Ulrich Benjamin Kaupp

Cells transform complex environmental stimuli into physiological responses. In dynamic environments, or when a motile cell moves in a static setting, stimuli change over time. Here we introduce a concept, “reverse opto-chemical engineering,” which uses temporal light patterns and photo-triggers to create virtual sensory landscapes for cells. This allows us to record their physiological responses and motor behavior in real time. Using this approach, we studied cyclic-nucleotide signaling in sperm cells and mapped their stimulus-response transfer function. The technique can be employed for remote control of motility by light. Exploiting the chemotactic signaling backbone, we enable sperm to navigate in light gradients, making them attracted to light. This method offers possibilities for uncovering the mechanisms and signaling molecules behind rapid cellular computations, helping to understand the wiring diagram of cellular networks.

In mammalian sperm, high HCO ₃ ⁻ concentrations in semen and the oviduct activate soluble adenylyl cyclase (sAC), which synthesizes cyclic AMP to regulate sperm motility. Here, we demonstrate that sAC orthologs in echinoderms and fish, species whose sperm fertilize eggs in aquatic environments with low HCO ₃ ⁻ , are activated by alkaline pH rather than HCO ₃ ⁻ . In human sAC, two charged residues coordinate HCO ₃ ⁻ and are essential for HCO ₃ ⁻ -mediated activation. In contrast, pH-regulated sACs and orthologs from 13 phyla have these residues replaced by neutral ones. These substitutions abolish the enzyme’s responsiveness to HCO ₃ ⁻ and suggest that pH regulation of sAC is widespread in nonmammalian metazoans. Furthermore, we show that in sea urchin sperm, a rise of pH during spawning stimulates cAMP synthesis, a key step in the activation of motility. An evolutionarily significant pattern is emerging: Across phyla, sAC regulation by pH or HCO ₃ ⁻ represents an adaptation to environments with low or high HCO ₃ ⁻ , respectively.

Activation of the sperm-specific Ca ²⁺ channel CatSper by progesterone evokes rapid changes in intracellular Ca ²⁺ in human sperm that are required for fertilization. However, the mechanisms regulating the progesterone-induced Ca ²⁺ signals have remained elusive. Here, we used quantitative kinetic fluorimetry with fast voltage-sensitive fluorescent indicators to investigate how progesterone affects the membrane potential (V _m ) of human sperm. Additionally, we employed the FAST ^M technique to simultaneously record at millisecond time resolution changes in both V _m and intracellular Ca ²⁺ . We show that progesterone evokes a rapid pulse-like depolarization and repolarization. The depolarization is caused by Ca ²⁺ influx through CatSper, which pulls V _m away from a resting membrane potential (V _rest ) of −65 mV set by the sperm-specific K ⁺ channel Slo3. We further show that V _m - and Ca ²⁺ -dependent mechanisms limit the CatSper-mediated Ca ²⁺ influx, thereby promoting repolarization and enabling K ⁺ efflux through Slo3 channels to restore V _rest . Our findings demonstrate that non-genomic progesterone signaling in human sperm is regulated by negative feedback on CatSper and involves a dynamic interplay between CatSper and Slo3 in controlling V _m . We anticipate that our novel kinetic, quantitative V _m recording and V _m /Ca ²⁺ -multiplexing techniques will reveal additional molecular mechanisms underlying CatSper-mediated Ca ²⁺ signaling in human sperm both in health and disease.

Cells transform complex environmental stimuli into physiological responses. For time-varying stimuli or motile cells, the perception of the environment depends on the temporal stimulus pattern and cell motion, respectively. Here we report a concept, reverse optochemical engineering (ROCE), that uses temporal light patterns and photo-triggers to expose cells to virtual sensory landscapes while recording in real time their physiological responses and motor behavior. We studied cyclic-nucleotide signaling in cell lines, sperm, olfactory neurons, and cardiomyocytes. The technique can be employed for remote control of motility by light. We reprogrammed sperm from a chemotactic to a phototactic cell that is attracted towards light. The method provides new opportunities to decipher the mechanisms and signaling molecules underlying rapid cellular computations, and thus reveal the wire diagram of cellular networks.

The reaction of CO₂ with H₂O to form bicarbonate (HCO₃⁻) and H⁺ controls sperm motility and fertilization via HCO₃⁻-stimulated cAMP synthesis. A complex network of signaling proteins participates in this reaction. Here, we identify key players that regulate intracellular pH (pH_i) and HCO₃⁻ in human sperm by quantitative mass spectrometry (MS) and kinetic patch-clamp fluorometry. The resting pH_i is set by amiloride-sensitive Na⁺/H⁺ exchange. The sperm-specific putative Na⁺/H⁺ exchanger SLC9C1, unlike its sea urchin homologue, is not gated by voltage or cAMP. Transporters and channels implied in HCO₃⁻ transport are not detected, and may be present at copy numbers < 10 molecules/sperm cell. Instead, HCO₃⁻ is produced by diffusion of CO₂ into cells and readjustment of the CO₂/HCO₃⁻/H⁺ equilibrium. The proton channel H_v1 may serve as a unidirectional valve that blunts the acidification ensuing from HCO₃⁻ synthesis. This work provides a new framework for the study of male infertility.

The eukaryotic flagellum propels sperm cells and simultaneously detects physical and chemical cues that modulate the waveform of the flagellar beat. Most previous studies have characterized the flagellar beat and swimming trajectories in two space dimensions (2D) at a water/glass interface. Here, using refined holographic imaging methods, we report high-quality recordings of three-dimensional (3D) flagellar bending waves. As predicted by theory, we observed that an asymmetric and planar flagellar beat results in a circular swimming path, whereas a symmetric and non-planar flagellar beat results in a twisted-ribbon swimming path. During swimming in 3D, human sperm flagella exhibit torsion waves characterized by maxima at the low curvature regions of the flagellar wave. We suggest that these torsion waves are common in nature and that they are an intrinsic property of beating axonemes. We discuss how 3D beat patterns result in twisted-ribbon swimming paths. This study provides new insight into the axoneme dynamics, the 3D flagellar beat, and the resulting swimming behavior. Graphic

The reaction of CO ₂ with H ₂ O to form HCO ₃ ^- and H ⁺ is one of the most important chemical equilibria in cells. In mammalian sperm, a soluble adenylyl cyclase (sAC) serves as cellular HCO ₃ ^- sensor that conveys the equilibrium state via cAMP synthesis to cAMP-signaling molecules. The function of sAC and cAMP in non-mammalian sperm is largely unknown. Here, we identify sAC orthologs in sea urchin and salmon sperm that, surprisingly, are activated by alkaline pH rather than HCO ₃ ^- . Two amino-acid residues required for HCO ₃ ^- binding of mammalian sAC are lacking in pH-regulated sAC. Orthologs identified in ten other phyla are also lacking either one of these key residues, suggesting that pH control is widespread among non-mammalian metazoan. The pH-sensitive sAC controls several functions of sperm from external fertilizers. Upon spawning, alkalization triggers cAMP synthesis and, thereby, activates motility of quiescent sperm. Egg-derived chemoattractants also alkalize sperm and elevate cAMP, which then-modulates pacemaker HCN channels to trigger a chemotactic Ca ²⁺ response. Finally, the sAC and the voltage- and cAMP-activated Na ⁺ /H ⁺ exchanger sNHE mutually control each other. A picture of evolutionary significance is emerging: motility and sensory signaling of sperm from both internal and external fertilizers rely on cAMP, yet, their sAC is regulated by HCO ₃ ^- or pH _i , respectively. Acidification of aquatic habitats due to climate change may adversely affect pH-sensing by sAC and thereby sexual reproduction in the sea. Statement of significance: Adenylyl cyclases synthesize cAMP, a prominent cellular messenger. A bicarbonate-sensitive AC family member, soluble AC (sAC), is tied to the chemical equilibrium: H ₂ O + CO ₂ ↔ HCO ₃ ^- (bicarbonate) + H ⁺ . The sAC is required for fertilization: Mammals lacking sAC are infertile and sperm immotile. We now identify a new sAC form in sperm of non-mammalian animals that reproduce in the sea. This novel sAC is activated at alkaline pH rather than bicarbonate. It controls sperm motility and chemotaxis. The switch from HCO ₃ ^- to pH rests on substitution of two amino-acids, which represents an adaptation to aquatic environments low in bicarbonate. Acidification of aquatic habitats due to climate change may adversely affect sAC activity and, thereby, fertilization.

The function of proteins is linked to their conformations that can be resolved with several high-resolution methods. However, only a few methods can provide the temporal order of intermediates and conformational changes, with each having its limitations. Here, we combine pulsed electron–electron double resonance spectroscopy with a microsecond freeze-hyperquenching setup to achieve spatiotemporal resolution in the angstrom range and lower microsecond time scale. We show that the conformational change of the C_α-helix in the cyclic nucleotide-binding domain of the Mesorhizobium loti potassium channel occurs within about 150 μs and can be resolved with angstrom precision. Thus, this approach holds great promise for obtaining 4D landscapes of conformational changes in biomolecules.

Significance: Understanding the evolution of sex determination mechanisms and sex chromosomes is of fundamental importance in biology. Here we have reconstructed the evolution of the sex-determining region in the Atlantic herring. The region is small and contains only three intact genes. The candidate sex-determining factor BMPR1BBY is an evolutionary innovation in the herring lineage. It encodes a truncated form of a BMP type I receptor, which originated by gene duplication and underwent rapid protein evolution. The receptor has maintained its kinase activity and has the potential to induce development of testis. The other two genes in the sex-determining region, CATSPERG and CATSPER3Y , are male beneficial genes because they encode proteins predicted to be essential for sperm to fertilize the egg.