The basal beat frequency of bPAC sperm was slightly lower compared to wild-type sperm 5. Light stimulation of bPAC sperm evoked a transient acceleration of the flagellar beat: a ms light pulse increased the beat frequency within 10 s about 2. The acceleration persisted for at least 10 s and sperm returned to basal beat frequency within 30 s Figure 2H. The light-stimulated acceleration of the flagellar beat was dose-dependent: the longer the light exposure, the faster the onset and higher the beat frequency Figure 2I,J. Local illumination of a small part of the flagellum also enhanced the beat frequency, albeit the increase was smaller and its kinetics slower compared to global illumination Figure 2K , Videos 3,4.
This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. The cell was tethered to the glass surface by lowering the BSA concentration 0. For clarity, the video is played with fps half original speed. The recording was performed as described for Video 1 using local illumination of the sperm flagellum for ms; the video is played with fps half of the original speed.
In this setting, the light flash is not visible and is, therefore, visualized with a circle. The analysis of the beat frequency is presented in Figure 2K.
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Video 3 shown at higher intensity for the frames containing the light flash. This allows to visualize the light flash. The profile of the light flash is indicated in an inset. Experiments analyzing the action of 8-Br-cNMPs on membrane currents in mouse sperm yielded inconsistent results Kirichok et al. Thus, this effect needs to be addressed by future studies.
Arrows indicate the addition of compounds. Signals were measured in 96 multi-well plates in a fluorescence plate reader. C Mean signals evoked by light stimulation, cyclic nucleotide analogs, and ionomycin. We explored the potential of optogenetics to compensate for the functional loss of SACY.
Furthermore, we examined whether in sperm lacking functional SACY, bPAC activation can restore motility and the ability to fertilize the egg. Male Slc9a null mice are infertile, because their sperm are largely immotile Wang et al. Thus, we tested whether motility can be restored by solely increasing cAMP levels with light. Sperm were immotile in darkness Figure 4C , left ; light stimulation restored both the flagellar beat and forward motility Figure 4C , right; Video 5 , demonstrating that restoring cAMP levels alone is sufficient to rescue motility.
In bPAC sperm, light stimulation is sufficient to restore tyrosine phosphorylation. The recording was performed as described for Video 1 , but without the additional 1. The video is shown in real time. In this study, we report on a transgenic mouse model designed to control cAMP signaling in sperm using optogenetics. Furthermore, we show that optogenetics can be used to restore fertility. Expression of bPAC in Slc9a null sperm allowed restoring motility after light stimulation and, most importantly, restoring fertilization in vitro.
Of note, in vitro fertilization IVF using light-stimulated sperm was successful using zona pellucida-intact oocytes, demonstrating that a cAMP increase, at least in vitro, is sufficient to restore fertility in Slc9a null mice. In contrast, incubation of Slc9a null sperm with cAMP analogs only restored fertilization of zona pellucida-free but not zona pellucida-intact oocytes Wang et al.
This discrepancy could be explained by 1 cAMP analogs not reaching sufficiently high levels in sperm, or 2 cAMP analogs being not as efficient as cAMP to stimulate downstream targets, or 3 non-specific effects compromising the interpretation of their action Brenker et al. Now that we have established the Prm1-bPAC mouse model and demonstrated that it can be used to analyze cAMP signaling in sperm, the next step will be to implement the spatio-temporal precision of this tool to study cAMP signaling in sperm.
The Prm1-bPAC mouse model allows deciphering these signaling events, if bPAC expression does not interfere with other signaling pathways. Signaling molecules in sperm flagella are highly compartmentalized Chung et al. The flagellar beat is controlled by cAMP-dependent phosphorylation of PKA targets in the axoneme Salathe, ; however, it is unknown whether cAMP signaling is restricted to microdomains or whether changes in cAMP propagate along the flagellum.
Compartmentalization of cAMP signaling confers specificity to this ubiquitous cellular messenger, for example, in cell-cycle progression or modulation of gene expression. Our results show that local illumination of the flagellum of bPAC sperm is sufficient to increase its beat frequency. Optogenetics using bPAC provides the spatial and temporal precision to increase cAMP levels in subcellular regions and specialized signaling compartments such as cilia and flagella, which allows to study complex signaling networks - not only in mammalian sperm but also any other ciliated cell type.
Finally, the optogenetic toolkit has been recently expanded by a light-activated phosphodiesterase LAPD that hydrolyses cyclic nucleotides upon stimulation by red light Gasser et al. These findings encourage future studies to explore the full potential of controlling cellular messengers by optogenetics.
Mice used in this study were 2—5 months of age. All animal experiments were in accordance with the relevant guidelines and regulations. To activate bPAC, sperm samples were illuminated for 90 min in suspension in a reaction tube using a halogen fiber optics illuminator Olympus, Tokio, Japan. For re-probing, membranes were incubated in stripping buffer For the study of protein phosphorylation, sperm were incubated in capacitating buffer containing 25 mM NaHCO 3 for 10 min PKA-dependent phosphorylation or 90 min tyrosine kinase-dependent phosphorylation.
Similarly, bPAC sperm were illuminated for either 10 or 90 min using a halogen fibre optics illuminator W, Intralux ; Volpi, Schlieren, Switzerland. The primary anti-HA antibody rat monoclonal; Roche was diluted in blocking buffer and incubated for 2 hr. Fluorescent secondary antibodies donkey anti-rat Alexa; Dianova was diluted in blocking buffer containing 0.
Red light was used during the isolation of bPAC sperm. Sperm were adjusted to a concentration of 1.
To activate bPAC, sperm samples were illuminated in suspension in a reaction tube using a halogen fiber-optics illuminator W, Intralux ; Volpi, Schlieren, Switzerland. Samples were frozen in liquid N 2 , thawed, and neutralized by addition of K 3 PO 4 0. The cAMP content in the supernatant was determined by a competitive immunoassay Molecular Devices, Sunnyvale, USA , including an acetylation step for higher sensitivity.
Calibration curves were obtained by serial dilutions of cAMP standards. Non-capacitated sperm were incubated in BSA buffer only. Images were acquired using a confocal laser scanning microscope FV; Olympus , a minimum of cells was analyzed per condition.
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Cells were tethered to the glass surface by adjusting the BSA concentration in the buffer to 0. For analysis, cells that had their head attached to the glass surface and that had a free beating flagellum were chosen. This resulted in a Gaussian-shaped UV spot R 2 of the fit 0. The total UV-light power delivered was 2.
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The software can be made available upon request. The program identified the best threshold for binarization followed by a skeleton operation to identify the flagellum. The flagellar beat parameters were determined within a time window of 0. For frames at the boundaries the beginning or the end of the video or flanking the UV flash , the time windows were asymmetric but contained the same number of frames.
We monitored the angle between the straight line connecting the middle of the flagellum with the sperm head and the axis of symmetry of the cell. This angle varied in a sinusoid-like manner in time. The beat frequency was obtained by fitting a sinus to this wave. Using defined thresholds, the image was binarized. From a user-defined region-of-interest centred at the cell head, the program determined the location of the head on subsequent frames using a registering procedure. The neck of the cell was identified by applying a mask with the shape of an annulus centred into the sperm head.
All frames were then rotated and superimposed with a rotation angle equal to the azimuth of the neck region on a reference system centred at the sperm head. For better visualization, representative datasets were smoothed using Graph Pad Prism 5. Superovulation in females was induced by intraperitoneal injection of 10 I.
Intergonan SimposiumVet, Lisbon, Portugal 3 days before the experiment.
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Ovogest SimposiumVet. Sperm were capacitated for 90 min in TYH medium supplemented as indicated above. Cumulus-enclosed oocytes were prepared from the oviducts of superovulated females and added to the drops. The number of 2-cell stages was evaluated after 24 hr. An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown.
cAMP Signaling: Methods and Protocols
Reviewers have the opportunity to discuss the decision before the letter is sent see review process. Similarly, the author response typically shows only responses to the major concerns raised by the reviewers. Your article has been favorably evaluated by a Senior editor, David Clapham as a guest Reviewing editor, and two peer reviewers.
The Reviewing editor and the reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.
follow url The full reviewers comments are shown at the end of this letter, but the points for revision boil down to the following:. Such a small change at this high dose is difficult to interpret. In the Kirichok et al. Thus, if you wish to support this statement, you could record mouse ICatSper changes with 8Br-cAMP and show that 8-Br-cAMP activation is nonspecific, perhaps by showing a dose response curve, comparing inside and outside perfusion of the analog, etc. Alternatively, for both points, you could refrain from overextending your conclusions and simply state the points you have definitively shown.
This would require removing the comments quoted above and alike comments in the discussion, and state without exaggeration the merits and current limitations of the experiments. This is an important new tool, and it is best to accurately state what is, and is not, possible to conclude. Further refinement of the indicator, or other indicators may enable you to definitively address these points in the future.
In this manuscript, Jansen et al. Light stimulation in the transgenic sperm is able to increase cAMP concentration, leads to the acrosome reaction and triggers sperm tail beating. The tool developed in the paper allowed the authors to directly test the function of cAMP. Previous experiments relied on the addition of bicarbonate, exogenously applied cell-permeable cAMP or cAMP UV-uncaging, which can be somewhat indirect.
The results from the paper largely confirmed previous findings: cAMP is important for all the major sperm physiology including capacitation, motility and fertilization.