Analysis of physiological signaling mechanisms in reproductive and neuronal systems

  • Analyse physiologischer Signalmechanismen in reproduktiven und neuronalen Systemen

Mundt, Nadine; Spehr, Marc (Thesis advisor); Lishko, Polina (Thesis advisor)

Aachen : RWTH Aachen University (2021, 2022)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2021


The molecular and cellular mechanisms that govern testicular transport of immotile spermatozoa are controversial, if not elusive. Here, I reveal that extracellular ATP serves as an important paracrine messenger to activate smooth muscle TPCs that surround seminiferous tubules. ATP triggers cytosolic calcium elevations via P2 receptors, coordinated TPC contractions, and eventually luminal sperm transport. My data suggests a stage-dependent directionality of sperm transport, while the underlying mechanism for said directionality remains to be identified. ATP-induced calcium elevations are confined to directly stimulated areas in isolated whole-mount tubules. Thus, the observed directionality of luminal transport must rely on anatomical / morphological features of the intratubular compartment. Once spermatozoa enter the fallopian tubes within the female reproductive tract, they must transition to hyperactivated motility to ascend through viscous fluids and eventually overcome the egg's protective vestments. A key component for hyperactivation is the sperm calcium channel CatSper, but the origin of membrane depolarization that facilitates CatSper opening, was undescribed. My work identified "DSper" - the depolarizing ion channel of human sperm - as the temperature-activated cation channel TRPV4. Under physiological conditions, TRPV4 conducts a sodium inward current that provides the necessary positive net charge for membrane potential depolarization. TRPV4 activity was increased upon capacitation in a temperature-sensitive range between 22°C and 37°C. My work, thus, contributes to a better understanding of human sperm physiology and reveals the molecular basis for sperm membrane depolarization and temperature-sensitivity. The sense of smell is a vital tool for many species to perceive and adapt to the outside world. Several highly complex olfactory subsystems have evolved to process the enormous magnitude of olfactory stimuli. Our current knowledge of the accessory olfactory system that is involved in social information processing, however, is rudimentary. Therefore, the overall aim of this work was to gain a more profound understanding of chemosensory signaling mechanisms in the accessory olfactory system. I established an optogenetic stimulation paradigm in the murine main olfactory bulb, that mimics peripheral sensory input and will facilitate future investigations of the first central olfactory processing stages in vitro. Surprisingly, I found that VSN sensory afferents were incapable of synaptic transmission in corresponding in vitro settings, which renders the optogenetic stimulation paradigm unsuitable for further AOB studies. In addition, my work focused on peripheral chemosensation in the VNO. Darcin is a potent pheromone that elicits sexually dimorphic responses in recipients. I aimed to verify (or falsify) V2R27 as the darcin receptor, which was previously predicted by a protein coevolution algorithm. I established AAV-driven gene transfer in VSNs for ectopic V2R27 expression. Preliminary results suggest a significantly higher response rate for infected V2R27-expressing VSNs, as compared to noninfected control groups. This work, thus, provides an experimental basis for future receptor deorphanization studies and preliminary evidence for V2R27 as a darcin receptor. In summary, this thesis addresses several questions about signaling mechanisms in both reproductive and neuronal tissues. My work, thus, provides substantial advances in our knowledge about these two essential physiological systems.