Ulrich Benjamin Kaupp

Direct action of endocrine disrupting chemicals on human sperm

Synthetic endocrine disrupting chemicals (EDCs), omnipresent in food, household, and personal care products, have been implicated in adverse trends in human reproduction, including infertility and increasing demand for assisted reproduction. Here, we study the action of 96 ubiquitous EDCs on human sperm. We show that structurally diverse EDCs activate the sperm‐specific CatSper channel and, thereby, evoke an intracellular Ca2+ increase, a motility response, and acrosomal exocytosis. Moreover, EDCs desensitize sperm for physiological CatSper ligands and cooperate in low‐dose mixtures to elevate Ca2+ levels in sperm. We conclude that EDCs interfere with various sperm functions and, thereby, might impair human fertilization.

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.

The computational sperm cell

Sperm are guided to the egg by a gradient of chemical attractants - a process called chemotaxis. The binding of the chemoattractant to receptors on the surface of the flagellum triggers a cascade of signaling events that eventually lead to an influx of Ca(2+) ions. Based on these Ca(2+) surges, which control the waveform of the flagellar beat, sperm adjust their swimming path toward the egg. In past years, many components of chemotactic signaling have been identified. Moreover, kinetic spectroscopy and imaging techniques unraveled the sequence of cellular events controlling swimming behavior. During navigation in a chemical gradient, sperm perform a surprising variety of computational operations. Here we discuss theoretical concepts of navigation strategies and the cellular underpinnings.

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.

Higher-Order Architecture of Rhodopsin in Intact Photoreceptors and Its Implication for Phototransduction Kinetics

The visual pigment rhodopsin belongs to the family of G protein-coupled receptors that can form higher oligomers. It is controversial whether rhodopsin forms oligomers and whether oligomers are functionally relevant. Here, we study rhodopsin organization in cryosections of dark-adapted mouse rod photoreceptors by cryoelectron tomography. We identify four hierarchical levels of organization. Rhodopsin forms dimers; at least ten dimers form a row. Rows form pairs (tracks) that are aligned parallel to the disk incisures. Particle-based simulation shows that the combination of tracks with fast precomplex formation, i.e. rapid association and dissociation between inactive rhodopsin and the G protein transducin, leads to kinetic trapping: rhodopsin first activates transducin from its own track, whereas recruitment of transducin from other tracks proceeds more slowly. The trap mechanism could produce uniform single-photon responses independent of rhodopsin lifetime. In general, tracks might provide a platform that coordinates the spatiotemporal interaction of signaling molecules.

Structural insights into conformational changes of a cyclic nucleotide-binding domain in solution from Mesorhizobium loti K1 channel

Cyclic nucleotide-sensitive ion channels, known as HCN and CNG channels, are activated by binding of ligands to a domain (CNBD) located on the cytoplasmic side of the channel. The underlying mechanisms are not well understood. To elucidate the gating mechanism, structures of both the ligand-free and -bound CNBD are required. Several crystal structures of the CNBD from HCN2 and a bacterial CNG channel (MloK1) have been solved. However, for HCN2, the cAMP-free and -bound state did not reveal substantial structural rearrangements. For MloK1, structural information for the cAMP-free state has only been gained from mutant CNBDs. Moreover, in the crystal, the CNBD molecules form an interface between dimers, proposed to be important for allosteric channel gating. Here, we have determined the solution structure by NMR spectroscopy of the cAMP-free wild-type CNBD of MloK1. A comparison of the solution structure of cAMP-free and -bound states reveals large conformational rearrangement on ligand binding. The two structures provide insights on a unique set of conformational events that accompany gating within the ligand-binding site.

Solution structure of the Mesorhizobium loti K1 channel cyclic nucleotide-binding domain in complex with cAMP

Cyclic nucleotide‐sensitive ion channels, known as HCN and CNG channels, are crucial in neuronal excitability and signal transduction of sensory cells. HCN and CNG channels are activated by binding of cyclic nucleotides to their intracellular cyclic nucleotide‐binding domain (CNBD). However, the mechanism by which the binding of cyclic nucleotides opens these channels is not well understood. Here, we report the solution structure of the isolated CNBD of a cyclic nucleotide‐sensitive K+ channel from Mesorhizobium loti. The protein consists of a wide anti‐parallel β‐roll topped by a helical bundle comprising five α‐helices and a short 310‐helix. In contrast to the dimeric arrangement (‘dimer‐of‐dimers’) in the crystal structure, the solution structure clearly shows a monomeric fold. The monomeric structure of the CNBD supports the hypothesis that the CNBDs transmit the binding signal to the channel pore independently of each other.

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.

Solid-state NMR [13C,15N] resonance assignments of the nucleotide-binding domain of a bacterial cyclic nucleotide-gated channel.

Channels regulated by cyclic nucleotides are key signalling proteins in several biological pathways. The regulatory aspect is conferred by a C-terminal cyclic nucleotide-binding domain (CNBD). We report resonance assignments of the CNBD of a bacterial mlCNG channel obtained using 2D and 3D solid-state NMR under Magic-angle Spinning conditions. A secondary chemical shift analysis of the 141 residue protein suggests a three-dimensional fold seen in earlier X-ray and solution-state NMR work and points to spectroscopic polymorphism for a selected set of resonances.

CRIS- a novel cAMP-binding protein controlling spermiogenesis and the development of flagellar bending

The second messengers cAMP and cGMP activate their target proteins by binding to a conserved cyclic nucleotide-binding domain (CNBD). Here, we identify and characterize an entirely novel CNBD-containing protein called CRIS (cyclic nucleotide receptor involved in sperm function) that is unrelated to any of the other members of this protein family. CRIS is exclusively expressed in sperm precursor cells. Cris-deficient male mice are either infertile due to a lack of sperm resulting from spermatogenic arrest, or subfertile due to impaired sperm motility. The motility defect is caused by altered Ca2+ regulation of flagellar beat asymmetry, leading to a beating pattern that is reminiscent of sperm hyperactivation. Our results suggest that CRIS interacts during spermiogenesis with Ca2+-regulated proteins that—in mature sperm—are involved in flagellar bending.