MOLECULAR REPRODUCTION AND DEVELOPMENT 25:400408 (1990)

Review Article

Effects of Gossypol on the Motility of Mammalian Sperm OFFIE PORAT Reproductive Immunology, McMaster University, Hamilton, Ontario, Canada The effects of the male contraABSTRACT ceptive gossypol on the motility of mammalian spermatozoa are reviewed. The role of sperm motility in the processes of fertilization and the effect of the drug on these processes determine its effectiveness as a contraceptive. The promising male contraceptive potential of gossypol is discussed in the context of the serious adverse effects of the agent.

MALE CONTRACEPTION

Male contraceptives can be operative a t various points along the male reproductive system. Contraceptive agents may interfere with the continual process of spermatogonia production by the seminiferous germinal epithelium or with the development and maturation of epididymal sperm (Hinton, 1980). In the search for a reversible male contraceptive, the focus of many investigators has been the suppression of sperm proKey Words: Sperm motility, Fertilization, Male duction (Harper, 1982; Jackson and Schnieden, 1982; contraception Aitken, 1983; Bernstein, 1984; Diczfalusy, 1985). Attempts a t pharmacological male-fertility control in laboratory animals and humans have mainly made use of hormones (Swerdloff and Heber, 1983; Lohiya and INTRODUCTION Sharma, 1985; Rousseau et al., 1985; Wong and Lee, The dramatic increase in world population calls for 1985). new and improved methods of fertility regulation, inAt present, the approaches to male fertility control cluding the development of male contraceptives as althat have been tested or are under consideration internatives to existing methods. Male contraceptive clude inhibition of the pituitary-gonadal axis by steagents should be safe and effective without impairing roids such a s testosterone, progestin-testosterone comlibido and potency and have no significant adverse effects or interference with body hormone balance, and binations, and luteinizing hormone-releasing hormone their reproductive effects should be reversible. Ideally, (LHRH) analogues, with or without testosterone subsuch agents should be easy to use, readily available, stitution, or selective inhibition of follicle-stimulating hormone (FSH) by antibodies or inhibin (Frick and Auand inexpensive. The last stages of spermiogenesis involve sperm mat- litzky, 1988). Combinations of hormonal steroids are uration and the acquisition of sperm motility. Mamma- not sufficiently effective (Drife, 1987) and are often aslian sperm needs to actively reach the oocyte-cumulus sociated with impaired libido and other side effects (Gocomplex and requires motility to penetrate the egg mbe, 1983; Lohiya and Sharma, 1985). The existing vestments in order to reach the oolema (Hinton, 1980; male contraception methods, condom and vasectomy, Mellinger and Goldstein, 1987). Occurring when the do not meet the requirements either of safety and respermatozoa have already been developed, interference liability or of easy and full reversibility. A different avenue towards optimal male contracepwith sperm motility may be the least likely step to affect other functions of the male reproductive system. tion is in identification of a substance that would supGossypol, a natural product of the cotton plant, has press sperm motility without suppressing spermatobeen suggested to affect the motility of mature spermatozoa without adversely affecting other male reproductive functions. This review examines the antifertility potential of gossypol on mammalian sperm in the September 10, 1989; accepted November 8, 1989. context of its known adverse effects; the relationship Received Address reprint requests to Offe Porat, Immunology 4H13 HSC, Mcbetween these two will determine its future application Master University, 1200 Main Street West, Hamilton, Ontario, Canas a male contraceptive. ada L8N 325.

0 1990 WILEY-LISS, INC.

EFFECTS OF GOSSYPOL ON THE MOTILITY OF MAMMALIAN SPERM genesis. Since motility is acquired at a stage when the spermatozoa are already genetically defined (Hammerstedt, 1981; Cosentino and Cockett, 1986),a n agent whose sole effect is in prevention of sperm motility would be expected to produce infertility without causing other reproductive side effects. Presently, sperm motility is clinically inhibited in the female genital tract by vaginal spermicides such as nonoxynol-9 (Drife, 1987).However, the safety of this method is not complete, and many couples resent its use a s interrupting the natural course of their intercourse. A new, attractive approach would therefore be to inhibit sperm motility soon after sperm production by pharmacological modulation of the male system.

SPERM MOTILITY AND ITS SIGNIFICANCE IN MALE CONTRACEPTION Spermatogenesis is a complex, dynamic, and continuous process, which takes about 70 days in humans. It involves cell divisions and differentiation, regulated by hormones and nonhormonal factors. Its endocrine control may be affected by various hormones, antihormones, or psychotropic drugs. When produced in the testes, spermatozoa are immature, immotile, and lack fertilizing capacity (Hinton, 1980;Hammerstedt, 1981). During their passage through the epididymis, the spermatozoa become motile and acquire nongenetic components, enabling mature sperm to fertilize oocytes (Ellis and Nemetallah, 1985;Cosentino and Cockett, 1986). The mechanisms by which spermatozoa acquire motility are still poorly understood (MacLennnan, 1983; Hidiroglou and Knipfel, 1984;Brokaw, 1987;Mellinger and Goldstein, 1987;Kamiya, 1988).The vitality of the spermatozoon is expressed by the movement of the flagellum; mammalian sperm must make use of their locomotion ability to ensure successful transport through the female reproductive tract to reach the site of fertilization and the oocyte-cumulus complex. Once a t the site of fertilization, motility is necessary primarily for the fertilization process (Hinton, 1980;Mellinger and Goldstein, 1987),and i t is impossible for spermatozoa to penetrate the zona pellucida without having vigorous motility (Kooij et al., 1985). Mammalian sperm surface molecules are known to influence sperm transport. Alterations in the plasma membrane are involved in sperm capacitation, in induction of the acrosome reaction, and in sperm binding to the zona pellucida (Yanagimachi, 1981; Bedford, 1983; Fraser and Ahuja, 1988). Similar membrane changes may be involved in altering membrane ion conductance, related to changes in flagellar motion. In the presence of antisperm antibodies, these surface molecules may interact with the antisperm antibodies to cause spermatozoa with vigorous motility to resist forward progression and thus prevent fertilization (Bronson et al., 1985).Once in the female reproductive tract, sperm motility is modulated by the epithelial surfaces and secretions of the female (Green, 1987;

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1.

/

cn3

\

?ti3

CH3

2.

CH3

3.

Fig. 1. Tautomeric structures of gossypol [(2,2’-binaphthalene)-8, 8‘-dicarboxaldehyde-1,1‘,6,6’,7,7’-hexahydroxy-5,5’-diisopropyl-3,3’dimethyll in aqueous solution. Compounds 1 and 3 are the keto and enol forms; compound 2 is the hemiacetal.

Katz et al., 1989).In addition, sperm motility appears to be modulated by the oocyte granulosa cell investments, which may coordinate the transport and union of the gametes.

GOSSYPOL Biochemical Properties Gossypol [(2,2’-binaphthalene)-8,8’-dicarboxaldehyde -1,1’,6,6’,7,7’-hexahydroxy-5,5’diisopropyl-3,3’dimethyll (Edwards, 1958)is a yellowish pigment found in cotton plants of the genus Gossypium (family Malvaceae). It is present mostly in the seeds and root bark (Kong et al., 1986;Zhou and Lin, 1988),and in nature it protects the plant against insect pests (Jones, 1985). Gossypol is a highly reactive polyphenolic dialdehyde with a molecular weight of 518.54Daltons (Fig. 1) (Adams et al., 1960;Abou-Donia, 1976).It exists in three tautomeric forms: aldehyde, ketone, and hemiacetal. Gossypol is a weak organic acid with pK, of 7.2 (AbouDonia, 1976). The chemistry of gossypol is very relevant to its unique actions. The two aldehyde groups can easily bind to proteins via aldehyde-amino group linkage. It is transported by the renal tubule through the classic organic anion system (Goldinger et al., 1985).There are marked differences in the disposition and metabolism between ( + )- and (-)-gossypol. The elimination half-life of ( + )-gossypol is much longer than that of the (- )-gossypol isomer. The higher rate of elimination of (-)-gossypol may be due to its lower rate of binding to tissue proteins, since the distribution volume of (-1gossypol is much smaller than that of the (+)-isomer (Yu, 1987). Being highly lipophilic, gossypol can interact directly with the phospholipid bilayer of biological mem-

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branes to alter membrane structure (De Peyster e t al., 1986), electrostatic charge (Reyes et al., 19861, and transmembrane ion fluxes (Huang and Urthaler, 1986), with consequent functional changes in membraneous organelles. In common with other aromatic phenols, gossypol is a n antioxidant (Adams et al., 1960; Janero and Burghardt, 1988). Gossypol has been found to inhibit several kinetic enzymes (Maugh, 1981; Hoffer, 1985; Lee et al., 1985; Mohri, 1985; Rovan et al., 1985; Sadykov et al., 1985; Tso, 1985; Tso et al., 1985; Moore et al., 1988). In addition, it inhibits a number of oxidoreductases (Gerez DeBurgos et al., 1984) as well a s arachidonate lipoxygenases (Hamasaki and Tai, 1985).

Spermicidal Effects of Gossypol The effect of gossypol on humans has been known for generations. It has been used in the Far East for the treatment of chronic bronchitis and cough (Qian and Wang, 1984) and a s a n abortifacient and menses inducer (Xue, 1985). The antifertility activity of gossypol was a recent observation made in China (National Coordinating Group, 1978; Kong et al., 1986). In the 1950s, in a general survey of common diseases, rural communities in two provinces were found to have subnormal fertility. This was linked to the use of crude cotton seed oil (Kong et al., 1986). In the 1960s, i t was deduced that gossypol, a constituent of cotton seed oil, caused the male sterility (Kong e t al., 1986). These observations have led to the hope that gossypol would serve as a n ideal male contraceptive. Subsequently, the antispermatogenic and spermicidal effects of gossypol have been investigated in various animal species and man (Qian and Wang, 1984; Kong et al., 1986). Gossypol has been reported to exert spermicidal activity upon spermatozoa in vitro (Poso, 1980; Cameron et al., 1982; Wichmann et al., 1983) and in vivo (Kennedy et al., 1982; Tso et al., 1985; Coutinho and Melo, 1988). Spermatozoa, spermatids, and spermatocytes of mid- and late stages were identified a s gossypol target cells by some investigators (Coutinho e t al., 1985; Frick and Danner, 1985; Kalla et al., 1985a; Liu et al., 1985; Ahluwalia e t al., 1986; Fei and Teng, 1988; Hoffer et al., 1988; Teng and Fei, 1988; Ueno et al., 1988). These cells were the most susceptible to damage by gossypol, the first to show detectable change, and the first to degenerate. Similar findings were obtained in both human and primate sperm (Shandilya et al., 1982; Fei and Teng, 1988; Teng and Fei, 1988). With increased duration of drug administration, alterations in the spermatid head cap acrosome system, including the distortion and lysis of the acrosomal vesicles and granules, were observed (Ahluwalia et al., 1986). No detectable damage to Leydig cells, Sertoli cells, or the epididymal epithelium were detected in rats fed antifertility doses of gossypol (Change et al., 1985; Chang and Segal, 1985; Huang et al., 1985; Coutinho and Melo, 1988).

Effects on the Seminiferous Epithelium The seminiferous epithelium is a tissue in which the cells divide continually (Hinton, 1980; Cosentino and Cockett, 1986). It is thus subject to the injurious effects of many agents that adversely affect rapidly dividing cell populations. Long-term treatment with gossypol may cause complete atrophy of the seminiferous epithelium in some animals species, with sterility a s the likely consequence (Coutinho e t al., 1985). The relative sensitivity of somatic and germ cells to gossypol determines whether its effects will favor antifertility or toxicity (Nomeir and Abou-Donia, 1985). The ratio between effective and toxic doses of gossypol differs among species and strains. Rats, monkeys, hamsters (Change et al., 1980; Shandilya et al., 1982; Kalla et al., 1985a), and humans (National Coordinating Group, 1978; Qian and Wang, 1984) are more sensitive to the antispermatogenic and spermicidal action of gossypol than mice, rabbits, and dogs (Change et al., 1980; Ypersele de Strihou e t al., 1988; Abou-Donia et al., 1989). The minimal effective dose is variable among different animals and among the different studies. The onset of infertility and the recovery of fertility after the treatment are dose-dependent and specific for the animal treated (Meng, 1988; Xu et al., 1988). Other species tested seem to be more sensitive to the generalized toxic effects of gossypol (Frick and Danner, 1985; Heywood, 1988; Wang et al., 1988). Various tissues and cell types respond differently to gossypol as revealed by autoradiographic studies in rats (Xue, 1985; Stumpf et al., 1988). Damage to testicular germ cells usually occurs prior to a toxic effect in somatic cells of organs such as liver (Ypersele de Strihou e t al., 19881, heart (Ye et al., 1987; Ypersele de Strihou et al., 19881, kidneys (Xue et al., 1988; Ypersele de Strihou et al., 19881, and testicular Sertoli cells (Kennedy et al., 1982; Frick and Danner, 1985; Fu et al., 1988). The interspecies acute toxicity of gossypol is related to its differential detoxification to carbon dioxide (AbouDonia, 1976) and the unstable and considerably less toxic apogossypol (Abou-Donia, 1976) a s well as its bioavailability. However, i t is possible that metabolic products of gossypol are responsible for some of the side effects associated with gossypol treatment (Qian and Wang, 1984; Bender e t al., 1988a,b). In Vitro Studies of the Effect of Gossypol on Motility Gossypol has a dose-dependent inhibitory effect on in vitro sperm motility in humans (Poso, 1980; Waller et al., 1980; Wichmann et al., 1983) and monkey (Cameron et al., 1982; Shandilya et al., 1982). Although the precise mechanism causing this inhibition is not known, it is evident that gossypol inhibits the activities of certain enzymes involved in the metabolic regulation of spermatozoa (Maugh, 1981; Rajpurohit and Giridharan, 1988; Nakamura et al., 1988). Cytoplasmic

EFFECTS OF GOSSYPOL ON THE MOTILITY OF MAMMALIAN SPERM membrane integrity is essential for membrane transport, electric potential, and cell survival. The functional integrity of the sperm membrane is a n important factor in sperm motility and is involved in the acrosome reaction, sperm capacitation, sperm metabolism, and the binding of the spermatozoon to the egg surface. Electron microscopy studies of gossypol incubated in vitro with hamster (Hoffer et al., 1988) and bull (Rovan et al., 1985) sperm have revealed marked structural defects in the cellular membrane system and in intracellular organelles (Rovan et al., 1985; Zamboni, 1987; Sonenberg et al., 1988; White et al., 1988). This can be attributed to the reaction of gossypol with membrane compounds and to inactivation of metabolic enzymes (Rovan et al., 1985). Several studies reported that essential kinetic enzymes, such as ATPase, lactate dehydrogenase, and lactate dehydrogenase isoenzyme X (a sperm-specific enzyme postulated to participate in a shuttle system transferring H' from the cytosol to the mitochondria) are partially inactivated by gossypol (Maugh, 1981; Hoffer, 1985; Lee et al., 1985; Mohri, 1985; Rovan et al., 1985; Sadykov et al., 1985; Tso, 1985; Tso et al., 1985; Moore et al., 1988). Gossypol inhibition of sperm acrosin (a hydrolytic enzyme of acrosomal origin important in oocyte penetration) has also been suggested as the mechanism leading to sperm immotility (Kennedy et al., 1982; Sadykov et al., 1985). In vitro incubation of gossypol with isolated germ cells causes impairment of the energy metabolism of these cells; gossypol markedly reduces oxygen consumption and increases anaerobic glycolysis (Robaire et al., 1985; Nakamura et al., 1988). Motility initiation of the rat caudal epididymal spermatozoa requires the presence of extracellular sodium and calcium but is inhibited by high concentrations of potassium and hydrogen ions (Hammerstedt, 1981; Cosentino and Cockett, 1986). Apparently, potassium leakage is not the cause of gossypol-induced antimotility in spermatozoa (Tso and Lee, 1982). Involvement of prostaglandin in the antifertility effects of gossypol has also been proposed (Kalla et al., 1986). Sertoli cells studied in vitro show morphological and functional impairment induced by gossypol (Zhuangh et al., 1983). These changes are reversible and are not related to impairment in energy metabolism, since oxygen consumption and lactate production in Sertoli cells are unaffected by in vitro treatment with the drug. The impairment of Sertoli cell function has also been demonstrated in vivo by transient inhibition of androgen binding protein production (Kalla et al., 198513; Tanphaichiter and Bellve, 1985). It is possible that gossypol provokes perturbation of Sertoli cell metabolism in a manner that could influence the subsequent motility of epididymal sperm, either by altering a late stage in spermatogenesis or by decreasing the production of a Sertoli cell factor required for sperm maturation (Fei and Teng, 1988). New techniques of culturing and the development of monoclonal antibod-

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ies specific for spermatozoa in certain stages of maturation will permit further investigation of spermatozoa response to pharmacological stimuli and identification of their general and motility functions.

Clinical Studies Gossypol has not been approved for clinical use outside China (Liu e t al., 1985, 1988; Frick and Danner, 1985; Meng, 1988; Xu et al., 1988) and Brazil (Coutinho e t al., 1985; Coutinho and Melo, 1988). Data coming from these countries could be of great scientific relevance but might lead to some problems. Not all the literature is available, and some of it, specifically the behaviorial data, may be interpreted differently in different cultures. With that in mind, interpretation of nonbehaviorial data is summarized. Peak concentrations of gossypol in the plasma of the human male following a single 20.0 mg dose (p.0.) is -2.0 pM, with a half-life of 10 days (Wang et al., 1985). Several studies to identify the smallest effective gossypol dose for male fertility control have been reported. In one trial, a dose of 10 mglday gossypol was administered to young adult males for 3 months. Forward sperm motility was severely affected (less than 4% forward motility), and sperm density was markedly decreased, with no side effects reported (Frick et al., 1988). In another study, volunteers were given 20 mgi day for 4 months. The dose was then reduced to 20 mg three times per week. A significant reduction in sperm motility was detected in all subjects, accompanied by severe oligospermia (Coutinho and Melo, 1988). The antispermatogenic effect of gossypol was in one study reported to be fully reversible after termination of treatment (Frick and Danner, 1985); another study reported irreversibility in 10-20% of treated men (Mellinger and Goldstein, 1987). Some men receiving a dose of 20 mglday remained azoospermic 2 years following discontinuation of the drug (Coutinho et al., 1985; Coutinho and Melo, 1988; Meng, 1988). Failure of recovery was associated with greater total dose and longer duration of gossypol administration (Duo et al., 1988; Meng, 1988). Studies conducted in male rats following a single intravenous or oral doses and subchronic oral administration suggest that the pharmacokinetics of ( 21-gossypol may play, a t least in part, a role in the reproductive toxicity of subchronic but not of single oral dosing (Othman and Abou-Donia, 1988; AbouDonia et al., 1989). Stereoselectivity of (-)-gossypol was suggested to explain its antispermatogenic effects; single intratesticular injection of 200 pg of (-)-gossypol caused a 70.4% decrease of the sperm count along with marked atrophy of the testes (Yu, 1987).However, neither a significant decrease in sperm count nor atrophy of the testes was observed after a similar injection of ( + 1-gossypol, suggesting that there is a strict stereochemical requirement for the interaction between gossypol and the testicular target molecules.

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A major side effect of administration of gossypol is the change in electrolyte status (Kennedy et al., 1982; Xue, 1985), in particular hypokalemia, the reduction in serum potassium levels (K less than 3.5 mMol/liter) (Chang et al., 1985; Liu e t al., 1985, 1988; Ypersele de Strihou et al., 1988). Of interest, potassium deficiency has been shown to enhance the antispermatogenic effect of gossypol (Qian, 1985).Among the more potentially serious effects of hypokalemia are cardiac arrhythmias and hypokalemic paralysis. In a survey of 8,806 volunteers taking gossypol as a male contraceptive, about 1%of the men had hypokalemic paralysis, the direct cause being renal potassium loss (Qian, 1985). In those to whom gossypol was given who did not show hypokalemia, serum potassium levels were within the normal range but were significantly lower than serum potassium levels in controls. In the majority of patients suffering from gossypolinduced hypokalemia, recovery has been prompt and complete following potassium repletion, but in some men there were recurrent attacks of hypokalemia during a period of several months to years after cessation of gossypol treatment, suggesting sustained renal damage. Some investigators found a downward trend in plasma potassium levels during the loading phase, but hypokalemic paralysis did not occur even after periods of 6-10 years of gossypol administration (Duo et al., 1988). A randomized and controlled study to evaluate the effect of K salt ingestion or a potassium blocker while using gossypol has indicated that supplementation of K salt does not cause a reversal of the effect of gossypol on serum potassium levels and that the inhibitor triamterene does not prevent loss of serum potassium (Liu et al., 1988). The renal mechanisms by which gossypol produces hypokalemia are not well understood. Some investigators have suggested that gossypol induces renal leakage of potassium both in animals and in humans (Wang and Yeung, 1985). The most likely mechanism is a direct toxic effect of gossypol on the renal tubules. Following studies of the effects of gossypol on the kidney, some investigators found no toxic effect (Ypersele de Strihou et al., 1988), whereas others suggested that gossypol is a specific and potent membrane active agent (Fu e t al., 1988; Liu et al., 1988). Gossypolone, the in vivo oxidation product of gossypol, may form a redox system with its corresponding hemiquinone, leading to free radical generation. Racemic gossypol stimulated superoxide free radical formation when incubated with either rat liver or kidney microsomes but not with those of the heart or testes (Yu, 1987). The suggested mechanisms for the development of gossypol-induced hypokalemia include inhibition of Na-K-ATPase activity, stimulation of prostaglandin biosynthesis, and morphological damage to the renal tubule, but further studies are warrented (Drayer and Reidenberg, 1988). Gossypol-induced hypokalemia appears to be diet-dependent, and there appears to be +

interracial variation in the effect of gossypol, with oriental men being more profoundly affected by hypokalemia (Coutinho and Melo, 1988). A potassium-deficient diet could be considered a contributing factor in the development of gossypol-induced hypokalemia (Braham and Bressani, 1985; Liu et al., 1985). The incidence of hypokalemic paralysis in gossypol takers shows distinct regional differences, being much higher in Nanjing, where the dietary potassium level of the inhabitants is low, than in Taian, where the dietary potassium level is high (Qian, 1985). Experiments conducted in rats have shown that, in animals fed a low-K fodder, gossypol was associated with reduction of intracellular Mg and K concentrations of the skeletal muscle, whereas in regularly fed rats, this effect of gossypol was not observed (Qian, 1985). The lack of gossypol effect on plasma concentrations of pituitary hormones and testosterone suggests that it does not act on the hypothalam+pituitary axis (Paz and Homonnai, 1985; Coutinho and Melo, 1988). There are studies reporting that gossypoI has the potential to lower sperm counts and suppress sperm motility without changing the hormonal balance and libido levels (Frick and Danner, 1985; Kalla et al., 1985a; Liu e t al., 1985). On the other hand, some Chinese studies have reported high rates of altered libido among Beijing participants, whereas very low rates were found among Nanjing participants (Liu et al., 1985). The reasons for these discrepancies may include differences in approach and interpretation of libido among the various investigators and the men who were studied.

DISCUSSION Drug-induced changes in male reproductive function are complex and may involve several elements of the system. The motility factor is one of several parameters determining infertility or fertilizing ability; consequently, enhancement of sperm motility does not necessarily prove fertilizing ability. In humans, malefactor infertility is commonly identified as being present when fewer than 50% of spermatozoa display forward progression within 60 minutes after semen collection, together with sperm density of less than 20 x 106/ml or normal morphology of less than 50% of the spermatozoa (WHO Laboratory manual, 1987). The complex processes of actively reaching the oocytecumulus complex and penetration of the egg vestments in order to reach the oolema are varied among the species and limit comparison. There are differences in the environmental, physical, and chemical characteristics a s well as in the distance sperm need to travel from the site of sperm deposition to the site of fertilization. Variation in sperm surface molecules contributes to different biochemical properties and behavior as well a s to differences in the physical properties of sperm of different species. Another complicating parameter is the involvement of the female reproductive tract in the locomotion of the spermatozoa (Green, 1987), a s well as

EFFECTS OF GOSSYPOL ON THE MOTILITY OF MAMMALIAN SPERM modulation of sperm motility by the epithelial surfaces and secretions of the female tract (Katz et al., 1989). Animal studies using gossypol have shown that various mammalian species react differently, even unpredictably, to similar treatments with the contraceptive. It is therefore difficult to identify the exact mechanism for the antifertility effects of gossypol. By using in vitro systems, i t might be possible to identify key steps in the process leading to the inhibition of fertilization by gossypol. In vitro studies have produced some very interesting data, but one must consider their limitations. They exclude the metabolism of the drug and do not take account of interference of other systems. It is also possible that sperm maturation in vivo proceeds in a different, perhaps more efficient, way from that in vitro, thus presenting a different milieu to the activity of gossypol. When using epididymal sperm for in vitro cultures, seminal vesicle and prostate fluids are excluded, leading to discrepancies in results. It is very difficult to compare sperm cell morphological changes during long-term in vivo experiments with brief in vitro studies in that the extent of cellular necrosis can be related to the incubation times of drug media (Waller et al., 1980; Hoffer et al., 1988). In addition, the amount of drug necessary to produce a n effect in vitro may be a great deal less than that necessary to cause a similar effect in exposed cells or tissue in vivo. There is no laboratory model that appears to be suitable for assessment of gossypol toxicity (Othman and Abou-Donia, 1988). Subhuman primates bear the closest physiological similarities to man. In particular, the spermatogenic cells and the spermatogenic cycle of the baboon closely resembles those of man, but the costs are high and availability limited; therefore, rats and hamsters have been more widely used. Some inconsistencies in published results have been attributed to differences between individuals or animal strains as well as to drug impurity, the use of different drug derivatives (Kong e t al., 19861, dosage, method of administration, and other differences in experimental conditions. In humans, the most serious side effect of gossypol ingestion was hypokalemia (Chang et al., 1985; Liu and Lyle, 1987; Liu e t al., 1987, 1988; Coutinho and Melo, 1988; Ypersele de Strihou et al., 1988). Some researchers claim that, with the appropriate diet, the problem of potassium loss can be overcome (Frick and Danner, 1985; Liu e t al., 1985). However, others claim that supplementation of potassium salt does not cause a reversal of the effect of gossypol on serum potassium levels (Liu and Lyle, 1987; Liu et al., 1987, 1988). Genotoxicity studies have produced conflicting data; increased sister chromatid exchange and DNA strand breaks have been reported in human cells, such a s lymphocytes, exposed to gossypol in vitro (Best and McKenzie, 1988; Wang et al., 1988). It is possible for drugs found in semen to be absorbed by the vagina and affect

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the female partner. A developmental toxicity evaluation of gossypol was analyzed in females and in utero in pregnancies that resulted from matings between gossypol-treated male rats and untreated females. There was no adverse effect of gossypol on the females or developing embryos (Beaudoin, 1985,1988; Weinbauer et al., 1985; Bender et al., 1988b). It has been suggested that selective action of gossypol on the testes is due not to its local concentration but to a higher sensitivity and vulnerability of germ and mature cells (Poso, 1980; Cameron et al., 1982; Wichmann et al., 1983; Xue, 1985). Gossypol may exhibit two categories of cytotoxic effects to different organs and tissues. This is indicated by stereoselectivity of (- )-gossypol to induce antispermatogenic effects, whereas there is equipotency between the two isomers in causing toxic effects to some of the somatic tissues. This suggests that one may be able to eliminate one effect while keeping the other. The future of gossypol as a male contraceptive may depend on the ability to separate its toxic from its antifertility properties. Further studies to elucidate the biochemical mechanisms involved in the reversible lesions caused by administration of gossypol as a n agent preventing sperm motility are called for. These investigations may also allow a better understanding of the structural and dynamic processes involved in sperm functions. Research on the relationship between sperm motility and gossypol may shed more light on these complex mechanisms, lead to improved sperm function, and provide novel approaches to controlling fertility by development of safe and reversible male contraceptive.

CONCLUSIONS To control fertility, or even reverse infertility, it is important to understand sperm cell activity and the mechanism whereby systemic and local stimuli induce the appropriate cellular responses. Gossypol appears to be a potential candidate for a male contraceptive. It is a natural substance, a byproduct of the cotton industry (currently considered a waste product), produced in large quantities throughout the world. It has a remarkable specificity for sperm cells. Concerns persist regarding the reversibility of its contraceptive actions and its toxicity. Sperm motility is a crucial requirement in the fertilization process. Understanding the antifertility effect of gossypol on mammalian sperm may assist in identification of the factors involved in sperm motility. This may lead to new techniques that can be applied clinically in male contraception and may assist in understanding the mechanisms of infertility due to poor sperm motility. Taking into consideration the danger in extrapolating animal data to man and the uncertainty in extrapolation of data from in vivo testing systems and especially in vitro models, there seems to be no direct correlation between the contraceptive effects of gossy-

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pol and its general toxicity. These may be two independent variables. It is possible that, with appropriate diet, chronic administration of gossypol a s a n antifertility agent will provide highly effective contraceptive protection. Under these conditions the action of gossypol is most likely to be reversible, and in most cases it does not appear to induce toxicity. However, in view of its cardjovascular side effects, including a direct negative inotropic effect and promotion of arrhythmia, and its nephrotoxicity, it is unlikely that gossypol will be approved a s a contraceptive agent. Extensive investigation and quantitative characterization of gossypol activity and general pharmacology are necessary before it can be safely used clinically.

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