Nachiket D. Kashikar

The CatSper channel mediates progesterone-induced Ca2+ influx in human sperm

In the oviduct, cumulus cells that surround the oocyte release progesterone. In human sperm, progesterone stimulates a Ca2+ increase by a non-genomic mechanism. The Ca2+ signal has been proposed to control chemotaxis, hyperactivation and acrosomal exocytosis of sperm. However, the underlying signalling mechanism has remained mysterious. Here we show that progesterone activates the sperm-specific, pH-sensitive CatSper Ca2+ channel. We found that both progesterone and alkaline pH stimulate a rapid Ca2+ influx with almost no latency, incompatible with a signalling pathway involving metabotropic receptors and second messengers. The Ca2+ signals evoked by alkaline pH and progesterone are inhibited by the Cav channel blockers NNC 55-0396 and mibefradil. Patch-clamp recordings from sperm reveal an alkaline-activated current carried by mono- and divalent ions that exhibits all the hallmarks of sperm-specific CatSper Ca2+ channels. Progesterone substantially enhances the CatSper current. The alkaline- and progesterone-activated CatSper current is inhibited by both drugs. Our results resolve a long-standing controversy over the non-genomic progesterone signalling. In human sperm, either the CatSper channel itself or an associated protein serves as the non-genomic progesterone receptor. The identification of CatSper channel blockers will greatly facilitate the study of Ca2+ signalling in sperm and help to define further the physiological role of progesterone and CatSper.

The CatSper channel: a polymodal chemosensor in human sperm

The sperm‐specific CatSper channel controls the intracellular Ca2+ concentration ([Ca2+]i) and, thereby, the swimming behaviour of sperm. In humans, CatSper is directly activated by progesterone and prostaglandins—female factors that stimulate Ca2+ influx. Other factors including neurotransmitters, chemokines, and odorants also affect sperm function by changing [Ca2+]i. Several ligands, notably odorants, have been proposed to control Ca2+ entry and motility via G protein‐coupled receptors (GPCRs) and cAMP‐signalling pathways. Here, we show that odorants directly activate CatSper without involving GPCRs and cAMP. Moreover, membrane‐permeable analogues of cyclic nucleotides that have been frequently used to study cAMP‐mediated Ca2+ signalling also activate CatSper directly via an extracellular site. Thus, CatSper or associated protein(s) harbour promiscuous binding sites that can host various ligands. These results contest current concepts of Ca2+ signalling by GPCR and cAMP in mammalian sperm: ligands thought to activate metabotropic pathways, in fact, act via a common ionotropic mechanism. We propose that the CatSper channel complex serves as a polymodal sensor for multiple chemical cues that assist sperm during their voyage across the female genital tract.

The rate of change in Ca2+ concentration controls sperm chemotaxis

Sperm navigate in a chemoattractant gradient by translating changes in intracellular calcium concentration over time into changes in curvature of the swimming path.

An Atypical CNG Channel Activated by a Single cGMP Molecule Controls Sperm Chemotaxis

The ability of a single molecule of cGMP to activate the K+-selective cyclic nucleotide–gated channel allows sea urchin sperm to find an egg. Finding an Egg in an Ocean Sperm of the sea urchin Arbacia punctulata, which are released into the ocean and must find their way to an egg before fertilization can take place, can sense and respond to a single molecule of the egg-derived chemoattractant resact. This response depends on the production of guanosine 3′,5′-monophosphate (cGMP) and the consequent activation of K+-selective cyclic nucleotide–gated (CNGK) channels, which leads to production of an intracellular calcium signal that regulates movement of the sperm flagellum and thereby the direction in which the sperm cell swims. After cloning the A. punctulata CNGK, Bönigk et al. combined mutational analysis with optical analysis and electrophysiology to explore the mechanisms responsible for this sensitivity. They found that, although CNGK contains four repeating regions, each of which resembles a cyclic nucleotide–gated (CNG) channel subunit and contains a cyclic nucleotide–binding domain, it is activated through binding of only a single molecule of cGMP. Using a compound that cages cGMP and becomes fluorescent after its release, they were able to calibrate the system and determine that fewer than 50 molecules of cGMP were required to mediate the Ca2+ response to a single molecule of resact. Sperm of the sea urchin Arbacia punctulata can respond to a single molecule of chemoattractant released by an egg. The mechanism underlying this extreme sensitivity is unknown. Crucial signaling events in the response of A. punctulata sperm to chemoattractant include the rapid synthesis of the intracellular messenger guanosine 3′,5′-monophosphate (cGMP) and the ensuing membrane hyperpolarization that results from the opening of potassium–selective cyclic nucleotide–gated (CNGK) channels. Here, we use calibrated photolysis of caged cGMP to show that ~45 cGMP molecules are generated during the response to a single molecule of chemoattractant. The CNGK channel can respond to such small cGMP changes because it is exquisitely sensitive to cGMP and activated in a noncooperative fashion. Like voltage-activated Cav and Nav channels, the CNGK polypeptide consists of four homologous repeat sequences. Disabling each of the four cyclic nucleotide–binding sites through mutagenesis revealed that binding of a single cGMP molecule to repeat 3 is necessary and sufficient to activate the CNGK channel. Thus, CNGK has developed a mechanism of activation that is different from the activation of other CNG channels, which requires the cooperative binding of several ligands and operates in the micromolar rather than the nanomolar range.

Caged progesterone: a new tool for studying rapid nongenomic actions of progesterone

Ketalization of the biomolecule progesterone with (6-bromo-7-hydroxycoumarin-4-yl)ethane-1,2-diol gives the photolabile progesterone derivatives 3 and 4. These compounds display dramatically reduced bioactivity and release progesterone upon irradiation with UV/vis or IR light. In particular, 4 can be used to perform concentration-jump experiments with high temporal and spatial resolution that allows one to study elegantly the mechanisms of rapid nongenomic cellular events evoked by progesterone. The usefulness of 4 was demonstrated by measurement of changes in swimming behavior of single human sperm caused by progesterone-induced Ca(2+) influx in the sperm flagellum.

The CatSper channel controls chemosensation in sea urchin sperm

Sperm guidance is controlled by chemical and physical cues. In many species, Ca2+ bursts in the flagellum govern navigation to the egg. In Arbacia punctulata, a model system of sperm chemotaxis, a cGMP signaling pathway controls these Ca2+ bursts. The underlying Ca2+ channel and its mechanisms of activation are unknown. Here, we identify CatSper Ca2+ channels in the flagellum of A. punctulata sperm. We show that CatSper mediates the chemoattractant‐evoked Ca2+ influx and controls chemotactic steering; a concomitant alkalization serves as a highly cooperative mechanism that enables CatSper to transduce periodic voltage changes into Ca2+ bursts. Our results reveal intriguing phylogenetic commonalities but also variations between marine invertebrates and mammals regarding the function and control of CatSper. The variations probably reflect functional and mechanistic adaptations that evolved during the transition from external to internal fertilization.

Temporal sampling, resetting, and adaptation orchestrate gradient sensing in sperm

Sperm use temporal sampling, resetting of intracellular calcium level, and adaptation of their sensitivity to respond to a wide range of chemoattractant concentrations during their voyage toward the egg.

High density and ligand affinity confer ultrasensitive signal detection by a guanylyl cyclase chemoreceptor

The sea urchin sperm guanylyl cyclase chemoreceptor achieves ultrasensitive signal detection and precise signal modulation through high receptor density, subnanomolar ligand affinity, and sequential dephosphorylation.

A Receptor Guanylyl Cyclase Reveals Auto-Phosphatase Activity

In sea urchin sperm of Arbacia punctulata, a guanylyl cyclase (GC) serves as chemotaxis receptor that enables sperm to respond to a single molecule of the chemoattractant, resact. The efficiency of resact capture is high, because GC covers about 50% of the flagellar surface and binds resact with picomolar affinity. Furthermore, the binding affinity is controlled by the level of occupancy of the receptor. At high occupancy the resact affinity is lowered through negative cooperativity among subunits of the trimeric GC complex. The lifetime of active GC is controlled by its phosphorylation state. At rest GC is phosphorylated at six serine residues. After activation by resact, the GC becomes dephosphorylated with a biphasic time course, whereas dephosphorylation strongly coincides with the decrease of cGMP synthesis. During the initial fast phase the amplitude of GC dephosphorylation increases with the occupancy level. However, the time constant of this phase is independent of receptor occupancy. Moreover, dephosphorylation is superstoichiometric: even if only 5% of the GCs are occupied by resact, approximately 70% become dephosphorylated. We conclude from these results, that the occupied GC inactivates by auto-dephosporylation (fast phase) and additionally can dephosphorylate adjacent non-occupied GCs (slow phase). We could show for the first time that a receptor GC is regulated by auto-dephosphorylation.