Understanding the signaling pathways and molecular components of sperm using stopped-flow

One of the key questions linked to a true understanding of the origin of life in multicellular organisms is how, exactly, do sperm locate eggs? This same question is highly pertinent in medical research for solutions related to infertility problems and for the development of the next generation of contraceptive techniques.

Chemotaxis is the movement of an organism in response to a chemical stimulus. It is an essential phenomenon in the early development of multicellular organisms. In humans (or mammals to a greater extent) sperm guidance is based on a complex interaction between chemotaxis, thermotaxis and rheotaxis. For echinoderms, like sea urchins, reproduction is external and based only on chemotaxis. The animal releases the sperm and the egg in the seawater so the sperm has to find its way by detecting chemical cues released by the egg. In this organism, receptors on the flagella are so sensitive they can detect a single molecule of chemoattractant to optimize its chance of success of spotting the egg. Such reasons explain why sea urchins have been used as a model to better understand the subtleties of reproductive processes.

In a recent article ‘Kinetic and photonic techniques to study chemotactic signaling in sea urchin sperm’ (Methods Cell Biol. 2019;151:487-517. doi: 10.1016/bs.mcb.2018.12.001) Professors Kaupp and Strünker describe in detail the signaling pathway and molecular components endowing sperm with single-molecule sensitivity. Chemotactic signaling and behavioral responses occur on a timescale of a few milliseconds to seconds, which requires rapid-mixing devices such as stopped-flow and ultra-sensitive detectors.  Bio-Logic’s SFM-4000 was used for these kinetics studies. The open design of the observation head of the stopped-flow allowed the team to develop a batch of methods to follow changes of Na⁺ and Ca²⁺ concentration as well as pH or membrane voltage using fluorescent indicators. These techniques included a combination of rapid mixing and photolysis where the sperm is first stimulated by a chemoattractant before being re-stimulated by a caged cellular messenger after light stimulation in the stopped-flow cuvette.

Sperm cells are delicate, pressure sensitive samples, thus sheer forces have to be fully controlled so the cells conserve their physical integrity and make these measurements possible. SFM Stopped-flow include independent stepping motors making them ideal systems for handling such sperm cells as the user can adjust and fully controls the injection speed in the mixing chamber. Sample consumption for these in vivo experiments are also important and this becomes a major consideration when  human research takes place.

The microvolume stopped-flow µSFM can reduce sample volumes by a factor of 10 without any loss on signal sensitivity. Professors Kaupp and Strünker in the above article report a 16 µl consumption but the volume per experiment has been reduced to 3 µl in other applications.

The stopped-flow is a key instrument to understand how the ions channels work, how sperm reacts to different chemoattractant and thus how the sperms moves to its target.