Effects Of Adenylate Kinase Ak4 Knockout On Germ Cell Apoptosis Of Male Zebrafish

The continuation and development of life rely on the breeding of creatures.Increasing attentions have been paid toward research of reproductive development.As a commonly used animal model,their fertilization and development are carried out in vitro;this makes them be easy to handle for the experiments.In addition,as a vertebrate,the genome of zebrafish is functionally similar to the human,and the structure of the tissues and organs is homologous.Therefore,zebrafish is broadly used in various aspects of life science research.Adenylate kinase(AK)is a monomeric enzyme being prevalent in microbes,plants and animals and is abundant in cells that require high levels of Adenosine triphosphate(ATP).Recently,the migration and alteration of enzyme activity of AK have been observed in the course of cellular apoptosis,and the metabolic signal defects of AK are directly related to many human diseases.It was reported that human AK1-4,6-7 are expressed in the testis and epididymis,AK5is only expressed in the epididymis,and AK6 is also expressed in the sperm tail.But the role of AK in the male reproductive system still remains unclear.Previous study from our collaborators found that AK genes abnormally express in patients with weak schistosomiasis.Hereto,in the study CRISPR/Cas9 technique was used to knock out zebrafish ak4 gene to invest its effect on AMP/ATP ratio in male reproductive system and its effect on male germ cell development and reproductive capacity,to provide theoretical support for further treating relevant diseases.Through sequence alignment and evolutionary analysis,it was found that the sequence identity of each Ak gene in zebrafish was merely 8.5%,indicating that the ak gene family of zebrafish had a great differentiation in sequence and function.The expression of each ak gene in various tissues or organs in the early stage of embryotic development and adult was analyzed by using qPCR assay,indicating that zebrafish ak7a has not only maternal expression,but also zygotic expression;The other ak genes are mainly zygotic expression;akl,ak4,ak6,ak7a,ak7b,ak8 and ak9 are expressed in the testis.This clarifies the expression status of ak subtypes in zebrafish and provides clues to perform gene knockout for specific functional studies.Then by using CRISPR/Cas9 technology,the zebrafish adenylate kinase ak4 gene was successfully knocked out,followed by screening to homozygous mutant offspring.The content of ATP,ADP,AMP and AMP/ATP ratio in the testis were determined by HPLC.The results showed that the content of ATP in the mutant was significantly decreased,the content of AMP did not change significantly,and the AMP/ATP ratio increased.This Indicated that the energy state of the cells in the testes significantly changed.The apoptosis was increased in testis of mutant zebrafish detected by TUNEL and flow cytometry assay(FCM).Based on the results of other literatures and this study,it is speculated that the energy change in cells led to the apoptosis of ak4 mutant male germ cells.At 8 h and 24 h,it was found that the survival rate of ak4 male mutant was decreased and the mortality rate was increased.It was presumably because that the increase of apoptosis caused the decrease of sperm quality after ak4 mutation.In the early development of mutant embryos,the yolk got large,the pigment was shallow,and the body was short,and the development got lagged.In the combination of the expression of ak4 in early development,suggesting that ak4 is also involved in early embryonic development.In addition,we also knock out ak6 and ak7a genes in zebrafish by using CRISPR/Cas9 technique,and generated FO mutant zebrafish.