Study On The Roles Of Cyp17a2 In Steroidogenesis And Gametogenesis In Nile Tilapia

Cytochrome p450s（Cytochrome P450 or CYP）comprise a superfamily of heme-thiolate proteins named by the spectral absorbance peak of their carbon-monoxide-bound species at 450 nm.Cyp17a（cytochrome P450,family 17 and subfamily A）gene belongs to the cytochrome P450 superfamily,locating the key position in the steroidogenic pathway for the production of C-18,-19 and-21 steroids in the gonads and head-kidney of fish.There are two cyp17a genes（cyp17a1 and cyp17a2）duplication in fish.Previous studies have shown that Cyp17a1,with both hydroxylase and lyase activity,expressed in gonads indicating its possible important role for the estrogen and androgen synthesis.While Cyp17a2,with only the hydroxylase,expressed in both head-kidney and gonads,suggesting that it might participate in the synthesis of cortisol in head-kidney and DHP（17α,20β,21-trihydroxy-4-pregnen-3-one）in gonads due to a up-regulation of cyp17a2 in follicular cells during gamete maturation in reproductive cycle of tilapia.So far,the functional studies of Cyp17a2 in steroidogenesis and gametogenesis of fish has not been reported.In this study,by using Nile tilapia（Oreochromis niloticus）as an experimental animal,we successfully established a cyp17a2 homozygous mutation line by CRISPR/Cas9 knockout system.Furthermore,the effects of cyp17a2 mutation on gonadal development,gametogenesis,steroidogenesis and sperm motility and fertility were investigated.The main results of this paper are as follows:1)The expression pattern and cellular localization of cyp17a gene in gonads and head-kidney of tilapiaThe Real-time PCR data illustrated that cyp17a1 and cyp17a2 were higher expressed in ovary than that in testis before 60 days after hatching（dah）,thereafter,the expression of cyp17a1 and cyp17a2 in testis was higher than that in ovary.IHC showed that Cyp17a1 was expressed mainly in somatic cells at 5 dah,and in interstitial cells and granulosa cells at later stage in ovary.In testis,Cyp17a1 started expressed in somatic cells at 10 dah.Later on,it was expressed in leydig cells.Cyp17a2 expression was detected in somatic cells of ovary at 10 dah,and expressed in interstiial cells and theca cells at later stage.In testis,Cyp17a2 started to express in leydig cells of testis at90 dah.In the head-kidney,Cyp17a2 but not Cyp17a1 expression was detected in the interrenal cells from 5 dah.2)Successful establishment of cyp17a2 gene mutation lineWe successfully obtained homozygous mutant of cyp17a2 gene by cross the heterzygous mutants by CRISPR/Cas9.The results of Sanger sequencing and PAGE showed that cyp17a2 mutant lines were successfully obtained.IHC and WB also showed that there was no positive signals of Cyp17a2 antibody in the mutant gonads.3)Effects of cyp17a2 gene mutation on steroid synthesis and steroidogenesisAt 90 and 180 dah,Real-time PCR showed no significant changes were detected in the expression level of cyp17a1 and cyp19a1a in ovaries,as well as cyp17a1 and cyp11b2 in testes in the mRNA and protein.And the serum of E2 level in female,11-KT level in male had no significant difference between the control and cyp17a2^-/-groups.These data suggested that Cyp17a2 might not be involved in estrogen and androgen synthesis in tilapia.Then,we measured the serum DHP level during the spawning cycle of mature female fish,and it peaked on the eighth days after spawning.In cyp17a2^-/-fish,the serum DHP level remain at a low level,and there was no significant increase as cyp17a2^+/+fish after hCG injection.No expression of Cyp17a2was detected in the head-kidney,and therefore the serum cortisol level was significantly decreased and extremely low in cyp17a2^-/-fish.These results suggested that Cyp17a2 in tilapia might be involved in the synthesis of DHP in gonad and cortisone in head-kidney.4)Effects of cyp17a2 gene mutation on gonadal development and oogenesis in female fish.At 90 and 120 dah,H.E.staining found that germ cells unable to enter meiosis,and remain in oogonia stage in the cyp17a2^-/-ovary.At 180 dah,stage III oocytes can be detected in the ovary of cyp17a2^-/-fish,indicated that cyp17a2 deficiency led to a delay the initiation of meiosis,and the delay would be restored at later stage.At 90 dah,there was no male special maker gene Dmrt1 and Cyp11b2 signals detected in cyp17a2^-/-ovary,indicated that cyp17a2 deletion would no resulted the sex reversal.The early stage oocytes marker 42sP50 and RA synthetase Aldh1a2 were much lower in cyp17a2^-/-than that in cyp17a2^+/+ovary,and the Real-time also illustrated the expression of aldh1a2 significantly decreased,the lyase of RA cyp26b1 significantly increased,the meiosis gene spo11 and sycp3 expression level were significantly decreased,and the expression of fshr and lhr were decreased in cyp17a2^-/-ovary.At180 dah,aldh1a2 and cyp26b1 expression level were no significant difference,spo11and sycp3 expression level were significantly increased,fshr expression level increased and lhr expression level also decreased in cyp17a2^-/-ovary.Indicated that Cyp17a2 is very important for the initiation of meiosis,at later stage,there was no significant effect on oocyte development,but it might play an important role in oocyte fully maturation and ovulation.5)Effects of cyp17a2 gene mutation on gonad development and spermatogenesis in male fish.H.E.staining showed that at 75 dah,spermatogenesis was observed in cyp17a2^+/+testis,while no meiotic germ cell was observed in the testis of cyp17a2^-/-.At 180 dah,the spermatogenesis in cyp17a2^-/-testis has caught up to that in cyp17a2^+/+testis.These data indicated that active Cyp17a2 are essential for the initiation of meiosis since the deletion of cyp17a2 led to a delay of meiotic initiation in early spermatogenesis.However,the delay will be restored at later stage.IHC and Real-time PCR results revealed that at 90 dah,germ cell marker vasa and piwil in cyp17a2^-/-testis were significant lower than those in cyp17a2^+/+testis even though no significant differences were found for somatic marker Dmrt1,Gsdf and Cyp11b2.Moreover,cyp26b1expression level significantly increased in cyp17a2^-/-testis and the expression level of spo11 and sycp3 were decreased in cyp17a2^-/-testis.While at 180 dah,there was no significant difference of cyp26b1 expression level in these two groups,and the expression level of spo11 and sycp3 was increased in cyp17a2^-/-fish.These all data illustrated that Cyp17a2 plays an important role in the initiation of meiosis for spermatogenesis in testis,and the deletion of cyp17a2 gene will delay the spermatogenesis in male fish.6)The effects of cyp17a2 gene mutation on sperm motility and fertility.Papanicolaou staining was used to detect the morphology of sperms,and no significant difference was observed between cyp17a2^+/+and cyp17a2^-/-fish.A significant decrease in the ratio of progressive,velocity of straight and curve line,while a increase in the proportion of inactive sperm were found in cyp17a2^-/-fish in comparison with control XY fish.In summary,we successfully established the homozygous mutant of cyp17a2 gene in Nile tilpia.Our work indicated that Cyp17a2 might not participate in the synthesis of estrogen and androgen,while play an important role in cortisol and DHP synthesis.Mutation of cyp17a2 resulted in a delay of the meiotic initiation,anovulation,and decrease of perm motility.