From head to tail: The remarkable 3D architecture of mammalian sperm cells across scales and species

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For most people, sperm cells have a very simple structure consisting of head and tail. This simplistic view is based on how the cells look under a light microscope and has been challenged by advances in electron microscopy. Dr Tzviya Zeev-Ben-Mordehai and her team from Utrecht University, Netherlands, use an advanced electron microscopy technique, known as cryo-electron tomography (cryo-ET), to reveal the complexity of sperm cells. The high-resolution imaging studies conducted by Dr Zeev-Ben-Mordehai provide vital information on how the molecular architecture of mammalian sperm cells supports cell motility through diverse fluid environments.

Despite their distinctive streamlined shape, sperm cells are the most diverse cell type among multicellular organisms. On the way to becoming streamlined, sperm cells lose most of their cellular organelles. As they are the smallest cell in our body, the question arises how the remaining organelles fit into their diminutive size. When observed under a light microscope, sperm from many species appear similar and very simple, consisting of a head and a tail. This simplistic view of sperm cells has long been challenged by electron microscopy. With the advent of advanced electron microscopy, scientists now reveal the molecular basis underlining species-dependent variations of these remarkably complex cells. The differences observed reflect the sheer range of fertilisation and reproductive arenas, from marine and freshwater media to the viscous fluids of the female reproductive tract for mammals.

A packing problem

Dr Tzviya Zeev-Ben-Mordehai and her team from Utrecht University, Netherlands, study the molecular architecture of mammalian sperm cells. The team takes advantage of cryo-electron tomography (cryo-ET), a cutting-edge technique that allows them to image cells in hydrated form without fixatives or stains. The researchers show how the different subcellular structures interact to form a compact and streamlined cell. By comparing the structural features of sperm cells from different species of mammals, the team demonstrated both conserved (identical or similar across species) and species-specific features underlining sperm cells.

This schematic shows the concept of electron tomography. A sample is imaged in a TEM as it is tilted to different angles, resulting in a ‘tilt-series’ of 2D images. wikipedia.org/wiki/Electron_cryotomography

Microtubules make up the basic architecture of the flagellum

Flagella, tail-like structures like those observed in sperm cells, are well-conserved molecular assemblies used by eukaryotic cells to propel themselves through fluid environments. The oscillatory wave-like motions associated with flagella are made possible by an intricate architecture built from a vast array of molecular structures that work together in a concerted fashion. The core of the flagella is conserved across eukaryotic cells, but flagella from different cells will generate different waveforms to reflect the wide range of fluid environments to which they are adapted.

The flagellar core is called axoneme and it is made of microtubules and hundreds of support and motor proteins. Microtubules are hollow filament structures made of a protein known as tubulin. Microtubules can be found as singlets or arranged into more complex patterns of doublets and triplets. The axoneme consists of nine doublet microtubules arrayed around a central pair of singlet microtubules. The bending of an axoneme is orchestrated by hundreds of motor proteins called dyneins that generate movement. At the base of the flagellum, the axoneme is anchored to the basal body, or centriole. In most cell types, the centrioles have a cylinder shape made of triplet microtubules. As mentioned, flagella from different cells will generate different waveforms. However, since their core is similar, this raises the question of how this basic core is modified to facilitate distinct waveforms.

“Despite their diminutive size, mammalian sperm cells have remarkably complex cellular structure.”

Mammalian sperm, with their natural diversity, is an excellent cell type to study axoneme adaption to facilitate different waveforms. The adaptions are in the nanoscale range, thus requiring high-resolution imaging. While conventional electron microscopy contributed greatly to our understanding of sperm and egg-cell biology, as reviewed in 2020 by Zeev-Ben-Mordehai’s team, it does not provide molecular resolution. The team thus developed protocols to use cryo-ET for the first time on mammalian sperm. Taking advantage of the multi-scale capabilities of cryo-ET, the team reports novel observations on mammalian sperm organelles and macromolecular complexes.

Unique axoneme accessory structures support motility in mammalian sperm flagellum

Using cryo-ET, Zeev-Ben-Mordehai’s team gains insights into the molecular structures that surround the axoneme in sperm cells. The researchers published their results in 2021, revealing how the conserved flagella core is ornamented to support motility through diverse fluid environments. Their comparative study of sperm cells from horse, mouse, and pig revealed both conserved and species-specific features underlining sperm cells. They found that the axoneme microtubules are reinforced by proteins that bind to the inside surface of the filaments in horse and pig, but not in mouse. The study also revealed that the structural elements known as outer dense fibres (ODFs) that surround the axoneme along most of the tail are directly coupled to the axonemal microtubule doublets in the distal part of the tail but not in the proximal, and this feature is conserved among all species studied. This study provided vital information on how the molecular architecture of mammalian sperm flagella directly affects sperm motility. It demonstrated that the unique molecular architecture imposes asymmetry and anisotropy on the flagellum which are of particular relevance to the different waveforms generated.

Architecture of mammalian sperm.

Zeev-Ben-Mordehai’s team also looked at the base of the flagellum in the region that connects the tail and head, known as the connecting piece. This region hosts the centrioles. It is crucial to transmit movement from the tail to the head and relevant for cell navigation. The 2021 study demonstrated that the base is anchored through a large, asymmetric chamber around the two centrioles, referred to as the proximal centriole for the one closer to the head and the distal centriole for the one further away from the head. Intriguingly, cryo-ET confirmed that the proximal centriole is composed of triplet microtubules in pig and horse sperm, but not all triplets are of the same length. The distal centriole on the other hand is a very atypical centriole composed of doublet microtubules (and not triplets) arrayed asymmetrically around a pair of singlet microtubules.

The explanation of the atypical structure of the distal centriole was revealed in a follow-up study that was published in 2021 together with the team’s collaborators. The distal centriole is the flagellum basal body and the team found that, unlike basal bodies in other cell types that are very rigid in order to be shock absorbent, the sperm distal centriole shows internal movement. Thus, the sperm distal centriole and its surrounding matrix form a dynamic basal complex (DBC) that facilitates a cascade of internal sliding deformations, coupling tail beating with asymmetric head kinking. The deformations, transmitted throughout the DBC, produce a head tilt to the left, generating a kinking motion. The researchers propose that the DBC may have evolved to serve as a mechanical transducer, coupling sperm head and tail into a single, self-coordinated system.

“The molecular architecture of mammalian sperm flagella directly affects sperm motility.”

Mitochondria and cytoskeleton in mammalian sperm cells

Zeev-Ben-Mordehai’s team has recently submitted a study for review describing the use of cryo-ET to image the sperm mitochondrial sheath in pig, mouse, and horse sperm cells. Mitochondria are the organelles where cellular respiration occurs. They can be described as the ‘powerhouse’ of the cell and although they exist in almost every cell type, mitochondria are present in bigger numbers in cells with a high demand of energy, such as muscle, liver, neural, and sperm cells. In sperm cells, mitochondria are strategically located in the flagellum but close to the head, defining a region called the midpiece. Perhaps due to the limited space and to facilitate coupling between mitochondria, in sperm cells mitochondria are arranged in a tight spiral around the axoneme and ODFs known as the mitochondrial sheath. In the recent study, Dr Zeev-Ben-Mordehai and her colleagues combined cryo-ET and in-cell cross-linking mass spectrometry to determine how the mitochondria sheath is held together and interacts with the cytoskeleton, a network of protein filaments that provide cells with structural support, stabilising their shape.

Their study highlighted unexpected diversity in the internal ultrastructure of mitochondria across mammalian species. These differences can be correlated with different rates of respiration, providing structural bases for interspecies differences in mitochondrial energetics. Despite this observed diversity in mitochondrial internal structure, Zeev-Ben-Mordehai’s team found that the molecular underpinnings the mitochondrial sheath architecture are conserved, at least in mammals. They found that novel linker proteins anchor neighbouring mitochondria to each other and an array of different proteins anchor them to the cytoskeleton.

By using in-cell cross-linking mass spectrometry, the team identified the proteins that mediate the interaction of the mitochondria to the cytoskeleton. The protein arrays consist of glycerol kinase (GK)-like proteins anchored on a network of voltage dependent anion channels (VDACs) embedded in the outer mitochondrial membrane. The team argues that these arrays may help maintain the integrity of the mitochondria-cytoskeleton interactions, stabilising them against shear stresses caused by high sperm motility.

Stained human sperm. Bobjgalindo, CC BY-SA 4.0, via Wikimedia Commons

Concluding remarks

The advances in cellular electron microscopy, in the form of cryo-ET, have allowed Dr Zeev-Ben-Mordehai and her team to image sperm cells from different mammals in their hydrated form and as close as possible to their native conditions for the first time, revealing how sperm cells’ molecular architecture is a design made for function, correlating both conserved and species-specific features with motility in different environments.

The data from the Zeev-Ben-Mordehai laboratory clearly show that mammalian sperm flagella are modified across different scales, from larger structures a few hundred nanometres wide – which increase the size and rigidity of the entire flagellar assembly – to smaller microtubule inner proteins that provide support to the microtubules.

The team also identified the proteins responsible for the interactions of mitochondria to the cytoskeleton of mammalian sperm cells. The researchers found that the binding is mediated by GK-like proteins and VDAC on the outer mitochondrial membrane. VDACs are ubiquitous mitochondrial proteins, prompting Dr Zeev-Ben-Mordehai and her colleagues to speculate whether they also regulate mitochondria-cytoskeleton interactions in other differentiated cells, such as neurones and skin cells.


Your knowledge of the structural biology of mammalian sperm cells and its role on sperm motility could inform other researchers looking for strategies to treat male infertility. Is such an application already included in the scope of your research?
Our research is fundamental research in nature, deepening our understanding of the cell biology of mammalian sperm. Nonetheless, our research can directly impact treatments for male infertility. To be able to find treatments for male infertility, we should first find the cause. More than 60% of male infertility is unexplained, known as idiopathic male infertility, showing a gap in diagnosis. Currently, routine semen analyses include mainly sperm concentration, motility and progression, and are based on inspections under a light microscope. Our studies highlight the power of using cryo-ET and provide detailed descriptions of healthy mammalian sperm. It was much easier for us to establish the approach to image with cryo-ET mammalian sperm from animals due to ease in getting samples. Having the approach established, we are expanding our research now to sperm from healthy humans as well as from patients with idiopathic male infertility.

 

References

  • Leung, M, Roelofs, M, Ravi, R, Maitan, P, Henning, H, Zhang, M, Bromfield, E, Howes, S, Gadella, B, Bloomfield-Gadêlha, H, Zeev-Ben-Mordehai, T, (2021). The multi-scale architecture of mammalian sperm flagella and implications for ciliary motility. The EMBO journal, 40(7), e107410. doi.org/10.15252/embj.2020107410
  • Leung, M, Chiozzi, R, Roelofs, M, Hevler, J, Ravi, R, Maitan, P, Zhang, M, Henning, H, Bromfield, E, Howes, S, Gadella, B, Heck, A, Zeev-Ben-Mordehai, T, (2021). In-cell structures of a conserved supramolecular array at the mitochondria-cytoskeleton interface in mammalian sperm. Pre-print. biorxiv.org/content/early/2021/02/17/2021.02.16.431372.abstract
  • Ravi, R, Leung, M, Zeev-Ben-Mordehai, T, (2020). Looking back and looking forward: contributions of electron microscopy to the structural cell biology of gametes and fertilization. Open biology, 10(9), 200186. doi.org/10.1098/rsob.200186
  • Khanal, S, Leung, M, Royfman, A, Fishman, E, Saltzman, B, Bloomfield-Gadêlha, H, Zeev-Ben-Mordehai, T, Avidor-Reiss, T, (2021). A dynamic basal complex modulates mammalian sperm movement. Nature communications, 12(1), 3808. doi.org/10.1038/s41467-021-24011-0
DOI
10.26904/RF-138-1809096403

Research Objectives

Dr Tzviya Zeev-Ben-Mordehai and her team use cryo-electron tomography (cryo-ET), to reveal the complexity of sperm cells.

Funding

NWO, Dutch Research Council. Grant 740.018.007

Collaborators

Bio

Tzviya Zeev-Ben-Mordehai is an associate professor at Bijvoet Centre for Biomolecular Research of the Utrecht University in the Netherlands. Tzviya was awarded her PhD in structural biology from the Weizmann Institute of Science, Israel. She did her postdoc at the University of Oxford, UK, where she specialised in cryo-electron tomography. In 2015 she established her own research group also at the University of Oxford. In 2017 she moved to the university of Utrecht where her group specialises in cellular structural biology of mammalian gametes.

Tzviya Zeev-Ben-Mordehai

Contact
David de Wied Building
Room 2.66, Universiteitweg 99
3584 CG Utrecht
Netherlands

E: z.zeev@uu.nl
T: 31 30 253 3178
W: www.uu.nl/en/research/structural-biochemistry/research/dr-tzviya-zeev-ben-mordehai
W: @TzviyaZBM

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