Subpopulation distribution of motile sperm relative to activation medium in steelhead (Oncorhynchus mykiss).

In the present study, steelhead sperm were activated in artificial tap water, ovarian fluid, activating saline, or in combinations of these media, and motility characteristics were determined using computer-assisted sperm analysis. Motility characteristics of individual sperm were then assessed to test the hypothesis that motile sperm are distributed among discrete subpopulations and that their distribution is influenced by the activation medium. Analysis with k-means clustering detected three discrete motile sperm subpopulations in steelhead semen, regardless of the activation medium. Based on multivariate analysis of variance, proportions of these subpopulations did not differ between sperm activated with ovarian fluid and activating saline, or any combination of these two media. However, subpopulation distributions for sperm activated with either ovarian fluid or activating saline were influenced by the level of dilution of these media in artificial tap water. There was an increase in the number of sperm in high velocity (curvilinear), high straightness, and high wobble subpopulation with increased levels of ovarian fluid or activating saline. The change in sperm motility characteristics with a change in activation medium may play a role in normal fertilization, as discharged sperm pass from seminal plasma and water through ovarian fluid en route to the egg.

Metabolism of motile zebrafish sperm.

As metabolism of motile fish sperm is not well understood, the current study examined the metabolism of saline-activated zebrafish (Danio rerio) sperm. Activation of sperm with inhibitors of oxidative phosphorylation (potassium cyanide, 2,4 dinitrophenol or carbonyl cyanide 3-cholorophenylhydrazone) negatively impacted sperm motility by 60-90 s postactivation. Incubation of quiescent sperm with 2,4 dinitrophenol prior to activation resulted in a 67% decrease in the percent motile sperm assessed 15s postactivation. Thus, production of ATP in quiescent sperm is important for motility upon activation and nascent ATP production via oxidative phosphorylation by motile sperm appears important at 60-90 s postactivation. Exposure of sperm to iodoacetamide, an inhibitor of creatine kinase, at activation was without effect. However, incubation of quiescent sperm with iodoacetamide prior to activation resulted in a 77% reduction in percent motile sperm and decreased velocity and wobble at 15s postactivation. These results suggest that creatine kinase and phosphocreatine shuttle are physiologically important at, or shortly after the initiation of motility. Finally, sperm were exposed to lactate, pyruvate, or acetate as well as to several monosaccharides upon activation. The results provided no evidence supporting any metabolic role of exogenous organics (potentially from the female via ovarian fluid) in sperm once motility has begun.

Influence of osmolality and ions on the activation and characteristics of zebrafish sperm motility.

Despite the prevalence of zebrafish as a model scientific organism, understanding sperm function in this species is essentially limited to observations that osmotic shock initiates motility. During natural spawning, sperm encounter a range of environmental salinities as well as freshwater mixed with egg-associated ovarian fluid (OF), thus sperm are likely to be exposed to saline prior to egg contact. Effects of saline on sperm function in this model species are unknown, but likely to be important. Using computer assisted sperm analysis, this study addressed the effects of osmolality of spawning media and ionic composition and pH on the proportion of sperm becoming motile at activation (motility), as well as sperm velocity and path. When activated with tap water, motility was maximal (80%) at 10s (earliest time measured), declining to 5% by 87 s postactivation. With activation at moderate osmolalities ( approximately 160-200 mmol/kg) initial motility was decreased relative to low osmolality, increased from 10 to 30s, and subsequently declined less rapidly (motility in 80 mM NaCl was 35%, 80%, and 60% at 10, 30 and 147 s, respectively). Thus, moderate osmolality increased duration, but introduced a temporal lag in motility onset. With moderate osmolalities, the rate of velocity decay was less than that with tap water activation. Sodium chloride and sucrose similarly impacted both motility and velocity. Replacement of NaCl with KCl, pH values ranging from 6.8 to 8.4, or the presence of gadolinium were without effect. Motility, but not velocity, was slightly supressed by Ca(2+). Therefore, whereas pH and concentrations of Ca(2+) or K(+) of OF are unlikely to impact fertility via sperm motility, the OF contribution to spawning media osmolality may have pronounced effects on motility and velocity of sperm, factors previously correlated with fertility in other species.

Initiation of motility by steelhead (Oncorhynchus mykiss) sperm: membrane ion exchangers and pH sensitivity.

Initiation of motility in salmonid sperm is sensitive to the pH of the extracellular medium, however, the basis of this sensitivity is not clear. Sperm incubated in an immobilization buffer (SI) at low pH ( approximately 7.1-7.2) become motile when diluted with activating medium (AM) at high ( approximately 8.5) but not low pH. Based on this observation, various agents were tested to determine whether the onset of steelhead sperm motility upon activation with high pH AM, following incubation with low pH SI, could be blocked by inhibiting membrane exchangers postulated to be important in intracellular pH (pHi) regulation. Amiloride (inhibitor of proton:sodium exchange), SITS and DIDS (inhibitors of anion exchange) and bafilomycin A 1 (inhibitor of H(+)-ATPase activity) were not effective in this experimental design. However, regardless of SI pH, DIDS was effective in blocking motility as was replacing chloride with thiocyanate or including the chloride channel blocker, niflumic acid, in SI suggesting that chloride efflux plays a key role in motility initiation. Nonetheless, the results of this study suggest that the rapid onset of sperm motility with activation at high pH following incubation at low pH is probably not based on rapid adjustment of pHi via membrane exchangers/transporters but rather due to an effect of pH on motility-associated processes at the extracellular surface of the sperm.