Positive correlation between the level of protein-carboxyl methylase in spermatozoa and sperm motility*

FERTILITY AND STERILITY Copyright © 1986 The American Fertility Society

Vol. 45, No.6, June 1986 Printed in U.8A.

Positive correlation between the level of protein-carboxyl methylase in spermatozoa and sperm motility*

Claude Gagnon, Ph.D. t:J: Eve de Lamirande, Ph.D. t Richard J. Sherins, M.D.§ Royal Victoria Hospital and Faculty of Medicine, McGill University, Montreal, Quebec, Canada, and Developmental Endocrinology Branch, National Institute of Child Health and Human Development, Bethesda, Maryland

Levels of protein-carboxyl methylase (PCM) activity were measured in spermatozoa from infertile patients with < 50% sperm motility and compared with those of normal fertile controls. When spermatozoa were washed by a standard centrifugation procedure, the level of PCM activity in a subgroup of patients with sperm motility ranging from 0% and 20% (24.0 ± 5.2 pmollmg protein, mean ± standard error of the mean) was significantly different from that of controls (35.9 ± 2.3 pmollmg). However, when the entire population of patients with sperm motility ranging from 0% to 50% (32.6 ± 62 pmollmg) was compared with controls, no significant difference was observed in sperm PCM levels. With this standard washing procedure no significant relationship (r = 028; P > 0.05) between sperm PCM activity and motility was observed. By contrast, when spermatozoa were washed on a Percoll gradient, to eliminate other cellular elements, both groups of patients with 0% to 20% (14.6 ± 2.5 pmollmg) and with 0% to 50% (21.5 ± 2.4 pmollmg) sperm motility had sperm PCM levels significantly lower than that of controls (34.7 ± 3.6). A highly significant relationship (r = 0.78; P < 0.001) was observed between the levels of sperm PCM activity and motility. Fertil SteriI45:847, 1986

Received November 13, 1985; revised and accepted February 18, 1986. *Supported by grants from the Medical Research Council of Canada, from the Population Council, New York, New York, and from the Ministere de l'enseignement superieur de la science et de la technologie, Quebec, Quebec, Canada. tUrology Research Laboratory, Royal Victoria Hospital and Faculty of Medicine, McGill University. :j:Reprint requests: Claude Gagnon, Ph.D., Urology Research Laboratory, Royal Victoria Hospital, 687 Pine Avenue West, Suite H6:46, Montreal, Quebec, Canada. §Developmental Endocrinology Branch, National Institute of Child Health and Human Development.

been shown to vary during epididymal transit. Whereas the methylase activity increases 3-fold, the methylesterase decreases 7- to 20-fold as spermatozoa acquire motility and mature from the caput to the cauda epididymidis. 1 , 2 In our previous study 3 comparing PCM levels in spermatozoa from control fertile volunteers with those of infertile patients with nonmotile spermatozoa, it was shown that the enzyme activity in the patient group was 4-fold lower than that of the fertile control group. In a recent communication,4 other investigators have confirmed the presence of a very low level of PCM activity in nonmotile spermatozoa. However, these investigators have also reported that there was no correlation between sperm

Vol. 45, No.6, June 1986

Gagnon et al. Protein methylation in spermatozoa

Protein-carboxyl methylase (PCM) and protein methylesterase, two enzymes that reversibly modify the net charge of sperm proteins, have

847

Table 1. Semen Analysis and Clinical Features of Infertile Men with Low Sperm Motility No.

Concentration

Controls (n = 44) Mean ± SEM Range

Cytology

1061ml

ml

%

% oval

76 67 40 38 29 27 22 19 18 10 5 5 2 1

4.3 2.6 1.5 2.9 1.7 2.3 1.7 1.6 4.3 1.1 2.2 3.1 3.0 2.6

36 34 11 34 41 35 0 49 21 34 9 23 10 10

85 75 78 79 75 67 84 79 80 76 65 72 77 61

26 ± 6 b 1-76

2.5 ± 0.3 1.1-4.3

25 ± 4 b 0-49

> 60 61--85

46 ± 10 20-200 n = 22

2.7 ± 0.2 1-5 n = 22

65 ± 5 50-90 n = 22

> 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 Patients (n = 14) Mean ± SEM Range

Semen analysisa Volume Motility

Clinical features

Varicocele Varicocelectomy (1981) Varicocelectomy (1982) Varicocele Varicocelectomy (1982) Varicocele Varicocele (1983) Congenital obstruction Varicocele

aMean value for each subject determined from at least five specimens collected on different days over a period of at least 18 months. bSignificantly different from controls; P < 0.05.

PCM activity and motility when spermatozoa from patients or controls with a wide range of motility were investigated. In this report, we confirm the results of Rogers and Nelson4 on the lack of correlation between the level of PCM activity and motility in spermatozoa washed twice by a standard centrifugation procedure and motility. However, we also report that when spermatozoa are separated from cellular debris, spermatids, leukocytes, and other cellular elements, a positive linear relationship exists between the percentage of total sperm motility and PCM activity.

MATERIALS AND METHODS SELECTION OF SUBJECTS

Fourteen infertile men were studied during this investigation. The laboratory features are summarized in Tables 1 and 2. The patients, 28 to 59 years of age, were fully virilized males in good general health. They were selected specifically because of suboptimal sperm motility. Each subject had undergone a complete general evaluation, and more than five sequential ejaculates 848

Gagnon et a1. Protein methylation in spermatozoa

were available for characterization of semen quality before inclusion of the subject in the study. None of the patients had bronchiectasis, drug or alcohol abuse, or known exposure to any toxin. Four subjects had a palpable varicocele, and four other men had had a prior varicocele repair but persistent poor semen quality. Of the remaining subjects, one had had an orchiopexy for congenital unilateral cryptorchidism, and another man had undergone vasoepididymostomy for congenital ductal obstruction. In four patients the poor semen quality was unexplained. Serum concentrations of follicle-stimulating hormone,5 luteinizing hormone,6 testosterone,7 and estradiol 8 were measured by specific radioimmunoassays to complete the characterization of testicular function (Table 2); these values were indistinguishable from those of normal men. Among the 14 infertile patients, sperm concentrations were within normallimits9 in 7 and < 20 x 106/ml in 7 patients. Cellular morphologic features were within normal limits, as determined from stained seminal smears (Table 1). Fresh semen samples from healthy, normal male volunteers with confirmed fertility were used as control samples. Semen samples from vasectomized volunteers were used to establish the Fertility and Sterility

Table 2. Testicular Size and Hormone Levels of Infertile Men with Low Sperm Motility Subject

Age

1 2 3 4 5 6 7 8 9 10 11 12 13 14

35 ± 2 28-59

Patients Cn = 14) Mean ± SEM Range

Testicular

Serum hormone levels b Testosterone Luteinizing hormone

Estradiol

sizea

Folliclestimulating hormone

yr

ml

mlUlml

mlUlml

nglml

pglml

37 32 33 36 31 36 59 33 34 28 33 35 32 36

60 33 25 28 25 55 23 24 16 19 55 50 20

3 4 9 9 15 5 12 8 8 40 17 5 6 20

8 11 12 10 11 6 9 10 4 18 11 4 6 10

3.4 4.1 3.6 6.7 5.9 3.6 8.8 5.1 5.1 3.7 14.1 3.6 3.5 5.7

22 23 29 48 24 33 31 28

33 ± 5 16--60

12 ± 3 3.6--40

8 ± 1 4.8-18.5

5.5 ± 0.8 3.4-14.1

28 ± 3 22-50

24 ± 1 15-35 n = 44

12 ± 1 4-25 n = 41

9 ± 1 4-25 n = 41

5.2 ± 0.3 2.5-12 n = 27

35 ± 3 12-58 n = 16

Controls Cn = 44) Mean ± SEM Range

27 29 27 50 18

"Testicular size was estimated by comparing gonadal size with a series of ovoids of calibrated volume. bMean value for each subject determined from at least five specimens collected on different days over a period of at least 18 months.

accessory gland contributions in the sperm pellets.

SPERM WASHING PROCEDURES

A 10-I.d portion of the undiluted liquefied semen was placed on a microscope slide and covered with a 484-mm2 coverslip (#0 thickness). From 150 to 250 spermatozoa were counted with an inverted microscope, and the percentage of total (progressive and nonprogressive) motility was calculated. Sperm motility in the ejaculate was determined at room temperature 30 to 60 minutes after collection.

After liquefaction, semen samples were filtered through cheesecloth and divided in two fractions. The first fraction (0.25 ml) was diluted 12-fold with HEPES balanced saline (HBS) buffer 10 (130 mM NaCI, 4 mM KCI, 1 mM CaCI 2 , 0.5 mM MgCI 2 , 14 mM fructose, and 10 mM N-2-hydroxyethylpiperazine-N' -2-ethanesulfonic acid [HE PES] adjusted to pH 8.0) containing 1 mg/ml of bovine serum albumin (ESA) (HBS + BSA buffer) and centrifuged at room temperature at 600 x g for 5 minutes. The pellets, which contained spermatozoa, other cellular elements, and debris, were washed twice by centrifugation in the same buffer and under the same conditions. The final pellet was resuspended in HBS + BSA buffer at a concentration of 2 x 108 spermatozoal ml. The remaining semen fraction was layered on a Percoll (Pharmacia Fine Chemicals, Dorval, Canada) gradient made of 0.5 ml of 95% Percoll in HBS + BSA buffer and of an 8-ml 15% to 65% linear Percoll gradient in the same buffer sitting on the 95% Percoll layer. IO The gradients were

Vol. 45, No.6, June 1986

Gagnon et aI. Protein methylation in spermatozoa

SEMEN COLLECTION

All semen samples were collected by masturbation after 36 hours' abstinence and were delivered to the laboratory within 1 hour after ejaculation. The samples from both the volunteers and the patients formed postejaculatory coagulums, and their liquefaction was normal. Two semen samples were provided by each subject or volunteer.

MOTILITY MEASUREMENTS

849

RESULTS

Table 3. Levels of PCM Activity in Human Spermatozoa Washed by a Standard Centrifugation Procedure Subjects

PCM activitya

CORRELATION BETWEEN SPERM MOTILITY AND LEVEL OF PROTEIN-CARBOXYL METHYLASE ACTIVITY IN

pmollmg protein

Controls (> 50% motility) Patients (0-50% motility) Patients (0-20% motility)

SPERMATOZOA WASHED BY A STANDARD

35.9 ± 2.3 (15) 32.6 ± 6.2 (15) 24.0 ± 5.2b (6)

aValues represent the mean ± SEM. bStatistically different from controls; P < 0.05

centrifuged at room temperature at 1300 x g for 45 minutes. Spermatozoa devoid of cellular elements and debris were recovered at the 65% to 95% Percoll interface and in the 95% Percoll layer. These two fractions were pooled, diluted 10fold with HBS + BSA buffer, and centrifuged at 600 x g·for 5 minutes. The pellet was resuspended in HBS + BSA buffer at a concentration of 2 x 10 8 spermatozoa/ml. PROTEIN·CARBOXYL METHYLASE ASSAY

PCM activity was measured in duplicate as follows: 5 j.Ll of sperm suspensions were incubated with 10 j.Ll of ovalbumin dialyzed against H 2 0 (50 mg/ml) , 10 j.Ll of 1% Triton X-100, 5 j.Ll of H 2 0, 10 j.Ll of 10 j.LM S-adenosyl eH-methyl) methionine (Amersham Canada Ltd., Oakville, Ontario, Canada) at 125 j.LCi/ml, and 10 j.Ll of 0.5 M sodium acetate, pH 6.0. After a 10-minute incubation at 37°C, the methylation reaction was stopped by the addition of 0.5 ml of 15% cold trichloroacetic acid. The extent of protein methylation was determined as previously described. 11 PCM activity is expressed as picomoles of methyl group transferred per milligram of protein per 10 minutes.

CENTRIFUGATION PROCEDURE

Levels of PCM activity in spermatozoa from patient and control groups were compared after washing by a standard centrifugation procedure (Table 3). With a specific activity of 32.6 ± 6.2 pmollmg protein (mean ± standard error of the mean), the PCM activity of the entire patient group (motility < 50%) was similar to that of controls (motility> 50%). However, PCM activity in spermatozoa of patients with < 20% sperm motility (24.0 ± 5.2 pmollmg protein) was significantly lower than that of the control group (35.9 ± 2.3 pmollmg protein, P < 0.05). When semen from vasectomized volunteers was processed under identical conditions, the 600 x g pellet containing the contributions from the auxiliary glands had an enzymatic activity of 6.6 ± 1.8 pmollmg protein. The levels of PCM activity from both control and patient groups were analyzed in relation to sperm motility, ranging from 0% to 48% for patients and 53% to 79% for controls. A correlation 90

•

80 c:

ec. 70

•

Q)

Cl

~ 60 "0

.§, 50

•

>-

•

•

:>

PROTEIN DETERMINATION

i=

STATISTICAL ANALYSIS

Regression curves were calculated by the leastsquares method, and the correlation coefficient, r, was calculated. Student's t-test (two-tailed) was used to determine whether two groups of data were significantly different. The 0.05 level of probability (P < 0.05) was used as the minimal criterion of significance. 850

Gagnon et al. Protein methylation in spermatozoa

40

u

<{

Protein concentrations were measured according to Lowry et al. 12 with BSA as the standard.

•

•

I-

::!: u 0..

30 20 10

o

10

20

30

40

50

60

70

80

MOTILITY (%)

Figure 1 Relationship between the level of PCM activity and motility in spermatozoa washed by a standard centrifugation procedure. Motility in patients range from 0% to 48% and in controls from 53% to 78% (r = 0.28; P > 0.05).

Fertility and Sterility

90

Table 4. Levels of PCM Activity .in Human Spermatozoa Washed Through a Percoll Gradient

•

80

'2

e

60

CI

"-

~

50

Controls (> 50% motility) Patients (0-50% motility) Patients (0-20% motility)

34.7 ± 3.6 (15) 21.5 ± 2.4 b (15) 14.6 ± 2.5 b (6)

aValuesrepresent the mean ± SEM. bStatistically different from controls; P < 0.001.

~

~ 40

e«

PCM activity" pmol/mg protein

Co

E

Subjects

•

'iii 70

>

30

were representative of those in the original semen, except that no cellular elements other than spermatozoa were present in these Percolllayers.

~ 20 Q.

10

•

o

10

20

30

40

50

CORRELATION BETWEEN SPERM MOTILITY AND THE

MOTILITY (%)

Figure 2 Relationship between the level of PCM activity and motility in patients' spermatozoa washed by a standard centrifugation procedure (r = 0.55; P < 0.05).

coefficient of 0.28 (P > 0.05) was obtained, indicating that there was no statistically significant correlation between PCM activity and sperm motility when all samples were considered (Fig. 1). However, when only the data from the patient group were analyzed, a statistically significant relationship (r = 0.55; P < 0.05) was observed (Fig. 2).

LEVEL OF PROTEIN·CARBOXYL METHYLASE ACTIVITY IN SPERMATOZOA WASHED THROUGH A PERCOLL GRADIENT

After a wash through the Percoll gradient, the levels of PCM activity in spermatozoa from control and patient groups were compared. A significantly lower enzyme activity was observed in the entire patient group, with sperm motility ranging from 0% to 50%, 21.5 ± 3.6 pmollmg protein, when compared with that of controls (34.7 ± 2.4 pmollmg protein) (Table 4). Patients with sperm motility ranging between 0% and 20% had even 80

WASHING OF SPERMATOZOA THROUGH A PERCOLL GRADIENT

Because germ cells such as spermatids and white blood cells are frequently present in semen and since both germ cells and white blood cells have a high level of PCM activity,13-15 the poor correlation between sperm motility and PCM activity could be due to contaminations by these types of cells. To investigate this possibility, we assessed the use of a Percoll gradient as a spermwashing procedure that would allow both the elimination of other types of cells and a high yield of sperm recovery. Sperm were washed on a Percoll gradient by a modification of the method of Lessley and Garner.lO The modification consisted of lowering the linear gradient from 15% to 75%, to 15% to 65%. This small decrease allowed an increase in sperm yield from 59% ± 6% to 87% ± 3% for the combined 65% to 95% Percoll interface and 95% Percolllayer. Thus, sperm from these two layers Vol. 45, No.6, June 1986

..e

c: 'iii

c.

70 60

Cl

~ 50

•

'0 E

•

E: 40 >~

:>

~

30

u

<{

~

20

u

•

a..

10

• 0

10

20

30

40

50

60

70

80

MOTILITY (%)

Figure 3 Relationship between the level of PCM activity and motility in spermatozoa washed through a Percoll gradient. Motility in patients range from 0% to 48% and in controls from 53% to 78% (r = 0.72; P < 0.001). Gagnon et al. Protein methylation in spermatozoa

851

1

40

• :5

~c.

30

CI

E "0 E

"-

~

20.

~

>

~c( ~

10

IL

•

o

10

20

30

40

50

MOTILITY (%1

Figure 4 Relationship between the level of PCM activity and motility in patients' spermatozoa washed through a Percoll gradient (r = 0.94; P < 0.001).

lower levels of PCM activity (14.6 ± 2.5 pmol/mg protein). With spermatozoa washed through the Percoll gradient, a significant relationship (r = 0.72; P < 0.001) was observed between the level of PCM activity and sperm motility of both control and patient groups (motility ranging from 0% to 79%) (Fig. 3). The correlation coefficient increased to 0.94 when only the data from patients (motility from 0% to 50%) were analyzed (Fig. 4). DISCUSSION

The data showing a low level ofPCM activity in spermatozoa with 0% to 20% motility washed by a standard centrifugation procedure support our initial observation on the low level ofPCM activity in immotile spermatozoa. 3 Recently, Rogers and Nelson4 confirmed the low level of PCM activity in. immotile spermatozoa but failed to observe a correlation between PCM activity and sperm motility. We also have noted this lack of correlation with spermatozoa washed by a standard centrifugation procedure, but now demon~ strate that these results can be explained by the presence of other cellular elements and debris in the washed sperm fraction tested. Since spermatids and white blood cells, two cell types frequently encountered in semen of patients, have a 852

Gagnon et .al. Protein methylation in spermatozoa

high level of PCM activity,13-l5 their preSence results in PCM overestimation. Two typical examplesof this overestimation are illustrated by the two highest values of PCM in Figure 1 (coordinates Xl Y1 = 35, 72 and X2Y2 = 38, 86) which after the Percoll washing procedure dropped to much lower values in Figure 3 (coordinates Xl Y1 = 35,25 andX 2Y2 = 38,29). In these two specific cases PCM levels were overestimated by almost threefold. Thus, Percoll gradient washing represents an adequate procedure for eliminating cellular elements other than spermatozoa before PCM determination; In our initial study with infertile patients with < 2% motile spermatozoa3 we used a standard centrifugation procedure to wash spermatozoa from seminal plasma. Even though the washed spermatozoa might have contained other types of cells and cellular debris, the fact that PCM activity was very low or absent inimmotile spermatozoa allowed us to observe a significant difference between the levels of PCM activity in patients and those in controls. Recently, Hatch et al. 16 studied the level of Triton-extractable PCM activity in spermatozoa (washed by the standard centrifugation procedure) from infertile patients. They observed that . infertile patients with more than 50% sperm motility had a normal level of enzyme activity when compared with controls, the levels of PCM activity being especially high when semen contained high proportions of immature spermatozoa. They also noted the low level of PCM activity in occasionally immotile spermatozoa from four patients, but this relationship was not constant. These findings support our results and emphasize the importance of eliminating immature forms of spermatozoa, especially those with large remnant cytoplasm, white blood cells, endothelial cells, and cell debris before determining PCM activity. These contaminants are all efficiently removed by the Percoll gradient. The observation that PCM activity was normal in spermatozoa from infertile men with motility above 50% may suggest that PCM levels are linked to motility rather than fertility potential. However, to differentiate between these two possibilities, a proper controlled study remains to be undertaken. PCM, like protein phosphorylation has been implicated in a variety of biologic processes, including sperm motilityP-2l Several pieces of evidence, in addition to the present results, support the involvement of protein methylation in sperm Fertility and Sterility

,

.

motility. Acquisition of motility during epididymal transit has been associated with a threefold increase in PCM activity.1 Incubation with membrane-permeable methylation inhibitors such as low concentrations of erythro-9-(3-[2-hydroxynonyl]) adenine (EHNA), adenosine, and homocysteine resulted in a time-dependent inhibition of sperm motility.22 Even though EHNA is not entirely specific at high concentration,23,24 the low concentrations used in the above study did not block dynein adenosine triphosphatase, because EHNA alone had no effect on motility.22 Immotile spermatozoa from infertile patients have very low levels of PCM activity. 1, 3 Finally, protein methylesterase, the enzyme hydrolyzing protein-methyl esters synthesized by PCM, decreases 7- to 20-fold as spermatozoa acquire motility during epididymal transit. 2 Thus, it seems that a high level of PCM activity and a low level of protein methyl esterase activity are prerequisites for sperm motility.

REFERENCES 1. Purvis K, Cusan L, Attramadal H, Ege A, Hansson V: Rat sperm enzymes during epididymal transit. J Reprod Fertil 65:381, 1982 2. Gagnon C, Harbour D, de Lamirande E, Bardin CW, Dacheux JL: Sensitive assay detects protein methylesterase in spermatozoa: decrease in enzyme activity during epididymal maturation. Bioi Reprod 30:953, 1984 3. Gagnon C, Sherins RJ, Phillips DM, Bardin CW: Deficiency of protein-carboxyl methylase in immotile spermatozoa of infertile men. N Engl J Med 306:821, 1982 4. Rogers BJ, Nelson J: The relationship of protein-carboxyl methylase to motility and fertility of human spermatozoa. J Androl 3:17, 1982 5. Cargille CM, Rayford PL: Characterization of antisera from human follicule-stimulating hormone radioimmunoassay. J Lab Clin Med 75:1030, 1970 6. Odell WD, Ross GT, Rayford PL: Radioimmunoassay for luteinizing hormone in human plasma and serum: physiological studies. J Clin Invest 46:248, 1967 7. Nieschlag E, Loriaux DL: Radioimmunoassay for plasma testosterone. Z Klin Chern Klin Biochem 10:164, 1972 8. Loriaux DL, Ruder HJ, Lipsett MB: The measurement of estrone sulfate in plasma. Steroids 18:463, 1971

Vol. 45, No.6, June 1986

9. Sherins RJ Howard SS: Male infertility. In Campbell's Urology, V~l. 1, Edited by JH Harrisson, RF Gittes, AD Perlmutter, TA Stamey, PC Walsh. Philadelphia, W. B. Saunders, 1977, p 715 10. Lessley BA, Garner DL: Isolation of motile spermatozoa by density gradient centrifugation in Percoll. Gamete Res 7:49,1983 . 11. Gagnon C, Axelrod J, Musto N, Dym M, Bardm CW: Protein carboxyl-methylation in X-ray-induced seminiferous tubule features. Endocrinology 105:1040, 1979 12. Lowry OH, Rosebrough NJ, FaIT AL, Randall RJ: Protein measurement with the Folin phenol reagent. J Bioi Chern 193:265, 1951 13. O'Dea RF, Viveros OH, Axelrod J, Aswanikumar S, Scheffmann E, Corcoran BA: Rapid stimulation of protein carboxyl methylation in leukocytes by a chemotactic peptide. Nature 272:462, 1978 14. Pike MC, Kredich NM, Snyderman R: Requirements of S-adenosyl-L-methionine-mediated methylation for human monocyte chemotaxis. Proc Nat! Acad Sci USA 75:3928, 1978 15. Gagnon C, Axelrod J, Musto N, Dym M, Bardin CW: Protein carboxyl-methylation in rat testes: a study of inherited and X-ray-induced seminiferous tubule failure. Endocrinology 105-1440, 1979 16. Hatch R, Harvey SE, Williams-Ashman HG: Protein 0carboxylmethyltransferase in spermatozoa from normal and infertile men. Fertil Steril 43:636, 1985 17. Gagnon C, Heisler S: Protein-carboxyl methylation: role in exocytosis and chemotaxis. Life Sci 25:993, 1979 18. Springer MS, Goy MF, Adler J: Protein methylation in behavioural control mechanisms and in signal transduction. Nature 280:279, 1979 19. Paik WK, Kim SK: Protein Methylation. New York, John Wiley & Sons, 1980, p 211 20. O'Dea RF, Viveros OH, Diliberto EJ Jr: Protein carboxymethylation: role in the regulation of cell functions. Pharmacology 30:1163, 1981 21. Diliberto EJ Jr: Protein carboxyl methylation: putative role in exocytosis and in the cellular regulation of secretion and chemotaxis. In Cellular Regulation of Secretion and Release, Edited by PM Conn. New York, Academic Press, 1982, p 147 22. Bardin CW, Gagnon C: The possible role of protein-carboxyl methylation in sperm motility and capacitation. In Physiopathology of Hypophysial Disturbances and Diseases of Reproduction, Edited by R Sato. New York, Allen R. Liss, Inc., 1982, p 217 23. Bouchard P, Penningroth SM, Cheung A, Gagnon C, Bardin CW: Erythro-9-[3(2-hydroxynonyl)] adenine is an in, hibitor of sperm motility that blocks dynein ATPase and protein carboxylmethylase activities. Proc Nat! Acad Sci USA 78:1033, 1981 24. Pimningroth SM, Cheung A, Bouchard P, Gagnon C, Bardin CW: Dynein ATPase is inhibited selectively in vitro by erythro-9-[3-(2-hydroxynonyll] adenine. Biochem Biophys ResCommun 104:234, 1982

Gagnon et al. Protein methylation in spermatozoa

853