Neutral α-1,4-glucosidase in human seminal plasma: molecular forms in varicocele and after vasectomy

FERTILITY AND STERILITY Copyright c 1982 The American Fertility Society

Vol. 38, No.3, September 1982 Printed in U.SA.

Neutral a-l,4-glucosidase in human seminal plasma: molecular forms in varicocele and after vasectomy

Roland R. Tremblay, M.D., Ph.D., F.R.C.P.(C), F.A.C.P.* Pierre Chapdelaine, B.Sc. Jean Y. DuM, Ph.D. Laboratory of Endocrinology and Metabolism, Department of Medicine, Laval University, Quebec, Quebec, Canada

Neutral 0.-1,4-glucosidase catalyzes the breakdown of oligosaccharides in several tissues including the reproductive organs, and we have demonstrated the presence of two molecular forms (Fl and F2) of the enzyme in human seminal plasma. The identification of these forms can be achieved by sucrose density gradient analysis andl or electrophoresis in the presence of detergents. Individuals with normal sperm analyses or affected by a varicocele, thus showing normo- or oligoasthenozoospermia, show a similar prevalence of Fl only and Fl + F2 forms, while the presence of F2 alone becomes obvious following vasectomy. In conclusion, molecular forms of the enzyme do not appear to be indicative of the presence of a varicocele, but they may reflect modifications in the secretory function of specific reproductive organs (prostate) or glands (seminal vesicles), as observed in the course of an obstructive abnormality at the epididymal or vas deferens level. Fertil SteriI38:344, 1982

Previous works from this laboratory have confirmed the presence of maltase or neutral 0.-1,4glucosidase in human seminal plasma (SP). In men this enzyme has a widespread organ distribution,1-11 and we have established that in the reproductive organs the highest specific activity of the cytosolic a-glucosidase was found in the epididymis, followed by the testis and the prostate. 12 The contribution of these organs to the SP pool is difficult to ascertain, but exclusion of both epididymis and testes by vasectomy leads to a 50% decrease of the specific activity of the enzyme. 13 This result appears to be impressive, because it reflects mainly the absence of epididymal secretions, which account for 5% to 10% of the volume of the ejaculate. 14 Moreover, among all

the glucosidases found in human semen,15 we have shown that the a-1,4-glucosidase activity was correlated with sperm density16 and proposed that it was a useful marker of male infertility. In the course of our attempts to purify a-1,4glucosidase from large volumes of human SP, it became evident that two peaks of enzyme activity could be observed in nondenaturing conditions by sucrose density gradient analysis (SDA) of partially purified material obtained by gel filtrationP Our objective then became to find optimal conditions in which to study these two molecular forms of the enzyme in individual aliquots of human SP and to explore the possible relationships of these components to male infertility. MATERIALS AND METHODS

Received November 17,1981; revised and accepted May 6, 1982. *Reprint requests: Roland R. Tremblay, Ph.D., Laboratory of Endocrinology and Metabolism, 2705 Boulevard Laurier, Ste-Foy, Quebec, Quebec, Canada G1V 4G2.

344

ENZYME SOURCE

Ejaculates from normal and infertile subjects were collected into sterile tubes. Three distinct

Tremblay et aI. Molecular forms of neutral a-l,4-glucosidase

Fertility and Sterility

groups of individuals were progressively constituted for the purpose of this study; in rare instances, vasectomized men served as their own controls. Their ejaculates were examined for morphologic features, motility, and sperm count with an improved Neubauer chamber (American Optical Co., Buffalo, NY), 0.1 mm deep, according to the standards of the World Health Organization. 18 The remaining semen was kept frozen at - 20° C before use. CHEMICALS

Acrylamide, bis-acrylamide, ammoniu)Il persulfate, and Coomassie blue R 250 were obtained from Bio-Rad Laboratories, Richmond, CA. Paranitrophenyl-a-D-glucoside (PNPG), Triton X100, sodium dodecyl sulfate (SDS), and bovine serum albumin (BSA) were purchased from Sigma Chemical Company, St. Louis, MO. Sucrose was a product of J. T. Baker Chemical Company, Phillisburg, NJ. DIALYSIS OF SEMINAL PLASMA

Aliquots of thawed semen (300 j.Ll) were diluted with five volumes of distilled water and centrifuged at 800 x g for 30 minutes. The pellet containing the spermatozoa was discarded, and the supernatant was referred to as SP. For partial purification purposes, SP was dialyzed for 3 days against four liters of distilled water in Spectrapor membrane tubing No. 2 (Spectrum Medical Industries Inc., Los Angeles, CA) with a molecular weight cutoff of 12,000 to 14,000. The precipitate formed in the dialysis tubing was recovered by centrifugation at 30,000 x g for 30 minutes at 4° C. In subsequent experiments, this material was referred to as the precipitate obtained by dialysis (POD). This step afforded a 7-fold purification of a-1,4-glucosidase with a yield of 80% from the starting material. SUCROSE DENSITY GRADIENT ANALYSIS (SDA)

The pellet obtained from POD was dissolved in 0.3 ml of 0.1 M potassium phosphate buffer, pH 6.8, containing 0.2% Triton X-100, and the solution was incubated overnight at 37° C with continuous agitation; then 0.2 ml was applied on top oflinear sucrose gradients (6% to 24% wt/wt) prepared in the same buffer as for the sample. The gradients were run for 18 hours at 200,000 x gin a L2-65B ultracentrifuge (Beckman Instruments Inc., Spinco Division, Palo Alto, CA) with the use Vol. 38, No.3, September 1982

of an SW 60 rotor. After· the centrifugation, the gradients were collected from the bottom of the tube with the use of a fraction recovery system (Beckman). Twenty-seven fractions of six drops (0.15 ml) were obtained, and sedimentation coefficients were determined by the procedure described by Griffith.19 BSA was also included as a marker. SDS POLYACRYLAMIDE GEL ELECTROPHORESIS

Preparation of samples and electrophoretic separation of the enzyme forms were done according to Shapiro et al. 20 The pellet from crude POD was dissolved in 250 j.LI of 0.01 M sodium phosphate buffer, pH 7.2, containing 1% SDS and incubated overnight at 37° C under continuous agitation. The sample applied on top of the gel (5.5 x 0.5 cm) contained 50 j.LI solubilized POD, 10 j.LI 1.4 M ~-mercaptoethanol, 10 j.LI glycerol, and 10 j.LI 0.02% bromophenol blue. The acrylamide concentration was 5% (2.6% cross-linker), and the buffer gel used was 0.1 M sodium phosphate, pH 7.2, containing 0.1 % SDS. After migration with a current of 8 rnA/gel during 3.5 hours, the gels were cut with a device composed of razor blades (1 mm apart). In all the electrophoretic runs, the enzyme activity was found in the first 20 slices. ENZYMATIC DETERMINATIONS

Neutral a-1,4-glucosidase activities were determined as previously reported,21 with the exception that sucrose gradient fractions and gel slices were incubated at 37° C in 0.6 ml citrate buffer, pH 5.8, with 0.1 ml ofPNPG solution* (3.3 mg). The potassium phosphate buffer was replaced by this last buffer because the enzyme activity was slightly higher. This modification was introduced uniquely for the evaluation of the enzyme forms. After the incubation, the reaction was stopped as follows: 0.3 ml of the mixture was deposited in 2.5 ml of 0.1 M glycine-NaOH buffer, pH 10.6. PROTEIN DETERMINATIONS

Protein concentrations were measured according to the method of Lowry et al. 22 *The stock solution of PNPG was prepared as follows: 100 mg PNPG in 3 ml distilled water was slightly heated; after complete dissolution of the synthetic substrate, 0.1-ml ali· quots of the solution were added to the incubation mixture at 37° C. Thus, PNPG remained soluble during the incubation period.

Tremblay et aI. Molecular forms of neutral a-l,4-glucosidase

345

1

FORMS 1&2 FORM 2 FORM 1 SUCROSE DENSITY GRADIENT WITH TAlTON X-l00

I

'A ~ J\

100 6

20

SDS POLYACRYLAMIDE GEL 5%

100

60 20

J[ ~ ~ 1

2

5

7

9 11 13 15 1

2

5

7

9

11 13 15 1

3

4

7

9 11 13 15

FRACTION OR SLICE NUMBER (mm)

Figure 1 Selected profiles of neutral !X-l,4-glucosidase in precipitate obtained after dialysis of SP from three individuals who presented either Fl, F2, or Fl + F2. The samples were analyzed by sucrose density gradient centrifugation in the presence of Triton X-lOO and by SDS-PAGE. The enzyme activity is expressed as a percentage of fraction or slice having the maximal activity. The relative mobility (Rr) of the enzyme is also shown for SDS gels. The protein concentration applied on each sucrose gradient and gel were 1.5 to 2.0 mg and 0.2 to 0.25 mg, respectively.

RESULTS MOLECULAR FORMS OF NEUTRAL a-l,4-GLUCOSIDASE IN HUMAN SEMINAL PLASMA

The identification of one or two molecular forms of neutral a-glucosidase was possible when crude POD was f:luhmitted to ultracentrifugation on sucrose density gradient with Triton X-100 or to SDS polyacrylamide gel electrophoresis (SDSPAGE). The addition of detergents to both procedures prevented aggregation as observed by the absence of enzyme activity in the bottom of sucrose gradients or on the top of gels. No loss of enzyme activity was measured in the presence of 0.2% Triton X-100, while a 20% decrease was found in buffers containing SDS. These molecular forms were observed with small individual aliquots of SP (0.3 ml), and a strict correspondence between the molecular forms was demonstrated by both procedures, based on molecular weight separation of proteins (Fig. 1). SDS-PAGE, however, allowed a much more effective separation of both forms than SDA. These forms were simply identified as F1 and F2 for further studies (no relation should be made between F1 and F2 molecular forms of the enzyme and the fractions B and C previously demonstrated in human seminal plasma by ion exchange chromatography, because different experimental conditions were used in our laboratory)P In the presence of Triton X-100, the enzyme forms sedimented in the 346

11S (F1) and 9S (F2) regions of the gradients. By reference to known molecular weight markers, the molecular weights of F1 and F2 were found to be 180,000 and 155,000, respectively, by SDSPAGE. Analysis of either form by SDS-PAGE revealed several Coomassie blue staining bands which were devoid of any a-1,4-g1ucosidase activity. The region with enzyme activity was only weakly stained with Coomassie blue, thus indicating that it was a minor constituent of the SP. REPRODUCIBILITY AND PREVALENCE OF Fl AND/OR F2 IN HUMAN SEMINAL PLASMA

In an attempt to check whether the observed molecular form(s) occurred repetitively over a long period of time in a given subject, three to four ejaculates of five normal individuals were obtained during a 1-year period at 2- to 3-month intervals, and the POD was analyzed by SDA and SDS-PAGE. The molecular form profile was constant over the period of observation. Either SDA or SDS-PAGE was used to study the prevalence of F1 and/or F2 in subjects whose sperm analysis was normal (but fertility was not necessarily proven) or revealed various degrees of oligoasthenozoospermia (OAZ), according to the criteria of Belsey et al. 18 ; all these patients had a varicocele classified as moderate to large, according to the criteria described by Dubin and Amelar,23 and properly identified by clinical and/or radiologic examination. 24 We selected this common condition to eliminate OAZ (zoospermia less than 30 x 106 spermatozoa/ml and motility less than 50% and 30% after 2 and 6 hours, respectively, at 37° C) of unknown origin. Vasectomized men whose surgery had been performed at least 2 to 4 months prior to the study were chosen to constitute the group of azoospermic subjects. As can be seen from Table 1, the prevalence of F1 and/or F2 was identified in control subjects (group 1) and in patients affected by a varicocele (group 2). It is interesting to note that in the latter group eight patients showed a normal sperm analysis (NS). The ratios of F1, F2, and F1 + F2 over the total number (N) of observations thus remained very similar. In vasectomized men, a 3-fold decrease in the ratio of FlIN and a drastic increase of F2/N were observed with either technique in groups of 14 and 18 individuals, respectively. DISCUSSION

This study provides evidence that human SP, as well as other human tissues,10 exhibits two

Tremblay et al. Molecular forms of neutral !X-l,4-glucosidase

Fertility and Sterility

Table 1. Prevalence of the Two Molecular Forms of Neutral a-l,4-Glucosidase in Human Seminal Plasma of Normal, Oligozoospermic, and Vasectomized Men Forms

Group 1 (normal subjects)a SDA SDS-PAGE Group 2 (varicocele) SDA-OAZ SDA-NS Group 3 (postvasectomy)a SDA SDS-PAGE

Ratio

N

Fl

F2

25 36

15 22

1 2

19 8

12 6

14 18

2 4

6 7

Fl + F2

FIIN

F2IN

Fl + F21N

9 12

0.6 0.6

0.04 0.06

0.36 0.33

7 2

0.6 0.7

6 7

0.14 0.22

0.36 0.25 0.42 0.38

0.42 0.38

QIn two of three groups, SDA was compared with SDS-PAGE. Different groups of individuals were used because of the frequent limitations in the volume of SP available for such analysis. Results are finally expressed as the prevalence of a given form (Fl or F2 alone or Fl + F2) over the total number of observations (N) in each group.

major molecular forms of neutral a-l,4-glucosidase. The use of PAGE appears to be a convenient way to process individual samples containing 0.2 to 0.25 mg proteins; detergents are essential, however, to prevent protein aggregates in a milieu which contains 200 to 300 proteins or protein subunits. 25 Detergents have been widely used for the study of a-glucosidases in human intestine 6,7 and more recently in rat liver microsomes. 26 The biologic role and the importance of these molecular forms of the enzyme remain to be established. If one considers that the distinct protein components of SP reflect to some extent the functioning of the male accessory organs or glands, our data indicate that in varicocele, a condition known to affect spermiogenesis and also the secretion of gonadal steroids,27 the molecular form pattern of a-l,4-glucosidase is similar to that observed in normal subjects. As previously demonstrated by our group,12 this condition can be associated with a decreased specific activity of the enzyme but without apparent changes in its molecular forms, according to this study. As such, the absence of alterations of the molecular form pattern is not particularly useful for clinical purposes, but it remains to be established, on a quantitative basis, to what extent the diminution of the total activity ofthe enzyme can be ascribed to the specific decrease of Fl or F2 when both are present in SP. If the enzyme forms remain unaffected in the course of a disease in constant progress, as is the varicocele, the possibility should then exist that a given pattern (Fl alone or Fl + F2) can reflect the genetic program of the secretory organs. As suggested by this study, the specific profile of normal men remains constant over long periods of Vol. 38, No.3, September 1982

observation. Thus, the hypothesis of genetic inheritance must be suspected for the molecular forms of a-l,4-glucosidase in SP, as is the case for the lymphoid cells in human beings. 10 The changes that characterize vasectomy cannot, however, be explained on this basis; indeed, in this new functional state of the reproductive system, the seminal vesicles and/or the prostate could contribute differently to the remaining al,4-glucosidase pool. Our previous work with human prostate cytosol suggests this possibility, 12 but further studies should be conducted to determine the a-l,4-glucosidase molecular forms in the accessory reproductive organs using the procedure described herein. Furthermore, changes in the ratio of Fl and F2 after vas ligation deserve more detailed study, where modifications of the enzyme forms are followed at regular time intervals before and after surgery; experiments with animal models will allow the exclusion of selected organs or glands to determine their influence on the molecular forms of a-l,4-glucosidase in SP.

Acknowledgments. The authors wish to thank Mrs. Diane Dorval and Mr. Jacques Renaud for their skillful technical assistance, and Mrs. Marcelle Fillion for typing the manuscript. REFERENCES 1. Gamklou R, Schersten T: Activity of a-l,4-glucosidase in

human serum. Scand J Clin Lab Invest 31:21, 1973 2. DeBurlet G, Vannier C, Giducelli J, Sudaka P: Neutral a-glucosidase from human kidney: molecular and immunological properties; relationships with intestinal glucoamylase. Biochimie 61:1177, 1979 3. Sheth AR, Rao SS: Maltase activity in human semen. Experientia 18:370, 1962 4. Sheth AR, Rao SS: Purification and properties of human seminal maltase. J Reprod Fertil 4:267,1962

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5. Torres HN, Olavarria J: Liver a-glucosidases. J BioI Chern 239:2427, 1964 6. Eggermont E, Hers HG: The sedimentation properties of the intestinal a-glucosidases of normal human subjects and of patients with sucrose intolerance. Eur J Biochem 9:485,1969 7. Maestracci D, Preiser H, Hedges T, Schmitz J, Crane RK: Enzymes of the human intestinal brush border membrane: identification after gel electrophoresis separation. Biochim Biophys Acta 382:147,1975 8. Franzini C, Bonini PA: a-glucosidases in human urine. Clin Chim Acta 17:505, 1967 9. Angelini C, Engel AG: Subcellular distribution of acid and neutral a-glucosidases in normal, acid maltase deficient, and myophosphorylase deficient human skeletal muscle. Arch Biochem Biophys 156:350, 1973 10. Martiniuk F, Hirschhorn R: Human neutral a-glucosidase C: genetic polymorphism including a null allele. Am J Hum Genet 32:497, 1980 11. Martiniuk F, Hirschhorn R: Characterization of neutral isozymes of human a-glucosidase. Biochim Biophys Acta 658:248, 1981 12. Chapdelaine P, Tremblay RR, DuM JY, St-Yves C, Mailhot J: Origin of maltase and variations in infertile men. Arch Androl 1:61, 1978 13. Tremblay RR, Chapdelaine P, Roy R, Thabet M: Correlation between L-carnitine and a-1,4-glucosidase activity in the semen of normal, infertile and vasectomized men. Infertility, July 1982. In press 14. Lundquist F: Aspects of the biochemistry of human semen. Acta Physiol Scand (Suppl 66) 19:7, 1949 15. Bostrom K, Ockerman PA: Glycosidases in human semen and male genital organs. Scand J Urol Nephrol 5:117, 1971 16. Tremblay RR, Chapdelaine P, Mailhot J: a-1,4-glucosidase activity in human semen: variations with number and motility of spermatozoa. Fertil Steril 31:592, 1979

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17. Chapdelaine P, Tremblay.RR-, DuM JY: Purification and properties of neutral a-1,4-glucosidase from human seminal plasma. Arch Androl 3:153, 1979 18. Belsey MA, Eliasson R, Gallegos AJ, Moghissi KS, Pauben CA, Prasard MRN: Laboratory Manual for the Examination of Human Semen and Semen-Cervical Mucus Interaction. Publication of World Health Organization, Singapore, Press Concern, 1980, p 7 19. Griffith OM: Instruction Manual. Palo Alto, California, Beckman Instruments, Inc., 1975, p 14 20. Shapiro AL, Vinuela E, Maizel JV: Molecular weight estimation of polypeptide chains by electrophoresis in SDSpolyacrylamide gels. Biochem Biophys Res Commun 28: 815, 1967 21. Chapdelaine P, Tremblay RR, DuM JY: p-Nitrophenola-D-glucopyranoside as substrate for measurement of maltase activity in· human semen. Clin Chern 24:208, 1978 22. Lowry OH, Rosenbrough NH, Farr AL, Randall RJ: Protein measurement with the Folin phenol reagent. J BioI Chern 193:265, 1951 23. Dubin L, Amelar RD: Etiologic factors in 1294 consecutive cases of male infertility. Fertil Steril 22:469, 1971 24. Tremblay RR, Mailhot J, Simard L: Diagnostic value of spermatic phlebography in the diagnosis of varicocele in men. L'Union Medicale du Canad.a 109:588, 1980 25. Edwards JJ, Tollaksen SL, Anderson NG: Proteins ofhuman semen: two-dimensional mapping of human seminal fluid. Clin Chern 27:1335,1981 26. Grinna LS, Robbins PW: Glycoprotein biosynthesis. J BioI Chern 254:8814, 1979 27. Rabach J, Starka L: Hormonal testicular activity in men with varicocele. Fertil Steril 22:152, 1971

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Fertility and Sterility