- Email: [email protected]
Reproductive toxicologic effects of gossypol on male rabbits: biochemical, enzymatic, and electrolytic properties of seminal plasma
Reproductive toxicologic effects of gossypol on male rabbits: biochemical, enzymatic, and electrolytic properties of seminal plasma Walaa F. Shaaban, M.Sc.,a Taha A. Taha, Ph.D.,a Farahat D. El-Nouty, Ph.D.,a Ahmed R. El-Mahdy, Ph.D.,b and Mohamed H. Salem, Ph.D.a a Department of Animal Production, and b Department of Food Science and Technology, Faculty of Agriculture (El-Shatby), Alexandria University, Alexandria, Egypt
Objective: To evaluate the effects of two sublethal doses of gossypol on biochemical, enzymatic, and electrolytic properties of male rabbit seminal plasma. Design: Retrospective study. Setting: A university-affiliated laboratory. Animal(s): Fifteen adult male New Zealand white rabbits. Intervention(s): Two sublethal doses of gossypol (4 and 20 mg per kilogram of body weight, every other day) were given orally. Main Outcome Measure(s): Biochemical, enzymatic, and electrolytic properties of male rabbit seminal plasma. Result(s): Seminal plasma total protein and albumin were statistically significantly increased by treatment with gossypol. Total lipids concentration was statistically significantly decreased by treatment with both doses of gossypol, whereas cholesterol was statistically significantly decreased by treatment with the low gossypol dose. Seminal plasma lactate dehydrogenase and alanine aminotransferase activities were statistically significantly decreased by treatment with the low dose of gossypol, whereas activities of these enzymes as well as of aspartate aminotransferase were statistically significantly increased by treatment with the high dose. Treatment with the high dose of gossypol statistically significantly reduced Kþ concentration in the seminal plasma, whereas it statistically significantly increased Naþ and the Naþ-Kþ ratio. Conclusion(s): Seminal plasma values of total protein, total lipids, cholesterol, aspartate aminotransferase, and Naþ concentration returned to control levels after withdrawal of gossypol, whereas the effect of this drug on other parameters continued during the recovery period. (Fertil Steril 2008;89:1585–93. 2008 by American Society for Reproductive Medicine.) Key Words: Gossypol, rabbit, seminal plasma proteins, lipids, enzymes, electrolytes
Seminal plasma is the secretion of the male accessory glands to provide a medium and vehicle for spermatozoal transportation. It is known to have high protein content, enzymes, lipids, electrolytes, and various other factors that may play significant roles in the metabolic regulation of spermatozoa. To our knowledge, no previous studies regarding the effect of gossypol on seminal plasma constituents have been reported. However, several investigations have been performed on the effect of gossypol on blood plasma constituents, from which its effect on seminal plasma constituents can be extrapolated because of the ability of gossypol to cross the blood–testis barrier (1). Because of the protein-binding property of gossypol, the cytotoxicity of gossypol can be alleviated by serum protein (2), and thus, an increase in protein concentration in the gossypol-treated animals can be considered a biological
Received December 9, 2006; revised and accepted May 9, 2007. Reprint requests: Taha A. Taha, Ph.D., Department of Animal Production, Faculty of Agriculture (El-Shatby), Alexandria University, Aflaton Street, Alexandria 21545, Egypt (FAX: 2035922780; E-mail: tahaataha@yahoo. com).
0015-0282/08/$34.00 doi:10.1016/j.fertnstert.2007.06.077
response, intended to counteract its deleterious effect. In addition, gossypol strongly interacts with various enzymes and interferes with many enzymatic reactions that are involved in the metabolic regulation of spermatozoa (3–5). Seminal plasma lipids also play significant roles in the membrane structure of spermatozoa, in sperm metabolism, in sperm capacitation, and in fertilization of the female gamete (6). Because it is highly lipophilic, gossypol can interact directly with the phospholipid bilayer of biological membranes to alter membrane structure, electrostatic charge, and transmembrane ion fluxes. A major side effect of gossypol administration is the change in electrolyte status, in particular the reduction in serum Kþ levels (7) that is accompanied by a marked increase in serum Naþ concentration (8). Daily ingestion of gossypol provokes infertility in various animal species, including human beings. The contraceptive effect of gossypol in human beings was discovered first in China and still continues to be tested as a favorable alternative for male contraception (9). In addition, our recent studies (10) clearly indicated that gossypol has pronounced hazardous effects on semen characteristics and blood hormonal parameters in male rabbits that may lead to deleterious
Fertility and Sterility Vol. 89, Suppl 3, May 2008 Copyright ª2008 American Society for Reproductive Medicine, Published by Elsevier Inc.
1585
productive and reproductive performance. Thus, the objectives of the present study were to evaluate the effects of gossypol on seminal plasma constituents in an attempt to correlate the disturbances in biochemical composition and in enzymatic and electrolyte content of seminal plasma with this gossypol-induced decline in rabbit semen characteristics (10). Such studies would help identify some of the mechanisms that are responsible for the role of gossypol as a potential male contraceptive. MATERIALS AND METHODS Animals and Management This study was performed at the Department of Animal Production, Faculty of Agriculture, Alexandria University. Experiments were performed after departmental approval, followed standard scientific procedures without compromising animal welfare rights, and were performed without any commercial profit purpose. Fifteen adult male New Zealand white rabbits, which were 6 to 8 months of age and weighed 2.76 0.39 kg (mean SE) at the beginning of the experiment, were used during the reproductive season (starting in September). The rabbits were individually housed in cages. Food and water were provided ad libitum. The animals were offered pellets consisting of (per kg) 330 g of berseem (Trifolium alexandrinum) hay, 170 g of soybean meal, 165 g of ground maize, 160 g of barley, 120 g of wheat bran, 38 g of molasses, 10 g of salt, 4 g of dibasic calcium phosphate, and 3 g of vitamins. The chemical analysis of the pellets according to the Association of Official Analytical Chemists (11) indicated that they contained (per kg) 175 g of crude protein, 140 g of crude fiber, and 27 g of fat. All animals were allowed to adjust to their new environment and were tested for semen quality for 3 weeks before the experiment commenced. Experimental Design Gossypol was extracted from cottonseeds and purified according to Boatner (12), as described by Taha et al. (10). The rabbits were divided randomly into three groups of five animals each and were assigned at random to one of the following treatments: the first group served as control (the animals were given an equivalent dose of the vehicle, consisting of maize oil þ acetone), and the second and third groups were used to study the effect of the low dose (1/100 of median lethal dose, 4 mg per kilogram of live weight) and high dose (1/20 of median lethal dose, 20 mg per kilogram of live weight) of gossypol. The correct dose for each rabbit was given orally by syringe, directly into the esopharyngeal region, every other day throughout the treatment period, which lasted for 8 weeks. This period was followed by an 8-week recovery period, during which all drugs were withdrawn. Sample Collection and Analysis Semen was collected weekly from all animals throughout the 16-week experimental period. Ejaculates were obtained by 1586
Shaaban et al.
Gossypol and rabbit seminal plasma
using an artificial vagina and a teaser doe. Seminal plasma was separated from ejaculates by centrifugation at 5,000 rpm for 10 minutes. The recovered seminal plasma fraction was further processed by centrifuge at 10,000 rpm for 15 minutes at 4 C, and the supernatant was stored at 20 C until analysis. Seminal plasma total protein (TP) was measured by the Biuret method, as described by Armstrong and Carr (13). Total albumin concentration was determined by the bromocresol green method of Doumas et al. (14). Seminal plasma total lipids were determined as described by Frings et al. (15), and total cholesterol concentration was measured by a colorimetric method (16). Activity of lactate dehydrogenase (LDH) was measured according to Buhl and Jackson (17). Transaminase activities (aspartate aminotransferase [AST] and alanine aminotransferase [ALT]) were measured by colorimetric methods, as described by Reitman and Frankel (18). Seminal plasma electrolytes (Naþ and Kþ) were determined by using commercial kits (Quimica Clinica Aplicada S.A., Ampsta, Spain), then Naþ-Kþ ratios were calculated. Statistical Analysis Data for each of the treatment and recovery periods were analyzed separately by using the general linear model procedure of the SAS Institute (19). The statistical model describing all parameters included the following: Yijk ¼ m þ Ti þ Wj þ ðTWÞij þ eijk ; where Yijk ¼ an observation of trait recorded on individual k; m ¼ the overall mean; Ti ¼ the fixed effect of the ith treatment (control, low dose, high dose); Wj ¼ the fixed effect of the jth week; (TW)ij ¼ the interaction between treatment and weeks; and eijk ¼ a random error assumed to be independent normally distributed, with mean ¼ 0 and variance ¼ s2e. Treatment means were compared by using the least significant difference procedure (20). The level of significance was P<.05. RESULTS Seminal Plasma Proteins, Total Lipids, and Cholesterol Means of seminal plasma TPs, albumin, total lipids, and cholesterol during treatment and recovery periods as influenced by gossypol are shown in Table 1, and the weekly mean values of these parameters, expressed as percentage of control, are presented in Figure 1. Treatment with the high dose of gossypol increased (P<.05) TP concentration when compared with the case of the control group. Withdrawal of gossypol during the recovery period abolished this effect on TP. Gossypol administration resulted in significant (P<.05) increases in albumin concentration in both the low- and high-dose groups when compared with the control group. Withdrawal of gossypol during the recovery period resulted in significant (P<.05) reductions in albumin Vol. 89, Suppl 3, May 2008
TABLE 1 Mean (±SEM) concentrations of TP, albumin, total lipids, and cholesterol in seminal plasma. Semen parameters Group Treatment Control LD HD Week (W) TW Recovery Control LD HD Week (W) RW
Albumin (g per 100 mL)
Total lipids (g/L)
2.09 0.06b 2.19 0.04b 2.44 0.03a NS NS
0.80 0.05b 1.00 0.03a 1.09 0.03a
1.27 0.03a 1.01 0.05b 1.10 0.05b
c
d
NS
NS
45.5 1.07a 39.4 1.38b 48.9 1.99a NS NS
2.24 0.05 2.11 0.04 2.09 0.03 NS NS
1.00 0.03a 0.81 0.03b 0.86 0.03b NS NS
1.07 0.04 1.03 0.03 0.93 0.03 NS NS
41.6 1.71 38.6 1.50 37.9 1.20 NS NS
TP (g per 100 mL)
Cholesterol (mg per 100 mL)
Note: In both the treatment and recovery groups, n ¼ 5. NS ¼ nonsignificant; LD ¼ low dose of gossypol; HD ¼ high dose of gossypol. a,b Within columns, within periods, means with one of these two superscript letters differ significantly from one another (P< .05). c P< .05. d P< .01. Shaaban. Gossypol and rabbit seminal plasma. Fertil Steril 2008.
concentration at the two doses used. Total lipids concentration was decreased (P<.05) by treatment with both doses of gossypol, compared with the case of the control, whereas cholesterol was decreased (P<.05) by treatment with the low dose. Withdrawal of gossypol resulted in complete recovery of seminal plasma total lipids and cholesterol. Enzymes Gossypol effects on seminal plasma concentration of LDH, AST, and ALT during treatment and recovery periods are shown in Table 2. The biweekly mean values of these parameters, expressed as percentage of control, are presented in Figure 2. Lactate dehydrogenase in the low-dose group (468.0 U/L) was significantly (P<.05) lower, whereas LDH in the high-dose group (1,855.5 U/L) was significantly (P<.05) higher, compared with the case in the control group (1,265.3 U/L). A similar trend was also noted during the recovery period. Moreover, enzyme ALT responded to gossypol in a manner similar to that of LDH during treatment and recovery periods. In addition, treatment with the high dose of gossypol increased (P<.05) AST concentration compared with the control group. Withdrawal of gossypol during the recovery period resulted in complete recovery in AST concentration at the two doses used. Electrolytes Mean values of seminal plasma Naþ, Kþ, and Naþ-Kþ ratio during treatment and recovery periods, as influenced by gosFertility and Sterility
sypol, are shown in Table 3. The tetraweekly mean values of these parameters, expressed as percentage of control, are presented in Figure 3. Treatment with the high dose of gossypol increased (P<.05) Naþ concentration when compared with the case of the control group, and this effect was abolished during the recovery period. Gossypol administration resulted in consistent reductions of Kþ concentration during the treatment period in the high- and low-dose–treated groups compared with the control group, but these reductions were significant (P<.05) only in the high-dose group. After gossypol cessation, this reduction in Kþ concentration was significant (P<.05) in the two doses used. The Naþ-Kþ ratio was increased (P<.05) by the high dose of gossypol during the treatment period and by the low- and high-dose groups during the recovery period.
DISCUSSION Effect of Gossypol on TP and Albumin Because of the ability of gossypol to cross the blood–testis barrier (1), the aldehyde groups that are present in the gossypol molecule may be bound readily to the free amino groups of the protein molecules to form a Schiff’s base (21). Because of the protein-binding property of gossypol, the cytotoxicity of gossypol can be alleviated by serum protein (2). Thus, the increase in protein and albumin concentrations in the gossypol-treated rabbits in the present study (Table 1 and Fig. 1) can be considered a biological 1587
FIGURE 1 Changes in seminal plasma TP, albumin, total lipids, and cholesterol during treatment and recovery of male rabbits with gossypol (control, B; gossypol low dose, :; and gossypol high dose, -).
Shaaban. Gossypol and rabbit seminal plasma. Fertil Steril 2008.
response, intended to counteract the cytotoxicity of gossypol. The increase in albumin concentration during the treatment period also may be attributed to the degenerative effect that gossypol administration exerts upon testicular tissues (22). Total Lipids and Cholesterol Seminal lipids play significant roles in the membrane structure of spermatozoa, sperm metabolism, sperm capacitation, 1588
Shaaban et al.
Gossypol and rabbit seminal plasma
and fertilization of female gametes (6). In the present study, gossypol treatment reduced seminal plasma total lipids in rabbits (Table 1), which was associated with reductions in sperm motility and sperm concentration (10). Studies elsewhere also showed concomitant decrease in sperm motility and sperm concentration with the decline in seminal plasma total lipids in bulls (23) or in total lipids and fructose in rams (24). During the recovery period, seminal plasma total lipids were restored to their normal concentration after gossypol withdrawal. Vol. 89, Suppl 3, May 2008
TABLE 2 Mean (±SEM) concentrations of LDH, AST, and ALT in seminal plasma. Semen parameters Group Treatment Control LD HD Week (W) TW Recovery Control LD HD Week (W) RW
LDH (U/L)
AST (U/mL)
ALT (U/mL)
1,265.3 84.7b 468.0 163.2c 1,855.5 56.6a
38.6 1.18b 44.0 1.70b 51.4 1.96a NS NS
12.0 0.31b 10.4 0.29c 16.2 0.64a NS NS
38.6 1.18 42.6 2.67 46.9 1.91 NS NS
12.0 0.31b 10.6 0.19c 14.1 0.46a NS NS
d d
1,624.1 150.1b 746.6 81.8c 2,060.8 92.6a NS NS
Note: In both the treatment and recovery groups, n ¼ 5. NS ¼ nonsignificant; LD ¼ low dose of gossypol; HD ¼ high dose of gossypol. a,b,c Within columns, within periods, means with one of these three superscript letters differ significantly from one another (P< .05). d P< .01. Shaaban. Gossypol and rabbit seminal plasma. Fertil Steril 2008.
FIGURE 2 Changes in seminal plasma LDH, AST, and ALT during treatment and recovery of male rabbits with gossypol (control, B; gossypol low dose, :; and gossypol high dose, -).
The hypocholesteremic effect of gossypol that was observed in the present study agrees with observations from studies conducted elsewhere on rats (25) and monkeys (26). The gossypol-induced reductions in blood plasma T3 and T concentrations that were observed in the study undertaken by Taha et al. (10) in rabbits would contribute to the hypocholesteremic response in the low-dose–treated animals (27). However, the high gossypol dose revealed no effect on seminal plasma cholesterol (Table 1), with concomitant marked increase in AST and ALT concentrations (Table 2), which reflects altered hepatic function. Consequently, cholesterol removal was reduced, resulting in cholesterol accumulation. Because cholesterol synthesis was inhibited by the decline in T3 and T concentration, the accumulated cholesterol did not exceed the control value. During the recovery period, seminal plasma cholesterol was restored to its control value. Effect of Gossypol on Some Enzymatic Activities Many investigators have reported that gossypol interferes with many enzymatic reactions that are involved in the metabolic regulation of spermatozoa, including those resulting from malate dehydrogenase (3), pyruvate dehydrogenase (4), and LDH (5). Concerning LDH activity, gossypol may act as an uncoupler of mitochondrial oxidative phosphorylation (28) and inhibit LDH-X, an enzyme that is postulated to participate in a shuttle system transferring Hþ from cytosol to mitochondria (29).
Shaaban. Gossypol and rabbit seminal plasma. Fertil Steril 2008.
Fertility and Sterility
In the present study (Table 2), a marked inhibitory effect of gossypol on LDH was observed in the low-dose group, in 1589
TABLE 3 Mean (±SEM) concentrations of NaD and KD in seminal plasma, as well as mean NaD-KD ratio. Semen parameters Group Treatment Control LD HD Week (W) TW Recovery Control LD HD Week (W) RW
NaD (mmol/L)
KD (mmol/L)
NaD-KD ratio
65.2 7.36b 53.5 7.15b 107.9 5.19a NS NS
29.5 2.33a 24.5 2.20a,b 22.0 1.25b NS NS
2.31 0.32b 2.26 0.35b 5.16 0.45a NS NS
34.4 1.80a 27.2 1.86b 26.0 1.43b NS NS
2.32 0.20b 3.15 0.59a 3.81 0.29a
79.4 6.49 85.4 15.75 96.3 3.33 d d
c d
Note: In both the treatment and recovery groups, n ¼ 5. NS ¼ nonsignificant; LD ¼ low dose of gossypol; HD ¼ high dose of gossypol. a,b Within columns, within periods, means with one of these two superscript letters differ significantly from one another (P< .05). c P< .05. d P< .01. Shaaban. Gossypol and rabbit seminal plasma. Fertil Steril 2008.
which LDH concentration was reduced 2.7-fold compared with in the control group. This dose of gossypol was also found to cause significant reduction in forward motility (10). This effect of gossypol on LDH activity was reported in human beings (30), monkeys (31), bovines (5), boars (32), rabbits (33), rats (34), hamsters (35), and mice (36). The present study, however, revealed a marked discrepancy between the effect of the low and high doses of gossypol on LDH activity, in that the high dose enhanced the activity of LDH (Table 2). Several mechanisms have been proposed to explain these inconsistent effects. Gossypol is known to promote the formation of reactive oxygen species (37), so at the ultrastructural level, gossypol has been shown to induce marked structural defects in the cellular membrane system and in the intracellular organelles (38), as well as to induce extensive mitochondrial damage (39). Collectively, rupture of plasma and acrosomal membranes and mitochondrial destruction are suggested to induce cytoplasmic leakage and subsequent increase in seminal plasma LDH concentration. Despite the inhibitory effect of gossypol on LDH, the released LDH from destroyed sperm could not be inhibited by gossypol because of the marked increase in extracellular proteins (Table 1), which counteracted the inhibitory effect of gossypol (2). The affinity of gossypol for albumin is several orders of magnitude higher than the affinity of gossypol for LDH, resulting in a low concentration of gossypol to which the enzymes are exposed (2).
Our findings on the same rabbits showed reduction of total functional sperm fraction concomitantly with an increasing percentage of altered acrosomes (10), reflecting marked damage of sperm cells in the high-dose group. Our studies using electron microscopy revealed that the high dose of gossypol caused marked destruction of sperm plasma acrosomal and mitochondrial membranes (40). In addition, seminal plasma TP concentration was higher in the high-dose group than in the low-dose and control groups (Table 1), which counteracted the cytotoxic effects of gossypol on LDH activity.
This view could explain the increasing activity of seminal plasma LDH in the high-dose–treated animals (Table 2).
The present study revealed a marked discrepancy between the effects of the low and high doses of gossypol on
1590
Shaaban et al.
Gossypol and rabbit seminal plasma
The concentration of transaminase enzymes (AST and ALT) in semen is a good indicator of semen quality because it measures sperm membrane stability (41). Thus, increasing the percentage of dead spermatozoa in a semen sample causes high concentrations of transaminase enzymes in the extracellular fluid as a result of sperm membrane damage and, consequently, the ease of leakage of enzymes from spermatozoa. As indicated for LDH in a previous paragraph, gossypol exerted its cytotoxic effect by the formation of reactive oxygen species (37), which may cause plasma membrane rupture and mitochondrial destruction of sperm (38–40), in addition to degeneration of cells lining seminiferous tubules (42, 43). These effects are expected to induce cytoplasmic leakage and subsequent increase in transaminase concentrations in extracellular fluid (seminal plasma).
Vol. 89, Suppl 3, May 2008
FIGURE 3 Changes in seminal plasma Naþ, Kþ, and Naþ-Kþ ratio during treatment and recovery of male rabbits with gossypol (control, B; gossypol low dose, :; and gossypol high dose, -).
Gossypol cessation revealed complete recovery of AST activity, whereas the effect of gossypol on ALT and LDH activities was extended during the recovery period. The extended inhibitory effect of the low dose of gossypol on ALT and LDH activities could be explained by preferential accumulation of gossypol within the mitochondria (46) and by the binding of gossypol to spermatozoa in an irreversible manner (31). In addition, the moderate reduction in total functional sperm fraction concentration and the increasing percentage of altered acrosome that were observed in the low-dose group during the recovery period (10) substantiated the extended deleterious effect of gossypol, which could not be eliminated because of the low albumin concentration that was recorded during the recovery period (Table 1). However, the enhancing effect of the high dose of gossypol on ALT and LDH activities that was noted during the treatment period also was extended during the recovery period (Table 2). Levels of ALT provide more reliable indications in acute, and AST levels, in chronic, tissue injury (47). Just as gossypol cessation minimizes the severe effect of gossypol, so the limited residue of gossypol appears to cause injury at the cytoplasmic level but not at the mitochondrial level, which is reflected in the extending effect of gossypol on ALT activity, whereas the effect on AST activity was moderate (insignificant increase). This suggestion may explain the continued effect of gossypol on ALT activity that was noted after gossypol cessation, whereas there was no significant effect on AST activity.
Shaaban. Gossypol and rabbit seminal plasma. Fertil Steril 2008.
transaminase activities. Several mechanisms have been proposed to explain these inconsistent effects. Concerning the high dose of gossypol, the increased activity of transaminases observed in the present study (Table 2) is consistent with results of studies elsewhere (44). The reason for such increase is the leakage of transaminases through the destroyed sperm plasma membrane and mitochondria, which is evident from the gossypol-induced decrease in the concentration of total functional sperm fraction that was reported by Taha et al. (10). Furthermore, despite the inhibitory effect of gossypol on transaminase activities, the released transaminases from destroyed sperm could not be inhibited by gossypol because of the presence of a marked increase in extracellular proteins (Table 1) that counteract the inhibitory effect of gossypol (2). However, when the low dose of gossypol was used, a mild degree of tissue injury appeared to occur, as reflected by the normal concentration of total functional sperm fraction (10). In this case, the moderately leaked transaminases may be inhibited in the presence of gossypol. This view is supported by the finding of Nomeir and Abou-Donia (45), who reported that gossypol may alter the activity of transaminases by reacting with the substrate and blocking the action of the enzyme and/or by combining with the enzyme itself. Fertility and Sterility
Effect of Gossypol on Seminal Plasma Electrolyte Contents The importance of the ionic and osmotic environment on sperm motility and metabolic activity is well established. Extremes of Kþ concentration were found to depress the motility and viability of spermatozoa (48). A major side effect of gossypol administration is the change in electrolyte status (49), in particular hypokalemia, the reduction in serum Kþ levels (7). Furthermore, Kþ deficiency has been shown to enhance the antispermatogenic effect of gossypol (50). Enzyme 11b-hydroxysteroid dehydrogenase in the kidney is known to oxidize the active hormone cortisol to the inactive cortisone (51). Thus, the active enzyme lowers apparent mineralocorticoid activity by inactivating cortisol and leaving only aldosterone as the active mineralocorticoid, whereas inhibition of this enzyme produces the effects of mineralocorticoid excess, hypokalemia (52). Several studies reported that gossypol inhibits 11bhydroxysteroid dehydrogenase in guinea pigs (53) and human beings (54). These findings provide an explanation for the hypokalemic effect of gossypol that was observed in the present study (Table 3), in which the gossypol-induced hypokalemia was more pronounced when the high dose of gossypol was used. In addition, the hypokalemic effect of this gossypol dose was accompanied by Naþ retention, as reflected by a marked increase of seminal plasma Naþ concentration, as well as by an elevation in Naþ-Kþ ratio. These observations 1591
coincide with those from a study conducted on monkeys (8). The hypokalemic effect of gossypol was extended after gossypol cessation in both the low and high doses, whereas seminal plasma Naþ concentration showed almost complete recovery. Despite restoration of a relatively normal level of Naþ, the Naþ-Kþ ratio remained higher than that in the control group, which reflected a continued hypokalemic effect of gossypol. In conclusion, sublethal doses of gossypol induced effects on rabbit seminal plasma TP, total lipids, cholesterol, AST, and Naþ that were reversible after withdrawal of gossypol, as well as effects on albumin, LDH, ALT, and Kþ that were irreversible.
REFERENCES 1. Wang JM, Tao L, Wu XL, Lin LX, Wu J, Wang M, et al. Differential binding of (þ) and () gossypol to plasma protein and their entry into rat testis. J Reprod Fertil 1992;95:277–82. 2. Haspel HC, Ren YF, Watanabe KA, Sonnenberg M, Corin RE. Cytocidal effect of gossypol on cultured murine erythroleukemia cells is prevented by serum protein. J Pharmacol Exp Ther 1984;229:218–25. 3. Montamat EE, Burgos C, Geres de Burgos NM, Rovai LE, Blanco A. Inhibitory action of gossypol on enzymes and growth of Trypanosoma cruzi. Science 1982;218:288–9. 4. Adeyemo O, Chang CY, Segal SJ, Koide SS. Gossypol action on the production and utilization of ATP in sea urchin spermatozoa. Arch Androl 1983;9:343–9. 5. Olgiati KL, Toscano WA. Kinetics of gossypol inhibition of bovine lactate dehydrogenase X. Biochem Biophys Res Commun 1983;115:180–5. 6. Mann T, Lutwak-Mann C. Biochemistry of spermatozoa: chemical and functional correlations in ejaculated semen. In: Mann T, LutwakMann C, eds. Male reproductive function and semen. Themes and trends in physiology and biochemistry and investigative andrology. Berlin: Springer-Verlag, 1981:185–268. 7. Liu GZ, Chiu-Hinton K, Cao JA, Zhu CX, Li BY. Effects of Kþ salt or a potassium blocker on gossypol-related hypokalemia. Contraception 1988;37:111–8. 8. Lohiya NK, Sharma K, Kumar M, Sharma S. Limitations in developing gossypol acetic acid as a male contraceptive. Contraception 1990;41: 519–32. 9. Cui GH, Xu ZL, Yang ZJ, Xu YY, Xue SP. A combined regimen of gossypol plus methyltestosterone and ethinylestradiol as a contraceptive induces germ cell apoptosis and expression of its related genes in rats. Contraception 2004;70:335–42. 10. Taha TA, Shaaban WF, EL-Mahdy AR, EL-Nouty FD, Salem MH. Reproductive toxicological effects of gossypol on male rabbits: semen characteristics and hormonal levels. Anim Sci 2006;82:259–69. 11. Association of Official Analytical Chemists. Official methods of analysis. 16th ed. Arlington, VA: Association of Official Analytical Chemists, 1995. 12. Boatner CH. Pigments of cottonseed. In: Bailey AE, ed. Cottonseed and cottonseed products. Their chemistry and chemical technology. New York: Wiley (Interscience), 1948:215–23. 13. Armstrong WD, Carr CW, eds. Physiological chemistry laboratory directions. 3rd ed. Minneapolis, MN: Burges Publishing Co, 1964. 14. Doumas BT, Watson WA, Biggs HG. Albumin standards and the measurement of serum albumin with bromocresol green. Clin Chim Acta 1977;31:87–96. 15. Frings CS, Fendley TW, Dunn RT, Queen CA. Improved determination of total serum lipids by the sulfo-phospho-vanillin reaction. Clin Chem 1972;18:673–4. 16. Watson D. A simple method for the determination of serum cholesterol. Clin Chim Acta 1960;5:637–8.
1592
Shaaban et al.
Gossypol and rabbit seminal plasma
17. Buhl SN, Jackson KY. Optimal conditions and comparison of lactate deydrogenase catalysis of the lactate-to-pyruvate and pyruvate-to-lactate reactions in human serum at 25, 30 and 37 degrees. Clin Chem 1978;24: 828–31. 18. Reitman S, Frankel S. A colorimetric method for the determination of serum glutamic oxaloacetic pyruvic transaminases. Am J Clin Pathol 1957;28:56–63. 19. SAS Institute. SAS user’s guide: statistics. Version 8 ed. Cary, NC: SAS Institute, 1999. 20. Fisher RA. The design of experiments. Edinburgh, United Kingdom: Oliver and Boyd, 1949. 21. Wu DF, Reidenberg MM. Stereoselective interaction between gossypol and rat plasma. Contraception 1990;41:377–88. 22. Arshami J, Ruttle JL. Effects of diets containing gossypol on spermatogenic tissues of young bulls. Theriogenology 1988;30:507–16. 23. Kelso KA, Redpath A, Noble RC, Speake BK. Lipid and antioxidant changes in spermatozoa and seminal plasma throughout the reproductive period of bulls. J Reprod Fertil 1997;109:1–6. 24. Taha TA, Abdel-Gawad EI, Ayoub MA. Monthly variations in some reproductive parameters of Barki and Awassi rams throughout 1 year under subtropical conditions. 1. Semen characteristics and hormonals levels. Anim Sci 2000;71:317–24. 25. Nwoha PU, Aire TA. Reduced level of serum cholesterol on low proteinfed Wistar rats administered gossypol and chloroquine. Contraception 1995;52:261–5. 26. Shandilya LN, Clarkson TB. Hypolipidemic effects of gossypol in cynomolgus monkeys (Macaca fascicularis). Lipids 1982;4:285–90. 27. Guyton AC. Textbook of medical physiology. 8th ed. Philadelphia: WB Saunders, 1991:754–64. 28. Abou Donia MB, Dieckert JW. Gossypol: uncoupling of respiratory chain and oxidative phosphorylation. Life Sci 1974;14:1955–63. 29. Lee CYG, Moon YS, Duleba A, Chen AF, Gomel V. Gossypol inhibition of LDH-X. In: Lobl TJ, Hafez ESE, eds. Male fertility and its regulation. London: MTP Press, 1985:193–9. 30. Wichmann K, K€apyaho K, Sinervirta R, J€anne J. Effect of gossypol on the motility and metabolism of human spermatozoa. J Reprod Fertil 1983;69:259–64. 31. Stephens DT, Critchlow LM, Hoskins DD. Mechanism of inhibition by gossypol of glycolysis and motility of monkey spermatozoa in vitro. J Reprod Fertil 1983;69:447–52. 32. Tso WW, Lee CS. Lactate dehydrogenase-X, an isoenzyme particularly sensitive to gossypol inhibition. Int J Androl 1982;5:205–9. 33. Eliasson R, Virji N. Effect of gossypol acetic acid on the activity of LDHC4 from human and rabbit spermatozoa. Int J Androl 1983;6:109–12. 34. Lin YC, Chitcharoenthum M, Rikihisa Y. Effect of gossypol on spermatozoal lactate dehydrogenase-X (LDH-X) in male rats. Contraception 1987;5:581–92. 35. KalIa NR, Bisnooduth U, Ranga A. Response of hamster to the antifertility effect of gossypol. Acta Eur Fertil 1990;1:25–37. 36. Lee CYG, Moon YS, Yuan JH, Chen AF. Enzyme inactivation and inhibition by gossypol. Mol Cell Biochem 1982;47:65–70. 37. Barhoumi R, Burghardt RC. Kinetic analysis of the chronology of patulin- and gossypol-induced cytotoxicity in vitro. Fundam Appl Toxicol 1996;30:290–7. 38. Zamboni L. The ultrastructural pathology of the spermatozoon as a cause of infertility: the role of electron microscopy in the evaluation of semen quality. Fertil Steril 1987;48:711–34. 39. Hoffer AP. Ultrastructural studies of spermatozoa and the epithelial lining of the epididymis and vas deferens in rats treated with gossypol. Arch Androl 1982;8:233–46. 40. Shaaban WF. Biological and biochemical seminal changes in rabbits treated with gossypol [M.Sc. thesis]. Egypt: Alexandria University, 2003. 41. Pace MM, Graham EF. The release of GOT from bovine spermatozoa as a test method of assaying semen quality and fertility. Biol Repro 1970;3: 140–6. 42. Hoffer AP, Agarwal A, Meltzer P, Herlihy P, Nagvi RH, Lindberg MC, et al. Ultrastructural, fertility and spermicidal studies with isomers
Vol. 89, Suppl 3, May 2008
43.
44. 45.
46.
47. 48.
and derivatives of gossypol in male hamsters. Biol Reprod 1987;37: 909–24. Chase CC, Bastidas P, Ruttle JL, Long CR, Randal RD. Growth and reproductive development in Brahman bulls fed diets containing gossypol. J Anim Sci 1994;72:445–52. Abou-Donia MB. Physiological effects and metabolism of gossypol. Residue Rev 1976;61:125–60. Nomeir AA, Abou-Donia MB. Toxicological Effects of Gossypol. In: Lobl TJ, Hafez ESE, eds. Male fertility and its regulation. London: MTP Press, 1985:111–33. Tso WW, Lee CS. Gossypol uncoupling of respiratory chain and oxidative phosphorylation in ejaculated boar spermatozoa. Contraception 1982;25:649–56. Clinical Laboratory. 11th edition of medico-chemical investigation methods. Darmstadt, Germany: Merck, 1970. McGrady AV, Nelson L, Ireland M. Ionic effects on the motility of bull and chimpanzee spermatozoa. J Reprod Fertil 1974;40:71–6.
Fertility and Sterility
49. Xue SP. Gossypol contraception and mechanism of action. In: Lobl TJ, Hafez ESE, eds. Male fertility and its regulation. London: MTP Press, 1985:175–84. 50. Qian SZ. Gossypol-hypokalaemia interrelationships. Int J Androl 1985;8:313–24. 51. Stewart PM, Krozowski ZS. 11 beta-Hydroxysteroid dehydrogenase. Vitam Horm 1999;57:249–324. 52. Stewart PM, Wallace AM, Valentino R, Burt D, Shackleton CHL, Edwards CRW. Mineralocorticoid activity of liquorice: 11-beta-hydroxysteroid dehydrogenase deficiency comes of age. Lancet 1987;10:821–4. 53. Sang GW, Lorenzo B, Reidenberg MM. Inhibitory effects of gossypol on corticosteroid 11-beta-hydroxysteroid dehydrogenase from guinea pig kidney: a possible mechanism for causing hypokalemia. J Steroid Biochem Mol Biol 1991;39:169–76. 54. Song D, Lorenzo B, Reidenberg MM. Inhibition of 11 beta-hydroxysteroid dehydrogenase by gossypol and bioflavonoids. J Lab Clin Med 1992;120:792–7.
1593
Objective: To evaluate the effects of two sublethal doses of gossypol on biochemical, enzymatic, and electrolytic properties of male rabbit seminal plasma. Design: Retrospective study. Setting: A university-affiliated laboratory. Animal(s): Fifteen adult male New Zealand white rabbits. Intervention(s): Two sublethal doses of gossypol (4 and 20 mg per kilogram of body weight, every other day) were given orally. Main Outcome Measure(s): Biochemical, enzymatic, and electrolytic properties of male rabbit seminal plasma. Result(s): Seminal plasma total protein and albumin were statistically significantly increased by treatment with gossypol. Total lipids concentration was statistically significantly decreased by treatment with both doses of gossypol, whereas cholesterol was statistically significantly decreased by treatment with the low gossypol dose. Seminal plasma lactate dehydrogenase and alanine aminotransferase activities were statistically significantly decreased by treatment with the low dose of gossypol, whereas activities of these enzymes as well as of aspartate aminotransferase were statistically significantly increased by treatment with the high dose. Treatment with the high dose of gossypol statistically significantly reduced Kþ concentration in the seminal plasma, whereas it statistically significantly increased Naþ and the Naþ-Kþ ratio. Conclusion(s): Seminal plasma values of total protein, total lipids, cholesterol, aspartate aminotransferase, and Naþ concentration returned to control levels after withdrawal of gossypol, whereas the effect of this drug on other parameters continued during the recovery period. (Fertil Steril 2008;89:1585–93. 2008 by American Society for Reproductive Medicine.) Key Words: Gossypol, rabbit, seminal plasma proteins, lipids, enzymes, electrolytes
Seminal plasma is the secretion of the male accessory glands to provide a medium and vehicle for spermatozoal transportation. It is known to have high protein content, enzymes, lipids, electrolytes, and various other factors that may play significant roles in the metabolic regulation of spermatozoa. To our knowledge, no previous studies regarding the effect of gossypol on seminal plasma constituents have been reported. However, several investigations have been performed on the effect of gossypol on blood plasma constituents, from which its effect on seminal plasma constituents can be extrapolated because of the ability of gossypol to cross the blood–testis barrier (1). Because of the protein-binding property of gossypol, the cytotoxicity of gossypol can be alleviated by serum protein (2), and thus, an increase in protein concentration in the gossypol-treated animals can be considered a biological
Received December 9, 2006; revised and accepted May 9, 2007. Reprint requests: Taha A. Taha, Ph.D., Department of Animal Production, Faculty of Agriculture (El-Shatby), Alexandria University, Aflaton Street, Alexandria 21545, Egypt (FAX: 2035922780; E-mail: tahaataha@yahoo. com).
0015-0282/08/$34.00 doi:10.1016/j.fertnstert.2007.06.077
response, intended to counteract its deleterious effect. In addition, gossypol strongly interacts with various enzymes and interferes with many enzymatic reactions that are involved in the metabolic regulation of spermatozoa (3–5). Seminal plasma lipids also play significant roles in the membrane structure of spermatozoa, in sperm metabolism, in sperm capacitation, and in fertilization of the female gamete (6). Because it is highly lipophilic, gossypol can interact directly with the phospholipid bilayer of biological membranes to alter membrane structure, electrostatic charge, and transmembrane ion fluxes. A major side effect of gossypol administration is the change in electrolyte status, in particular the reduction in serum Kþ levels (7) that is accompanied by a marked increase in serum Naþ concentration (8). Daily ingestion of gossypol provokes infertility in various animal species, including human beings. The contraceptive effect of gossypol in human beings was discovered first in China and still continues to be tested as a favorable alternative for male contraception (9). In addition, our recent studies (10) clearly indicated that gossypol has pronounced hazardous effects on semen characteristics and blood hormonal parameters in male rabbits that may lead to deleterious
Fertility and Sterility Vol. 89, Suppl 3, May 2008 Copyright ª2008 American Society for Reproductive Medicine, Published by Elsevier Inc.
1585
productive and reproductive performance. Thus, the objectives of the present study were to evaluate the effects of gossypol on seminal plasma constituents in an attempt to correlate the disturbances in biochemical composition and in enzymatic and electrolyte content of seminal plasma with this gossypol-induced decline in rabbit semen characteristics (10). Such studies would help identify some of the mechanisms that are responsible for the role of gossypol as a potential male contraceptive. MATERIALS AND METHODS Animals and Management This study was performed at the Department of Animal Production, Faculty of Agriculture, Alexandria University. Experiments were performed after departmental approval, followed standard scientific procedures without compromising animal welfare rights, and were performed without any commercial profit purpose. Fifteen adult male New Zealand white rabbits, which were 6 to 8 months of age and weighed 2.76 0.39 kg (mean SE) at the beginning of the experiment, were used during the reproductive season (starting in September). The rabbits were individually housed in cages. Food and water were provided ad libitum. The animals were offered pellets consisting of (per kg) 330 g of berseem (Trifolium alexandrinum) hay, 170 g of soybean meal, 165 g of ground maize, 160 g of barley, 120 g of wheat bran, 38 g of molasses, 10 g of salt, 4 g of dibasic calcium phosphate, and 3 g of vitamins. The chemical analysis of the pellets according to the Association of Official Analytical Chemists (11) indicated that they contained (per kg) 175 g of crude protein, 140 g of crude fiber, and 27 g of fat. All animals were allowed to adjust to their new environment and were tested for semen quality for 3 weeks before the experiment commenced. Experimental Design Gossypol was extracted from cottonseeds and purified according to Boatner (12), as described by Taha et al. (10). The rabbits were divided randomly into three groups of five animals each and were assigned at random to one of the following treatments: the first group served as control (the animals were given an equivalent dose of the vehicle, consisting of maize oil þ acetone), and the second and third groups were used to study the effect of the low dose (1/100 of median lethal dose, 4 mg per kilogram of live weight) and high dose (1/20 of median lethal dose, 20 mg per kilogram of live weight) of gossypol. The correct dose for each rabbit was given orally by syringe, directly into the esopharyngeal region, every other day throughout the treatment period, which lasted for 8 weeks. This period was followed by an 8-week recovery period, during which all drugs were withdrawn. Sample Collection and Analysis Semen was collected weekly from all animals throughout the 16-week experimental period. Ejaculates were obtained by 1586
Shaaban et al.
Gossypol and rabbit seminal plasma
using an artificial vagina and a teaser doe. Seminal plasma was separated from ejaculates by centrifugation at 5,000 rpm for 10 minutes. The recovered seminal plasma fraction was further processed by centrifuge at 10,000 rpm for 15 minutes at 4 C, and the supernatant was stored at 20 C until analysis. Seminal plasma total protein (TP) was measured by the Biuret method, as described by Armstrong and Carr (13). Total albumin concentration was determined by the bromocresol green method of Doumas et al. (14). Seminal plasma total lipids were determined as described by Frings et al. (15), and total cholesterol concentration was measured by a colorimetric method (16). Activity of lactate dehydrogenase (LDH) was measured according to Buhl and Jackson (17). Transaminase activities (aspartate aminotransferase [AST] and alanine aminotransferase [ALT]) were measured by colorimetric methods, as described by Reitman and Frankel (18). Seminal plasma electrolytes (Naþ and Kþ) were determined by using commercial kits (Quimica Clinica Aplicada S.A., Ampsta, Spain), then Naþ-Kþ ratios were calculated. Statistical Analysis Data for each of the treatment and recovery periods were analyzed separately by using the general linear model procedure of the SAS Institute (19). The statistical model describing all parameters included the following: Yijk ¼ m þ Ti þ Wj þ ðTWÞij þ eijk ; where Yijk ¼ an observation of trait recorded on individual k; m ¼ the overall mean; Ti ¼ the fixed effect of the ith treatment (control, low dose, high dose); Wj ¼ the fixed effect of the jth week; (TW)ij ¼ the interaction between treatment and weeks; and eijk ¼ a random error assumed to be independent normally distributed, with mean ¼ 0 and variance ¼ s2e. Treatment means were compared by using the least significant difference procedure (20). The level of significance was P<.05. RESULTS Seminal Plasma Proteins, Total Lipids, and Cholesterol Means of seminal plasma TPs, albumin, total lipids, and cholesterol during treatment and recovery periods as influenced by gossypol are shown in Table 1, and the weekly mean values of these parameters, expressed as percentage of control, are presented in Figure 1. Treatment with the high dose of gossypol increased (P<.05) TP concentration when compared with the case of the control group. Withdrawal of gossypol during the recovery period abolished this effect on TP. Gossypol administration resulted in significant (P<.05) increases in albumin concentration in both the low- and high-dose groups when compared with the control group. Withdrawal of gossypol during the recovery period resulted in significant (P<.05) reductions in albumin Vol. 89, Suppl 3, May 2008
TABLE 1 Mean (±SEM) concentrations of TP, albumin, total lipids, and cholesterol in seminal plasma. Semen parameters Group Treatment Control LD HD Week (W) TW Recovery Control LD HD Week (W) RW
Albumin (g per 100 mL)
Total lipids (g/L)
2.09 0.06b 2.19 0.04b 2.44 0.03a NS NS
0.80 0.05b 1.00 0.03a 1.09 0.03a
1.27 0.03a 1.01 0.05b 1.10 0.05b
c
d
NS
NS
45.5 1.07a 39.4 1.38b 48.9 1.99a NS NS
2.24 0.05 2.11 0.04 2.09 0.03 NS NS
1.00 0.03a 0.81 0.03b 0.86 0.03b NS NS
1.07 0.04 1.03 0.03 0.93 0.03 NS NS
41.6 1.71 38.6 1.50 37.9 1.20 NS NS
TP (g per 100 mL)
Cholesterol (mg per 100 mL)
Note: In both the treatment and recovery groups, n ¼ 5. NS ¼ nonsignificant; LD ¼ low dose of gossypol; HD ¼ high dose of gossypol. a,b Within columns, within periods, means with one of these two superscript letters differ significantly from one another (P< .05). c P< .05. d P< .01. Shaaban. Gossypol and rabbit seminal plasma. Fertil Steril 2008.
concentration at the two doses used. Total lipids concentration was decreased (P<.05) by treatment with both doses of gossypol, compared with the case of the control, whereas cholesterol was decreased (P<.05) by treatment with the low dose. Withdrawal of gossypol resulted in complete recovery of seminal plasma total lipids and cholesterol. Enzymes Gossypol effects on seminal plasma concentration of LDH, AST, and ALT during treatment and recovery periods are shown in Table 2. The biweekly mean values of these parameters, expressed as percentage of control, are presented in Figure 2. Lactate dehydrogenase in the low-dose group (468.0 U/L) was significantly (P<.05) lower, whereas LDH in the high-dose group (1,855.5 U/L) was significantly (P<.05) higher, compared with the case in the control group (1,265.3 U/L). A similar trend was also noted during the recovery period. Moreover, enzyme ALT responded to gossypol in a manner similar to that of LDH during treatment and recovery periods. In addition, treatment with the high dose of gossypol increased (P<.05) AST concentration compared with the control group. Withdrawal of gossypol during the recovery period resulted in complete recovery in AST concentration at the two doses used. Electrolytes Mean values of seminal plasma Naþ, Kþ, and Naþ-Kþ ratio during treatment and recovery periods, as influenced by gosFertility and Sterility
sypol, are shown in Table 3. The tetraweekly mean values of these parameters, expressed as percentage of control, are presented in Figure 3. Treatment with the high dose of gossypol increased (P<.05) Naþ concentration when compared with the case of the control group, and this effect was abolished during the recovery period. Gossypol administration resulted in consistent reductions of Kþ concentration during the treatment period in the high- and low-dose–treated groups compared with the control group, but these reductions were significant (P<.05) only in the high-dose group. After gossypol cessation, this reduction in Kþ concentration was significant (P<.05) in the two doses used. The Naþ-Kþ ratio was increased (P<.05) by the high dose of gossypol during the treatment period and by the low- and high-dose groups during the recovery period.
DISCUSSION Effect of Gossypol on TP and Albumin Because of the ability of gossypol to cross the blood–testis barrier (1), the aldehyde groups that are present in the gossypol molecule may be bound readily to the free amino groups of the protein molecules to form a Schiff’s base (21). Because of the protein-binding property of gossypol, the cytotoxicity of gossypol can be alleviated by serum protein (2). Thus, the increase in protein and albumin concentrations in the gossypol-treated rabbits in the present study (Table 1 and Fig. 1) can be considered a biological 1587
FIGURE 1 Changes in seminal plasma TP, albumin, total lipids, and cholesterol during treatment and recovery of male rabbits with gossypol (control, B; gossypol low dose, :; and gossypol high dose, -).
Shaaban. Gossypol and rabbit seminal plasma. Fertil Steril 2008.
response, intended to counteract the cytotoxicity of gossypol. The increase in albumin concentration during the treatment period also may be attributed to the degenerative effect that gossypol administration exerts upon testicular tissues (22). Total Lipids and Cholesterol Seminal lipids play significant roles in the membrane structure of spermatozoa, sperm metabolism, sperm capacitation, 1588
Shaaban et al.
Gossypol and rabbit seminal plasma
and fertilization of female gametes (6). In the present study, gossypol treatment reduced seminal plasma total lipids in rabbits (Table 1), which was associated with reductions in sperm motility and sperm concentration (10). Studies elsewhere also showed concomitant decrease in sperm motility and sperm concentration with the decline in seminal plasma total lipids in bulls (23) or in total lipids and fructose in rams (24). During the recovery period, seminal plasma total lipids were restored to their normal concentration after gossypol withdrawal. Vol. 89, Suppl 3, May 2008
TABLE 2 Mean (±SEM) concentrations of LDH, AST, and ALT in seminal plasma. Semen parameters Group Treatment Control LD HD Week (W) TW Recovery Control LD HD Week (W) RW
LDH (U/L)
AST (U/mL)
ALT (U/mL)
1,265.3 84.7b 468.0 163.2c 1,855.5 56.6a
38.6 1.18b 44.0 1.70b 51.4 1.96a NS NS
12.0 0.31b 10.4 0.29c 16.2 0.64a NS NS
38.6 1.18 42.6 2.67 46.9 1.91 NS NS
12.0 0.31b 10.6 0.19c 14.1 0.46a NS NS
d d
1,624.1 150.1b 746.6 81.8c 2,060.8 92.6a NS NS
Note: In both the treatment and recovery groups, n ¼ 5. NS ¼ nonsignificant; LD ¼ low dose of gossypol; HD ¼ high dose of gossypol. a,b,c Within columns, within periods, means with one of these three superscript letters differ significantly from one another (P< .05). d P< .01. Shaaban. Gossypol and rabbit seminal plasma. Fertil Steril 2008.
FIGURE 2 Changes in seminal plasma LDH, AST, and ALT during treatment and recovery of male rabbits with gossypol (control, B; gossypol low dose, :; and gossypol high dose, -).
The hypocholesteremic effect of gossypol that was observed in the present study agrees with observations from studies conducted elsewhere on rats (25) and monkeys (26). The gossypol-induced reductions in blood plasma T3 and T concentrations that were observed in the study undertaken by Taha et al. (10) in rabbits would contribute to the hypocholesteremic response in the low-dose–treated animals (27). However, the high gossypol dose revealed no effect on seminal plasma cholesterol (Table 1), with concomitant marked increase in AST and ALT concentrations (Table 2), which reflects altered hepatic function. Consequently, cholesterol removal was reduced, resulting in cholesterol accumulation. Because cholesterol synthesis was inhibited by the decline in T3 and T concentration, the accumulated cholesterol did not exceed the control value. During the recovery period, seminal plasma cholesterol was restored to its control value. Effect of Gossypol on Some Enzymatic Activities Many investigators have reported that gossypol interferes with many enzymatic reactions that are involved in the metabolic regulation of spermatozoa, including those resulting from malate dehydrogenase (3), pyruvate dehydrogenase (4), and LDH (5). Concerning LDH activity, gossypol may act as an uncoupler of mitochondrial oxidative phosphorylation (28) and inhibit LDH-X, an enzyme that is postulated to participate in a shuttle system transferring Hþ from cytosol to mitochondria (29).
Shaaban. Gossypol and rabbit seminal plasma. Fertil Steril 2008.
Fertility and Sterility
In the present study (Table 2), a marked inhibitory effect of gossypol on LDH was observed in the low-dose group, in 1589
TABLE 3 Mean (±SEM) concentrations of NaD and KD in seminal plasma, as well as mean NaD-KD ratio. Semen parameters Group Treatment Control LD HD Week (W) TW Recovery Control LD HD Week (W) RW
NaD (mmol/L)
KD (mmol/L)
NaD-KD ratio
65.2 7.36b 53.5 7.15b 107.9 5.19a NS NS
29.5 2.33a 24.5 2.20a,b 22.0 1.25b NS NS
2.31 0.32b 2.26 0.35b 5.16 0.45a NS NS
34.4 1.80a 27.2 1.86b 26.0 1.43b NS NS
2.32 0.20b 3.15 0.59a 3.81 0.29a
79.4 6.49 85.4 15.75 96.3 3.33 d d
c d
Note: In both the treatment and recovery groups, n ¼ 5. NS ¼ nonsignificant; LD ¼ low dose of gossypol; HD ¼ high dose of gossypol. a,b Within columns, within periods, means with one of these two superscript letters differ significantly from one another (P< .05). c P< .05. d P< .01. Shaaban. Gossypol and rabbit seminal plasma. Fertil Steril 2008.
which LDH concentration was reduced 2.7-fold compared with in the control group. This dose of gossypol was also found to cause significant reduction in forward motility (10). This effect of gossypol on LDH activity was reported in human beings (30), monkeys (31), bovines (5), boars (32), rabbits (33), rats (34), hamsters (35), and mice (36). The present study, however, revealed a marked discrepancy between the effect of the low and high doses of gossypol on LDH activity, in that the high dose enhanced the activity of LDH (Table 2). Several mechanisms have been proposed to explain these inconsistent effects. Gossypol is known to promote the formation of reactive oxygen species (37), so at the ultrastructural level, gossypol has been shown to induce marked structural defects in the cellular membrane system and in the intracellular organelles (38), as well as to induce extensive mitochondrial damage (39). Collectively, rupture of plasma and acrosomal membranes and mitochondrial destruction are suggested to induce cytoplasmic leakage and subsequent increase in seminal plasma LDH concentration. Despite the inhibitory effect of gossypol on LDH, the released LDH from destroyed sperm could not be inhibited by gossypol because of the marked increase in extracellular proteins (Table 1), which counteracted the inhibitory effect of gossypol (2). The affinity of gossypol for albumin is several orders of magnitude higher than the affinity of gossypol for LDH, resulting in a low concentration of gossypol to which the enzymes are exposed (2).
Our findings on the same rabbits showed reduction of total functional sperm fraction concomitantly with an increasing percentage of altered acrosomes (10), reflecting marked damage of sperm cells in the high-dose group. Our studies using electron microscopy revealed that the high dose of gossypol caused marked destruction of sperm plasma acrosomal and mitochondrial membranes (40). In addition, seminal plasma TP concentration was higher in the high-dose group than in the low-dose and control groups (Table 1), which counteracted the cytotoxic effects of gossypol on LDH activity.
This view could explain the increasing activity of seminal plasma LDH in the high-dose–treated animals (Table 2).
The present study revealed a marked discrepancy between the effects of the low and high doses of gossypol on
1590
Shaaban et al.
Gossypol and rabbit seminal plasma
The concentration of transaminase enzymes (AST and ALT) in semen is a good indicator of semen quality because it measures sperm membrane stability (41). Thus, increasing the percentage of dead spermatozoa in a semen sample causes high concentrations of transaminase enzymes in the extracellular fluid as a result of sperm membrane damage and, consequently, the ease of leakage of enzymes from spermatozoa. As indicated for LDH in a previous paragraph, gossypol exerted its cytotoxic effect by the formation of reactive oxygen species (37), which may cause plasma membrane rupture and mitochondrial destruction of sperm (38–40), in addition to degeneration of cells lining seminiferous tubules (42, 43). These effects are expected to induce cytoplasmic leakage and subsequent increase in transaminase concentrations in extracellular fluid (seminal plasma).
Vol. 89, Suppl 3, May 2008
FIGURE 3 Changes in seminal plasma Naþ, Kþ, and Naþ-Kþ ratio during treatment and recovery of male rabbits with gossypol (control, B; gossypol low dose, :; and gossypol high dose, -).
Gossypol cessation revealed complete recovery of AST activity, whereas the effect of gossypol on ALT and LDH activities was extended during the recovery period. The extended inhibitory effect of the low dose of gossypol on ALT and LDH activities could be explained by preferential accumulation of gossypol within the mitochondria (46) and by the binding of gossypol to spermatozoa in an irreversible manner (31). In addition, the moderate reduction in total functional sperm fraction concentration and the increasing percentage of altered acrosome that were observed in the low-dose group during the recovery period (10) substantiated the extended deleterious effect of gossypol, which could not be eliminated because of the low albumin concentration that was recorded during the recovery period (Table 1). However, the enhancing effect of the high dose of gossypol on ALT and LDH activities that was noted during the treatment period also was extended during the recovery period (Table 2). Levels of ALT provide more reliable indications in acute, and AST levels, in chronic, tissue injury (47). Just as gossypol cessation minimizes the severe effect of gossypol, so the limited residue of gossypol appears to cause injury at the cytoplasmic level but not at the mitochondrial level, which is reflected in the extending effect of gossypol on ALT activity, whereas the effect on AST activity was moderate (insignificant increase). This suggestion may explain the continued effect of gossypol on ALT activity that was noted after gossypol cessation, whereas there was no significant effect on AST activity.
Shaaban. Gossypol and rabbit seminal plasma. Fertil Steril 2008.
transaminase activities. Several mechanisms have been proposed to explain these inconsistent effects. Concerning the high dose of gossypol, the increased activity of transaminases observed in the present study (Table 2) is consistent with results of studies elsewhere (44). The reason for such increase is the leakage of transaminases through the destroyed sperm plasma membrane and mitochondria, which is evident from the gossypol-induced decrease in the concentration of total functional sperm fraction that was reported by Taha et al. (10). Furthermore, despite the inhibitory effect of gossypol on transaminase activities, the released transaminases from destroyed sperm could not be inhibited by gossypol because of the presence of a marked increase in extracellular proteins (Table 1) that counteract the inhibitory effect of gossypol (2). However, when the low dose of gossypol was used, a mild degree of tissue injury appeared to occur, as reflected by the normal concentration of total functional sperm fraction (10). In this case, the moderately leaked transaminases may be inhibited in the presence of gossypol. This view is supported by the finding of Nomeir and Abou-Donia (45), who reported that gossypol may alter the activity of transaminases by reacting with the substrate and blocking the action of the enzyme and/or by combining with the enzyme itself. Fertility and Sterility
Effect of Gossypol on Seminal Plasma Electrolyte Contents The importance of the ionic and osmotic environment on sperm motility and metabolic activity is well established. Extremes of Kþ concentration were found to depress the motility and viability of spermatozoa (48). A major side effect of gossypol administration is the change in electrolyte status (49), in particular hypokalemia, the reduction in serum Kþ levels (7). Furthermore, Kþ deficiency has been shown to enhance the antispermatogenic effect of gossypol (50). Enzyme 11b-hydroxysteroid dehydrogenase in the kidney is known to oxidize the active hormone cortisol to the inactive cortisone (51). Thus, the active enzyme lowers apparent mineralocorticoid activity by inactivating cortisol and leaving only aldosterone as the active mineralocorticoid, whereas inhibition of this enzyme produces the effects of mineralocorticoid excess, hypokalemia (52). Several studies reported that gossypol inhibits 11bhydroxysteroid dehydrogenase in guinea pigs (53) and human beings (54). These findings provide an explanation for the hypokalemic effect of gossypol that was observed in the present study (Table 3), in which the gossypol-induced hypokalemia was more pronounced when the high dose of gossypol was used. In addition, the hypokalemic effect of this gossypol dose was accompanied by Naþ retention, as reflected by a marked increase of seminal plasma Naþ concentration, as well as by an elevation in Naþ-Kþ ratio. These observations 1591
coincide with those from a study conducted on monkeys (8). The hypokalemic effect of gossypol was extended after gossypol cessation in both the low and high doses, whereas seminal plasma Naþ concentration showed almost complete recovery. Despite restoration of a relatively normal level of Naþ, the Naþ-Kþ ratio remained higher than that in the control group, which reflected a continued hypokalemic effect of gossypol. In conclusion, sublethal doses of gossypol induced effects on rabbit seminal plasma TP, total lipids, cholesterol, AST, and Naþ that were reversible after withdrawal of gossypol, as well as effects on albumin, LDH, ALT, and Kþ that were irreversible.
REFERENCES 1. Wang JM, Tao L, Wu XL, Lin LX, Wu J, Wang M, et al. Differential binding of (þ) and () gossypol to plasma protein and their entry into rat testis. J Reprod Fertil 1992;95:277–82. 2. Haspel HC, Ren YF, Watanabe KA, Sonnenberg M, Corin RE. Cytocidal effect of gossypol on cultured murine erythroleukemia cells is prevented by serum protein. J Pharmacol Exp Ther 1984;229:218–25. 3. Montamat EE, Burgos C, Geres de Burgos NM, Rovai LE, Blanco A. Inhibitory action of gossypol on enzymes and growth of Trypanosoma cruzi. Science 1982;218:288–9. 4. Adeyemo O, Chang CY, Segal SJ, Koide SS. Gossypol action on the production and utilization of ATP in sea urchin spermatozoa. Arch Androl 1983;9:343–9. 5. Olgiati KL, Toscano WA. Kinetics of gossypol inhibition of bovine lactate dehydrogenase X. Biochem Biophys Res Commun 1983;115:180–5. 6. Mann T, Lutwak-Mann C. Biochemistry of spermatozoa: chemical and functional correlations in ejaculated semen. In: Mann T, LutwakMann C, eds. Male reproductive function and semen. Themes and trends in physiology and biochemistry and investigative andrology. Berlin: Springer-Verlag, 1981:185–268. 7. Liu GZ, Chiu-Hinton K, Cao JA, Zhu CX, Li BY. Effects of Kþ salt or a potassium blocker on gossypol-related hypokalemia. Contraception 1988;37:111–8. 8. Lohiya NK, Sharma K, Kumar M, Sharma S. Limitations in developing gossypol acetic acid as a male contraceptive. Contraception 1990;41: 519–32. 9. Cui GH, Xu ZL, Yang ZJ, Xu YY, Xue SP. A combined regimen of gossypol plus methyltestosterone and ethinylestradiol as a contraceptive induces germ cell apoptosis and expression of its related genes in rats. Contraception 2004;70:335–42. 10. Taha TA, Shaaban WF, EL-Mahdy AR, EL-Nouty FD, Salem MH. Reproductive toxicological effects of gossypol on male rabbits: semen characteristics and hormonal levels. Anim Sci 2006;82:259–69. 11. Association of Official Analytical Chemists. Official methods of analysis. 16th ed. Arlington, VA: Association of Official Analytical Chemists, 1995. 12. Boatner CH. Pigments of cottonseed. In: Bailey AE, ed. Cottonseed and cottonseed products. Their chemistry and chemical technology. New York: Wiley (Interscience), 1948:215–23. 13. Armstrong WD, Carr CW, eds. Physiological chemistry laboratory directions. 3rd ed. Minneapolis, MN: Burges Publishing Co, 1964. 14. Doumas BT, Watson WA, Biggs HG. Albumin standards and the measurement of serum albumin with bromocresol green. Clin Chim Acta 1977;31:87–96. 15. Frings CS, Fendley TW, Dunn RT, Queen CA. Improved determination of total serum lipids by the sulfo-phospho-vanillin reaction. Clin Chem 1972;18:673–4. 16. Watson D. A simple method for the determination of serum cholesterol. Clin Chim Acta 1960;5:637–8.
1592
Shaaban et al.
Gossypol and rabbit seminal plasma
17. Buhl SN, Jackson KY. Optimal conditions and comparison of lactate deydrogenase catalysis of the lactate-to-pyruvate and pyruvate-to-lactate reactions in human serum at 25, 30 and 37 degrees. Clin Chem 1978;24: 828–31. 18. Reitman S, Frankel S. A colorimetric method for the determination of serum glutamic oxaloacetic pyruvic transaminases. Am J Clin Pathol 1957;28:56–63. 19. SAS Institute. SAS user’s guide: statistics. Version 8 ed. Cary, NC: SAS Institute, 1999. 20. Fisher RA. The design of experiments. Edinburgh, United Kingdom: Oliver and Boyd, 1949. 21. Wu DF, Reidenberg MM. Stereoselective interaction between gossypol and rat plasma. Contraception 1990;41:377–88. 22. Arshami J, Ruttle JL. Effects of diets containing gossypol on spermatogenic tissues of young bulls. Theriogenology 1988;30:507–16. 23. Kelso KA, Redpath A, Noble RC, Speake BK. Lipid and antioxidant changes in spermatozoa and seminal plasma throughout the reproductive period of bulls. J Reprod Fertil 1997;109:1–6. 24. Taha TA, Abdel-Gawad EI, Ayoub MA. Monthly variations in some reproductive parameters of Barki and Awassi rams throughout 1 year under subtropical conditions. 1. Semen characteristics and hormonals levels. Anim Sci 2000;71:317–24. 25. Nwoha PU, Aire TA. Reduced level of serum cholesterol on low proteinfed Wistar rats administered gossypol and chloroquine. Contraception 1995;52:261–5. 26. Shandilya LN, Clarkson TB. Hypolipidemic effects of gossypol in cynomolgus monkeys (Macaca fascicularis). Lipids 1982;4:285–90. 27. Guyton AC. Textbook of medical physiology. 8th ed. Philadelphia: WB Saunders, 1991:754–64. 28. Abou Donia MB, Dieckert JW. Gossypol: uncoupling of respiratory chain and oxidative phosphorylation. Life Sci 1974;14:1955–63. 29. Lee CYG, Moon YS, Duleba A, Chen AF, Gomel V. Gossypol inhibition of LDH-X. In: Lobl TJ, Hafez ESE, eds. Male fertility and its regulation. London: MTP Press, 1985:193–9. 30. Wichmann K, K€apyaho K, Sinervirta R, J€anne J. Effect of gossypol on the motility and metabolism of human spermatozoa. J Reprod Fertil 1983;69:259–64. 31. Stephens DT, Critchlow LM, Hoskins DD. Mechanism of inhibition by gossypol of glycolysis and motility of monkey spermatozoa in vitro. J Reprod Fertil 1983;69:447–52. 32. Tso WW, Lee CS. Lactate dehydrogenase-X, an isoenzyme particularly sensitive to gossypol inhibition. Int J Androl 1982;5:205–9. 33. Eliasson R, Virji N. Effect of gossypol acetic acid on the activity of LDHC4 from human and rabbit spermatozoa. Int J Androl 1983;6:109–12. 34. Lin YC, Chitcharoenthum M, Rikihisa Y. Effect of gossypol on spermatozoal lactate dehydrogenase-X (LDH-X) in male rats. Contraception 1987;5:581–92. 35. KalIa NR, Bisnooduth U, Ranga A. Response of hamster to the antifertility effect of gossypol. Acta Eur Fertil 1990;1:25–37. 36. Lee CYG, Moon YS, Yuan JH, Chen AF. Enzyme inactivation and inhibition by gossypol. Mol Cell Biochem 1982;47:65–70. 37. Barhoumi R, Burghardt RC. Kinetic analysis of the chronology of patulin- and gossypol-induced cytotoxicity in vitro. Fundam Appl Toxicol 1996;30:290–7. 38. Zamboni L. The ultrastructural pathology of the spermatozoon as a cause of infertility: the role of electron microscopy in the evaluation of semen quality. Fertil Steril 1987;48:711–34. 39. Hoffer AP. Ultrastructural studies of spermatozoa and the epithelial lining of the epididymis and vas deferens in rats treated with gossypol. Arch Androl 1982;8:233–46. 40. Shaaban WF. Biological and biochemical seminal changes in rabbits treated with gossypol [M.Sc. thesis]. Egypt: Alexandria University, 2003. 41. Pace MM, Graham EF. The release of GOT from bovine spermatozoa as a test method of assaying semen quality and fertility. Biol Repro 1970;3: 140–6. 42. Hoffer AP, Agarwal A, Meltzer P, Herlihy P, Nagvi RH, Lindberg MC, et al. Ultrastructural, fertility and spermicidal studies with isomers
Vol. 89, Suppl 3, May 2008
43.
44. 45.
46.
47. 48.
and derivatives of gossypol in male hamsters. Biol Reprod 1987;37: 909–24. Chase CC, Bastidas P, Ruttle JL, Long CR, Randal RD. Growth and reproductive development in Brahman bulls fed diets containing gossypol. J Anim Sci 1994;72:445–52. Abou-Donia MB. Physiological effects and metabolism of gossypol. Residue Rev 1976;61:125–60. Nomeir AA, Abou-Donia MB. Toxicological Effects of Gossypol. In: Lobl TJ, Hafez ESE, eds. Male fertility and its regulation. London: MTP Press, 1985:111–33. Tso WW, Lee CS. Gossypol uncoupling of respiratory chain and oxidative phosphorylation in ejaculated boar spermatozoa. Contraception 1982;25:649–56. Clinical Laboratory. 11th edition of medico-chemical investigation methods. Darmstadt, Germany: Merck, 1970. McGrady AV, Nelson L, Ireland M. Ionic effects on the motility of bull and chimpanzee spermatozoa. J Reprod Fertil 1974;40:71–6.
Fertility and Sterility
49. Xue SP. Gossypol contraception and mechanism of action. In: Lobl TJ, Hafez ESE, eds. Male fertility and its regulation. London: MTP Press, 1985:175–84. 50. Qian SZ. Gossypol-hypokalaemia interrelationships. Int J Androl 1985;8:313–24. 51. Stewart PM, Krozowski ZS. 11 beta-Hydroxysteroid dehydrogenase. Vitam Horm 1999;57:249–324. 52. Stewart PM, Wallace AM, Valentino R, Burt D, Shackleton CHL, Edwards CRW. Mineralocorticoid activity of liquorice: 11-beta-hydroxysteroid dehydrogenase deficiency comes of age. Lancet 1987;10:821–4. 53. Sang GW, Lorenzo B, Reidenberg MM. Inhibitory effects of gossypol on corticosteroid 11-beta-hydroxysteroid dehydrogenase from guinea pig kidney: a possible mechanism for causing hypokalemia. J Steroid Biochem Mol Biol 1991;39:169–76. 54. Song D, Lorenzo B, Reidenberg MM. Inhibition of 11 beta-hydroxysteroid dehydrogenase by gossypol and bioflavonoids. J Lab Clin Med 1992;120:792–7.
1593