Functional Analysis Of MiR-200 Family In Zebrafish Reproductive Development

Zebrafish miR-200 family(miR-200s)is consist of six members(miR-200 a,miR-200 b,miR-200 c,miR-141,miR-429 a and miR-429b)and distributed on chromosome 6 and chromosome 23.Our previous studies have revealed that the miR-200 s affect the embryos’ body size during the development in zebrafish.However,reports about miR-200 s in adult zebrafish are rare.The aims of this study were to investigate the function of miR-200 family in the reproductive process of mature zebrafish.We knocked out the miR-200 s through deleting their six precursors and generated two mutated lines by the technology of CRISPR/CAS9.The mutanted line 1(hereinafter referred to as chr6-miR-200-KO)which loses miR-200 members(miR-141,miR-429 b,miR-200c)in chromosome 6 has no obviously different phonotype from wild type zebrafish(WT),while The mutanted line 2(hereinafter referred to as chr23-miR-200-KO)which loses miR-200 members(miR-200 b,miR-200 a,miR-429a)in chromosome 23 has a significant advantage in sperm motlility over WT in male,and was sterile in female.In the male zebrafish,there was no significant difference in testis morphology and histology between wildtype and chr6-miR-200-KO mutants or chr23-miR-200-KO mutants.Interestingly,compared with wildtype zebrafish,chr6-miR-200-KO zebrafish had no difference on sperm motility,while chr23-miR-200-KO zebrafish showed significantly improved sperm motility.Then we screened out several candidate sperm motility-related target genes,such as amh,wt1 a and srd5a2 b by online targetscan prediction.The three genes have been confirmed as direct targets of miR-200 s on chromosome23 through comparative expression of these genes between WT and chr23-miR-200-KO zebrafish and the evidence that miR-200 a,miR-200 b,miR-429 a could bind to the 3’UTR of amh,wt1 a and srd5a2 b by dual luciferase system in vitro.Consistently,ectopic expression of miR-429 a,miR-200 a and miR-200 b that are located in the miR-200 cluster on chromosome 23,significantly reduced the sperm motility and downregulated amh,wt1 a and srd5a2 b.Gradient 17α-ethynylestradiol(EE2)exposure resulted in gradiently upregulated expression of miR-429 a and p53 in testis and impairment of sperm motility.Previous study has confirmed that p53 could directly regulate miR-200 s,then we detected an extremely lower expression levels of miR-200 a,miR-200 b and miR-429 a and accompanied by a significant stimulation of sperm motility compared with WT zebrafish.Moreover,when p53 mutants were treated with the same EE2 explosure,the up-regulation of miR-429 a was abolished in testis in p53 mutants.Meanwhile,the impairment of sperm activity by EE2 treatment was also eliminated when p53 was mutated.Together,we draw a conclusion that miR-200 cluster controls sperm motility in a p53-dependent manner: EE2 could regulated miR-200 a,miR-200 b and miR-429 through activating p53 pathway and then miR-200 s on chromosome 23 affects sperm motility by targeting amh、wt1a and srda2 b.In female zebrafish,we found that chr23-miR-200-KO mutants was unfertile,while the chr6-mir-200-KO mutant could spawn as wild type zebrafish.Comparing the anatomy and histologic section in sexually mature zebrafish,there was no obvious differences among wild type,chr6-miR-200-KO and chr23-miR-200-KO zebrafish ovary and there were also no differences between WT and chr23-miR-200-KO mutant ovary: both included various oocytes in different developmental stage.For further investigation,we found that chr23-miR-200-KO mutants’ oocytes could undergo VI stage and also could be induced matured oocyte maturation(FOM)in vitro.When rescuing the reproductive failure,we found that h CG could partly rescue the mutants and parts of follicle cells in all induced mutants got transparent and waited for ovulation,but most of the matured oocyte can not be detached from follicular cells and fall off into the ovarian cavity,resulting in ovulation failure.Gonadotropin is consist of LH and FSH,while fshb mutant female has no reproductive barrier,so we focus our attention on the potentially pivotal function of LH.Then we detected that chr23-miR-200-KO mutants exhibited a significantly lower expression of lhb when comparing with WT.As we confirmed wt1 a was the target gene of miR-200 s on chromosome 23 and then verified that protein of Wt1 a could effectively inhibited the promoter activity of lhb in vitro.We put forward that lhb was regulated by the transcription of downstream target gene wt1 a of miR-200 s.But how did LH affect the oocytes’ maturation and ovulation in zebrafish? We hypothesized that reproductive defects were caused by the insufficiency of LH downstream progesterone and prostaglandins.Then series of experiments were conducted to verify the function of progesterone and prostaglandins.The hormone content of DHP reached the peak around 5:30am before ovulation,then we successfully rescued partial mutants by injecting DHP,which confirming our suppose that mutants’ reproductive barrier was was partially caused by LHdepedent progesterone insufficiency.On the other hand,we injected prostaglandins(PGE2 and PGF2)to rescue chr23-mir-200-KO mutants before ovulation when ptgs2 a reached a high level but failed.Meanwhile,we generated ptgs2 a knockout zebrafish,the homozygote female could spawn and had no reproductive barrier.So we put forward the view that prostagin was not involved in the pathway that miR-200 s affected oocytes’ maturation and ovulation through regulating LH.Summarize the role of miR-200 s in females,we draw a conclusion that miR-200 cluster in 23 chromosome(miR-200b/200a/429a)could affect oocytes’ maturation and ovulation by regulating lhb and its downstream hormone DHP by targeting the transcription repressor wt1 a.In conclusion,the miR-200 cluster in 23 chromosome(miR-200 b,200a,429a)plays an important role in productive process in mature zebrafish,affecting the male sperm motility and female oocytes’ maturation and ovulation.