Functional Analysis Of Autophagy In Fish Sperm Motility

Sperm motility is one of the most critical indexes for evaluating semen quality that is the main factor affecting fish fertilization rate.How to improve sperm motility,semen quality and fertilization rate has become an important issue in fish reproduction.Some reports indicated that autophagy related genes were involved in human sperm motility.Therefore,we investigated whether autophagy would participate and regulate fish sperm motility by using autophagy inhibitor.The results demonstrated that chloroquine could improve sperm motility parameters including the average path velocity(VAP),straight-line velocity(VSL)and curvilinear velocity(VCL)using intraperitoneal injection in male yellow catfish.A concentration of 100?M chloroquine is the best to improve sperm motility,while 1000?M chloroquine inhibited the sperm motility of yellow catfish.Subsequently,we assessed sperm motility,fertilization rate and hatching rate by artificial reproduction when 100?M chloroquine was used.It was found that chloroquine treatment could significantly improve the fertilization rate of yellow catfish without affecting hatching rate under the same conditions.Meanwhile,we found that chloroquine affected the autophagy signaling in the yellow catfish testes when improving sperm motility.Then we performed deep sequence using the Illumina platform on yellow catfish testes.The sequence data showed that there were 4686 different expressed unigenes in the treatment group,including 2144 up-regulated unigenes and 2542 down-regulated unigenes.Interestingly,GO enrichment analysis indicated that many down-regulated unigenes were annotated to the toll-like receptor signaling pathway.KEGG enrichment analysis revealed that the PI3K/AKT signaling pathway was significantly up-regulated.Therefore,chloroquine improved sperm motility of yellow catfish possibly by regulating autophagy,PI3K/AKT signaling pathway and toll-like receptor signaling pathway.As the genetic information and molecular basis of yellow catfish is very weak,it is difficult for us to carry out deeper studies.Therefore,we took the model animal zebrafish,which is easier to operate and has very clear genetic information,as the research object to explore the effects of autophagy and PI3K/AKT signaling pathway on sperm motility.Chloroquine isn't a common medicine in aquaculture,so we hope we can find a safe and effective chemical which functions as same as chloroquine for fish reproduction.Previous research has found that many kinds of amino acid modulated the process of autophagy.Our results showed that the leucine,tryptophan and phenylalanine could significantly promote the zebrafish sperm motility,whereas valine and cysteine suppressed zebrafish sperm motility using computer-aided sperm analysis method.Subsequently,we used leucine with the best promotion effect in the following study.The experimental results indicated that leucine could inhibit the fusion of autophagosomes and lysosomes,thus inhibiting autophagy.This suggests that leucine can promote sperm motility of zebrafish by inhibiting the late process of autophagy.However,its function in sperm motility is similar to that of chloroquine.To investigate the function of PI3K/AKT signaling pathway in sperm motility,inhibitor and activity of PI3K/AKT pathway were used for zebrafish testis.When treated with the compound LY294002(the inhibitor of PI3K/AKT signal),PI3K/AKT signaling pathway and autophagy induction was inhibited and sperm motility was reduced.When treated with IGF1 in zebrafish testis,PI3K/AKT signaling pathway was activated and sperm motility was increased.When treated with leucine in testis,PI3K/AKT signaling pathway and sperm motility were elevated.These data suggest that PI3K/AKT signaling pathway regulate sperm motility.Generally,when the formation of autophagosome is restricted,sperm motility will be reduced.However,sperm motility will be promoted in the zebrafish and yellow catfish when leucine and chloroquine were used to inhibit autophagy and fusion of autophagosome and lysosome.These could be explained that higher lever of LC3 can guarantee the sperm activity,andthe PI3K/AKT signaling pathway plays an important role in this process.Moreover,chloroquine as a commercial agent and leucine as a common nutrient molecule can be used as a convenient drug additive in the artificial reproduction of yellow catfish or other fish species.