Effects Of Heterochromatin Protein Coding Gene Cbx3a Mutation On Gametogenesis And Fertility Of Nile Tilapia

Heterochromatin protein 1γ（HP1γ）is a conserved component of eukaryotic constitutive heterochromatin,encoded by the cbx3 gene.HP1γparticipates in transcriptional regulation of genes by affecting epigenetic modification of histones,thereby affecting biological processes such as cell differentiation and growth.Cbx3deletion in mice resulted in massive apoptosis of male germ cells and spermatogenesis obstruction,resulting in male infertility and most female infertility.Studies of HP1γin lower vertebrates,especially in teleost fish,are rarely reported.It has been reported that cbx3 was highly expressed in testis and ovary tissues in Nile tilapia（Oreochromis niloticus）and swamp eel（Monopterus albus）,but the specific cell expression type and gene function need further study.Most teleosts have two genes encoding HP1γ,namely cbx3a and cbx3b,due to the unique third round of genomic duplication.This study analyzed the expression patterns and cell expression types of cbx3a in male and female gonads at different developmental stages.cbx3a homozygous mutants were successfully mutated by CRISPR/Cas9 technology,and the phenotypes of the gonad,gene expression and fertility were analyzed.The main results are as follows:1.Expression of cbx3 in tilapia gonads.Transcriptome analysis showed that cbx3a was expressed in both male and female tilapia gonads at 5,30,90,180 and 300dah（Days after hatching,dah）.cbx3a was highly expressed in male and female gonads,while cbx3b was extremely low.In this study,the Cbx3a polyclonal antibody of Nile tilapia was prepared,and the cell expression types in gonads at 5,30,90 and 180 dah were analyzed by immunofluorescence.In females,cbx3a was expressed in the nuclei of oogonia and I-IV phase oocytes of ovaries at different developmental stages,as well as in granulosa cells arounding II-IV phase oocytes.In male fish,Cbx3a was specifically expressed in the nuclei of germ cells of the testis at different developmental stages,such as spermatogonia,primary and secondary spermatocytes,and spermatids.2.Establishment of Nile tilapia cbx3a mutant line.Nile tilapia cbx3a gene was mutated by CRISPR/Cas9 technology,and the target site was designed on the second exon.F0 positive males were mated with wild type（WT）females to obtain F1heterozygous mutants with 2 bp deletion.Then the F1 generation male and female mutants were mated to obtain homozygous mutants.The homozygous mutants were verified by heteroduplex motility assay（HMA）,Sanger sequencing,and Western blotting.3.Effect of cbx3a mutation on oogenesis.At 30 dah,histological analysis of the ovaries revealed that the germ cells in the WT were mainly oogonia and with few phase I oocytes,while only a few oogonia were observed in the cbx3a^-/-ovaries.At 90dah,there were more phase I and II oocytes in WT ovaries,and oogonia were also presented.Two gonadal phenotypes in the ovaries of cbx3a^-/-fish were observed.The development of some of the ovaries（5/14,type I）was similar to that of WT,while some of the ovaries（9/14,type II）were significantly smaller than that of WT,the development of germ cells was lagging,and there were only a large number of oogonia.At 180 dah,oocytes of different developmental stages（I-IV）were present in WT ovaries and cbx3a^-/-Type I ovaries.While ovarian development in cbx3a^-/-Type I ovaries still laged behind that of WT,but the gonadosomatic index（GSI）was not significantly different from that of the wild type.However,in cbx3a^-/-Type II ovaries,there were a large number of oogonia and few phase I oocytes,and the GSI was significantly lower than that of WT.Statistical analysis of the number of follicles in 90and 180 dah ovaries showed that the number of oogonia in Type II ovaries was significantly higher than that of WT,and the number of oocytes in other phases was significantly lower than that of WT.PCNA staining showed that the proliferation of oogonia in cbx3a^-/-fish increased significantly.Gene expression analysis revealed that mutations in cbx3a resulted in significant up-regulation of genes related to cell proliferation and cell cycle,as well as ribosome biosynthesis and oocyte meiotic recovery-related genes,and significant down-regulation of apoptosis-related lysosomal signaling pathway genes.4.Effects of cbx3a mutation on male germ cell development.At 90 dah,the histological analysis of the gonads showed that there were spermatogenic cells at different developmental stages in the WT testis,and the testes were full in shape.However,in the cbx3a^-/-testis,different degrees of germ cell deletion were observed.Both spermatogonia and spermatocytes were observed in some cbx3a^-/-testis（8/15Type I）,and only a few spermatogonia were observed in other cbx3a^-/-testis（7/15,Type II）.At 180 dah,compared with WT,the germ cells in cbx3a^-/-testis also degenerated to different degrees,or even completely disappeared,the testis appeared to be hydrated and transparent to different degrees,and the semen concentration was significantly reduced.Sperm motility analysis showed that there was no significant difference in the curvilinear velocity（VCL）,straight-line velocity（VSL）,progressive sperms（PR）and other sperm motility indicators of Type I mutants compared with that of WT,while significantly decreased in Type II mutants.Staining with the live cell dye PKH26 revealed that most of the Type I sperm were deformed,with significantly shorter tails.Transcriptome data of 2-month-old cbx3a^-/-testis showed that the trypsin gene,which is closely related to the meiotic process of teleost germ cells in the neuroactive ligand-receptor interaction pathway,was significantly down-regulated compared with WT.Immunofluorescence results showed that the expressions ofγH2AX and Sycp3 were significantly down-regulated in the testis of cbx3a^-/-fish.TUNEL apoptosis assay detected a large number of apoptotic germ cells in the testis of cbx3a^-/-fish.In conclusion,the present study demonstrated that Cbx3a was expressed in the nuclei of male and female germ cells of Nile tilapia at different developmental stages.cbx3a was successfully mutated using CRISPR/Cas9 technology,and homozygous mutants were obtained.Homozygous mutation of cbx3a resulted in increased proliferation of oogonia,abnormal meiosis of male and female germ cells and arrested folliculogenesis,and apoptosis of a large number male germ cells or even germ cell deletion.Our results indicated that cbx3a plays an important role in the proliferation,maintenance and development of germ cells in tilapia.This study deepens and broadens the understanding of the role of heterochromatin proteins in teleost gametogenesis and the regulation of vertebrate gametogenesis.