- Title
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Morpho-histological and Transcriptome Analysis Reveal the Unreduced Sperm Formation Mechanism in cdk1-Depletion Zebrafish
- Authors
- Zhang, Y., Li, R., Li, H., Huang, Y., Mei, Y., Zheng, Y., Guo, Y., Zhou, Z., Yong, Z., Zhao, Y., Dong, W., Gao, J., Cao, X.
- Source
- Full text @ Mar. Biotechnol.
cdk1 mutation leads to delayed development of testes in zebrafish. A The expression levels of cdk1 gene in different tissues of zebrafish. B Immunofluorescence analysis of Cdk1 localization in zebrafish testes. Scale bar = 10 µm or 5 µm. C Schematic diagram of CRISPR/Cas9 target sites for zebrafish cdk1 gene knockout. D A representative chromatogram of PCR-product sequencing in cdk1+/−. E Anatomical examination of the testes of WT and cdk1+/− zebrafish. Scale bar = 0.5 cm. F Body weight analysis of male WT and cdk1+/− zebrafish (n = 6). G GSI of male WT and cdk1+/− zebrafish (n = 6). H Histological observations of the testes of WT and cdk1+/− zebrafish. Scale bar = 10 µm or 1 µm. I The proportions of testis cells at different stages in WT and cdk1+/− zebrafish, evaluated based on the area ratio. cdk1, cyclin-dependent kinase 1; DAPI, 4,6-diamidino-2-phenylindole; SG, spermatogonia; SC, spermatocytes; SZ, spermatozoa; WT, wild-type zebrafish; cdk1+/−, zebrafish with heterozygous mutation of Cdk1; GSI, gonadosomatic indexes; different lowercase letters indicated significant differences (p < 0.05). *p < 0.05 was considered significant, while **p < 0.01 indicated a very significant difference |
Analysis of sperm quality of WT and cdk1+/− zebrafish. A Sperm dynamic trajectory curves of WT and cdk1+/− zebrafish. Scale bar = 20 µm. B, C, D, E, F Evaluation of sperm motility. BCF, beat cross frequency; VAP, velocity average path; VSL, straight-line velocity; VCL, curvilinear velocity; LIN, linearity. WT, wild-type zebrafish; cdk1, cyclin-dependent kinase 1; cdk1+/−, zebrafish with heterozygous mutation of cdk1. *p < 0.05 was considered significant PHENOTYPE:
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cdk1 mutation leads to the production of polyploid and aneuploid zebrafish. A Chromosome spreads of cells of 24-hpf embryos of WT zebrafish. Scale bar = 5 µm. B Chromosome spreads of cells of 24-hpf embryos of WT ♀ × cdk1+/− ♂ zebrafish. Scale bar = 5 µm. C Ploidy detection of 24-hpf embryos of WT zebrafish. D Ploidy detection of 24-hpf embryos of WT ♀ × cdk1+/− ♂ zebrafish. cdk1, cyclin-dependent kinase 1; WT, wild-type zebrafish; cdk1+/−, zebrafish with heterozygous mutation of cdk1; hpf, hour post fertilization; n, number; 2N, diploid; 3N, triploid; 3N + , hypertriploid; 5N, pentaploid; ♀, female; ♂, male PHENOTYPE:
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Bivalent chromosome analysis and sperm ploidy detection of WT and cdk1+/− zebrafish. A Ploidy detection of sperms of WT zebrafish. B Ploidy detection of sperms of cdk1+/− zebrafish. C Assessment of sperm morphology of WT and cdk1+/− zebrafish. The black arrows indicated large-head sperms. Scale bar = 5 µm. D Measurement of sperm head of WT and cdk1+/− zebrafish. E Observations on the bivalent chromosome of WT zebrafish. Scale bar = 5 µm. F Observations on the bivalent chromosome of cdk1+/− zebrafish. Scale bar = 5 µm. cdk1, cyclin-dependent kinase 1; WT, wild-type zebrafish; cdk1+/−, zebrafish with heterozygous mutation of cdk1; n, number; 1N, haploid; 2N, diploid; 3N, triploid; 4N, tetraploid. ***p < 0.001 indicated an extremely significant difference PHENOTYPE:
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Electron microscopic observation of WT and cdk1+/− zebrafish spermatogonia and transcriptome analysis of testis. A The ultrastructures of spermatogonia from WT and cdk1+/− zebrafish. Scale bar = 2 µm. B Measurement of spermatogonia diameter of WT and cdk1+/− zebrafish. C The numbers of up-regulated and down-regulated DEGs of cdk1+/− vs WT zebrafish. D KEGG pathway enrichment analysis of DEGs between WT and cdk1+/− zebrafish. E Expression heatmap of cell cycle-related genes. F Network interaction analysis of DEGs corresponding proteins. G Expression levels of Smc1a, Wee1, Smc3, Espl1, and Pp1 protein in the testes of WT and cdk1+/− zebrafish. cdk1, cyclin-dependent kinase 1; WT, wild-type zebrafish; cdk1+/−, zebrafish with heterozygous mutation of cdk1; DEGs, differentially expressed genes; KEGG, Kyoto Encyclopedia of Genes and Genomes PHENOTYPE:
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Patterns of DNA replication-related genes expression and unreduced sperm production in the early testis of cdk1+/− zebrafish. A Expression heatmap of DNA replication-related genes. B Network interaction analysis of DNA replication-related DEGs corresponding proteins. C Pattern diagram of unreduced gametes production in cdk1+/− zebrafish. cdk1, cyclin-dependent kinase 1; WT, wild-type zebrafish; cdk1+/−, zebrafish with heterozygous mutation of cdk1; DEGs, differentially expressed genes |