructed from 7-day larvae RNA of the marine teleost Sparus aurata. The full length cDNA is 1350 nucleotides (nt) long and contains an ORF of 1128 nt [encoding 376 amino acid (aa) residues]. Identity of 82% was found with human class III ADH (305 of 372 aa compared), and only 62% identity with a fish (cod) ADH (234 of 375 aa compared). Northern hybridization analysis with the cDNA revealed a transcript of about 1.4-1.5 kb, which is expressed in all tissues from adult fish studied: skeletal muscle, heart muscle, kidney, gill filaments and liver, with the highest levels found in the kidney. The expression of ADH mRNA was determined also during early development of Sparus aurata by Northern blot analysis. ADH transcripts were detected in eggs and in embryos 4, 8 and 12 h after fertilization, as well as on all days post-hatching studied. The levels of expression decreased during early embryonal development, but increased 4-fold from day 1 to day 21 after hatching. The size of the transcript was identical to that of hepatic ADH. Our results suggest that maternal ADH mRNA is present in the eggs and embryos, which decreases as divisions and development occur, while after hatching ADH mRNA is expressed by the larval tissues.

Cloning and sequencing of the gilthead seabream (Sparus aurata) growth hormone-encoding cDNA.

The cDNA clones encoding gilthead seabream (gsb) (Sparus aurata) growth hormone (GH) have been isolated from a cDNA library prepared from seabream pituitary gland poly(A)+ RNA. The cDNA library was screened using red seabream and rainbow trout GH cDNAs. The complete nucleotide (nt) sequence of gsbGH has been determined. The cDNA sequence codes for a polypeptide of 204 amino acids (aa), including a putative signal peptide of 17 aa. The 5'- and 3'-untranslated regions of the message are 55 and 236 nt long, respectively. The predicted aa sequence of gsbGH revealed 97% homology with red seabream GH, 95% with tuna GH, 85% with yellowtail GH, and 65% with rainbow trout GH.

Are vitellin and vitellogenin coded by one gene in the marine shrimp Penaeus semisulcatus?

A cDNA clone encoding a female-specific ovarian protein (presumably vitellin, Vt) has been isolated from a cDNA library prepared from poly (A)+ RNA extracted from vitellogenic ovaries of the shrimp Penaeus semisulcatus. The cDNA library was constructed and screened using a major cDNA band which was observed following analysis of total cDNA products by gel electrophoresis. This band, as well as the cDNA insert purified from the library, was estimated to have 1.1 kb. Both hybridized to mRNA prepared from ovaries or hepatopancreas (HEP) of vitellogenic females and showed a faint signal with ovaries from non-vitellogenic females, but did not hybridize to HEP from non-vitellogenic females or to HEP from males or testes. The size of the transcripts from the ovary and HEP was estimated to be 1.1 kb, similar to that of the cDNA insert, suggesting that a full length cDNA had been synthesized. Furthermore, the identical sizes of the transcripts from ovary and HEP and the ability of the ovarian cDNA to detect a transcript in HEP mRNA suggest that Vt from the ovary and vitellogenin (Vg) from HEP are the gene products of one gene. Alternatively, the homology between Vt and Vg is very high.

Developmental and tissue-regulated expression of IGF-I and IGF-II mRNAs in Sparus aurata.

Recent studies have shown that homologues of the mammalian IGF-I and -II genes are also found in teleosts. We report here the cDNAs coding for IGF-I and IGF-II cloned from the gilthead seabream, Sparus aura ta. Sequence comparisons revealed that both IGFs have been well conserved among teleosts, although Sparus IGF-I is shorter bv three amino acid residues due to truncated B-and C-domains. Using the cloned cDNAs as probes, the relative expression of IGF-I and IGF-II mRNAs were assayed in different Sparus tissues. Sparus liver clearly contained the highest level of IGF-I mRNA while relatively high levels of IGF-II mRNA were found in liver, heart and gill using the ribonuclease protection assay. After GH administration the amount of IGF-I mRNA was increased by 220% in liver but no changes in IGF-II mRNA levels were detected in any tissue. We also assayed the expression of IGF-I and IGF-II in Sparus during early development. The IGF-II mRNA level was highest in larva I day after hatching and decreased thereafter. In contrast, IGF-I mRNA was detected in 1-day-old larva but there was an increase in expression in 12- and 16-day-old larva. These results demonstrated that the expression of IGF-I and IGF-II is highly regulated in teleosts and suggest that they play distinct roles during growth and development.

Growth hormone increases plasma levels of insulin-like growth factor (IGF-I) in a teleost, the gilthead seabream (sparus aurata).

A heterologous radioimmunoassay (RIA) was applied for the determination of immunoreactive (IR)--insulin-like growth factor (IGF-I) in a teleost, the gilthead seabream (Sparus aurata). Serial dilutions of the fish plasma gave a linear curve when added to constant amounts of 125I-labelled human IGF-I(53-70) and antiserum prepared against this fragment. The RIA was used to study the effect of GH on plasma levels of IR-IGF-I in S. aurata. A single injection of human recombinant GH (1 micrograms/g) resulted in a significant increase in IR-IGF-I at 29, 48 and 72 h, when compared with saline-injected fish. This novel observation suggests that in fish, as in mammals, circulating IGF-I levels are modulated by GH.

Expression and cellular localization of insulin-like growth factor-II protein and mRNA in Sparus aurata during development.

The spatial localization of IGF-II protein and mRNA was investigated during larval and postlarval developmental stages of the gilthead sea bream (Sparus aurata) by immunohistochemistry and in situ hybridization, using specific antisera and riboprobes. Steady-state levels of IGF-II mRNA in larvae were determined by Northern blot analysis and were found to be increased. Immunoreactivity towards IGF-II was found in larval skin, muscle, gills, gut, olfactory epithelium and kidney. After metamorphosis, the strongest immunoreactivity was found in red skeletal muscle. Positive reaction with IGF-II antibodies was also found in the olfactory epithelium and in the epithelia of pharynx, oesophagus, stomach and kidney. In the adult, the most intense signal was observed in the red and pink musculature and in heart musculature. Immunostaining was also found in saccus vasculosus, thymus, spleen and ovary. IGF-II mRNA was detected by in situ hybridization in the brain, olfactory epithelium, eye, pharynx, skeletal musculature and liver. The spatial distribution of IGF-II shown in this study is consistent with previous findings on the cellular localization of IGF type 1 receptor in the sea bream and supports a role for IGF-II during development and growth of sea bream. Furthermore, these results suggest that IGF-II acts in an autocrine/paracrine manner.

Recombinant gilthead seabream (Sparus aurata) insulin-like growth factor-I: subcloning, expression in Escherichia coli, purification and characterization.

Gilthead seabream (Sparus aurata) insulin-like growth factor-I (gsIGF-I) cDNA coding for the mature protein was cloned in a pGEM-3Z vector, and then transferred into prokaryotic expression vector pET-11a and expressed in Escherichia coli BL21(DE3) cells upon induction with isopropyl thiogalactoside. The expressed protein contained within the inclusion-body pellet was solubilized in 4.5 M urea, refolded for 24 h at pH 11.3 in the presence of catalytic amounts of cysteine and purified to over 98% purity, as a monomeric methionyl-gsIGF-I. Amino acid composition and N-terminal sequence confirmed the identity to be the predicted protein. Binding assays of the 125I-gsIGF-I to gilthead seabream or carp (Cyprinus carpio) sera resulted in high specific binding, indicating the existence of one or more IGF-binding proteins. In binding experiments to crude gilthead seabream brain homogenate, using human (h) IGF-I as a ligand, the respective IC50 value of hIGF-I was about fourfold lower than that of gsIGF-I. Recombinant gsIGF-I exhibited mitogenic activity in a mouse mammary gland-derived MME-L1 cell line which was approximately 200-fold lower than that of hIGF-1. Binding experiments to intact MME-L1 cells suggests that this difference most likely results from a correspondingly lower affinity for IGF-I receptor in these cells. In contrast, the activities of gsIGF-I and hIGF-I measured by 35S uptake by gill arches from the goldfish (Carassius auratus) were identical, indicating that the recombinant gsIGF-I is biologically active.

Developmental expression of the growth hormone gene in the gilthead sea bream Sparus aurata.

Expression of growth hormone (GH) gene during early stages of larval development of the teleost Sparus aurata was determined by Northern blot analysis. Poly(A+) RNA was prepared from a pool of larvae collected on different days after hatching. When hybridized to Sparus aurata GH cDNA, GH specific mRNA was first seen on day 6 post-hatching. In contrast, the levels of beta-actin mRNA, which was used to normalize for RNA amounts, were already high on the day of hatching. Our results suggest that expression of the GH gene is very low immediately after hatching, and increases dramatically within 6 days.

LH/hCG receptors and stimulation of testosterone biosynthesis in the rat testis: changes during foetal development in vivo and in vitro.

The development of gonadotropin receptors to LH/hCG in the foetal rat testis from 14 to 20 days of gestation was monitored by quantitative binding assays using [125I]hCG and compared to testosterone secretion under basal and stimulated conditions in vitro. Specific hCG binding was first detected on day 15. Thereafter the binding increased gradually with advancement in gestational age and correlated with LH-stimulated secretion of testosterone in vitro. On day 18 of gestation the KA was 0.82 x 10(10) M-1 and the binding capacity was 0.57 fmoles per testis. No binding was detectable in the female gonads at this age. The differentiation of hCG receptors obtained in vitro was very low, although it was sufficient to give a full response to LH with testosterone biosynthesis. The results of the present study suggest that functional receptors do not appear before the capacity to synthesize testosterone is expressed and that their appearance is not dependent on factors extrinsic to the testis. However, additional factors could be necessary for a full development of the receptors.

Cloning of putative piscine (Sparus aurata) transthyretin: developmental expression and tissue distribution.

cDNA encoding putative transthyretin (prealbumin, TTR) was cloned from liver of the marine fish Sparus aurata. The cDNA contains an open reading frame of 453 nt, encoding for a TTR precursor of 151 amino acids. The deduced amino acid sequence of S. aurata TTR shows identity of 54, 57.3 and 54.1% with lizard, chicken and rat TTR, respectively. Northern blot analysis revealed a TTR transcript of about 700 nt, highly expressed in liver, but also in skin. Low expression was detected in 12 other tissues by using RT-PCR. The ontogeny of TTR expression during early stages of larval development of S. aurata was examined by Northern blot analysis using poly(A+)RNA from larvae collected on different days after hatching. TTR mRNA was seen already on the first day after hatching and its steady-state levels increased from Day 15 onwards. Molecular cloning of a TTR-like cDNA from fish suggests that TTR evolved earlier in vertebrate development than previously thought. Furthermore, its expression in liver exceeds by several-fold that found in brain, yet high expression is also found in skin. These results suggest that in fish, liver is the main site of TTR synthesis, but that TTR may have an important function in fish skin.

Contrasting effects of estrogen on transthyretin and vitellogenin expression in males of the marine fish, Sparus aurata.

A partial cDNA encoding for the C-terminus of vitellogenin (VTG) was cloned from liver of Sparus aurata male treated with 17beta-estradiol (E(2)). E(2) treatment of S. aurata males resulted in increased synthesis and secretion of VTG protein into the plasma, determined by a specific enzyme-linked immunosorbent assay (ELISA) in a time-dependent manner. While VTG mRNA was induced by E(2) treatment, transthyretin (TTR) mRNA levels were reduced. These data provide the first demonstration that estrogen exhibits contrasting effect on VTG and on TTR gene expression in teleosts.

Piscine (Sparus aurata) alpha subunit of the G-protein transducin is homologous to mammalian cone and rod transducin.

A novel cDNA encoding alpha subunit of the GTP-binding protein, transducin, has been cloned from a marine fish, Sparus aurata. The cDNA contains an open reading frame of 1050 nt (encoding 350 amino acid residues). A high degree of identity was found with known mammalian transducin proteins of cones (Gt2 alpha) or rods (Gt1 alpha): human Gt2 alpha (80.2%), bovine Gt2 alpha (79.3%), mouse Tt1 alpha (78.2%), mouse Gt2 alpha (78%) and bovine Gt1 alpha (77.9%). Northern blot analysis of different tissues revealed a transcript of about 2.5 kb, which is expressed only in the fish eye and not in other tissues from adult fish, supporting its identification as transducin. Ontogeny of transducin mRNA expression during early development of Sparus aurata, determined by Northern blot analysis, showed very low levels in larvae 3 days after hatching but not earlier. Levels increased 3- and 6-fold on days 4 and 6 (respectively) compared with those on day 3 and remained essentially unchanged thereafter, until day 21 after hatching (the last day studied). Our results suggest that in fish only one alpha subunit of transducin is found, which shows similar identity with cone and rod alpha subunits of mammals.

Developmental expression, tissue distribution and hormonal regulation of fish (Sparus aurata) serum retinol-binding protein.

Retinol-binding protein (RBP) is the specific carrier of retinol in vertebrates and forms a 1:1 complex with transthyretin (TTR). A cDNA encoding serum RBP was cloned from liver and 7-day larvae of the marine fish Sparus aurata. The mature protein is 176 amino acids long and shows sequence identity of 77-78%, 56%, 63% and 62% with rainbow trout, Xenopus, chicken and human RBP, respectively. Northern blot analysis of hepatic RBP revealed two transcripts: a major one of approximately 1.4-1.5 kb and a minor of approximately 0.7 kb. Distribution of RBP mRNA in various tissues was studied by RT-PCR and showed high expression in liver and skin, and low expression in brain, kidney and gill filament (20-35% of the level in liver). RBP expression in intestine, pyloric caeca, muscle and pituitary was estimated to be approximately 7-14% of the level in liver. The ontogeny of RBP expression in S. aurata was examined in unfertilized eggs, embryos and larvae by using RT-PCR followed by hybridization with a specific probe. RBP transcript was found in all larval stages studied. Very low levels of RBP mRNA were detected in unfertilized eggs and in embryos 8 h after fertilization with a gradual increase at 12 h and 15-16 h post-fertilization. A single injection of estradiol-17beta to S. aurata immature, bisexual fish or to adult males reduced steady-state levels of hepatic RBP by 37 and 25%, respectively. The same treatment induced vitellogenin expression. The present data suggest that in fish, liver is the main site of RBP synthesis, but that RBP may have an important function in fish skin. RBP is expressed early in embryonic development and in fish its expression can be down regulated by estrogen.

Cellular localization of insulin-like growth factor-II protein in the sea bass (Dicentrarchus labrax) from hatching to adult.

The cellular localization of IGF-II protein was investigated during larval and postlarval developmental stages of sea bass (Dicentrarchus labrax) by immunohistochemistry using antisera raised against Sparus aurata IGF-II. At hatching, IGF-II immunoreactivity was already present in the skin, developing intestine and skeletal muscle. During larval life IGF-II protein was also observed in heart musculature, in kidney and gill epithelia as well as in liver. In fry skeletal muscle a moderate IGF-II immunostaining was detected in red fibres, whereas white muscle fibres exhibited a faint immunoreactivity. In adults, a marked IGF-II immunostaining was observed in red muscle fibres. A moderate immunoreactivity was also present in white fibres as well as in heart striated myocardial fibres. These results are in agreement with previous findings on the spatial localization of IGF-II and IGF type 1 receptor in S. aurata and Umbrina cirrosa, confirming the role of IGF system during development and growth of fish.

Characterization and functional analysis of the 5' flanking region of myosin light chain-2 gene expressed in white muscle of the gilthead sea bream (Sparus aurata).

The promoter region ( approximately 1400 bp) of myosin light chain 2 gene of fast skeletal muscle from the marine fish Sparus aurata was cloned, sequenced and characterized. It contains a consensus sequence for TATA box, six perfect E-boxes known as binding sites to myogenic basic helix-loop-helix transcription factors and four putative MEF2-binding sites. Three genomic fragments (truncated at their upstream region) of 244, 650 and 1400 bp showed promoter activity evidenced by muscle-specific reporter gene activity using transient expression of green fluorescent protein in microinjected zebrafish embryos and in skeletal muscle of S. aurata fry following intramuscularly injection of plasmid DNA. The three genomic fragments also directed luciferase activity in skeletal muscle of S. aurata fry following intramuscularly injection of plasmid DNA showing a 60 to 150-fold higher luciferase activity compared to that obtained with pGL3-Basic. These experiments show that the three genomic fragments are functional muscle-specific promoters which will be useful for directing myostatin and follistatin expression in fish muscle in order to study their effect on fish muscle growth.