| N6-methyladenosine（m6A）modification is a ubiquitous reversible epigenetic RNA modification existing on the 6th nitrogen（N）atom of adenine（A）in m RNA.In recent years,it has been demonstrated as a critical post-transcriptional regulatory mechanism and thus plays a key role in regulation of gene expression regulation.More and more studies have found that m6A modification plays an important role in the embryo development,reproduction and disease,etc.m6A’s regulation of m RNA transcription,splicing and translation depends on its recognition protein（m6A reader）.As an important m6A reader,Ythdc2 has been studied in mammalian mice（Mus musculus）,in which control the transition from mitosis to meiosis of germ cells.During the course of this study,it was reported that the loss of ythdc2 caused sterility in zebrafish（Danio rerio）,but the expression pattern and function of ythdc2 in other fish species were not clear.In this study,a cultured fish,Nile tilapia（Oreochromis niloticus）,was selected as the animal model.By studying the expression and function of ythdc2,the role of ythdc2 in regulating the meiosis initiation in tilapia was investigated.The results of this study are listed as follows:1.Isolation and identification of ythdc2 gene.The Ythdc2 was isolated from the genome of Nile tilapia,human,mouse,chicken,python,tropical xenopus,coelacanth,elephant shark,zebrafish,medaka.Collinear and phylogenetic analysis were performed.The results showed that Ythdc2 is highly conserved among tetrapods and relatively conserved in fish.The analysis of its structure revealed that there was a highly conserved YTH domain and a helicase domain in Ythdc2 that was not found in other YTH family proteins.2.Expression of ythdc2 in Nile tilapia.The expression pattern of ythdc2 in adult tissues was detected by RT-PCR,and the results showed that ythdc2 was mainly expressed in the testis,while its expression level was very low in the ovaries.Gonadal transcriptomic data showed that ythdc2 was highly expressed in the testis at 90 dah（75-90 dah（days after hatching）is the key period for the meiosis initiation of male individuals）,and its expression gradually decreased thereafter.q RT-PCR was used to detect the expression level of ythdc2 in the testis at 60,75,90 and 120 dah.The results showed that the expression level of ythdc2 gradually increased before meiosis（90 dah）and then decreased.Chemical in situ hybridization showed that ythdc2 was low expressed in testis at 60 dah,while high expressed in testis at both 90 and 180 dah,and was localized in the spermatocytes undergoing meiosis.Fluorescence in situ hybridization combined with antibody staining of the meiosis marker gene Sycp3further confirmed the expression of ythdc2 in spermatocytes.3.Establishment of ythdc2 homozygous mutant in Nile tilapia and histological examination.Using CRISPR/Cas9,the ythdc2 homozygous mutants were established.8 bases was deleted in the third exon of ythdc2-/-,resulting in frameshift mutation.RT-PCR results showed that the ythdc2 m RNA was not detected in the gonads of the ythdc2-/-fish.There is no difference in body length and weight betweent he ythdc2-/-and wild-type（WT）at 90 dah.Compared with the WT fish,the gonadosomatic index（GSI）of the ythdc2-/-fish was decreased at 90 and 180 dah.Gonadal histological results showed that the testis of XY-ythdc2-/-had no spermatocytes,but with spermatogonia in the testis,which is different from the WT-XY fish with a large number of spermatocytes in the testis at 90 dah.Spermatogonia,spermatocytes and spermatids were observed in WT-XY fish,while only spermatogonia was observed in ythdc2-/-at 180 dah.The results of chromosome spreading showed that Sycp3 did not express in testis of the ythdc2-/-,while remained in the leptotene.The chromosome morphology of the leptotene was different between the ythdc2-/-and the WT,indicating that meiosis of spermatogonia was blocked.In addition,compared with the XX-WT,there were no oocytes in the ovaries of XX-ythdc2-/-fish at 90 and 180 dah,while the XX-WT fish had a large number of oocytes.These results suggest that loss of ythdc2 disrupt meiosis initiation in XX/XY fish.4.Gene expression analysis of ythdc2-/-gonads.Immunofluorescence results showed that there was no significant change in the expression of germ cell specific marker Vasa in the testis of ythdc2-/-fish at 90 dah compared with the WT fish.Consistent with the phenotype,spermatocyte marker Sycp3 and spermatocyte/spermatid marker Sox30 were not expressed in the testis of ythdc2-/-fish at 90 dah.Compared with the WT fish,the expression of somatic cell marker gene gsdf in the testis of ythdc2-/--XY fish was up-regulated,while the positive signal of androgen synthetase Cyp11c1 and Amh was not changed.In addition,transcriptomic data at 90 dah showed that the expressions of meiosis related genes rec8a,rec8b,sycp3,spo11,meioc and xrn1were significantly decreased in the ythdc2-/-testis.And the expression of RA lysis enzyme encoding gene cyp26a1 was significantly up-regulated,while the expression of RA synthetase encoding gene aldh1a2 was not changed.In addition,transcriptome data were further confirmed by q RT-PCR,which showed decreased expression of genes associated with meiosis initiation.Furthermore,oocyte marker 42sp50 was not expressed in the ovaries of ythdc2-/--XX fish,while Gsdf,which（was mainly expressed in somatic cells around oogonia）,was significantly increased in ythdc2-/-ovary.In summary,we isolated the ythdc2 of Nile tilapia.It is highly expressed in the spermatocyte and oocyte in testis and ovary.The ythdc2 homozygous mutant was established by using CRISPR/Cas9 technology.Phenotype and gene expression analysis showed that meiosis of the ythdc2-/-fish was disrupted.ythdc2 regulate meiosis initiation by controlling genes associated with meiosis.This study has broadened the understanding of the role and significance of m6A reader in the gametogenesis of teleost fish and contributed to controlling fertility of cultured fish by controlling the expression of this gene. |
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