| Chinese mitten crab(Eriocheir sinensis)belongs to the crustacean,Decapoda,and shorttail,and is commonly known as river crab.Compared with mammalian sperm,its sperm is very different.Chinese mitten crab sperms has the characteristics of no flagella,no movement,and non-condensed nucleus.Mammalian sperm need to be energized before fertilization can be completed.So does the sperm of mitten crab have a capacitation process? Before completion of fertilization,the sperm of Chinese mitten crab are stored in the seminal vesicles after mating,temporarily stored in the seminal vesicles,and sperm are attached to the eggs during ovulation.Complete fertilization.In this study,the spermatozoa of Chinese mitten crabs at different stages before completion of fertilization were used as research objects.The physical and chemical properties of sperm were investigated.The extracted sperm sacs and egg water were used to stimulate the mature sperm in the sperm sac and explore the mature sperm physiological and biochemical changes before fertilization,and then recognize its capacitation characteristics.First,chlortetracycline was used to stain differently treated spermatozoa.The results showed that after the spermatozoa of the seminal vesicles entered the seminal vesicles,calcium ions on the inner membrane of the acrosome would accumulate on the inner membrane of the acrosome tube.It shows that the sperm is preparing for acrosome reaction.At the same time,the pH value of sperm cells treated with different treatments has been tested.The results show that the internal environment of sperm cells in the seminal vesicles is neutral,and changes after treatment with the seminal vesicle secretion.It is acidic and becomes alkaline after treatment with egg water.The acrosome response induced by calcium ionophore A23187 was tested on different treated sperm.The results showed that the acrosome response ability of sperm treated with seminal receptacle secretions decreased,indicating that the internal environment of acidic sperm cells would inhibit the acrosome of crab sperm.reaction.Decreased cholesterol / phospholipid data will improve membrane fluidity,promote the fusion of sperm membrane and egg membrane,and improve sperm-egg fusion efficiency.The verification of the total cholesterol content of sperm cells under different treatments showed that the mature sperm of crabs gradually reduced the total cholesterol content of sperm as they passed through the seminal vesicles,the seminal vesicles,and the egg water.Shows an outflow of cholesterol.The tyrosine phosphorylated proteins of different sperm samples were verified by western blot.The results showed that mature spermatozoa had different molecular weights of tyrosine phosphorylated proteins after treatment with the seminal receptacle secretion and egg water.The samples were subjected to immunofluorescence detection,and the results showed that the aggregation position of tyrosine phosphorylated protein had changed.Among mature sperm,only the acrosome capsule showed tyrosine phosphorylation and after treatment,it also showed in the acrosome tube.Tyrosine phosphorylation.It shows that tyrosine phosphorylation has an important role in the fertilization process;tyrosine phosphorylated protein was enriched by coimmunoprecipitation(CIP Co-Immunoprecipitation),and the enriched protein was subjected to LC-MS/MS)analysis,the results identified 236 kinds of tyrosine phosphorylated proteins in seminal vesicles,204 kinds of seminal vesicles,and 222 kinds of seminal vesicles treated seminal vesicles.From a molecular point of view,it was determined that cholesterol efflux also occurred in river crab sperm.At the same time,the function of tyrosine phosphorylated protein before fertilization was preliminary classified.This study preliminarily believes that there is a capacitation process in Chinese mitten crab sperm. |
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