| Eukaryotic translation elongation factor 1A?eEF1A?is one of the most abundant protein synthesis factors in eukaryotic cells.eEF1A play a central role in protein biosynthesis.In its GTP-liganded form,eEF1A catalyzes the first step of the elongation cycle during peptide synthesis.During elongation,aminoacyl-tRNA are delivered to the A site of the ribosome by eEF1A.Once a codon/anticodon match is detected,eEF1A deposits the aminoacyl-tRNA and itself released from the ribosome.Gametogenesis is a complex process,during which numerous proteins are synthesized in the testis and ovary.Although eEF1A is reported to be ubiquitously expressed in all tissues examined,its role in gonads remains to be elucidated.In mammals,two eEF1A genes named as eEF1A1 and eEF1A2 have been identified,and only eEF1A1 is expressed in the male and female gonads.In teleost,five eEF1A genes,referred to as eEF1A1-4 and 42Sp50,have been isolated,and their relative expressions are different in tissues.Moreover,different eEF1A genes are expressed in the testis and ovary.Our previously study demonstrated that eEF1A1b and 42Sp50 are XY-enhanced and XX-specific genes in Nile tilapia.In the present study,we analyzed the expression pattern and cellular localization of eEF1A1b and 42Sp50 in gonad by Western blot and immunohistochemistry?IHC?.Further,we generated eEF1A1b and 42Sp50 mutant lines using CRISPR/Cas9 and analyzed the gonadal phenotype,gene expression,serum E2 and 11-KT levels.The main results are as follows:1)Expression profile and cellular localization of eEF1A1bin developing gonads.Western blot analyses revealed that eEF1A1b was expressed in both testis and ovary,with significantly higher expression in the testis than in the ovary from 90 dah,peaking at approximately 120 dah,and decreased to a much lower level at 300 dah.IHC analysis revealed that eEF1A1b was expressed exclusively in the spermatogonia of the testis at 5,30,90 and 180dah,and was expressed in the oogonia of the ovary at 5 dah,later in the oogonia and phase I oocyte of the ovary at 30 to 180 dah.2)Effects of eEF1A1b mutation on spermatogenesis and fertility.We successfully mutated eEF1A1b by CRISPR/Cas9 and obtained eEF1A1b heterozygous F1 offspring.Consistent with the phenotype of F0 generation,eEF1A1b+/-XY fish also displayed spermatogenesis arrest,abnormal spermiogenesis,suppression of steroidogenesis and male infertility.The size of the eEF1A1b+/-testis was significantly smaller than that of the wide-type testis as demonstrated by gonadal somatic index?GSI,gonad weight/body weight×100%?.By histology,abundant spermatocytes,spermatids and spermatozoa were observed in the wide-type testis at 120,150 and 180 dah.In contrast,in the eEF1A1b+/-testis,just a few spermatocytes were observed at 120 dah,and a markedly reduced number of spermatocytes,spermatids and spermatozoa were observed at 150 and 180 dah.Meanwhile,significantly lower serum 11-KT level was detected in the eEF1A1b+/-XY fish compared with that of the wide-type XY fish.Fertility test showed that eEF1A1b+/-XY fish were infertile.Morphological and biochemical analyses of sperm revealed that compared with that of wide-type XY fish,the sperm of eEF1A1b+/-XY fish were flagella-less.Therefore,the ratio of sperm with morphological abnormalities to the total sperm was significantly higher in eEF1A1b+/-XY fish than that in the wide-type XY fish.Consistently,the activity of sperm flagella was significantly lower in eEF1A1b+/-XY fish than that in wide-type XY fish.Consequently,the sperm forward motility was lost in the eEF1A1b+/-XY fish.Transcriptome analysis revealed that the key elements of spermatogenesis?aspm,cdk16,suz12a,usp26and spo11?were significantly down-regulated in the eEF1A1b+/-testis.All these genes were co-expression with eEF1A1b.We speculated that mutation of eEF1A1b led to blockaged of translation proceed of genes expressed in spermatogonia,followed by spermatogenesis arrest.Moreover,the genes involved in flagellum formation?tuba1b,tuba3d and tuba8?were also significantly dowm-regulated in the eEF1A1b+/-testis,which may be a main reason for abnormal spermiogenesis in the eEF1A1b+/-testis.Furthermore,steroidogenesis related genes?sf-1,cyp11a1,star and cyp11b2?were significantly down-regulated in the eEF1A1b+/-testis,consequently led to decreased of serum 11-KT level.Nevertheless,it is an indirect effect of the eEF1A1b mutation as eEF1A1b was not expressed in the Leydig cells of tilapia.We speculate that the Leydig cells in the testis might be influenced by signals from the male germ cells.Additionally,overexpression of eEF1A1b rescued the spermatogenesis arrest phenotype of the eEF1A1b+/-testis,indicating that the spermatogenesis arrest phenotype was exclusively caused by eEF1A1b haploinsufficiency.On the other hand,although eEF1A1b was expressed in oogonia and phase I oocytes of the ovary,mutation of eEF1A1b had no effects on oogenesis of the ovary.3)Expression profile and cellular localization of 42Sp50 in developing gonads.Western blot analysis revealed that 42Sp50 was expressed in the ovary with high expression at 60 and 90 dah,and relatively low expression at 30 and 180 dah,but absent in the testis.By fluorescence immunohistochemistry?FIHC?,42Sp50 was found to be expressed in the phase I and II oocytes of the ovary,but absent in the testis.4)Effects of 42Sp50 mutation on oogenesis and fertility.Loss of 42Sp50 caused folliculogenesis arrest,suppression of steroidogenesis and infertility.At 120 dah,the 42Sp50-/-ovary showed no difference from the wild-type ovary at either gross anatomical or histological level.The wild-type and 42Sp50-/-ovary both contained oogonia,phase I and II oocytes.In addition,the GSI displayed no significant difference between the wild-type and 42Sp50-/-XX fish.Further,oocyte counting showed that there was no difference in oocytes number between the wild-type and 42Sp50-/-ovary.At 180 dah,anatomical analysis showed the 42Sp50-/-ovary was smaller than that of wide-type XX,and the GSI of 42Sp50-/-fish was significantly lower than that of wide-type fish.Histological analysis showed that phase III and IV oocytes were already present in the wide-type ovary,as demonstrated by the appearance of cortical alveolus and yolk granule,respectively.In contrast,only phase I and II oocytes were observed in the 42Sp50-/-ovary.Consistently,the expression of the oocyte growth markers gdf9,bmp15 and figla were significantly down-regulated in the42Sp50-/-ovary.DAPI staining showed that the follicular layer,consisting of cuboidal granulosa cells at the inner,surrounded with theca cells was observed in the wide-type ovary,while a monolayer of flat pre-granulosa cells was observed in the 42Sp50-/-ovary.Consistently,the expression of the follicle cells specific marker amhr2,were significantly down-regulated in the 42Sp50-/-ovary.Oocyte counting showed that compared with the wide-type ovary,a significantly increased number of phase I and II oocytes was observed in the 42Sp50-/-ovary.Transcriptome analysis revealed that the genes involved in folliculogenesis,oocyte growth and steroidogenesis were significantly down-regulated in the 42Sp50-/-ovary.Moreover,the activity of mammalian target of rapamycin complex 1?mTORC1?was impaired in the42Sp50-/-ovary as demonstrated by the disorganized expression of genes involved in the mTORC1 signaling pathway.It is well documented that mTORC1 signaling pathway is indispensible for folliculogenesis.To provide more evidences,we injected mTORC1 signaling pathway inhibitor rapamycin into wide-type XX fish.Rapamycin treatment phenocopied the folliculogenesis arrest and suppression of steroidogenesis obtained with the 42Sp50 mutant.Combining the loss of function study of 42Sp50 and rapamycin treatment experiment,we speculated that the inactivity of mTORC1 signaling pathway may be one of the reasons of folliculogenesis arrest in the 42Sp50-/-ovary.42Sp50 may be responsible for proteins synthesis of the genes involved in mTOR signaling pathway,or may regulate mTOR signaling pathway directly or indirectly.However,the further mechanisms involved remain to be elucidated.On the other hand,mutation of 42Sp50 had no effects on spermatogenesis of the testis.In summary,eEF1A1b and 42Sp50 were indispensible for the male and female germ cells in Nile tilapia,respectively.Mutation of eEF1A1b resulted in spermatogenesis arrest and male infertility,while it had no effects on oogenesis of the ovary.In contrast,loss of 42Sp50 caused folliculogenesis arrest and female infertility,while it had no effect on spermatogenesis of the testis.Taken together,eEF1A genes were indispensible for gametogenesis,while,different eEF1A isoforms were used for spermatogenesis and oogenesis in the Nile tilapia.These data enrich the understanding of the role of eEF1A genes in the gametogenesis in teleosts,even in vertebrates. |
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