Morphology And Differential Proteins Of Cranoglanis Bouderius Sperm And Investigation Of DMRT1 And VASA

Cranoglanis bouderius(Richardson) is a rare economic cultivar fish and just to be acclimated in recent years. So far, the resource of wild species had been exhausted, and there is not a breakthrough in the artificial reproduction. C. bouderius can propagate in the natural situation, but the problem of mating and the low ratio of insemination existed in the progress of artificial reproduction. The present study depends on biological characteristics of sperm in C. bouderius, and we performed separation, purification, morphology,differential proteins, and the relative genes about the sex determination and differentiation(DMRT1 and VASA). So we can take the information about C. bouderius sperm to support the artificial reproduction, and the methodology of research can be the reference to other kinds of fish with the difficulties of reproduction. The results are presented as follow:The present study showed that the application of Percoll discontinuous gradient to separate the semen with angle-head rotors, and a three-layer discontinuous Percoll gradient column(30%, 60% and 70%) was an effective method to separate C. spermatozoa, and the activity of separated sperm was retained and the cytomembrane was intact.According to the results of morphology, the sperm of C.bouderius was classified into head, midpiece and flagellum. Under the electron microscope, Spermic head has a hippocrepiform-shaped nucleus occupied most area. The nucleus showed an implantation fossa and has nuclear vacuoles. There was no acrosome or acrosomal vesicles in front of nucleus. Spermic midpiece was filled with cytoplasm and organelles. The cytoplasmic channel could be found between the flagellum and cytomembrane. A vesicle fused with the axoneme appeared at cytoplasmic channel which might take part in transport and assembly of microtubules. The centriolar complex was situated at median of implantation fossa which was made up of a proximal and a distal centriole. The centrioles were arranged at an obtuse angle to each other. The flagellum has a classical 9+2 axoneme structure. C.bouderius spermatozoa have only one flagellum without lateral fin. The spermatogenesis of C.bouderius was categorized in type Ⅲ.We analyzed the differential proteins between the sperm and spermatid of C.bouderius by the technology of i TRAQ. It obtained 1941 proteins in total, which were 361 of differential proteins between the sperm and spermatid, 157 of up-regulation proteins, 204 of down-regulation proteins. Annotation for subcellular localization, molecule function and biological process of differential proteins were supplied by the Gene Ontology bank. Signal pathway of differential proteins was annotated by KEGG Pathway, which included 235 of pathways. The pathway of TCA(IDH\PEPCK\OGDH\DLST\DLD) played an important role on energy metabolism of sperm, and the pathway of EABB(PLCγ\PAK\Raf\GSK-3)provided information for explaining the mechanism of spermatogenesis.The full-length of DMRT1 and VASA gene in C.bouderius were obtained by RACE.We reported that three isoforms of DMRT1 were generated in gonads by multiple alternative splicing, named cb DMRT1 a, cb DMRT1 b and cb DMRT1 c, and the mainly difference among them were 3′UTR. DM-domain in DMRT1 was highly conservative.There were two isoforms of VASA, named cb VASAa and cb VASAb, which revealed the difference between 5′UTR. DEAD-box domain was highly conservative. The alternative spliced isoforms might participant in some biological regulation. The expression pattern of the gene DMRT1 was analyzed by Q-PCR, indicating that the DMRT1 gene was expressed in testis specifically, and the expression was at a higher level than other kinds of tissues. In order to investigate the situation of the expression pattern, three isoforms of the DMRT1 and the VASA were analyzed by FISH, which revealed the expression of three isoforms of the DMRT1 was highly expressed in testicle, including spermatozoa and spermatogonium.While the cb DMRT1 b and cb DMRT1 c had strong hybridization signal in other tissues besides testicle and cb DMRT1 c was mainly expressed in spermatogonium. These three isoforms of the DMRT1 were weakly expressed in oocyte, the tendency of expression was that the signal transfer from cytoplasm to nucleus following the development of oocyte.The VASA gene had high expression level in the gonad of C.bouderius, which was highly expressed in stage I, Ⅱof ovary and the VASA gene was well-distributed in the cytoplasm.Along with the development of oocyte, the hybridization signal was decreasing and dismissing. There was a similar tendency present in the testis, the hybridization signal in spermatocyte was strong but dismissed in the spermatozoa. All the information indicated that these two genes might involve in early stage of reduplication and development in the germ cell, and regulated the later development and maturation in the germ cell.