Research On Three Spermatogenesis Related Genes From The Crayfish Cherax Quadricarinatus

Reproductive biology is one of the most important researches in biology, it involves many important biological problems, such as germ cells ontogenesis, development, sex determination, genetics and evolution, these mechanisms of biological processes has been arisen many researchers'concerns at home and abroad. Currently the molecular mechanism for germ cell ontogenesis mainly focused on humans and other common model animals, and there are little related molecular mechanism reports on the aquatic organism especially in crustaceans shrimps and crabs, and the molecular mechanisms on male germ cells is even rare. Therefore in our present study, we selected the freshwater crayfish Cherax quadricarinatus, which is a commercially important aquatic organism, as the experimental materials and to study the molecular mechanism on ontogenesis and spermatogenesis.The "red claw" crayfish Cherax quadricarinatus (von Martens,1898)(Astacida, Parastacidae) is an important species for culture since1985for consumption and acquarism. At present, it is cultured intensively and semi intensively in many countries including Australia, United States, China, Ecuador, Mexico and Argentina. As C. quadricarinatus is concerned, the knowledge about the reproductive biology of males is limited to the anatomy of the reproductive system and formation and transference of the spermatophore. Therefore, a better understanding of the molecular mechanism of spermatogenesis has become a research priority at the moment.Hence in this paper, the major contents are as follows:(1) molecular cloning of three spermatogenesis closely related genes DDX5, HSP70, Prohibitin, and their expression patterns at different spermatogenesis phase;(2) reproduction performance and temporal expression patterns of Cq-DDX5after unilateral eyestalk-ablation;(3) localization of the Cq-HSP70protein in testes was studied by immunohistochemical analyses;(4) immunofluorescence analysis on localization of the Cq-PHB protein in testes (5) HSP70and Prohibitin protein expression analysis on different testes development stage by Western Blot; and (6) localization of Cq-PHB in sperm mitochondria by immunogold electron microscopy to find out its biological function in mitochondria membrane proteins and explore the mitochondria maternal inheritance mechanism in invertebrate.The mainly results of this paper are as following:1. Using degenerated PCR amplification strategy, DDX5, HSP70and Prohibitin cDNA fragments were cloned from the crayfish testes, and their length are368bp,493bp,302bp respectively. After RACE PCR,2,258bp,2,366bp,1,472bp full-length cDNA of DDX5, HSP70and Prohibitin are obtained, which containing the open reading frame (ORF) encoded522aa (1,569bp),652aa (1,959bp),275aa (828bp) respectively.2. BLAST search against the databases revealed that the deduced amino acid sequence shows high similarity to other similar protein sequence. Cq-DDX5, Cq-HSP70and Cq-PHB are highly conserved multigene superfamily. The deduced amino acid sequence of Cq-DDX5has a53-90%similarity to DDX5of other eukaryotic species, and Cq-HSP70has73-95%similarity with others, and Cq-PHB has55-92%similarity. A neighbor-joining (NJ) phylogenetic tree was constructed using MEGA software version4.0. Phylogenetic analysis results also showed that C. quadricarinatus clustered with other invertebrates, and most closely to that of the gene products encoded by other shrimp or crab species, which supports the traditional taxonomic relationships.3. Using InterPro searches the predicted amino acid sequences showed:the Cq-DDX5comprise nine consensus sequence characteristic of the DEAD-box proteins, Q-motif of dead-box RNA helicase profile and DNA/RNA helicase (DEAD/DEAH box) domain. Three HSP70family signature motifs were identified in the Cq-HSP70protein sequence, many potential phosphorylation sites, a calcium-binding domain profile (EF-HAND-2), a glycine-rich region profile motif (GLY-RICH), a nebulin repeat profile (NEBULIN) and a bipartite nuclear localization signal profile (NLS-BP) were also identified. But the primary structure of Cq-PHB protein lacks motifs typical for signal sequences, nuclear localization signals, ATP-binding sites or transcription factors. The comparison between PHB genes in other species discovers eight highly conserved regions with four of them corresponding to binding sites of known transcriptional control proteins (CCAAT box,'SV40'sites and two Spl sites) were identified in the Cq-PHB protein sequence.4. The mRNA transcript of Cq-DDX5, Cq-HSP70and Cq-PHB were expressed universally in all the organs investigated, including the brain, eyestalk, gill, thoracic ganglia, heart, muscle, hemocytes, hepatopancreas, stomach, intestine, testes and ovaries. Expression was at the high level in high differentiation organizations such as gonads, hepatopancreas and hemocytes. Further, the temporal expression patterns on each gene transcripts are as follows:(1). During the course of embryonic development, the expression of Cq-DDX5is very low in the fertilized egg stage but significantly up-regulate at the cleavage stage, and maintain at high level at gastrula stage and sharply up-regulate to peak at the nauplii stage and maintained at high level even in one-day larvae stage. In testes developmental period, the expression of Cq-DDX5was significantly low during absence period and resting phases when compared to the developmental period and multiplication phase, and a highest level was seen at the developmental phase and a lowest level at the resting level. In the mature male testes seasonal/annual cycle, the Cq-DDX5transcript up-regulate sharply and peak at the prespawning phase and maintain high level at the spawning phase, and then down-regulate and decline drastically during post-spawning/regressed phase. After eyestalk ablation, the relative testicular weight of destalked males was higher than that of intact males, but decreased sharply after the second week and these differences were not statistically significant. Interestingly, a gradual increase in Cq-DDX5expression is seen from0d to3d time interval, and significantly increase at6d and peak at12d after eyestalk ablation treatment group then declined drastically at18d.(2). In the testes developmental cycle, Cq-HSP70mRNA was up-regulated to peak expression during the developmental phase, and high expression was maintained during the spermatogonial multiplication period. Then the expression was down-regulated during the mature sperm stage, and significantly lower expression levels were found during the resting phase (P<0.05). In the seasonal/annual reproductive cycle, significantly greater expression (P＜0.05) was found in the preparatory (January-March), pre-spawning (April) and spawning (May-August) phases compared with the post-spawning/regressed phases (November-January). Overall, Cq-HSP70mRNA transcripts were maintained at high levels during the spermatogenesis and spawning stages.(3). In the testes developmental cycle, Cq-PHB mRNA was up-regulated during the developmental phase, and high expression was maintained during the spermatogonial multiplication period. Then the expression was down-regulated during the mature sperm stage, and lower expression levels were found during the resting phase (P<0.05).5. Western Blot results showed that a distinct single band characteristic of Cq-HSP70was observed on the immunoblot when the protein extracts were transferred to a nitrocellulose membrane and immunoprobed with anti-HSP70. The anti-HSP70recognized bands of approximately70kDa, which matches well with the calculated molecular mass for CqHSP70. Furthermore the Immunohistochemistry (IHC) results showed that immunoreactive positive signals (in brown color) for the HSP70protein were detected in spermatocytes, spermatids and spermatozoa of normal mature testes. Within the testes, the strongest signals for Cq-HSP70were found in spermatogonia, with lower positive signals in secondary spermatocytes, and weak or absent signals in mature sperm. Moreover, the Cq-HSP70protein was concentrated mainly in the cytoplasm of developmental sperm cells.6. When the protein extracts were transferred to a nitrocellulose membrane and immunoprobed with anti-PHB, a series of ladder bands characteristic of Cq-PHB was observed on the immunoblot. The anti-PHB recognized bands of approximately from180kDa to30kDa. Further the Immunofluorescence (IF) results showed that immunoreactive positive signals (blue fluorescence) for the PHB protein were detected in spermatocytes, spermatids and spermatozoa of normal mature testes. Within the testes, the strongest signals for Cq-PHB were found in spermatogonia, with lower positive signals in secondary spermatocytes, and weak or absent signals in mature sperm. Moreover, the Cq-PHB protein was concentrated mainly in the cytoplasm of developmental sperm cells and the peripheral'sertoli cells'. The Immunogold Electron Microscopy (IEM) results showed that Prohibitin was found mainly on the mitochondrial inner membrane of the spermatozoa, with some labeling in the matrix and free mitochondria particles. Prohibitin immunoreactivity was also observed on the spermatozoa nucleus but not any signals in negative preparations with omission of first antibody.Taken together the above results, we first reported Cq-DDX5, Cq-HSP70and Cq-PHB are new members in'DEAD-box'protein family, HSP superfamily and Band-7protein family respectively. Our findings demonstrated that Cq-DDX5might have an essential role in ontogenesis and spermatogenesis. The Cq-HSP70mRNA transcript levels changed during the sperm development process, which was in accordance with the immunoreactive signals of Cq-HSP70in the testes. These observations suggest that Cq-HSP70is critical for spermatogonial multiplication and spermatogenesis. The high levels of Cq-HSP70mRNA observed in the spermatogonial multiplication period may imply that Cq-HSP70is also an essential promoter in crayfish spermatogenesis, in turn, could be involved in high spermatogenic efficiency, which would support earlier findings in vertebrates. The results in Cq-PHB showed that it is related to the maternal inheritance of mtDNA and Cq-PHB also has a similar biological function involved in ubiquitination process in crustacean even in the invertebrate animals.Overall, this study provides the forwarding step towards understanding molecular mechanism about ontogenesis and spermatogenesis in marine invertebrates, which will contribute towards improving the quality and quantity of aquaculture. In the future, the molecular mechanism(s) linking DDX5or HSP70functions to spermatogenesis and ontogenesis need to be determined, particularly if these genes are to be exploited as a molecular biomarker in further studies of development. As to the Cq-PHB's function, it is also therefore necessarily to find out the mitochondrial inheritance mechanism in invertebrate animals for further research.