| Thrombospondins（Tsps）were evolutionarily conserved matricellular glycoproteins.Mammalian Tsp played an important role in biological processes such as angiogenesis,wound healing and synaptogenesis.Thrombospondin1（Tsp1）was one of the five members of the Tsp family.Tsp1 was expressed in mammalian lung,skeletal muscle,kidney,ovary and other tissues,and involved in multiple biological processes including the activation of TGF-β（transforming growth factor-β）signaling pathways and the regulation of angiogenesis during wound repair and tumor growth.In teleosts,two copies of tsp1（tsp1a and tsp1b）were found due to the teleosts specific genome duplication.Previously,in situ hybridization analysis showed that the expressions of tsp1a and tsp1b were different in the tilapia gonads.tsp1a was expressed in ovarian granulosa cells,and tsp1b was expressed in the epithelial cells of the efferent duct in the testis and the ovarian theca cells in N ile tilapia.Currently,studies on the role of Tsp1 in gonadal development and gametogenesis in vertebrates were limited to mice in which Tsp1 knockout led to changed ovarian morphology and reduced fertility,and the effect of tsp1a/b mutant chimera on spermatogenesis in tilapia,and its function in reproductive development,especially in oogenesis of teleosts,remains to be elucidated.In the present study,homozygous mutants of tsp1a and tsp1b were established in tilapia based on the F0 mutants.Effects of tsp1a and tsp1b deficiency on ovary development and oogenesis of female tilapia were analyzed.The main results were as follows:1)Expression patterns of tsp1a and tsp1b in tilapia ovaries.q RT-PCR（quantitative real-time PCR）analysis revealed that tsp1a m RNA was detected in different developmental stages of the ovaries.tsp1a m RNA was detected in the gonads from 5 dah（days after hatching）and increased with the development of the ovaries,with the highest expression level at 180 dah（the vitellogenic stage）.In order to study the protein expression of tsp1a,Tsp1a polyclonal antibody was produced to characterize the cellular localization in tilapia ovaries.By IF（Immunofluorescence）,Tsp1a was detected in the oogonia and the granulosa cells of the ovaries.Expression of Tsp1a in the granulosa cells was low in primary growth follicle,and then increased in secondary growth follicle.Fluorescence in situ hybridization analysis showed that tsp1b was expressed in the somatic cells of undifferentiated gonads and theca cells of differentiated ovaries.This indicated that there were significant differences in the expression patterns of tsp1a and tsp1b.2)Effects of tsp1a mutation on oogenesis.On the basis of the existing F0generation chimera,the F2 generation mutant fish with tsp1a homozygous mutation were successfully obtained,and the ovarian phenotypes of the homozygous mutant fish were analyzed.At 60 dah,histological analysis revealed no difference in ovary development and oogenesis between the tsp1a-/-and WT（wild type）.At 120 dah,histological observation showed that the number of oogonia in the tsp1a-/-ovaries was significantly higher than that of the WT ovaries,and the number of phase II follicles was significantly lower than that of the W T ovaries,while no significant difference was observed in the number of phase I follicles.The GSI（gonadosomatic index）of the tsp1a-/-fish was similar to that of the WT fish.At 180 dah,the GSI of the tsp1a-/-fish was significantly lower than that of the WT fish.Morphological analysis showed that the ovaries of the WT fish contained plenty of vitellogenic oocytes while the ovaries of the tsp1a-/-fish contained a few vitellogenic oocytes.Histological and statistical analyses showed that the number of oogonia and phase I and II follicles was significantly higher,and the number of phase III and IV follicles in the tsp1a-/-ovaries was significantly lower than that of the WT ovaries.Expression of the cell proliferation marker PCNA was significantly up-regulated in the oogonia of the mutant ovaries.Consistently,transcriptomic analysis revealed that expressions of DNA replication related genes were significantly up-regulated,while c AMP（cyclic adenosine monophosphate）and MAPK（mitogen-activated protein kinase）signaling pathway genes which inhibited cell proliferation and promoted cell differentiation were significantly down-regulated.In addition,expression of aromatase（Cyp19a1a）and serum 17β-estradiol（E2）concentration were significantly decreased in the mutants.These results indicated that lacking tsp1a resulted in increased proliferation and inhibited differentiation of oogonia,which in turn,resulted in increased oogonia,reduced secondary growth follicles and decreased E2.3)Effects of tsp1b mutation on oogenesis.Similarly,on the basis of the existing F0 generation mutant chimera,the F2 generation mutant fish with tsp1b homozygous mutation were successfully obtained,and the ovarian phenotypes of the homozygous mutant fish were analyzed.At 90 dah,histological analysis revealed no difference between the tsp1b-/-and WT ovaries.At 180 dah,morphological analysis showed that the ovaries of the WT fish contained plenty of vitellogenic oocytes while the ovaries of the tsp1b-/-fish contained a few vitellogenic oocytes.Histological analysis revealed the number of phase I and II follicles was significantly higher,and the number of phase III and IV follicles in the tsp1b-/-ovaries was significantly lower than that of the WT ovaries.But the GSI of the tsp1b-/-fish showed no significant difference compared with that of the WT fish.Immunofluorescence results showed that the expressions of granulosa cell marker genes amh,cyp17a1 and cyp19a1a in ovaries of tsp1b-/-fish were not significantly different from those of WT fish,and serum E2 level was similar to that of the WT fish at 90 dah.At 180 dah,the positive signals of amh,cyp17a1 and cyp19a1a in ovaries of tsp1b-/-fish were significantly lower than that of WT fish,and serum E2 level was also significantly down-regulated.In order to systematically study the changes of gene expression after tsp1b gene knockout,transcriptomic analysis was performed at 90 dah.No significant difference in the expressions of tsp family related genes,indicating that other tsp members had no compensatory effect after tsp1b knockout.Significant down-regulation of chromatin assembly related genes was observed in the tsp1b-/-ovaries.Significant up-regulation of cell adhesion and focal adhesion related genes was observed in the tsp1b-/-ovaries.Down-regulation of follicular cell expressed genes amh,cyp17a1 and cyp19a1a,which were essential for follicle development,and down-regulation of chromatin assembly-related genes related to follicular growth and differentiation,might be the reason for the arrested follicle development and decreased E2 in tsp1b-/-ovaries.Cell adhesion molecules,which are important factors involved in the recognition of ovarian endocrine cells and intercellular interactions,were significantly up-regulated in tsp1b-/-ovaries.These results indicated that the tsp1b mutation had no effect on the proliferation of oo gonia and the development of primary follicles,which might be due to the compensatory effects of cell adhesion molecules and focal adhesion related genes.In summary,this study analyzed the expression pattern of tsp1a/b in the ovaries of tilapia,and confirmed that the expressions of tsp1a and tsp1b in the ovaries of N ile tilapia were differentiated.tsp1a was expressed in the granulosa cells,and tsp1b was expressed in the theca cells of the ovaries.Loss of function studies had shown that tsp1a mutation resulted in increased oogonia and primary follicles,decreased secondary follicles,and delayed follicle development.The tsp1b mutation had no effect on the proliferation of oogonia,while caused the failure of transition of primary growth follicle to secondary growth fo llicles.Therefore,both tsp1a and tsp1b homozygous mutation affected the oogenesis process of tilapia,but there were differences in phenotypes.This study revealed the function of the duplicated genes tsp1a/b in teleosts oogenesis through gene expression and mutation analysis,which was helpful to broaden and deepen our basic knowledge of Tsp1 in oogenesis of vertebrates,and provided theoretical support for fish to obtain higher quality eggs. |
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