Mutagenicity of gossypol analyzed by induction of meiotic micronuclei in vitro

Chromosome breakage caused by mutagens in male germ cells can be analyzed by micronucleus induction during meiotic division. This can be followed in vitro by culturing seminiferous tubular segments from stages of the epithelial cycle that contain late pachytene and diakinetic primary spermatocytes. We studied the mutagenic potential of a male contraceptive, gossypol, in this test system using adriamycin (10 ng/ml) as a reference mutagen. A small but significant increase in the frequency of micronuclei was induced with concentrations of 10 and 20 micrograms/ml of gossypol, while cytotoxic effects appeared at concentration of 20 micrograms/ml and were evident at 50 micrograms/ml. Analysis of meiotic micronucleus induction in vitro seems to be a sensitive test system of male germ-cell mutagenesis, but further studies on the possible mutagenic effects of gossypol are needed.     

Ultrastructural, fertility, and spermicidal studies with isomers and derivatives of gossypol in male hamsters

The effects of fourteen new, orally administered synthetic analogs of gossypol on testicular ultrastructure and fertility in hamsters and the spermicidal properties of these compounds, as well as of the optical isomers of gossypol against hamster and human sperm in vitro, are reported in this study. Test compounds were administered to adult male hamsters by daily gavage for 9 weeks at doses ranging from 15 to 50 mg/kg. The results of this study have demonstrated that the fourteen new gossypol analogs evaluated herein are not effective as male antifertility agents and their in vitro activity or lack of activity as spermicides is unrelated to their in vivo contraceptive potential. In addition, the results of the study suggest that (1) the isopropyl moiety of the gossypol molecule, like the aldehyde group, is essential for its mechanism of action and (2) the pathognomonic defect in the mitochondrial sheath induced by gossypol appears to be related to its unique activity as a male antifertility agent. The significance of these findings is discussed.     

Cottonseed feeding delivers sufficient quantities of gossypol as a male deer contraceptive

To define the quantitative and qualitative effects of gossypol (GP) on deer (Cervus elaphus) semen, the animals were fed cottonseed (CS). Adult stags each received 350 g of CS for 109 days. Animals received 15 mg of gossypol per kilogram body weight per day. Quantitative and qualitative parameters of experimental ejaculates (n = 182) were compared to ejaculates (n = 571) of control animals (n = 5) collected during three previous natural reproductive seasons. Ejaculate fractions were evaluated by classical methods used in domestic animals. In this paper, we show that mature male deer fed CS exhibited morphological changes and decreased motility of spermatozoa and abnormalities in spermatogenesis. Radioimmunoassay measured concentrations of various steroid hormones (T-testosterone, A₄-androstenedione, and E₂-estradiol 17β) in separated ejaculate fractions of the CS group were compared to a control group of stags. Generally, mean steroid concentrations in CS-treated deer decreased during the entire sampling period in examined ejaculate fractions. These changes resulted in decreased semen quality with no detectable side effects in the animals. It seems that gossypol fed to the deer in the form of CS serves as an efficient male contraceptive.     

Modification of the positive inotropic effects of catecholamines, cardiac glycosides and Ca2+ by the orally active male contraceptive, gossypol, in isolated guinea-pig heart

Gossypol is an orally active male contraceptive with cardio-depressant side effects. To understand the mechanism of its cardiac actions, the interaction of gossypol with positive inotropic drugs was examined in isolated atrial muscle preparations obtained from guinea-pig heart. Gossypol delayed the onset of arrhythmias caused by digoxin. In the presence of gossypol, the positive inotropic effect of isoproterenol declined rapidly, and the effect of isoproterenol to increase tissue cyclic AMP concentrations was smaller. Pretreatment of atrial muscle with the combination of gossypol and isoproterenol markedly reduced effects of isoproterenol on developed tension and cyclic AMP concentrations when these effects were tested after the washout of the first dose of isoproterenol. These effects, however, were not specific to isoproterenol. The gossypol-isoproterenol pretreatment reduced the positive inotropic effect of ouabain or extracellular Ca2+. These results indicate that gossypol has pharmacodynamic interactions with several positive inotropic agents that are known to enhance developed tension by increasing intracellular Ca2+ transients.     

肌肽棉酚对绵羊精子运动及超微结构的影响

肌肽促进绵羊精子运动增强,直线运动精子数目增多;棉酚则强烈抑制精子运动并使精子的运动方式发生改变;肌肽与棉酚混合处理精子,肌肽可不同程度抵消棉酚对精子产生的有害作用。但当棉酚完全抑制精子运动后,再加入肌肽则不能使精子恢复运动。电镜观察,肌肽对绵羊精子超微结构无任何不良影响;棉酚同造成精子严重损伤,导致生物膜系统损害、轴丝崩解、线粒体螺旋鞘紊乱等。本文通过肌肽,棉酚加入的时序不同,探讨两者对绵羊精子     

Reversibility of the reproductive toxicity of gossypol in peripubertal bulls

Gossypol has been shown to impair sperm production in male ruminants. The purpose of this study was to determine if the adverse effects of gossypol on spermatogenesis in peripubertal bulls were reversible. Twenty-eight crossbred Angus bulls were allocated into treated and control groups at 11 months of age. For 8 weeks, treated bulls were fed a ration containing 8 mg of free gossypol per kilogram of body weight per day while control bulls were fed a soybean meal ration free of gossypol. At 28-days intervals, scrotal circumference was measured and semen collected to assess sperm motility and morphology. Seven control and seven treated animals were castrated 56 days after the start of the experiment and the testes were examined histologically. The remaining bulls were fed a gossypol-free diet for 210 days prior to castration. There were significant increases in primary and secondary sperm abnormalities in treated bulls 28 and 56 days after gossypol feeding. The number of sperm with proximal droplets was significantly higher in gossypol-treated bulls, suggesting testicular degeneration. There was no significant effect on the sperm motility, scrotal circumference, or histopathological characteristics of the testes. Four weeks after the end of gossypol feeding, primary and secondary abnormalities were still increased in gossypol-treated bulls, however in subsequent collection periods the percentage of abnormalities were similar between groups. At 210 days, there was no treatment effect on scrotal circumference, and histological characteristics of the testes were not different between groups. The deleterious effects of gossypol on the morphological characteristics of spermatozoa were reversible. Gossypol (8 mg/kg per day for 56 days) increased sperm abnormalities but the effects were reversible.     

Effect of gossypol on the ultrastructure of rat spermatozoa

Summary Gossypol was found to induce sterility in male rats when administered orally. A reduction in the number of spermatozoa in the epididymis from the gossypol-treated rats was observed when compared to the control animals. An examination of the spermatozoa from the treated rats showed the following ultrastructural modifications: disorganization of the mitochondrial sheath and missing cell membrane from the middle piece, broken cell membrane and missing members of both outer fibers and inner microtubules of the principal piece, and broken cell membrane of the sperm head. Serial mating experiments proved that gossypol-treated males were indeed sterile. The results suggest that gossypol at low concentrations is able to affect the motility of spermatozoa, thus contributing to its contraceptive action.     

Experimental analysis of gossypol and chloroquine interaction in serum and in liver of rat.

Interactive effects of gossypol and chloroquine as determined by activities of serum alanine transaminase (ALT), aspartate transaminase (AST) and liver lactate dehydrogenase (LDH), alkaline phosphatase (ALK-pase), glucose-6-phosphatase (G-6-pase) and cholesterol level were investigated in rats. Administration of gossypol for eight weeks, at a concentration of 20 mg per kg body wt. per day with or without chloroquine had no effect on the serum enzymes and glucose-6-phosphatase activities. When chloroquine at a concentration of 5 mg per kg body wt. thrice a week was administered alone, there was a marked decrease in total protein content and ALK-pose activities, while a significant increase in LDH activity was observed. Administration of either gossypol or chloroquine decreased the level of cholesterol. A greater decrease was recorded when both were given together. It is suggested that gossypol can be employed as a male contraceptive among malaria-infected populations.     

Cytotoxic effect of gossypol on colon carcinoma cells

Gossypol, a male contraceptive drug extracted from cottonseeds, has been found to have antiproliferative activity on tumour cells and is thought to be a potential anticancer drug. The aim of this study was to investigate the mechanisms of gossypol-induced cell death on two colon carcinoma cell lines, HT29 and LoVo. Firstly, we studied the effect of gossypol on the colony forming ability of these tumour cells, which is the main target of chemotherapeutic drugs. Using clonogenic assays, flow cytometry and DNA gel electrophoresis techniques, we have found that gossypol not only inhibited colony forming ability of these tumour cells, but we also observed cellular internucleosomal DNA fragmentation in the cells treated with 3 doses of gossypol and this was accompanied by the appearance of a sub-G1 apoptotic peak and morphological characteristics of apoptosis. Our results suggest that the gossypol induced cell death is via an apoptotic pathway and the effect of gossypol may not be cell cycle specific. Using Western blotting analysis, we found that the gossypol-induced apoptosis may not be involved in the regulation of p53 but possibly associated with the regulation of bcl-2 and Bax expression. Our evidence indicates that gossypol may provide a potential therapeutic benefit for the treatment of colon carcinoma and understanding the mechanisms of gossypol-induced cytotoxicity on tumour cells is essential for including this drug in clinical use.     

Loss of calcium in human spermatozoa via EPPIN, the semenogelin receptor

The development of a new male contraceptive requires a transition from animal model to human and an understanding of the mechanisms involved in the target's inhibition of human spermatozoan fertility. We now report that semenogelin (SEMG1) and anti-EPPIN antibodies to a defined target site of 21 amino acids on the C terminal of EPPIN cause the loss of intracellular calcium, as measured by Fluo-4. The loss of intracellular calcium explains our previous observations of an initial loss of progressive motility and eventually the complete loss of motility when spermatozoa are treated with SEMG1 or anti-EPPIN antibodies. Thimerosal can rescue the effects of SEMG1 on motility, implying that internal stores of calcium are not depleted. Additionally, SEMG1 treatment of spermatozoa decreases the intracellular pH, and motility can be rescued by ammonium chloride. The results of this study demonstrate that EPPIN controls sperm motility in the ejaculate by binding SEMG1, resulting in the loss of calcium, most likely through a disturbance of internal pH and an inhibition of uptake mechanisms. However, the exact steps through which the EPPIN-SEMG1 complex exerts its effect on internal calcium levels are unknown. Anti-EPPIN antibodies can substitute for SEMG1, and, therefore, small-molecular weight compounds that mimic anti-EPPIN binding should be able to substitute for SEMG1, providing the basis for a nonantibody, nonhormonal male contraceptive.     

Characterization of zebra mussel (Dreissena polymorpha) sperm motility: duration of movement, effects of cations, pH and gossypol

We have examined effects of time after activation, pH, sodium and potassium, and gossypol concentrations on sperm motility of zebra mussel (Dreissena polymorpha). Zebra mussel spermatozoa appeared to have remarkable viability in the fresh water in comparison with freshwater fish sperm. Duration of sperm motility in fresh water is possibly one of the longest among freshwater animals, since it was not significantly changed 3 h after incubation at room temperature (20 °C) or 24 h of incubation at ±0 °C. High osmotic pressure suppresses sperm motility and effects of sodium and potassium are similar. Spermatozoa were inactive at acid pH and became gradually motile when exposed to pH 6.0–9.0. Gossypol appeared to be a very potent spermicidal agent and inhibited motility. This compound also inhibited fertilization. We observed some differences in gossypol effects on spermatozoa between North American and European zebra mussels. These data on zebra mussel sperm biology may be useful for better handling of gametes under laboratory conditions.     

Hydrolysis of phospholipid monolayers by human spermatozoa. Inhibition by male contraceptive gossypol

Monomolecular films of phospholipid were used to study the interaction of intact human spermatozoa with model membranes. Exclusively with negatively charged phosphatidylglycerol monolayers rapid penetration of spermatozoa into the monolayer with subsequent hydrolysis of the lipid was triggered by the addition of 5 mM calcium into the medium. The results suggest the localization of a calcium-dependent phospholipase A2 at the outer acrosomal or plasma membrane of human spermatozoa with its active site exposed to the external environment. Preincubation of the cells with 100 microM gossypol completely abolished the ability of human spermatozoa to hydrolyze or penetrate monolayers of phosphatidylglycerol. The inhibition of the phospholipase activity by gossypol may contribute to the unknown contraceptive mechanisms of this non-steroidal male antifertility agent.     

Gossypol inhibits estrogen binding to rat alpha-fetoprotein

We find that gossypol, a male anti-fertility compound, is a reversible competitive inhibitor of estrogen binding to rat alpha-fetoprotein (AFP). The Kd of gossypol for rat AFP is 1.75 microM, which is similar to gossypol's affinity for lactate dehydrogenase isozyme X, the putative site where gossypol exerts its anti-fertility effects. Reacting sodium cyanoborohydride with gossypol reduces its affinity for AFP, showing that intact aldehyde groups on gossypol are important for binding to rat AFP and indicating that gossypol is specifically inter-acting with a nucleophilic site on AFP that influences estrogen binding.     

Gossypol-induced inhibition of guinea pig sperm capacitation in vitro

The effect of gossypol acetate at various concentrations (10(-6) to 10(-4) M) on guinea pig sperm forward progressive movement, capacitation, and the acrosome reaction was explored in vitro. We found that 10(-4) M gossypol completely abolished the forward progressive motility of the sperm, and that this inhibition of motility was proportional to the concentration of gossypol used. Also, a dose-dependent decrease in acrosome reactions occurred with concentrations of the agent as low as 5.0 X 10(-6) M. However, we observed that such prevention of the acrosome reaction apparently happens at the capacitation stage rather than during the acrosome reaction itself. Inhibition of capacitation by gossypol was reversible--once the spermatozoa were capacitated in gossypol-free medium, the compound did not block the reaction.     

Molecular mechanisms of gossypol action on sperm motility

1. Gossypol acetic acid inhibits collective motility of ejaculated ram spermatozoa. 2. Oxygen consumption was stimulated at low gossypol concentrations and inhibited as the concentrations are increased. 3. Gossypol inhibits respiration of permeabilized spermatozoa supported by durohydroquinome, which indicates a direct inhibition of mitochondrial electron transport chain. 4. The rapid reduction of mitochondrial dependent motility, high uncoupling effect and almost complete inhibition of mitochondrial calcium accumulation, indicate that gossypol inhibits motility in a mechanism by which mitochondrial uncoupling is involved.     

Binding of gossypol to purified tubulin and inhibition of its assembly into microtubules

Gossypol is a polyphenolic pigment, which is employed as a male antifertility drug. It inhibits, among other reported effects, the growth of cultured mammalian cells, spermiogenesis, flagellar motility in Trypanosoma and sperm, dynein ATPase and the lactate dehydrogenase X (LDH-X) isozyme. We have characterized the non-covalent binding of gossypol to purified calf brain tubulin in 10 mM phosphate buffer, 0.1 mM GTP pH 7.0 at 25 degrees C. Equilibrium measurements were performed by difference spectroscopy. A peak at 435 nm was produced by the perturbation of gossypol light absorption upon binding to tubulin. The experimental isotherm was fitted by 1.96 +/- 0.06 gossypol binding sites per tubulin molecule, with identical apparent equilibrium binding constants of (7.5 +/- 1.1) X 10(4) M-1. The complex formed could be separated from free gossypol by gel chromatography. Binding of gossypol was independent of the presence of 0.1 mM GTP in the buffer. Gossypol did not affect the binding of ligands to the colchicine site. Gossypol interacted with vinblastine but apparently did not bind to the vinblastine sites of tubulin. Gossypol did not displace anilinonaphthalene sulphonate (ANS) bound to tubulin, but caused a strong (fivefold) quenching of its fluorescence. This indicated that gossypol probably binds in the vicinity of the ANS site of tubulin. Gossypol inhibited in vitro microtubule assembly at the same concentration range employed in the binding studies. An increase in the critical protein concentration required for polymerisation was observed, most simply interpreted by a stoichiometric mechanism. Gossypol did not induce any noticeable distortion of the microtubules observed under the electron microscope. This compound constitutes a new tubulin ligand and an inhibitor of microtubule assembly in vitro.     

Antifertility investigations on the crude chloroform extract of Carica papaya Linn. seeds in male albino rats.

Crude chloroform extract of C. papaya seeds (5 mg/animal/day, po, for 20, 40 and 60 days) was investigated for contraceptive efficacy and related side effects in male albino rats. The crude extract reduced fertility to zero per cent by 40 to 60 days of treatment. Suppression of cauda epididymal sperm motility was the most pronounced effect of the drug administration. Scanning electron microscopic observations revealed treatment induced abnormalities in sperms. Cauda epididymal and testicular sperm counts decreased following treatment. Clinical parameters did not show any alterations. Results suggest that the contraceptive effects of chloroform extract of papaya seeds are mainly post-testicular in nature without influencing toxicological profile and libido of the animals.     

Gossypol isomers bind specifically to blood plasma proteins and spermatozoa of rainbow trout fed diets containing cottonseed meal

We investigated the role of gossypol isomers binding to blood plasma, seminal plasma and spermatozoa to elucidate gossypol anti-fertility action in the teleost fish, rainbow trout (Oncorhynchus mykiss). Growth and hematological indicators of males were depressed when fish meal protein in diets was completely replaced with cottonseed meal. The cottonseed meal contained equal proportions of (-) (47.8+/-1.6%) and (+) gossypol isomers. Concentrations of spermatozoa were decreased with increasing proportions of gossypol in diets (from 0.22% to 0.95%); however, sperm motility and fertilizing ability were not affected. In contrast to mammals, steroid hormone concentrations were not suppressed in fish given diets with gradual increase of gossypol level. Gossypol concentrations were 100-fold higher in blood plasma than in seminal plasma, confirming a barrier in gossypol transfer between the general circulation and the testis. Spermatozoa accumulated predominantly (+) enantiomer (65-75%) with decreasing proportions as dietary gossypol concentrations increased. Spermatozoa bound most of the gossypol contained in the semen; however, this did not result in impairment of the sperm motility apparatus. Teleost fish sperm rely on ATP stores that accumulate during maturation as a source of energy during activation. In addition, the duration of sperm movement is short in these fish. As such, we hypothesize that the major action of gossypol on mammalian sperm, which is uncoupling of oxidative phosphorylation, does not impair the energy supply required for flagellar beating in fish spermatozoa.     

Inhibition of human sperm motility by contraceptive anti-eppin antibodies from infertile male monkeys: effect on cyclic adenosine monophosphate

Epididymal protease inhibitor (eppin [official symbol, SPINLW1]) is of interest as a male contraceptive target because of its specificity and location on the human sperm surface. We have examined the effect of anti-eppin antibodies from infertile male monkeys and the effect of recombinant human semenogelin on human sperm motility. Anti-eppin antibodies significantly decreased the progressive motility of human spermatozoa as measured by decreased total distance traveled, decreased straight-line distance, and decreased velocity. Anti-eppin treatment of spermatozoa significantly increased the amount of cAMP present in nonprogressive spermatozoa; however, approximately 25% of antibody-treated spermatozoa could be rescued by the addition of cAMP-acetoxymethyl ester, indicating that anti-eppin-treated spermatozoa have a compromised ability to utilize cAMP. Addition of recombinant human semenogelin has a concentration-dependent inhibitory effect on progressive motility (increased tortuosity and decreased velocity). We tested the hypothesis that anti-eppin antibodies bound to eppin would subsequently block semenogelin binding to eppin. Anti-eppin antibodies from infertile monkeys inhibited eppin from binding to semenogelin. Addition of affinity-purified antibodies made to the dominant C-terminal epitope of eppin had an inhibitory effect on progressive motility (increased tortuosity, decreased velocity, and straight distance). Our results suggest that the eppin-semenogelin binding site is critical for the removal of semenogelin in vivo during semen liquefaction and for the initiation of progressive motility. We conclude that the eppin-semenogelin binding site on the surface of human spermatozoa is an ideal target for a nonsteroidal male contraceptive.