3D in situ imaging of female reproductive tract reveals molecular signatures of fertilizing spermatozoa in mice

  1. Lukas Ded
  2. Jae Yeon Hwang
  3. Kiyoshi Miki
  4. Huanan F Shi
  5. Jean-Ju Chung  Is a corresponding author
  1. Yale School of Medicine, United States
  2. Boston Children's Hospital, United States

Abstract

Out of millions of ejaculated sperm, only a few reach the fertilization site in mammals. Flagellar Ca2+ signaling nanodomains, organized by multi-subunit CatSper calcium channel complexes, are pivotal for sperm migration in the female tract, implicating CatSper-dependent mechanisms in sperm selection. Here, using biochemical and pharmacological studies, we demonstrate that CatSper1 is an O-linked glycosylated protein, undergoing capacitation-induced processing dependent on Ca2+ and phosphorylation cascades. CatSper1 processing correlates with protein tyrosine phosphorylation (pY) development in sperm cells capacitated in vitro and in vivo. Using 3D in situ molecular imaging and ANN-based automatic detection of sperm distributed along the cleared female tract, we demonstrate that all spermatozoa past the UTJ possess intact CatSper1 signals. Together, we reveal that fertilizing mouse spermatozoa in situ are characterized by intact CatSper channel, lack of pY, and reacted acrosomes. These findings provide molecular insight into sperm selection for successful fertilization in the female reproductive tract.

Data availability

All data generated or analysed during this study are included in the manuscript, supplementary and source data files.

Article and author information

Author details

  1. Lukas Ded

    Department of Cellular and Molecular Physiology, Yale School of Medicine, New Haven, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Jae Yeon Hwang

    Department of Cellular and Molecular Physiology, Yale School of Medicine, New Haven, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Kiyoshi Miki

    Boston Children's Hospital, Boston, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Huanan F Shi

    Department of Cellular and Molecular Physiology, Yale School of Medicine, New Haven, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3710-5917
  5. Jean-Ju Chung

    Department of Cellular and Molecular Physiology, Yale School of Medicine, New Haven, United States
    For correspondence
    jean-ju.chung@yale.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8018-1355

Funding

National Institutes of Health (R01HD096745)

  • Jean-Ju Chung

Yale School of Medicine (Start-up funds)

  • Jean-Ju Chung

Yale University (a Yale Goodman-Gilman ScholarAward-2015)

  • Jean-Ju Chung

Male Contraceptive Initiative (Postdoctoral fellowship)

  • Jae Yeon Hwang

Czech Science Foundation (GJ20-17403Y)

  • Lukas Ded

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Animal experimentation: Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All the mice were treated in accordance with guidelines approved by Yale (20079) Animal Care and Use Committees (IACUC).

Reviewing Editor

  1. Merritt Maduke, Stanford University School of Medicine, United States

Publication history

  1. Received: August 12, 2020
  2. Accepted: October 19, 2020
  3. Accepted Manuscript published: October 20, 2020 (version 1)
  4. Accepted Manuscript updated: October 22, 2020 (version 2)
  5. Version of Record published: December 1, 2020 (version 3)

Copyright

© 2020, Ded et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 3,583
    Page views
  • 441
    Downloads
  • 14
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Lukas Ded
  2. Jae Yeon Hwang
  3. Kiyoshi Miki
  4. Huanan F Shi
  5. Jean-Ju Chung
(2020)
3D in situ imaging of female reproductive tract reveals molecular signatures of fertilizing spermatozoa in mice
eLife 9:e62043.
https://doi.org/10.7554/eLife.62043
  1. Further reading

Further reading

    1. Cell Biology
    Lu Zhu et al.
    Research Article

    Nedd4/Rsp5 family E3 ligases mediate numerous cellular processes, many of which require the E3 ligase to interact with PY-motif containing adaptor proteins. Several Arrestin-Related Trafficking adaptors (ARTs) of Rsp5 were self-ubiquitinated for activation, but the regulation mechanism remains elusive. Remarkably, we demonstrate that Art1, Art4, and Art5 undergo K63 linked di-Ubiquitination by Rsp5. This modification enhances the PM recruitment of Rsp5 by Art1 or Art5 upon substrate induction, required for cargo protein ubiquitination. In agreement with these observations, we find that di-ubiquitin strengthens the interaction between the Pombe orthologs of Rsp5 and Art1, Pub1 and Any1. Further, we discover that the HECT domain exosite protects the K63 linked di-Ubiquitin on the adaptors from cleavage by the deubiquitination enzyme Ubp2. Together, our study uncovers a novel ubiquitination modification implemented by Rsp5 adaptor proteins, underscoring the regulatory mechanism of how adaptor proteins control the recruitment and activity of Rsp5 for the turnover of membrane proteins.

    1. Cell Biology
    2. Structural Biology and Molecular Biophysics
    Ritvija Agrawal et al.
    Research Article Updated

    Dynein harnesses ATP hydrolysis to move cargo on microtubules in multiple biological contexts. Dynein meets a unique challenge in meiosis by moving chromosomes tethered to the nuclear envelope to facilitate homolog pairing essential for gametogenesis. Though processive dynein motility requires binding to an activating adaptor, the identity of the activating adaptor required for dynein to move meiotic chromosomes is unknown. We show that the meiosis-specific nuclear-envelope protein KASH5 is a dynein activating adaptor: KASH5 directly binds dynein using a mechanism conserved among activating adaptors and converts dynein into a processive motor. We map the dynein-binding surface of KASH5, identifying mutations that abrogate dynein binding in vitro and disrupt recruitment of the dynein machinery to the nuclear envelope in cultured cells and mouse spermatocytes in vivo. Our study identifies KASH5 as the first transmembrane dynein activating adaptor and provides molecular insights into how it activates dynein during meiosis.