Skip to main content
SpringerLink
Log in

Surrogate production of genome-edited sperm from a different subfamily by spermatogonial stem cell transplantation

  • Research Paper
  • Published:
Science China Life Sciences Aims and scope Submit manuscript

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Anders, S., and Huber, W. (2010). Differential expression analysis for sequence count data. Genome Biol 11, R106.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Beer, R.L., and Draper, B.W. (2013). Nanos3 maintains germline stem cells and expression of the conserved germline stem cell gene nanos2 in the zebrafish ovary. Dev Biol 374, 308–318.

    Article  CAS  PubMed  Google Scholar 

  • Bellaiche, J., Lareyre, J.J., Cauty, C., Yano, A., Allemand, I., and Le Gac, F. (2014). Spermatogonial stem cell quest: nanos2, marker of a subpopulation of undifferentiated A spermatogonia in trout testis. Biol Reprod 90, 79.

    Article  PubMed  CAS  Google Scholar 

  • Brinster, R.L. (2002). Germline stem cell transplantation and transgenesis. Science 296, 2174–2176.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Burgess, S., Reim, G., Chen, W., Hopkins, N., and Brand, M. (2002). The zebrafish spiel-ohne-grenzen (spg) gene encodes the POU domain protein Pou2 related to mammalian Oct4 and is essential for formation of the midbrain and hindbrain, and for pre-gastrula morphogenesis. Development 129, 905–916.

    Article  CAS  PubMed  Google Scholar 

  • Chang, N., Sun, C., Gao, L., Zhu, D., Xu, X., Zhu, X., Xiong, J.W., and Xi, J.J. (2013). Genome editing with RNA-guided cas9 nuclease in zebrafish embryos. Cell Res 23, 465–472.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chen, Q., Yan, W., and Duan, E. (2016). Epigenetic inheritance of acquired traits through sperm RNAs and sperm RNA modifications. Nat Rev Genet 17, 733–743.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Cinalli, R.M., Rangan, P., and Lehmann, R. (2008). Germ cells are forever. Cell 132, 559–562.

    Article  CAS  PubMed  Google Scholar 

  • Ciruna, B., Weidinger, G., Knaut, H., Thisse, B., Thisse, C., Raz, E., and Schier, A.F. (2002). Production of maternal-zygotic mutant zebrafish by germ-line replacement. Proc Natl Acad Sci USA 99, 14919–14924.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Cossetti, C., Lugini, L., Astrologo, L., Saggio, I., Fais, S., and Spadafora, C. (2014). Soma-to-germline transmission of RNA in mice xenografted with human tumour cells: possible transport by exosomes. PLoS ONE 9, e101629.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Goto, R., and Saito, T. (2019). A state-of-the-art review of surrogate propagation in fish. Theriogenology 133, 216–227.

    Article  PubMed  Google Scholar 

  • Gratacap, R.L., Wargelius, A., Edvardsen, R.B., and Houston, R.D. (2019). Potential of genome editing to improve aquaculture breeding and production. Trends Genet 35, 672–684.

    Article  CAS  PubMed  Google Scholar 

  • Hammerschmidt, M., Pelegri, F., Mullins, M.C., Kane, D.A., van Eeden, F. J., Granato, M., Brand, M., Furutani-Seiki, M., Haffter, P., Heisenberg, C.P., et al. (1996). Dino and mercedes, two genes regulating dorsal development in the zebrafish embryo. Development 123, 95–102.

    Article  CAS  PubMed  Google Scholar 

  • Houston, R.D., Bean, T.P., Macqueen, D.J., Gundappa, M.K., Jin, Y.H., Jenkins, T.L., Selly, S.L.C., Martin, S.A.M., Stevens, J.R., Santos, E.M., et al. (2020). Harnessing genomics to fast-track genetic improvement in aquaculture. Nat Rev Genet 21, 389–409.

    Article  CAS  PubMed  Google Scholar 

  • Huang, D.W., Sherman, B.T., and Lempicki, R.A. (2009). Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 4, 44–57.

    Article  CAS  Google Scholar 

  • Ivell, R., and Bathgate, R.A.D. (2002). Reproductive biology of the relaxin-like factor (RLF/INSL3). Biol Reprod 67, 699–705.

    Article  CAS  PubMed  Google Scholar 

  • Iwasaki-Takahashi, Y., Shikina, S., Watanabe, M., Banba, A., Yagisawa, M., Takahashi, K., Fujihara, R., Okabe, T., Valdez Jr, D.M., Yamauchi, A., et al. (2020). Production of functional eggs and sperm from in vitro-expanded type A spermatogonia in rainbow trout. Commun Biol 3, 308.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Jin, Y.H., Robledo, D., Hickey, J.M., McGrew, M.J., and Houston, R.D. (2021). Surrogate broodstock to enhance biotechnology research and applications in aquaculture. Biotechnol Adv 49, 107756.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kimmel, C.B., Ballard, W.W., Kimmel, S.R., Ullmann, B., and Schilling, T. F. (1995). Stages of embryonic development of the zebrafish. Dev Dyn 203, 253–310.

    Article  CAS  PubMed  Google Scholar 

  • Kubota, H., and Brinster, R.L. (2018). Spermatogonial stem cells. Biol Reprod 99, 52–74.

    Article  PubMed  PubMed Central  Google Scholar 

  • Lauter, G., Söll, I., and Hauptmann, G. (2011). Multicolor fluorescent in situ hybridization to define abutting and overlapping gene expression in the embryonic zebrafish brain. Neural Dev 6, 10.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Leal, M.C., Cardoso, E.R., Nóbrega, R.H., Batlouni, S.R., Bogerd, J., França, L.R., and Schulz, R.W. (2009). Histological and stereological evaluation of zebrafish (Danio rerio) spermatogenesis with an emphasis on spermatogonial generations. Biol Reprod 81, 177–187.

    Article  CAS  PubMed  Google Scholar 

  • Lehmann, R. (2012). Germline stem cells: Origin and destiny. Cell Stem Cell 10, 729–739.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Li, M., Hong, N., Xu, H., Song, J., and Hong, Y. (2016). Germline replacement by blastula cell transplantation in the fish medaka. Sci Rep 6, 29658.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Li, Q., Fujii, W., Naito, K., and Yoshizaki, G. (2017). Application of dead end-knockout zebrafish as recipients of germ cell transplantation. Mol Reprod Dev 84, 1100–1111.

    Article  CAS  PubMed  Google Scholar 

  • Li, X., Zhang, F., Wu, N., Ye, D., Wang, Y., Zhang, X., Sun, Y., and Zhang, Y.A. (2020). A critical role of foxp3a-positive regulatory T cells in maintaining immune homeostasis in zebrafish testis development. J Genet Genomics 47, 547–561.

    Article  PubMed  Google Scholar 

  • Liang, X., and Zha, J. (2016). Toxicogenomic applications of chinese rare minnow (Gobiocypris rarus) in aquatic toxicology. Comp Biochem Physiol Part D-Genomics Proteomics 19, 174–180.

    Article  CAS  PubMed  Google Scholar 

  • Lubzens, E., Young, G., Bobe, J., and Cerdà, J. (2010). Oogenesis in teleosts: How fish eggs are formed. Gen Comp Endocrinol 165, 367–389.

    Article  CAS  PubMed  Google Scholar 

  • Luo, S., Jin, S., Su, L., and Wang, J. (2017). Effect of water temperature on reproductive performance and offspring quality of rare minnow, Gobiocypris rarus. J Thermal Biol 67, 59–66.

    Article  Google Scholar 

  • Morita, T., Kumakura, N., Morishima, K., Mitsuboshi, T., Ishida, M., Hara, T., Kudo, S., Miwa, M., Ihara, S., Higuchi, K., et al. (2012). Production of donor-derived offspring by allogeneic transplantation of spermatogonia in the yellowtail (Seriola quinqueradiata). Biol Reprod 86.

  • Nasevicius, A., and Ekker, S.C. (2000). Effective targeted gene ‘knockdown’ in zebrafish. Nat Genet 26, 216–220.

    Article  CAS  PubMed  Google Scholar 

  • Nóbrega, R.H., Greebe, C.D., van de Kant, H., Bogerd, J., de França, L.R., and Schulz, R.W. (2010). Spermatogonial stem cell niche and spermatogonial stem cell transplantation in zebrafish. PLoS ONE 5, e12808.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Octavera, A., and Yoshizaki, G. (2019). Production of donor-derived offspring by allogeneic transplantation of spermatogonia in Chinese rosy bitterling. Biol Reprod 100, 1108–1117.

    Article  PubMed  Google Scholar 

  • Ohta, H., Wakayama, T., and Nishimune, Y. (2004). Commitment of fetal male germ cells to spermatogonial stem cells during mouse embryonic development. Biol Reprod 70, 1286–1291.

    Article  CAS  PubMed  Google Scholar 

  • Okutsu, T., Suzuki, K., Takeuchi, Y., Takeuchi, T., and Yoshizaki, G. (2006). Testicular germ cells can colonize sexually undifferentiated embryonic gonad and produce functional eggs in fish. Proc Natl Acad Sci USA 103, 2725–2729.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Okutsu, T., Shikina, S., Kanno, M., Takeuchi, Y., and Yoshizaki, G. (2007). Production of trout offspring from triploid salmon parents. Science 317, 1517.

    Article  CAS  PubMed  Google Scholar 

  • Pérez-Cadahía B., Drobic, B., and Davie, J.R. (2009). H3 phosphorylation: dual role in mitosis and interphase. Biochem Cell Biol 87, 695–709.

    Article  PubMed  CAS  Google Scholar 

  • Rodríguez-Marí, A., Yan, Y.L., Bremiller, R.A., Wilson, C., Cañestro, C., and Postlethwait, J.H. (2005). Characterization and expression pattern of zebrafish anti-Müllerian hormone (amh) relative to sox9a, sox9b, and cyp19a1a, during gonad development. Gene Expr Patterns 5, 655–667.

    Article  PubMed  CAS  Google Scholar 

  • Saito, T., Fujimoto, T., Maegawa, S., Inoue, K., Tanaka, M., Arai, K., and Yamaha, E. (2006). Visualization of primordial germ cells in vivo using GFP-nos1 3′UTR mRNA. Int J Dev Biol 50, 691–699.

    Article  CAS  PubMed  Google Scholar 

  • Sawatari, E., Shikina, S., Takeuchi, T., and Yoshizaki, G. (2007). A novel transforming growth factor-β superfamily member expressed in gonadal somatic cells enhances primordial germ cell and spermatogonial proliferation in rainbow trout (Oncorhynchus mykiss). Dev Biol 301, 266–275.

    Article  CAS  PubMed  Google Scholar 

  • Schulte-Merker, S., Lee, K.J., McMahon, A.P., and Hammerschmidt, M. (1997). The zebrafish organizer requires chordino. Nature 387, 862–863.

    Article  CAS  PubMed  Google Scholar 

  • Schulz, R.W., de França, L.R., Lareyre, J.J., Le Gac, F., LeGac, F., Chiarini-Garcia, H., Nobrega, R.H., and Miura, T. (2010). Spermatogenesis in fish. Gen Comp Endocrinol 165, 390–411.

    Article  CAS  PubMed  Google Scholar 

  • Slanchev, K., Stebler, J., de la Cueva-Méndez, G., and Raz, E. (2005). Development without germ cells: The role of the germ line in zebrafish sex differentiation. Proc Natl Acad Sci USA 102, 4074–4079.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sun, Y., and Zhu, Z. (2019). Designing future farmed fishes using genome editing. Sci China Life Sci 62, 420–422.

    Article  PubMed  Google Scholar 

  • Sun, Y., Zhang, B., Luo, L., Shi, D.L., Wang, H., Cui, Z., Huang, H., Cao, Y., Shu, X., Zhang, W., et al. (2020). Systematic genome editing of the genes on zebrafish chromosome 1 by CRISPR/Cas9. Genome Res 30, 118–126.

    Article  CAS  PubMed Central  Google Scholar 

  • Tajima, A., Naito, M., Yasuda, Y., and Kuwana, T. (1993). Production of germ line chimera by transfer of primordial germ cells in the domestic chicken. Theriogenology 40, 509–519.

    Article  CAS  PubMed  Google Scholar 

  • Takeuchi, Y., Yoshizaki, G., and Takeuchi, T. (2003). Generation of live fry from intraperitoneally transplanted primordial germ cells in rainbow trout. Biol Reprod 69, 1142–1149.

    Article  CAS  PubMed  Google Scholar 

  • Takeuchi, Y., Yoshizaki, G., and Takeuchi, T. (2004). Surrogate broodstock produces salmonids. Nature 430, 629–630.

    Article  CAS  PubMed  Google Scholar 

  • Thisse, B., Heyer, V., Lux, A., Alunni, V., Degrave, A., Seiliez, I., Kirchner, J., Parkhill, J.P., Thisse, C. (2004). Spatial and temporal expression of the zebrafish genome by large-scale in situ hybridization screening. Methods Cell Biol 77, 505–519.

    Article  CAS  PubMed  Google Scholar 

  • Weidinger, G., Stebler, J., Slanchev, K., Dumstrei, K., Wise, C., Lovell-Badge, R., Thisse, C., Thisse, B., and Raz, E. (2003). Dead end, a novel vertebrate germ plasm component, is required for zebrafish primordial germ cell migration and survival. Curr Biol 13, 1429–1434.

    Article  CAS  PubMed  Google Scholar 

  • Wong, T.T., Saito, T., Crodian, J., and Collodi, P. (2011). Zebrafish germline chimeras produced by transplantation of ovarian germ cells into sterile host larvae. Biol Reprod 84, 1190–1197.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ye, D., Zhu, Z.Y., and Sun, Y.H. (2015). Fish genome manipulation and directional breeding. Sci China Life Sci 58, 170–177.

    Article  PubMed  Google Scholar 

  • Ye, D., Zhu, L., Zhang, Q., Xiong, F., Wang, H., Wang, X., He, M., Zhu, Z., and Sun, Y. (2019). Abundance of early embryonic primordial germ cells promotes zebrafish female differentiation as revealed by lifetime labeling of germline. Mar Biotechnol 21, 217–228.

    Article  CAS  Google Scholar 

  • Yoshikawa, H., Ino, Y., Shigenaga, K., Katayama, T., Kuroyanagi, M., and Yoshiura, Y. (2018). Production of tiger puffer Takifugu rubripes from cryopreserved testicular germ cells using surrogate broodstock technology. Aquaculture 493, 302–313.

    Article  CAS  Google Scholar 

  • Yoshikawa, H., Morishima, K., Fujimoto, T., Saito, T., Kobayashi, T., Yamaha, E., and Arai, K. (2009). Chromosome doubling in early spermatogonia produces diploid spermatozoa in a natural clonal fish. Biol Reprod 80, 973–979.

    Article  CAS  PubMed  Google Scholar 

  • Yoshikawa, H., Ino, Y., Kishimoto, K., Koyakumaru, H., Saito, T., Kinoshita, M., and Yoshiura, Y. (2020). Induction of germ cell-deficiency in grass puffer by dead end 1 gene knockdown for use as a recipient in surrogate production of tiger puffer. Aquaculture 526, 735385.

    Article  CAS  Google Scholar 

  • Yoshikawa, H., Takeuchi, Y., Ino, Y., Wang, J., Iwata, G., Kabeya, N., Yazawa, R., and Yoshizaki, G. (2017). Efficient production of donor-derived gametes from triploid recipients following intra-peritoneal germ cell transplantation into a marine teleost, nibe croaker (Nibea mitsukurii). Aquaculture 478, 35–47.

    Article  Google Scholar 

  • Yuan, L., Liu, J.G., Zhao, J., Brundell, E., Daneholt, B., and Höög, C. (2000). The murine scp3 gene is required for synaptonemal complex assembly, chromosome synapsis, and male fertility. Mol Cell 5, 73–83.

    Article  CAS  PubMed  Google Scholar 

  • Zhang, F. H., Wang, H.P., Huang, S. Y., Xiong, F., Zhu, Z. Y., and Sun, Y.H. (2016). A comparison of the knockout efficiencies of two codon-optimized Cas9 coding sequences in zebrafish embryos. Hereditas 38, 144–154.

    CAS  PubMed  Google Scholar 

  • Zhang, F., Li, X., He, M., Ye, D., Xiong, F., Amin, G., Zhu, Z., and Sun, Y. (2020). Efficient generation of zebrafish maternal-zygotic mutants through transplantation of ectopically induced and Cas9/gRNA targeted primordial germ cells. J Genet Genomics 47, 37–47.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We sincerely thank Ms Ming Li at Institute of Hydrobiology, CAS for providing Gobiocypris rarus embryos and we thank Kuoyu Li at the China Zebrafish Resource Center for zebrafish and Gobiocypris rarus rearing. We also thank Ms Zhixian Qiao, Fang Zhou and Yuan Xiao at analytical and testing center of Institute of Hydrobiology, CAS for providing technical support in RNA-seq, confocal microscope, SEM and TEM analysis. This work was supported by the National Natural Science Foundation of China (32025037 and 31721005), the National Key R&D Project of China (2018YFA0801000 and 2018YFD0901205), Chinese Academy of Sciences (XDA24010108), and State Key Laboratory of Freshwater Ecology and Biotechnology (2019FBZ05).

Author information

Authors and Affiliations

  1. State Key Laboratory of Freshwater Ecology and Biotechnology, Hongshan Laboratory, Institute of Hydrobiology, Innovation Academy for Seed Design, Chinese Academy of Sciences, Wuhan, 430072, China

    Fenghua Zhang, Yongkang Hao, Xianmei Li, Yi Li, Ding Ye, Ru Zhang, Xiaosi Wang, Mudan He, Houpeng Wang, Zuoyan Zhu & Yonghua Sun

  2. College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, Beijing, 100049, China

    Fenghua Zhang, Yongkang Hao, Xianmei Li, Yi Li, Ding Ye, Ru Zhang, Xiaosi Wang, Mudan He, Houpeng Wang, Zuoyan Zhu & Yonghua Sun

Authors
  1. Fenghua Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yongkang Hao
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Xianmei Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yi Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ding Ye
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ru Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Xiaosi Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Mudan He
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Houpeng Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Zuoyan Zhu
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Yonghua Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Yonghua Sun.

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhang, F., Hao, Y., Li, X. et al. Surrogate production of genome-edited sperm from a different subfamily by spermatogonial stem cell transplantation. Sci. China Life Sci. 65, 969–987 (2022). https://doi.org/10.1007/s11427-021-1989-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11427-021-1989-9

Keywords

Search

Navigation