Enzyme Distribution in “Naturally-decapitated” Spermatozoa : Acetylcholinesterase, Adenyl- pyrophosphatase and Adenosine-

Bull

triphosphatase ’ LEONARD NELSON Department of Physiology, Emory University, Atlanta, Georgia

ABSTRACT Naturally-decapitated spermatozoa were separated into motile flagella and head and immotile flagella by differential and density gradient centrifugation. In preparations microscopically free of cross-contamination after repeated centrifuga- tion, the heads appeared to be enzymatically inert, while there was virtually no change in the specific activity of the immotile flagella which had been subjected to as much manipulation as the heads. The non-motile flagella had almost twice the acetylcholinesterase and about one-third the apyrase activity of the motile flagella. The flagella appear to contain a structurally-bound adenosinetriphosphatase which may be identical with the “spermosin” extracted from bull sperm.

Apparently all the spermatozoa in the ejaculate of a Guernsey bull with a con- genital reproductive tract defect are de- capitated. Mann (’58) and Masaki and Hartree (’62) have discussed the advan- tages of the use of this semen (containing free sperm heads and free flagella, a high proportion of which are vigorously motile) for investigations on the distribution of chemical components and metabolic ac- tivity. Studies on mechanically disrupted spermatozoa obtained from normal bulls (Henle, Henle and Chambers, ’38; Zittle and O’Dell, ’41; Mann, ’49; Nelson, ’54, ’64) indicate that the fine fragmentation of the spermatozoa in the homogenates renders it difficult to free the individual fractions from cross-contamination and that variable lengths of the flagella may remain attached to the base of the sperm heads. However, gentle centrifugation of the Guernsey semen in a suitable density gradient can reproducibly yield cleanly separated heads, nonmotile and motile flagella. Spermatozoa prepared under these conditions thus provide material for more critical analysis of enzyme distribu- tion.

MATERIALS AND METHODS

The Guernsey semen was obtained by means of an artificial vagina and allowed to cool slowly to room temperature (18-

J. CELL. PHYSIOL., 68: 113-116.

20°C). Initial separation of the seminal components was carried out at room tem- perature by centrifuging the semen sam- ple diluted with an equal volume of 0.12 M Tris buffer, pH 7.4, at 70 g for 30 min- utes. The cloudy supernatant of motile flagella was carefully removed by means of a fine-tipped pipette. A mixture of heads and immotile flagella, comprising the residue was resuspended in 3-4 ml of 0.12 M Tris buffer, pH 7.4, and layered over 1.5 ml of 1.8 M sucrose (sp. gr. 1.23) 4- 1.5 ml of 2.4 M sucrose (sp. gr. 1.30). After centrifugation at 1,400 g for 30 min- utes, the heads appeared in the residue along with some clumps of immotile flagella, while the bulk of the immotile flagella remained at the interface between the two concentrations of sucrose. Next, the various fractions were resuspended in the Tris buffer, washed several times and then concentrated by centrifugation at 1,400 g. All the flagella could be removed from the head fraction by carefully re- peated centrifugation of the broken up clumps in a similar density gradient.

Cholinesterase, previously identified as “true specific acetylcholinesterase” (Nel-

Received Feb. 25, ’66. Accepted April 18, ’66. 1 This work was undertaken at the Physiological

Laboratory, University of Cambridge, England, under Research Career Development Award X3-GM-15 193 and with the aid of a grant from the Commonwehlth Fund, New York. Publication no. 813 of Division of Basic Health Sciences, Emory University.

113

114 LEONARD N E L S O N

son, ’641, was assayed by the method of Hestrin (’49). Adenylpyrophosphatase (ad- enosinetriphosphatase + adenylate kinase ) was estimated as previously described (Nel- son,’64). The specific activities of the en- zymes refer to micrograms of acetylcho- line hydrolyzed or inorganic phosphate liberated per milligram of protein per hour. Protein determinations were per- formed according to Lowry et al. (’51). Incubations in acetylcholine took two hours at room temperature and those in ATP, ten minutesa

RESULTS

When only a single density gradient centrifugation was performed, similar re- sults to those previously reported were ob- tained, both for the cholinesterase and adenylpyrophosphatase partition between heads and tails (Nelson, ’54, ’64). If, how- ever, the “separated components were re- suspended and washed in the buffer, and the density gradient centrifugation care- fully repeated so that microscopic exam- ination showed no cross-contamination of the fractions, the heads were clearly inert with respect to these two enzyme systems (see table 1) . Under these conditions, the non-motile flagella fraction was subjected to as much dilution and manipulation as the head fraction but underwent virtually no change in specific activity.

In a separate series of experiments, it was possible to demonstrate that the flagella contained what may be regarded as an “adenosinetriphosphatase.” After re- peated washing in Tris buffer to remove the seminal plasma, the flagella were re- suspended and washed three more times each in 20 volumes of glass distilled water, which caused them to undergo marked swelling. The incubation medium, con- taining 7.7 X M ATP in lo-’ M Tris buffer, pH 8.6, included 2.5 X M cy- steine hydrochloride and M CaCL as activator. Prolonged incubation of the osmotically shocked ffagella at 19.5”C re- sults in the liberation of no more than one mole of inorganic phosphate per mole of ATP as shown in figure 1.

These data suggest that the flagellar enzyme hydrolyzes only the terminal phos- phate from the adenosinetriphosphate. Under these conditions, viz., alkaline pH,

M I N Fig. 1 Adenosinetriphosphatase activity of os-

motically shocked bull sperm flagella. The in- cubation medium contains 7 x M adenosine- triphosphate in lop2 M Tris buffer, pH 8.6, 10-3 M CaCL, 2.5 X M cysteine hydrochloride. Incubation temperature, 19.5”C.

calcium activation in the presence of free sulfhydryl groups, the “structural” enzyme of the flagellum appears to be either the same adenosinetriphosphatase identified as “spermosin” by Burnasheva (’58) or else closely associated with it.

DISCUSSION

The sperm head apparently exercises little, if any, control over sperm move- ment, and certainly as evidenced by the motility of the decapitated flagella, coordi- nated movement can occur independently of the head. Not only does it contribute negligibly to the power output demands on the moving cell (Carlson, ’59), but the sperm head also seems to be devoid of enzymes directly concerned with movc- ment.

Evidently, the acetylcholinesterase is confined to the motile organelle, the flagel- lum, of the bull spermatozoon. The pre- cise role of this enzyme in sperm motility remains to be determined: i.e., whether it is involved in initiation of flagellation, propagation of the flagellar wave or co- ordination of the sequential contraction of the longitudinal flagellar fibers. The fact that the specific activity of the enzyme in intact cells approximates that of mechan- ically disrupted sperm, supports the sug- gestion that the enzyme is quite stable and

2 Acetylcholine chloride was purchased from Nu- tritional Biachemicals Co., and 99-100% dlso+um adenosinetnphosphate was obtained from the Sigma Chemical C6.

ENZYMES I N NATURALLY-DECAPITATED SPERM 115

is either at or near the flagellar surface (Nelson, '63) or that the externally ap- plied substrate rapidly diffuses to the site of hydrolysis. The immotile, partially de- generating flagella appear to have lost some morphological or physiological diffu- sion barrier since the specific activity of this fraction is nearly double that of the motile flagella (see table 1). In general, acetylcholinesterase appears to be highly stable and not readily brought into solu- tion, nor is it easily separable from the particulate fraction of homogenates (Nach- mansohn and Wilson, '55).

On the other hand, the immotile flagella exhibit a lower adenylpyrophosphatase ac- tivity than the motiIe flagella. The de- crease in activity from motile to non-motile flagella, which also occurs on storage, could perhaps partially be attributed to in- stability or loss of a component enzyme along with loss of morphological integrity. The absence of an intact plasma mem- brane in non-motile flagella, observed in the electron microscope tends to confirm the latter suggestion (unpublished obser- vations).

Extraction of adenosinetriphosphatase from muscle usually requires treatment with salt solutions of ionic strength of about 0.6, while myokinase and sperm re- laxing factor are soluble in more dilute salt solutions (Kinoshita, '59). Overnight treatment with distilled water reduces the apyrase activity of homogenized bull sperm tails by about one-third, but 0.6 M KCl is required to solubilize the bulk of the flagel- lar apyrase (Nelson, '54). Therefore, al- though spermatozoa are notoriously leaky

even when sIightly insulted, it does not appear likely that leakage alone could ac- count for the complete absence of acetyl- cholinesterase. and apyrase from the rela- tively gently washed head fraction. In fact the non-motile flagella, which had been subjected to the same amount of washing and manipulation as the heads, showed no marked change in enzyme ac- tivity. Moreover, pooled motile and non- motile flagella, stored overnight in the cold, showed the same apyrase activity (table 1) as freshly prepared non-motile flagella. If the adenylate kinase compo- nent (Kinoshita, '59) of the apyrase system should diffuse out of the deteriorat- ing or aged flagella, leaving only struc- turally-bound ATP-ase, this would account in part for the difference in the ATP-split- ting ability of the non-motile vs. motile flagella.

With respect to acetylcholinesterase, one may now account for the variable, low en- zyme activity ascribed to the "head" frac- tion of sonically disrupted bull and Myti lus spermatozoa (Nelson, '64; Applegate and Nelson, '62). This activity apparently arose from contamination of the heads by incompletely detached or small fragments of flagella.

The results of these experiments differ considerably from what might have been predicted on the basis of other work on sper- matozoa obtained from the same bull. Ma- saki and Hartree ('62) assigned the respira- tory activity and anaerobic fructolysis of the '%head-immotile tail" fraction primarily to the heads. However, respiratory activity of cells in general is typically ascribed to

TABLE 1

No. of centrifugations

Enzyme ( H and NMF)

Specific activities - y/mg protein jhour 1

Heads flagella flagella n ( H ) (MF) (NMF)

Motile Non-motile

AChEase 1 ( 5 ) 72.9t 6.5 176 .2e 10.1 321.22 9.7 323.0f 7.9 AChEase 2 ( 3 ) 0 1 6 7 . 7 2 12.9

300.4 -C- 28.2 99.2 -C- 10.8 Ad-pyrase 1(5) 17.4-C-1.6 Ad-pyrase 2 ( 3 ) 0 310.0% 5.1 114.0C 2.0

~

1 Specific activity: y acetylcholine hydrolyzed per milligram of protein per hour; y inorganic phosphate liberated per milligram of .protein hour, & standard error of the mean.

The cholinesterase medium contained 8.5 mM acetylcholine chloride in, 0.2 mM N a acetate, 0.06 M Tris buffer, pH 7.4, 0.85 m M MgC12, 0.6 M NaC1; total volume 12 ml, incuhatlon temperature 20°C. 1.3 ml aliquots .plpetted into 0.2 ml ice-cold 10% trichloroacetlc acid at zero time and after two hours mcubatlon.

Adenylpyrophosphatase and ATP-ase media contained I mM ATP, 1 mhl CaCk, 2.5 mM cyste+e, 35 mM Trrs bpffer, pH 8.6; .total volume , l . O ml, including 0.4 ml sperm fractlon suspension. Reactions termmated by addihon of 1.0 ml ice-cold 10% trichloroacetic acid.

I16 LEONARD NELSON

the mitochondria1 fraction. Admixture of even a relatively small number of mito- chondria-bearing non-motile but enzymati- cally active tails with the so-called ‘?lead fraction” could conceivably throw off both the calculated and measured enzyme ac- tivities and other parameters.

ACKNOWLEDGMENT

The author gratefully acknowledges the generosity and cooperation of Dr. C . R. Austin. The equipment was obtained through Ford Foundation grants to Prof, A. S. Parkes, who kindly provided the facilities for this work. The bull semen was kindly supplied by Dr. Hector Dott of the Agricultural Research Council Unit of Reproductive Physiology and Biochemistry, Huntingdon Road, Cambridge. The author also thanks Dr. T. Mann, Director of the ARC Unit, for his many kindnesses; and Dr. R. Edwards for many stimulating dis- cussions.

LITERATURE CITED Applegate, A., and L. Nelson 1962 Acetylcho-

linesterase in Mytilus spermatozoa. Biol. Bull., 123: 475.

Burnasheva, S. A. 1958 Properties of spermo- sin, a contractile protein, in sperm cells. Bio- khimia, 23: 558-563.

Carlson, F. D. 1959 The motile power of a swimming spermatozoon. In: Proceedings of the First National Biophysics Conference. Yale University Press, New Haven, Conn., pp. 443- 449.

Henle, W., G. Henle and L. A. Chambers 1938 Studies on the antigenic structure of some mammalian spermatozoa. J. Exp. Med., 68:

Hestrin, S. 1949 The reaction of acetylcholine and other carboxylic acid derivatives with hy- droxylamine, and its analytical application. J. Biol. Chem., 180: 249-261.

Kinoshita, S. 1959 On the identity of the motil- ity inducing factor of flagellum and the re- laxing factor of muscle. J. Fac. Sci., Univ. Tokyo, Sect. IV, Vol. 8: 427-437.

Lowry, 0. H., N. J. Rosebrough, A. L. Farr and R. J. Randall 1951 Protein measurement with the Folin phenol reagent. J. Biol. Chem.,

Mann, T. 1949 Metabolism of semen. Advan. Enzymol., 9: 329-390.

1958 Recherches rkcentes sur la bio- chimie du sperme. SOC. Chim. Biol., 40: 745- 758.

Masaki, J., and E. F. Hartree 1962 Distribution of metabolic activity, phospholipid and hyalu- ronidase between the heads and tails of bull spermatozoa. Biochem. J., 84: 347-353.

Nachmansohn, D., and I. B. Wilson 1955 Ace- tylcholhesterase. In: Methods in Enzymol. Vol. I, S. P. Colowick and N. 0. Kaplan, editors. Academic Press, New York, pp. 642-651.

Nelson, L. 1954 Enzyme distribution in frag- mented bull spermatozoa. I. Adenylpyrophos- phatase. Biochim. Biophys. Acta, 16: 494-501.

1963 Spermatozoan cholinesterase and coordination of the flagellar wave. Biol. Bull., 125: 352-353.

1964 Acetylcholinesterase in bull sper- matozoa. J. Reprod. Fertil., 7: 65-71.

Zittle, C. H., and R. A. O’Dell 1941 Chemical studies of bull spermatozoa. J. Biol. Chem., 140: 899-907.

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