Reorganization of the Flagellum Scaffolding Induces a Sperm Standstill During Fertilization

Reviewer #1 (Public Review):

Summary:

This important work advances our understanding of sperm motility regulation during fertilization by uncovering the midpiece/mitochondria contraction associated with motility cessation and structural changes in the midpiece actin network as its mode of action. The evidence supporting the conclusion is solid, with rigorous live cell imaging using state-of-the-art microscopy, although more functional analysis of the midpiece/mitochondria contraction would have further strengthened the study. The work will be of broad interest to cell biologists working on the cytoskeleton, mitochondria, cell fusion, and fertilization.

Strengths:

The authors demonstrate that structural changes in the flagellar midpiece F-actin network are concomitant to midpiece/mitochondrial contraction and motility arrest during sperm-egg fusion by rigorous live cell imaging using state-of-art microscopy.

Weaknesses:

Many interesting observations are listed as correlated or in time series but do not necessarily demonstrate the causality and it remains to be further tested whether the sperm undergoing midpiece contraction are those that fertilize or those that are not selected. Further elaboration of the function of the midpiece contraction associated with motility cessation (a major key discovery of the manuscript) would benefit from a more mechanistic study.

Reviewer #2 (Public Review):

The authors used various microscopy techniques, including super-resolution microscopy, to observe the changes that occur in the midpiece of mouse sperm flagella. Previously, it was shown that actin filaments form a double helix in the midpiece. This study reveals that the structure of these actin filaments changes after the acrosome reaction and before sperm-egg fusion, resulting in a thinner midpiece. Furthermore, by combining midpiece structure observation with calcium imaging, the authors show that changes in intracellular calcium concentrations precede structural changes in the midpiece. The cessation of sperm motility by these changes may be important for fusion with the egg. Elucidation of the structural changes in the midpiece could lead to a better understanding of fertilization and the etiology of male infertility. The conclusions of this manuscript are largely supported by the data, but there are several areas for improvement in data analysis and interpretation. Please see the major points below.

1. It is unclear whether an increased FM4-64 signal in the midpiece precedes the arrest of sperm motility. This needs to be clarified in order to argue that structural changes in the midpiece cause sperm motility arrest. The authors should analyze changes in both motility and FM4-64 signal over time for individual sperm.

2. It is possible that sperm stop moving because they die. Figure 1G shows that the FM4-64 signal is increased in the midpiece of immotile sperm, but it is necessary to show that the FM4-64 signal is increased in sperm that are not dead and retain plasma membrane integrity by checking sperm viability with propidium iodide or other means.

3. It is unclear how the structural change in the midpiece causes the entire sperm flagellum, including the principal piece, to stop moving. It will be easier for readers to understand if the authors discuss possible mechanisms.

4. The mitochondrial sheath and cell membrane are very close together when observed by transmission electron microscopy. The image in Figure 9A with the large space between the plasma membrane and mitochondria is misleading and should be corrected. The authors state that the distance between the plasma membrane and mitochondria approaches about 100 nm after the acrosome reaction (Line 330 - Line 333), but this is a very long distance and large structural changes may occur in the midpiece. Was there any change in the mitochondria themselves when they were observed with the DsRed2 signal?

5. In the TG sperm used, the green fluorescence of the acrosome disappears when sperm die. Figure 1C should be analyzed only with live sperm by checking viability with propidium iodide or other means.

Reviewer #3 (Public Review):

While progressive and also hyperactivated motility are required for sperm to reach the site of fertilization and to penetrate the oocyte's outer vestments, during fusion with the oocyte's plasma membrane it has been observed that sperm motility ceases. Identifying the underlying molecular mechanisms would provide novel insights into a crucial but mostly overlooked physiological change during the sperm's life cycle. In this publication, the authors aim to provide evidence that the helical actin structure surrounding the sperm mitochondria in the midpiece plays a role in regulating sperm motility, specifically the motility arrest during sperm fusion but also during earlier cessation of motility in a subpopulation of sperm post acrosomal exocytosis.

The main observation the authors make is that in a subpopulation of sperm undergoing acrosomal exocytosis and sperm that fuse with the plasma membrane of the oocyte display a decrease in midpiece parameter due to a 200 nm shift of the plasma membrane towards the actin helix. The authors show the decrease in midpiece diameter via various microscopy techniques all based on membrane dyes, bright-field images and other orthogonal approaches like electron microscopy would confirm those observations if true but are missing. The lack of additional experimental evidence and the fact that the authors simultaneously observe an increase in membrane dye fluorescence suggests that the membrane dyes instead might be internalized and are now staining intracellular membranes, creating a false-positive result. The authors also propose that the midpiece diameter decrease is driven by changes in sperm intracellular Ca2+ and structural changes of the actin helix network. Important controls and additional experiments are needed to prove that the events observed by the authors are causally dependent and not simply a result of sperm cells dying.