Growth hormone receptors in the atherinid Odontesthes bonariensis: characterization and expression profile after fasting-refeeding and growth hormone administration.

In order to improve the understanding of pejerrey Odontesthes bonariensis, growth hormone (Gh)-insulin-like growth factor-1(Igf1) axis, O. bonariensis growth hormone receptor type 1 (ghr1) and type 2 (ghr2) mRNA sequences were obtained. Both transcripts were ubiquitously expressed except in kidney, encephalon and anterior intestine. Alternative transcripts of both receptors were found in muscle. Interestingly, two different ghr2 transcripts with alternative polyadenylation (APA) sites located in the long 3' untranslated region (UTR-APA) were also found in liver. Hepatic ghr1, ghr2 and insulin-like growth factor type 1 (igf1) transcript levels were examined under two different metabolic conditions. In the first experimental condition, fish were fasted for 2 weeks and then re-fed for another 2 weeks. Despite igf1 mRNA relative expression did not show significant differences under the experimental period of time examined, both ghr transcripts decreased their expression levels after the fasting period and returned to their control levels after re-feeding. In the second treatment, recombinant O. bonariensis growth hormone (r-pjGh) was orally administered once a week. After 4 weeks of treatment, liver igf1, ghr1 and ghr2 mRNA relative expression increased (13, 4·5 and 2·1 fold, P < 0·05) compared to control values. These results add novel information to the growth hormone-insulin-like growth factor system in teleosts.

Evaluation of the genetic diversity of microsatellite markers among four strains of Oreochromis niloticus.

Different strains of Nile tilapia can be found worldwide. To successfully use them in breeding programs, they must be genetically characterized. In this study, four strains of Nile tilapia - UFLA, GIFT, Chitralada and Red-Stirling - were genetically characterized using 10 noncoding microsatellite loci and two microsatellites located in the promoter and first intron of the growth hormone gene (GH). The two microsatellites in the GH gene were identified at positions -693 to -679 in the promoter [motif (ATTCT)8 ] and in intron 1 at positions +140 to +168 [motif (CTGT)7 ]. Genetic diversity was measured as mean numbers of alleles and expected heterozygosity, which were 4 and 0.60 (GIFT), 3.5 and 0.71 (UFLA), 4.5 and 0.57 (Chitralada) and 2.5 and 0.42 (Red-Stirling) respectively. Genetic differentiation was estimated both separately and in combination for noncoding and GH microsatellites markers using Jost's DEST index. The UFLA and GIFT strains were the least genetically divergent (DEST  = 0.10), and Chitralada and Red-Stirling were the most (DEST  = 0.58). The UFLA strain was genetically characterized for the first time and, because of its unique origin and genetic distinctness, may prove to be an important resource for genetic improvement of Nile tilapia. This study shows that polymorphisms found in coding gene regions might be useful for assessing genetic differentiation among strains.

Isolation and characterization of 20 polymorphic microsatellite loci in the migratory freshwater fish Leporinus obtusidens (Characiformes: Anostomidae) using 454 shotgun pyrosequencing.

Twenty polymorphic microsatellite markers were developed for the Neotropical fish Leporinus obtusidens using a next generation sequencing approach and tested in two other characifomes species, Schizodon platae and Prochilodus lineatus. Microsatellite loci alleles in L. obtusidens ranged between 2 and 20 alleles per locus (mean = 5·7), with expected heterozygosity values ranging from 0·097 to 0·956 (mean = 0·578) and observed heterozygosity values ranging from 0·000 to 0·800 (mean = 0·400) in a sample of 20 specimens from the lower Paraná River (Argentina). Most of these markers will be a valuable tool for captive breeding and stocking programmes, as well as for analyses of population connectivity and genetic structure in this broadly distributed Neotropical migratory fish.

Molecular cloning, expression and immunological characterization of pejerrey (Odontesthes bonariensis) growth hormone.

Growth hormone is an essential polypeptide required for normal growth and development of vertebrates. The pejerrey fish, Odontesthes bonariensis, is a South American atherinid freshwater fish considered as a promising species for aquaculture. Although growth hormone has been characterized in a number of fish, there are no published data on the structure of this hormone in atherinids, except that of a related species Odontesthes argentinensis. In this paper, the molecular cloning, expression and immunological characterization of pejerrey growth hormone (pjGH) is described. The predicted amino acid sequence of pjGH cDNA consisted of 204 amino acid residues with an estimated molecular mass of 23 kDa. Amino acid sequence was highly conserved among the two Atheriniformes where the growth hormone sequences are known (99% aa identity), highly to moderately conserve (75-92% aa identity) when compared to the other members of Acantopterigii superorder and clearly less conserved (49-66% identity) when compared to Salmoniformes (Protacanthopterygii), Cypriniformes and Siluriformes (Ostariophysi). A phylogenetic tree depicting the relationship of various teleost GH nucleotide sequences was inferred. Pejerrey GH was produced using recombinant DNA technology in a bacterial system, representing the first time an atherinid growth hormone protein was expressed as a recombinant protein in Escherichia coli. A specific antiserum of this hormone was raised in rabbits and its specificity tested by using Western blot and immunocytochemistry. The distribution of pjGH mRNA was also studied by RT-PCR and Southern blot analysis. The transcript was detected not only in the pituitary gland but also in the testis.

Diffusible highly glycosylated protein from Bufo arenarum egg-jelly coat: biological activity.

L-HGP is a highly glycosylated protein from Bufo arenarum egg-jelly coat that diffuses into the surrounding medium when the strings of oocytes are incubated in saline solutions. L-HGP was purified from egg water and the estimated percentage of L-HGP/total protein in egg water was estimated in 30%. In the present study we examine, by indirect immunofluorescence, the effect of L-HGP on acrosome status of homologous spermatozoa. A high percentage (77%) of sperm lost the acrosome when incubated in 10% Ringer solution buffered with 10 mM Tris-HCl, pH 7.6, during 60 min, a condition that resembles egg-jelly osmolarity. The addition of purified L-HGP to the incubation medium prevents acrosome breakdown. The acrosome integrity is maintained for at least 1 hr. This effect is specific for L-HGP at concentration ranging from 0.01 to 0.1 mg/ml since neither BSA nor fetuin seems to have similar activity at similar concentrations. The same effect was observed when spermatozoa were incubated in egg water. Preliminary results suggest that L-HGP binds to B. arenarum spermatozoan membranes.

Identification of mRNA-binding proteins during development: characterization of Bufo arenarum cellular nucleic acid binding protein.

Ultraviolet irradiation was used to covalently cross-link poly(A)+RNA and associated proteins in eggs and embryos of the toad Bufo arenarum. Four major proteins with apparent sizes of 60, 57, 45 and 30-24 kDa were identified. It was observed that the same mRNA-binding proteins were isolated from eggs to gastrula and neural stages of development. The 30 kDa polypeptide, p30, appeared as the main ultraviolet (UV) cross-linked protein in the developmental stages analyzed. By means of polyclonal antibodies, it was determined that this polypeptide has a cytoplasmic localization and it was detected in liver, eggs and embryos. The presence of p30 was also analyzed by western blot during oogenesis and development. The 30 kDa polypeptide was present in all stages analyzed but it could not be detected in stages I-II of oogenesis. At the neural stage, the relative amount of p30 began to decrease, reaching its lowest levels after stages 26-30 (tail-bud in Bufo arenarum). On the basis of purification, immunoprecipitation and western blot assays the 30 kDa protein was identified as the Bufo arenarum cellular nucleic acid binding protein.

Bufo arenarum egg jelly coat: purification and characterization of two highly glycosylated proteins.

Egg jelly coats from Bufo arenarum are formed by components secreted along the oviduct. These secretion products overlay the oocytes as they transit along the different oviductal portions. In this study, we have isolated two highly glycosylated proteins of the jelly coat, which are secreted almost all the way along the oviduct. Both glycoproteins [designated as highly glycosylated protein (HGP) and low-molecular-mass highly glycosylated protein (L-HGP)] were purified to homogeneity, from the secretion of the caudal oviduct portion, by CsCl density gradient ultracentrifugation. HGP is a high-molecular-mass protein with mucin-like characteristics: high viscosity, a high content of serine and threonine, about 70% carbohydrate by weight, and a protease-resistant domain. Cleavage of disulphide bridges with reducing agents resulted in the release of a single subunit (300000 Da). L-HGP is also a disulphide-cross-linked protein with lower apparent monomeric molecular mass, in the range 100-120 kDa and containing 50% carbohydrate by weight. HGP contains galactose, fucose, N-acetylgalactosamine and sialic acid, but no mannose, suggesting the presence of O-linked oligosaccharides exclusively. The secretion ratio of HGP increases from cephalic (16% of total protein in pars preconvoluta) to caudal (40% of total protein in pars convoluta) oviductal portions. It appears to be the major structural component of the jelly coat. Our purification data suggest that HGP is non-covalently linked to the other egg jelly proteins. Polyclonal antiserum to each purified glycoprotein from secretion was raised in rabbits and used to localize both glycoproteins in the different oviductal portions, total egg jelly and the aqueous medium where oocyte strings were incubated. HGP forms a stable fibre matrix around the oocyte. L-HGP is present in the jelly coat and is released into the incubation medium.