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Imaging sperm as they travel through the female reproductive tract has revealed new details about fertilization at the molecular level., Competing Interests: KK, AC No competing interests declared, (© 2020, Komondor and Carlson.)

The four sperm-specific CatSper ion channel proteins are required for hyperactivated motility and male fertility, and for Ca(2+) entry evoked by alkaline depolarization. In the absence of external Ca(2+), Na(+) carries current through CatSper channels in voltage-clamped sperm. Here we show that CatSper channel activity can be monitored optically with the [Na(+)](i)-reporting probe SBFI in populations of intact sperm. Removal of external Ca(2+) increases SBFI signals in wild-type but not CatSper2-null sperm. The rate of the indicated rise of [Na(+)](i) is greater for sperm alkalinized with NH(4)Cl than for sperm acidified with propionic acid, reflecting the alkaline-promoted signature property of CatSper currents. In contrast, the [Na(+)](i) rise is slowed by candidate CatSper blocker HC-056456 (IC(50) approximately 3 microM). HC-056456 similarly slows the rise of [Ca(2+)](i) that is evoked by alkaline depolarization and reported by fura-2. HC-056456 also selectively and reversibly decreased CatSper currents recorded from patch-clamped sperm. HC-056456 does not prevent activation of motility by HCO(3) (-) but does prevent the development of hyperactivated motility by capacitating incubations, thus producing a phenocopy of the CatSper-null sperm. When applied to hyperactivated sperm, HC-056456 causes a rapid, reversible loss of flagellar waveform asymmetry, similar to the loss that occurs when Ca(2+) entry through the CatSper channel is terminated by removal of external Ca(2+). Thus, open CatSper channels and entry of external Ca(2+) through them sustains hyperactivated motility. These results indicate that pharmacological targeting of the CatSper channel may impose a selective late-stage block to fertility, and that high-throughput screening with an optical reporter of CatSper channel activity may identify additional selective blockers with potential for male-directed contraception.

Among several candidate Ca(2+) entry channels in sperm, only CatSper1 and CatSper2 are known to have required roles in male fertility. Past work with CatSper1 null sperm indicates that a critical lesion in hyperactivated motility underlies the infertility phenotype and is associated with an absence of depolarization-evoked Ca(2+)entry. Here we show that failure of hyperactivation of CatSper2 null sperm similarly correlates with an absence of depolarization evoked Ca(2+) entry. Additional shared aspects of the phenotypes of CatSper1 and -2 null sperm include unperturbed regional distributions of conventional voltage-gated Ca(2+) channel proteins and robust acceleration of the flagellar beat by bicarbonate. Further study reveals that treatment of both wild-type and CatSper2 null sperm with procaine increases beat asymmetry, a characteristic of the flagellar waveform of hyperactivation. This partial rescue of the loss-of-hyperactivation phenotype suggests that an absence of CatSper2 precludes hyperactivation by preventing delivery of needed Ca(2+) messenger rather than by preventing flagellar responses to Ca(2+). CatSper2 null sperm also have an increased basal cAMP content and beat frequency. Protein kinase A inhibitor H89 lowers beat frequency to that of wild-type sperm, suggesting that CatSper2 is required for protein kinase A-mediated, tonic control of resting cAMP content. Relative to wild-type testis, CatSper1 and -2 null testes contain normal amounts of CatSper2 and -1 transcripts, respectively. However, CatSper1 null sperm lack CatSper2 protein and CatSper2 null sperm lack CatSper1 protein. Hence, stable expression of CatSper1 protein requires CatSper2 and vice versa. This co-dependent expression dictates identical loss-of-function sperm phenotypes for CatSper1 and -2 null mutants.

CatSper family proteins are putative ion channels expressed exclusively in membranes of the sperm flagellum and required for male fertility. Here, we show that mouse CatSper1 is essential for depolarization-evoked Ca2+ entry and for hyperactivated movement, a key flagellar function. CatSper1 is not needed for other developmental landmarks, including regional distributions of CaV1.2, CaV2.2, and CaV2.3 ion channel proteins, the cAMP-mediated activation of motility by HCO3-, and the protein phosphorylation cascade of sperm capacitation. We propose that CatSper1 functions as a voltage-gated Ca2+ channel that controls Ca2+ entry to mediate the hyperactivated motility needed late in the preparation of sperm for fertilization.

At mating, mammalian sperm are diluted in the male and female reproductive fluids, which brings contact with HCO(3)(-) and initiates several cellular responses. We have identified and studied two of the most rapid of these responses. Stop-motion imaging and flagellar waveform analysis show that for mouse epididymal sperm in vitro, the resting flagellar beat frequency is 2-3 Hz at 22-25 degrees C. Local perfusion with HCO(3)(-) produces a robust, reversible acceleration to 7 Hz or more. At 15 mM the action of HCO(3)(-) begins within 5 seconds and is near-maximal by 30 seconds. The half-times of response are 8.8+/-0.2 seconds at 15 mM HCO(3)(-) and 17.5+/-0.4 seconds at 1 mM HCO(3)(-). Removal of external HCO(3)(-) allows a slow return to basal beat frequency over approximately 10 minutes. Increases in beat symmetry accompany the accelerating action of HCO(3)(-). As in our past work, HCO(3)(-) also facilitates opening of voltagegated Ca(2+) channels, increasing the depolarization-evoked rate of rise of intracellular Ca(2+) concentration by more than fivefold. This action also is detectable at 1 mM HCO(3)(-) and occurs with an apparent halftime of approximately 60 seconds at 15 mM HCO(3)(-). The dual actions of HCO(3)(-) respond similarly to pharmacological intervention. Thus, the phosphodiesterase inhibitor IBMX promotes the actions of HCO(3)(-) on flagellar and channel function, and the protein kinase A inhibitor H89 blocks these actions. In addition, a 30 minute incubation with 60 micro M cAMP acetoxylmethyl ester increases flagellar beat frequency to nearly 7 Hz and increases the evoked rates of rise of intracellular Ca(2+) concentration from 17+/-4 to 41+/-6 nM second(-1). However, treatment with several other analogs of cAMP produces only scant evidence of the expected mimicry or blockade of the actions of HCO(3)(-), perhaps as a consequence of limited permeation. Our findings indicate a requirement for cAMP-mediated protein phosphorylation in the enhancement of flagellar and channel functions that HCO(3)(-) produces during sperm activation.