Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Mitopherogenesis, a form of mitochondria-specific ectocytosis, regulates sperm mitochondrial quantity and fertility

Nature Cell Biology volume 25, pages 1625–1636 (2023)Cite this article

Subjects

Abstract

Mitochondrial export into the extracellular space is emerging as a fundamental cellular process implicated in diverse physiological activities. Although a few studies have shed light on the process of discarding damaged mitochondria, how mitochondria are exported and the functions of mitochondrial release remain largely unclear. Here we describe mitopherogenesis, a formerly unknown process that specifically secretes mitochondria through a unique extracellular vesicle termed a ‘mitopher’. We observed that during sperm development in male Caenorhabditis elegans, healthy mitochondria are exported out of the spermatids through mitopherogenesis and each of the generated mitophers harbours only one mitochondrion. In mitopherogenesis, the plasma membrane first forms mitochondrion-embedding outward buds, which then promptly bud off and thereby result in the generation of mitophers. Mechanistically, extracellular protease signalling in the testis triggers mitopher formation from spermatids, which is partially mediated by the tyrosine kinase SPE-8. Moreover, mitopherogenesis requires normal microfilament dynamics, whereas myosin VI antagonizes mitopher generation. Strikingly, our three-dimensional electron microscopy analyses indicate that mitochondrial quantity requires precise modulation during sperm development, which is critically mediated by mitopherogenesis. Inhibition of mitopherogenesis causes accumulation of mitochondria in sperm, which may lead to sperm motility and fertility defects. Our findings identify mitopherogenesis as a previously undescribed process for mitochondria-specific ectocytosis, which may represent a fundamental branch of mechanisms underlying mitochondrial quantity control to regulate cell functions during development.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Spermatids generate mitophers to export mitochondria into the extracellular space.
Fig. 2: Illustration of the dynamics of mitopherogenesis.
Fig. 3: Extracellular protease signalling triggers mitopherogenesis during sperm development, which is partially dependent on SPE-8.
Fig. 4: The motor protein myosin VI negatively regulates mitopherogenesis, whereas actin filaments are required for mitopher generation.
Fig. 5: Mitopherogenesis exports healthy mitochondria.
Fig. 6: Mitopherogenesis critically modulates mitochondrial quantity and fertility of sperm.

Similar content being viewed by others

Data availability

Source data are provided with this paper. All other data supporting the findings of this study can be obtained from the corresponding authors on reasonable request.

Code availability

The code used for acquiring ODT images in this study can be found at https://github.com/dashandong/ODTDemo.

References

  1. Jiao, H. et al. Mitocytosis, a migrasome-mediated mitochondrial quality-control process. Cell 184, 2896–2910 (2021).

    CAS  PubMed  Google Scholar 

  2. Melentijevic, I. et al. C. elegans neurons jettison protein aggregates and mitochondria under neurotoxic stress. Nature 542, 367–371 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Davis, C. H. et al. Transcellular degradation of axonal mitochondria. Proc. Natl Acad. Sci. USA 111, 9633–9638 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Hayakawa, K. et al. Transfer of mitochondria from astrocytes to neurons after stroke. Nature 535, 551–555 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Torralba, D., Baixauli, F. & Sanchez-Madrid, F. Mitochondria know no boundaries: mechanisms and functions of intercellular mitochondrial transfer. Front. Cell Dev. Biol. 4, 107 (2016).

    PubMed  PubMed Central  Google Scholar 

  6. Zhu, Q., An, Y. A. & Scherer, P. E. Mitochondrial regulation and white adipose tissue homeostasis. Trends Cell Biol. https://doi.org/10.1016/j.tcb.2021.10.008 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  7. Crewe, C. et al. Extracellular vesicle-based interorgan transport of mitochondria from energetically stressed adipocytes. Cell Metab. 33, 1853–1868 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Nakajima, A., Kurihara, H., Yagita, H., Okumura, K. & Nakano, H. Mitochondrial extrusion through the cytoplasmic vacuoles during cell death. J. Biol. Chem. 283, 24128–24135 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Nicolas-Avila, J. A. et al. A network of macrophages supports mitochondrial homeostasis in the heart. Cell 183, 94–109 (2020).

    CAS  PubMed  Google Scholar 

  10. Ma, L. et al. Discovery of the migrasome, an organelle mediating release of cytoplasmic contents during cell migration. Cell Res. 25, 24–38 (2015).

    CAS  PubMed  Google Scholar 

  11. Kurihara, Y. et al. Mitophagy plays an essential role in reducing mitochondrial production of reactive oxygen species and mutation of mitochondrial DNA by maintaining mitochondrial quantity and quality in yeast. J. Biol. Chem. 287, 3265–3272 (2012).

    CAS  PubMed  Google Scholar 

  12. Palikaras, K., Lionaki, E. & Tavernarakis, N. Balancing mitochondrial biogenesis and mitophagy to maintain energy metabolism homeostasis. Cell Death Differ. 22, 1399–1401 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Youle, R. J. & Narendra, D. P. Mechanisms of mitophagy. Nat. Rev. Mol. Cell Biol. 12, 9–14 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Varuzhanyan, G. & Chan, D. C. Mitochondrial dynamics during spermatogenesis. J. Cell Sci. 133, jcs235937 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Moretti, E., Pascarelli, N. A., Federico, M. G., Renieri, T. & Collodel, G. Abnormal elongation of midpiece, absence of axoneme and outer dense fibers at principal piece level, supernumerary microtubules: a sperm defect of possible genetic origin? Fertil. Steril. 90, 1201.e3–1201.e8 (2008).

  16. Shakes, D. C. & Ward, S. Initiation of spermiogenesis in C. elegans: a pharmacological and genetic analysis. Dev. Biol. 134, 189–200 (1989).

    CAS  PubMed  Google Scholar 

  17. Dietert, S. E. Fine structure of the formation and fate of the residual bodies of mouse spermatozoa with evidence for the participation of lysosomes. J. Morphol. 120, 317–346 (1966).

    Google Scholar 

  18. Shakes, D. C. & Ward, S. Mutations that disrupt the morphogenesis and localization of a sperm-specific organelle in Caenorhabditis elegans. Dev. Biol. 134, 307–316 (1989).

    CAS  PubMed  Google Scholar 

  19. Winter, E. S. et al. Cytoskeletal variations in an asymmetric cell division support diversity in nematode sperm size and sex ratios. Development 144, 3253–3263 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Wang, Q. et al. Membrane contact site-dependent cholesterol transport regulates Na+/K+-ATPase polarization and spermiogenesis in Caenorhabditis elegans. Dev. Cell 56, 1631–1645 (2021).

    CAS  PubMed  Google Scholar 

  21. Dong, D. et al. Super-resolution fluorescence-assisted diffraction computational tomography reveals the three-dimensional landscape of the cellular organelle interactome. Light Sci. Appl. 9, 11 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Sung, Y. et al. Optical diffraction tomography for high resolution live cell imaging. Opt. Express 17, 266–277 (2009).

    CAS  PubMed  Google Scholar 

  23. Zhou, Q. et al. Mitochondrial endonuclease G mediates breakdown of paternal mitochondria upon fertilization. Science 353, 394–399 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Nishimura, H. & L’Hernault, S. W. Spermatogenesis-defective (spe) mutants of the nematode Caenorhabditis elegans provide clues to solve the puzzle of male germline functions during reproduction. Dev. Dyn. 239, 1502–1514 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Smith, J. R. & Stanfield, G. M. TRY-5 is a sperm-activating protease in Caenorhabditis elegans seminal fluid. PLoS Genet. 7, e1002375 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Varga, K., Jiang, Z. J. & Gong, L. W. Phosphatidylserine is critical for vesicle fission during clathrin-mediated endocytosis. J. Neurochem. 152, 48–60 (2020).

    CAS  PubMed  Google Scholar 

  27. Stanfield, G. M. & Villeneuve, A. M. Regulation of sperm activation by SWM-1 is required for reproductive success of C. elegans males. Curr. Biol. 16, 252–263 (2006).

    CAS  PubMed  Google Scholar 

  28. Liu, Z., Wang, B., He, R., Zhao, Y. & Miao, L. Calcium signaling and the MAPK cascade are required for sperm activation in Caenorhabditis elegans. Biochim. Biophys. Acta 1843, 299–308 (2014).

    CAS  PubMed  Google Scholar 

  29. Alvau, A. et al. The tyrosine kinase FER is responsible for the capacitation-associated increase in tyrosine phosphorylation in murine sperm. Development 143, 2325–2333 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Muhlrad, P. J., Clark, J. N., Nasri, U., Sullivan, N. G. & LaMunyon, C. W. SPE-8, a protein-tyrosine kinase, localizes to the spermatid cell membrane through interaction with other members of the SPE-8 group spermatid activation signaling pathway in C. elegans. BMC Genet. 15, 83 (2014).

    PubMed  PubMed Central  Google Scholar 

  31. Frokjaer-Jensen, C. et al. Single-copy insertion of transgenes in Caenorhabditis elegans. Nat. Genet. 40, 1375–1383 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Hu, J. et al. Distinct roles of two myosins in C. elegans spermatid differentiation. PLoS Biol. 17, e3000211 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Heissler, S. M. et al. Kinetic properties and small-molecule inhibition of human myosin-6. FEBS Lett. 586, 3208–3214 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Dunn, K. W., Kamocka, M. M. & McDonald, J. H. A practical guide to evaluating colocalization in biological microscopy. Am. J. Physiol. Cell Physiol. 300, C723–C742 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Sweeney, H. L. & Houdusse, A. What can myosin VI do in cells? Curr. Opin. Cell Biol. 19, 57–66 (2007).

    CAS  PubMed  Google Scholar 

  36. Nelson, G. A., Roberts, T. M. & Ward, S. Caenorhabditis elegans spermatozoan locomotion: amoeboid movement with almost no actin. J. Cell Biol. 92, 121–131 (1982).

    CAS  PubMed  Google Scholar 

  37. Tajima, T. et al. Proteinase K is an activator for the male-dependent spermiogenesis pathway in Caenorhabditis elegans: its application to pharmacological dissection of spermiogenesis. Genes Cells 24, 244–258 (2019).

    CAS  PubMed  Google Scholar 

  38. Rengan, A. K., Agarwal, A., van der Linde, M. & du Plessis, S. S. An investigation of excess residual cytoplasm in human spermatozoa and its distinction from the cytoplasmic droplet. Reprod. Biol. Endocrinol. 10, 92 (2012).

    PubMed  PubMed Central  Google Scholar 

  39. Watson, P. F. The causes of reduced fertility with cryopreserved semen. Anim. Reprod. Sci. 60-61, 481–492 (2000).

    CAS  PubMed  Google Scholar 

  40. Petrella, L. N. Natural variants of C. elegans demonstrate defects in both sperm function and oogenesis at elevated temperatures. PLoS ONE 9, e112377 (2014).

    PubMed  PubMed Central  Google Scholar 

  41. Ellis, R. E. & Kimble, J. The fog-3 gene and regulation of cell fate in the germ line of Caenorhabditis elegans. Genetics 139, 561–577 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Menkveld, R., Holleboom, C. A. & Rhemrev, J. P. Measurement and significance of sperm morphology. Asian J. Androl. 13, 59–68 (2011).

    PubMed  Google Scholar 

  43. Ficarro, S. et al. Phosphoproteome analysis of capacitated human sperm. Evidence of tyrosine phosphorylation of a kinase-anchoring protein 3 and valosin-containing protein/p97 during capacitation. J. Biol. Chem. 278, 11579–11589 (2003).

    CAS  PubMed  Google Scholar 

  44. Nishimura, H. & L’Hernault, S. W. Spermatogenesis. Curr. Biol. 27, R988–R994 (2017).

    CAS  PubMed  Google Scholar 

  45. Manning, L. & Richmond, J. High-pressure freeze and freeze substitution electron microscopy in C. elegans. Methods Mol. Biol. 1327, 121–140 (2015).

    CAS  PubMed  Google Scholar 

  46. Zhang, S. & Kuhn, J. R. Cell isolation and culture. WormBook https://doi.org/10.1895/wormbook.1.157.1 (2013).

  47. Loessner, D. et al. Functionalization, preparation and use of cell-laden gelatin methacryloyl-based hydrogels as modular tissue culture platforms. Nat. Protoc. 11, 727–746 (2016).

    CAS  PubMed  Google Scholar 

  48. Lee, J. S. et al. Actin-related protein 2/3 complex-based actin polymerization is critical for male fertility. Andrology 3, 937–946 (2015).

    CAS  PubMed  Google Scholar 

  49. Valm, A. M. et al. Applying systems-level spectral imaging and analysis to reveal the organelle interactome. Nature 546, 162–167 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank the Caenorhabditis Genetics Center for strains; L. Miao (Institute of Biophysics, Chinese Academy of Sciences) and X. Wang (Institute of Biophysics, Chinese Academy of Sciences) for providing strains and reagents; the Microscopy Core and the Flow Cytometry Core facilities in Westlake University as well as the High-performance Computing Platform of Peking University for support; Z. Yu, Y. Zhang, Y. Wang, G. Fang, Y. Gao, F. Xiao and X. Ding for technical support; Y. Sun (Ultra-Pathology Research Office of Beijing Institute of Neurosurgery) for help with the TEM analysis of microfilaments; M. Jiang, L. Miao, C. Yang, X. Wang, Q. Chen, M. Han and D. Li for comments and editing; and all of the members of the Tang laboratories for comments and suggestions. H.T. was funded by the National Key Research and Development Program of China (grant number 2019YFA0802900), National Natural Science Foundation of China (grant numbers 32070565 and 31871465), HRHI programme (grant numbers 202109007 and 202209003) of Westlake Laboratory of Life Sciences and the Westlake Education Foundation. X.H. was funded by the National Science and Technology Major Project Program (grant numbers 2021YFA1100201 and 2022YFF0712503), the National Natural Science Foundation of China (grant numbers 32170691 and 92150301), the Lingang Laboratory (grant number LG-QS-202206-06), the Beijing Nova Program (grant number 20220484033), Clinical Medicine Plus X-Young Scholars Project of Peking University, the Fundamental Research Funds for the Central Universities and the High-performance Computing Platform of Peking University.

Author information

Author notes

  1. These authors contributed equally: Peng Liu, Jing Shi, Danli Sheng.

  2. These authors jointly supervised this work: Xiaoshuai Huang, Hongyun Tang.

Authors and Affiliations

  1. Key Laboratory of Growth Regulation and Translational Research of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, China

    Peng Liu, Jing Shi, Danli Sheng, Jie Guo, Lei Gao, Xiaoqing Wang, Shaofeng Wu, Yanwen Feng & Hongyun Tang

  2. Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, China

    Peng Liu, Jing Shi, Danli Sheng, Jie Guo, Xiaoqing Wang, Shaofeng Wu, Yanwen Feng & Hongyun Tang

  3. Institute of Biology, Westlake Institute for Advanced Study, Hangzhou, China

    Peng Liu, Jing Shi, Danli Sheng, Jie Guo, Xiaoqing Wang, Shaofeng Wu, Yanwen Feng & Hongyun Tang

  4. Biomedical Engineering Department, Peking University, Beijing, China

    Wenqing Lu & Xiaoshuai Huang

  5. International Cancer Institute, Peking University, Beijing, China

    Wenqing Lu & Xiaoshuai Huang

  6. State Key Laboratory for Mesoscopic Physics and Frontiers Science Center for Nano-optoelectronics, School of Physics, Peking University, Beijing, China

    Dashan Dong

Authors
  1. Peng Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jing Shi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Danli Sheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Wenqing Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jie Guo
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Lei Gao
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Xiaoqing Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Shaofeng Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Yanwen Feng
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Dashan Dong
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Xiaoshuai Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Hongyun Tang
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

H.T. and P.L. conceptualized the study. P.L., J.S. and D.S. conceived, designed and performed most of the experiments as well as analysed the data. H.T., P.L., J.S. and D.S. wrote the paper, with input from all authors. X.H., P.L., J.S., W.L. and J.G. performed the ODT imaging and analyses, and D.D. developed the code for the ODT data acquisition. L.G. provided technical support for the EM experiments with worms. S.W., Y.F. and X.W. performed part of the experiments for the mitopher FACS analyses. H.T. supervised the study.

Corresponding authors

Correspondence to Xiaoshuai Huang or Hongyun Tang.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Cell Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Workflow for SBF-SEM analysis and 3D reconstruction.

(a) Worms were frozen with a high-pressure freezing machine (Leica EM ICE) and then freeze-substituted in a Leica EM AFS2 system using osmium-thiocarbohydrazide-osmium (OTO). After positioning the targeted worms with a Versa 510 X-Ray microscope (XRM-510, Zeiss), the samples were trimmed and mounted on SBF-SEM pin. The specimens were sputter coated with gold-palladium and imaged using a Gemini scanning electron microscope (Zeiss) equipped with a 3View2XP and OnPoint backscatter detector (Gatan). (b) Serial SBF-SEM images were segmented using Amira software to reconstruct 3D models of plasma membrane (blue), mitochondria (green), MOs (orange) and nucleus (yellow) of spermatids and mitophers (bright blue). (c-e) The SBF-SEM images and bar graph revealing that individual mitophers contain a single mitochondrion. The EM image displayed in (c) denotes four boxed regions, indicating the locations with mitophers. An enlargement of the boxed region in (c) is presented in (d), which depicts three mitophers located in the extracellular space of spermatids in the male germline, and only one single mitochondrion was observed in each of these three mitophers (d). The mitochondrial number of each mitopher was evaluated through 3D reconstruction of the EM images, and a total of 311 mitophers from three males were quantified (e). For all of these mitophers, each of them carries only one single mitochondrion (e). Source numerical data in (e) are available in source data.

Source data

Extended Data Fig. 2 Fluorescent signals indicate the mitochondrial, plasma membrane, MOs and nucleus appearance under ODT imaging.

(a-i) The 3D microscopic images presenting three different angles of the spermatid displayed in Fig. 2a(i)-(vi) (0.00 s). The images in (d-f) and (g-i) were obtained by rotating images in (a-c) 40-degrees clockwise and 184-degrees counterclockwise along the y-axis, respectively. The spermatids were marked by GFP::CATP-4 (green, plasma membrane) and MTDR (yellow, mitochondria). The rose dashed lines distinguish two separate spermatids. These images indicate that there were no extracellular structures nearby the spermatids before mitopherogenesis (0.00 s). (j-l) Images showing colocalization of MTDR (red) with the large, round, bright dots (white) under ODT imaging mode. The yellow arrows indicate several typical mitochondria. (m-o) Images demonstrating the colocalization of the nucleus-located spe-11p::mCherry::histone with the largest, round, and bright-white dots (in the center of the cell) observed in ODT images. Red arrow, nucleus. (p-r) Images displaying the cell shape of the spermatid captured by ODT imaging mode. The overall white signals within the spermatids detected by ODT imaging (q) depict the cell border, as demonstrated by the localization of GFP::CATP-4 (p). PM, plasma membrane. (s-u) Images showing that GFP::CATP-4-marked MOs colocalize with the lighter white signals in the spermatozoon head (u). After sperm activation, the GFP::CATP-4 signals localizes to the fused MOs as previously described20. Yellow arrow, mitochondria; blue dashed lines, cell border; green arrow, MOs in focal plane; purple arrow, MOs off the focal plane. (v) A representative TEM image of a spermatozoon. Yellow arrow, mitochondria; red arrow, nucleus; blue dashed lines, cell border; green arrow, MOs in focal plane; purple arrow, MOs off the focal plane. (w,x) Images of the spermatid corresponding to Fig. 2f at Z-12 focal plane indicating that the budding mitopher and the cell body remain connected at 277.2 s (w) and 278.4 s (x). The yellow box highlights the region where the budding mitopher (blue arrow) connects with the spermatid, and the enlarged images are shown in the bottom left corner. Scale bar, 500 nm (inset). Three biologically independent experiments were performed in (j–u).

Extended Data Fig. 3 3D-EM analysis of ratio of mitopher/spermatid in the male testis.

Bar graph showing the ratio of testis mitophers to the spermatids calculated from the 3D-EM analysis. Approximately 1,000 consecutive EM slices of the spermatid region of male germline arms were reconstructed to analyse the quantity of the mitophers and spermatids. The average ratio of mitopher/spermatid is around 1.36. n = 3 biologically independent samples. Source numerical data are available in source data.

Source data

Extended Data Fig. 4 Identification and quantitative analyses of in vivo-generated mitophers by FACS.

(a–k) FACS analyses, TEM images and microscopic analyses showing the identification of mitophers according to the combination of three features: diameter, presence of the interior mitochondrion, and externalized phosphatidylserine (PS) on the mitopher membrane. (a) Shows the procedures for dissecting worms. (b–d,h) Show the gating strategies. The forward scatter signals (FSC) from the reference microspheres (Beads, 0.45 μm, 0.5 μm, 1 μm, 2 μm, 3 μm) were used to estimate the size range of samples (b) and according to the size, the sample was gated into four groups (c). Annexin V with Alexa Fluor 647 was used to label the externalized PS, and MTG was used to label mitochondria. A representative TEM image (e) and the corresponding bar graph (f) indicated that structures from R2-Q2 region in (d) were all mitophers (n = 21). Confocal images indicate that the mitophers displayed Annexin V signals on the surface and interior MTG signals (g). The structures from the R1-Q3 region were all spermatids, confirmed by TEM analysis (i,j) and the DIC microscopy analyses (k). (l,m) Bar graph and representative image showing that approximately 290 sperm per male (n = 8 biologically independent experiments) and 295 sperm per male (n = 14 biologically independent worms) were detected by FACS and microscopic imaging, respectively. Each dot represents the spermatids number in one male worm. (n) Bar graph showing the total number of mitophers per male in a day-1 adult N2 male. Each dot represents the average mitopher number per male in one experiment. (o) Bar graph indicating the number of extracellular mitophers normalized to the number of spermatids in N2 males. n = 40,000 spermatids were analysed. Each dot represents the average number of mitophers normalized to one spermatid in one independent experiment. The number of biologically independent experiments is 3 in (a–k), 6 in (n), 8 in (o). Source numerical data (f,j,l,n,o) are available in source data.

Source data

Extended Data Fig. 5 Role of SPE-8-group genes in mitopherogenesis.

(a) A graph showing that sperm-specific expression of SPE-8 reverses the reduction in mitophers number in the spe-8(lf). Is[spe-11p::SPE-8];spe-8(lf) males show increased mitophers generation relative to the spe-8(lf) mutant males. Promoter of spe-11 drives the expression of the transgene specifically in the sperm. n = 30,000 spermatids were analysed. (b,c) Bar graphs showing that spe-27(lf);spe-29(lf) and spe-29(lf) mutations exhibit no obvious effect on number of in vivo mitopher in the male testis. n = 15,000 spermatids in (b) and n = 30,000 spermatids in (c) were analysed. The number of biologically independent experiments is 6 in (a,c), 3 in (b). Source numerical data are available in source data.

Source data

Extended Data Fig. 6 Procedure of analysing effects of chemicals on mitopherogenesis and quantification of microfilaments diameter in C. elegans spermatids.

(a) Schematic depiction of FACS-based methods for analysing effect of chemicals on mitophers generation in the isolated spermatids. The detailed methods are described in Methods. Created with BioRender.com. (b) Graph showing the diameter of microfilaments (approximately 7 nm) in spermatids, which is consistent with the typical size of actin filaments in other cell types. Calculated from TEM images of 10 spermatids (Fig. 4r,s) by Fiji software. Each dot represents the diameter of one microfilament and n = 98 microfilaments were analysed. (c) A graph showing that inhibiting actin polymerization represses mitopherogenesis. CK-636 was used to treat spermatids isolated from the male germline, followed by treatment with/without pronase. n = 25,000 spermatids each group were analysed from 5 biologically independent experiments. Source numerical data are available in source data.

Source data

Extended Data Fig. 7 Roles of MMP and ROS in mitopherogenesis.

(a–c) Microscopic images and corresponding bar graph showing that N2 spermatids treated with FCCP can still be activated to form mature spermatozoa in the presence of pronase (a,b). The activation rate of the sperm from the solvent control and the FCCP treatment group are 100% and 99%, respectively (c). This result supports the notion that the inhibition of mitopherogenesis by FCCP shown in Fig. 5c is not a result of reduced spermatid viability. Each dot represents the percentage of mature spermatozoa in one experiment. 200 cells from 3 biologically independent experiments were analysed. Green arrow, spermatozoa. (d) Box-and-whisker graph indicating that antioxidants treatment showed no effect on basal level of mitopherogenesis and pronase-induced mitopherogenesis. The antioxidants, tempol that scavenges superoxide anion O2− and acetylcysteine (NAC), mainly scavenging hydrogen peroxide H2O2 and hydroxyl radical OH−, were used to reduce ROS levels in the isolated spermatids, followed by treatment with/without pronase. The number of mitophers were analysed by FACS. n = 25,000 spermatids per group from 5 biologically independent experiments were analysed. Source numerical data (c,d) are available in source data.

Source data

Extended Data Fig. 8 Procedure for analysing mitophers number released by newly-generated spermatids.

(a) Schematic diagrams showing the procedure to analyse the capacity of a spermatid generating mitophers. Briefly, the spermatocytes were released by dissecting the gonad arm and micromanipulation were carried out to collect the spermatocytes with budding spermatids, which were then differentiated in vitro to obtain the newly-formed spermatids as previously described33. Pronase was added to trigger mitopherogenesis and the resulted generation of mitophers from these newly-generated spermatids were analysed by FACS. Similar number of matured spermatozoa were also collected and the released mitophers from these spermatozoa were calculated as the negative control. The quantity of mitophers normalized to one spermatid is used to show the capacity of a spermatid to release mitophers. Created with BioRender.com. (b) Representative images showing the budding spermatids collected by micromanipulation and the newly-generated spermatids in vitro differentiated from these isolated budding spermatids. The newly-generated spermatids used for analysing mitopher-generation capacity were pretested with sperm-activation assay to indicate the normal activity of these spermatids. RB, residual body.

Extended Data Fig. 9 Sperm activation and fertility analyses in spe-8(lf) males.

(a–d) Microscopic images showing that when cultured at 25 °C, spermatids from spe-8(lf) males can be activated in the presence of pronase. When grown at 20 °C, spe-8(lf) spermatids treated with pronase exhibit spikes and therefore fail to be activated (b), in accordance with previous findings16,37. In contrast, the spermatids from spe-8(lf) male grown at 25 °C can be activated to form normal pseudopodia in the presence of pronase (d), as that observed in the N2 wild-type positive control (a,c). The blue arrow indicates the pseudopodia and the white arrow indicates the spikes. (e) The bar graph showing that there is no significant difference in the length of pseudopodia between the motile spe-8(lf) sperm and non-motile spe-8(lf) sperm. Each dot represents the length of one pseudopod. 176 sperm with poor motility and 181 sperm with normal motility were analysed. (f–i) Microscopic images indicating that the sperm derived from spe-8(lf) males display increased distribution in the uterus by the vulva of fog-3(lf) females after mating. Sperm in one of the spermatheca and the uterus of fog-3(lf) females mated with males with indicated genotypes cultured at 25 °C were imaged. DAPI staining was exploited to indicate the sperm. The statistical data were shown in Fig. 6r. (j) Bar graph showing that spe-8(lf) and wild-type males produce similar number of sperm. n value is labelled on the column. Three biologically independent experiments were performed. Source numerical data (e,j) are available in source data.

Source data

Extended Data Fig. 10 A proposed model for the generation of mitophers in C. elegans spermatids.

During sperm development in male C. elegans, spermatids generate mitophers through plasma outward budding to export healthy mitochondria into the extracellular space to reduce mitochondrial quantity. During mitopherogenesis, the mitopher is released immediately upon the formation of the bud that contains one mitochondrion. The mitopher is a membrane-bound vesicle sized around 490–1,100 nm that is featured by its cargo, only one single mitochondrion, and the biogenesis via quick plasma membrane budding-off. In the testis, the extracellular protease signal triggers mitopherogenesis partially through SPE-8. Moreover, normal microfilaments dynamics are required for generating mitophers, and myosin VI negatively regulate mitopher generation. Mitopherogenesis represents a previously-unknown process to specifically export mitochondria out of cells, which is critical for modulating sperm mitochondrial quantity that may be important for producing motile male gametes. Created with BioRender.com.

Supplementary information

Reporting Summary

Peer Review File

Supplementary Table 1

Supplementary Table 1. Comparison of mitophers to other characterized EVs that carry organelles. Supplementary Table 2. Worm strain list.

Supplementary Video 1

3D-EM reconstruction showing two released mitophers in the extracellular space and one spermatid that is connected to four budding mitophers (related to Fig. 1). Reconstruction and 3D view of the plasma membrane (blue), mitochondria (green), membranous organelles (orange) and nucleus (yellow) of a spermatid and mitophers (bright blue). The 3D reconstruction shows that a mitopher harbours only one mitochondrion and the mitopher membrane is from the outward-budding plasma membrane of spermatids. Moreover, multiple mitophers can be generated by a spermatid at the same time, as evidenced by the presence of four budding mitophers that remain linked to the same spermatid.

Supplementary Video 2

Time-lapse analysis of mitopher generation from a spermatid captured by light-sheet microscopy imaging (related to Fig. 2a–e). Live imaging of spermatids, marked with cell membrane-localized GFP::CATP-4 (green) and the mitochondrial stain MTDR (yellow) indicates the quick generation of a mitopher. The MTDR channel presents the mitochondrial signal in the budding mitopher (yellow arrow) and then in the released mitopher (blue arrow). In the merged channel, in one cell, the plasma membrane outward bud containing one mitochondrion is first initiated to form a budding mitopher (yellow arrow) and the mitopher is subsequently rapidly released from the spermatid (blue arrow). This mitopherogenesis takes approximately 3 s to complete. The first 22 s of the video shows the context of the spermatid displayed in Fig. 2a–e, which indicates no mitopher signals in the extracellular space at 0 s (before mitopherogenesis). This observation further supports that the mitopher observed at 2.58 s is newly generated by the indicated mitopherogenesis. Note that, due to the lighting angle of 30° and the imaging angle of 60° employed by the lattice light-sheet microscope, the resulting 3D volume displays the shape of a parallelogram, rather than a rectangle.

Supplementary Video 3

Dynamics of mitopher formation by spermatids visualized by dual-colour imaging (related to Fig. 2a–e). Similar to Supplementary Video 2, this dual-colour imaging video captured by light-sheet microscopy also illustrates the rapid generation of a mitopher from a freshly isolated spermatid. The cell membrane and mitochondria are marked with GFP::CATP-4 (green) and MTDR (yellow), respectively. This video detects the initiation of a budding mitopher (yellow arrow), characterized as a plasma-membrane bud containing a mitochondrion and subsequently, its swift release from the spermatid to form a mitopher (blue arrow). This mitopher generation process is finished within a few seconds. The red dashed lines separate the three individual cells. The first 21 s of the video show the context of the spermatid that will produce a mitopher before mitopherogenesis; no mitopher signals were observed in the extracellular space at 0 s.

Supplementary Video 4

Dynamics of mitopherogenesis from a spermatid analysed by ODT imaging (related to Fig. 2f). Time-lapse video of label-free visualization of the complete generation of a mitopher from a freshly isolated spermatid in hydrogel. The video depicts the outward translocation of a mitochondrion (green arrow) in an upward-diagonal direction, followed by its quick release to form the mitopher (blue arrow).

Supplementary Video 5

Dynamics of mitopher release inside a dissected germline (related to Fig. 2g). Time-lapse video from ODT imaging showing the biogenesis process of a mitopher inside a dissected germline. The video illustrates the initiation of a budding mitopher (orange arrow), which is followed by the rapid release of this mitochondrion-embedding bud from the spermatid, resulting in the formation of a mitopher (blue arrow). The released mitopher eventually moves away. The yellow dashed lines outline the cell boundary. The whole mitopher generation process takes only a few seconds to finish.

Supplementary Video 6

Time-lapse analysis of mitopherogenesis inside a dissected germline (related to Fig. 2g). Another time-lapse video captured by ODT imaging indicates the rapid biogenesis of a mitopher from spermatids within a dissected germline. It visualizes the initiation of a budding mitopher (orange arrow) and its quick disconnection from the spermatid, resulting in the formation of a mitopher (blue arrow). The released mitopher eventually moves away. The entire mitopherogenesis process is completed within only a few seconds. The yellow dashed lines depict the cell boundary of the region in which mitopherogenesis occurs.

Supplementary Video 7

Motility of in vitro-activated sperm from wild-type N2 and spe-8(lf) worms (related to Fig. 6). Time-lapse videos displaying the motility of pronase-activated sperm with featured pseudopodia (blue arrow) from N2 and spe-8(lf) males grown at 25 °C. Orange arrows point to sperm with normal motility and red arrows point to sperm with pseudopodia exhibiting poor motility. Our video shows the motility defect in part of the spe-8(lf) sperm with pseudopodia.

Source data

Source Data Fig. 1

Statistical source data.

Source Data Fig. 3

Statistical source data.

Source Data Fig. 4

Statistical source data.

Source Data Fig. 5

Statistical source data.

Source Data Fig. 6

Statistical source data.

Source Data Extended Data Fig. 1

Statistical source data.

Source Data Extended Data Fig. 3

Statistical source data.

Source Data Extended Data Fig. 4

Statistical source data.

Source Data Extended Data Fig 5

Statistical source data.

Source Data Extended Data Fig. 6

Statistical source data.

Source Data Extended Data Fig. 7

Statistical source data.

Source Data Extended Data Fig. 9

Statistical source data.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Liu, P., Shi, J., Sheng, D. et al. Mitopherogenesis, a form of mitochondria-specific ectocytosis, regulates sperm mitochondrial quantity and fertility. Nat Cell Biol 25, 1625–1636 (2023). https://doi.org/10.1038/s41556-023-01264-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41556-023-01264-z

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing