CatSperζ regulates the structural continuity of sperm Ca²⁺ signaling domains and is required for normal fertility

Thank you for submitting your article "CatSperζ Regulates the Structural Continuity of Sperm Ca²⁺ Signaling Domains and is Required for Normal Fertility" for consideration by eLife. Your article has been reviewed by three peer reviewers, one of whom, Kenton Swartz, is a member of our Board of Reviewing Editors, and the evaluation has been overseen by and Richard Aldrich as the Senior Editor. The following individual involved in review of your submission has agreed to reveal his identity: Christopher J Lingle (Reviewer #3).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

In this very interesting manuscript, Chung et al. report the discovery and the function of two novel subunits of the CatSper complex, CatSperε and CatSperζ. The CatSper channel has been known for >10 years now to form the most important Ca²⁺ channel in sperm tail and is absolutely required for male fertility. However, the complex has been resistant to detailed functional studies by biophysicists because it somehow doesn't form "functional" channel in heterologous expression system. One explanation is that we don't have all the subunits in the complex yet despite the 4 pore-forming (CatSper1-4) and 3 "auxiliary" (CatSperβ, γ, δ) reported thus far. The discovery of another two subunits described in this paper is a major step forward. Intriguingly, CatSperζ doesn't have a transmembrane-spanning domain and is yet membrane-anchored (perhaps in a fashion similar to the β subunits of CaVs). The authors show that during evolution, CatSperζ was "added" to the complex in mammals and it presumably reflects an adaptation to the changes of environment of the site of fertilization in the female reproductive tract. The authors convincingly demonstrate that the new proteins are associated with CatSper (using biochemical methods), colocalized within the CatSper nanodomains (with STROM and conventional immuno-staining) and, for CatSperζ, are required for the continuity of the CatSper structural stripes along the sperm tail. Finally, the authors demonstrate that CatSperζ is required for normal sperm hyperactivated motility, egg penetration and male fertility (sub-fertile in the KO).

The findings in the paper are novel, interesting and convincing. The studies are remarkably comprehensive, and include data from the identification of new subunits to the biochemical characterization of interaction and whole-organism function. The following are suggestions the authors should carefully consider in preparing a revised manuscript.

Suggested revisions:

1) "Auxillary" subunits in other channels (e.g. CaV β and NaV β) usually affect the channel's voltage dependence of activation/inactivation kinetics. In Figure 3C, the authors present the averaged amplitudes of I_CatSper in the KO and WT. It would be nice to have the averaged G-V relationships reconstructed from data in Figure 3A and B. In addition, it's useful to show the traces recorded with square wave stimulation, in addition to the ones with ramp protocols, to check whether there is any inactivation in the KO sperm. Finally, does the channel without CatSperζ have the same pH-dependence?

2) There was a concern that the authors have not carefully distinguished to what extent the sperm motility deficits in the zeta KO arise from reductions in I_CatSper)vs. structural changes in localization and association of the CatSper complex. In the minor comment 2 below, there is a suggestion for potential tests of this issue. At a minimum, some cautionary remarks on this potential distinction are required.