Study On The Gene Cloning,differential Expression And The Expression Of Related Receptors Of Vitellogenin In Turbot (Scophthalmus Maximus)

The production of high-quality gametes is the foundation of effective reproduction in animals.For oviparous animals,the energy necessary for embryonic development is derived from the advanced accumulation by the mother prior to the maturation of the egg.The nutrients includes proteins,lipids,sugars,vitamins,minerals,and so on.Among them,the Vtg is the precursor form of Yp,storing as energy substance in the egg.The production process of Yp in bony fish is complicated,which presents obvious species specificity,so it is necessary to carry out extensive research among teleost species.In China,as an important economic species of marine aquaculture industry,turbot lacks basic research on the process of egg yolk generation.Therefore in this study,we identified the multiplicity of the vtg genes,analyzed the class A form Vtgs and their derivative Yp in the liver,blood and ovary during the breeding season,discussed the endogenous synthesis of Vtg and the expression of receptors relating to endocytosis within exogenous synthesis pathways,and the results showed that:1.During the breeding season,oocytes in the ovary of female turbot was derived from oogonia and then experienced several key events,including the primary growth,the accumulation of nutrients,the maturation process,the degeneration process,and so on.On this basis,the oocytes could be divided into several types,which were oocytes at the chromatin nucleolar stage,the early perinucleolus stage,the late perinucleolus stage,the cortical alveoli stage,the early-vitellogenic stage,the late-vitellogenic stage,the early mutration stage,the late mutration stage and the degradation stage.At the same time,five ovarian developmental stages were identified based on their dominant oocyte type,which were the pre-vitellogenic stage,the early-vitellogenic stage,the late-vitellogenic stage,the post-vitellogenic stage,and the degradation stage.The division of ovigenetic and ovarian development stages during the breeding season made a foundation for the subsequent molecular biology experiments.2.There were three vtg genes in turbot,VtgAa and VtgAb presented the typical complete form,consisting of all five Yp domains,while VtgC was much smaller and encoded only two Yp domains（LvH-LvL）,as was typical for VtgC in other fish species.The phylogenetic analysis showed that VtgAa and VtgAb were the closest modern relatives of each other,while VtgC was the most primitive Vtg subtypes.Therefore,the polygene system of Vtg in turbot followed the“three vtg model”in Acanthomorpha.3.The expression of all three vtg genes in the liver was far higher than that in the other tissues,including the ovary,which suggested that the ovary of turbot accumulate Yp mostly through the exogenous synthesis pathway.During the accumulation of Yp,the total ratio of the relative transcript quantities expressed in the liver was 11:21:1（vtgAa:vtgAb:vtgC）,and this fluctuated slightly among different developmental stages.This suggested that the exogenous synthesis of turbot Yp should be dominated by class A Vtgs,first VtgAb,then VtgAa,and the expression of VtgC should be low.4.Tissue distribution analysis found that the expression of vtgAb in the ovary was the highest except the liver.Then we made a comparative analysis of the expression between the liver and ovary,and we found that both expression profile was in accordance with the observed changes in the concentrations of E₂.Notably,the concentration of E₂ in the ovary was significantly higher than that in the liver during vitellogenesis,while the expression of all three vtg genes in the ovary was far lower than that in the liver.This inferred the regulation of E₂.Further,we found that the expression site of vtgAb in the ovary was oogonia and primary oocytes,and obvious signals of Yp derived from this gene’s transcripts were also detected in these cells.Interestingly,in our IHC results the signals detected in oogonia and primary oocytes presented homogeneous distributions,which was obviously distinct from that detected in vitellogenic oocytes.This suggested that these proteins formed via different modes,and the oogonia and primary oocytes of turbot might possess the ability to perform autosynthesis of Vtg.5.For 2 class A Vtg proteins synthesized through the exogenous pathway（liver）,molecular weights in the liver and serum were both identified as 180.2 kDa and 175.6kDa,and one of them experienced partially proteolysis to 164.8 kDa in the ovary.The LvL domain of class A Vtg proteins formed through the first maturational disassembly were 28.6 kDa and 24.5 kDa（ovary）,while a main band of 22.7 kDa and a weak band of 20.9 kDa were formed through the second maturational proteolysis（mature egg）.Besides,there were also several proteins identified in the ovary as LvL domains partially separated from others,which were 87.7 kDa,53.8 kDa and 49.9 kDa.In addition,we analysed the dynamic changes of Vtg in the liver,serum and ovary during the breeding season.On one hand,there was a weak signal in the serum at the PreVG stage,indicating that the liver just began to synthesize Vtg and most oocytes basically completed the accumulation of Yp at the LVG stage.On the other hand,at the PostVG stage,the main Vtg bands in the serum were significantly enhanced,and in the detection of ovary,the main LvL bands in mature eggs appeared.These two phenomena were consistent with the reproduction strategy in turbot,which was group-synchronous development.6.Both receptors involved in the endocytosis of Vtgs in turbot,VLDLR and LRP13,were members of LDLR superfamily.On one hand,vldlr presented two splicing variants,lr8-and lr8+,and was dominated by lr8-,which played a role mainly in egg yolk accumulation.On the other hand,tissue distribution of lrp13 was more specific,by contrast,which expressed almost exclusively in the ovary.The expression patterns of lr8-and lrp13 were similar in the ovary,and the results of ISH showed that they were both mainly expressed in the oocytes of perinucleolus stage and completely disappeared when the vitellogenesis began,suggesting the recycling of receptor proteins during endocytosis.Besides,quantitative results showed that there were still a certain amounts of expression after the peak,indicating the diversity of oocyte type at each stage,which was in accordance with the reproduction strategy in turbot.