Retinol binding protein in rainbow trout: molecular properties and mRNA expression in tissues.

Retinoids are important regulatory signaling molecules during embryonic development. The molecular properties of rainbow trout (Oncorhynchus mykiss) retinol-binding protein (rtRBP), the specific retinol carrier in vertebrate plasma, were studied to elucidate its role in transporting retinols to developing fish oocytes. A 954-nucleotide rtRBP cDNA was cloned from the liver coding for a 176-amino-acid (aa) mature protein, with an estimated molecular mass of 20,267 Da. The nucleotide sequence suggests a putative 16-aa signal peptide and shows all the aa residues that were previously identified as critical for the retinol binding pocket. Five of the eight amino acid residues that are associated with the interaction of RBP and transthyretin in mammalian and non-mammalian species are conserved. The deduced aa sequence of rtRBP shows 60-66% identity with zebrafish, chicken, mouse, rat, horse, bovine, and human RBPs and 56% identity with Xenopus RBP. Northern blot analysis revealed a approximately 1.1-kb hepatic mRNA transcript. RBP is highly expressed in the liver, but low levels were also detected in the spleen, kidney, ovary, and brain. In the rainbow trout, 17beta-estradiol treatment led to a decrease in the RBP mRNA signal relative to that of the controls. The efficacy of the 17beta-estradiol treatment was verified by an induction of vitellogenin (VTG) mRNA expression in the liver and occurrence of VTG in the plasma.

Tilapia glycoprotein hormone alpha subunit: cDNA cloning and hypothalamic regulation.

The cDNA encoding the glycoprotein alpha (GPalpha) subunit of tilapia (Oreochromis mossambicus) was partially cloned using RACE-polymerase chain reaction (PCR) technique. The amplified cDNA was found to be 583 bases long, and to consist of a portion of the signal peptide, the full sequence encoding the mature peptide (94 amino acids) and the 3' untranslated region. Northern blot analysis revealed a single band of approximately 600 bp. Alignment of the deduced amino acids of the mature protein showed that the tilapia GPalpha subunit shares more than 80% identity with that of other perciform fish (i.e. striped bass, sea bream and yellowfin porgy) and less than 70% with that of more taxonomically remote fish and other vertebrates. Exposure of dispersed tilapia pituitary cells to salmon gonadotropin-releasing hormone (sGnRH) elevated GPalpha mRNA levels via both PKC and cAMP-protein kinase A (PKA) pathways. The transcript levels were also regulated by pituitary adenylate cyclase activating polypeptide (PACAP) and neuropeptide Y (NPY), both acting through PKC and PKA pathways. Moreover, a combined treatment of PACAP or NPY with GnRH seems to have an additive effect on the GPalpha subunit gene transcription. These results suggest that in tilapia the expression of GPalpha subunit is regulated by GnRH mainly via PKC and PKA pathways. Furthermore, PACAP and NPY can elevate the GnRH-stimulated GPalpha subunit transcription and can directly affect the subunit mRNA levels, via the same transduction pathways.

Characterization of tilapia FSHbeta gene and analysis of its 5' flanking region.

The objective of the current study was to unveil molecular mechanisms underlying transcriptional regulation of the FSHbeta gene expression in the pituitary of tilapia (Oreochromis mossambicus). The full-length sequence of tilapia FSHbeta (tFSHbeta) gene was determined. Its transcriptional unit (2.7 kb) exhibits the conserved genomic organization, i.e. three exons and two introns. Primer extension and RT-PCR analysis revealed heterogeneity of the tFSHbeta transcripts, due to alternate mRNA splicing and multiple initiation sites for transcription. Examination of the 5' flanking region (5'FR) of the tFSHbeta gene identified potential CAAT and TATA promoter proximal elements as well as several sequences of cis-acting motifs known to dictate inducible and tissue-specific transcriptional regulation in other gonadotropin genes. Chimeric constructs containing 1.7 kb of the tFSHbeta 5'FR fused to a luciferase (LUC) reporter gene were transiently transfected into primary culture of tilapia pituitary cells. The tFSHbeta-LUC construct was efficiently expressed under basal conditions and was rapidly induced by GnRH stimulation. Our data indicate that the 5'FR contains a functional promoter, which is responsive to GnRH treatment. In addition, 5' deletion analysis showed that the 1.7 kb, DNA sequence of the FSHbeta 5'FR encompasses both positive and negative regulatory elements.

Molecular components related to egg viability in the gilthead sea bream, Sparus aurata.

In pelagic egg spawners, the production of large numbers of sinking eggs, unable to develop into embryos, represents one of the major limiting factors in controlled reproduction. The aim of this study is to elucidate the molecular differences between floating and nonfloating eggs at cytoplasmic and nuclear level. Comparison of analyses between floating and nonfloating sea bream Sparus aurata eggs evidenced differences in vitelline envelope protein components, such differences being probably related with the hydration process but not with fertilization as supported by the assessment of DNA that doubled after in vitro insemination. These data clearly indicated that the absence of embryo development in nonfloating eggs is not due to lack of fertilization. The cytoplasmic composition was also different, the number of protein components being higher in floating eggs, and these extra components may generate the appropriate osmotic pressure at the base of the hydration process. Some lysosomal enzymes, such as cathepsin D and L both involved in yolk proteolysis, in virgin nonfloating eggs were significantly higher with respect to floating ones; the levels of these two enzymes significantly increased in the latter after fertilization. On the contrary, in nonfloating eggs cathepsin L significantly decreased after fertilization. These changes may be related with a series of metabolic processes vital for the production of viable offspring. The capacity of egg transcription and the protein synthesis in these two types of eggs, indicated by the RNA/DNA and RNA/protein ratios, evidenced that the status of cell transcription rate and protein synthesis capacity is significantly higher in floating eggs. This, in turn, suggested that the lack of embryo development may be due to low levels of proteins involved in cell cycle regulation.

Levels of the native forms of GnRH in the pituitary of the gilthead seabream, Sparus aurata, at several characteristic stages of the gonadal cycle.

Brains of the gilthead seabream, Sparus aurata, contain three different forms of gonadotropin-releasing hormone (GnRH): seabream (sb) GnRH, chicken (c) GnRH-II, and salmon (s) GnRH. In the present study, we developed three specific enzyme-linked-immunosorbent assays (ELISA) for sbGnRH, cGnRH-II, and sGnRH and used them to measure the levels of each GnRH form in the pituitary of male and female seabream at different stages of gametogenesis. The sensitivity was 6 pg/well for the sbGnRH assay, 7 pg/well for the cGnRH-II assay, and 2 pg/well for the sGnRH assay. Levels of each of the three GnRH forms were measured in pituitaries from fish sampled at the beginning of gonadal recrudescence and during the spawning season. Of the three forms, only sbGnRH and cGnRH-II were detected in the pituitary, irrespective of reproductive state or sex. Recrudescent fish had similar levels of sbGnRH and cGnRH-II in the pituitary. In sexually mature fish, the levels of sbGnRH were higher than those in recrudescent fish while pituitary cGnRH-II content remained unchanged. Consequently, sbGnRH levels were 3- to 17-fold higher than cGnRH-II levels in mature fish. Positive correlations also existed between pituitary sbGnRH content and pituitary and plasma gonadotropin (GtH) II levels. Surprisingly, mature 1-year-old males had significantly higher levels of sbGnRH in the pituitary than mature 3-year-old males, while pituitary and plasma GtH II levels were similar between these two groups. Although the reason for this difference in sbGnRH levels is unclear, a possible role of sbGnRH in the processes of puberty or sex-inversion is implied. Based on the present results, it can be suggested that in the gilthead seabream, sbGnRH is the most relevant form of GnRH in the control of reproduction.

Preovulatory changes in the levels of three gonadotropin-releasing hormone-encoding messenger ribonucleic acids (mRNAs), gonadotropin beta-subunit mRNAs, plasma gonadotropin, and steroids in the female gilthead seabream, Sparus aurata.

Gilthead seabream females undergo daily cycles of final oocyte maturation (FOM), ovulation, and spawning throughout their spawning season. FOM consists of lipid droplet and yolk granule coalescence, germinal vesicle (GV) migration, and GV breakdown. Plasma maturational gonadotropin (GtH-II) levels fluctuate throughout the day, reaching a peak at 8 h before spawning, when the GV is at the periphery of the oocyte. The preovulatory GtH-II surge is accompanied by an increase in the plasma levels of 17alpha,20beta-dihydroxy-4-pregnen-3-one and estradiol, while testosterone and 17alpha,20beta,21-trihydroxy-4-pregnen-3-one levels remain unchanged. Concurrent with the preovulatory GtH-II surge, there is an increase in pituitary GtH-II beta subunit mRNA levels followed by an increase in GtH-Ibeta mRNA levels. Gilthead seabream brain contains three different forms of GnRH: salmon (s)GnRH, seabream (sb)GnRH, and chicken (c)GnRH-II. All three GnRH-encoding mRNAs fluctuate throughout the day, reaching highest levels 8 h before spawning, concurrent with the preovulatory GtH-II surge. On the basis of these correlations and of the anatomical organization of the three GnRH systems, it is hypothesized that in the daily-spawning gilthead seabream females, preovulatory GtH-II secretion, and probably synthesis, are induced by a surge of sbGnRH secretion. The involvement of the other two GnRH forms, sGnRH and cGnRH-II, in the control of ovulation and spawning is presumed, on the basis of the elevation of their mRNA levels at the time of the preovulatory GtH-II secretion and spawning.

Differential effect of components of the extracellular matrix on differentiation and apoptosis.

BACKGROUND: Epithelial cells are closely associated with a basement membrane, but the intimate relationships that affect growth, differentiation and survival remain enigmatic. We have previously reported that granulosa cells adjacent to the basement membrane of the ovarian follicle have a higher degree of differentiation compared with cells located distal to the basement membrane. By contrast, granulosa cells distal to the basement membrane are the first to undergo apoptosis during follicular atresia. Moreover, growth of granulosa cells in vitro on a naturally produced basement-membrane-like extracellular matrix (ECM) enhances progesterone production and the cellular response to gonadotropic hormones by an undefined mechanism. RESULTS: To investigate the effect of the ECM on granulosa cell differentiation and death, primary granulosa cells were cultured on ECMs that lacked or contained bFGF (basic fibroblast growth factor). These otherwise identical ECMs were deposited by HR9 mouse endodermal cells, which do not synthesize bFGF, or by HR9 cells transfected with the bFGF gene. Both ECMs provided protection against apoptosis in serum-free medium, but only the bFGF-containing ECM maintained expression of the steroidogenic P450scc enzyme system and the production of progesterone. Moreover, culturing the cells on this ECM enhanced the expression of the 30 kDa steroid acute regulatory protein which plays a key role in steroid hormone biosynthesis. Laminin, but not fibronectin, was able to replace the ECM in protecting the cells from apoptosis; but not in maintaining steroidogenesis, whereas bFGF was able to enhance steroidogenesis without protecting the cells against apoptosis. Cells cultured on both ECMs or laminin had a well-developed actin cytoskeleton compared with cells cultured on non-coated dishes, which underwent apoptosis. CONCLUSIONS: Cellular responses to ECM are mediated by the combined action of macromolecular constituents and regulatory molecules, such as bFGF, that are sequestered and stored in the ECM. ECM or laminin protects against cell death by interacting with specific integrin receptors and maintaining a well-developed actin cytoskeleton. ECM-bound bFGF provides differentiation signals for granulosa cells, which are in intimate contact with the ECM. Thus, a clear distinction can be made between the survival activity and the differentiation stimulus exerted by the ECM on epithelial cells.

Gonadotropins beta-GtHI and beta-GtHII from the gilthead seabream, Sparus aurata.

Full-length cDNA sequences encoding the beta-subunits of the gonadotrophins GtHI and GtHII were isolated, cloned, and sequenced from a single gilthead seabream (Sparus aurata) pituitary using RACE PCR. Beta-GtHI and beta-GtHII degenerate PCR primers were designed according to regions of high amino acid sequence homology between the chum salmon and the bonito beta-GtHI or beta-GtHII. DNA sequence analysis of the cloned PCR products confirmed the presence of the predicted complete coding region as well as 5' and 3' untranslated sequences. The deduced amino acid sequences of beta-GtHI and beta-GtHII from the gilthead seabream were compared to GtH sequences from a number of teleosts. Seabream beta-GtHI shows the highest homology to the bonito beta-GtHI (76%) and the striped bass (74%), while seabream beta-GtHII is 98% homologous to the yellow fin porgy, 87% homologous to the bonito, and 93% homologous to the striped bass beta-GtHII. Northern blot analysis showed the transcripts of the gilthead seabream beta-GtHI and beta-GtHII to be about 700 and 740 bases, respectively. During the spawning season, beta-GtHI is expressed at higher levels in males than in females while beta-GtHII is expressed at similar levels in both sexes.