Research Topics & Main Achievements

Navigation mechanisms in bacteria and sperm

Bacterial chemotaxis

One of the most common navigation mechanisms in nature is chemotaxis, namely, moving up or down a concentration gradient of a chemoattractant or a chemorepellent. We studied chemotaxis of Escherichia coli as a model system. In this system, changes in the environment, sensed by receptors, are translated into a change in the direction of flagellar rotation (Figure 1).

Figure 1. A simplified scheme of signal transduction in bacterial chemotaxis of E. coli. Changes in the environment, sensed by the receptors, modulate the phosphorylation level of the excitatory response regulator, CheY, by a histidine kinase, CheA, and a phosphatase, CheZ, both being part of the receptor complex. Phosphorylated CheY dissociates from the complex and binds to the switch at the base of the flagellar motor, with a resultant change in the rotation direction from the default, counterclockwise, to clockwise. These translate into the swimming mode of the bacteria. Black arrows stand for regulated protein-protein interactions. The scheme is not drawn to scale.

Among our numerous, major contributions to this research field, the highlights were:

By mean of a unique in vitro system of flagellated, cytoplasm-free envelopes of E. coli and Salmonella, which we had developed, we provided direct evidence that the motor of these bacteria can only rotate in one rotation, counterclockwise, which is its default direction, and that for rotating in the other direction, the response regulator CheY (Figure 1) has to interact with the switch of the motor (Ravid & Eisenbach 1984; Ravid et al. 1986).
We identified the main binding site of CheY at the switch — the N-terminus of the switch protein FliM (FliM_N), and demonstrated that phosphorylation of CheY increases this binding and, consequently, enhances clockwise rotation (Barak & Eisenbach 1992a; Welch et al. 1993; Bren & Eisenbach 1998).
We found that the clockwise-generating activity of CheY is regulated not only by phosphorylation but also by acetylation (Barak et al. 1992, 1998; Barak & Eisenbach 2001).
We revealed the molecular mechanism of CheY acetylation as well as the essential function that acetylated CheY fulfills in the motor’s switching mechanism (Baraket al. 2004; Barak & Eisenbach 2004; Baraket al. 2006; Yan et al.2008; Liarzi et al. 2010; Fraiberg et al. 2015; Afanzar et al. 2021).
We uncovered the switching mechanism of the flagellar motor by identifying the processes that occur there subsequent to CheY binding to FliM_N (Bren & Eisenbach 2001; Sagi et al. 2003; Afanzar et al. 2021).
We discovered that fumarate is a switching factor of E. coli, we identified its target — the enzyme fumarate reductase, we demonstrated that fumarate reductase binds to the switch protein FliG and forms a complex with the switch of the flagellar motor, and we revealed the molecular mechanism by which fumarate binding to this complex generates clockwise rotation and switching (Barak & Eisenbach 1992b, Prasad et al. 1998, Cohen-Ben-Lulu et al. 2008; Koganitsky et al. 2019).

Sperm navigation in mammals

In view of the very large number of spermatozoa ejaculated into the female genital tract, which is a space of defined, limited volume, it was believed until not too long ago that there is no need for sperm navigation in mammal. Contrary to this dogma, however, only small numbers of the ejaculated spermatozoa enter the Fallopian tube. This indicated to us that these few spermatozoa must be guided in order to make the remaining long, obstructed way to the egg. We then discovered that mammalian spermatozoa are, indeed, actively guided to the egg, and by this we opened a novel research field — sperm navigation in mammals. We demonstrated that the active navigation is done by chemotaxis and thermotaxis, which are short- and long-range processes, respectively (Figure 2). We made key contributions to the understanding of these two processes at the molecular, physiological and behavioral levels.

Figure 2. A scheme of the female genital tract demonstrating the locations of sperm thermotaxis and chemotaxis.

Our major contributions to this new research field were:

We found that human spermatozoa accumulate in diluted follicular fluid and that there is a remarkably strong correlation between the ability of follicular fluid from a particular follicle to cause sperm accumulation and the ability of the egg, obtained from the same follicle, to be fertilized (Ralt et al. 1991).
We provided the first evidence that sperm accumulation in follicular fluid is the result of chemotaxis, accompanied by chemokinesis, and defined criteria for distinguishing chemotaxis from other processes that might cause sperm accumulation (Ralt et al. 1994).
We discovered that only a small fraction of the spermatozoa (~10% in humans) is chemotactically responsive and that the responsive spermatozoa are the capacitated ones (spermatozoa that reached a maturation stage at which they can penetrate the egg and fertilize it) (Cohen-Dayag et al. 1994, 1995).
We found that the capacitated state is temporary (50-240 min for human spermatozoa in vitro), that there is a continuous process of replacement of capacitated spermatozoa within the sperm population, and that cells that stopped being capacitated are phagocytized by macrophages (Cohen-Dayag et al. 1995, Eisenbach 2003, Oren-Benaroya et al. 2007).
We hypothesized, and then provided indirect evidence, that the physiological role of this continuous replacement of capacitated spermatozoa in humans is to prolong the availability of these spermatozoa to the egg during the relatively short time window at which it resides in the fertilization site (Cohen-Dayag et al. 1995, Giojalas et al. 2004).
We showed that there is lack of species-specificity among humans, rabbits and bovine with respect to sperm chemotaxis, suggesting that at least one of the chemoattractants is common, or very similar, in these species (Sun et al. 2003).
We provided the first evidence that, subsequent to ovulation (i.e., outside the follicle), both the mature egg and its surrounding cumulus cells secrete chemoattractants (Sun et al. 2004).
We provided direct evidence that progesterone is the main chemoattractant secreted from human cumulus cells (Oren-Benaroya 2008).
We characterized the human oocyte-derived sperm chemoattractant as a hydrophobic molecule associated with a carrier protein in an aqueous environment (Armon et al. 2014).
We discovered that human, rabbit and mouse spermatozoa can navigate in a temperature gradient by thermotaxis and that, as in chemotaxis, only capacitated cells are thermotactically active (Bahat et al. 2003, Pérez-Cerezales et al. 2015).
We found that, at ovulation, a temperature gradient of 0.016°C/mm is established in the rabbit’s oviduct as a result of a temperature drop at the storage site (Figure 2) (Bahat et al. 2005).
We found that the temperature sensitivity of human spermatozoa for thermotaxis is extraordinarily high, being able to detect gradients shallower than 0.014°C/mm within a wide temperature range. This means that as a spermatozoon swims through its entire body length, it can astonishingly respond to a temperature difference of less than 0.0006°C (Bahat et al. 2012).
We showed that hyperactivation (a vigorous motility type, essential for fertilization, with characteristic large amplitudes of head displacement) is part of the sperm navigation mechanism (Armon & Eisenbach 2011).
We demonstrated that human spermatozoa detect chemoattractant concentration gradients and temperature gradients temporally rather than spatially (namely, by comparing consecutive time points rather than different locations) (Armon & Eisenbach 2011, Boryshpolets et al. 2015).
We revealed the behavioral mechanisms of both human sperm chemotaxis and thermotaxis, and showed that both are similar and based on modulation of the frequency of turns and hyperactivation events according to the gradient (Armon & Eisenbach 2011, Boryshpolets et al. 2015).
We found that the thermosensors in the temperature-sensing system of mammalian spermatozoa are opsins, known to act as photosensors in vision (Pérez-Cerezales et al. 2015).
We provided evidence for two signaling pathways in sperm thermotaxis: the cyclic-nucleotide pathway, triggered by rhodopsin, and the phospholipase-C pathway, triggered by melanopsin and, likely, by other opsins (Bahat & Eisenbach 2010, Pérez-Cerezales et al. 2015, Royet al. 2020).