Organization and promoter analysis of a tiger shrimp Penaeus monodon single WAP domain-containing protein gene

The main function of the single whey acidic protein domain (SWD)-containing protein in shrimp is unknown. To elucidate the function of the SWD-containing protein in vivo, the SWD-containing protein gene was isolated and characterized. A 9.3-kb shrimp SWD-containing protein gene, and a 3.9-kb 3′-flanking region. The shrimp SWD-containing protein gene contained three exons and two introns. Different fragments of the shrimp SWD-containing protein 5′-flanking region were transfected into HeLa cells. The promoter activities were assayed by basal human chorionic gonadotropin (HCG), luteinizing hormone-releasing hormone (LRH), and gonadotropin-releasing hormone (GnRH) treatments. The in vitro actions of the SWD-containing protein promoter expression pattern were studied by transfection of an SWD-containing protein promoter (1 kb)-driven green fluorescent protein (GFP) encoding the GFP cDNA transgene into the HeLa cell line, which was then microinjected into zebrafish Danio rerio embryos. These results indicate that the shrimp SWD-containing protein promoter might play an important role in gene regulation of sex hormones in mammalian cell lines and in gene regulation of developmental stages in zebrafish.     

Molecular cloning,characterization and expression of FSH and LH beta subunits from grass carp (<Emphasis Type="Italic">Ctenopharyngodon idella</Emphasis>)

Follicle-stimulating hormone beta subunit (FSHβ) and luteinizing hormone beta subunit (LHβ) have been cloned and characterized from the pituitary of grass carp (Ctenopharyngodon idella). The full length of FSHβ and LHβ cDNA was 393 bp and 441 bp, with open reading frame encoding proteins of 130 and 146 amino acids, respectively. Phylogenetic analysis of the proteins of FSHβ and LHβ showed a high homology with other fishes. Homology analysis also indicated that LHβ has higher conservation than FSHβ. The expression analysis of grass carp FSHβ and LHβ by RT-PCR suggested that they were only expressed in the pituitary. Real-time quantitative PCR protocols were developed and validated to measure FSHβ and LHβ mRNAs during ovarian development. The FSHβ and LHβ mRNA level was very low in the pituitaries of early-pubertal fish and significantly increased during the ovulation period. These results suggested that in grass carp the gonadotropins synthesized synchronously in order for asynchronous oogenesis to take place.     

Immunoreactive changes in pituitary FSH and LH cells during seasonal reproductive and spawning cycles of female chub mackerel <Emphasis Type="Italic">Scomber japonicus</Emphasis>

The physiological functions of pituitary gonadotropins (GtHs) are well established in higher vertebrates, whereas those in teleosts are still poorly understood. To describe the role of GtHs during gonadal development of female chub mackerel Scomber japonicus, changes in follicle-stimulating hormone (FSH) and luteinizing hormone (LH) cells were investigated immunohistochemically during the seasonal reproductive and spawning cycles. FSH and LH cells were identified in the different cell types of the proximal pars distalis (PPD); FSH cells were located in the central PPD, whereas LH cells were localized along the border of the pars intermedia. To examine changes in FSH and LH cells, the percentage of FSH or LH cell-occupying area in the PPD was evaluated and represented as FSHβ-immunoreactive (ir) or LHβ-ir levels, respectively. FSHβ-ir levels increased significantly from immature to the completion of vitellogenesis, whereas LHβ-ir levels were maintained at high levels from early vitellogenesis to post-spawning. During the spawning cycle, which consisted of four stages from just after spawning to the next oocyte maturation, both FSHβ-ir and LHβ-ir levels showed no significant changes among different stages; however, LHβ-ir levels remained relatively high, and FSHβ-ir levels were constantly low. These results suggest that both FSH and LH may be involved in vitellogenesis and LH may act at final oocyte maturation in female chub mackerel, although the role of FSH during the spawning cycle is still unclear.     

Cloning,characterization, and expression analysis of a CC chemokine gene from turbot (<Emphasis Type="Italic">Scophthalmus maximus</Emphasis>)

The chemokines are a superfamily of chemotactic cytokines playing an important role in leukocyte chemotaxis. Here, a turbot head kidney cDNA library was constructed in which KC70 was identified as a CC chemokine. Unknown 5′ and 3′ parts of the cDNA were amplified by 5′ and 3′ rapid amplification of cDNA ends (RACE). The complete cDNA of KC70 contains a 59-bp 5′ UTR, a 336-bp ORF, and a 152-bp 3′ UTR. Four exons and three introns were identified in KC70. Phylogenetic analysis showed that KC70 was similar to CCL19. In normal turbot KC70 was expressed in all tissues except brain and skin. Infection of turbot with pathogenic bacteria significantly increased expression of KC70 in the liver. Expression of KC70 in head kidney first increased and then decreased after bacterial challenge. No significant change was observed in the spleen after bacterial challenge. During embryonic development, KC70 was highly expressed after the gastrula stage. These results indicated KC70 plays important and multiple roles in turbot immune response.     

Molecular cloning of cDNA of mammalian and chicken II gonadotropin-releasing hormones (mGnRHs and cGnRH-II) in the beluga (<Emphasis Type="Italic">Huso huso</Emphasis>) and the disruptive effect of methylmercury on gene expression

Two gonadotropin-releasing hormone (GnRH) isoforms were identified in the beluga (Huso huso) brain by cDNA sequencing: prepro-mammalian GnRH (mGnRH) and prepro-chicken GnRH-II (cGnRH-II). The nucleotide sequences of the beluga mGnRH and cGnRH-II precursors are 273 and 258 base pairs (bp) long, encoding peptides of 91 and 86 amino acids, respectively. To investigate the effect of methylmercury (MeHg) on GnRH gene expression, animals were fed with four diets containing increasing levels of MeHg (0 mg kg⁻¹ [control]; 0.76 mg kg⁻¹ [low]; 7.8 mg kg⁻¹ [medium]; 16.22 mg kg⁻¹ [high]) for 32 days. The effects of MeHg on brain GnRH mRNA levels were evaluated by real-time PCR. A significant decrease in brain mGnRH and cGnRH-II mRNA levels were detected in fish receiving high dietary MeHg dose compared to controls on day 11 (P < 0.05). On day 18 and 32, all treatment groups had significantly lower brain mGnRH and cGnRH-II mRNA levels compared to the control group (P < 0.05). These findings demonstrate a disruptive role of MeHg on the level of brain mGnRH and cGnRH-II mRNAs in immature beluga.     

The duality of teleost gonadotropins

The duality of salmon gonadotropins has been proved by biochemical, biological, and immunological characterization of two chemically distinc gonadotropins. GTH I and GTH II were equipotent in stimulating estradiol production, whereas GTH II appears to be more potent in stimulating maturational steroid synthesis. The ratio of plasma levels and pituitary contents of GTHs and the secretory control by a GnRH suggest that GTH I is the predominant GTH during vitellogenesis and early stages of spermatogenesis in salmonids, whereas GTH II is predominant at the time of spermiation and ovulation. GTH I and GTH II are found in distinctly separate cells. In trout, GTH I is expressed first in ontogeny, whereas GTH II cells appear coincident with the onset of spermatogenesis and vitellogenesis, and increase dramatically at the time of final reproductive maturation. Comparison of the amino acid sequences of polypeptides and the base sequences of cDNA revealed that salmon GTH I β is more similar to bovine FSHβ than bovine LHβ and salmon GTH II β shows higher homology to bovine LHβ than to bovine FSHβ. The existence of two pituitary gonadotropins in teleosts as well as tetrapods suggests that the divergence of the GTH gene took place earlier than the time of divergence of teleosts from the main line of evolution leading to tetrapods.     

Isolation and characterization of the rbcS genes from a sterile mutant of Ulva pertusa (Ulvales,Chlorophyta) and transient gene expression using the rbcS gene promoter

We isolated two different genomic DNAs (UprbcS1 and UprbcS2) encoding the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase and portions of the 5′- and 3′-flanking regions from sterile Ulva pertusa Kjellman. The UprbcS1 and UprbcS2 genes had three introns in the coding region. Each predicted UprbcS polypeptide was a 180-amino-acid (AA) residue including a 38-AA transit peptide, although the 104th AA residue was replaced. The nucleotide sequences of UprbcS cDNAs isolated from a cDNA library corresponded to that of the UprbcS1 gene, suggesting that the UprbcS1 gene was predominantly expressed in sterile U. pertusa compared to UprbcS2. Southern blot analysis showed that each UprbcS gene was a single-copy gene in the sterile U. pertusa genome. Northern hybridization indicated that the expression of UprbcS was induced and repressed by dark and light treatments, respectively. When sterile U. pertusa cells were transformed with an expression vector containing the UprbcS1 promoter and terminator sequences fused with the green fluorescent protein (GFP) gene, GFP fluorescence was observed in the cells transformed. These results suggest that the UprbcS1 gene promoter is light regulated and highly active in the sterile U. pertusa cells and is available for genetic transformation system in the alga.     

Selective ligand-binding determinants in catfish and human gonadotropin receptors

In mammals, the specificity of FSH–FSH receptor (FSHR), LH–LH receptor (LHR) and TSH–TSH receptor couples is such that no cross-activation occurs under normal physiological conditions. The interactions between fish gonadotropins and their receptors, however, appear to be less discriminatory. For example, the catfish FSHR is highly responsive to both catfish LH and catfish FSH, while the catfish LHR is specific for its cognate LH. Comparative structure–function studies aimed at elucidating the molecular basis of ligand promiscuity (in fish) and ligand selectivity (in mammals) are described in this paper.