Effects Of EAP1(Enhanced At Puberty 1) On Adolescent Development And Fertility Of Female Zebrafish

EAP1 is a transcriptional regulator originally discovered by Sabine Heger et al.in studies of hypothalamus development in female monkeys.Since its expression level continues to increase during puberty,it was named enhanced at puberty 1-EAP1.In the hypothalamus of non-human primates and rodents,the expression of EAP1 is selectively increased during adolescence.Lentivirus-mediated EAP1 siRNA targeting rodents inhibit the expression of EAP1 in the hypothalamus,leading to delayed puberty,periodic estrus destruction,and abnormal ovarian development.Therefore,exploring the function of EAP1 has important implications for the study of adolescent and female reproductive cycles in mammals.In our previous study,we used CRISPR-Cas9 to knock out the zebrafish EAP1 gene and obtain homozygous mutants.A preliminary study found no obvious abnormal phenotype in male fish.Therefore,this paper focuses on the study of adolescence and reproductive ability of female zebrafish.After knocking out the zebrafish EAP1 gene,it was found that the sex ratio of the offspring of the homozygous mutant was severely imbalanced,with females accounting for about 20%,and in wild-type zebrafish reared under the same conditions for the same period,females accounted for about 46%.The qPCR test revealed that the expression of EAP1 gene was stable during early gonad development and expressed in mature testes and ovaries.It was speculated that the EAP1 gene may be involved in the zebrafish sex determination process.By selecting the same age of male and female zebrafish for mating,the first time of oviposition,the number of eggs laid,the number of dead eggs,6-48hpf embryo mortality,ovarian development,and oviposition cycle were counted.The first oviposition time of the mutant offspring was delayed:the wild-type average was 94 days and the homozygous mutant averaged 108 days;ovarian development was delayed;the number of eggs laid was reduced:the wild-type average was 160,homozygous mutants had an average of 123 individuals;0-48h embryonic mortality was higher than wild-type;spawning cycle was delayed:average spawning cycle averaged 4.025 days,and EAP1 homozygous mutants produced an average of 4.775 days of ovipositional cycle.There were studies have suggested that EAP1 is an upstream gene of KISS1(protein coding gene,Kisspeptin is encoded by the metastasis suppressor gene KISS1,which is expressed in a variety of endocrine and gonadal tissues,KISS gene has two forms of KISS1 and KISS2 in zebrafish)and GnRH(gonadotropin-releasing hormone,in zebrafish there are GnRH2 and GnRH3 in fish).The expression of EAP1 inhibited KISS 1 and inhibited its own expression.KISS 1 can promote the secretion of GnRH.The expressions of EAP1,KISS1,and GnRH were localized in the fish brain.After the EAP1 gene knockout,the expression levels of KISS 1 and KISS2 were all inhibited to varying degrees by qPCR test,and the expression levels of GnRH2 and GnRH3 were decreased.The time delay for the first oviposition of the zebrafish is consistent with the delay in ovarian development.At the same time,EAP1 also had an effect on the apoptosis of ovarian cells.The homozygous mutant cell apoptosis was found to be higher than the wild type by flow cytometry,but there was no statistically significant difference.This study shows that EAP1 is involved in the regulation of puberty in zebrafish and affects the reproductive function of female zebrafish.