Zbl. Vet. Med. A, 26, 810-814 (1979) @ 1979 Verlag Paul Parey, Berlin und Hamburg ISSN 0300-871 l/ASTM-Coden: ZVRAAX

Deprzrtment of Biochemistry, College of Veterinary, Science and Animal Husbandry, Jabalpur ( M . P.),India

Adenosine Triphosphatase and Fructose-l,6-Diphosphatase Activity in Buffalo Semen BY

R. S. DHANOTIYA and R. K. SRIVASTAVA With one table (Received f o r publication M a y 6 , 1979)

The enzyme adenosine triphosphatase catalyses the hydrolysis of adenosine triphosphate into adenosine diphosphate and inorganic phosphate, with the release of a large amount of free energy. This energy is used as the immediate source of energy for flagellar contraction and other metabolic activities of spermatozoa. (1966) has reported the activity of ATPase in the Although NELSON different cell parts of the spermatozoa and the differences in the activity of the enzyme in the immotile and motile spermatozoa, there are no reports on the distributions of these enzymes in the different fractions of buffalo spermatozoa. The enzyme fructose 1 : 6 diphosphatase catalyses the hydrolysis of fructose 1 : 6 diphosphate into fructose 6 phosphate and inorganic phosphate. The substrate cycle of phosphofructokinase and FDPase is important i n a 1967). However, number of metabolic events (UNDERWOOD and NEWSHOLME, the distribution of the enzyme FDPase in the different cell parts of the buffalo spermatozoa and its precise role in mammalian semen are not known. The spatial distribution of these two enzymes among the structural components of the buffalo spermatozoa may provide a useful insight into its possible role as a mediator between mechano-chemical transduction, particularly in the ATP-ATPase plasticizer activity. In the present communication we report on the distribution of FDPase and ATPase in the seminal plasma and in the head, midpiece and tail fraction of buffalo spermatozoa. In the case of ATPase a comparative study has also been made with bull spermatozoa. U. S. Copyright Clearance Center Code Sratemenr:

0300-871 1/79/2610-0810$02.50/0

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Adenosine Triphosphatase

Material and Methods Experiments were conducted with t w o replicate semen samples from t w o buffalo bulls a n d one red Dane bull in the case of ATPase and six replicates from four buffalo bulls in the case of FDPase. Semen was centrifuged to separate the seminal plasma from the sperm pack. The sperm pack was washed twice with distilled water to remove traces of seminal plasma. Fractionation of sperm into head, midpiece and tail was accomplished by the (1976). The washed sperm pack was diluted three-fold method of RAJPUT and SRIVASTAVA with distilled water and disintegrated with the aid of a vibronic Ultrasonicator (Modcl VPL-P 1) for 60 seconds at a frequency of 25 kilocycles per second. A density gradient of sucrose was generated by layering 1 ml. each of 5On/i0, 40°/o, 30°/o and 20 O / o sucrose in ascending order in a centrifuge tube. An amount of 0.7-1.0 ml. of ultrasonicated sperm was layered on top of the sucrose gradient and centrifuged a t 1,050 xg. for 8 min. T w o distinct layers were formed and a sizable amount settled at the bottom of the centrifuge tube. The t w o layers were carefully removed with a pasteur pipette. The top layer comprised tails; the middle layer consisted of midpieces and the head fraction settled at the bottom. The purity of the fractions was ascertained by cytorhemical assay of succinic dehydrogenase activity. (1966). 1 ml. of the assay The enzyme ATPase was assayed by the method of NELSON mixture consistcd of 0.1 ml. of 10 mM ATP, 0.1 ml. of 10 mM CaCI,, 0.1 ml. of 25 mM cysteine-HCI, 0.3 ml. of 90 mM Tris buffer ( p H 8.6) and 0.4 ml. of enzyme source after appropriate dilution. The reaction mixture was incubated for 10 min. a t 37 ' C and thc reaction was stopped with 1 ml. of 10 O / o trichloroacetic acid. The tubes were centrifuged and 0.5 ml. of the aliquot was used for Pi estimation. (1952). The enzyme FDPase was assayed by the method of POGELLand MCGILVERY 1 ml. of the assay mixture consisted of 0.1 ml. of 0.05 M F D P ( p H 7-7.4) 0.1 ml. of 0.05 M MgSO,, 0.1 ml. of enzyme source and 0.7 ml. of 0.05 M borate buffer ( p H 9.5). After 30 min. of incubation at 37 "C the reaction was stopped by the addition of 1 ml. of 10°/o tricfiloroacetic acid. P i was estimated from the aliquot by the method of CHENet al., (1956). Protein was estimated by the method of LOWRYet al., (1951).

Results The enzyme activity in the seminal plasma and the head midpiece and tail fractions is presented in Table 1. Table I Adenosine triphosphatase and fructose 1 : 6 diphosphatase activity in bovine semen

Bull

Buffalo AT Pase

FDPase

ATPase

Seminal Plasma

0.476

3.78

0.215

Head

0.967 (8.981

Nil

0.793 (6.43)

Midpiece

3.70 (34.42)

Nil

3.75 (30.4)

Tail

6.09 (56.601

1.05

7.78 163.15)

~

~

FDPase

~

Figs. in parenthesis present the percentage distribution.

The ATPase activity was present maximally in the tail fraction, followed by midpiece, and a very low activity in the head fraction. There was no statistically significant difference in the distribution of the enzyme in different fraction of the spermatozoa of the two species. All the FDPase activity appeared in the tail fraction only. Substantial activity of the two enzymes,

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was also present in the buffalo seminal plasma, although the activity of FDPase was much higher.

Discussion The relative distribution of the enzyme ATPase is comparable to that reported by NELSON et al. (1970). The experimental manipulation of the spermatozoa may lead to certain et al. deviations from the normal distribution of the enzyme in vivo. NELSON (1970) reported that the enzyme is completely absent from the naturally decapitated head of bull spermatozoa but shows activity in ultrasonicated samples. This was ascribed to the difficulty of completely removing minute tail residues from the heads. They further reported that the enzyme activity appeared consistently greater in tail fraction in ultrasonicated than in the naturally decapitated specimens. Although we have determined the distribution of total ATPase in the different fractions, we have not been able to partition the soluble and parti(1971) reported that bull spermatoculate forms of the ATPase. HARRISON zoa contain only 12 O / o soluble ATPase of the total enzyme activity. O n the contrary, phosphofructokinase showed a very high concentration of the soluble form. Extraction of the total sperm enzyme activity, therefore, is difficult since they are held within organelles o r bound within membrane vesicles and are extracted relatively slowly, even after ultrasonication. An interrelationship between ATPase and protein phosphorylation has and CASILLAS (1975). A high ATPase activity in been reported by HOSKINS the subcellular particle reportedly interfered with soluble protein phosphorylation. The functional importance of ATP/ATPase, cyclic AMP and cyclic AMP phosphodiesterase inhibitors in spermatozoa1 motility has been reviewed (1971). GARBERS et al. (1973) demonstrated that cyclic AMP by HARRISON phosphodiesterase inhibitor caused a rise in the intracellular level of cyclic AMP with a resultant increase in motility accompanied by a decrease in intracellular ATP level. Filipin, which modifies the plasma membrane of the spermatozoa and permits leakage of cyclic AMP, inhibited fructolysis and 1971). There was motility in monkey spermatozoa (HOSKINSand STEPHENS, a dramatic fall of ATP level. Exogenous ATP, although, it partially restored Fructolysis, did not apparently restore motility. Thus the effect of cyclic AMP is direct on the motility, while the effect on metabolism is mediated via changes in ATP and the energy charge ratio (ATP)/(ADP AMP). When motility increases, the rate of ATP breakdown through the ATPase also increases and the intracellular ATP level is lowered, with a resultant decrease in the energy charge ratio which relieves the inhibition of glycolysis and respiration and stimulates metabolism. Certain sperm protein kinases which are ATPases in nature have been demonstrated as an integral component of the flagellar protein complex which and CASILLAS, links ATP breakdown with contractile processes (HOSKINS 1975). QUINN and WHITE(1968) reported that although mammalian spermatozoa exhibited respectable basal ATPase activity in the absence of cations, the activity was enhanced by the addition of cations Na, K and Mg. Calcium was as effective as Mg2+ as an activator of the enzyme and maximum activity was observed under the stoichiometry of equivalent substrate and activator concentration. It has also been reported that approximately 30 O / o of the ATPase was oubain-sensitive and is involved in active transport through the and WHITE,1968). membrane (QUINN

+

Adenosine Triphosphatase

813

The FDPase activity was present in the tail fraction only. It appears that this enzyme is present in highly solubilised form and therefore appears in the tail fraction, which happens to be the supernatant fraction of the ultrasonicated sperm on the sucrose gradient. Both enzymes were present in the seminal plasma. However, it is difficult to ascertain whether these enzymes are actually constituents of the seminal plasma or whether they have arisen as a result of leakage of the enzyme from the spermatozoa, as has been reported in the case of other enzymes (1967) has reported that the enzyme (GRAHAMand PACE,1967). NELSON is relatively washable and is liable to be washed from the flagella during the course of manipulation. Summary The enzymes ATPase and FDPase were assayed in the head, midpiece and tail fractions of buffalo spermatozoa and seminal plasma. The distribution of ATPase in the different fractions of the spermatozoa of the bull was also studied. The highest activity of ATPase was observed in the tail fraction, followed by the midpiece, and a very low activity in the head fraction. The FDPase was localized in the tail fraction. While ATPase is mostly particulate-bound, FDPase appeared to be in a highly soluble form. No significant differences were observed in the distribution of ATPase in the different fraction of the spermatozoa of bull and buffalo bull. Acknowledgement Our thanks are due to the Indian Council of Agriculture Research for providing the funds. Thanks are also due to Dr. M. R. PATEL,Dean for providing the facilities and keen interest in the work. We are indebted to for his help in many ways in the execution of the work. Dr. R. G. AGRAWAL Zusammenfassung Aktivitat von Adenosin-Triphosphatase und Fruktose-1,6-Diphosphatase im Biiffelsperma Im Kopf, Mittelstuck und Schwanz der Spermatozoen sowie im Plasma von Buffel- und Bullensperma wurde die Aktivitat der ATPase und FDPase bestimmt. Die groi3te Aktivitat der ATPase wurde im Schwanz gefunder,. Wesentlich geringer war die ATPase-Aktivitat im Mittelstuck und sehr klein im Kopf. Die FDPase war im Schwanz lokalisiert. Wahrend die ATPase zum grogten Teil an Partikel gebunden ist, durfte die FDPase vor allem in geloster Form vorkommen. Signifikante Unterschiede hinsichtlich des Auftretens der ATPase in den verschiedenen Teilen der Spermien von Buff eln und Bullen wurden nicht gefunden.

RCsum6 ActivitC de l’adhosine-triphosphatase et de la fructase-1,6-diphosphatase dans le sperme du buffle L’activitk de 1’ATPase et de la FDPase a ktk dkterminke dans la t k e , le corps moyen et la queue des spermatozoydes ainsi que dans le plasma d u sperme de buffles et de taureaux. La plus grande activitk de 1’ATPase a Ctk trouvke dans la queue. L’activitk de 1’ATPase a ktk nettement plus faible dans le corps moyen et tr& faible dans la t&te. La FDPase a ktk localiske dans la queue. Alors que 1’ATPase est like en grande partie A des particules, la FDPase apparakrait avant tout en solution. O n n’a pas trouvk de diffkrences

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DHANOTIYA and SRIVASTAVA

significatives en ce qui concerne l’apparition d’ATPase dans les diffkrentes parties des spermatozoydes des buffles et des taureaux.

Resumen Actividad de la adenosintrifosfatasa y fructasa-1,6-difosfatasa en el esperma de blifalos Se valoraron las actividades de la ATFasa y FDFasa en las fracciones cabeza, pieza media y cola de 10s espermatozoides y en el plasma seminal de bhfalos y toros. La actividad mls elevada de la ATFasa se ha116 en la cola. Bastante menor era la actividad de la ATFasa en la pieza media y muv escasa en la cabeza. La FDFasa se hallaba localizada en la cola. Mientras que la ATFasa estl ligada en gran parte a particulas, la FDFasa suele presentarse, ante todo, en forma disuelta. No se hallaron diferencias significantes con respecto a la aparici6n de la ATFasa en las distintas partes de 10s esperm3tozoides de blifalos y toros. References CHEN,P. S., Jr., J. Y. TORIBARA, and H. WERNER,1956: Microdetermination of phospho;us. Anal. Chem. 28, 1756. GARBERS, D. L., N. L. FIRST,and H. A. LARDY,1973: The stimulation of bovine epididymal sperm metabolism by cyclic nucleotide phosphodiesterase inhibitors. Biol. Reprod. 8, 589. GRAHAM,E. F., and M. M. PACE, 1967: Some biochemical changes in spermatozoa due to t’reezing. Cryobiology, 4, 75. HARRISON, R. A. P., 1971: Glycolytic enzymes in mammalian spermatozoa. Biochem. J. 124, 741. HARRISON, R. A. P., 1975: Aspects of the mammalian spermatozoa. In: The Biology of Male J., and 7 . A. RACEY. gamete. Ed. by DUCKETT, HOSKINS, D. D., and E. R. CASILLAS,1975: Function of cyclic nucleotides in mammalian spermatozoa. In: Handbook of physiology. Sect 7, Vol. 5, pp. 453. HOSKINS,D. D., and D. T. STEPHENS,1971: Filipin-induced inhibition of fructolysis and motility in monkey spermatozoa. Biol. Reprod., 5 , 330. LOWRY,0. H., N . S. ROSEBROUGH, A. L. FARR,and R. J. RANDALL, 1951 : Protein measurement with fohn phenol reagent. J. Biol. Chem. 193, 265. NELSON,L., 1966: Enzyme distribution: Naturally decapitated Bull spermatozoa: Acetylcholinesterase, Adenylpyrophasphatase and Adenosinetriphosphatase. J. Cell. Physiol., 68, 113. NELSON,L., 1967: Sperm motility. In: Fertilization. Ed. by METZ, C. B., and ALBERTO MONROY.Academic Press. N Y London. NELSON, L., 1975: Spermatozoan motility. In: Handbook of Physiology. Sect. 7, Vol. 5, pp. 421. NELSON,L., A. V. MACGRADY, and M. E. FANGBONER, 1970: Control of flagellar movement. In: Comparative Spermatology. Ed. by BACCIOBACCETTI.Academic Press N Y London, 465. POGELL, B. M., and R. W. MCGILVERY,1952: The proteolytic activities of fructose 1 : 6 diphosphatase. J. Biol. Chem. 197, 293. QUINN,P. J., and I. G. WHITE, 1968: Distribution of ademosine triphosphatase activity in ram and bull spermatozoa. J. Reprod. Fert. 1 5 , 449. RAJPUT,Y. S., and R. K. SRIVASTAVA, 1976: A density gradient method for fractionation of buffalo spermatozoa. Curr. Sci. 4 5 , 181. UNDERWOOD, H . A., and E. A. NEWSHOLME, 1967: Control of glycolysis and gluconeogenesis in rac kidney cortex slices. Biochem. J. 104, 300. Address of author: Dr. R. S. DHANOTIYA,Department of Biochemistry, College of Veterinary, Science and Animal Husbandry, Jabalpur. M. P., India.

Adenosine triphosphatase and fructose-1, 6-diphosphatase activity in buffalo semen.

Zbl. Vet. Med. A, 26, 810-814 (1979) @ 1979 Verlag Paul Parey, Berlin und Hamburg ISSN 0300-871 l/ASTM-Coden: ZVRAAX Deprzrtment of Biochemistry, Col...
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