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

We report that the Gm7068 (CatSpere) and Tex40 (CatSperz) genes encode novel subunits of a 9-subunit CatSper ion channel complex. Targeted disruption of CatSperz reduces CatSper current and sperm rheotactic efficiency in mice, resulting in severe male subfertility. Normally distributed in linear quadrilateral nanodomains along the flagellum, the complex lacking CatSperζ is disrupted at ~0.8 μm intervals along the flagellum. This disruption renders the proximal flagellum inflexible and alters the 3D flagellar envelope, thus preventing sperm from reorienting against fluid flow in vitro and efficiently migrating in vivo. Ejaculated CatSperz-null sperm cells retrieved from the mated female uterus partially rescue in vitro fertilization (IVF) that failed with epididymal spermatozoa alone. Human CatSperε is quadrilaterally arranged along the flagella, similar to the CatSper complex in mouse sperm. We speculate that the newly identified CatSperζ subunit is a late evolutionary adaptation to maximize fertilization inside the mammalian female reproductive tract. DOI: http://dx.doi.org/10.7554/eLife.23082.001

Sperm rheotax against Fallopian tubular and isthmus fluid flow (Miki and Clapham, 2013). Rheotactic turning to reorient to directional flow depends on flagellar rolling, not the sperm head or its geometry, as demonstrated by the rheotaxis of headless mouse sperm (Miki and Clapham, 2013). CatSper channels form unique Ca 2+ signaling domains in linearly quadrilateral arrays along the principal piece of sperm flagella. The integrity of these domains is necessary to time and/or maintain hyperactivated motility (Chung et al., 2014). Thus, CatSper1-null sperm cannot rheotax due to defects in rolling (Miki and Clapham, 2013), and presumably exert less lateral force in escaping from epithelial walls (Ho et al., 2009) or in pushing cumulus cells aside. In general, however, there is a lack of understanding of the steps between CatSper-mediated calcium entry, Ca 2+ -modified phosphorylation cascades, and the resulting structural changes underlying orchestrated flagellar movement.
Here, we reveal that the murine Gm7068 (C1orf101-like) and Tex40 genes encode two new subunits of the CatSper ion channel complex, CatSper epsilon (e) and zeta (z), respectively. In this study, we focus primarily on CatSperz's function. Genetic disruption of mammalian-specific CatSperz reduces the CatSper current in the sperm flagellum and hyperactivated motility, resulting in severe subfertility. We use high speed video microscopy and digital image analysis to determine swimming trajectory and the flagellar waveform in detail. Surprisingly, abrogation of CatSperz renders the proximal flagellum inflexible but preserves overall motility, thus resulting in restriction of the 3D flagellar envelope, inefficient sperm rheotaxis in vitro, and delayed sperm migration in vivo. Using eLife digest Male mammals ejaculate millions of sperm cells each time they mate with a female.
Only a few of these cells manage to travel up the female's reproductive tract to reach the egg, and usually only one sperm fertilizes it. Freshly ejaculated sperm are incapable of fertilizing eggs and have to undergo several changes within the female to become able to do so. One crucial change occurs in the sperm tail, which starts to beat vigorously in a whip-like motion. This type of movement -known as hyperactivated motility -enables the sperm to swim towards the egg, push through a sticky coating that surrounds it, and then burrow into it.
Hyperactivated motility is triggered when calcium ions enter the sperm cell via a specific channel protein known as CatSper, which is found in the membrane that surrounds the cell. CatSper channels form groups (known as complexes) with several other proteins that are arranged in a unique pattern of four straight 'stripes' running down the tail of the sperm. This arrangement is necessary for hyperactivated motility and mutations in the genes that encode these proteins can lead to infertility in males. The CatSper channel complex is known to contain seven proteins: four that form a pore through which calcium ions can enter, and three accessory proteins whose roles in hyperactivated motility are less clear. Chung et al. identified two genes in mice that encode new accessory proteins in the CatSper channel complex named CatSper epsilon and CatSper zeta. Further experiments analyzed the role of CatSper zeta in more detail. Mutant males that lack CatSper zeta have fragmented patterns of CatSper stripes in the tails of their sperm. Moreover, fewer calcium ions were able to pass through the channels to enter the cell. Together, this made the sperm tail more rigid, which prevented it from moving efficiently within the female, resulting in reduced fertility. Chung et al. also found that the mutant sperm were less able to penetrate the egg than normal sperm.
During evolution, the gene that encodes CatSper zeta appeared first in mammals and may represent an adaptation that improved the chances of a sperm fertilizing the egg inside the reproductive tract of female mammals. Future challenges will be to explore how the CatSper channel assembles on the membrane of sperm and find out exactly how calcium ions trigger hyperactivated motility.
super-resolution microscopy, we demonstrated that the structurally distinct CatSper Ca 2+ signaling domains along the flagellum (Chung et al., 2014) becomes fragmented in the absence of CatSperz. We demonstrate that IVF failure of CatSperz-null spermatozoa is partially rescued by using ejaculated sperm recovered from the uterus of mated females, explaining the discrepancy between in vitro and in vivo fertilizing ability. Finally, we show that mouse and human spermatozoa have a similar macroscopic organization of the CatSper complex.

Results
CatSper e and z: Two new accessory proteins in the CatSper channel complex We previously identified seven protein components of the CatSper channel complex (CatSper1-4, b, g, and d) from mouse testis using tandem affinity purification (Chung et al., 2011). As the most biochemically complex ion channel known to date, it has not been possible to express functional CatSper channels in heterologous systems. This includes many attempts in many cell types, including simultaneous injection of all 7 CatSper mRNAs into Xenopus oocytes (data not shown). Therefore, we continued to seek potential additional components to more thoroughly understand CatSper channel assembly and trafficking. We identified a mouse homolog of human C1orf101 (C1orf101like, currently Gm7068) ( Figure 1A CatSper e and z localize at quadrilateral Ca 2+ signaling domains in sperm flagella Mouse CatSper proteins form a unique pattern of four linear ('racing stripes') Ca 2+ signaling domains running down the four quadrants of the principal piece of the flagellum (Chung et al., 2014). We examined whether e and z share this distinctive compartmentalization. The antibodies, anti-he31, recognizing the N-terminal extracellular region of human CatSpere, and anti-mz174, against the very C-terminus of mouse CatSperz, were suitable for 3D stochastic optical reconstruction microscopy (STORM) ( Figure

CatSperz-null male mice have severely impaired fertility
The lack of functional expression of CatSper channels in heterologous systems requires that genetic manipulation be used to study the function of each component. CatSpere has the same ancient origin at the root of early eukaryotic evolution as those of CatSpers1-4, b, and g and the same pattern of extensive lineage-specific gene loss as CatSperb and g through metazoan evolution (   homology and conservation, we anticipated that deletion of CatSpere would likely be the same as the existing knockout of other CatSper subunits, but deletion of CatSperz might provide new insights into spermatozoan adaptations to changes concomitant with the evolution of mammalian fertilization. To test this idea, we began by generating a CatSperz-null mouse line from Tex40 gene targeted ES cell clones. Tex40 is a small gene composed of 5 exons that spans only~3 kb on chromosome 11 ( CatSperz-null mutant mice are indistinguishable from their wt or heterozygous (het) littermates in appearance, gross behavior, or survival. In addition, no morphological differences were observed by histological examination of testis and epididymis (data not shown). Sperm morphology and epididymal sperm number from CatSperz-null mice were not significantly different from those of 2-3 month old paired heterozygous littermates ( . However, when CatSperz-null male mice were mated with wt or het females, they were severely subfertile: 20% (5/25) CatSperz-null males were completely infertile over six months (Figure 2A), and progeny of the fertile paternal CatSperz-null mice were significantly fewer in number ( Figure 2B and Figure 2-figure supplement 2B). The latency from pair formation to the birth of these offspring from CatSperz-null males was !10 days compared to those from wt or het males (data not shown).
We examined the number of sperm within cumulus oocyte complexes (COCs) after copulation and checked in vivo fertilization rates by isolating the COCs and/or embryos from the female ampullae. At 8 hr after coitus, no sperm was found in the COCs when mated with CatSperz-null male mice ( Figure 2C). In contrast, the majority of the COCs from CatSperz-het mated females had one or more sperm cells within the complex. When mated with CatSperz-het males, more 2 cell eggs were observed over time after coitus, while the fertilization rate by CatSperz-null males did not change significantly ( Figure 2D and Figure 2-figure supplement 2C). These data suggest that CatSperznull sperm migration is delayed in the female reproductive tract.

CatSperz-null sperm cells have rigid proximal flagella
To understand why CatSperz-null spermatozoa did not efficiently migrate in the female reproductive tract, we first investigated sperm motility using computer assisted sperm analysis (CASA) (Figure 2figure supplement 2D). The percentage of motile spermatozoa was not significantly different and most motility parameters of z-null spermatozoa were comparable to those of z-het sperm cells. However, the characteristic increase of lateral head displacement upon capacitation was not observed in z-null spermatozoa (Figure 2-figure supplement 2D), suggesting that hyperactivated motility was reduced. Ninety minutes after capacitation, there was a less marked difference in swimming trajectories of z-null spermatozoa compared to z-het spermatozoa, supporting this notion ( Figure 2E and Video 1). Further analysis of flagellar amplitude and waveforms of tethered spermatozoa revealed a striking rigidity of z-null spermatozoa from their midpiece to midway down the principal piece ( Figure 2F and Video 2). This phenotype was also observed from hyperactivation-deficient CatS-per2-null patients (Smith et al., 2013). After incubation under capacitating conditions for 90 min, we observed that z-null spermatozoa beat only at the very distal end of a flagellum (Video 3). Moreover, CatSperz-null spermatozoa remain bent in the anti-hook direction (Ishijima et al., 2002) ( Figure 2F, Videos 2 and 3). The anti-hook bend predominates as the pro-hook bend (initiated by the CatSper- Source data 1. Impaired male fertility in CatSperz-/-mice: pregnancy rate, litter size, sperm number per egg, and in vivo fertilization rate. DOI: 10.7554/eLife.23082.008 mediated Ca 2+ signaling pathway (Chang and Suarez, 2011) is dysregulated in CatSperz-null spermatozoa.

Reduced CatSper current in CatSperz-null spermatozoa
To examine how Ca 2+ signaling in CatSperz-null spermatozoa is impaired, we first examined I CatSper , the sperm-specific Ca 2+ -selective ion current. Since Ca 2+ has high affinity to calcium-selective pores (Almers et al., 1984), CatSper permeation of monovalents increases when external calcium is removed (Kirichok et al., 2006;Navarro et al., 2007). In divalent-free (DVF) solutions, wt spermatozoan I CatSper conducts a large Na + current, which is completely absent in mice lacking CatSpers1, 2, 3, 4, or d (Chung et al., 2011;Kirichok et al., 2006;Qi et al., 2007). However, in CatSperz-null spermatozoa, monovalent CatSper current is present but reduced to~60% of normal (À426 ± 50 pA at À100 mV; Figure 3A) compared to control CatSperz-het spermatozoa (À683 ± 77 pA at À100 mV; Figure 3B). Thus, in the absence of CatSperz, the CatSper channel complex is still targeted to the flagellar membrane and forms functional channels. We hypothesize that the reduction in CatSper current reflected decreased protein expression levels.
P2X receptors are nonselective ion channels gated by purines such as ATP. The ATP-activated cation-nonselective current in the midpiece of murine sperm is mediated by the P 2 X 2 purinergic receptor . In CatSperz-null spermatozoa, I ATP did not differ substantially from heterozygous spermatozoa ( Figure 3A and B), supporting the assumption that there is selective down regulation of CatSper channels. Smaller I CatSper explains, in part, the attenuated hyperactivated motility, delayed sperm migration, and male subfertility ( potentiated and delocalized in CatSper knockout mice (Chung et al., 2014) or when Ca 2+ influx is pharmacologically blocked (Navarrete et al., 2015). Upon capacitation, P-Tyr was more prominent in CatSperz-null spermatozoa than wt, but to a lesser extent than CatSper1-null spermatozoa (Figure 2-figure supplement 2E), consistent with the reduced calcium current. It is, however, also possible that an altered arrangement of the CatSper complex and/or its interaction with target proteins in the linear domains could have contributed to these functional deficits.

Abrogation of CatSperz retards targeting of the CatSper complex to flagella
To better understand why I CatSper is reduced in CatSperz-null spermatozoa, we examined levels of protein expression in CatSperz-null spermatozoa ( Figure 4A). Expression of other CatSper subunits was detected in CatSperz -null spermatozoa, albeit at 30-60% lower levels than that of wt ( Figure 4B), consistent with reduced I CatSper (Figure 3). This contrasts with the complete absence of other CatSper subunits in CatSper1-and CatSperd-null spermatozoa. mRNA and protein levels of other CatSper subunits were not reduced in the testis of CatSperz-null mice ( Figure 4C and D), suggesting that the defect occurs during or after assembly of the protein complex.
CatSperz is essential in maintaining the continuity of linear flagellar CatSper Ca 2+ domains Loss of CatSperz resulted in fragmentation of CatSper1 staining on the flagellar membrane and these defects are large enough to be resolved by confocal imaging ( Figure 5A). These gaps were not observed in wt/het ( Figure 1H and Figure 2-figure supplement 1E) or previous wt and CatSper knockout studies (Chung et al., 2011;Liu et al., 2007;Ren et al., 2001). 3D STED and 3D STORM super-resolution microscopies clearly demonstrate that structural continuity is interrupted in CatSperz-null spermatozoa -each 'stripe' of the CatSper domains is fragmented, while the overall quadrilateral structure is maintained ( Figure 5B  The Fourier transform shows a fundamental frequency of (800 nm) À1 (Figure 5-figure supplement 1F). We assume this thinning of one or more linear domains reflects an underlying structural periodicity that regulates CatSper complex trafficking or membrane insertion.

CatSperz-null spermatozoa rheotax inefficiently with reduced torque
Thus far, our results show that CatSperz-null sperm have reduced I CatSper , dysregulated structural continuity of the CatSper Ca 2+ signaling domains, beat in an atypical pattern, and are delayed in migrating in the female reproductive tract, resulting in reduced male fertility. Rheotactic guidance for sperm over long distances requires rotational motion during CatSper-mediated hyperactivated motility (Miki and Clapham, 2013). We thus measured rheotactic parameters and the rotation rate of CatSperz-null spermatozoa with a particular focus on whether their proximal tail rigidity and subsequent low amplitude lateral movement ( Figure 2) affects sperm movement. In flow-directed capillary tubes (Miki and Clapham, 2013), we observed that the rheotactic ability of CatSperz-null spermatozoa was significantly reduced ( Figure 6A and B and Figure 6-figure supplement 1A). At all flow rates tested, most motile CatSperz-null spermatozoa were unable reorient to swim against the flow and were swept out of the tube (Video 4). In contrast, 85% of motile heterozygous spermatozoa displayed rheotactic behaviors by maintaining their position or swimming upstream for more than 2 s of the 9 s period of recording ( Figure 6B and Video 4).
We next examined the rotational motion of CatSperz-null spermatozoa. At high viscosities (0.3% methyl cellulose (MC), cP = 6.7), uncapacitated z-het spermatozoa swim in circles ( Figure 6C, left and Video 5), while capacitated z-het spermatozoa swim in a more linear path as they rotate around a longitudinal axis, like wt spermatozoa (Video 6) (Miki and Clapham, 2013). Interestingly, z-null spermatozoa exhibit linear migration as they can rotate along the tail axis regardless of capacitating conditions, even at higher viscosities ( Figure 6C, right and Videos 5 and 6). Indeed, uncapacitated CatSperz-null spermatozoa rotate~50% faster than z-het spermatozoa ( Figure 6D). This indicates that CatSperz-null spermatozoa have less lateral motion and are subject to less torque by the moving stream. Spatially, the spermatozoa trace out a less conical 3D envelope ( Figure 2F and Figure 6-figure supplement 1B). In short, the rigidity of the CatSperz-null sperm proximal tail constrains its motion to that of a propeller-driven rod.
Compromised Ca 2+ signaling alters the sperm's 3D flagellar envelope and movement We next examined the relation between external calcium entry and sperm function. First, we tested whether increasing extracellular [Ca 2+ ] could rescue z-null sperm motility. After incubation for 90 min with a two-fold greater [Ca 2+ ], most CatSperz-null sperm remain bent in the anti-hook orientation with a rigid proximal tail ( Figure 6-figure supplement 1C, z(-/-) middle, and Video 7). A few z-null spermatozoa partially recover, bending occasionally in the pro-hook direction with hyperactivated motility (Figure 6-figure supplement 1C, z(-/-) right, and Video 7). Conversely, a 20-fold reduction , and nominally divalent-free solution (3, DVF) at À100 mV (gray circles) and +100 mV (black circles). I CatSper in CatSperz-null sperm cells is~60% of that recorded from wt. Inward I ATP current induced by 100 mM ATP is similar in both phenotypes and indistinguishable from previously published wt I ATP . (C) Average I CatSper measured from CatSperz+/-(À683 ± 77 pA) and CatSperz-/-(À426 ± 50 pA) sperm cells at À100 mV. Data are mean ± SEM. p=0.0297. Cartoon shows the standard pipette solution (mM); internal Cs used to block K + currents. DOI: 10.7554/eLife.23082.012 The following source data is available for figure 3: A transient Ca 2+ pulse induced by Ca 2+ ionophore, A23187, significantly reduces the time required for wt sperm to develop hyperactivated motility (Tateno et al., 2013). Moreover, a short (10 min) exposure to A23187 can rescue defects in hyperactivated motility and the fertilizing capability of CatSper1-null sperm in vitro (Navarrete et al., 2016). We tested the relation of calcium transients to hyperactivated motility in CatSperz-het and null sperm. In z-het spermatozoa, an A23187induced Ca 2+ pulse followed by washout, enables full hyperactivation, characterized by wide lateral displacement with large midpiece a angle within 30 min ( Figure 6-figure supplement 1D and Videos 9 and 10). However, in z-null sperm, the same treatment improved the flexibility of the proximal flagella, particularly in the principal piece, but the midpiece remained largely inflexible (Figure 6figure supplement 1D and Videos 9 and 10). Building on our previous work, the present study suggests that calcium entry through CatSper channels has time-dependent, complex effects on the    3) males after incubation in HTF. The sperm rotation rate is calculated as previously reported (Miki and Clapham, 2013). Data are mean ± SEM. ****p<0.0001. (E and F) IVF with epididymal and/or ejaculated CatSperz+/-and CatSperz-/-spermatozoa. 2 cell stage eggs were counted 24 hr after insemination. (E) IVF rate with cumulus-intact oocytes from CatSperz+/-(epididymal, 71 ± 6) and CatSperz-/-(epididymal, 0.7 ± 0.7; ejaculate, 20 ± 6, p=0.0051). (F) IVF rate of cumulus-free/ZP-intact eggs with (CatSperz+/-, 80 ± 8; CatSperz-/-, Figure 6 continued on next page

CatSperz-null sperm cells inefficiently penetrate the egg cumulus
We performed in vitro fertilization (IVF) to determine how the low rotational torque generated by CatSperz-null spermatozoa affects sperm-egg interactions. We found that these spermatozoa cannot fertilize cumulus-intact oocytes ( Figure 6E), but could dissociate the cumulus cell layers and bind to the ZP (data not shown). Cumulus removal did not change the fertilization rate of ZP intact oocytes by CatSperz-null spermatozoa. Furthermore, this rate was only marginally enhanced by destabilization of the ZP by 2 mM glutathione ( Figure 6F) (Miyata et al., 2015). This indicates that reduced hyperactivated motility alone does not explain the failure of CatSperz-null spermatozoa in IVF. One possibility is that the kinetics of capacitation in vitro is different from that in vivo, resulting from fluctuations in timing or amplitude of known factors (e.g., HCO 3 , pH) or from unknown factors present in seminal and/or female fluids. We then compared IVF rates with ejaculated and epididymal spermatozoa of CatSperz-null mice. When ejaculated spermatozoa flushed from the uterus of the mated females were used in IVF trials, 20% of oocytes incubated with z-null spermatozoa developed into two-cell embryos ( Figure 6E). This compares to 50% of oocytes incubated with z-het ejaculated (data not shown), or epididymal sperm. Thus, additional factors may be functionally relevant in in vivo fertilization.

Discussion
Complex protein composition and conservation of compartmentalization in mammals Sperm hyperactivation and normal fertility in mammals requires the unique CatSper channel complex. With four distinct pore-forming gene products (CatSper 1-4) and, now, five accessory subunits (b, g, d, e, and z), the CatSper channel is the most complex of known ion channels. This may reflect the relatively high evolutionary pressure on spermatozoan evolution (Swanson and Vacquier, 2002;Torgerson et al., 2002), and various adaptations to different modes of fertilization. Like many gamete-specific proteins and the other CatSper proteins reported so far (Cai and Clapham, 2008;Chung et al., 2011), mouse and human CatSper e and z show signs of rapid evolutionary change with only 50% and 45% amino acid sequence identity, respectively. In particular, the sequence regions outside TM segments and the pore loop of CatSper proteins are poorly conserved across species, indicating these regions possibly convey species-specific modulation of flagellar motility (Miller et al., . This is illustrated by striking differences in progesterone-elicited I CatSper responses in mouse and human (Lishko et al., 2011). Here we have shown that CatSper e and z are components of the highly organized CatSper complex, that CatSperz is required for proper continuity of this complex along the flagellum, and that loss of z alters hyperactivation waveforms and reduces fertilizing capacity.
The conservation pattern of the lineage-specific gain and loss of the CatSpere gene is identical to those of b and g, suggesting that they likely belonged to an ancient CatSper channel Ca 2+ signaling network before the divergence of unikonts and bikonts. Since their protein expression is strictly interdependent, we speculate that CatSpere-null mice will have the phenotype of CatSper1-4, or dnull mice. In contrast, CatSperz is conserved only in mammals, suggesting that this protein imparts some adaptation, perhaps as a method enabling rheotaxis in the mammalian female reproductive tract.
Interestingly, although CatSperz has no putative transmembrane domains, it is localized in the same quadrilateral pattern as other CatSpers, but is not present elsewhere in sperm. An intriguing aspect of our observations is that, unlike CatS-per1-4 and d-null mice, which produce complete infertility, CatSperz-null males exhibit an incomplete loss of fertility. The CatSper current is reduced in CatSperz-null spermatozoa, and may have similar permeation properties (likely dominated by the CatSper1-4 pore subunits), but the effects of CatSperz on channel gating remain to be determined in future studies. This is reminiscent of the non-spermatozoan, voltage-gated Ca v channel auxiliary subunits, which are not required for expression but modulate expression levels and gating (Catterall et al., 2005). Most tantalizing is the thinning and disruption of the linear CatSper signaling domains at repeat intervals in the absence of z. Further detailed examination via mutagenesis experiments has been stymied by our inability to heterologously express functional CatSper channels. New rapid Video 5. Movement of CatSperz +/-and -/-sperm in viscous medium; uncapacitated, related to Figure 6. Uncapacitated spermatozoa were allowed to disperse for 10 min pre-incubation in a 37˚C chamber containing HEPES-HTF supplemented with 0.3% methylcellulose; swimming sperm cells were recorded within the next 5 min; video rate 50 fps, 2 s movies. genome editing techniques should enable more mice to be generated that will further the study of CatSper trafficking, subunit interactions, and localized signaling pathways.

Traffic into the linear domains of sperm flagella
Functional Ca 2+ signaling domains are common adaptations in many biological systems, such as synapses and muscle. They enable specific and fast triggering of downstream events (Clapham, 2007). CatSper channels are compartmentalized into a unique multilinear arrangement and form Ca 2+ signaling nanodomains with other Ca 2+ signaling molecules along the sperm flagellum (Chung et al., 2014). The mechanisms involved in the delivery of the CatSper channels to these specific domains are currently unknown, and we suspect will be as interesting and complex as those in primary and motile cilia (Sung and Leroux, 2013). We found that abrogation of CatSperz not only retards targeting of the CatSper complex to flagella, but also disrupts continuity of the linear domains, resulting in repeated fragmented domains with~800 nm periodicity. In order for CatSper domains to form and function properly, interactions are needed between the CatSper channel complex in the flagellar membrane and the underlying cytoskeletal proteins. One speculation is that CatSperz might adapt to cytoskeletal structures that traffic, distribute, and enable membrane insertion of CatSper.
The fibrous sheath (FS), a cytoskeletal structure unique to the mammalian sperm flagellum, defines the extent of the tail's principal piece, in which all the CatSper proteins are specifically localized. The FS closely lies under the plasma membrane and its two longitudinal columns are connected by circumferential ribs. Immunogold electron microscopy demonstrated that the CatSper channels are distributed on the end of ribs, where they merge with the column (Chung et al., 2014). It seems likely that the timing of occurrence and localization of CatSper Ca 2+ signaling domains is coordinated with the assembly of FS proteins along the axoneme.
The column appears early in spermiogenesis, forming from the distal tip of the tail along the axoneme, followed by subsequent rib formation in the opposite direction (Oko, 1998;Oko and Clermont, 1989). Based on scanning electron micrographs (Danshina et al., 2010;Miki et al., 2004), we find that the distance between ribs is about 800 nm in mouse spermatozoa. Thus, it seems likely that the repeated disruption in the absence of CatSperz is related to rib spacing of the FS.

Ca 2+ regulation of the flagellar envelope in sperm rheotaxis and egg penetration
Genetic abrogation of CatSper disrupts hyperactivated motility as manifested by changes in movement symmetry, amplitude, and rolling (Carlson et al., 2003;Chung et al., 2011;Miki and Clapham, 2013;Qi et al., 2007). Here we report that the flagellar envelope is significantly altered in the absence of CatSperz, in part due to the inflexibility of the proximal tail. We previously reported that the catalytic subunit of calcineurin, PP2B-Ag, expresses throughout the tail but localized to the CatSper quadrilateral structures and axoneme (Chung et al., 2014). In CatSper1-null spermatozoa, PP2B-Ag remains localized primarily to the axoneme but disappears from the quadrilateral structures.
Recently, a similar but not identical phenotype (inflexible midpiece, reduced hyperactivated motility, and impaired ZP penetration) was reported in testis-specific calcineurin Ppp3cc-null and Ppp3r2-null spermatozoa (Miyata et al., 2015). Note that the principal piece of both CatSper1-null and Ppp3ccnull spermatozoa are not rigid. The integrity and distribution of CatSper channels in Ppp3cc-null spermatozoa remain to be examined and may clarify midpiece/principal piece disparities. In any case, inflexibility in the proximal regions of flagellum results in a flagellar envelope approximated as a rod with a distal propeller. The sperm can rotate faster but the smaller lateral deviation reduces torque. This limits the sperm's ability to orient into the flow, as well as penetrate the cumulus and ZP.

Physiological modulation of CatSper in sperm function and fertility
Gene-manipulated mice highlight the importance of in vivo observations and have reshaped the landscape of fertilization science (Okabe, 2015). In vitro capacitation and fertilization systems underpin much of the study of sperm motility and fertilization potential. While ejaculated sperm are preferred for fertilization studies in larger animals and humans, epididymal sperm are commonly used in genetically tractable mouse studies. Notably, these sperm are not exposed to accessory sex gland secretions and female fluids. This may explain why CatSperz-null spermatozoa are completely infertile in an IVF setting (COCs), but in vivo are merely subfertile. Perhaps natural modulators, absent in epididymal sperm IVF studies, partially rescue the fertilizing potential of CatSperz-null spermatozoa by activating Ca 2+ signaling activity. Interestingly, a transient pulse of Ca 2+ can greatly reduce the capacitation time required for wt sperm to develop hyperactivated motility (Tateno et al., 2013). Moreover, Navarrete et al recently demonstrated that a short exposure to A23187 rescued the defects in motility and fertilizing capability of CatSper1-null sperm in vitro (Navarrete et al., 2016). These independent studies were interpreted to mean that the initial priming by Ca 2+ influx, perhaps above a certain threshold, is essential for sperm function. However, the linear quadrilateral CatSper complexes are not present in CatS-per1-null spermatozoa and in CatSperz-null spermatozoa are disrupted by gaps. We hypothesize that the linear quadrilateral structure in vivo likely maintains, regulates, and distributes CatSper Ca 2+ signaling during hyperactivated motility. But it is important to point out that alterations in the structure should also result in changes in mechanical properties, movement of the flagellum, distribution of entering calcium, and downstream kinase activity and the motor elements they regulate. This complexity is illustrated in vivo sperm swimming trajectories, which are modulated by switching between pro-and anti-hook beating patterns. In the absence of CatSperz, anti-hook beating predominates. Pro-hook motions are associated with intact CatSper-mediated Ca 2+ signaling pathways (Chang and Suarez, 2011). Finally, ejaculated sperm display more pro-hook hyperactivation than epididymal sperm (Li et al., 2015).
Future areas for investigation are the functional positioning of the remaining accessory subunits of the CatSper channel in assembly and domain organization, the testing of potential modifiers present in accessory sex gland secretions that may activate CatSper channels, and the determination of Ca 2+ dependent molecules in the axoneme which eventually determine flagellar bending and its envelope. CatSperz-null mice, which are hypomorphic to the null-mutation of other CatSper genes with abrogated hyperactivation, and newly expanding animal models from recent advances in genome editing will serve as a foundation to this end. Advanced imaging techniques with higher time and spatial resolution will be necessary to carry this out. The present results also suggest that alterations of Ca 2+ current and/or dysregulated downstream Ca 2+ signaling affecting dynamic structures may be sufficient to compromise sperm function. CatSper's unique composition and central role in hyperactivated motility make it an ideal target for contraception.

Materials and methods
Details of source and identifier are provided in the Key Resources Table as a supplementary file.

Capacitation in varying external [Ca 2+ ]
To test the effect of external [Ca 2+ ] on the development of hyperactivated motility, 2 mM CaCl 2 in standard HTF was replaced with 4 mM, 0.5 mM, and 0.1 mM CaCl 2 and sperm cells were incubated for 90 min under capacitating conditions (37˚C, 5% CO 2 ).

Human sperm preparation
All experiments using human samples were approved by the Committee of Clinical Investigation, Boston Children's Hospital CCI/IRB (IRB-P00000538). Human semen samples were obtained from fertile donors. Human spermatozoa were collected by the swim-up method with the use of modified human tubal fluid medium (HTF).

Cell line origin and authentication
HEK293T cells were purchased from ATCC. In this study, they were used to overexpress recombinant human CatSperz in order to test antibodies. The cells were tested negative for mycoplasma and validated as of human origin. The identity was authenticated by confirming their negative expression of testis-specific genes including CatSper. The cell line was cultured in DMEM/F12 containing 10% FBS.

Genomic database search
Annotated orthologs in the NCBI gene database (http://www.ncbi.nlm.nih.gov/gene/) and/or homologous amino acid sequences of reported protein databases were screened in 17 eukaryotes for the presence of genes for CatSper auxiliary subunits. Non-annotated orthologs in the NCBI gene database were identified by comparing sequences of the annotated orthologs to those in the protein database of species by Phmmer implemented on HMMER 3.1 (default option, http://hmmer.org/). The longest amino acid sequences among all the isoforms of the orthologs annotated in each species and protein sequence databases from 15 eukaryotes, except human and mouse, were downloaded from the NCBI Genome database ( Multiple tissue RT-PCR PCR was performed according to standard protocols using a commercial multiple panel cDNA template (MTC), Clontech). PCR primers amplified Gm7068 (forward: 5 0 -CTATGGCTCAAGTGTAA TGACC-3 0 , reverse: 5 0 -GCTCTTATTGAATCCTCGAACC-3 0 ), Tex40 (forward: 5 0 -GAAACAGGA TTCGCAAGTACAG-3 0 , reverse: 5 0 -TCGTGGACCTATATGTGATGAG-3 0 ) using mouse GAPDH (forward: 5 0 -TGAAGGTCGGTGTGAACGGATTTGGC-3 0 , 5 0 -ATGTAGGCCATGAGGTCCACCAC-3 0 ) as a control.

RNA in situ hybridization
In situ hybridization experiments were carried out with an RNAscope (Advanced Cell Diagnostics, Newark, CA). Testes from three month old wild-type mice were fixed in 10% (vol/vol) neutral-buffered formalin at room temperature for 24 hr, dehydrated, and embedded in paraffin. Paraffin sections (10 mm thick) were processed according to the manufacturer's instructions for in situ detection in the Rodent Histopathology Core Facility at Harvard Medical School. Sequences of the probes used in this study are: Gm7068 (XM_982472.3, 645-1072) and Tex40 (NM_001039494.2, 41-456). After the DAB (3,3, -diaminobenzidine) reaction, slides were counterstained using hematoxylin.

Preparation of mouse testis microsome
Testes (200 mg, normally two testicles) from 8-to 12-wk-old male mice were homogenized on ice using a Dounce homogenizer in 2 mL 0.32 M sucrose solution with protease inhibitor cocktails (Roche). The tissue suspension was centrifuged at 300 g for 10 min at 4˚C and the supernatant was then transferred to an ultra-speed centrifuge tube. The microsome faction was isolated by centrifuging the tube at 105,000 g for 60 min.

Sperm immunocytochemistry
Caudal epididymal mouse sperm cells attached to glass coverslips were fixed in 4% paraformaldehyde (PFA) in PBS, permeabilized with 0.1% TrixonX-100 for 10 min. Human sperm cells from swimup purification were fixed 4% PFA in PBS for 10 min followed by 100% MeOH. Fixed human sperm cells were permeabilized in 0.1% saponin for 10 min. Permeabilized sperm cells were washed in PBS and blocked with 10% goat serum for 1 hr. Mouse samples were stained overnight with primary antibody against CatSper1 (10 mg ml À1 ) and CatSperz (mz174, 20 mg ml À1 ) as were human samples with primary antibodies against CatSpere (he31, 20 mg ml À1 ) and CatSperz (hz11, 10 mg ml À1 ), in 10% goat serum in PBS, 4˚C. After PBS wash, goat-anti-rabbit Alexa488 conjugate (Invitrogen) served as the secondary antibody. Images were acquired on laser scanning confocal microscopes (Olympus Fluoview 1000; Figure 1G and H, Figure 1-figure supplement 2G, and Figure 2-figure supplement 1E and Leica TCS SP8; deconvolved image in Figure 5A).

Super-resolution imaging 3D STED imaging
For analysis of CatSper nanodomain organization, CatSper1 images were acquired with Leica TCS SP8 gated stimulated emission and depletion (STED 3Â) microscopy using an HCX PL APO 100Â/ 1.40 oil objective lens (Leica Microsystems, Germany). Samples were prepared as described in Sperm Immunocytochemistry with slight modifications. After incubation with primary antibody, cells were washed with PBS and incubated with goat anti-rabbit IgG coupled to Alexa Fluor 546 (Invitrogen, 1:100) for 1 hr at room temperature. Coverslips were mounted with Prolong Gold (Invitrogen) and cured for 24 hr before image acquisition. Within each experiment, identical settings for laser power, STED power, and gating were used to acquire images. The wavelength of the STED depletion laser was 660 nm and was adjusted to 50% of power. z-stacks of 17 optical sections with a step size of 0.1 mm were deconvolved using Huygens Software.

3D STORM imaging
3D STORM experiments were performed as previously described (Chung et al., 2014). Imaging buffer was prepared in 60% (wt/wt) sucrose solution, increasing imaging depth to 1 mm (Chung et al., 2014). Imaging buffer was supplemented with 100 mM mercaptoethylamine (pH 8.5) as a switching agent as well as an O 2 scavenger (5% glucose (wt/vol), 0.5 mg/ml glucose oxidase, and 40 mg/ml catalase) to reduce the rate of photobleaching. The sample was illuminated at 657 nm for imaging the photoswitchable reporter molecules (Alexa 647), and 405 or 532 nm for facilitating the activation of Alexa 647 from the dark state. For 3D localization, a cylindrical lens (focal length = 1 m) was inserted into the detection path to enable determination of z positions from the ellipticities of the molecular images and the x and y positions from the centroid positions (Huang et al., 2008). Image analysis and rendering was performed and angular profiles were constructed as previously described (Chung et al., 2014).

Fourier transform and autocorrelation
Fourier transform and autocorrelation analyses of 3D STORM images were performed as previously described (Xu et al., 2013;Zhong et al., 2014). A Fourier transform of the 1D projection localization distribution yielded a main peak that corresponds to a spatial period of~800 nm for the Cat-Sperz-/-spermatozoa. The autocorrelation curve for the CatSperz-/-spermatozoa showed a periodic modulation with the first peak at~850 nm.

Sperm migration assay and in vivo fertilization
For timed coitus, females were introduced to single-caged CatSperz-het or -null males for 1 hr and checked for the presence of a vaginal plug. To examine sperm migration to the fertilization site in vivo, ampullae were removed from the mated females at 8 hr after coitus and COCs were released. A series of z-stacked images (2 mm step size) of the COCs was taken and number of sperm within each COC was recorded according to the presence of a sperm head. To calculate in vivo fertilization rate, eggs were gently flushed from oviducts and ampullae from the mated females at 20 hr and 27-30 hr after coitus. The total number of eggs and the number of 2 cell eggs were counted.

Fertility test and in vitro fertilization
Two females were caged with each male for three months to track pregnancy and litter production. For IVF assays, oocytes were recovered from superovulated 5-6-week-old B6D2F1 female mice 13 hr after injection of 5 U human chorionic gonadotropin. For standard IVF, sperm were collected from the cauda epididymis. For ejaculate IVF, sperm were retrieved from the uterus of a 1 hr windowtimed coitus. Both epididymal and ejaculated sperm were capacitated in vitro at 37˚C for 1 hr, and coincubated with eggs at~10 5 sperm/mL. After about 4.5 hr, unbound sperm were washed away. After 24 hr incubation the embryos were observed under light microscopy (Olympus IX-70) to check for development of the two-cell stage.

Flagellar waveform analysis
Spermatozoa from the dissected cauda epididymis (swim up method) were collected in HEPES buffered saline (HS) media. Spermatozoa were plated on 35 mm fibronectin-coated coverslips for 15 min (22˚C); unattached sperm were removed by the gentle pipette wash (time 0) and basal motility recorded. Activated motility was recorded within the first 10 min after adding pre-warmed human tubal fluid (HTF)-capacitating medium (Millipore). To induce hyperactivation, attached sperm cells were incubated in HTF media for 90 min at 37˚C (5% CO 2 ). All subsequent images were recorded at 37˚C. The flagellar waveform was analyzed by stop-motion digital imaging collected at 200 fps (HC Image software, Hamamatsu Photonics or Zen Blue, Zeiss; 2 s movies). Overlay of flagellar traces from two complete flagellar beats were generated by hyperstacking binary images using opensource FIJI software (Schindelin et al., 2012) and time coded in color.

Sperm motility analysis
Cauda epididymal spermatozoa were suspended and incubated in non-capacitating M2 medium (Specialty Media, Millipore) or in HTF medium for capacitation. Sperm motility was then measured using the IVOS sperm analysis system (Hamilton Thorne Biosciences, Beverly, MA) in an 80 mm (depth) chamber to obtain various parameters (Figure 2-figure supplement 2D). Sperm motility was also analyzed with an Olympus IX-70 microscope equipped with a high-speed sCMOS camera (Orca-Flash4.0) and a 10x objective. 1-2 Â 10 5 mouse sperm before and after capacitation were added to the 37˚C chamber (Delta T culture dish controller; In some experiments, the medium was supplemented with methylcellulose (MC) (M0512, 4000 cP in 2% solution; Sigma) at 0.3%, 0.4%, or 0.5% (w/v). Sperm swimming 3-5 mm from the rim were recorded after a 10 min preincubation period that allowed spontaneous dissociation of sperm clumps. To inhibit convective flow, 1 ml of medium was overlaid by 1 ml of mineral oil and covered by a heated glass lid (Bioptechs). Sperm motility at 37˚C was videotaped at 100 fps. Images (HC Image software, Hamamatsu Photonics) were analyzed for swimming trajectory from a 1 s playback movie at 1/5 speed, by head tracing via Computer Assisted Sperm Analysis (CASA; http://rsbweb.nih.gov/ij/plugins/casa.html). To track swimming trajectory in viscous medium, the sperm motility was videotaped at 50 fps. The images were analyzed using Fiji software (Schindelin et al., 2012) by assembling overlays of the flagellar traces generated by hyperstacking binary images of 20 frames of 2 s movies coded in a gray intensity scale.

In-capillary sperm rheotaxis
Mouse sperm incubated in HTF medium for 90 min at 2 Â 10 6 /ml yielded capacitated sperm. Capacitated sperm were transferred and concentrated for capillary loading by centrifugation at 900 g for 3 min. The loose sperm pellet at the bottom of the microcentrifuge tube was resuspended in HEPES-HTF at 4 Â 10 6 /ml, and loaded into the capillary by suction via an air-pressure microinjector (IM-5B; Narishige; 22 C,~200 mm/s). While applying gentle positive pressure, the sperm in the tip of the capillary were moved out of the sperm drop. The tip of the capillary is transferred to a 37˚C chamber (Delta T culture dish controller; Bioptechs) and placed into a 50 ml drop of HEPES-HTF medium covered with mineral oil. Negative pressure was applied slowly and sperm cells swimming against the flow and down to the H-HTF drop was video-recorded at 33 fps.

Quantification and statistical analysis
All the experiments are repeated at least three times. Sample size and number of replicates are described in each figure and the figure legends. Statistical analyses were performed using Student's t-test unless indicated; e.g. F-test in one-way ANOVA. Differences were considered significant at *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. When ****p<0.0001, actual P value is not indicated.