| In vertebrates,steroidogenesis occurs in specific tissues and cells in the body and is regulated by a series of steroidogenic enzymes.It is well known that Steroidogenic acute regulatory protein?StAR?transports cholesterol,the substrate for steroid synthesis,from the outer to the inner membranes of mitochondria.And StAR is thought as the rate-limiting factor in the biosynthesis of most steroid hormones.Our previous research revealed that there were two different StAR isoforms,named as StAR1 and StAR2 in non-mammalian vertebrates.The research showed that StAR1 is a direct homologous gene of mammulain StARs,while StAR2 is a teleost specific gene,which might attribute to the fish specific genome duplication.In Nile tilapia,StAR1 was dominantly expressed in Leydig cells of testis and the interrenal cells of head kidney,while StAR2 was expressed exclusively in the gonads.We speculate that StAR1is likely to be required for cortisol production in the head kidney,and StAR2 is probably involved in estrogen production during early sex differentiation in XX gonads.In contrast,both StAR1 and StAR2 might be required for androgen production in testes.So far,there is no report about the mediated roles of StAR1 and StAR2 in steroidogenesis during sex differentiation and gonadal development in fish.To clarify the molecular mechanisms of StAR1 and StAR2 in fish steroidogenesis,in vivo genes knockout of StAR1 and StAR2 by CRISPR/Cas9 was carried out in Nile tilapia?Oreochromis niloticus?.The roles of StAR1 and StAR2 in steroidogenesis were studied based on the knockout lines.At the same time,the serum E2?estradiol-17??and 11-KT?11-ketotestosterone?levels,gonad histological phenotype and the expression of the genes involved in sex determination and differentiation were checked in the StAR1 and StAR2 mutant fish.Our final goal is to clarify the molecular mechanisms of StAR1 and StAR2 in regulating fish steroidogenesis and then gonadal differentiation and gametogenesis.The main results are as follows:1)The establishment of StAR1 and StAR2 knockout lines in tilapia.Using the CRISPR/Cas9 targeted gene knockout technology,StAR1 and StAR2 were knocked out respectively in tilapia,and the StAR homozygous knockout lines were established basis on the F0 generation positive fish.The target mutant sites were designed on the second exon of StAR1 and StAR2 adjacent to PAM region.Two restriction Enzymes Bsl I and Pst I were selected for the screening of StAR1 and StAR2 deficient F0 generation fish,respectively.And,the StAR1 and StAR2 chimeric XY fish were incrossed with the wild-type XX fish to obtain F1 offsprings.Subsequently,the heterozygous StAR1+/-and StAR2+/-fish were screened by enzyme digestion,the polyacrylamide gel electrophoresis?PAGE?and Sanger sequencing from F1 offspring.The homozygous StAR2-/-?-8bp?F2 fish were obtained by incrossing the heterozygous XY fish with the heterozygous XX fish that had the same type of mutation.2)Histological studies and immunohistochemistry?IHC?studies were carried out to detect theStAR1chimeric mutant F0 generation fish.At 90 dah?days after hatching?,the normally developed spermatocytes,spermatocytes,spermatids and spermatozoa were observed in the testis of the StAR1+/+XY fish.While only a few primary germ cells were observed in the StAR1 deficient XY gonads.The result revealed that deficiency of StAR1deficiency resulted in gonadal dysgenesis,delayed spermatogenesis,disappeared spermatocytes in XY fish.By IHC,the Cyp11b2?the key enzyme for the synthesis of androgen?and Vasa?the marker of germ cells?were decreased in the gonad of StAR1 deficient XY fish.By real-time PCR,StAR1 deficiency caused the down-regulation of germ cell related genes of spo11,piwil1,dazl and Scp3,and the androgen synthesis related genes of cyp11b2.At the same time,serum 11-KT level was downregulated.These results indicate that StAR1 might play an important role in regulating androgen synthesis.We speculated that StAR1 deficiency might affect the synthesis of androgen,which in turn caused the delay of spermatogenesis in XY fish,indicating that StAR1 may play an important regulated role at the early stage of spermatogenesis.By 180dah H.E.staining revealed that the StAR1 deficient XY gonads restored spermatogonia proliferation and spermatogenesis,and had normally developed spermatocytes,spermatids and spermatozoa.The survival rate of the offsprings obtained by crossing StAR1 chimeric mutant XY fish with wile-type XX fish,was about 40%suggested that the StAR1 gene mutant individuals were fertile.3)Histological studies and IHC detection in StAR2 histological mutant F1 generation fish.H.E.staining was carried out to analyze the characteristics of testicular morphology in StAR2+/-XY fish at 90 dah.The results showed that StAR2 heterozygous mutation resulted in the developmental disorder of seminiferous lobules,and abnormal spermatogenesis compared with StAR2+/+XY fish.The IHC and Real-time PCR were carried out to examine the effects of StAR2 deficiency on the expression of male pathway genes.The results showed that Cyp11b2and Vasa were decreased and Dmrt1 positive Sertoli cells were disordered in the testes of StAR2+/-XY fish.These results demonstrate that StAR2 heterozygous mutation resulted in the retardation of spermatogenesis in XY fish.At 300 dah,most StAR2+/-XY gonads had almost catch up with the developmental stage of the control,with the appearance of normal spermatogenesis and the normal expression PCNA?marker gene of cell proliferation?.Interestingly,we found that Cyp11b2 and Cyp17a2 positive Leydig cells were clustered near the efferent duct.Real-time PCR results showed that significantly increased expression of cyp11b2 and cyp17a2 was found in the StAR2+/-XY gonads compared with that in the control XY testis.This result indicates that the testicular morphology development had not completely return to normal.About the StAR2-/-XY fish,StAR2 homozygous mutation led to the blockage of meiosis initiation and spermatogenesis at 70 and 90 dah.Moreover,StAR2 deficiency in XY tilapia resulted in disorganized seminiferous lobules structure and the abnormally efferent duct.By IHC,we found a fewer Vasa positive spermatogonia and spermatocytes in the gonad StAR2-/-XY fish at 90 dah.StAR2 homozygous mutation caused the down-regulation of germ cell related genes of vasa,dazl and sycp3,and the androgen synthesis related gene of cyp11b2,while the expression of steroidogenesis related genes of cyp11a1,cyp17a1 and 3?-HSD-I did not change in XY fish at 90 dah.At the same time,serum 11-KT level was downregulated.We hypothesized that StAR2 mutation can affect the germ cells proliferation and spermatogenesis and StAR2 minght be involved in the androgen biosynthesis and the initiation of steroidogenesis.4)Effects of StAR2 homozygous mutation on estrogen synthesis and ovarian development.At 90 dah,phase I and II oocytes were observed in the gonad of the StAR2-/-XX fish,the number of oocytes were reduced,at the same time,Cyp19a1a,the key enzyme for the synthesis of estrogen,was expressed normally in the gonad of the StAR2-/-XX fish.Interestingly,Cyp11b2?testis specific Leydig cell marker gene?and StAR1?which were not expressed in the ovary?was ectopically expressed in the interstitial cells of the 90 and 120 dah StAR2-/-XX fish ovaries.Real-time PCR result showed that the expression levels of germ cell related genes of vasa and spoll were significantly decreased in the StAR2-/-XX gonad.On the other hand,the steroidogenesis related genes cyp19a1a expression was decreased,but the StAR1 and cyp11b2 expression were increased significantly in the StAR2-/-XX ovaries.Consistently,the cyp19a1a,and E2?estradiol-17??level were remarkably down-regulated in the XX StAR2-/-XX fish compared with that of StAR2+/+XX fish at 90 dah.Our current research results showed that knockout of StAR2 would cause the masculinization of somatic cells in the ovaries,but not female-to-male sex reversal in the XX fish.We speculated that the upregulation of StAR1 may compensate for the deficiency of StAR2,regulating the synthesis of estrogen and development of the StAR2-deficient ovary to“catch up”.However,the mechanisms need further investigations.It is reasonable to conclude that StAR2 might play an important role in the regulation of estrogen synthesis and ovarian development.In summary,we generated StAR1 deficient F1 and StAR2 homozygous mutation F2 tilapia using CRISPR/Cas9,and studied the function of StAR1 and StAR2 in steroidogenesis.The study found that the deficiency of StAR1 and StAR1 will affect testicular development and spermatogenesis at an early stage.We speculate that both StAR1 and StAR2 may be involved in the androgen synthesis and spermatogenesis in XY male fish.The homozygous mutant of StAR2 only led to masculinization but did not cause sex reversal in XX fish.Therefore,it is urgent to establish a dual-knockout model of the StAR1 and StAR2 to further analyze the role of StAR in estrogen and androgen synthesis and gonadal development. |
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