Omega-3 polyunsaturated fatty acids(n-3 PUFAs),particularly docosahexaenoic acid(22:6n-3,DHA),play crucial roles in the reproductive health of vertebrates,including humans.Nevertheless,the underlying mechanism related...Omega-3 polyunsaturated fatty acids(n-3 PUFAs),particularly docosahexaenoic acid(22:6n-3,DHA),play crucial roles in the reproductive health of vertebrates,including humans.Nevertheless,the underlying mechanism related to this phenomenon remains largely unknown.In this study,we employed two zebrafish genetic models,i.e.,elovl2^(-/-)mutant as an endogenous DHAdeficient model and fat1(omega-3 desaturase encoding gene)transgenic zebrafish as an endogenous DHA-rich model,to investigate the effects of DHA on oocyte maturation and quality.Results show that the elovl2^(-/-)mutants had much lower fecundity and poorer oocyte quality than the wild-type controls,while the fat1 zebrafish had higher fecundity and better oocyte quality than wildtype controls.DHA deficiency in elovl2^(-/-)embryos led to defects in egg activation,poor microtubule stability,and reduced pregnenolone levels.Further study revealed that DHA promoted pregnenolone synthesis by enhancing transcription of cyp11a1,which encodes the cholesterol side-chain cleavage enzyme,thereby stabilizing microtubule assembly during oogenesis.In turn,the hypothalamic-pituitary-gonadal axis was enhanced by DHA.In conclusion,using two unique genetic models,our findings demonstrate that endogenously synthesized DHA promotes oocyte maturation and quality by promoting pregnenolone production via transcriptional regulation of cyp11a1.展开更多
鱼类的性腺分化与配子发生受到多种因子的调控。蛋白磷酸酶(protein phosphatase,PP)具有催化生物体中蛋白去磷酸化的功能。蛋白磷酸酶6(protein phosphatase type 6,PP6)作为蛋白磷酸酶家族的一员,在性腺发育及配子发生过程中发挥重要...鱼类的性腺分化与配子发生受到多种因子的调控。蛋白磷酸酶(protein phosphatase,PP)具有催化生物体中蛋白去磷酸化的功能。蛋白磷酸酶6(protein phosphatase type 6,PP6)作为蛋白磷酸酶家族的一员,在性腺发育及配子发生过程中发挥重要功能。ppp6r3编码蛋白磷酸酶6调节亚基3,其在性腺分化和配子发生方面的作用还不清楚。本研究通过CRISPR/Cas9技术构建了两个斑马鱼ppp6r3突变品系,发现ppp6r3缺失后导致斑马鱼仅发育为雄鱼,且该突变体不能使野生型卵子受精。进一步研究发现,ppp6r3突变体精巢中精母细胞向成熟精子转换失败,精子发生受阻。综上所述,本研究构建了ppp6r3突变的斑马鱼模型,表现出性腺分化和配子发生的缺陷,为后续研究Ppp6r3调控生殖细胞增殖、分化及性别决定的机制提供了模型和参考。展开更多
The clustered regularly interspaced short palindromic repeats(CRISPR)/Cas9 technology has been widely utilized for knocking out genes involved in various biological processes in zebrafish. Despite this technology is e...The clustered regularly interspaced short palindromic repeats(CRISPR)/Cas9 technology has been widely utilized for knocking out genes involved in various biological processes in zebrafish. Despite this technology is efficient for generating different mutations, one of the main drawbacks is low survival rate during embryogenesis when knocking out some embryonic lethal genes. To overcome this problem, we developed a novel strategy using a combination of CRISPR/Cas9 mediated gene knockout with primordial germ cell(PGC) transplantation(PGCT) to facilitate and speed up the process of zebrafish mutant generation, particularly for embryonic lethal genes. Firstly, we optimized the procedure for CRISPR/Cas9 targeted PGCT by increasing the efficiencies of genome mutation in PGCs and induction of PGC fates in donor embryos for PGCT. Secondly, the optimized CRISPR/Cas9 targeted PGCT was utilized for generation of maternal-zygotic(MZ) mutants of tcf7l1a(gene essential for head development), pou5f3(gene essential for zygotic genome activation) and chd(gene essential for dorsal development) at F1 generation with relatively high efficiency. Finally, we revealed some novel phenotypes in MZ mutants of tcf7l1 a and chd, as MZtcf7l1 a showed elevated neural crest development while MZchd had much severer ventralization than its zygotic counterparts. Therefore, this study presents an efficient and powerful method for generating MZ mutants of embryonic lethal genes in zebrafish. It is also feasible to speed up the genome editing in commercial fishes by utilizing a similar approach by surrogate production of CRISPR/Cas9 targeted germ cells.展开更多
Zebrafish serve as a valuable model organism for studying germ cell biology and reproductive processes.The AB strain of zebrafish is proposed to exhibit a polygenic sex determination system,where most males initially ...Zebrafish serve as a valuable model organism for studying germ cell biology and reproductive processes.The AB strain of zebrafish is proposed to exhibit a polygenic sex determination system,where most males initially develop juvenile ovaries before committing to male fate.In species with chromosomal sex determination,gonadal somatic cells are recognized as key determinants of germ cell fate.Notably,the loss of germ cells in zebrafish leads to masculinization,implying that germ cells harbor an intrinsic feminization signal.However,the specific signal triggering oogenesis in zebrafish remains unclear.In the present study,we identified foxl2l as an oocyte progenitor-specific gene essential for initiating oogenesis in germ cells.Results showed that foxl2l-knockout zebrafish bypassed the juvenile ovary stage and exclusively developed into fertile males.Further analysis revealed that loss of foxl2l hindered the initiation of oocyte-specific meiosis and prevented entry into oogenesis,leading to premature spermatogenesis during early gonadal development.Furthermore,while mutation of the pro-male gene dmrt1 led to fertile female differentiation,simultaneous disruption of foxl2l in dmrt1 mutants completely blocked oogenesis,with a large proportion of germ cells arrested as germline stem cells,highlighting the crucial role of foxl2l in oogenesis.Overall,this study highlights the unique function of foxl2l as a germ cell-intrinsic gatekeeper of oogenesis in zebrafish.展开更多
Zebrafish(Danio rerio)has been used as a promising animal model to study gonadal development and gametogenesis.Although previous studies have identified critical molecules participating in zebrafish gonad differentiat...Zebrafish(Danio rerio)has been used as a promising animal model to study gonadal development and gametogenesis.Although previous studies have identified critical molecules participating in zebrafish gonad differentiation,a landscape view of the biological processes involved in this process is still lacking.Here we isolated intact zebrafish differentiating gonads,at 25 days post-fertilization(dpf)and 30 dpf and conducted RNA-seq analyses on the juvenile gonads that tended to develop into ovaries or testes.Our study demonstrates that the juvenile ovary and testis at 25 dpf and 30 dpf are different at the biological process level.During ovary differentiation,the biological processes related to metabolic activities in the production of energy and maternal substances,RNA degradation,and DNA repair were enriched.During testis differentiation,the biological processes related to cell proliferation,differentiation,and morphogenesis were enriched,with a total of 15 signaling pathways.Notably,we reveal that the immune-related processes are extensively involved in the regulation of testis development.Overall,this study provides a landscape of differentiated biological processes and novel insights into the initiation of sex differentiation in zebrafish.展开更多
Nanog,a homeobox-containing transcription factor,is essential for pluripotent maintenance and self-renewal of embryonic stem(ES)cells.Studies in zebrafish have demonstrated a central role for Nanog in regulating early...Nanog,a homeobox-containing transcription factor,is essential for pluripotent maintenance and self-renewal of embryonic stem(ES)cells.Studies in zebrafish have demonstrated a central role for Nanog in regulating early embryonic development.Nanog activates maternal-to-zygotic transition(MZT)by associating with Pou5f3 and SoxB1[1,2],and Nanog is required for suppression of maternalβ-catenin activity in the early embryo[3].展开更多
The surrogate reproduction technique,such as inter-specific spermatogonial stem cells(SSCs)transplantation(SSCT),provides a powerful tool for production of gametes derived from endangered species or those with desirab...The surrogate reproduction technique,such as inter-specific spermatogonial stem cells(SSCs)transplantation(SSCT),provides a powerful tool for production of gametes derived from endangered species or those with desirable traits.However,generation of genome-edited gametes from a different species or production of gametes from a phylogenetically distant species such as from a different subfamily,by SSCT,has not succeeded.Here,using two small cyprinid fishes from different subfamilies,Chinese rare minnow(gobiocypris rarus,for brief:Gr)and zebrafish(danio rerio),we successfully obtained Gr-derived genome-edited sperm in zebrafish by an optimized SSCT procedure.The transplanted Gr SSCs supported the host gonadal development and underwent normal spermatogenesis,resulting in a reconstructed fertile testis containing Gr spermatids and zebrafish testicular somatic cells.Interestingly,the surrogate spermatozoa resembled those of host zebrafish but not donor Gr in morphology and swimming behavior.When pou5f3 and chd knockout Gr SSCs were transplanted,Gr-derived genome-edited sperm was successfully produced in zebrafish.This is the first report demonstrating surrogate production of gametes from a different subfamily by SSCT,and surrogate production of genome-edited gametes from another species as well.This method is feasible to be applied to future breeding of commercial fish and livestock.展开更多
基金supported by the Strategic Priority Research Program of the Chinese Academy of Sciences(Precision Seed Design and Breeding,XDA24010108)National Natural Science Foundation of China(31972780&31721005)+1 种基金National Key R&D Program of China(2018YFA0801000)State Key Laboratory of Freshwater Ecology and Biotechnology(2019FBZ05)。
文摘Omega-3 polyunsaturated fatty acids(n-3 PUFAs),particularly docosahexaenoic acid(22:6n-3,DHA),play crucial roles in the reproductive health of vertebrates,including humans.Nevertheless,the underlying mechanism related to this phenomenon remains largely unknown.In this study,we employed two zebrafish genetic models,i.e.,elovl2^(-/-)mutant as an endogenous DHAdeficient model and fat1(omega-3 desaturase encoding gene)transgenic zebrafish as an endogenous DHA-rich model,to investigate the effects of DHA on oocyte maturation and quality.Results show that the elovl2^(-/-)mutants had much lower fecundity and poorer oocyte quality than the wild-type controls,while the fat1 zebrafish had higher fecundity and better oocyte quality than wildtype controls.DHA deficiency in elovl2^(-/-)embryos led to defects in egg activation,poor microtubule stability,and reduced pregnenolone levels.Further study revealed that DHA promoted pregnenolone synthesis by enhancing transcription of cyp11a1,which encodes the cholesterol side-chain cleavage enzyme,thereby stabilizing microtubule assembly during oogenesis.In turn,the hypothalamic-pituitary-gonadal axis was enhanced by DHA.In conclusion,using two unique genetic models,our findings demonstrate that endogenously synthesized DHA promotes oocyte maturation and quality by promoting pregnenolone production via transcriptional regulation of cyp11a1.
文摘鱼类的性腺分化与配子发生受到多种因子的调控。蛋白磷酸酶(protein phosphatase,PP)具有催化生物体中蛋白去磷酸化的功能。蛋白磷酸酶6(protein phosphatase type 6,PP6)作为蛋白磷酸酶家族的一员,在性腺发育及配子发生过程中发挥重要功能。ppp6r3编码蛋白磷酸酶6调节亚基3,其在性腺分化和配子发生方面的作用还不清楚。本研究通过CRISPR/Cas9技术构建了两个斑马鱼ppp6r3突变品系,发现ppp6r3缺失后导致斑马鱼仅发育为雄鱼,且该突变体不能使野生型卵子受精。进一步研究发现,ppp6r3突变体精巢中精母细胞向成熟精子转换失败,精子发生受阻。综上所述,本研究构建了ppp6r3突变的斑马鱼模型,表现出性腺分化和配子发生的缺陷,为后续研究Ppp6r3调控生殖细胞增殖、分化及性别决定的机制提供了模型和参考。
基金supported by the National Key R&D Project of China (2018YFA0801000 and 2018YFD0901205)National Natural Science Foundation of China (Nos. 31721005, 31671501 and 31222052)+1 种基金the Youth Innovation Association of CASthe State Key Laboratory of Freshwater Ecology and Biotechnology (No. 2019FBZ05).
文摘The clustered regularly interspaced short palindromic repeats(CRISPR)/Cas9 technology has been widely utilized for knocking out genes involved in various biological processes in zebrafish. Despite this technology is efficient for generating different mutations, one of the main drawbacks is low survival rate during embryogenesis when knocking out some embryonic lethal genes. To overcome this problem, we developed a novel strategy using a combination of CRISPR/Cas9 mediated gene knockout with primordial germ cell(PGC) transplantation(PGCT) to facilitate and speed up the process of zebrafish mutant generation, particularly for embryonic lethal genes. Firstly, we optimized the procedure for CRISPR/Cas9 targeted PGCT by increasing the efficiencies of genome mutation in PGCs and induction of PGC fates in donor embryos for PGCT. Secondly, the optimized CRISPR/Cas9 targeted PGCT was utilized for generation of maternal-zygotic(MZ) mutants of tcf7l1a(gene essential for head development), pou5f3(gene essential for zygotic genome activation) and chd(gene essential for dorsal development) at F1 generation with relatively high efficiency. Finally, we revealed some novel phenotypes in MZ mutants of tcf7l1 a and chd, as MZtcf7l1 a showed elevated neural crest development while MZchd had much severer ventralization than its zygotic counterparts. Therefore, this study presents an efficient and powerful method for generating MZ mutants of embryonic lethal genes in zebrafish. It is also feasible to speed up the genome editing in commercial fishes by utilizing a similar approach by surrogate production of CRISPR/Cas9 targeted germ cells.
基金supported by the National Natural Science Foundation of China(32025037)Ministry of Science and Technology of China(2023YFD2401603)+2 种基金Ministry of Agriculture and Rural Affairs of China(NK2022010207)Science and Technology Special Fund of Hainan Province(ZDYF2024XDNY256)Natural Science Foundation of Wuhan。
文摘Zebrafish serve as a valuable model organism for studying germ cell biology and reproductive processes.The AB strain of zebrafish is proposed to exhibit a polygenic sex determination system,where most males initially develop juvenile ovaries before committing to male fate.In species with chromosomal sex determination,gonadal somatic cells are recognized as key determinants of germ cell fate.Notably,the loss of germ cells in zebrafish leads to masculinization,implying that germ cells harbor an intrinsic feminization signal.However,the specific signal triggering oogenesis in zebrafish remains unclear.In the present study,we identified foxl2l as an oocyte progenitor-specific gene essential for initiating oogenesis in germ cells.Results showed that foxl2l-knockout zebrafish bypassed the juvenile ovary stage and exclusively developed into fertile males.Further analysis revealed that loss of foxl2l hindered the initiation of oocyte-specific meiosis and prevented entry into oogenesis,leading to premature spermatogenesis during early gonadal development.Furthermore,while mutation of the pro-male gene dmrt1 led to fertile female differentiation,simultaneous disruption of foxl2l in dmrt1 mutants completely blocked oogenesis,with a large proportion of germ cells arrested as germline stem cells,highlighting the crucial role of foxl2l in oogenesis.Overall,this study highlights the unique function of foxl2l as a germ cell-intrinsic gatekeeper of oogenesis in zebrafish.
基金supported by grants from National Natural Science Foundation of China(31872550,31721005,and 31871305)National Key R&D Program of China(2018YFD0901205)+4 种基金Strategic Priority Research Program of the Chinese Academy of Sciences(XDA24010108)State Key Laboratory of Freshwater Ecology and Biotechnology(2019FBZ05,2020FB08)Fundamental Research Funds for the Central Universities(2662019PY003,2662020PY001)HZAU-AGIS Cooperation Fund(SZYJY2021010)Huazhong Agricultural University Scientific&Technological Self-innovation Foundation(2016RC011).
文摘Zebrafish(Danio rerio)has been used as a promising animal model to study gonadal development and gametogenesis.Although previous studies have identified critical molecules participating in zebrafish gonad differentiation,a landscape view of the biological processes involved in this process is still lacking.Here we isolated intact zebrafish differentiating gonads,at 25 days post-fertilization(dpf)and 30 dpf and conducted RNA-seq analyses on the juvenile gonads that tended to develop into ovaries or testes.Our study demonstrates that the juvenile ovary and testis at 25 dpf and 30 dpf are different at the biological process level.During ovary differentiation,the biological processes related to metabolic activities in the production of energy and maternal substances,RNA degradation,and DNA repair were enriched.During testis differentiation,the biological processes related to cell proliferation,differentiation,and morphogenesis were enriched,with a total of 15 signaling pathways.Notably,we reveal that the immune-related processes are extensively involved in the regulation of testis development.Overall,this study provides a landscape of differentiated biological processes and novel insights into the initiation of sex differentiation in zebrafish.
基金supported by the National Natural Science Foundation of China(32025037 and 32403018)the Strategic Priority Research Program of Chinese Academy of Sciences(CAS)(XDB0730300)+5 种基金the Natural Science Foundation of Hubei Province(2025AFA053)the Natural Science Foundation of Wuhan(2024040701010069)the Science and Technology Special Fund of Hainan Province(ZDYF2024XDNY256)the State Key Laboratory of Breeding Biotechnology and Sustainable Aquaculture(2024BBSA01)the China Postdoctoral Science Foundation(2023M733701)the Special Research Assistant Project CAS and the Postdoctoral Innovation Research Position Fund of Hubei Province。
文摘Nanog,a homeobox-containing transcription factor,is essential for pluripotent maintenance and self-renewal of embryonic stem(ES)cells.Studies in zebrafish have demonstrated a central role for Nanog in regulating early embryonic development.Nanog activates maternal-to-zygotic transition(MZT)by associating with Pou5f3 and SoxB1[1,2],and Nanog is required for suppression of maternalβ-catenin activity in the early embryo[3].
基金supported by the National Natural Science Foundation of China(32025037 and 31721005)the National Key R&D Project of China(2018YFA0801000 and 2018YFD0901205)+1 种基金Chinese Academy of Sciences(XDA24010108)State Key Laboratory of Freshwater Ecology and Biotechnology(2019FBZ05)。
文摘The surrogate reproduction technique,such as inter-specific spermatogonial stem cells(SSCs)transplantation(SSCT),provides a powerful tool for production of gametes derived from endangered species or those with desirable traits.However,generation of genome-edited gametes from a different species or production of gametes from a phylogenetically distant species such as from a different subfamily,by SSCT,has not succeeded.Here,using two small cyprinid fishes from different subfamilies,Chinese rare minnow(gobiocypris rarus,for brief:Gr)and zebrafish(danio rerio),we successfully obtained Gr-derived genome-edited sperm in zebrafish by an optimized SSCT procedure.The transplanted Gr SSCs supported the host gonadal development and underwent normal spermatogenesis,resulting in a reconstructed fertile testis containing Gr spermatids and zebrafish testicular somatic cells.Interestingly,the surrogate spermatozoa resembled those of host zebrafish but not donor Gr in morphology and swimming behavior.When pou5f3 and chd knockout Gr SSCs were transplanted,Gr-derived genome-edited sperm was successfully produced in zebrafish.This is the first report demonstrating surrogate production of gametes from a different subfamily by SSCT,and surrogate production of genome-edited gametes from another species as well.This method is feasible to be applied to future breeding of commercial fish and livestock.
