The Research Of Spermiogenesis In Gekko Japonicus And The C-terminal Motor Protein KIFC1 In This Process

The experimental model in this research,Gekko japonicus,belongs to Reptilia,Squamata,Lacertilia,Gekkonidae,Gekko,which is a small squamata species.In this research,we first studied the ultrastructure of spermiogenesis and the mature spermatozoa of G.japonicus.The gene sequence,protein sequence,different tissue expression characteristic of kifc1 from the testis of G.japonicus were determined by means of degenerate PCR,RACE and qPCR technology.We also concentrated on the spatial-temporal expression pattern of kifc1 through fluorescence in situ hybridization.Thought all these experiments,we were aiming to figure out what probable function of kifc1 during the spermiogenesis of G.japonicus,which could improve scientific basis for furture research on molecular mechanism of spermiogenesis of G.japonicus.1.The ultrastructure of spermiogenesis and the mature spermatozoa of G.japonicus The spermiogenesis process of G.japonicus can be devided into five stages.The early spermatid:the nucleus was round andGerm cells were connected by cytoplasmic junctions.StageI: The nucleus formed a concavity to accommodate the proacrosomal vesicle.The acrosomal vesicle and nuclear fossa took shape.Stage?: The acrosomal vesicle was pushed into a flattened appearance.Stage ?: The head of the spermatid looked like a cone.The acrosomal vesicle and subacrosomal cone were muture.Stage ?: The perforator and epinuclear lucent zone were observed.The spermatid tail reached it's complete development.The sperm morphology and basic structure of G.japonicus are in conformity with other repitles.While the neck cylinder with mitochondria and dense bodies arranged in grid with mitochondria are autapomorphies of Gekkonidae.2.Gene sequence,protein sequence and it's expression in different tissues of kifc1 gene Used the degenerate primers designed by comparation of DNA and ammo acid homologous sequences,the full-length sequence of kifc1 was cloned and sequenced.the length of kifc1 c DNA was 2321 bp,which contained a168 bp 5'-untranslated region(5'-UTR),a 134 bp 3'-untranslated region(3'-UTR)and a2321 bpopen reading frame(ORF)and encoded 672 ammo acids.The caculated molecular weight of the putative KIFC1 was about 74.5kD.The molecule of KIFC1 was consist of head,stalk and tail through protein structure prediction.The result of phylogenetic tree suggested that the KIFC1 protein was closely with thdt of Plestiodon chinensis and Anoliscarolinensis.qPCR analysis showed that kifc1 expressed in all tissues and in testis the expression quantity was highest,which had significant difference from those of heart,liver,muscle,epididymis and oviduct(P<0.01)only except for ovary.3.Temporal and spatial expression pattern of kifc1 mRNA during the spermiogenesis of G.japonicus Through fluorescence in situ hybridization to track the temporal and spatial expression patterns of kifc1 mRNA,we found that kifc1 mRNA mainly distributed in the cytoplasm around the nucleus,the acrosomal complex and the nucleus.In the early stage spermatid,there was a very slight expression of kifc1 mRNA signal in the nucleus;State?,kifc1 mRNA signal was centralized at the top of the nucleus,the location of acrosomal formation.And also faintly expressed in the nucleus;State?,kifc1 mRNA signal was centralized at the location of acrosomal formation.The signal began to increase in the nucleus;State?,the signal was concentrated mainly in the acrosome and further increasing in the nucleus;State?,the signal was concentrated mainly in the acrosome and the nucleus;In the mature sperm,kifc1 mRNA signal only appeared in the acrosome.Thus,kifc1 mainly distributed in the acrosomal complex and the nuclues,and it may play important roles in nuclear morphogenesis and acrosome formation during spermiogenesis of G.japonicus.