Histological Analysis Of Oogenesis And Expression Analysis Of Vasa Of The Freshwater Crab

In order to study the cellular and molecular mechanisms of oogenesis in the freshwater crab,Sinopotamon henanense,the study tracked the process of oogenesis and ovarian development through histological observations,and described the cytological regularity of oogenesis and ovarian development in detail.Based on the vasa sequence that was obtained in the present study,the results revealed the temporal and spatial expression characteristics of vasa.The study is mainly divided into three parts as follows:1.Histological analysis of oogenesis and ovarian development of the freshwater crab,Sinopotamon henanense.The paper studied the cytological regularity of oogenesis and ovarian development through histological and cytological observations.Based on the shape and size of the oocytes,the formation and accumulation of yolk granules,the state of nucleus and cytoplasm and the maturity stage of oocytes,we classified the developing oocytes of S.henanense into four distinct stages that altogether include six phases:oogonium stage,the first phase(OI)and the second phase(OII)comprising the previtellogenic stage,the third phase(OIII),the fourth phase(OIV)and the fifth phase(OV),comprising the vitellogenic stage and the sixth phase representing the mature stage.Ovarian development was classified into six stages:proliferation stage,small growth stage,large growth stage,pre-maturation stage,mature stage and spawning stage,and ovaries varied in size and color during each developmental stage.The results show that there were cases that ovary developmental stages were substantially and oocytes at two or more phases were present at each stage different.Furthermore,examining and analyzing the gonadosomatic indices showed that the developmental cycle of the ovary was closely related to season,and indicated that the breeding season of S.henanense was between May and June.2.Prokaryotic expression,antibody preparation and immunological identification of VASA protein of S.henanense.We selected a specific segment of vasa of S.henanense(Shvasa),constructed the prokaryotic expression vector pET32a-Shvasa.SDS-PAGE analysis showed that the fusion protein,which was about 47 kDa,mainly existed in the supernatant.This study obtained the polyclonal antibody of VASA of S.henanense.The titer of anti-VASA polyclonal antibody reached 1:10~5 in ELISA assay.Furthermore,we identified the specificity of the antibody gained in this study.Immuno-adsorption and Western blot analysis indicated that the produced anti-VASA polyclonal antibody could specifically bind to the fusion protein as well as the nature VASA protein extracted from ovary of S.henanense.3.The expression patterns of vasa during the oogenesis in S.henanense.In this paper,the expression level of the vasa gene in oocytes at different developmental stages was detected by SqRT-PCR.The results showed that vasa was highly expressed at the oogonium stage and previtellogenic stage,the expression level of vasa began to decline at the vitellogenic stage(OIV),with oocytes development,the expression level of vasa gene gradually decreased.The tissue distribution of VASA was examined by Western Blot and revealed that VASA protein was specifically expressed in ovary and testis.Furthermore,the dynamic distribution of VASA protein in oocytes at differentdevelopmentalstageswasobservedbyfluorescence immunohistochemistry.The results showed that VASA protein uniformLy distributed in the cytoplasm of oogonia;VASA protein distributed in the cytoplasm and gradually accumulated around the nucleus at previtellogenic stage(OI and OII).At the early and medium vitellogenic stage(OIII and OIV),VASA protein mainly distributed around the nucleus;and at the medium vitellogenic stage(OIV),the VASA protein signal weakened.In the paper,we studied the cytological mechanisms of oogenesis and ovarian development of S.henanense,and described the divisions and characteristics of related developmental stages,providing data for the reproduction biology of decapoda.On this basis,in order to further study the molecular mechanism of the reproductive development of S.henanense,the reproductive marker gene vasa was selected to study.The preparation of VASA protein special polyclonal antibodies and the analysis of expression characteristics of VASA protein,which provided an effective means for identifying the germ cells of S.henanense and other crabs,and provided a molecular basis for further analysis of the functions of VASA protein in decapoda.