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Studying biology of sperm provides valuable information to optimize artificial reproduction and is crucial for sustainable aquaculture. Here, we investigated morphology of spermatozoon in Atlantic cod (Gadus morhua) using transmission and scanning electron microscopy. Furthermore, spermatozoa motility kinetics at different osmolalities were studied using computer-assisted sperm analysis software. The spermatozoon lacked an acrosome and consisted of a head, midpiece, and flagellum. The head of spermatozoa was round, oval, and rather elongated in shape, showing high variations in dimensions. There were up to 6 mitochondria that encircled the proximal part of the flagellum. The proximal and distal centrioles were located within the nuclear notch and arranged orthogonal to each other. The axoneme had a typical 9 + 2 microtubule structure. The flagellar length of spermatozoon was 66.94 ± 0.46 μm. Spermatozoa were immotile in the seminal plasma. Dilution of sperm with natural seawater (1100 mOsmol/kg) resulted in initiation of motility for 91.0 ± 3.4% of spermatozoa with average velocity of 86.2 ± 2.3 μm/s and beating frequency of 52 Hz. The duration of spermatozoa motility was > 6 min; however, the percentage of motile spermatozoa decreased at 60 s post-activation. When osmolality of natural seawater was modified using distilled water or NaCl, spermatozoa motility was not initiated at ≤ 400 and ≥ 2500 mOsmol/kg, and the highest percentage of motility was observed at 730-1580 mOsmol/kg. In a sucrose solution, spermatozoa motility was initiated and suppressed at 600 and 1500 mOsmol/kg, respectively, and highest percentage of motility was observed at 800-1100 mOsmol/kg. Spermatozoon morphology comparisons within Gadiformes showed differences in dimensions of head and mitochondria, flagellar length, and number of mitochondria. The present study provides valuable data that can be used for phylogenetic implications based on spermatozoon morphology and for development of artificial fertilization and sperm cryopreservation protocols based on sperm motility.

Though bivalve mollusks are keystone species and major species groups in aquaculture production worldwide, gamete biology is still largely unknown. This review aims to provide a synthesis of current knowledge in the field of sperm biology, including spermatozoa motility, flagellar beating, and energy metabolism; and to illustrate cellular signaling controlling spermatozoa motility initiation in bivalves. Serotonin (5-HT) induces hyper-motility in spermatozoa via a 5-HT receptor, suggesting a serotoninergic system in the male reproductive tract that might regulate sperm physiology. Acidic pH and high concentration of K + are inhibitory factors of spermatozoa motility in the testis. Motility is initiated at spawning by a Na + -dependent alkalization of intracellular pH mediated by a Na + /H + exchanger. Increase of 5-HT in the testis and decrease of extracellular K + when sperm is released in seawater induce hyperpolarization of spermatozoa membrane potential mediated by K + efflux and associated with an increase in intracellular Ca 2+ via opening of voltage-dependent Ca 2+ channels under alkaline conditions. These events activate dynein ATPases and Ca 2+ /calmodulin-dependent proteins resulting in flagellar beating. It may be possible that 5-HT is also involved in intracellular cAMP rise controlling cAMP-dependent protein kinase phosphorylation in the flagellum. Once motility is triggered, flagellum beats in asymmetric wave pattern leading to circular trajectories of spermatozoa. Three different flagellar wave characteristics are reported, including "full", "twitching", and "declining" propagation of wave, which are described and illustrated in the present review. Mitochondrial respiration, ATP content, and metabolic pathways producing ATP in bivalve spermatozoa are discussed. Energy metabolism of Pacific oyster spermatozoa differs from previously studied marine species since oxidative phosphorylation synthetizes a stable level of ATP throughout 24-h motility period and the end of movement is not explained by a low intracellular ATP content, revealing different strategy to improve oocyte fertilization success. Finally, our review highlights physiological mechanisms that require further researches and points out some advantages of bivalve spermatozoa to extend knowledge on mechanisms of motility., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Fish spermatozoa acquire potential for motility in the sperm duct where they are immotile. Osmolality of the seminal plasma is a key factor to maintain spermatozoa in the quiescent state in either freshwater or marine fishes. However, potassium (K + ) ions prevent spermatozoa motility in salmonid and sturgeon fishes, while CO 2 inhibits spermatozoa motility in flatfishes. Once, spermatozoa are released at spawning, their motility is initiated in hypo-osmotic and hyper-osmotic environments in freshwater and marine fishes, respectively. Some substances produced by the testes (a progestin), or released from oocytes (peptides) induce spermatozoa hypermotility in some marine fishes including the Atlantic croaker and Pacific herrings, respectively. Duration of spermatozoa motility is short, lasting for a few seconds to few minutes in most fishes due to rapid depletion of energy required for the beating of the motility apparatus called axoneme. In the osmotic-activated spermatozoa, K + and water effluxes occur in freshwater and marine fishes, respectively, which trigger spermatozoa motility signaling. In general, initiation of axonemal beating is associated with an increase in intracellular calcium (Ca 2+ ) ions in spermatozoa of both freshwater and marine fishes and a post- or pre-increase in intracellular pH, while cyclic adenosine monophosphate (cAMP) remains unchanged. However, axonemal beating is cAMP-dependent in demembranated spermatozoa of salmonid and sturgeon fishes. Calcium from extracellular environment or intracellular stores supply required Ca 2+ concentration for axonemal beating. Several axonemal proteins have been so far identified in fishes that are activated by Ca 2+ and cAMP, directly or mediated by protein kinase C and protein kinase A, respectively. The present study reviews differences and similarities in complex regulatory signals controlling spermatozoa motility initiation in fishes, and notes physiological mechanisms that await elucidation., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Motility is a characteristic function of the male gamete, which allows spermatozoa to actively reach and penetrate the female gamete in organisms with internal and external fertilization. Sperm motility is acquired under the control of many extrinsic and intrinsic factors and is based on a specialized structure of the sperm flagellum called "axoneme". An overview of how the sperm flagellum is organized, and it operates to support cell motility is presented, with special focus on the molecular mechanisms and factors involved in the development, maintenance and control of motility. Data obtained in aquatic organisms with external fertilization, such as sea urchins, ascidians or fishes are critically analyzed because they constitute model species on which most of the present day understanding of sperm motility function is based. In most animal species, sperm motility is dependent on a long appendage called flagellum. Flagella are essential organelles found in most eukaryotic cells; their basic structure is the axoneme, which consists of a scaffold of microtubules and is responsible for movement in an autonomous manner if ATP-energy is present. Flagellar beat propels the cell through the medium which surrounds sperm cells and is responsible of the translational drive of spermatozoa. The present paper includes: (1) an introduction to typical sperm morphology and ultrastructure in most aquatic species, (2) the motility apparatus or axoneme of the spermatozoa: the axoneme, (3) the structural and biochemical composition of the axoneme, (4) the axonemal motor or dynein, and its operation, (5) the regulation of motility at axoneme and cell membrane levels, including several effectors such as Ca 2+ ions, (6) biophysical features of the wave propagation mechanism in motile spermatozoa, (7) the energy production and consumption, and (8) the building of a flagellum. Flagellar beating in aquatic animals is illustrated using several examples in figures and video-clips. These types of data are also used for computer simulation of various aspects of the modulation of sperm motility of marine animals., (Copyright © 2019 Elsevier Inc. All rights reserved.)

All extant groups of Elasmobranches have internal fertilization and the structure of the male reproductive organs is very specific: sperm passes from the internal organs via the cloaca, but the male copulating organ (clasper) is distant from the cloaca. This suggests that sperm can contact the surrounding medium before fertilization. Because of this involvement with the environment, external signaling in sperm motility activation could occur in these species even though their fertilization mode is internal. In this case, spermatozoa of Elasmobranches should hypothetically possess a specific structure and membrane lipid composition which supports physiological functions of the sperm associated with environmental tonicity changes occurring at fertilization. Additionally, sperm motility properties in these taxa are poorly understood. The current study examined sperm lipid composition and motility under different environmental conditions for the ocellate river stingray, Potamotrygon motoro, an endemic South America freshwater species. Sperm samples were collected from six mature males during the natural spawning period. Sperm motility was examined in seminal fluid and fresh water by light video microscopy. Helical flagellar motion was observed in seminal fluid and resulted in spermatozoon progression; however, when diluted in fresh water, spermatozoa were immotile and had compromised structure. Lipid class and fatty acid (FA) composition of spermatozoa was analyzed by thin layer and gas chromatography. Spermatozoa FAs consisted of 33 ± 1% saturated FAs, 28 ± 1% monounsaturated FAs (MUFAs), and 41 ± 1% polyunsaturated FAs (PUFAs), and a high content of n-6 FAs (32 ± 2%) was measured. These results allowed us to conclude that sperm transfer from P. motoro male into female should occur without coming into contact with the hypotonic environment so as to preserve potent motility. In addition, this unusual reproductive strategy is associated with specific spermatozoa structure and lipid composition. Low level of docosahexaenoic acid and relatively low PUFA/MUFA ratio probably account for the relatively low fluidity of freshwater stingray membrane and can be the main reason for its low tolerance to hypotonicity., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Results of previous studies with different fish species, mostly from temperate- or cold-water habitats, indicate a species-specific diversity regarding the relationship between environmental temperature and values for sperm motility variables. In the current study, there was appraisal of environmental temperature effects on sperm motility of tilapia Oreochromis niloticus, a tropical fish species selected because of its aquaculture importance and capacity to reproduce in a broad range of water temperatures. Effects of environmental temperature on the spermatozoa motility characteristics were studied by temperature-controlled video-microscopy and CASA analysis at temperature range from 5 to 50 °C. It appeared that the Nile tilapia spermatozoa exhibit an unexpected capacity to express very different velocity characteristics over this temperature range. In the lower temperature range (5-10 °C), the percentage of motile cells was markedly variable among males. An abrupt increase in the linearity index was observed between 15 and 20 °C suggesting a physiological threshold in sperm movement at about 20 °C which is the minimum temperature for reproduction in the Nile tilapia. With faster spermatozoa velocity, there was a reduction of the motility duration at the greater temperatures. Initially, there is an increase in sperm velocity as the temperature increased until the maximal velocity occurred at 40 to 50 °C which is a temperature beyond that which occurs in natural spawning conditions. Results of the present study clearly indicate the importance of considering ambient temperature when charactering sperm motility and in determining optimal temperature conditions for fertilization in fish., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Motility analysis of spermatozoa relies on the investigation of either head trajectories or flagellum characteristics. Those two sets of parameters are far from being independent, the flagellum playing the role of motor, whereas the head plays a passive role of cargo. Therefore, quantitative descriptions of head trajectories represent a simplification of the complex pattern of whole sperm cell motion, resulting from the waves developed by the flagellum. The flagellum itself responds to a large variety of signals that precisely control its axoneme to allow activation, acceleration, slowing down or reorientation of the whole spermatozoon. Thus, it is obvious that analysis of flagellum characteristics provides information on the original source of movement and orientation of the sperm cell and presents additional parameters that enrich the panoply of quantitative descriptors of sperm motility. In this review, we briefly describe the methodologies used to obtain good-quality images of fish spermatozoa (head and especially flagellum) while they move fast and the methods developed for their analysis. The paper also aims to establish a link between classical analyses by computer-aided sperm analysis (CASA) and the descriptors generated by fish sperm flagellum analysis, and emphasises the information to be gained regarding motility performance from flagellum motion data.

Spermatozoa of externally fertilizing freshwater fish possess several different modes of motility activation. Spermatozoa of common carp ( Cyprinus carpio L.) are activated by hypoosmolality, whereas spermatozoa of sterlet ( Acipenser ruthenus ) require Ca2+ and low concentration of K+ for motility activation. Intracellular signaling differs between these two species as well, particularly in terms of utilization of secondary messengers (cAMP and Ca2+), and kinase activities. The current study was performed in order to determine the importance of protein phosphorylation and protein kinases for activation of sperm motility in carp and sterlet. Treatment with kinase inhibitors indicates that protein kinases A and C (PKA and PKC) participate in spermatozoa motility of both species. Immunodetection of phospho-(Ser/Thr) PKA substrates shows that phosphorylated proteins are localized differently in spermatozoa of carp and sterlet. Strong phosphorylation of PKC substrate was observed in flagella of sterlet spermatozoa, whereas in carp sperm, PKC substrates were lightly phosphorylated in the midpiece and flagella. Motility activation induced either phosphorylation or dephosphorylation on serine, threonine and tyrosine residues of numerous proteins in carp and sterlet spermatozoa. Proteomic methods were used to identify proteins whose phosphorylation state changes upon the initiation of sperm motility. Numerous mitochondrial and glycolytic enzymes were identified in spermatozoa of both species, as well as axonemal proteins, heat shock proteins, septins and calcium-binding proteins. Our results contribute to an understanding of the roles of signaling molecules, protein kinases and protein phosphorylation in motility activation and regulation of two valuable fish species, C. carpio and A. ruthenus ., (© 2017 Society for Reproduction and Fertility.)

Alternative reproductive tactics (ARTs) are prevalent in nature, where smaller parasitic males typically have better sperm quality than larger territorial guard males. At present, it is unclear what is causing this phenomenon. Our objective was to gain insights into sperm form and function by examining flagellar beating patterns (beat frequency, wave amplitude, bend length, bend angle, wave velocity) and biomechanical sperm metrics (velocity, hydrodynamic power output, propulsive efficiency) of wild spawning Chinook salmon ARTs. Ovarian fluid and milt were collected to form a series of eight experimental blocks, each composed of ovarian fluid from a unique female and sperm from a unique pair of parasitic jack and guard hooknose males. Sperm from each ART were activated in river water and ovarian fluid. Flagellar parameters were evaluated from recordings using high-speed video microscopy and biomechanical metrics were quantified. We show that ART has an impact on flagellar beating, where jacks had a higher bend length and bend angle than hooknoses. Activation media also impacted the pattern of flagellar parameters, such that beat frequency, wave velocity and bend angle declined, while wave amplitude of flagella increased when ovarian fluid was incorporated into activation media. Furthermore, we found that sperm from jacks swam faster than those from hooknoses and required less hydrodynamic power output to propel themselves in river water and ovarian fluid. Jack sperm were also more efficient at swimming than hooknose sperm, and propulsive efficiency increased when cells were activated in ovarian fluid. The results demonstrate that sperm biomechanics may be driving divergence in competitive reproductive success between ARTs., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

In fish, sperm quality is frequently associated with sperm motility variables. The response of sperm motility to different temperatures varies among species and plasma membrane lipid composition may contribute to variations in findings in previous research. In the present study, sperm motility and lipid composition were analysed between motile or immotile carp Cyprinus carpio sperm at different in vitro temperatures (4, 14 and 24°C). The duration of the period over which sperm motility is sustained was longer at 4°C compared with 14 and 24°C; while sperm velocity was greatest at 24°C. Motile sperm had lesser proportions of 18:3 (n-3) and 22:6 (n-3) fatty acids at 24°C relative to immotile sperm. There was no difference in fatty acid composition of motile and immotile sperm at 4 and 14°C. The total phospholipid content was less in motile than in immotile sperm at 24°C. At 24°C, phosphatidylcholine and phosphatidylserine proportions were less in motile than immotile sperm. It is concluded that lipid composition of motile carp sperm is affected by temperature, with greater temperatures associated with reduced lipid content, elevation of sperm curvilinear velocity and a decreased duration of the period over which motility is sustained., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The present study examined the lipid composition of plasma membranes in carp sperm with different post-thaw motility. The approach adapted for carp sperm cryopreservation, which involves the selection of the most effective protocol for individual males by comparing two cryoprotective media, was applied to the cryopreservation procedure. Sperm motility prior to freezing was greater than 80% but decreased to 40% in one group and to 10% in another group following cryopreservation. Lipid content of fresh sperm in all groups was analysed by thin layer chromatography and gas chromatography, with significant differences in phospholipid content, cholesterol and free fatty acids detected between groups, whereas the cholesterol/phospholipid ratio was extremely similar between groups (0.52 ± 0.038 and 0.52 ± 0.022). Increasing concentrations of saturated fatty acids, monounsaturated acids and decreasing concentrations of polyunsaturated n-6 fatty acids were negatively correlated (P < 0.05) with post-thaw motility of the carp sperm., (Copyright © 2016 Elsevier Inc. All rights reserved.)

In most teleost fishes, sperm cells are quiescent in the seminal plasma and are activated by either a drop (fresh water fish) or an increase in osmolality (marine fish) when released in the water. It is most interesting to examine how the mechanisms of sperm motility activation can adapt to a broad range of salinities, as applies to some euryhaline species, and particularly to the tilapia Sarotherodon melanotheron heudelotii, which can reproduce at salinities from 0 up to 120 in the wild. Here, the gonado-somatic index, semen characteristics, and the osmotic and ionic requirements of sperm motility activation were compared in S. m. heudelotii reared in fresh water (FW), sea water (SW), or hypersaline water (HW; salinities of 0, 35, and 70, respectively). No salinity-dependent differences were found in gonado-somatic index or semen characteristics, except for an increase of seminal plasma osmolality with increasing salinity (from 318 to 349 mOsm kg(-1) in FW and HW fish, respectively). The osmolality range allowing the highest percentages of sperm activation broadened and shifted toward higher values with increasing fish ambient salinity (150-300, 300-800, and 500-1200 mOsm kg(-1), for FW, SW, and HW fish, respectively). Nevertheless, at the three fish rearing salinities, sperm could be activated in media that were hypotonic, isotonic, or hypertonic relative to the seminal plasma, at least when some calcium was present above a threshold concentration. The [Ca(2+)] required for the activation of S. m. heudelotii sperm is (1) higher in fish reared at a higher salinity (2) higher in hypertonic than that in hypotonic activation media, whatever the fish rearing salinity, and (3) higher in the presence of Na(+) or K(+), the negative effects of which increased with an increase in fish rearing salinity. The [Ca(2+)]/[Na(+)] ​ ratios allowing for maximal sperm motility in SW or HW fish are close to those observed in natural environments, either in sea or hypersaline waters. In comparison to most teleosts with external fertilization, the total duration of sperm motility in S. m. heudelotii was exceptionally long (>2 hours regardless the fish rearing salinities). The decrease in sperm activity with increasing time since activation did not result from limiting energy reserves, as the addition of calcium in the activation medium caused most spermatozoa to become motile again. The comparison of sperm characteristics of S. m. heudelotii acclimated from FW to SW or HW with those of fish maintained all lifelong at their native salinity showed that adaptive responses were completed within 2 months or less., (Copyright © 2016 Elsevier Inc. All rights reserved.)

As spermatozoon motility duration differs significantly among fish species, the mechanism of ATP generation-regeneration and its distribution along the flagellum may be species-dependent. The present study compared the role of creatine kinase (CK) with that of adenylate kinase (AK) in ATP regeneration during motility of demembranated spermatozoa of taxonomically distant fish species, sterlet, and common carp, allowing investigation for the presence of the creatine-phosphocreatine (PCr) shuttle in sterlet spermatozoa. The flagellar beat frequency of demembranated spermatozoa was measured in reactivating media in the presence or absence of ATP, ADP, PCr, and CK and AK inhibitors. After demembranation, AK, CK, and total ATPase activity was measured in spermatozoon extracts. Beat frequency of demembranated spermatozoa was found to be positively correlated with ATP levels in reactivating medium and to reach a plateau at 0.8 mM and 0.6 mM ATP for carp and sterlet, respectively. It was shown for the first time that sterlet axonemal dynein ATPases have a higher affinity for ATP than do those of carp. Supplementation of reactivating medium with ADP and PCr without ATP resulted in beat frequencies comparable to that measured with 0.3 to 0.5-mM ATP for both studied species. The presence of the PCr-CK phosphagen system and its essential role in ATP regeneration were first confirmed for sturgeon spermatozoa. The inhibition of CK exerted a high impact on spermatozoon energy supply in both species, whereas the inhibition of AK was more pronounced in sterlet than in carp. This was confirmed by the quantification of enzyme activity in spermatozoon extracts. We concluded that spermatozoa of these taxonomically distant species use similar systems to supply energy for flagella motility, but with different efficacy., (Copyright © 2016 Elsevier Inc. All rights reserved.)

For teleost fish fertilisation, sperm must proceed through a small opening on the egg surface, referred to as the micropyle. In this paper, we have used boundary element simulations to explore whether the hydrodynamic attraction between sperm and a fish egg can be a sperm guidance cue. Hydrodynamical egg-sperm interactions alone do not increase the chances of an egg encounter, nor do they induce surface swimming for virtual turbot fish sperm across smooth spheres with a diameter of 1mm, which is representative of a turbot fish egg. When a repulsive surface force between the virtual turbot sperm and the egg is introduced, as motivated by surface charge and van-der-Waals interactions for instance, we find that extended surface swimming of the virtual sperm across a model turbot egg occurs, but ultimately the sperm escapes from the egg. This is due to the small exit angle of the scattering associated with the initial sperm-egg interaction at the egg surface, leading to a weak drift away from the egg, in combination with a weak hydrodynamical attraction between both gametes, though the latter is not sufficient to prevent eventual escape. The resulting transience is not observed experimentally but is a detailed quantitative difference between theory and observation in that stable surface swimming is predicted for eggs with radii larger than about 1.8mm. Regardless, the extended sperm swimming trajectory across the egg constitutes a two-dimensional search for the micropyle and thus the egg is consistently predicted to provide a guidance cue for sperm once they are sufficiently close. In addition, the observation that the virtual turbot sperm swims stably next to a flat plane given repulsive surface interactions, but does not swim stably adjacent to a turbot-sized egg, which is extremely large by sperm-lengthscales, also highlights that the stability of sperm swimming near a boundary is very sensitive to geometry., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Spermatozoa are stored in a quiescent state in the male reproductive tract and motility is induced in response to various environmental stimuli, such as change of osmolality (general case) and a decrease of extracellular K+ in fish from Acipenseridae family. This study was aimed to investigate the relationship between osmolality and extracellular K+ concentration in controlling sperm motility in sturgeon. Pre-incubation of sturgeon sperm for 5s in hypertonic solutions of glycerol, NaCl, or sucrose (each of 335 mOsm/kg osmolality) prepares sturgeon spermatozoa to become fully motile in presence of high concentration of K+ ions (15 mM), which has previously been demonstrated to fully repress motility. Furthermore, presence of 0.5mM KCl during the high osmolality pre-incubation exposure completely prevented subsequent spermatozoa activation in a K+-rich media. Manipulating the transport of K+ ions by the presence of K+ ionophore (valinomycin), it was concluded that once an efflux of K+ ions, the precursor of sturgeon sperm motility activation, is taking place, spermatozoa then become insensitive to a large extracellular K+ concentration., (Copyright © 2015 Elsevier B.V. All rights reserved.)

In the present study, for the first time in fish spermatozoa, we describe the precise chronology of motility initiation of sterlet (sturgeon) sperm from completely immotile flagella to regular full wave propagation. The successive activation steps were investigated by high-speed video microscopy, using specific experimental situation, where sperm motility initiation was delayed in time up to several seconds (10 ± 2.68 seconds). Starting from fully immotile, the flagellum shows some trembling for a brief period, soon followed by appearance of the first real bend (so-called "principal bend") with a large wave amplitude 4.28 ± 0.65 μm, then by the "reverse bend," the latter presenting a lower (P < 0.05) wave amplitude (1.14 ± 0.32 μm). This couple of first bends formed at the basal region begins to propagate toward the flagellar tip but gradually fades when reaching the midflagellum, wherein consequently the sperm cell remains nonprogressive. This behavior repeats several times until a stage where the amplitude of the reverse bend gradually reaches a value similar that of the principal bend: The larger amplitude of this couple of bends finally leads to sustain a real "takeoff" of the sperm cell characterized by a full flagellar wave propagation generating an active forward displacement similar to that occurring during regular steady state motility (several seconds after activation). Starting from the earliest stages of motility initiation, the wave propagation along the flagellum and formation of new waves proceeded in a helical manner leading to a 3-dimensional rotation of the whole spermatozoon. Eventually, we estimated that the time period needed from the activation signal (contact with fresh water) to full wave propagation ranges from 0.4 to 1.2 seconds., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The effect of reactive oxygen species production on the motility parameters, DNA integrity, acid phosphatase activity, and protein tyrosine phosphorylation in spermatozoa of the common carp (Cyprinus carpio) was investigated. Spermatozoa were exposed to different concentrations of xanthine and xanthine oxidase (X-XO) either in the presence or absence of antioxidants for 15 and 60 min. A dose- and time-dependent reduction in spermatozoa motility and velocity was observed. Comet assays showed a dramatic increase in DNA fragmentation after 15 min. Changes in tyrosine phosphorylation of spermatozoa proteins were observed by Western blotting with anti-phosphotyrosine antibodies, and proteins of interest were identified by mass spectrometry. After a 60 min exposure to X-XO, O-linked N-acetylglucosamine transferase, isoform 4 was phosphorylated and septin-8-A was dephosphorylated. Acid phosphatase activity also decreased in a dose-dependent manner after a 60 min exposure to oxidative stress. The results demonstrate that oxidative stress impaired functional variables (sperm motility, velocity, DNA integrity) of carp spermatozoa, and altered intracellular signalling pathways through changes in tyrosine phosphorylation and acid phosphatase activity., (© 2014 Wiley Periodicals, Inc.)

We review the phylogeny, sperm competition, morphology, physiology, and fertilization environments of the sperm of externally fertilizing fish and amphibians. Increased sperm competition in both fish and anurans generally increases sperm numbers, sperm length, and energy reserves. The difference between the internal osmolarity and iconicity of sperm cells and those of the aquatic medium control the activation, longevity, and velocity of sperm motility. Hypo-osmolarity of the aquatic medium activates the motility of freshwater fish and amphibian sperm and hyperosmolarity activates the motility of marine fish sperm. The average longevity of the motility of marine fish sperm (~550 seconds) was significantly (P < 0.05) greater than that of freshwater fish sperm (~150 seconds), with the longevities of both marine and freshwater fish being significantly (P < 0.05) lower than that of anuran sperm (~4100 seconds). The average velocity of anuran sperm (25 μm/s) was significantly (P < 0.05) lower than that of marine fish (140 μm/s) or freshwater fish (135 μm/s) sperm. The longevity of the sperm of giant salamanders (Cryptobranchoidea) of approximately 600 seconds was greater than that of freshwater fish sperm but much lower than anuran sperm. Our research and information from the literature showed that higher osmolarities promote greater longevity in anuran sperm, and some freshwater fish sperm, and that anuran and cryptobranchid sperm maintained membrane integrity long after the cessation of motility, demonstrating a preferential sharing of energy reserves toward the maintenance of membrane integrity. The maintenance of the membrane integrity of anuran sperm in fresh water for up to 6 hours showed an extremely high osmotic tolerance relative to fish sperm. The very high longevity and osmotic tolerance of anuran sperm and high longevity of cryptobranchid sperm, relative to those of freshwater fish, may reflect the complex fertilization history of amphibian sperm in general and anurans reversion from internal to external fertilization. Our findings provide a greater understanding of the reproductive biology of externally fertilizing fish and amphibians, and a biological foundation for the further development of reproduction technologies for their sustainable management.

The protective influence of seminal plasma and the antioxidants catalase (CAT), superoxide dismutase (SOD), and glutathione (GTH) on quality parameters, oxidative stress indices, and antioxidant activity was studied in common carp (Cyprinus carpio) spermatozoa exposed to the xanthine-xanthine oxidase (X-XO) system. Fish spermatozoa were incubated for 5 and 20 min at 4 °C with X-XO concentrations of 1 mM X-0.1 U/mL, 0.6 mM X-0.05 U/mL, 0.3 mM X-0.025 U/mL, and 0.1 mM X-0.0125 U/mL. A dose-dependent reduction in spermatozoa motility and velocity was observed at concentrations of 0.1 mM X-0.0125 U/mL to 1 mM X-0.1 U/mL XO. Increase in spermatozoa motility parameters was recorded following treatment with antioxidants and seminal plasma. The level of the oxidative stress indices lipid peroxidation (LPO) and carbonyl derivatives of proteins (CP) was significantly reduced after addition of CAT, SOD, or GTH along with seminal plasma. Significant differences in SOD, glutathione reductase, and glutathione peroxidase activity were seen in spermatozoa incubated with, compared to that without, seminal plasma at all studied X-XO concentrations. The data demonstrated that CAT, SOD, or GTH in combination with SP can reduce reactive oxygen species stress in fish spermatozoa and improve spermatozoa quality.

The role of environmental ion composition and osmolality in Ca(2+) signaled activation was assessed in spermatozoa of brook trout Salvelinus fontinalis. Milt from ten mature males was obtained by abdominal massage. Spermatozoa motility was evaluated in 0, 100, and 300 mOsm/kg NaCl or sucrose solutions, buffered by 10 mM Tris-HCl pH 8.5. For investigation of spermatozoa reaction to external Ca(2+) concentration, 2 mM ethylene glycol tetraacetic acid (EGTA) was added to the activation media as a calcium ions chelator. For investigation of the effect of external Na(+) concentration in conditions of low external Ca(2+), 100 µM amiloride was added to the EGTA-containing solutions as a Na(+) transport blocker. Low motility was observed in sucrose (Na(+) free) solutions containing 2 mM EGTA but not in Na(+) solutions containing 2 mM EGTA. Addition of amiloride led to significantly increased motility (P < 0.05) compared with sucrose (Na(+) free) solutions containing 2 mM EGTA. We conclude that Na(+) transport in Ca(2+)-free solutions plays a regulatory role in brook trout spermatozoa activation. The influence of competitive Na(+) and Ca(2+) transport on the control of spermatozoa activation requires further study with respect to its application for improvement of artificial activation and storage media.

The role of environmental ion composition and osmolality in calcium ion (Ca(2+) ) signalling of spermatozoa activation over the course of the spawning period of brook trout Salvelinus fontinalis was investigated. Motility at the end of spawning was low (mean ± s.d. of 5 ± 2% motile spermatozoa with curvilinear velocity of 25 ± 8 µm s(-1) ). Addition of 10 mM Ca(2+) to the activation medium resulted in values similar to those recorded during the middle of the spawning period (mean ± s.d. of 95 ± 6% motile spermatozoa with curvilinear velocity of 130 ± 15 µm s(-1) )., (© 2014 The Fisheries Society of the British Isles.)

For successful fertilization, spermatozoa must access, bind, and penetrate an egg, processes for which activation of spermatozoa motility is a prerequisite. Fish spermatozoa are stored in seminal plasma where they are immotile during transit through the genital tract of most externally fertilizing teleosts and chondrosteans. Under natural conditions, motility is induced immediately following release of spermatozoa from the male genital tract into the aqueous environment. The nature of an external trigger for the initiation of motility is highly dependent on the aquatic environment (fresh or salt water) and the species' reproductive behavior. Triggering signals include osmotic pressure, ionic and gaseous components of external media and, in some cases, egg-derived substances. Extensive study of environmental factors influencing fish spermatozoa motility has led to the proposal of several mechanisms of activation in freshwater and marine fish. However, the signal transduction pathways initiated by these mechanisms remain clear. This review presents the current knowledge with respect to (1) membrane reception of the activation signal and its transduction through the spermatozoa plasma membrane via the external membrane components, ion channels, and aquaporins; (2) cytoplasmic trafficking of the activation signal; (3) final steps of the signaling, including signal transduction to the axonemal machinery, and activation of axonemal dyneins and regulation of their activity; and (4) pathways supplying energy for flagellar motility., (Copyright © 2014 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.)

The aim of the study was to examine sperm maturation in sturgeon and to establish the localization of the maturation. We demonstrated that sperm maturation occurs in sturgeon outside the testes via dilution of sperm by urine. The process involves the participation of high molecular weight (>10kDa) substances and calcium ions., (Copyright © 2014 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.)

Egg-derived sperm-activating factors and attractants activate sperm motility and attract the sperm, respectively. These phenomena constitute the first communication signaling between males and females in the process of fertilization in many animals and plants, and in many cases, these are species-specific events. Thus, sperm motility activation and chemotaxis may act as a safety process for the authentication between conspecific egg and sperm, and help to prevent crossbreeding. Here, we examine species-specificity of sperm motility activation and chemotaxis in the ascidians belonging to the order Phlebobranchiata: Ciona intestinalis, Ciona savignyi, Phallusia mammillata, Phallusia nigra, and Ascidia sydneiensis. Cross-reactivity in both motility activation and chemotaxis of sperm was not observed between C. savignyi and P. mammillata, or between A. sydneiensis and Phallusia spp. However, there is a "one way" (no reciprocity) cross-reaction between P. mammillata and P. nigra in sperm activation, and between C. savignyi and A. sydneiensis in sperm chemotaxis. Furthermore, the level of activity is different, even when cross-reaction is observed. Thus, sperm motility activation and chemotaxis are neither "species-" nor "genus-" specific phenomena among the ascidian species. Moreover, the interaction between the sperm-activating and sperm-attracting factors (SAAFs) in the ascidian species and the SAAF receptors on the sperm cells are not all-or-none responses.

Here we report for the first time the possibility of sequential sperm motility activation in sturgeon (sterlet, Acipenser ruthenus), a fish with external fertilization, through changes either in osmolality (global solute concentration) or in the Ca(2+) concentration of the medium surrounding the spermatozoa. Sperm motility was initiated in any of three solutions containing buffer and sucrose at 80, or 40 or 10mM (called S80, S40, S10, respectively); S80 is hypertonic relative to sterlet seminal fluid, while S40 is isotonic and S10 is hypotonic. After cessation of sperm movement at the end of this first motility period, a second and then a third, subsequent motile phase were observed. The second motility period was induced at cessation of motility in S80 by imposing a two-fold decrease in osmolality. After arrest of motility in this half-diluted S80, a third motility period could be initiated by addition of CaCl2 to 1mM final concentration. At the end of a first motility period in either S40 or S10, subsequent motility re-activation episodes were achieved only by addition of 1mM CaCl2. Depending on conditions in which sperm samples were activated, significant differences in curvilinear velocity, percent motile spermatozoa, motility duration time, and specific external features of spermatozoa flagella were observed. Altogether, these observations on the ability of sturgeon spermatozoa to sustain sequential activation episodes by experimental adjustment of their environmental conditions represent a potent model for deeper investigations on the sperm motility activation mechanisms., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The aim of this study was to describe spermatozoa volume changes during the motility period of fish species with either osmotic (common carp Cyprinus carpio) or with ionic (sterlet Acipenseri ruthenus and brook trout Salvelinus fontinalis) modes of motility activation. Nephelometry, light microscopy, and spermatocrit methods were used for quantitative assessment of cell volume changes in media of different osmolalities. Significant correlation (R(2) = 0.7341; P < 0.001) between parameter of volume changes measured using nephelometry and light microscopy methods confirmed nephelometry as a sufficiently sensitive method to detect changes of spermatozoa volume. The spermatocrit alteration method resulted in a large proportion of damaged and potentially immotile spermatozoa in media of osmolality less than 150 mOsm/kg in carp and osmolalities from 10 to 300 mOsm/kg in sterlet and brook trout. Therefore, this method is not reliable for assessing spermatozoa swelling in hypotonic solutions, because the integrity of the cells is not fully preserved. Increase in carp spermatozoa (osmotic activation mode) volume occurred during the motility period in hypotonic conditions, but no indications of volume changes were found in sterlet and brook trout spermatozoa (ionic activation mode) associated with environmental osmolality alteration. Accordingly, we conclude that sperm volume changes are differentially involved in the motility activation process. Species-specific differences in spermatozoa volume changes as a response to a hypotonic environment during the motility period are discussed in relation to their potent physiological role., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The flagella of sturgeon sperm have an ultrastructure comprising paddle-like fins extending along most of their length. These fins are seen in several other marine and freshwater fish. The sperm of these fish are fast swimmers and are relatively short lived: it is therefore tempting to think of these fins as having evolved for hydrodynamic advantage, but the actual advantage they impart, at such a small length scale and slow speed, is unclear. The phrase "the fins improve hydrodynamic efficiency" is commonly found in biological literature, yet little hydrodynamic analysis has previously been used to support such conjectures. In this paper, we examine various hydrodynamic models of sturgeon sperm and investigate both swimming velocity and energy expenditure. All of the models indicate a modest hydrodynamic advantage of finned sperm, in both straight line swimming speed and a hydrodynamic efficiency measure. We find a hydrodynamic advantage for a flagellum with fins, over one without fins, of the order of 15-20% in straight line propulsive velocity and 10-15% in a hydrodynamic efficiency measure., (Copyright © 2012 Wiley Periodicals, Inc.)

Spermatozoa tend to swim near surfaces. Such attraction toward surface vicinity was approximated by the force-dipole theoretical approach and hydrodynamic modeling, but the physical parameters of surfaces have not usually been included in these models and their effect on sperm mobility remains unknown. In spermatozoa, changes in wave parameters, together with rotation around their longitudinal axis and circling appear when movement takes place close to surfaces. Here we show, by analysis of microscopy images (including high-speed video), a strong influence of the liquid-solid interface on sterlet spermatozoa motility characteristics compared with motility near the liquid-gas interface. Sperm cells swam at 16% lower velocity near a liquid-solid interface, rotating at a stable frequency of 25 Hz, each 180° rotation corresponding to one beat cycle and circling clockwise (when observed from top). In case of spermatozoa close to a water-air interface, rotation and circling were sporadic and irregular. Sterlet spermatozoa movement near a surface affects their velocity and possibly causes rotation. These behaviors are highly dependent on the level of suppleness of the interface, as has been previously predicted by modeling. Our results enhance the understanding of how surfaces influence fish spermatozoa motility. These insights on the effects of surfaces on fish spermatozoa motility imply that widely used methods rating sperm motility, such as computer-assisted sperm analysis, might lead to erroneous results. Further study of sperm motility near surfaces is urgently needed to correct our rating methods and better understand sperm behavior in natural conditions. Improved evaluation of sperm motility behavior near surfaces could be used to determine physical properties of aquatic interfaces with various surfaces composed of different materials., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Motility of Pinctada margaritifera (Linnaeus, 1758); var: cumingii (Jameson, 1901) (P. margaritifera) spermatozoa collected from gonads are not immediately activated at spawning in seawater (SW) but motility occurs when spermatozoa are transferred into alkaline seawater (pH ranging from 9.0 to 11.4). This motility-activating effect of alkaline pH is reversed when pH is shifted back to more acidic values. In both cases, activity of sperm (% motile cells) increases gradually after alkaline pH activation then lasts for several minutes. The characteristics of these fully motile spermatozoa are described in details at the level of flagella: the wave amplitude and wave-length range 5 to 6 μm and 15 μm respectively, while the flagellar beat frequency is approximately 49 Hz. The velocity of sperm displacement is from 220 to 230 μm/sec. The general swimming pattern is almost circular: the head trajectories describe portions of circles intercalated with small linear segments. Spermatozoa saved in natural seawater at 4°C retain potent motility for several days and can be subsequently activated by alkaline seawater. Respiration and ATP concentration were measured in 3 conditions: regular seawater (pH 7.8), artificial diluent (pH 8.2), and alkaline Tris-buffered seawater (pH 10.5). Results show that sperm respiration rates are higher whereas ATP levels are lower in the latter two media., (Copyright © 2012 Elsevier Inc. All rights reserved.)

In most species, fish spermatozoa activate their motility on contact with the external medium (sea or fresh water depending of their reproductive habitat). Their flagella immediately develop waves propagated at high beat frequency (up to 70 beats s(-1)), which propel these sperm cells at high velocity (6-10 mm min(-1)), but for a quite short period of time, usually limited to minutes. Their specific inability to restore their energy content (mostly adenosine triphosphate) fast enough relatively to their high rate of energy consumption by flagellar contributes mainly to the activity arrest of motility, as the spermatozoa need to rely on early accumulated energy prior to activation. This review of the published data explains the present understanding of physico-chemical mechanisms by which flagellar motility is activated (mostly through osmotic and ionic regulation) and then propels sperm cells at speed. It aims also to describe the gradual arrest of their motility much of which occurs within a few minutes.

Northern pike (Esox lucius L.) spermatozoa are uniflagellated cells differentiated into a head without acrosome, a midpiece and a flagellar tail region flanked by a fin structure. Total, flagellar, head and midpiece lengths of spermatozoa were measured and show mean values of 34.5, 32.0, 1.32, 1.17 microm, respectively, with anterior and posterior widths of the midpiece measuring 0.8 and 0.6 microm, respectively. The osmolality of seminal plasma ranged from 228 to 350 mOsmol kg(-1) (average: 283.88+/-33.05). After triggering of sperm motility in very low osmolality medium (distilled water), blebs appeared along the flagellum. At later periods in the motility phase, the tip of the flagellum became curled into a loop shape which resulted in a shortening of the flagellum and a restriction of wave development to the proximal part (close to head). Spermatozoa velocity and percentage of motile spermatozoa decreased rapidly as a function of time postactivation and depended on the osmolality of activation media (P<0.05). In general, the greatest percentage of motile spermatozoa and highest spermatozoa velocity were observed between 125 and 235 mOsmol kg(-1). Osmolality above 375 mOsmol kg(-1) inhibited the motility of spermatozoa. After triggering of sperm motility in activation media, beating waves propagated along the full length of flagella, while waves appeared dampened during later periods in the motility phase, and were absent at the end of the motility phase. By increasing osmolality, the velocity of spermatozoa reached the highest value while wave length, amplitude, number of waves and curvatures also were at their highest values. This study showed that sperm morphology can be used for fish classification. Sperm morphology, in particular, the flagellar part showed several changes during activation in distilled water. Sperm motility of pike is inhibited due to high osmolality in the seminal plasma. Osmolality of activation medium affects the percentage of motile sperm and spermatozoa velocity due to changes in flagellar wave parameters.

After a long period of spermatogenesis (several weeks to months), marine fish spermatozoa are delivered at male spawning in seawater (SW) at the same time as ova. In some fish species, as the ova micropyle closes quickly after release, these minute unicells, the spermatozoa, have to accomplish their task of reaching the micropyle within a very brief period (several seconds to minutes), for delivery of the haploid male genetic information to the ova. To achieve this goal, their high-performance motile equipment, the flagellum, must fully activate immediately on contact with the SW and then propel the sperm cell at an unusually high initial velocity. The cost of such 'hyperactivity' is a very rapid consumption of intracellular ATP that outstrips the supply. The spermatozoa become rapidly exhausted because mitochondria cannot compensate for this very fast flagellar energy consumption. Therefore, any spermatozoon ends up with two possibilities: either becoming exhausted and immotile or reaching the egg micropyle within its very short period of forward motility (in the range of tens of seconds) before micropyle closure in relation to both contact of SW and cortical reaction. The aim of the present review is to present step by step the successive events occurring in marine fish spermatozoa from activation until their full arrest of motility. The present knowledge of activation mechanisms is summarized, as well as a description of the motility parameters characterizing the motility period. As a complement, in vitro results on axonemal motility obtained after demembranation of flagella bring further understanding. The description of the sperm energetic content (ATP and other high energy compounds) and its evolution during the swimming period is also discussed. A general model aiming to explain all the successive cellular events occurring immediately after the activation is presented. This model is proposed as a guideline for understanding the events governing the sperm lifespan in the marine fish species that reproduce through external fertilization.

In this study we describe acrosome staining and motility characteristics of fresh and cryopreserved sterlet (Acipenser ruthenus L.) spermatozoa using soybean trypsin inhibitor-Alexa conjugate fluorescent staining and computer-aided sperm analysis (CASA), respectively. Methanol or dimethylsulfoxide (DMSO) were used as cryoprotectants. After cryopreservation a decline in sperm motility characteristics occurred, but no differential effect between cryoprotectant was observed. Cryopreservation caused a significant increase in the percentage of spermatozoa with acrosome stained by SBTI-Alexa for samples cryopreserved using DMSO compared to methanol. These data suggest that the low usefulness of DMSO for cryopreservation of sturgeon spermatozoa is related to its harmful specific effect towards the acrosome, probably by causing its precocious triggering, much before any egg contact.

The spermatozoa of most fish species are immotile in the testis and seminal plasma. Therefore, motility is induced after the spermatozoa are released into the aqueous environment during natural reproduction or into the diluent during artificial reproduction. There are clear relationships between seminal plasma composition and osmolality and the duration of fish sperm motility. Various parameters such as ion concentrations (K+, Na+, and Ca2+), osmotic pressure, pH, temperature and dilution rate affect motility. In the present paper, we review the roles of these ions on sperm motility in Salmonidae, Cyprinidae, Acipenseridae and marine fishes, and their relationship with seminal plasma composition. Results in the literature show that: 1. K+ is a key ion controlling sperm motility in Salmonidae and Acipenseridae in combination with osmotic pressure; this control is more simple in other fish species: sperm motility is prevented when the osmotic pressure is high (Cyprinidae) or low (marine fishes) compared to that of the seminal fluid. 2. Cations (mostly divalent, such as Ca2+) are antagonistic with the inhibitory effect of K+ on sperm motility. 3. In many species, Ca2+ influx and K+ or Na+ efflux through specific ionic channels change the membrane potential and eventually lead to an increase in cAMP concentration in the cell, which constitutes the initiation signal for sperm motility in Salmonidae. 4. Media that are hyper- and hypo-osmotic relative to seminal fluid trigger sperm motility in marine and freshwater fishes, respectively. 5. The motility of fish spermatozoa is controlled through their sensitivity to osmolality and ion concentrations. This phenomenon is related to ionic channel activities in the membrane and governs the motility mechanisms of axonemes.

Sperm motility is a key factor in allowing us to determine semen quality and fertilizing capacity. Motility in semen is mainly controlled by K+ in salmonids, and probably also in sturgeons, and by osmotic pressure in other freshwater and seawater fish species, but other factors, such as concentration of surrounding metabolites and ions (Ca2+, Mg2+, etc.), pH and temperature also influence motility characteristics. In the present study, we have mainly reviewed and summarized the effects of temperature and pH on the motility of spermatozoa in three fish species: salmonids, cyprinids and sturgeons. Data in the literature show that motility, fertilizing ability and velocity of spermatozoa, as well as the duration of the motility period, depend on the temperature of the assay medium and also of that of the brood fish holding tank. In contrast, the pH of the swimming medium, and thus the intracellular pH of spermatozoa, has less influence on sperm motility parameters in cyprinids, salmonids and sturgeons.

Sperm motility is a prerequisite factor determining semen quality and fertilizing capacity. The effects of environmental factors including pH, cations and osmolality as well as the role of dilution rate on sperm motility parameters in Acipenser persicus were studied. The best pH and dilution rate for activation of spermatozoa were pH 8.0 and dilution ratio 1:50. Ionic factors can stimulate the initiation of sperm activation. The maximum percentage of motile sperm and total duration of sperm motility were observed in solutions containing 25 mM NaCl, 0.2 mM KCl, 3 mM CaSO4, 10 mM MgSO4 and sucrose with an osmolality of 50 mosmol kg(-1). The present study provides us with some basic knowledge about sturgeon spermatozoa biosensitivity to ionic and osmolality effects. A sensitivity of A. persicus sperm was observed after induction of activation of sperm motility in solution containing cations or sucrose with high osmolality. Concentrations more than 50 mM Na+, more than 1 mM K+, more than 3 mM Ca2+ and more than 10 mM Mg2+ had negative effects on sperm motility. Also, osmolality more than 100 mosmol kg(-1) had an inhibitory effect. It is clear that ions and osmolality stimulate the motility of spermatozoa by changes in the properties of the plasma membrane including its potential and its ionic conductance. The inhibitory role of high osmolality of the swimming medium (more than 100 mosmol kg(-1)) and insufficient osmolality of the seminal plasma to inhibit semen motility suggested that osmolality is not the principal factor preventing sperm motility in seminal fluid but that K+ is a major inhibitory factor of sperm motility in seminal plasma.

In most detailed studies, sea urchin sperm movement has been analyzed mainly from observations of spermatozoa swimming at the interface between two media: water/air or water/glass. When spermatozoa are placed on a microscope slide, they rapidly appear to swim near those interfaces. The aim of this article is to determine how they become confined to the vicinity of surfaces. High-speed observations of moving spermatozoa reveal blurred portions in the flagellum images that propagate from base to tip, suggesting that flagellar waves contain an out-of-plane component. The model we have developed depicts how this tri-dimensional component tends to keep spermatozoa close to interfaces and, as a consequence, increases the time of contact between the egg surface and spermatozoa., (Copyright 2003 Wiley-Liss, Inc.)

This study investigated the effects of different environmental conditions on the motility parameters of paddlefish (Polyodon spathula) spermatozoa. Paddlefish spermatozoa demonstrated the following characteristics: (i) all spermatozoa were motile 10 s after activation with a velocity of 130-160 microm s(-1); (ii) after 2 min, velocity decreased to 80-130 microm s(-1); and (iii) motility was maintained for up to 9 min. Concentrations of 0.5-5.0 mmol KCl l(-1) prevented activation of spermatozoa. After transfer into a swimming medium (20 mmol Tris l(-1), pH 8.2 and 1 mg BSA ml(-1)) containing 0.5 mmol KCl l(-1) (combined with 5 mmol NaCl or MgCl(2) l(-1)), 80-100% of cells were motile with a velocity of about 120-150 microm s(-1). MgCl(2) significantly improved the velocity of spermatozoa at 10, 40, 50 and 60 s after activation and the stable velocity of spermatozoa was about 140 microm s(-1). Very low concentrations of CaCl(2) (0.125 mmol l(-1)) combined with 0.5 mmol KCl l(-1) initiated motility in 20% of spermatozoa, whereas all spermatozoa were activated after 2 min with 0.25 mmol CaCl(2) l(-1) in similar medium for the full period of swimming with velocity of about 120 microm s(-1). This study demonstrated that potassium (5-15 mmol l(-1)) inhibits demembranated spermatozoa. Thus, initiation of movement in paddlefish spermatozoa is under the reciprocal control of potassium and calcium ion concentrations.

In this study, fish sperm cryopreservation methods were elaborated upon for ex situ conservation of nine strains of Bohemian common carp. Common carp sperm were diluted in Kurokura medium and chilled to 4 degrees C and dimethyl sulfoxide was added. Cryotubes of sperm with media were then cooled from +4 to -9 degrees C at a rate of 4 degrees C min(-1) and then from -9 to -80 degrees C at a rate of 11 degrees C min(-1), held for 6 min at -80 degrees C, and finally transferred into liquid N(2). The spermatozoa were thawed in a water bath at 35 degrees C for 110 s and checked for fertilization yield, hatching yield of embryos, and larval malformations. Fresh and frozen/thawed sperm were evaluated for the percentage and for the velocity of motile sperm from video frames using image analysis. The percentage and velocity of sperm motility at 15 s after activation of frozen/thawed sperm was significantly lower than that of fresh sperm (nine males). ANOVA showed a significant influence of fresh vs frozen/thawed sperm on fertilization rate (P < 0.0001), but differences in hatching rate and in larval malformation (0-6.8%) were not significant, and different males had a significant influence on fertilization and hatching rate (P < 0.003 and P < 0.007, respectively). Multiple range analysis (LSD) showed significant differences between fresh and frozen/thawed sperm regarding fertilization rate (68 +/- 11 and 56 +/- 10%, respectively) and insignificant differences between fresh and frozen/thawed sperm on the hatching rate (50 +/- 18 and 52 +/- 9%, respectively). The percentage and velocity of fresh sperm motility were correlated, respectively, with the fertilization yield of frozen/thawed sperm at the levels r = 0.51 and r = 0.54., (Copyright 2001 Academic Press.)

31P-NMR and (1)H-NMR were used to monitor changes of several compounds with high-energy bonds and metabolites prior to and after the initiation of motility of turbot spermatozoa (Psetta maxima). The obtained (31)P-NMR spectra revealed the presence of phosphomonoesters, phosphodiester, intracellular inorganic phosphate (Pi), phosphocreatine (PCr), and free nucleotide triphosphate. Following the activation of motility, the di- and tri-phosphate nucleotides, PCr, phosphomonoesters levels dropped while Pi levels increased. A significant increase of lactate was also seen at the end of the swimming phase. The compositions of seminal fluid and urine were also determined. Lipoproteins, formic acid, amino acid, and citric acid were detected in seminal fluid. Dimethyl amine, trimethylamine, and trimethylamine oxyde were found in urine. These data suggest that at least a part of the energy required during the swimming phase results from anaerobic fermentation and oxidative phosphorylation. J. Exp. Zool. 286:513-522, 2000., (Copyright 2000 Wiley-Liss, Inc.)

The interdependence between motility, respiration, ATP production, and utilization was investigated in intact spermatozoa of turbot (Psetta maxima), a marine teleost. When spermatozoa were diluted in a hyperosmotic medium (>300 mOsmol/kg), they immediately became motile, and the intracellular concentration of ATP as well as the adenylate energy charge ratio dropped concomitant with the straight-line velocity. The ADP and AMP levels increased from 1.4 to 8.0 nmole/10(8) cells and from 0.6 to 6.0 nmole/10(8) cells, respectively. Moreover, 31P-NMR spectra recorded prior to the swimming phase revealed the presence of phosphomonoesters (PMEs) and phosphodiesters (PDEs), intracellular inorganic phosphate (Pi), and phosphocreatine (PCr). At the end of the motility period, PCr, PDE, and PME decreased, while the Pi level increased markedly. Following initiation of motility, O2 consumption of spermatozoa increased from 34.9 to 124.8 O2 nmole/10(9) spermatozoa/min. FCCP, an uncoupler of oxidative phosphorylation, did not significantly affect the respiratory rate of motile spermatozoa. Ouabain, a specific inhibitor of (Na+/K+)/ATPase, slightly decreased the respiration rate of motile spermatozoa, indicating that the major part of ATP catabolism was linked to dynein ATPase. Inhibitors of the respiratory chain (KCN, NaN3, NaHCO3-, oligomycin) reduced sperm respiration, percentage of motile cells, velocity, and adenylate contents. Following the reactivation of motility of demembranated spermatozoa, KCN, NaN3, NaHCO3- altered the flagellar beat frequency, demonstrating that these respiratory inhibitors possess action sites other than mitochondria. Mitochondrial oxidative phosphorylation is highly requested to produce energy required during motion. Nevertheless it is insufficient to maintain endogenous ATP stores. A second phase of motility was induced by a transfer of exhausted spermatozoa into an ionic medium of low osmolality (200 mOsmol/kg) for 30 min. Spermatozoa, once reactivated in AM, recovered 55% of initial motility and 31% of initial fertilization rate. In hypo-osmotic medium, mitochondrial oxidative phosphorylation also induced ATP regeneration. Following activation of movement, several morphological changes were observed in the mitochondria and the midpiece.

Sea bass spermatozoa are maintained immotile in the seminal fluid, but initiate swimming for 45 s at 20 degrees C, immediately after dispersion in a hyperosmotic medium (1100 mOsm kg-1). The duration of this motile period could be extended by a reduction of the amplitude of the hyperosmotic shock. Five seconds after the initiation of motility, 94.4 +/- 1.8% of spermatozoa were motile with a swimming velocity of 141.8 +/- 1.2 microns s-1, a flagellar beat frequency of 60 Hz and a symmetric type of flagellar swimming, resulting in linear tracks. Velocity, flagellar beat frequency, percentage of motile cells and trajectory diameter decreased concomitantly throughout the swimming phase. After 30 s of motility, the flagellar beat became asymmetric, leading to circular trajectories. Ca2+ modulated the swimming pattern of demembranated spermatozoa, suggesting that the asymmetric waves produced by intact spermatozoa after 30 s of motility were induced by an accumulation of intracellular Ca2+. Moreover, increased ionic strength in the reactivation medium induced a dampening of waves in the distal portion of the flagellum and, at high values, resulted in an arrest of wave generation in demembranated spermatozoa. In non-demembranated cells, the intracellular ATP concentration fell immediately after transfer to sea water. In contrast, the AMP content increased during the same period, while the ADP content increased slightly. In addition, several morphological changes affected the mitochondria, chromatin and midpiece. These results indicate that the short swimming period of sea bass spermatozoa is controlled by energetic and cytoplasmic ionic conditions and that it is limited by osmotic stress, which induces marked changes in cell morphology.

Flagellar motility is the result of specific interactions between axonemal microtubular proteins and the dynein motors. Tubulin, the main component of microtubule, is a very polymorphic protein resulting from the expression of several isogenes and from the existence of various post-translational modifications. In order to characterize tubulin isoforms and tubulin domains that are important for flagellar movement, we prepared monoclonal antibodies against axonemal proteins from whole sea-urchin sperm tails. The monoclonal antibodies obtained were screened for their potency to inhibit demembranated-reactivated sperm models and for their monospecific immunoreactivity on immunoblot. Among the different antibodies we obtained, D66 reacted specifically with a subset of beta-tubulin isoforms. Limited proteolysis, HPLC, peptide sequencing, mass spectroscopy and immunoblotting experiments indicated that D66 recognized an epitope localized in the primary sequence Gln423-Glu435 of the C-terminal domain of Lytechinus pictus beta2-tubulin, and that this sequence belongs to class IVb. The use of synthetic peptides and immunoblotting analysis further narrowed the amino acids important for antibody recognition to Asp427-Glu432. Because the primary effect of this antibody on sperm motility is to decrease the flagellar beat frequency, we suggest that this sequence is involved in the tubulin-dynein head interaction.

Monoclonal antibodies raised against axonemal proteins of sea urchin spermatozoa have been used to study regulatory mechanisms involved in flagellar motility. Here, we report that one of these antibodies, monoclonal antibody D-316, has an unusual perturbating effect on the motility of sea urchin sperm models; it does not affect the beat frequency, the amplitude of beating or the percentage of motile sperm models, but instead promotes a marked transformation of the flagellar beating pattern which changes from a two-dimensional to a three-dimensional type of movement. On immunoblots of axonemal proteins separated by SDS-PAGE, D-316 recognized a single polypeptide of 90 kDa. This protein was purified following its extraction by exposure of axonemes to a brief heat treatment at 40 degrees C. The protein copurified and coimmunoprecipitated with proteins of 43 and 34 kDa, suggesting that it exists as a complex in its native form. Using D-316 as a probe, a full-length cDNA clone encoding the 90-kDa protein was obtained from a sea urchin cDNA library. The sequence predicts a highly acidic (pI = 4.0) protein of 552 amino acids with a mass of 62,720 Da (p63). Comparison with protein sequences in databases indicated that the protein is related to radial spoke proteins 4 and 6 (RSP4 and RSP6) of Chlamydomonas reinhardtii, which share 37% and 25% similarity, respectively, with p63. However, the sea urchin protein possesses structural features distinct from RSP4 and RSP6, such as the presence of three major acidic stretches which contains 25, 17, and 12 aspartate and glutamate residues of 34-, 22-, and 14-amino acid long stretches, respectively, that are predicted to form alpha-helical coiled-coil secondary structures. These results suggest a major role for p63 in the maintenance of a planar form of sperm flagellar beating and provide new tools to study the function of radial spoke heads in more evolved species.

Live trout spermatozoa initiate flagellar motility for a short period of time (30 s at 18 degrees C), during which their mean beat frequency (BF) decreases steadily from 60 to 20 Hz; motility then stops abruptly. When demembranated, the motility of axonemes lasts much longer, up to 20 min, with high beat frequency, provided that ATP (millimolar concentration) and cAMP (micromolar) are added. In the present paper, the motility of demembranated trout sperm was investigated in the absence of added ATP in various incubation conditions relative to other substrates. Without the addition of exogenous creatine kinase, the addition of phosphocreatine (PCr) and ADP shows the appearance of a progressive activation of all sperm models with BF increasing with time up to high values. Without the addition of cAMP, the BF increases to lower values but flagella propagated poorly coordinated waves for only a few min. Similar progressive activation is also observed when only ADP is added (without any previous in vivo activation) and BF increases up to moderate values. In this latter case, no activation occurs without addition of cAMP. The respective roles of creatine kinase and adenylate kinase in this process were investigated by addition of specific inhibitors such as fluorodinitrobenzene and P1,P5-di(adenosine-5')pentaphosphate in the above described conditions. We conclude from these observations that all the elements necessary for a coupling between ADP/PCr/creatine kinase on one hand and ATP/ADP/dynein on the other appear to be present in trout spermatozoa: thus the existence of a shuttle sustaining this coupling is strongly suggested.

The mechanism by which a hypo-osmotic shock activates motility of carp spermatozoa was studied. The direct role of osmolality at the axoneme was investigated after demembranation of spermatozoa with Triton X-100 and reactivation in various ionic or anionic solutions containing Mg-ATP: demembranated spermatozoa remain motile in solutions of osmolality up to 550 mOsm kg-1 while non-demembranated spermatozoa are immotile when osmolality rises above 250 mOsm kg-1 with the same salt solutions as well as in non-ionic solutions. Suspension in hypo-osmotic saline solutions triggered the swelling of native carp spermatozoa. No motility or swelling occurred above 200-300 mOsm kg-1 and this osmolality is probably that of the cytosol. The swelling of carp spermatozoa is the result of an entrance of water but this was not affected by pCMBS, an inhibitor of the aquaporin CHIP28, or by various inhibitors of the co-transport of water with ions. Various pharmacological agents that affect the motility of different sperm species had no effect on carp sperm motility when used under similar conditions. However, prolonged exposure to a solution devoid of K+ or Cl- affects the activation of motility in a reversible manner, suggesting that these ions have a role in the perception or transduction of the osmotic signal. Altering the concentration of intracellular second messengers such as Ca2+ and cAMP, and the pH did not affect the motility of carp spermatozoa. However, DMSO at 1-20% (400-3200 mOsm kg-1) affects the motility of carp spermatozoa 3-4 min after mixing. These results show that the activation signal of carp sperm motility differs from that known for spermatozoa of other species of fish such as trout. Our results indicate that the activation mechanism may involve a co-transport of ions or specific 'stretch-activated channels' that are sensitive to osmotic pressure.

We have generated a series of monoclonal antibodies against axonemal proteins from sea urchin spermatozoa in order to identify novel proteins involved in the regulation of flagellar motility. The monoclonal antibody D405-14 inhibited the motility of demembranated-reactivated sperm models at low concentrations and recognized a single polypeptide of 33 kDa (p33) on immunoblots of sea urchin axonemal proteins. Fractionation of the axonemes with high salt solutions, heat, and detergent resulted in the selective extraction of p33 into a 0.6 M NaCl-soluble and a 0.5% sodium lauryl sarcosinate (Sarkosyl)-soluble form. Both forms of p33 were purified to apparent homogeneity by immunoaffinity chromatography on monoclonal antibody D405-14-Sepharose. We have also isolated and sequenced a full-length cDNA clone encoding the 33-kDa protein. The sequence predicts a polypeptide of 260 amino acids having a mass of 29,730 Da and an isoelectric point of 9.3. Sequence comparison indicates that p33 is 66% identical (74% similar) to the p28 light chain of axonemal inner dynein arm of Chlamydomonas reinhardtii. Taken together, these results suggest that we have identified a p28 light chain homolog in sea urchin sperm axoneme and that this protein may play a dynamic role in flagellar motility.

Two monoclonal antibodies, AXO 49 and TAP 952, probed with carboxy-terminal peptides from Paramecium axonemal tubulin and with polyglycylated synthetic peptides, are found to recognize differently tubulin polyglycylation, the most recently identified posttranslational modification discovered in Paramecium axonemal tubulin. With these antibodies, we show that tubulin polyglycylation is widely distributed in organisms ranging from ciliated protozoa to mammals; it arose early in the course of evolution, but seems to be absent in primitive protozoa such as the Euglenozoa. Tubulin polyglycylation is the last posttranslational modification which takes place in the course of Drosophila spermatogenesis and its occurrence corresponds to the end of spermatozoan maturation. An involvement of polyglycylated tubulin in axoneme motility is suggested since AXO 49 and TAP 952 specifically inhibit the reactivated motility of sea urchin spermatozoa.

A panel of monoclonal antibodies (mAbs) has been generated against sea urchin sperm axonemes and selected for their ability to inhibit the motility of sea urchin sperm models. The mAb C9 recognized a 50 kDa protein on blots of sea urchin sperm axonemes. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that C9 recognized isoforms of beta-tubulin. Low concentrations of C9 (0.1-1.0 microgram/ml) blocked the motility of sea urchin sperm models by decreasing the sliding velocity and frequency of flagellar beating to less than 1 Hz and by modifying the shear angle along the axoneme, especially the distal end. Other antitubulin antibodies had little effect on motility at concentrations 100-fold higher than those effective for C9. The effects on motility were not restricted to flagella of sea urchin spermatozoa. Flagellar beating of the dinoflagellate Oxyrrhis marina was completely blocked by C9 in a manner reminiscent of that of sea urchin sperm flagella. The mAb also inhibited the motility of human spermatozoa and Chlamydomonas reinhardtii. Immunofluorescence techniques revealed that C9 stains the whole axoneme of sea urchin spermatozoa and O. marina flagella together with the cortical network of O. marina cell body. C9 is the first antitubulin antibody reported to interfere with flagellar beat frequency. The observation that this antibody arrests the motility of flagella from sea urchin sperm along with that of dinoflagellate, algae, and human sperm flagella suggests that the epitope recognized by C9 is conserved over a long period of evolution and plays an important role in sperm motility.

Studies on the flagellar movement of carp spermatozoa induced by dilution in distilled water allowed us to describe a sequence of early, rapid morphological and kinetic changes which begin at the very tip of the flagellum. They cause the progressive folding of the axoneme which ends stuck to the head within 90-120 seconds after the initiation of motility. However, the axonemal machinery remains functional as the folding can be reversed after transfer back into a high osmolality medium and partially folded flagella were able to propagate efficient waves along the non-folded proximal portion of the axoneme. The data also revealed that the membrane area of the terminal piece exhibits strong sensitivity to hypotonicity. These results suggest that in the normal freshwater medium, the brief swimming period allowing fertilization of oocytes is limited by the osmotic stress induced coiling of the carp sperm tail and not by ATP stores.