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Eggs from Bufo arenarum are surrounded by four layers of jelly that are needed for fertilization. The mechanism of sperm penetration through these layers is unknown and should not involve acrosome content, as the acrosome reaction seems to occur at the vitelline envelope. The jelly coat is composed of a gel-forming mucin and soluble proteins. One of the main soluble protein is L-HGP, a mucin-like protein. L-HGP is a disulfide-linked heterodimer with O-linked oligosaccharides and estimated [M.sub.r] between 200 and 250. L-HGP expression was detected only in the oviduct. Immunolocalization studies indicated that this protein is present in the apical region of secretory epithelial cells all along the oviduct. Immunoblot analyses on tissue homogenates and secretions of different oviductal portions showed that L-HGP is expressed in all of them and may be present in the four jelly layers. However, two isoforms were detected in different portions. The variability in molecular mass observed between them is due to a difference in the peptide core. This could be related to the gain-loss of some function of the protein depending on the jelly layer localization. Binding assays showed that L-HGP interacts with spermatozoa membranes. Moreover, incubation of spermatozoa with this protein prevents acrosome from breakdown. These combined results suggest that L-HGP may contribute to the migration of spermatozoa through the jelly layers providing antiadhesive properties and preventing acrosome reaction until the spermatozoa reach the vitelline envelope.

Abstract: Many fish species display compensatory growth (CG), a phenomenon by which fasted fish grow faster during refeeding. However, most studies use a group‐housed fish approach that could be problematic in social fish when interaction between individuals is not considered or eliminated. Additionally, the growth hormone (GH)/insulin‐like growth factors’ (IGF‐1 and IGF‐2) axis is implicated in postnatal growth in vertebrates, but its relevance in CG is not fully understood. Thus, the aim of this work was to determine whether CG occurs in a social fish, Cichlasoma dimerus, using an individually held fish approach and secondly, to evaluate the GH/IGFs expression profile during refeeding by 3 days and 3 weeks. C. dimerus showed partial CG. The feed conversion efficiency (FCE) was higher in three‐day‐refed fish, which presented higher GH plasma and mRNA levels than controls but shown no differences in liver and muscle GH receptors (GHR1 and GHR2) and IGFs mRNA levels. Surprisingly, three‐week‐refed fish exhibited GHR1 and IGF‐2 increments, but a reduction in GHR2 expression in muscle. These results show a strong association between GH levels, growth rate and FCE during refeeding, and a long‐lasting effect of refeeding on muscular expression of GHRs and IGF‐2. [ABSTRACT FROM AUTHOR]

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.

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., (Copyright 2000 Wiley-Liss, Inc.)

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.

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.