Primary Studies On The Regulation Mechanism Of Gonad Development In Triploid Shrimp, Fenneropenaeus Chinensis

Triploidy manipulation is one important approach in genetic improvement for aquaculture animals, which plays important roles in quantity and quality improvement. Gonad development and sex ratio of triploid shrimp show significant differences with that of diploid counterparts. In this study, primary studies were carried out on the molecular mechanism of gonad development in triploid shrimp, and the results have established a firm basis for further dissection on the mechanism of gonad development and sex determination in crustacean. Some important progresses are as follows:Comparative studies on meiosis behavior of spermatocytes between diploid and triploid males were carried out by synaptonemal complex (SC) analysis. Spermatocytes of diploid shrimp showed typical morphological characteristics of eukaryote SC, with complete synapsis of bivalents. No recognizable bivalent associated with sex chromosomes was observed in spermatocytes of diploid shrimp. However, differences in morphology of SC, including unsynapsed univalents, bivalents, totally paired trivalents with non-homologous synapsis, partner switches and triple synapsis were identified at early pachytene stage of triploid spermatocytes. Triple synapsis was especially common at late pachytene stage in spermatocytes of triploid shrimp. The abnormal synapsis behavior in triploid shrimp may be responsible for the production of triploidy sperms.To identify key molecular changes involved in ovary development of triploid shrimp, two subtracted cDNA libraries between triploid and diploid shrimp ovaries were constructed using suppression subtractive hybridization (SSH) technique. For the forward library (triploid ovary as tester), 54 genes were identified. For the reverse library (diploid ovary as tester), 16 genes were identified. Using semi-quantitative RT-PCR, the differential expression profiles of eleven selected genes were confirmed in the ovaries of triploid and diploid. These genes fell in gene categories with a wide range of functions. The results indicated that triploidy affects the dynamic gene regulatory network in triploid ovary. This study established a firm basis for future investigation on characterization of crucial molecular events for normal ovarian development in shrimp.To further dissect exact gene functions for gonad development of shrimp, three differentially expressed genes between diploid and triploid ovary, PCNA (proliferating cell nuclear antigen), CAS/CSE1 (cellular apoptosis susceptibility protein/chromosome segregation 1) and SSRF (spermatogonial stem-cell renewal factor) were characterized on certain aspects. These results have set firm basis for further functional analysis of these genes in ovary development and gene regulatory mechanism for triploids.FcPCNA expressed especially high in proliferating tissues of shrimp such as gonad and haematopoietic tissue (HPT). It showed significant expression level in diploid ovary compared to the triploid counterparts. Different expression profiles of FcPCNA in HPT were obtained after challenged by two pathogens, indicating different defense mechanisms of shrimp against these two pathogens. FcPCNA showed high identity and conservation with PCNAs reported in other species, which suggests that it may have conserved functions in shrimp. It has been reported that PCNA can be served as a marker for cell proliferation activity, which can be very helpful for studies in shrimp development, and also it is helpful for comparative development studies between diploid and triploid.CAS/CSE1 expressed especially high in diploid ovary, and its mRNA can be detected in cytoplasm and around nucleus in the oocytes. It can be served as the maternal factor which is necessary for early embryo development. The conserved domain for importin-βfamily proteins can be found at N terminal of its amino acid sequence, which suggests that the role in nucleocytoplasmic transport is important for this gene in development regulation. In addition, CAS/CSE1 was successfully expressed in E. coli, which will be utilized for further functional studies.SSRF expressed especially high in triploid ovary. For the diploid normal tissues, it showed the highest expression level in nerve, indicating the potential role of this gene for neuron development. The amino acid sequence of this gene shows high identity with TP (thymine phosphorylase) of other organisms. In addition, SSRF was successfully expressed in E. coli, which will be utilized for further functional studies; researches on the enzyme activity and function dissection are required to be carried out.