Effects of tyrosine kinase inhibitor on the motility and ATP concentrations of fowl spermatozoa

The possible role of tyrosine kinase in the regulation of fowl sperm motility was investigated by using a stable analogue of erbstatin, methyl 2,5-dihydroxycinnamate (2,5-MeC), a specific inhibitor of tyrosine kinase. This inhibited the motility of intact spermatozoa at 30°C in a dose-dependent manner. In contrast, the motility of demembranated spermatozoa was not inhibited by the same concentrations of 2,5-MeC. At 40°C, both intact and demembranated spermatozoa were almost immotile with or without 2,5-MeC. Additionally, intact spermatozoa, stimulated by the addition of Ca²⁺ or calyculin A, a specific inhibitor of protein phosphatases, lost their motility with the subsequent addition of 2,5-MeC at 40°C. However, unlike the motility, the ATP concentrations of spermatozoa were maintained in about 30–35 nmol ATP/10⁹ cells during these incubation periods. The activity of tyrosine kinase of spermatozoa at 30°C, estimated by measuring the phosphorylation of a synthetic peptide substrate, RR-SRC, was 0.17 pmol/min per milligram of protein. This activity was lower than that of fowl testes or chick brain but higher than that of chick liver. These results suggest that tyrosine kinase activity, which is not retained in the axoneme and/or accessory cytoskeletal components, may be involved in the maintenance of flagellar movement of fowl spermatozoa at 30°C. Mol. Reprod. Dev. 49:196–202, 1998. © 1998 Wiley-Liss, Inc.     

Glycolytic enzymes of fowl and turkey spermatozoa

• 1.1. It was confirmed that, under anaerobic conditions, fowl spermatozoa formed lactate from glucose thirteen times faster than turkey spermatozoa.
• 2.2. The profiles of glycolytic enzyme activities were similar for spermatozoa from both species; however fowl spermatozoal activities were generally 2- to 4-fold higher.
• 3.3. Exceptions were glycerophosphate mutase and lactate dehydrogenase activities which were respectively 9.5 and 41 times greater in fowl spermatozoa.
• 4.4. In both species, spermatozoal glyceraldehyde-3-phosphate dehydrogenase had the lowest activity of the glycolytic enzymes.

     

The phospholipid-bound fatty acids of fowl and turkey spermatozoa

The composition of the phospholipid-bound fatty acids in the spermatozoa of the turkey, Meleagris gallopavo, and fowl, Gallus domesticus, was studied. The types of fatty acids were similar in the two birds. The ratio of polyunsaturated : saturated fatty acids was generally low but slightly higher in the turkey than in the fowl. The significance of the findings in relation to the origin of the semen collected in these gallinaceous birds and the greater difficulty of freezing turkey spermatozoa was discussed.     

Excess nuclear DNA in spermatozoa of guinea fowl

The proportion of spermatozoa with elongated nuclei in ejaculates from a strain of guinea fowl was estimated, subjectively, to range from approximately 1 to 6%. It was confirmed by image analysis that in an ejaculate from one male, the distribution of nuclear lengths was bimodal, with a distinct population comprising 10% of spermatozoa having a mean nuclear length that was 52% greater than that of the remaining 90%. Furthermore, the mean DNA content of the 'large-nuclei' population was 1.85 times (not significantly different from twice) that of the main sperm population. The proportion of large-nuclei spermatozoa that was motile was less than that of normal sperm (31% versus 59%) and the velocity of motile spermatozoa was also less (24 microm/s versus 72 microm/s). The poor motility of the large-nuclei spermatozoa in vitro was reflected in their limited performance in vivo, since only 1.1% were found associated with the egg outer perivitelline layer. This is the first report to quantify the occurrence of, presumed, polyploid spermatozoa in a domestic bird. The incidence of such spermatozoa in commercial guinea fowl and other domestic poultry and the genesis and effects on fertility of such spermatozoa may be significant.     

The effect of anoxia on fowl spermatozoa: Recovery of fertilizing ability,motility, and cellular concentrations of potassium and ATP

Fowl semen when diluted in a glutamate-based medium without glucose, gradually lost its fertilizing ability during 4 hr of anaerobic incubation at 30°C. This incubation regime offered a system by which various in-vitro tests of spermatozoal viability could be assessed for their usefulness as monitors of fertilizing ability. Widely used tests such as spermatozoal enzyme leakage, dye exclusion, and morphology as assessed by light microscopy showed no change in spermatozoal status as the fertilizing ability declined. However the ability of sperm, during a short aerobic incubation to restore their motility and ATP and K⁺ concentrations, declined as did their fertilizing ability. When glucose was added to this re-aeration medium, spermatozoal motility, K⁺ and ATP concentrations, and fertilizing ability were restored to optimal levels. Thus the fertilizing ability of fowl sperm, following anaerobic storage at 30°C, appeared to be related to their ability to restore ATP and K⁺ concentrations and motility. An initial event in the loss of fertilizing ability was a loss in the ability of sperm to oxidise endogenous substrates. This could be restored by the addition of glucose.     

Interdependence of mitochondrial ATP production and extramitochondrial ATP utilization in intact spermatozoa

The dependence of both respiration and total activity of ATP-consuming reactions on the cellular adenine nucleotide pattern was investigated in intact bovine spermatozoa. ATP consumption was manipulated by inhibition with vanadate and activation with caffeine, leading to a decrease or increase in the rate of respiration up to 70% or 20%, respectively. Oligomycin blocked the respiration to the same extent as did vanadate, suggesting that the total extramitochondrial ATP-consuming activity is vanadate-sensitive. The major part of ATP utilization must be linked to dynein ATPase, since inhibition of (Na⁺, K⁺) ATPase by ouabain showed only a small effect on respiration (−17%). Being a potent inhibitor of dynein ATPase, vanadate drastically reduced the amount of motile cells, whereas caffeine tended to increase the intensity of motion. The effects of vanadate or caffeine on respiration were paralleled by changes in cellular ATP, reflecting the response of mitochondrial respiration on the cellular ATP/ADP ratio. Respiration was found to depend on changes in the ATP/ADP ratio in the range from about 3 (+ caffeine) to 9 (+ vanadate). The range of response of ATP consumption to the ATP/ADP ratio was determined by varying the mitochondrial ATP production via the concentration of lactate which was used as substrate. The measured effects on both respiratory rate and ATP/ADP ratio suggested that ATP consumption was markedly dependent on ATP/ADP ratios below 5. It is concluded that lactate concentrations above 1 mM sufficiently supply bovine spermatozoa with substrate and the energy turnover is mainly limited by the activity of dynein ATPase rather than by the capacity of mitochondrial oxidative phosphorylation.     

In vitro effects of beta-carotene for the motility, ATP, and intracellular free Ca(2+) concentrations of fowl spermatozoa

The motility of intact fowl spermatozoa was vigorous at 25 degrees C, but decreased gradually following the addition of 0-100 microM beta-carotene in a dose-dependent manner. Even in the presence of stimulators of fowl sperm motility, such as Ca(2+) or calyculin A, the motility of intact spermatozoa at both 25 and 40 degrees C remained inhibited following the addition of beta-carotene. Under all of these circumstances, sperm ATP concentrations were not reduced by the addition of beta-carotene. Moreover, the motility of demembranated spermatozoa was not inhibited by the addition of the same concentrations of beta-carotene. No changes in intracellular free Ca(2+) concentrations, measured by means of a fluorescent Ca(2+) indicator, fura-2, were observed in intact beta-carotene -treated spermatozoa. These results suggest that beta-carotene is involved in the inhibition of the flagellar movement of fowl spermatozoa without change in energy production, and that the target of beta-carotene might be present in the cytoplasmic matrix and/or the plasma membrane, but not retained in the axoneme and/or accessory cytoskeletal components.     

Flagellar movement in demembranated preparations of ejaculated fowl spermatozoa

Triton X-100 at a concentration of 0.1% in the extraction medium was optimal for demembranating fowl spermatozoa. The most suitable conditions for reactivation were obtained when a medium composed of 0.5 mM-ATP, 25 mM-potassium glutamate, 10(-7) M-CaCl2, 20 mM-Tris-HCl(pH 7.9), 1 mM-MgSO4, 1 mM-dithiothreitol and 0.2 M-sucrose was used. More than 60% motile spermatozoa were obtained under these conditions. The addition of 1 or 10 microM-cAMP did not appreciably affect motility. Intact and demembranated spermatozoa were immotile at 40 degrees C, whilst at 30 degrees C motility was restored.     

Regulation of the motility of fowl spermatozoa by calcium and cAMP

Using an objective light-scattering technique, it was confirmed that washed fowl spermatozoa become immotile as the temperature is raised from 30 degrees C to the normal body temperature of 40-41 degrees C. Motility of washed spermatozoa was restored at 40 degrees C by the addition of caffeine or calcium, both stimulating motility to a maximum in a dose-dependent manner. Neither effector stimulated the motility of spermatozoa at 30 degrees C. Caffeine, but not calcium, caused an increase in sperm cAMP levels at 40 degrees C. The concentrations of calcium and cAMP in untreated spermatozoa were not significantly different in samples incubated at 30 degrees C or 40 degrees C.     

Temperature-mediated regulation of calcium flux and motility in fowl spermatozoa.

The motility of fowl spermatozoa at various temperatures was shown to be a function of their intracellular calcium content, measured after hypotonic lysis of the cells. Retention of calcium by spermatozoa, with consequent enhancement of motility, increased as the temperature was lowered from 40 degrees to 30 degrees C. Raising the temperature within this range subsequently reduced calcium retention and motility again. The temperature-dependent retention of calcium was a function of the rate of calcium efflux rather then influx. The temperature-sensitive efflux mechanism appeared to involve a Ca2+ ATPase which was relatively inactive at 30 degrees C, but active at 40 degrees C.     

Intracellular free Ca2+ concentration in fowl spermatozoa and its relationship to motility and respiration in spermatozoa.

The ability of fowl spermatozoa to accumulate and de-esterify the intracellular fluorescent Ca2+ indicator fura-2 was established. The cytosolic Ca2+ concentrations, measured by this technique, did not change after the addition of 1 mmol EGTA l-1. Subsequently, addition of the calcium ionophore A23187 caused a reduction in cytosolic Ca2+ concentrations, presumably by efflux of Ca2+ from the spermatozoa. Intracellular free Ca2+ concentrations were then significantly increased by the addition of 1 mmol CaCl2 l-1. The motility of demembranated spermatozoa gradually decreased after the addition of EGTA alone or EGTA with A23187, but was instantly restored by the addition of CaCl2 in the presence of both EGTA and A23187. Unlike demembranated spermatozoa, intact spermatozoa maintained their motility, even after the addition of EGTA, but their motility was reduced by the addition of A23187 in the presence of EGTA. The addition of A23187 also reduced the rate of oxygen consumption, but not the ATP concentrations in intact spermatozoa. These results demonstrate that the motility and respiration of fowl spermatozoa are strongly influenced by their intracellular Ca2+ concentrations.     

Disintegration of spermatozoa in the infundibular sperm-host glands of the fowl

Summary The disintegration of spermatozoa in the infundibular sperm-host glands of the fowl was investigated by electron microscopy. After the 15th day following artificial insemination, secretory granules in the epithelial cells of the sperm-host glands increase in number and size, and subsequently the contents of the granules are released into the glandular lumen, so that the electron density of the lumen increases. At this stage, spermatozoa stored in the glands begin to undergo degenerative changes starting from the head. The heads become distended and chromatin of the nucleus begins to disperse as small masses, simultaneously with the destruction of the acrosome. As the dispersion of chromatin progresses, mitochondria of the middle piece become distended and irregular in shape, and then disintegrate. At the last stage, most of the organelles have disappeared, but the fibrous sheath and axial-filament complex are still identified.The authors are greatly indebted to Assoc. Prof. Osamu Koga for his invaluable advice. The authors also wish to thank Mr. Takayuki Möri for his helpful suggestions and technical advice. This investigation was supported by a grant from the Ministry of Education of Japan (156185)     

The mechanisms of reversible immobilization of fowl spermatozoa at body temperature

Intact fowl spermatozoa became almost immotile at 40 degrees C, but motility increased significantly at 30 degrees C. The oxygen consumption at both temperatures was 8-11 microliters O2/10(10) spermatozoa.min-1. The ATP concentration at 40 degrees C was higher than that at 30 degrees C but ADP concentration at 30 degrees C was higher than that at 40 degrees C. Consequently, the ATP/ADP ratio at 30 degrees C (1.9-2.2) increased to 3.5-3.7 at 40 degrees C. The motility of intact spermatozoa at 40 degrees C was effectively restored by 2 mM-Ca2+, 10% seminal plasma and 10% peritoneal fluid taken at the time of ovulation. In contrast, these effectors did not restore the motility of demembranated spermatozoa at 40 degrees C. Motility of demembranated spermatozoa was restored at 30 degrees C. These results suggest that the immobilization of fowl spermatozoa at 40 degrees C occurs due to a decrease in flagellar dynein ATPase activity. Furthermore, the action of effectors for motility such as Ca2+ may not be directly on the axoneme, but mediated by solubilized substances which have been removed by demembranation of the spermatozoa.     

The relationship between ATP content and motility of bovine spermatozoa

The ATP content of bovine semen was measured using a bioluminescent technique. No ATP was detected in seminal plasma or diluent. The mean concentration of ATP (+/- sd) in ejaculated semen was 76.5+/-38.21 n moles ATP/10(8) spermatozoa and in diluted, frozen and thawed semen 10.00+/-4.74 n moles ATP/10(8) spermatozoa. The observed motilities of ejaculated and of diluted, frozen and thawed semen were linearly related to the ATP content r=0.652; p<0.01 and r=0.466; p<0.001 respectively). The mean ATP content and motility calculated for each bull were similarly related (r=0.857; p<0.02 and r=0.607; p<0.05). The technique shows promise in providing an objective measure of spermatozoan activity.     

Fertilizing capacity and ultrastructure of fowl and turkey spermatozoa before and after freezing.

The fertilizing capacity, motility and ultrastructure of fowl and turkey spermatozoa were examined at various stages of the freezing process. For both species, fertility and motility were depressed after equilibration with dimethyl-sulphoxide at 5 degrees C. After freezing, motility was maintained at 55% for fowl spermatozoa and 40% for turkey spermatozoa; however, fertility was 55% for the fowl and 0% for the turkey. Qualitatively, the damage to the spermatozoa of both species was nearly identical, as revealed by scanning and transmission electron microscopy. The plasmalemma was the primary site of damage. 'Bent' spermatozoa, coiled tails and swollen mitochondria were also present. Damage to the acrosome was only observed in spermatozoa which had been frozen to -180 degrees or -196 degrees C. These changes were attributed to adverse osmotic conditions. Binding of cationic ferritin to the plasmalemma of spermatozoa from both species remained unaltered.