Study On Vitellogenin (Vg) And Vitellogenin-like (Vg-like)in The Mud Crab, Scylla Paramamosain

Vitellogenin (Vg) is the precursor of the major yolk protein (YP), vitellin (Vn), which is a high density lipoglycoprotein and widely exists in the oviparous animals. Vg not only provides some protein, carbohydrates, lipids, and other nutritional supplies to the developing embryo, but also transports lipids and metallic ions even participates in immune defense, and so on.We had cloned the gDNA and cDNA sequence of SpVg, and analyzed the temporal and spatial expression of SpVg in the mud crab Scylla paramamosain. Therefore, in this paper, we do some further study of SpVg based on the previous research. Our main results are as follows:1) Western blot analysis revealed that SpVg in the hemolymph of female crab was composed of two subunits of-80kDa and-207kDa, and Vn in the ovary was composed of three subunits of-80kDa,-100kDa and-107kDa. We supposed that the processing pathway of SpVg included two cleavage. Initial cleavage happened in hepatopancreas where Vg was synthesized, and SpVg was cleaved at the RERR consensus cleavage site by a subtilisin-like endoprotease to form two subunits of-80kDa and-207kDa. Then these two subunits were excreted into hemolymph and transported into ovary. The second cleavage was assumed to take place in ovary. The subunits of-207kDa was split by an unknown enzyme in ovary to bring two subunits of-100kDa and-107kDa, resulting in vitellin.2) The results obtained in the in situ hybridization and immunohistochemistry indicated that the site of expression and translation of SpVg were the epithelial cells of hepatopancreatic tubules and the follicle cells of ovary. SpVg synthesized in the two sites was absorbed by the oocyte in the end, and cleaved to form the Vn.Vg is usually considered a female specific protein, while it can be induced in male. But interestingly, we detected the expression of SpVg-like in the reproductive system of normal physiological male S. paramamosain, and compared it with SpVg in the female. The main contents are as follows:1) We cloned the full-length cDNA (8316bp) of SpVg-like from the testis, and compared it with the gDNA and cDNA of SpVg from female crab. It revealed that the cDNA of SpVg-like was almost the same as SpVg, while SpVg-like retained the first three intron of SpVg gDNA. This phenomenon was called intron retention which was a type of alternative splicing.2) We detected the expression of SpVg-like mRNA in all the tissues of the male S. paramamosain, and the result indicated that this gene was expressed only in the testis.3) A band of-210kDa was detected in the seminal vesicle of male crab by western blot analysis, and we deduced that it was the SpVg-like protein. Moreover, its molecular weight was nearly as the big subunit of SpVg in the hemolymph of female crab, so we suggested that SpVg-like protein might be the same with the big subunit of SpVg in amino acid sequence.4) Using immunohistochemistry, SpVg-like protein could be detected in the sperm/spermatophore of the testis, seminal vesicle and seminal receptacle of the mud crab. However, SpVg-like mRNA only appeared at the sperm of testis by in situ hybridization. Our results indicated that SpVg-like protein was stored in the sperm/spermatophore for a long time after synthesized in the sperm of testis, and not be consumed or utilized before fertilizing. So we predicted that SpVg-like protein might participate in the sperm maturation or fertilization.In conclusion, this paper enriches the research of SpVg at the protein aspect, and find SpVg-like in the reproductive system of male mud crab. Our finding will facilitate the understanding of Vg in reproduction.