SNIFFING SPERM
How do sperm find their way to the egg before fertilization? Heaven knows that from the sperm’s perspective this is not a trivial question, as finding the egg is like looking for a needle in a haystack. To successfully locate the egg, sperm have to possess some navigation system. In metazoan organisms, sperm navigate by chemotaxis, moving along a concentration gradient of an attractant released by the egg.
In 1992, a large mammalian gene family was described that encodes approximately 1000 different odorant receptors, mostly expressed in the cilia of olfactory receptor neurons. However, several distinct odorant receptors have also been found to be expressed either predominantly or exclusively in sperm flagella. Since the flagellum provides the propulsive force for sperm locomotion, it was speculated that these testicular odorant receptors are directly involved in navigating the sperm towards the egg. In a recent Science paper, Marc Spehr and his colleagues investigated a previously undescribed testicular odorant receptor, termed hOR17-4, in order to uncover its role in human sperm physiology. By combining molecular biology, pharmacological studies and behavioral assays, they were able to demonstrate for the first time that a testicular odorant receptor directly functions in sperm chemotaxis.
First, the scientists decided to test which odors the receptor ‘smelled’. They expressed the recombinant receptor protein in human embryonic kidney cells, treated the cells with a complex mixture of odorants and measured the receptor-induced calcium release. By subdividing
They also tested the cells’ responses to related substances and found that a broad spectrum of substances, including bourgeonal, activated the receptor.
After finding active odorants, Hans Hatt’s team wanted to see how their receptor functioned in live human sperm. They tested whether the identified ligands could induce a calcium response to odorant molecules in sperm by measuring cytosolic calcium levels in the cells. The sperm responded to bourgeonal and other active ligands but with a significantly higher sensitivity then when the receptor was expressed in the kidney cells.
They also confirmed chemotactile behavior of human sperm, by tracing sperm locomotion in microcapillaries loaded with different bourgeonal gradients. The results of these locomotion assays clearly demonstrated that the sperm were navigating up the gradient, towards the area of highest bourgeonal concentration.
In summary, the outlined experiments suggest that hOR17-4 may be a crucial component in the fertilization process as it may be directly involved in helping the sperm navigate towards the egg. Although the follicular factors that attract
mammalian sperm are not yet known, identification of a receptor that mediates sperm chemotaxis in vitro may pave the way to ascertain their molecular identity, which could help in the development of new therapies to help childless couples. It is also hoped that perturbation of hOR17-4-mediated navigation may lead to the development of new contraceptives with fewer adverse reactions than the current generation of contraceptives.
10.1242/jeb.00455
Spehr, M., Gisselmann, G., Poplawski, A., Riffell, J. A., Wetzel, C. H., Zimmer, R. K. and Hatt, H. (2003). Identification of a testicular odorant receptor mediating human sperm chemotaxis. Science 299, 2054-2058.
Hans Merzendorfer, University of Osnabrueck, merzendorfer@biologie.uni-osnabrueck.de
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EAT WELL TO LIVE LONG
The theory that trade-offs exist between longevity and performance is a pervasive paradigm in analyses of life-history evolution. However, in the case of the naturally occurring indy mutant Drosophila, lifespan has been doubled without decreases in fitness; these flies are both healthy and fertile during their entire lives. The name indy is short for mutations in a gene named I’m not dead yet, which encodes a protein that transports Krebs cycle intermediates needed forintermediary metabolism. The performance trade-offs in long-lived indy flies will only be present in the flies when food is scarce, as a reduction of INDY protein expression levels reduces the efficiency by which the insect utilises calories from food.
Marden and co-authors hypothesized that performance trade-offs in indy flies may be conditional and may depend on levels of environmental stress, including the quality and quantity of food available. To test this hypothesis, the authors measured mortality rates, metabolic rates, flight performance, fecundity and eclosion in two independent indy mutant strains of fly under normal conditions and when the flies’ caloric intake was restricted.
The team measured mortality rates for well-fed wild-type and mutant flies at 25°C and confirmed that all of the indy mutant flies had significantly lower mortality rates than the rates for normal-lifespan flies. Metabolic rates of flies were measured by CO2emissions of resting flies in
flow-through respirometry chambers. There was no indication of any significant difference between the metabolic rates of indy mutant and normal-lived flies, although the flies’ measured metabolic rates were affected by temperature, respirometry chamber, body mass and fly age. Marden and co-workers
also assessed the flies’ performances by measuring their flight velocities from three-dimensional video recording of flight paths and found no significant differences between flight velocities of the two indy mutant strains and the two normal-lived strains.
Having established that there appeared to be no trade-off between a variety of physiological traits and the mutant flies’ longevity, the team tested the insect’s reproductive fitness under normal circumstances and stressful conditions. Age-specific fecundity was determined by counting the daily egg production rate of individual females. Under high calorie conditions, indy mutant flies were significantly more fecund than normal-lived flies. However, when the indy mutant flies were reared on a calorie-restricted diet, their fecundity fell. This pattern held for eclosion as well; greater numbers of adults eclosed from indy mutant fly eggs than from normal life expectancy fly eggs under normal conditions, while fewer indy mutant eggs hatched under low-calorie conditions. Under stressful conditions, the mutant flies had traded off fertility in return for a greater life expectancy.
These results suggest that trade-offs between lifespan and performance in indy mutant flies exist, but that they are conditional and dependent on food availability. Although long-lived indy mutant strains will have an advantage over wild-type flies under normal circumstances, spatial and temporal variation in the quality and quantity of nutritional resources may be great enough to decrease the fitness of natural indy mutant flies, preventing them from swamping the wild-type population. Thus, the ability to eat well will have an impact on life-history evolution in this naturally occurring mutant population.
10.1242/jeb.00448
Marden, J. H., Rogina, B., Montooth, K. L. and Helfand, S. (2003). Conditional tradeoffs between aging and organismal performance of
indy long-lived mutant flies. Proc. Natl. Acad. Sci. USA 100, 3369-3373.
Jonathon H. Stillman Stanford University JStillman@stanford.edu
DON’T MIND THE GAP!
Unable to compromise on its intense energy consumption, most vertebrate brains go into energy failure within minutes of being deprived of oxygen, resulting in the loss of ionic gradients, depolarization and a cascade of pathological changes resulting eventually in neuronal death. A few vertebrate species, however, including freshwater turtles of the genera Trachemys and Chrysemys, have brains able to survive at least 48 h of anoxia at 25°C and up to 3–4 months during winter hibernation in frozen ponds. The animals are able to tolerate such long periods without oxygen by lowering energy metabolism to a bare minimum, where brain energy needs can be fully met by anaerobic glycolysis. As a result, the turtle brain is able to maintain ATP levels and ionic gradients during anoxia and thus avoid the fatal consequences of energy failure.Among other adaptations, one means by which this balance of decreased ATP production and energy utilization can be achieved is by a reduction in membrane ion permeability, termed channel arrest, which provides important energy savings for the anoxia-tolerant brain by reducing the costs of ion pumping to maintain homeostasis. Previous studies have indicated a significant and acute decrease in whole-cell conductance during anoxia, which is the result of decreased potassium flux, a decrease in the density of voltage-gated Na+channels and a downregulation
of NMDA receptor calcium channels; these reductions in ion leakage permit a
simultaneous decrease in Na+/K+-ATPase
activity to conserve ATP. But besides decreases in ion leakage currents, another potential way to save energy is by changing neuronal gap junction permeability, a possibility addressed by Shin and co-workers in their recent
LO
NGEVIT
Y
FISH MUSCLE IS STILL
HELICAL
Fish axial muscle is weird. It’s so weird that Sven Gemballa and Felix Vogel, in their December paper in Comparative
Biochemistry and Physiology, are the first to attempt to explain its three-dimensional structure since R. McNeill Alexander in 1969. Its complexity has long frustrated fish biologists because there’s been no direct way to link muscle properties to body bending. Alexander described a spiral pattern in muscle fibre orientation, which might allow all fibres to shorten the same amount. His study, however, was fairly theoretical and did not indicate how the muscle force would be transmitted to the skeleton. Gemballa and Vogel’s study, covering 26 species spanning the range of fish diversity, is the first to offer detailed evidence for a general bending mechanism in fishes.
Unlike in tetrapods, whose muscles tend to be separate units with fairly consistent fibre angles, fish white muscle is arranged down the body in nested W-shaped blocks, with the centre of the Wpointing forward. The blocks, called myomeres, are separated by collagenous sheets called myosepta. Between the myosepta, the muscle fibres themselves also have a complex arrangement.
Using techniques ranging from electron microscopy to manual microdissections, Gemballa and Vogel tracked muscle fibres from one myoseptum to the next. They then dissected out the myosepta and used polarized light microscopy to visualize regions of parallel collagen fibres that indicate force direction and how those forces might be transmitted to the skeleton. This process produces an integrated model of the bending mechanism.
Although Gemballa and Vogel found essentially the same arrangement that
Alexander described, with fibres spiralling down the body, they interpret it differently. Alexander hypothesized that the spiral allowed all muscle fibres to contract by about the same amount. Gemballa and Vogel instead divide it into two parts that wrap around each other like DNA’s double helix. The outside portion of the helices – one strand of the ‘DNA’ – which they term the helical muscle fibre arch, forms an arch from the body’s centre line out to the left or right side and back again. Passing under that arch is a central portion of the helix – the other ‘DNA’ strand – called the crossing muscle fibres. They hypothesize that the crossing muscle fibres, like a pulley, support the helical muscle arch when it bends the fish’s body.
Paradoxically, these arches are located anteriorly, but they cause bending mostly near the tail. In explanation, Gemballa and Vogel describe different fibre and tendon orientations near the tail that could help transmit bending forces posteriorly. The fibres lose their helical pattern and instead, like a pinnate muscle, attach to tendons running towards the tail to efficiently transfer force backwards.
Gemballa and Vogel’s most important contribution, however, may be their description of myoseptal tendons. These tendon-like thickenings in the myosepta may help muscle fibres from consecutive myomeres to act together as helices. By observing the tendon orientations, they estimate force directions in the myosepta. One tendon, the lateral band, approximately follows the helical muscle fibre arch, indicating that forces are indeed produced along that trajectory. This lateral band may also transfer force from the arch fibres towards the vertebral column during locomotion.
Because of the complex arrangement of myomeres, myosepta and muscle fibres, the mechanical linkage between muscle shortening and body bending in swimming fish has been very unclear. By relating muscle fibre and myoseptal tendon orientations, Gemballa and Vogel start to make the link between muscle structure and function in the biomechanics of fish swimming.
10.1242/jeb.00445
Gemballa, S. and Vogel, F. (2002). Spatial arrangement of white muscle fibers and myoseptal tendons in fishes. Comp. Biochem.
Physiol. A 133, 1013-1037.
Eric Tytell Harvard University tytell@oeb.harvard.edu
Comparative Biochemistry and Physiology A paper. Gap junctions are structural elements present in a variety of vertebrate and invertebrate tissues that provide a low-resistance, high-speed pathway between adjacent cells.
Les Buck and his group measured whole cell capacitance in normoxic and anoxic cortical sheets from the turtle Chrysemys picta under a variety of conditions known to affect gap junction permeability, including high calcium, hypo-osmotic shock, cold shock and exposure to a variety of neuroactive compounds including isoproterenol, a nitric oxide donor, and adenosine. To visually inspect whether gap junction permeability changed in the turtle brain, neurons were loaded with Lucifer yellow, a dye that shows gap junction coupling to adjacent cells.
Anoxia alone did not change cellular capacitance, nor did calcium or adenosine perfusion, although decreases in whole-cell conductance were observed under these conditions. In fact, perfusion with hypo-osmotic artificial cerebrospinal fluid was the only protocol consistently altering capacitance, and that was in the direction of an apparent decrease in gap junction permeability, resulting in reduced cell-to-cell communication. While it was
determined that gap junctions were present in the turtle cortex, they proved to be very difficult to open, leading the researchers to conclude that decreases in cellular conductance are due almost exclusively to decreases in leak channel permeability, rather than any changes in gap junctions, and that the possession of very low permeability gap junctions may be another identifying characteristic of a good vertebrate facultative anaerobe.
10.1242/jeb.00447
Shin, D. S., Ghai, H., Cain, S. W. and Buck, L. T. (2003). Gap junctions do not underlie changes in whole cell conductance in anoxic turtle brain. Comp. Biochem. Physiol. A 134, 181-194.
Sarah Milton Florida Atlantic University smilton@fau.edu
FROM MOLECULES TO
MORSE CODES
Noses, mouthparts and antennae are just some of the body parts used to detect smells in nature. What all these structures have in common is sensilla – hair-like structures that contain one to a few olfactory neurones, which respond to odours. Olfactory neurones sport a variety of receptor proteins on their cell surfaces, which bind to molecules in the
environment. When enough odour molecules have bound to the receptor proteins, the neurone fires, and sends a signal that is interpreted by the animal as an odour. Olfactory sensilla, the neurones within them, and the protein receptors on the neurones’ surfaces represent three levels of odour coding. Each level can be grouped into a variety of classes depending on morphology and function.
Unfortunately, the relationship between these three levels is complicated: it was previously thought that a single class of receptor proteins does not fall into a single class of olfactory neurones, and a single class of olfactory neurones does not fall into a single class of olfactory sensilla. In other words, the coding relationship was thought to be more complicated than a simple 1:1:1 ratio. That is until Dobritsa and colleagues proved everyone wrong.
In a paper published in Neuron, Dobritsa and co-workers have combined anatomical, electrophysiological and genetic techniques in a study of fruit fly antennae, to
challenge all three levels of the troublesome coding cascade. First, the team decided to map the location of two receptor proteins. They designed antibodies to the Or22a and Or22b receptor proteins, which they took as two examples of the full range of possible receptors to label cells that carried the receptor.
Electrophysiological techniques were then
used on the labelled cells to measure their sensitivities to a range of odours. By looking at the distribution of labelled cells, the team found that both receptors were restricted to a single morphological class of sensilla (LB-1) and a single functional class of sensilla (ab3).
Next, Dobritsa and colleagues found that the two receptor proteins were restricted to a single olfactory neurone type when they combined electrophysiological recording techniques with gene mutation studies. The team showed that when Or22a and Or22b are not expressed, the response of one type of olfactory neurone is affected (ab3A). Usually the ab3A neurone responds to a broad range of different odours and is most sensitive to ethyl butyrate. Interestingly, when Or22a was knocked-out, all ab3A activity disappeared. This means that the Or22a receptor is the only active receptor on the ab3A neurone and is fully responsible for its whole spectrum of odour-response properties. Because ab3A can respond to a range of odours, the presence of many different receptor protein types was expected. Therefore, finding that ab3A, a single neurone class, uses only one receptor protein (Or22a) was a surprising result. By combining this discovery with the fact that the ab3A neurone is restricted to one class of sensilla, Dobrista et al. propose a novel 1:1:1 ratio to describe the relationship between three coding levels; receptor to neurone to sensillum. Further studies of other olfactory receptor genes will be required to determine whether this is a general principle.
10.1242/jeb.00446
Dobritsa, A. A., van der Goes van Naters, W., Warr, C. G., Steinbrecht, R. A. and Carlson, J. R. (2003). Integrating the molecular and cellular basis of odor coding in the Drosophila antenna. Neuron 37, 827-841.
Keri Page University of Cambridge kp231@cam.ac.uk
PERIPHERAL PRECISION
FOR DISCRIMINATING
FEMALES
Finding and deciding upon a potential mate is a difficult decision for many female animals. Females are justifiably cautious – the wrong choice could cost them a whole year of successful breeding. In many animals, including insects, females are attracted to a mate by his song. Male grasshoppers (Chorthippus sp.) advertise to females using a species-specific song, where each male’s song is a variation upon the species-specific theme. These songs potentially signal male quality and so it may be important for female grasshoppers to discriminate between males to ensure they choose the most suitable mate.
Machens and colleagues wanted to find out if the female grasshopper auditory system can discriminate between fine differences in male songs. To discriminate between males, a female’s auditory system must be able to encode the differences between male songs. Differences between male songs could be present in single auditory nerve fibres (sensory neurones) or in the combined activity of many auditory fibres. To test this, they played songs from different males to female grasshoppers whilst recording the responses of single auditory fibres. They asked whether the responses of single auditory fibres are sufficient to allow discrimination between males.
The team found that male songs generated precise spike trains in female auditory nerve fibres, so they decided to test whether these spike trains were sufficient to allow other neurones (auditory interneurones) in the female nervous system to discriminate between male songs. When a spike is transmitted from an auditory nerve fibre to an interneurone
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ACTION
DISCRIMINA
reliable discrimination between male songs, whereas those with intermediate durations did. Discrimination was also improved as the length of the songs increased.
From these experiments, Machens and colleagues concluded that there is sufficient information within single auditory fibres to allow females to distinguish reliably between males, even on the basis of the fine structure of their songs. This is surprising since it might be expected that spike trains from many auditory fibres (there are ~50 receptors per ear) would have to be combined to enable females to distinguish between male songs.
Although Machens and colleagues show that females could potentially discriminate between male songs based on spike trains from single auditory fibres, this relies
upon auditory interneurone properties. However, real auditory interneurones may have quite different properties from the model interneurones and clearly need to be identified. Future studies are sure to provide more fascinating insights into mechanisms underlying discrimination and mate choice found not only in
grasshoppers but also in frogs, birds and mammals.
10.1242/jeb.00449
Machens, C. K., Schütze, H., Franz, A., Kolesnikova, O., Stemmler, M. B., Ronacher, B. and Herz, A. V. M. (2003). Single auditory neurons rapidly discriminate conspecific communication signals. Nat. Neurosci. 6, 341-342.
Jeremy E. Niven University of Cambridge jen22@hermes.cam.ac.uk
via a synapse, it is converted to a graded change in the membrane potential of the interneurone (a graded synaptic input). Knowing that interneurones are thought to perform the discrimination between songs, Machens and colleagues suspected that the inputs to these neurones might be a better measure of a female’s ability to discriminate between males. Since they didn’t know exactly which interneurones were responsible for discrimination, they designed a mathematical model to convert the auditory fibre spike trains into synaptic inputs. They suspected that the duration of each synaptic input would affect the resolution of the interneurone responses and, therefore, whether a female could distinguish between two very similar songs, so they varied input duration in their model to determine which was best for discrimination. Long or short input durations did not allow
