Inhibition of sialyl transferase activity by gossypol acetic acid in human seminal plasma

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ANDROLOGIA

ACCEPTED: JUNE 23, 1990

23, 159-161 (1991)

Inhibition of sialyl transferase activity by gossypol acetic acid in human seminal plasma H. Levinsky, R. Singer, M. Sagiv, N. Lehrer and D. Allalouf Key words. Seminal plasma

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sialyl transferase

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gossypol

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Summary. Seventeen aliquots of 15 pl seminal plasma of human origin were incubated'with 25 pg, 15 pg, 5 pg and 2.5 pg gossypol acetic acid (GAA), dissolved in BWW containing 0.9% sodium chloride and 0.9% benzyl alcohol, at 37 "C for 60 minutes. Following incubation, activity of sialyl transferase (S.T.) was determined by a procedure involving incorporation of radioactive sialic acid into asialofetuin. The activity of S.T., expressed as cpm per hour per 15 pl seminal plasma was compared to controls consisting in incubation of sperm in BWW containing 0.9% benzyl alcohol and 0.9% sodium chloride. The activity of S.T. in controls represented 61.3 f 8.0% - 67.7 f 8.9% of the activity obtained by incubation with BWW only. GAA was found to exert a dose-dependent inhibition of S.T. activity, ranging from 38.3 f 20.6% to 53.4 f 19.4% (with 25 pg) and from 11.3 f 14.8% to 21.9 f 14.8% (with 2.5 pg). Introduction There is a considerable amount of data claiming that sialic acid is involved in a variety of biological processes (Town & Stanford, 1979; Liu et al., 1982; Park et al., 1982; Schmell et al., 1982) including maturation, function and structural integrity of sperm (Arora et al., 1975; Hafez & Prasad, 1976; Base & Prasad, 1975; Rajalakshmi et al., 1976; Levinsky et al., 1983). Sialyl transferase (EC 2.4.99.1) (S.T.) is the enzyme involved in the transfer of sialic acid to glycoproteins and glycolipids in the course of their biosynthesis. In a previous work (Singer et al., 1988) we have estimated the activity of S.T. in The Male Fertility Laboratory, The Connective Tissue Research Unit, Beilinson and Hasharon Medical Centers, Petah Tikva, Israel. Correspondence: Dr Rina Singer, Male Fertility Laboratory, Beilinson Medical Center, Petah Tikva 49 100, Israel.

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human seminal plasma obtained from semen containing and devoid of sperm. The differences between the latter groups with respect to enzyme activity lacked statistical significance. Assessment of S.T. activity in seminal plasma obtained from split semen revealed significantly lower activity in the first fraction as compared to the second one, indicating that S.T. in semen originates mainly from the seminal vesicles. Gossypol, a polyphenolic compound isolated from cotton seed, has been given a great deal of attention due to its ability to serve as an antifertility agent. Experimental work carried out mostly in recent years revealed that gossypol may interfere with a variety of enzyme activities (Wong et al., 1972; Kalla & Vasudev, 1981; Lee & Malling, 1981; Tso & Lee, 1981; Tso et al., 1982; Rovan et al., 1984; McClarty et al., 1985; Vongsorasak & Svasti, 1986; Shi, 1986; Giridharan et al., 1987). However, to the best of our knowledge, the effect of gossypol on S.T. activity has not been examined so far. Our purpose was to examine whether the S.T. activity in human seminal plasma may be inhibited by gossypol in vitro. By using seminal plasma obtained from semen with various sperm densities we also intended to find out whether the inhibition of enzyme activity by gossypol may be affected by the quality of the examined aliquots.

Materials and methods Seventeen semen samples were obtained from patients after 4 days of abstinence. Immediately after liquefaction each specimen was routinely examined as reported elsewhere (Singer et al., 1980). A portion of semen was centrifuged at 10,000 rpm. at 0 "C for 15 minutes and the seminal plasma separated. Gossypol acetic acid (GAA), purchased from Sigma (anhydrous, m.w. 578.6, a crystalline complex consisting of equimolar quantities of gossypol and acetic acid) was dissolved in BWW (Biggers et al.,

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1971) containing 0.9% sodium chloride and 0.9% benzyl alcohol and the pH adjusted to 7.4 To 15 p1 seminal plasma were added 25 pg, 15 pg, 5 pg and 2.5 pg of G A A in a constant volume of 10 p1. Incubation of the latter as well as of appropriate blanks and controls consisting of samples without G A A in the presence or absence of solvent were carried out at 37°C for 60 minutes. Enzyme activity expressed as cpm per hour per 15 pl seminal plasma was determined by a previously reported procedure (Singer et al., 1988) involving the incorporation of radioactive sialic acid from CMP ('*C sialic acid) into asialofetuin. Of the 17 semen samples examined sperm counts of 6 ranged from 29 x lo6 to 11 1 x lo6 per ml, 7 had sperm counts of 1 x lo6 - 19 x lo6 per ml and 4 specimens were devoid of sperm (azoospermic). Statistical examination of results was carried out by the Student's t-test . Results

S.T. activity in control experiments consisted of incubation of seminal plasma originating from semen with various sperm counts in the presence of BWW containing 0.9% sodium chloride and 0.9% benzyl alcohol. This activity represented 61.3 f 8.0% (normal sperm counts), 67.7 f 8.9% (sperm counts ranging from 1 to 19 x 106/ml) and 66.0 f 19.6% (sperm devoid specimens) of total activities in seminal plasma which were 910 f 229, 908 k 284 and 839 k 284 cpm per hour per 15 pl correspondingly. As presented in Figure 1 GAA was found to exert a dose dependent inhibition of S.T. activity as com-

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GAA pg

Figure 1. The percentile inhibition of sialyl transferase activity in human seminal plasma by gossypol acetic acid. Inhibition was calculated by comparison with enzyme activities in the presence of solvent (0.9% sodium chloride and 0.9% benzyl alcohol in BWW). These were 61.3 8.0, 67.7 & 8.9% and 66.0 f 19.6y0 of the activity in the presence of BWW only. Vertical lines represent standard deviations.

pared to activities in the presence of solvent. Comparison of inhibition between the successive doses of G A A (25 pg vs 15 pg; 15 pg vs 5 pg; 5 pg vs 2.5 pg) lacked statistical significance. However, the differences between most extreme doses (25 pg vs 2.5 pg) were always of statistical significance (P < 0.05). Differences between groups (low sperm counts, high sperm counts and azoospermic specimens) with respect to the GAA enzyme-inhibiting activities were found to be statistically non significant. Discussion

There is a dose-dependent inhibition of the activity of S.T. in seminal plasma by GAA. This actually could have been anticipated due to similar effect of G A A on the activity of other enzymes involved with the physiology of sperm (Kalla & Vasudev, 1981; Lee & Malling, 1981; Tso & Lee, 1981; Tso et al., 1982; Rovan et al. 1984; Shi, 1986; Giridharan et al. 1987). S.T. may be secondarily absorbed from the semen to the sperm cell (Durr et al., 1977; Daunter & Newlands, 1981). Thus the inhibition of S.T. activity which may occur also in vivo, would interfere with the transfer of sialic acid to glycoproteins and glycolipids in the sperm cell, having a deteriorating effect on its development and biological functions.

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Lee, C. Y. & M. V. Malling (1981): Selective-inhibition of sperm-specific lactate dehydrogenase-X by an anti-fertility agent gossypol. Fed. Proc. 40:718. Levinsky, H., R. Singer, M. Barnet, M. Sagiv & D. Allalouf (1983): Sialic acid content ofhuman spermatozoa and seminal plasma in relation to sperm counts. Arch. Androl. 10: 4 5 4 6 . Liu, D. Y., K. D. Petschek, H. G. Remold & J. R. David (1982): Isolation of guinea-pig macrophage glycolipids with the properties of the putative migration inhibitory factor receptor. J. Biol. Chem. 257:159-162. McClarty, G. A., A. K. Chan, D. C. Creasey & J. A. Wright (1985): Ribonucleotide reductase; an intracellular target for the male antifertility agent, gossypol. Biochem. Biophys. Res. Comm. 133:30@-305. Park, L. S., M. S. Kuhlenschmidt & S. Roseman (1982): Specific interaction between chicken hepatocytes and an immobilized chicken liver adhesion factor. Fed. Proc. 42:2129. Rajalakshmi, M., R. Arora & T. K. Base (1976): Physiology of the epididymis and induction of functional sterility in the male. J. Reprod. Fertil. (Suppl.) 24:71-94. Rovan, E., N. R. Kalla, J. Frick & H. Adam (1984): Effect of gossypol on bull spermatozoa in vitro. Urol Res. 12:187-192. Schmell, E., C. W. Slife, M. S. Kuhlenschmidt & S. Roseman (1982): Studies on the intercellular adhesion of rat and chicken hepatocytes. Conditions for stimulation by liver plasma membranes. J. Biol. Chem. 257:3 17 1-3 176.

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