Studies On StAr2 During Steroidogenesis And Sex Differentiation In Nile Tilapia

Steroid hormones are produced by cholesterol as a substrate and catalyzed by a series of steroid synthase enzymes,playing a pivotal role in maintaining sex differentiation,gonadal development and endocrine homeostasis.Cholesterol needs to be transported from the cytoplasmic matrix to the mitochondrial inner membrane to participate in the reaction,and this first rate-limiting transportation step in the steroid hormone synthesis depends on the StAR（Steroidogenic acute regulatory protein）.There is only one type of StAR gene in mammals,and its role has been reported in human and mice.StAR gene replication was found in non-mammalian vertebrates.The two StAR genes were named StAR1 and StAR2 genes,which need to be studied more deeply on mechanisms of gonadal development,gametogenesis and steroidogenesis.Two StAR genes were isolated from the gonads of Nile tilapia（Oreochromis niloticus）in this laboratory previously.StAR2 gene was expressed from 30 dah（days after hatching）in male testes and from 5 dah in female ovaries.StAR2 gene was already knocked out by CRISPR/Cas9（Clustered regularly interspaced short palindromic repeats（CRISPR）-associated enzyme Cas9）technique,and homozygous mutant line was established in experimental animal of Nile tilapia.A series of our previous studies showed that delayed meiotic initiation and impaired spermatogonia proliferation were detected in StAR2^-/-XY testes,and masculinization was observed in StAR2^-/-XX ovaries.On this basis,in order to further reveal the functions and mechanisms of StAR2in steroidogenesis and sex differentiation in teleost fish,our study on effects of StAR2gene knockout on the testis morphology and development,testis steroidogenesis and viability in male tilapia and gonadal development,serum hormone levels in female tilapia were carried out.Our experimental results are as follows:1.Testis morphology and development in StAR2^-/-XY.Histological observation of StAR2^-/-XY and StAR2^+/+XY at 120 and 300 dah was stained by H.E.（Hematoxylin-eosin）.Spermatogenic cells at all stages without spermatic ducts were observed in the StAR2^-/-XY fish testes at 120 dah,implying StAR2 gene mutation caused delayed spermatic duct development.However,well-developed spermatic ducts were observed in both the control and StAR2-deficient XY testes at 300 dah.Distinct from the control fish,the irregularly arranged seminiferous lobules were found in the testes of StAR2^-/-XY fish.2.Steroidogenesis was blocked in StAR2^-/-XY testes.Germ cell counting basing on H.E.staining showed that the number of Leydig cells in the testes of StAR2^-/-XY fish was significantly lower than that of the control XY fish at 300 dah.IHC（Immunohistochemistry）also showed that Leydig cell marker gene positive cells were irregularly arranged in StAR2^-/-XY testis seminiferous lobules at this time.And the signals of Leydig cell marker 3β-HSD-I and Cyp17a1 were significantly weakened.Real-time PCR demonstrated that the expressions of downstream steroid synthase genes of cyp11a1,3β-HSD-I,3β-HSD-II,cyp17a1,cyp17a2 and hsd17β3 were significantly decreased in the StAR2^-/-XY fish testes.WB（Western blotting）and densitometric analysis results further showed that the expressions of 3β-HSD-I and Cyp17a1 were significantly decreased in StAR2^-/-XY testes.EIA（Enzyme-linked immunosorbent assay）experimental results showed that the serum androgen levels in the StAR2^-/-XY were significantly lower than those of the control group.It is speculated that decreased Leydig cell numbers may cause the down-regulated expressions of steroid synthase genes in StAR2^-/-XY testes,thereby affecting steroid hormone syntheses.3.Effects of StAR2 gene mutation on testis cholesterol transportation in male fish.Large area of lipid accumulation was not observed in StAR2^-/-XY fish testes by oil red O staining.Real-time PCR showed that there were no significant differences in the expressions of tspo（translocator protein）gene and vdac2（voltage dependent anion channel 2）gene related to cholesterol transportation between StAR2^+/+XY and StAR2^-/-XY testes.The above results perhaps because of the way without steroid transportation or other cholesteryl transfer proteins and fatty acid-binding proteins.4.Effect of StAR2 gene mutation on male fish fertility.IHC and WB showed a large number of Vasa positive signals in StAR2^-/-XY and control testes at 300 dah.Real-time PCR showed that there were no significant differences in the expressions of the germ cell marker gene（vasa）and meiosis-related genes（sycp3 and dazl）between mutant male fish and control male fish at this time.In addition,the normal gonadal morphology was observed by dissecting StAR2^-/-XY fish compared with StAR2^+/+XY fish at 300 dah.Correspondingly,there was no significant difference in GSI between StAR2^+/+and StAR2^-/-XY fish.Our results revealed that the sperm morphology,sperm motility and VSL（Straight line velocity）of the StAR2^-/-XY fish did not seem to change significantly compared with the StAR2^+/+XY fish.In other words,Real-time PCR and sperm motility indicators showed that spermatogenesis of StAR2^-/-XY had basically returned to normal,and sperm morphology and motility were not affected at 300 dah.Interestingly,as mentioned above,the androgen levels in StAR2^-/-were significantly reduced at 300 dah.Subsequent experiments showed that expressions of ar（androgen receptor）genes in the gonads and gn（gonadotropin）genes in the pituitaries were up-regulated in StAR2^-/-XY at this time.It is speculated that although androgen levels were decreased,the ar genes and gn genes were feedbackally up-regulated,thereby ensuring normal spermatogenesis and sperm motility in StAR2^-/-XY at 300 dah.At 540 dah,a large amount of semen can be squeezed out by gently pressing the abdomen of the control male fish.However,the sperm production of StAR2^-/-XY individuals decreased sharply,and semen could hardly be squeezed out.The thin and degenerating connections between the gonad and genital mastoid were found in StAR2^-/-XY fish by histological observation.Moreover,abnormal testis morphology and disordered spermatogenic cell arrangement were observed by H.E.staining in StAR2^-/-XY fish testes.Furthermore,compared with StAR2^-/-XY fish,a large area of strong green apoptotic signals from spermatogenic cells were detected in StAR2^-/-XY testes using TUNEL（Td T-mediated d UTP Nick-End Labeling）.In summary,we supposed that the thin and degenerating connections between the gonad and genital mastoid may cause semen could not be squeezed out in StAR2^-/-XY at 540 dah.As a result,a large area of apoptotic spermatogenic cells in StAR2^-/-XY testes were detected.Therefore,the fertility of StAR2^-/-XY was severely affected.5.Female fish gonadal development and hormone levels were affected by StAR2 gene mutation.Various phenotypes（sexual reversal,can not spawn and can spawn）were observed in StAR2^-/-XX fish.Some sex reversed StAR2^-/-XX were screened by PAGE（Polyacrylamide gel electrophoresis）and agarose gel electrophoresis of molecular marker.The gonads of these sex reversed StAR2^-/-XX have developed into complete testes producing semen normally.Meanwhile,the spermatogenic cells and male-specific Cyp11b2（Cytochrome P450,family 11,subfamily B,polypeptide 2）positive signals were observed in sex reversed StAR2^-/-XX by H.E.staining and IHC,respectively.As for other StAR2^-/-XX without sexual reversal,Enlarged bellies and masculinized genital mastoids were observed.At the same time,obvious blood vessels,accumulated green fluid,germ cells at all stages were discovered in these StAR2^-/-XX ovaries.In addition,these StAR2^-/-XX ovaries with significantly increased GSI can not spawn.However,the other StAR2^-/-XX can spawn normally.Furthermore,the androgen 11-KT（11-ketotestosterone）and estrogen E2（17β-estradiol）levels in StAR2^-/-XX of different phenotypes and the control fish were measured by EIA.The results showed that serum 11-KT level in StAR2^-/-XX（can not spawn）was significantly higher than StAR2^-/-XX（can spawn）and significantly lower than StAR2^-/-XX（sexual reversal）;serum E2 level in StAR2^-/-XX（can not spawn）was significantly higher than StAR2^-/-XX（sexual reversal）and significantly lower than StAR2^-/-XX（can spawn）.In short,even though the molecular mechanism of StAR2^-/-XX with multiple phenotypes needs further study,the differences in phenotypes may be closely related to the serum estrogen and androgen levels.In summary,the effects of StAR2 gene mutation on reproductive development and sex differentiation of tilapia were studied by CRISPR/Cas9 genetic editing technique,basing on the construction of StAR2 gene homozygous knockout line before.Our paper showed that the delayed development,the abnormal seminiferous lobule morphology and the blocked steroid syntheses were detected in StAR2^-/-XY testes without lipid accumulation.Compared with fertile StAR2^-/-XY at 300 dah,strong apoptosis signals can be observed in infertile StAR2^-/-XY at 540 dah.In addition,for female individuals,three different phenotypes appeared in StAR2^-/-XX.And the different phenotypes in StAR2^-/-XX may be related to different estrogen and androgen levels.