Effects Of Tsp1 Mutant On Gametogenesis In Nile Tilapia,Oreochromis Niloticus

Gametogenesis,including spermatogenesis and oogenesis,is a main part of sexual reproduction,by which diploid or haploid precursor cells undergo cell division and differentiation to form mature haploid gametes,sperm or egg.Gametogenesis is regulated by a lot of factors,including many genes,transforming growth factors?TGF?and hormones.Thrombospondin1?Tsp1?,a member of TSP gene family,was expressed in many tissues including ovary,skin,bone,heart,skeletal muscle,kidney and other tissues,functioning as an inhibitor of angiogenesis and activator of TGF-?signaling pathways.Tsp1 deficient mice produced fewer litters,suggesting their reproductive dysfunction,but its mechanism is not clear.Our previous study showed that two types of tsp1?tsp1a and tsp1b?exist in Nile tilapia?Oreochromis niloticus?.By in situ hybridization,tsp1a was detected in granulosa cells,while tsp1b was detected in the theca cells in the ovary and the epithelial cells of the efferent duct in the testis.Real-time PCR analysis showed that the expression pattern of both tsp1a and tsp1b was positively correlated with the serum E2?17?-estradiol?level in XX tilapia.These results suggested a possible role of tsp1a and tsp1b in gametogenesis of tilapia.So far,there has been no reports on the functional study of tsp1a and tsp1b in teleosts.In the present study,the expression profiles of tsp1a and tsp1b genes in the gonads tilapia were analyzed.CRISPR/Cas9 genome editing technology was used to mutate tsp1a and tsp1b.The gonadal phenotype,gene expression,serum E2 levels the F₀ tsp1a and tsp1b mutant fish were analyzed.The main results are listed as follows:1.Expression pattern of tsp1a and tsp1b in gonads.Real-time PCR showed that tsp1a was expressed at a low level in the XX gonad at 5,30,60 and 90 dah?days after hatching?and in the XY gonad at all stages examined.while it was abundantly expressed in the XX gonads at 180 dah and 540 dah.tsp1b was expressed in both the XX and XY gonads at similar expression level and up-regulated gradually from 5 to 540dah.tsp1a and tsp1b expression was increased gradually during the ovarian and testicular development.2.Efficient site-directed disruption of tsp1a and tsp1b by CRISPR/Cas9.Enzyme digestion analysis revealed that for tsp1a and tsp1b 14 and 15 positive mutants were obtained from 16 fish screened,respectively.The mutation frequencies induced by CRISPR/Cas9 calculated from 50 dah mutants were approximately 22%to 52%and 36%to 70%for tsp1a and tsp1b targeting sites,respectively.Frame-shift and in-frame mutations were identified by Sanger sequencing.The ratio of frame-shift mutations was68.5%and 54.8%in tsp1a and tsp1b mutants,respectively.3.Sperm morphology of F₀ mutants.Papanicolaou staining showed that the sperm counts of the tsp1a and tsp1b mutants were reduced when compared with that of the wild-type males.About 77%sperms with normal morphology in the tsp1a mutants and 18%in the tsp1b mutants were observed,whereas around 91%normal sperms found in the wild-type males.Acephalic,double-tail,harpin-tail,no-tail sperms were observed in tsp1b mutants.The straightness of the tsp1a mutant sperms?47%?and the tsp1b mutant sperms?13%?was significantly lower than the wild-type sperms?95%?.The curvilinear velocity of the tsp1a mutant sperms?98.71?m/sec?and the tsp1b mutant sperms?180.90?m/sec?was significantly faster than the wild-type sperms?56.73?m/sec?.The Mean Angular Displacement of the tsp1b mutant sperms?128.21degree/sec?was significantly faster than those of the tsp1a mutant?27.86 degree/sec?and wild-type sperms?23.67 degree/sec?,while there was no significant difference between the tsp1a mutant and wild-type sperms.The Beat Cross Frequency of the tsp1b mutant sperms?12.22 times/sec?were significantly faster than those of the tsp1a mutant?2.36 times/sec?and wild-type sperms?2.26 times/sec?,while there was no significant difference between the tsp1a mutant and wild-type sperms.When compared with the control sperms,bolder and longer sperm mid-piece was observed in the tsp1a mutant sperms,shorter and bent sperms tail was observed in tsp1b mutant sperms by scanning electron microscope.Moreover,hypogonadism were observed in the tsp1a and tsp1b mutants,and the GSI?gonadal somatic index?of tsp1a and tsp1b mutants was significantly lower than the wild-type XY fish.4.Histological observation and apoptosis assay of the tsp1a and tsp1b mutant testes.The number of sperm in the tsp1a and tsp1b mutant testes was significantly reduced when compared with the wild-type testes.Positive TUNEL signals were observed in the germ cells of tsp1b mutant testes at 6 M?months?and 18 M,and in the tsp1a mutant testes at 18 M.Real-time PCR analyses showed that the mRNA levels of tsp1b,cd36,caspase3 were up-regulated,while the mRNA levels of tsp1a,integrin,tsp2 remained unchanged in the tsp1a mutant testes when compared with the wild-type testes.In tsp1b mutant testes,the mRNA levels of tsp1a,cd36,caspase3 were up-regulated,while the mRNA levels of tsp1b,integrin,tsp2 were not changed when compared with the wild-type testes.Immunohistochemical analyses showed that Caspase3?apoptosis marker?expression was up-regulated in the spermatids of tsp1b mutant testes at 6 M and 18 M and in the tsp1a mutant testes at 18 M when compared with the wild-type testes.The expression of Pcna?proliferating cell marker?,?h2ax?meiosis marker?,Aldh1a2?RA synthetase?,Cyp11b2 and Cyp17a1?androgen synthetase?were remained unchanged in the wild type testis by immunohistochemistry.Disruption of tsp1a and tsp1b led to gonadal agenesis,reduced number of spermatids.Increasing apoptosis of germ cells was observed during the development of mutant testes.5.Gene expression,apoptosis assay and measurement of serum E2concentration in the tsp1b mutant ovaries.When compared with the control XX fish,down-regulated Cyp19a1a expression in ovaries and decreased serum E2 concentration was observed in the tsp1b mutant XX fish at different stages.Positive TUNEL signals were observed in the theca cells of tsp1b mutant ovaries 18 M.In summary,in this study,CRISPR/Cas9 was employed to mutant tsp1a and tsp1b genes in Nile tilapia.Disruption of tsp1a and tsp1b in the XY fish leads to spermatids apoptosis,reduced number of spermatids and fertility.tsp1b is more important than tsp1a in tilapia spermatogenesis as the tsp1b mutant fish displayed more serious gonadal phenotype.On the other hand,disruption of tsp1b in the XX fish leads to theca cells apoptosis,reduced concentration of E2.