AS meeting / San Sebastian

Transcription

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2 AS meeting / San Sebastian PLASTICITY OF FLAGELLA SHAPE DURING FISH SPERM MOTILITY Jacky Cosson and Galina Prokopchuk University of South Bohemia in České Budějovice, Faculty of Fisheries and Protection of Waters, Research Institute of Fish Culture and Hydrobiology, Vodnany, Czech Republic

3 Plasticity of flagella shape To day s lecture intends to present a few examples of adaptative ability of flagella when fish spermatozoa are submitted to a large variety of physical constraints imposed by the surrounding water during their short life span (Ref below). Example in turbot sperm recorded from 20 sec to 1 min after activation Ref: Regulation of axonemal wave parameters of fish spermatozoa by ionic factors. (1999) J. Cosson, C. Dreanno, R. Billard, M. Suquet and C. Cibert. Book chapter in The Male Gamete: from Basic Knowledge to Clinical Applications. C. Gagnon Ed., Cache River Press pp

4 Material and methods Most of our observations of fish sperm flagella in movement make use of video records by optical microscopy at high magnification (40 or 100X lenses) and high image rate (1000 images/sec), both obtained by highspeed video techniques or stroboscopic illumination (Cosson, 2008). For observation of the different physical agents described below, we have used: 1- ionic or sugar solutions to control osmolality combined with DMSO (dimethylsufoxide) as example, 2- glass slide, cover slip, interface air/swimming solution or egg surface allowed to observe and quantify the variety of effects on flagella swimming parameters, 3- viscosity was adjusted by various concentrations of methylcellulose, 4- in case of burbot (Lota lota), sperm movement was recorded using a temperature controlled microscope, 5- gas effect was observed following application of a gentle stream of CO 2 using a micro capillary. Here are some examples (non-exhaustive list) of fish species that we investigated: carp (Cyprinus carpio), trout (Oncorhynchus mykiss), sturgeon (Acipenser ruthenus), turbot (Scophthalmus maximus).

5 In fish spermatozoa, motility activation occurs at spawning In fish species with external fertilization, immotile spermatozoa are delivered at spawning into the surrounding medium: dilution of spermatozoa and their accompanying seminal fluid immediatly activates motility of their flagella (Ref below). xample in sea-bass sperm Ref: Morisawa M Initiation mechanism of sperm motility at spawning in teleost Zool. Sci

6 After initiation, motility lasts for a short period Because they are submitted to various interactions with surrounding medium, motility lasts for short period, ranging minutes or tens of seconds (Ref. below) Sea bass sperm from 4 to 40 sec postactivation >>>>>>> Ref : Cosson, J. (2008b). The motility apparatus of fish spermatozoa. In Fish Spermatology (Alavi, S. M. H., Cosson, J. J., Coward, K. & Rafiee, G., eds) pp Alpha Science Oxford (UK).

7 Activation signals The most ubiquitous signal for fish spermatozoa is the osmotic signal (Refs below). Osmolality signal may combine with other signals such as the concentration of K + in case of trout/salmonids (Ref 1, 2 & 3) and sturgeon/chondrosteans (Ref 1). Ref 1 : Alavi, S. M. H. & Cosson, J. (2006). Sperm motility in fishes: (II) Effects of ions and osmotic pressure: a review. Cell Biology International 30, Ref 2 : Morisawa M & Suzuki K 1980 Osmolality and potassium ion: their role in initiation of sperm motility Science

8 Osmotic environment as signaling constraint As emphasized above, the main signal activating fish sperm motility is osmotic. At the same time, extreme osmotic choc received by sperm cells leads to serious damage to membranes and ends up with arrest of motility: flagella shape is highly affected by such stress which consists blebs and in hyper-curling of the distal flagellum (ref below). Ref: Morphological and kinetic changes of carp (Cyprinus carpio) spermatozoa after initiation of motility in distilled water. (1996) Perchec G., Cosson M-P., Cosson J., Jeulin C. and Billard R. Cell Mot. & the Cytosk. 35: (22).

9 Tail curling limits the motility period Because sperm cells react to external osmolality, a loop appears in the distal flagellum, which prevents waves propagation. This looping process is reversible. (ref below) Example in carp (SEM) ===>>>> a & b = 5s, c = 15s, d = 45s, e = 60s Ref: Morphological and kinetic changes of carp (Cyprinus carpio) spermatozoa after initiation of motility in distilled water. (1996) Perchec G., Cosson M-P., Cosson J., Jeulin C. and Billard R. Cell Mot.& the Cytosk. 35:

10 Osmotic environment as signaling constraint Reaction of flagellum with respect to osmotic signal has been quite well documented regarding its biochemical aspects but this activation step is very rapid and in any case occurring during the period when sperm cells are dispersed in a swimming medium. It is only recently that, by use of osmotic agents such as DMSO as example, we could induce a delay of several seconds after which the first and next waves could be observed at the head-tail junction (ref below). Video showing activation delay due to DMSO. Ref: Motility initiation in fish spermatozoa: description of the propagation of very first initial waves. Gala Prokopchuk, Boris Dzuba, Sergey Boryspolet, Otomar Linhart and Jacky Cosson (submitted).

11 Swimming in the vicinity of surfaces A general property of minute swimming cells such as spermatozoa, but also bacteria, is that while reaching vicinity to close-by surfaces, they remain swimming as "trapped" very close to this surface. We observed that detailed waves properties in the vicinity of surfaces are highly dependent on the suppleness on surfaces. In addition, such accumulation of swimming spermatozoa is clearly of biological importance in situation where sperm approach the egg surface. Our recent investigation of this question uses simulation/modelisation approach as a complement to experimental analysis of flagella when sperm swim on the surface of an egg. (refs below) Videos: vicinity of air/water interface (top) or glass slide vicinity (bottom) Ref 1: Different swimming behaviours of sterlet (Acipenser ruthenus) spermatozoa close to solid and free surfaces. Boryshpolets, S., Cosson, J., Bondarenko, V., Gillies, E., Rodina, M., Dzyuba, B. and Linhart O. Theriogenology (2013) 79(1): Ref 2: Fins improve the swimming performance of fish sperm: a hydrodynamic analysis of the Siberian sturgeon Acipenser baerii. Gillies E.A., Bondarenko V., Cosson J. & Pacey A. A. (2013) Cytoskeleton 70(2):

12 Viscosity constraints This property of any fluid is crucial to allow a spermatozoon to progress forwardly while it develops waves moving towards flagella tip and thus pushes efficiently on the surrounding medium (even in plain water at minimal viscosity) but is of biological importance in situation where sperm cells are swimming in the follicular or ovarian fluid surrounding eggs. Increase of viscosity by introduction of viscous long polymers such as methyl-cellulose in the swimming medium drastically changes waves properties in many respects (ref below) videos showing viscosity effect of Methyl Cellulose => Ref: Regulation of axonemal wave parameters of fish spermatozoa by ionic factors. (1999) J. Cosson, C. Dreanno, R. Billard, M. Suquet and C. Cibert. Book chapter in The Male Gamete: from Basic Knowledge to Clinical Applications. C. Gagnon Ed., Cache

13 Temperature effects This physical parameter is important, especially when considering fish species that reproduce at low temperature such as, for example, trout (4-8 C) or even lower such as burbot (Lota lota, 4 C). (ref below) Burbot sperm in seminal fluid at increasing temperature (real time) Ref: Sperm motility and seminal fluid composition in the burbot, Lota lota. Lahnsteiner et al. J. Appl. Ichthyol. 13 (1997)

14 Temperature effects This physical parameter is important, especially when considering fish species that reproduce at low temperature such as, for example, trout (4-8 C) or even lower such as burbot (Lota lota, 4 C). (ref below) Burbot sperm in seminal fluid at increasing temperature (real time)

15 Temperature effects Thermodynamic aspects in relation to physiological constraints are of special interest when mechano-chemical properties of flagella are affected differentially by temperature. (ref below) Full activation of waves in < 50 ms Control sperm after activation (20X slower than actual)

16 Effect of contact with gas (CO 2 ) In some fish species (flatfishes) such as turbot, we could show that, CO 2 as a gas, can block transiently flagella waves propagation but that such effect is fully reversible when CO 2 is removed. In the latter case, we could observe initiation of motility in terms of appearance of first bending waves in case of marine fish species where the use of DMSO cannot be applied as above to generate such opportunity to observe and record motility initiation sequence. (ref below) video of turbot sperm with CO 2 effect >>>> Ref: Control of flatfish sperm motility by CO 2 and carbonic anhydrase. Inaba K., Dreanno C. and Cosson J. (2003) Cell Mot. & Cytosk. 55:

17 Waves dampening In addition to various physical factors, a damping effect appears as a function of the time elapsed during the motility period, which consists in a fading of waves amplitude relative to the distance from head along the flagellum. Exhaustion of ATP by the flagella motors (dynein-atpase) could partly explain such effects. wave damping versus time in the motility period Ref: The ionic and osmotic factors controlling motility of fish spermatozoa. (2004) Jacky COSSON, Aquaculture International 12:69-85.

18 Conclusions Our aim is to describe quantitatively how various physical properties of the surrounding medium encountered by fish spermatozoa are able to inflict flagella waves in their form and efficiency. It is clear that very different physical and biochemical factors affect flagella wave shape, velocity, amplitude, beat frequency, etc, in a different fashion: all factors contribute to the efficiency of progressive swimming, which is globally infered into the classical parameter called velocity as CASA systems generate.

19 Many thanks to : Organizers of the present meeting To all colleagues in our Institute, To Volodymyr and Galina for video records obtained by high magnification microscopy using the high-speed video camera recording facility. To all colleagues all over the world with whom I enjoy to collaborate

20 Thank for your attention and for your interest to fish sperm flagella motility 20

21 Videos on U-tube Sturgeon video on U tube : Salmon sperm in River Water on U tube : Trout sperm in a Swimming Solution on U tube: Whitefish sperm swimming in pond water on U tube:

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