Expression and functional analysis of Fushi Tarazu transcription factor 1 (FTZ-F1) in the regulation of steroid hormones during the gonad development of Fujian Oyster, Crassostrea angulata.

Crassostrea angulata, a major shellfish cultivated in Southern China, has experienced a notable surge in commercial value in recent years. Understanding the molecular mechanisms governing their reproductive processes holds significant implications for advancing aquaculture practices. In this study, we cloned the orphan nuclear receptor gene, Fushi Tarazu transcription factor 1 (FTZ-F1), of C. angulata and investigated its functional role in the gonadal development. The full-length cDNA of FTZ-F1 spans 2357 bp and encodes a protein sequence of 530 amino acids. Notably, the amino acid sequence of FTZ-F1 in C. angulata shares remarkable similarity with its homologues in other species, particularly in the DNA-binding region (>90%) and ligand-binding region (>44%). In C. angulata, the highest expression level of FTZ-F1 was observed in the ovary, exhibiting more than a 200-fold increase during the maturation stage compared to the initiation stage (P < 0.001). Specifically, FTZ-F1 was mainly expressed in the follicular cells surrounding the oocytes of C. angulata. Upon inhibiting FTZ-F1 gene expression in C. angulata through RNA interference (RNAi), a substantial reduction in the expression of genes involved in the synthesis of sex steroids in the gonads, including 3ß-HSD, Cyp17, and follistatin, was observed. In addition, estradiol (E2) and testosterone (T) levels also showed a decrease upon FTZ-F1 silencing, resulting in a delayed gonadal development. These results indicate that FTZ-F1 acts as a steroidogenic factor, participating in the synthesis and regulation of steroid hormones and thus playing an important role in the reproductive and endocrine systems within oysters.

Vitellogenin of Fujian oyster, Crassostrea angulata: Synthesized in the ovary and controlled by estradiol-17ß.

In this study, we cloned a full-length cDNA encoding vitellogenin (Vg) in the Fujian oyster Crassostrea angulata. The complete Vg cDNA consists of 5160 nucleotides with a long open reading frame encoding 1641 amino acid residues. The deduced amino acid sequence shared high similarity with the Vgs of other mollusc, fish, nematode and arthropod species, particularly in the N-terminal region. We analyzed the spatiotemporal expression of caVg transcripts by Real-time Quantitative PCR. In common with other mollusc Vgs, the caVg gene was expressed primarily in the ovary, and the levels were 348 and 177 times higher in maturation and ripeness stages (P<0.01), respectively, than in the partially spent stage. There was negligible expression in male oysters. In situ hybridization analysis further localized caVg mRNA to the follicle cells (also named auxiliary cells) surrounding the oocytes in the ovary. Moreover, in vivo waterborne exposure experiments in early gametogenesis oysters showed that estradiol-17ß (E2) administration resulted in a significant increase in caVg mRNA expression. We conclude that caVg is synthesized in the follicle cell surrounding the vitellogenic oocyte in C. angulata, and directly passed to oocytes through the extracellular space without mediation through hemolymph. Also, we hypothesize that this process is mediated by E2 in a dose dependent.

Comparison of species sensitivity distributions constructed with predicted acute toxicity data from interspecies correlation estimation models and measured acute data for benzo[a]pyrene.

Benzo[a]pyrene (BaP) is a priority Polycyclic Aromatic Hydrocarbon (PAH), which is toxic to aquatic organisms and has been widely detected in the environment. However, ecological risk assessment for BaP is hard to perform because of the absence of water quality criteria (WQC) and lack of toxicity data for this chemical. To fill in the data gaps, a interspecies correlation estimation (ICE) model was developed by USEPA to predict toxicity values for multiple species from the toxicity estimate for one species. In order to validate the applicability of the ICE model for BaP, measured-based-species sensitivity distributions (SSDs) generated using eight Chinese native aquatic species were compared with ICE-based-SSDs generated using the data predicted from three surrogate species (Lepomis macrochirus, Cyprinus carpio and Daphnia magna). The results showed that there were no significant differences between the two SSD curves and the two hazardous concentrations for the 5% of species (HC5) derived from measured acute toxicity data and ICE-based predicted data. The ICE model was verified as a valid approach for generating SSDs with limited toxicity data.

Expression of glycogen synthase (GYS) and glycogen synthase kinase 3ß (GSK3ß) of the Fujian oyster, Crassostrea angulata, in relation to glycogen content in gonad development.

To investigate the regulation of glycogen metabolism at the mRNA level in Crassostrea angulata, we cloned and characterized glycogen synthase and glycogen synthase kinase 3ß cDNAs (Ca-GYS and Ca-GSK3ß, respectively), which encode the primary enzymes involved in glycogen storage. We examined their expression profiles in different tissues and during different reproductive stages. The full-length cDNA of GYS was 4771 bp in length with a 2023 bp open reading frame (ORF), predicted to encode a protein of 674 aa. The full-length GSK3ß cDNA was 2333 bp long, with an ORF of 1242 bp. High expression levels of both genes were observed in the gonad and the adductor muscle, as compared to the mantle, gill, or visceral mass, which correlates well with the ability to store glucose. The regulation of both genes was correlated with glycogen content via qPCR and in situ hybridization and was dependent upon the stage of the reproductive cycle (initiation stage, maturation stage, ripeness stage). Thus, it appears that the expression of Ca-GYS and Ca-GSK3ß is driven by the reproductive cycle of the oyster, reflecting the central role played by glycogen in energy storage and gametogenic development in C. angulata. We suggest that Ca-GYS and Ca-GSK3ß can be used as useful molecular markers for identifying the stages of glycogen metabolism and reproduction in C. angulata.

Cloning and characterization of the dopamine like receptor in the oyster Crassostrea angulata: expression during the ovarian cycle.

We cloned and characterized a complete cDNA encoding a dopamine receptor (DAR) named Ca-DA1R from Fujian oyster, Crassostrea angulata. The 2843 bp long cDNA sequence includes a 916-bp 5'-UTR, the 1197 bp ORF which encodes a putative protein of 399 amino acids, and a 729 bp 3'-UTR. The Ca-DA1R sequence possesses typical characteristics of a D1 receptor: two main features being a short third intracellular loop and a long inner COOH-terminal tail domain. Using a real-time PCR approach, expression profiles of Ca-DA1R were analyzed in adult tissues and during the four stages of ovarian development. Ca-DA1R was expressed ubiquitously, although transcript levels varied between tissues, with higher mRNA levels detected in the ovary, labial palps and mantle. During the four stages of ovarian development, Ca-DA1R mRNA expression level was higher in the proliferation stage than in the other three stages during the ovary cycle. In situ hybridization results reveal that the Ca-DA1R mRNA is mainly expressed in the epithelium of the gonoducts. These observations suggest that Ca-DA1R binding of DA probably plays an important role in early ovarian development and via regulating oocyte locomotion cooperates with the 5-HT receptor system during the ovarian cycle in C. angulata.

Cloning and characterization of the follistatin gene from Crassostrea angulata and its expression during the reproductive cycle.

Follistatin is an activin-binding protein that prevents activin from binding to its receptor and neutralizes its activity. Follistatin plays a key role in regulating folliculogenesis and the development of ovary. However, limited information on follistatin genes from molluscs is available until now. By using Race, real-time PCR, in situ hybridization and in silico analysis, a full-length cDNA of follistatin of the Portuguese oyster Crassostrea angulata was acquired. The full-length (1297 bp) cDNA of Ca-follistatin encodes a peptide of 241 amino acids. The similarity of its deduced amino acid sequence to these of other invertebrate species was about 60%. Ca-follistatin mRNA transcript was most abundantly expressed in ovary (p<0.05), and it was also expressed in testis, adductor muscle, mantle, gill and visceral mass. In situ hybridization revealed that the expression and distribution of Ca-follistatin gene were expressed exclusively in granulosa cells, neither in cumulus oophorus nor in oocytes. During the reproductive cycle of female oyster (initiation stage, maturation stage, ripeness stage and partially spent stage), the expression of Ca-follistatin in the ovary continuously increased from initiation to ripeness stages attaining its highest value (p<0.05), then the expression level decreased sharply to the lowest point in the partially spent stage (p<0.05), whereas the Ca-follistatin mRNA transcript of male oyster in the testis maintained a relatively stable low level during the first three stages, and also noticeably decreased thereafter (p<0.05). These findings suggest that follistatin is likely to play an important role in the ovary development of oysters by autocrine signaling.