Biology subjects

Brenker, C.

Publications and source records attributed to Brenker, C..

4 recordsLinked to original sources

bioRxiv2025

Membrane potential and feedback dynamics regulate CatSper-mediated progesterone signaling in human sperm

Activation of the sperm-specific Ca2+ channel CatSper by progesterone evokes rapid changes in intracellular Ca2+ in human sperm that are required for fertilization. However, the mechanisms regulating the progesterone-induced Ca2+ signals have remained elusive. Here, we used quantitative kinetic fluorimetry with fast voltage-sensitive fluorescent indicators to investigate how progesterone affects the membrane potential (Vm) of human sperm. Additionally, we employed the FASTM technique to simultaneously record at millisecond time resolution changes in both Vm and intracellular Ca2+. We show that progesterone evokes a rapid pulse-like depolarization and repolarization. The depolarization is caused by Ca2+ influx through CatSper, which pulls Vm away from a resting membrane potential (Vrest) of -65 mV set by the sperm-specific K+ channel Slo3. We further show that Vm- and Ca2+-dependent mechanisms limit the CatSper-mediated Ca2+ influx, thereby promoting repolarization and enabling K+ efflux through Slo3 channels to restore Vrest. 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 Vm. We anticipate that our novel kinetic, quantitative Vm recording and Vm/Ca2+-multiplexing techniques will reveal additional molecular mechanisms underlying CatSper-mediated Ca2+ signaling in human sperm both in health and disease.

cell biology↗

bioRxiv2024

ABHD2 activity is not required for the non-genomic action of progesterone on human sperm

Study questionIs the hydrolase ABHD2 required for progesterone-induced Ca2+ influx via CatSper and the resulting motility responses in human sperm? Summary answerProgesterone-induced Ca2+ influx via CatSper and the resulting motility responses in human sperm do not require ABHD2 activity. What is known alreadySperm motility is tightly regulated by signalling pathways that are activated as sperm ascend the female reproductive tract, including progesterone triggering Ca2+ influx via the CatSper channel and inducing hyperactivated motility needed for fertilization. This process is thought to involve ABHD2, which may hydrolyse the endogenous CatSper inhibitor 2-arachidonoylglycerol (2-AG), thereby relieving inhibition and enabling calcium entry into the flagellum. Study design, size, durationPotent small molecule inhibitors of ABHD2 activity were synthesized, characterized, and used as tools to scrutinize the role of ABHD2 in activation of CatSper and regulation of sperm motility. Participants/materials, setting, methodsDerivatives of published ABHD2 inhibitors were optimised for in vitro potency and cellular activity and subsequently tested in human sperm motility and Ca2+ influx assays. Main results and the role of chanceProgesterone does not activate ABHD2 in vitro. In addition, inhibition of ABHD2 in human sperm has no effect on progesterone-induced Ca2+ influx through CatSper nor on basal or progesterone-induced hyperactivated motility. This demonstrates that ABHD2 activity is, in fact, not required for the non-genomic action of progesterone on human sperm. Large scale datanone Limitations, reasons for cautionWe examined the effects of inhibition of the enzymatic activity of ABHD2. We cannot exclude that ABHD2 functions as a part of a larger multiprotein complex, in which it may play a structural role independent of its hydrolase activity. Wider implications of the findingsThis study presents conclusive evidence that ABHD2 does not bind progesterone and that its hydrolase activity is not required for progesterone activation of CatSper and resulting changes in motility of human sperm. These results highlight the need for further research to elucidate the mechanism underlying the non-genomic action of progesterone on human sperm. Study funding/competing interest(s)This publication is based on research funded by the Gates Foundation. The findings and conclusions contained within are those of the authors and do not necessarily reflect positions or policies of the Gates Foundation. LT, CB, and TS were supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) - project numbers 329621271 (CRU326; CB, TS), and 404595355 (Research Training Group "Chemical biology of ion channels (Chembion); LT, TS). This work was funded by the German Federal Ministry of Education and Research (BMBF) within the framework of Contraception Research, grant number 01GR2501A and 01GR2502A. A.A., K.V., A.T., N.D., A.H., M.W. and R.L. are employees of Nuvisan ICB GMBH, Berlin, Germany. Nuvisan is recipient of a Gates Foundation grant. A.A is associate editor of Human Reproduction Open.

cell biology↗

bioRxiv2022

Cylicins are a structural component of the sperm calyx being indispensable for male fertility in mice and human

Cylicins are testis-specific proteins, which are exclusively expressed during spermiogenesis. In mice and humans, two Cylicins, the gonosomal X-linked Cylicin 1 (Cylc1/CYLC1) and the autosomal Cylicin 2 (Cylc2/CYLC2) genes have been identified. Cylicins are cytoskeletal proteins with an overall positive charge due to lysine-rich repeats. While Cylicins have been localized in the acrosomal region of round spermatids, they resemble a major component of the calyx within the perinuclear theca at the posterior part of mature sperm nuclei. However, the role of Cylicins during spermiogenesis has not yet been investigated. Here, we applied CRISPR/Cas9-mediated gene-editing in zygotes to establish Cylc1- and Cylc2-deficient mouse lines as a model to study the function of these proteins. Cylc1 deficiency resulted in male subfertility, whereas Cylc2-/-, Cylc1-/y Cylc2+/-, and Cylc1-/y Cylc2-/- males were infertile. Phenotypical characterization revealed that loss of Cylicins prevents proper calyx assembly during spermiogenesis. This results in decreased epididymal sperm counts, impaired shedding of excess cytoplasm, and severe structural malformations, ultimately resulting in impaired sperm motility. Furthermore, exome sequencing identified an infertile man with a hemizygous variant in CYLC1 and a heterozygous variant in CYLC2, displaying morphological abnormalities of the sperm including the absence of the acrosome. Thus, our study highlights the relevance and importance of Cylicins for spermiogenic remodeling and male fertility in human and mouse, and provides the basis for further studies on unraveling the complex molecular interactions between perinuclear theca proteins required during spermiogenesis.

developmental biology↗

bioRxiv2020

Shedding light on the control of CatSper Ca2+ channels by cAMP and chemicals used to probe cAMP signaling

The sperm-specific CatSper channel controls the influx of Ca2+ into the flagellum and, thereby, the swimming behavior of sperm. A hallmark of human CatSper is its polymodal activation by membrane voltage, intracellular pH, and oviductal hormones. Whether CatSper is also activated by signaling pathways involving an increase of cAMP and ensuing activation of protein kinase A (PKA) is, however, a matter of controversy. Here, using kinetic ion-sensitive fluorimetry and patch-clamp recordings, we study transmembrane Ca2+ flux and membrane currents in human sperm from healthy donors and from patients that lack functional CatSper channels. We show that human CatSper is neither activated by intracellular cAMP directly nor indirectly by the cAMP/PKA-signaling pathway. Moreover, we demonstrate that non-physiological concentrations of cAMP and membrane-permeable cAMP analogs used to mimic the action of intracellular cAMP activate human CatSper from the outside via a previously unknown extracellular cyclic nucleotide-binding site. Finally, we demonstrate that the effects of common PKA inhibitors on human CatSper rest on off-target drug actions on CatSper itself rather than on inhibition of PKA. We conclude that the concept of an intracellular cAMP/PKA-activation of CatSper is primarily based on unspecific effects of chemical probes used to interfere with cAMP signaling. Altogether, our findings solve several controversial issues, which has important bearings on future studies of cAMP and Ca2+ signaling and the ligand-control of CatSper in sperm.

cell biology↗