Fish Sperm Motility Research Articles

Studying sperm motility in marine fish: an overview on the state of the art

Summary This contribution reviews existing literature and some new own findings on teleost sperm motility and factors controlling it, emphasizing selected marine species. In marine teleosts with external fertilization (halibut, turbot, sea bass, hake, cod and tuna serving as examples), mainly the osmolality controls sperm motility: movement is activated by transfer from the seminal fluid into sea water, representing a large upward step in osmolality. The exception are flatfishes (such as halibut or turbot) where CO2 is responsible for flagellar immotility in seminal fluid. In all cases, the duration of motility is short and limited to minutes ranges due to partial exhaustion of the ATP energy and to increase of internal ionic concentration as suggested by studies with de-membranated/ATP reactivated flagellae. In this overview, we compare motility characteristics (percentage of active spermatozoa, velocity, linearity), flagellar waves parameters (wave length and amplitude, number of waves) and energy content (respiration and ATP concentration) within species where these data have been established. All parameters show a rapid decrease after activation; therefore progressive forward movement needed by the sperm to effectively reach the egg surface, is limited to a short initial period following activation. In two species (turbot and sea bass) the rapid decrease of sperm motility is reflected by a corresponding decrease of the fertilizing ability. Exposure to external environments (sea water) at activation also leads to local defects of the sperm flagella posing additional limitations on motility duration. However, minor flagellar damages as well as energetic exhaustion are reversible: after a resting period in a non-swimming solution at the end of the motility period, spermatozoa can be re-activated for a second motility period. From these results and from additional data obtained from de-membranated/ATP re-activated spermatozoa, a paradigm has been developed which establishes a link between external osmolality (sea water), internal ionic concentration and control of axonemal activity.

Calcium‐induced quiescence of sperm motility in the bluegill (Lepomis macrochirus)

Before dilution in hypoosmotic media sperm of freshwater fish are maintained quiescent by a range of factors including osmolality, K+ and pH, and the onset of motility is generally associated with an increase in cytoplasmic Ca2+. In contrast, Ca2+ in conjunction with osmolality was found to inhibit motility of intact bluegill sperm. Consistent with seminal plasma composition, 0.16 mmol/L Ca2+ and greater, in conjunction with an osmotic concentration of 290 mOsm/kg, inhibited the onset of bluegill sperm motility; sperm diluted in saline at 290 mOsm/kg without Ca2+ became motile. Cations Mn2+ and Sr2+, in conjunction with osmolality, had an inhibitory effect on initiation of sperm motility similar to that of Ca2+. Sperm motility was inhibited by Ca2+ channel blockers nimodipine and nifedipine, the mitochondrial Ca2+ uniporter inhibitor ruthenium red and the calmodulin inhibitors W-7 and trifluoperazine dihydrochloride. These results provide evidence that elevated cytoplasmic Ca2+ inhibits sperm motility and yet low levels permit or promote motility. This study demonstrates a unique inhibitory action of Ca2+ on the motility of intact fish sperm at physiologically relevant levels.

Assessment of Short-Term Exposure to Nonylphenol in Japanese Medaka Using Sperm Velocity and Frequency of Motile Sperm

Nonylphenol is one of the compounds believed to cause endocrine disruption and affect sperm quality in mammals. However, there is little information on its effects on sperm motility in fish or other forms of wildlife. We examined the effects of nonylphenol on the motility of spermatozoa of medaka (Oryzias latipes) using an in vitro bioassay. Sperm was activated in aqueous media containing no nonylphenol (solvent control, 0.1% ethanol) or 1 or 100 micromol/L nonylphenol and immediately loaded into a sperm motility observation chamber. The ratio of motile spermatozoa and their swimming speeds were analyzed by computer-assisted image analysis at 30 and 60 s after activation. A decrease in swimming speed or the ratio of motile spermatozoa was observed in spermatozoa exposed for 60 s to 100 micromol/L nonylphenol. Our results suggest that nonylphenol causes a reduction in sperm viability in teleost fish in the short period between ejaculation and fertilization.

Long‐term exposure to environmentally relevant doses of methyl‐tert‐butyl ether causes significant reproductive dysfunction in the zebrafish (Danio rerio)

Methyl-tert-butyl ether (MTBE), an anthropogenic chemical used as a gasoline additive, is being detected at an increasing frequency in the environment. The acute lethal concentration that kills 50% of the fish test population and the chronic effects of exposure to MTBE were investigated in the zebrafish (Danio rerio). Chronic exposure over three weeks to effective MTBE conceritrations as low as 0.11 mg/L induced a significant increase in the vitellogenin concentration of male fish. The impact of a chronic, eight-week exposure at effective concentrations ranging from 0.44 to 220 mg/L had no significant effect on fecundity, fertilization, or hatch rate but highly significant impacts on sperm motility. Spermatozoa of all MTBE-exposure groups showed a significantly lower straight-line velocity and lower average path velocity compared to those of the nonexposed group. These results suggest that chronic exposure to MTBE negatively affects fish sperm motility at concentrations that are environmentally relevant and several orders of magnitude lower than concentrations inducing acute effects.

Will rising salinity in the Murray - Darling Basin affect common carp (Cyprinus carpio L.)?

Salinisation in the Murray–Darling Basin, Australia, may affect aquatic flora and fauna, including the common carp, an alien species that has become the most common fish in the river system. This study describes the responses of juvenile carp (31–108 mm total length) to salinity levels that prevail in some wetlands of the lower reaches of the River Murray. Carp are moderately tolerant of salinity (direct transfer LC50: 11 715 mg L–1), particularly after slow acclimation (LC50: 13 070 mg L–1), but sub-lethal effects are evident at lower salinities. These include effects on osmoregulation (>7500 mg L–1), behaviour (7500–12 500 mg L–1) and sperm motility in mature fish (150–300 mm) (8330 mg L–1). Salinities in some Murray–Darling Basin wetlands already approach half seawater (17 500 mg L–1) and carp populations in these important nursery areas could be impacted through sub-lethal effects on adults and lethal effects on juveniles, eggs and sperm.

Influence of Visible Light and Ultraviolet Irradiation on Motility and Fertility of Mammalian and Fish Sperm

The effects of visible light irradiation on sperm motility, fertility, and reactive oxygen species (ROS) formation were investigated and compared in ram and fish (tilapia). Low-energy visible light has previously been found to modulate various processes in different biological systems. In the literature, it is accepted that the first step following visible light irradiation is the formation of ROS by endogenous cellular photosensitizers. Sperm of ram and tilapia were irradiated with various light sources (400-800 nm white light, 660 nm red light, 360 nm blue light, 294 nm UV), and their motility and fertility rates were measured. The amount of ROS generated by irradiation was estimated using electron paramagnetic resonance (EPR) technique. Sperm taken from tilapia showed higher motility and fertility following red and white light irradiation. In contrast, the motility and fertility of ram sperm were slightly increased only by red light. A negative effect on motility and fertility of sperm of both species was obtained following irradiation with UV and blue light. The amount of ROS produced in irradiated tilapia sperm was much higher than that of ram sperm. The results show that different wavelengths differentially affect tilapia and ram sperm motility and fertilization. The difference in response to the various light sources might be explained by the different amounts of ROS formation by ram and tilapia, which are in agreement with the physiology of fertilization appropriate to each of these species. Based on these results, it is suggested that in vitro fertilization in mammals should be performed in darkness or at least under red light.

Sperm motility in fishes. (II) Effects of ions and osmolality: A review

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.

Metal Levels in Blood Samples From Male Great Lakes Fish Consumers

The Effect of Organochlorines on Male Infertility Study (FINS) is an NIH-funded project investigating the effects of exposure to organochlorine compounds (OC) and metals via fish consumption on male infertility. In the Great Lakes (GL) basin, a potential source of exposure to heavy metals, such as mercury, is through the consumption of sport-caught GL fish. The study population is men participating in FINS who submit semen specimens for analysis and provide blood samples for OC and metals testing. Cros-sectional study. The first 184 whole blood specimens collected from FINS participants were analyzed for 12 metals by the Michigan Department of Community Health Bureau of Laboratories. The mean values and ranges () in micrograms per liter were: Arsenic, 9.5 (5.8 - 25.5); Cadmium, 0.3 (0.0 - 2.8); Chromium, 0.21 (0.0 - 3.6); Copper, 928.4 (700.0 - 1397.0); Lead, 2.0 (0.5 - 16.2); Manganese, 13.5 (8.0 - 30.0); Mercury, 1.8 (0.0 - 14.5; Molybdenum, 0.4 (0.0 - 2.0); Selenium, 187.7 (130.0 - 260.0); Thallium, 0.0 (0.0 - 0.0); Uranium, 0.0 (0.0 - 0.02); Zinc, 6940.0 (5077.0 - 9251.0). In a preliminary analysis of questionnaire data from144 FINS participants for whom complete data existed for metal levels, fish consumption history and sperm concentration, motility and morphology, metal levels were examined for associations with semen parameters and fish consumption using correlation analysis and univariate logistic regression models. In these samples, no association was found between sperm concentration or motility and any metal. High mercury level (above the 75th percentile) was positively associated with consumption of any fish within 12 months (odds ratio = 8.14, 95% CI:[1.848, 35.814], p = 0.006), but not consumption of Great Lakes fish exclusively (odds ratio = 2.35, 95% CI:[0.915, 6.032], p = 0.08). Consumption of ocean fish within the past 3 months was most strongly associated with high mercury level (odds ratio = 10.34, 95% CI:[2.358, 45.351], p = 0.002). Mean serum values of heavy metals in infertile men have now been simultaneously determined. Increased fish consumption correlates with serum mercury levels. Serum levels of heavy metals levels did not correlate with semen parameters in this sample.

Chaotropic ions and multivalent ions activate sperm in the viviparous fish guppy Poecilia reticulata

The control system of sperm motility in the fishes with internal fertilization still remains to be studied. In the present study, we examined the sperm activation in the viviparous fish guppy Poecilia reticulata as a function of solute composition. The degree of sperm activation by inorganic and organic solutes correlated well with the lyotropic or Hofmeister series. The solutes that induced sperm activation corresponded to those that have been termed "chaotropes" or macromolecule-destabilizing solutes. In addition to this Hofmeister rule, multivalent cations and anions had valency-dependent activating effects at low concentrations. We investigated the cell signaling triggered by chaotropes and divalent cations and found an intracellular cAMP concentration increase upon the activation of sperm motility. From the present findings, we propose a novel regulation mechanism of sperm motility in the viviparous fish.

Sperm motility in fishes. I. Effects of temperature and pH: a review

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.

The effect of inorganic and organic pollutants on sperm motility of some freshwater teleosts

Using four different sperm types from brown trout Salmo trutta fario(Salmonidae), chub Leuciscus cephalus(Cyprinidae), burbot Lota lota(Gadidae) and African catfish Clarias gariepinus(Clariidae) the effect of inorganic (cadmium, copper, mercury, lead, zinc and nitrite) and organic (cyclohexane and 2,4‐dichlorophenol) environmental pollutants on sperm motility was investigated. Spermatozoa were activated in double distilled water containing the different test substances and the motility was compared to controls of similar pH. From the investigated motility variables the sperm motility rate and swimming velocity reacted most to the environmental pollutants whereby the changes depended on the species and on the test substance. African catfish spermatozoa were the most resistant, chub and burbot spermatozoa showed medium resistance and brown trout spermatozoa were the most sensitive to the pollutants. With exception of 2,4‐dichlorophenol and zinc the effective concentrations of the tested pollutants exceeded the recommendation for surface waters 100–10·000‐fold and were in a range lethal for the fish themselves. Therefore, it was concluded that fish sperm motility is not a suitable marker for risk assessment of environmental pollutants.

The Ionic and Osmotic Factors Controlling Motility of Fish Spermatozoa

This review presents actual knowledge about energetic, ionic, osmotic and gaseous control of fish sperm motility and its duration. Right after they are activated, fish spermatozoa of most species swim for a short period of time, in the range of one to several minutes. What determines the activation process? Is it due to the new ionic, gaseous and/or osmotic environment? Why is the duration of motility so short? Is it resulting from a fast exhaustion of energy stores (ATP, ADP, AMP, PCr) combined with the above-mentioned ionic/osmotic stress leading to morphological alterations? The motility criteria (flagellar beat frequency, head displacement velocity, flagellar waves morphology, etc.) used to characterize fish sperm movement and sperm flagella will be described. Most parameters change very rapidly during the brief motility period of fish sperm. Then will be considered the main environmental factors, ionic and/or osmotic signals, responsible of the activation of fish sperm motility. Then the metabolic compounds involved in cell energetics will be considered as their concentrations also rapidly change during the motility phase. An additional feature will then be discussed concerning the mechanisms by which fish sperm cell can be revived for a second motility round at the end of the first motility period. A model is proposed to explain how external osmolarity can control internal ionic composition, the latter being the key factor controlling flagellar activity.

Gadolinium, a mechano-sensitive channel blocker, inhibits osmosis-initiated motility of sea- and freshwater fish sperm, but does not affect human or ascidian sperm motility.

Exposure to hypo-osmotic or hyperosmotic environment triggers the initiation of fish sperm motility. In this article, we report that calcium and potassium channel blockers do not influence motility of puffer fish sperm but calmodulin antagonists reversibly decrease it, suggesting that calmodulin-Ca(2+) interactions are prerequisite for the initiation of sperm motility in this species. Gadolinium (a stretch activated ion channel blocker) decreased the motility of puffer fish sperm from 92 +/- 3% to 6 +/- 3% and that of carp sperm from 91 +/- 7% to 3.5 +/- 4.3% in a dose-dependent manner (10-40 micro M). The effect of gadolinium was reversible, suggesting that stretch activated ion channels participate in the initiation of sperm motility of the two species. Gadolinium inhibits changes in the isoelectric point of certain proteins of puffer fish sperm, which occur when sperm motility is initiated in a hypertonic solution. Anisotropy measurements showed that hypo-osmotic treatment, which initiates carp sperm motility, increased membrane fluidity. When hypo-osmotic treatment was given in the presence of gadolinium, the sperm membrane remained as rigid as in quiescent cells, while motility was blocked. By contrast, gadolinium did not influence the motility parameters of Ciona or human sperm. Based on these lines of evidence, we suggest that conformational changes of mechanosensitive membrane proteins are involved in osmolality-dependent but not osmolality-independent sperm.

Characterization of a cAMP-dependent protein kinase catalytic subunit from rainbow trout spermatozoa

The cyclic AMP-dependent phosphorylation of proteins is essential for the initiation of sperm motility in salmonid fishes. This study isolated cDNA for the catalytic subunit of a cAMP-dependent protein kinase (PKA-C) from rainbow trout testis. The deduced amino acid sequence shows 75–80% identity to sequences previously reported in other organisms. However, the N-terminal regions of PKA-C from the testis as well as ovary in the trout appear slightly shorter than those from other tissues, suggesting that small PKA-C might be specific to germ cells. An immunofluorescence study using polyclonal antibody against trout testis PKA-C shows that it localizes along sperm flagellum. Furthermore, immunoelectron microscopy revealed that PKA-C is anchored to the outer arm dynein of flagellar axonemes. These results suggest that PKA-C is involved in regulating the flagellar motility of sperm via phosphorylation of a subunit of the outer arm dynein.

Purification and Characterization of the 15-kDa Protein from the Sperm Flagella of Salmonid Fishes

Cyclic AMP-dependent phosphorylation of tyrosine residues in a protein with a molecular mass of 15-kDa is thought to play a key role in the initiation of sperm motility in salmonid fishes (7). In this study, flagellar proteins were solubilized with 7 M urea and fractionated by sucrose densitygradient isoelectrie focusing. A band relevant to 15-kDa was detected in the fraction at pH 5 on electrophoresis and analyzed using phosphotyrosine antibody. Analysis of the band by reversed phase chromatography showed a single peak, suggesting that the 15-kDa protein was completely purified. Amino acid sequence of His—Ile-Phe at the N-terminus was detected by analysis using peptide sequencer, and the fourth base could not be determined. Further analysis of the molecular structure of the protein by LC—MS/MS showed that partial amino acid sequence of the 15-kDa protein from tryptic fragments were similar to parts of the amino acid sequence of tubulin. The protein was not detected in inununoblots using anti-tubulin polyclonal antibody, suggesting that the 15-kDa protein may be not debris of the tubulin but a novel protein with a common partial sequence of tubulin.

Initiation of sperm motility depending on a change in external osmotic pressure in the noncopulatory marine cottid fish Gymnocanthus herzensteini

The initiation of sperm motility in a noncopulatory marine cottid fish, Gymnocanthusherzensteini, was examined. The spermatozoa, which were immotile in seminal plasma, initiated motility at osmolalities of more than 500 mOsm kg−1 in NaCl solution and 400 mOsm kg−1 in KCl and mannitol solutions, indicating that the initiation of sperm motility depends on changes in external osmolality, in contrast with that of the sperm of other marine cottid fish, which are motile in seminal plasma. This study revealed that there are plural manner of initiation of sperm motility in marine cottid fish, which are oviparous but include both copulatory and noncopulatory modes.

Proteasomes regulate the motility of salmonid fish sperm through modulation of cAMP-dependent phosphorylation of an outer arm dynein light chain.

Proteasomes are involved in ATP-dependent regulation of sperm motility in salmonid fish. We have demonstrated here by immunoelectron microscopy that proteasomes are located at the structure of the chum salmon sperm flagellum that attaches at the base of the outer arm dynein and extends toward the plasma membrane. Furthermore, substrates and inhibitors of proteasome inhibit the cAMP-dependent phosphorylation of a 22 kDa axonemal protein in chum salmon sperm. The 22 kDa phosphoprotein was solubilized by treatment of the axoneme with a high salt solution and subsequent sucrose density gradient centrifugation of the extract revealed that it cosedimented with 19 S outer arm dynein, indicating that it is a dynein light chain. These results suggest that proteasomes modulate the activity of outer arm dynein by regulating cAMP-dependent phosphorylation of the 22 kDa dynein light chain.

Use of computer assisted sperm analysis (CASA) for monitoring the effects of pollution on sperm quality of fish; application to the effects of heavy metals

Computer assisted sperm analysis (CASA) has been used to analyse the effects of cadmium and zinc on the motility of fish sperm treated either for 24 h after partial dilution in an extender, or during the final dilution step on the microscope stage during video recording. The progressive motility of catfish sperm, which was not affected by storage for 24 h, was decreased after exposure to 100 ppm cadmium or 2000 ppm zinc in extender for 24 h. Only 1000 ppm cadmium or 2000 ppm zinc had any effect on motility when added to sperm at the final dilution stage. These results suggest that concentrations of cadmium which have been found to be present in fish gonads as a result of bioaccumulation from the water or diet may decrease the motility of sperm during maturation or storage in the testis. Cadmium toxicity was significantly decreased in the presence of an equal amount of zinc, suggesting an equilibrium between zinc and cadmium at binding sites in the sperm. Computer assisted sperm analysis is shown for the first time to provide a rapid and quantitative measure of the effects of environmental pollutants on the quality of fish sperm. It is suggested that the methodology described may form the basis for a standard toxicity test to assess the potential hazards of environmental pollutants on reproduction.

Implication that potassium flux and increase in intracellular calcium are necessary for the initiation of sperm motility in salmonid fishes.

Flux of K+ and changes in intracellular Ca2+ in the sperm of salmonid fishes were measured with spectrophotometry, ion electrode, microscopic fluorometry, and radioisotope accumulation. Release of K+ occurred at the initiation of sperm motility which is induced by decrease in external K+ and the K+ efflux and sperm motility were inhibited by K+ channel blockers. Intracellular Ca2+ increased within a short period in K(+)-free condition, and the accumulation of 45Ca in sperm cells was higher in motile sperm than that in immotile sperm. The efflux of K+ and the increase in intracellular Ca2+ were suppressed when external K+ concentration increased, i.e., sperm remained immotile. These results suggest that efflux of K+ through K+ channel and subsequent increase in intracellular Ca2+ are prerequisite for the initiation of sperm motility.

The role of hormones in the acquisition of sperm motility in salmonid fish.

In salmonid fish, spermatozoa taken from the testes are immotile, but acquire motility during their passage through the sperm duct. Using male masu salmon (Oncorhynchus masou), we found that gonadotropin-induced testicular production of 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (17 alpha, 20 beta-DP), the oocyte maturation-inducing hormone of salmonid fish, is responsible for the acquisition of sperm motility. However, neither testosterone (T) nor 11-ketotestosterone (11-KT), the two major androgens in teleost fish, were effective. We also present evidence that 17 alpha, 20 beta-DP action is mediated through an increase in sperm duct pH, which in turn increases the cAMP content of sperm allowing the acquisition of motility.