P-097 Studying epididymal sperm using cryo-electron microscopy
Authors: Roelofs M , Zeev-Ben-Mordehai T 
Published: July 2024

Study question

What are the structural changes in epididymal sperm leading to gain of motility?

Summary answer

We resolved the contents of the cytoplasmic droplet in immature sperm. We further found that the outer dynein arms are in a pre-power stroke conformation.

What is known already

Cells exiting the testis are morphologically complete but incapable of fertilization. They first need to mature in the epididymis, where sperm gain progressive motility. Intriguingly, during this epididymal maturation the cytoplasmic droplet, a cytoplasmic remnant from spermiogenesis, migrates distally from the connecting piece to the principal piece.

At the heart of the sperm’s flagellum is the axoneme, the molecular motor of the cell. It is a microtubule-based structure that houses protein complexes with different functions. For instance, it has radial spokes for signaling and rigidity. It also houses dyneins, motor proteins attached to neighboring doublets that ultimately generate flagellar beating.

Study design, size, duration

Bovine testes were acquired from a slaughterhouse, and sperm were isolated from different regions of the epididymis. After a washing step, these cells were directly used for imaging.

Participants/materials, setting, methods

Cells were prepared for imaging by rapid freezing on cryo-Electron Microscopy (cryo-EM) grids. Frozen cells were either imaged directly or first thinned by Focused Ion Beam (FIB) – milling. We applied the image analysis approaches subtomogram averaging and single particle analysis.

Main results and the role of chance

We resolved the structure of the 96-nm repeat of the axonemal doublet from immature sperm and showed a complete axoneme, containing radial spokes and different sets of dynein arms. Focusing our refinement on a specific set of dyneins, the outer dynein arms, revealed that they are in the pre-power stroke state. In this primed state the dyneins have ATP bound and are active. Interestingly, these cells display no movement. This could indicate that a different blocking mechanism is present in the axoneme.

Furthermore, we used 3D cryo-EM to elucidate the contents of the cytoplasmic droplet. We showed that it contains many vesicles, some display a shape that is reminiscent of an autophagosome. We also find vesicles displaying a double membrane with a highly repetitive density connecting in between. This suggests that sperm cells bring along auxiliary proteins and molecules in the cytoplasmic droplet that may be required for proper maturation.

Altogether we show that cryo-EM can be used to study immature sperm cells in detail, with a focus on structures important in the gain of movement.

Limitations, reasons for caution

Cryo-EM imaging requires frozen cells and uses electrons as imaging source, therefore we lack information on cell state (e.g. viability/motility) or protein identity. By complementing our visual data with other techniques such as CASA and mass spectrometry, we aim to get a full picture.

Wider implications of the findings

Elucidating protein conformation or the structural mechanism behind sperm activation could open doors into developing new therapeutics for diseases on one hand. On the other hand, understanding how sperm motility arises (and how to potentially put it to a halt) could also lay ground for developing male contraception.

Trial registration number

not applicable