Mechanisms involved in reproductive damage caused by gossypol in rats and protective effects of vitamin E

BACKGROUND: Gossypol is a chemical present in the seeds of cotton plants (Gossypium sp.) that reduces fertility in farm animals. Vitamin E is an antioxidant and may help to protect cells and tissues against the deleterious effects of free radicals. The aim of this study was to evaluate the mechanisms of reproductive toxicity of gossypol in rats and the protective effects of vitamin E. Forty Wistar rats were used, divided into four experimental groups (n = 10): DMSO/ saline + corn oil; DMSO/saline + vitamin E; gossypol + corn oil; and gossypol + vitamin E. RESULTS: Fertility was significantly reduced in male rats treated with gossypol in that a significant decrease in epididy-mal sperm count was observed (P < 0.05) and the number of offspring was significantly reduced in females mated with them (P < 0.05). This dysfunction was prevented by vitamin E. Gossypol caused a significant increase in the activity of the enzymes glutathione peroxidase (P < 0.01) and glutathione reductase (P < 0.01), but vitamin E did not reduce the enzyme activities (P > 0.05). The levels of reduced glutathione and pyridine nucleotides in testis homogen-ate were significantly reduced by gossypol (P < 0.05 and P < 0.01, respectively) and this reduction was accompanied by increased levels of oxidized glutathione (P < 0.05). Vitamin E showed a preventive effect on the changes in the levels of these substances. Gossypol significantly increased the levels of malondialdehyde (P < 0.01), a lipid peroxida-tion indicator, whereas treatment with vitamin E inhibited the action of the gossypol. Vitamin E prevented a decrease in mitochondrial ATP induced by gossypol (P < 0.05). CONCLUSIONS: This study suggests that the reproductive dysfunction caused by gossypol may be related to oxidative stress and mitochondrial bioenergetic damage and that treatment with vitamin E can prevent the infertility caused by the toxin.

Morfologia testicular e qualidade espermática de touros da raça Nelore, submetidos à dieta contendo gossipol / Testes morphology and sperm quality of Nelore bulls fed a gossypol-enriched diet

Com o objetivo estudar o efeito de dieta contendo gossipol sobre a qualidade seminal e morfologia dos testículos de touros da raça Nelore, utilizaram-se 16 animais, distribuídos em dois tratamentos: T1, oito touros submetidos a dieta contendo 20 por cento de caroço de algodão e T2, oito touros submetidos a dieta isenta de gossipol. Realizaram-se coletas de sêmen para avaliar a qualidade seminal. Os testículos dos animais foram retirados ao final dos 73 dias de experimento, fragmentos dos mesmos foram preparados para serem analisados em microscopia eletrônica de transmissão e outros fixados em lâminas e corados por hematoxilina-eosina. O consumo médio de caroço de algodão/animal/dia de 1,71kg interferiu na morfologia dos testículos; os túbulos seminíferos apresentaram menor espessura de parede, redução no número de camadas de células espermatogênicas e no volume dos núcleos da célula de Sertoli em relação aos do T2. Por microscopia eletrônica observouse que os touros do T1 apresentavam células de Sertoli com membrana citoplasmática enrugada, acúmulo de lipídeos no citoplasma, mitocôndrias deformadas e algumas espermátides estavam em apoptose. Touros do T1 apresentaram redução na motilidade espermática, aumento no percentual de defeitos espermáticos maiores e totais em relação aos animais do T2. O gossipol alterou a morfologia testicular e reduziu a qualidade espermática dos touros...

We studied the effect of a gossypol-enriched diet on the quality of semen and the morphology of testes of Nelore bulls. Sixteen animals were scheduled for 2 treatments: T1, 8 bulls fed a diet containing 20 percent cottonseed and T2, 8 bulls fed a gossypol-free diet. Semen samples were collected in order to evaluate the semen quality. At the end of the 73-day period of treatment, the testes were removed and tissue fragments were prepared for transmission electron microscopy or fixed on slides and stained with haematoxylin-eosin for light microscopy. The mean daily intake of cottonseed by T1 bulls was 1.71 kg/animal, which interfered on testes morphology; the seminiferous tubules had less thick walls, fewer layers of the spermatogenic lineage cells and reduced volumes of Sertoli cell nuclei with regard to T2 bulls. Electron microscopy analyses revealed that the Sertoli cells from T1 animals had a rough cytoplasmic membrane, lipid accumulation in the cytoplasm, deformed mitochondriae; in addition, some apoptotic spermatids were seen. T1 bulls showed reduction of sperm motility and an increase of the percentage of major and total sperm defects in relation to the animals of T2 group. It is concluded that a gossypol-enriched diet promoted alterations in the testicles morphology and it reduced the sperm quality of bulls...

Morfologia testicular e qualidade espermática de touros da raça Nelore, submetidos à dieta contendo gossipol / Testes morphology and sperm quality of Nelore bulls fed a gossypol-enriched diet

Com o objetivo estudar o efeito de dieta contendo gossipol sobre a qualidade seminal e morfologia dos testículos de touros da raça Nelore, utilizaram-se 16 animais, distribuídos em dois tratamentos: T1, oito touros submetidos a dieta contendo 20 por cento de caroço de algodão e T2, oito touros submetidos a dieta isenta de gossipol. Realizaram-se coletas de sêmen para avaliar a qualidade seminal. Os testículos dos animais foram retirados ao final dos 73 dias de experimento, fragmentos dos mesmos foram preparados para serem analisados em microscopia eletrônica de transmissão e outros fixados em lâminas e corados por hematoxilina-eosina. O consumo médio de caroço de algodão/animal/dia de 1,71kg interferiu na morfologia dos testículos; os túbulos seminíferos apresentaram menor espessura de parede, redução no número de camadas de células espermatogênicas e no volume dos núcleos da célula de Sertoli em relação aos do T2. Por microscopia eletrônica observouse que os touros do T1 apresentavam células de Sertoli com membrana citoplasmática enrugada, acúmulo de lipídeos no citoplasma, mitocôndrias deformadas e algumas espermátides estavam em apoptose. Touros do T1 apresentaram redução na motilidade espermática, aumento no percentual de defeitos espermáticos maiores e totais em relação aos animais do T2. O gossipol alterou a morfologia testicular e reduziu a qualidade espermática dos touros [...]

We studied the effect of a gossypol-enriched diet on the quality of semen and the morphology of testes of Nelore bulls. Sixteen animals were scheduled for 2 treatments: T1, 8 bulls fed a diet containing 20 percent cottonseed and T2, 8 bulls fed a gossypol-free diet. Semen samples were collected in order to evaluate the semen quality. At the end of the 73-day period of treatment, the testes were removed and tissue fragments were prepared for transmission electron microscopy or fixed on slides and stained with haematoxylin-eosin for light microscopy. The mean daily intake of cottonseed by T1 bulls was 1.71 kg/animal, which interfered on testes morphology; the seminiferous tubules had less thick walls, fewer layers of the spermatogenic lineage cells and reduced volumes of Sertoli cell nuclei with regard to T2 bulls. Electron microscopy analyses revealed that the Sertoli cells from T1 animals had a rough cytoplasmic membrane, lipid accumulation in the cytoplasm, deformed mitochondriae; in addition, some apoptotic spermatids were seen. T1 bulls showed reduction of sperm motility and an increase of the percentage of major and total sperm defects in relation to the animals of T2 group. It is concluded that a gossypol-enriched diet promoted alterations in the testicles morphology and it reduced the sperm quality of bulls [...]

Effect of amino acids on inhibition of lactate dehydrogenase-X by gossypol

Gossypol acetic acid (GAA) has been shown to have male antifertility effects, but there are pronounced differences among animal species. In the search of endogenous effector molecules, which interfere with the functions of GAA, we have studied the in vitro effect of various amino acids on the inhibition of the purified LDH-X by GAA. Histidine, cysteine and glycine were shown to block the effect of GAA. The effects of these amino acids were concentration dependent. Histidine and glycine protection was found to be complex type in which both the Km and Vmax were decreased compared to control. Arginine, glutamic acid, phenylalanine and valine were found to be ineffective against the inhibitory action of GAA.

Entamoeba histolytica: inhibition of malic enzyme and alcohol dehydrogenase by (ñ)-,(+)-,and (-)- gossypol

Gossypol, a natural racemic mixture with action on NADP- and NAD-oxidoreductases from diverse species, has been proposed as a possible antiamebic medication considering several of its pharmacological properties. In this study it was found that malic enzyme and alcohol dehydrogenase from Entamoeba histolytica are strongly inhibited by (ñ)-gossypol, and both (+)- and (-)- enantiomers. The inhibition was of the noncompetitive type among their respective substrates in all cases. The (ñ)-, (+)-, (-)-gossypol half-maximal inhibitory concentrations (IC 50) for the malic enzyme were 3.71, 13.37 and 1.03 µM, and againts the alcohol dehydrogenase 79.64, 124.43 and 42.56 µM, respectively. Therefore, the (-) enantiomer resulted 3.6 and 13.0 times more potent than the racemic mixture and (+)- gossypol, respectively, to inhibit the malic enzyme, and 1.9 times and 2.9 times more potent than the racemic mixture and (+)-gossypol, respectively, against the alcohol dehydrogenase. Accordingly, one possible mechanism of the antiamebic affect of gossypol could be the inhibition of vital NADP-dependent enzymes as those analyzed in this study

Effect of gossypol on tissue levels of glutathione and thiobarbituric acid reactive products in rats.

Gossypol was administered in pubertal and adult rats and lipid peroxide formation and GSH levels were estimated in different tissues like liver, testis, heart and kidney. Gossypol caused low generation of lipid peroxides, measured as thiobarbituric acid reactive products (TBAR), without causing significant changes in tissue glutathione (GSH) levels. This effect was more pronounced in liver and testis as compared to other tissues. In vitro effect of gossypol to inhibit lipid peroxidation as observed in vivo suggested that binding of gossypol to plasma membranes may result in inhibition of lipid peroxide generation.

Effect of chlorpromazine and gossypol on Ca(2+)-ATPase activity in the microsomal membranes of rat testes.

The microsomal membranes isolated from rat testes have been found to contain a Mg(2+)-dependent and a Mg(2+)-independent Ca(2+)-ATPase. The enzyme activities were inhibited by two contraceptive drugs--gossypol and chlorpromazine. The inhibition by the former was affected by the presence of ligand(s) and not the substrate in the incubation medium, whereas ligand(s)/substrate did not affect the inhibition by chlorpromazine. This may be explained from the fact that the binding of chlorpromazine and ligand(s)/substrate to the enzyme are independent of each other whereas in case of gossypol the ligand(s) compete with the drug at the binding site of the enzyme.

In vitro effects of gossypol and lactic acid on rat uterus and ovary during implantation and antiimplantation.

ATPase activity of uterus and ovary was markedly elevated in presence of gossypol and decreased in presence of lactic acid indicating activation and inhibition of energy metabolism by gossypol and lactic acid respectively. The elevated levels of glycogen in uterus indicate inhibition of glycogenolysis as supported by phosphorylase activity. Whereas in ovary the glycogen depletion indicates activation of glycogenolysis supported by phosphorylase activity. The activity levels of aldolase and G-6-PDH decreased in the uterus in presence of gossypol and increased in presence of lactic acid. The same were elevated in ovary indicating the activation of hexose mono and diphosphate pathways. Lactic acid accumulated in presence of both gossypol and lactic acid with a depletion in level of pyruvic acid in both the tissues. This situation in the uterus indicates the condition of anti-implantation in presence of both gossypol and lactic acid. The NAD-LDH activity was inhibited in presence of gossypol and activated in presence of lactic acid in both tissues.

Effect of gossypol on few testicular enzymes in mature rats.

Effect of oral administration of gossypol acetic acid (15 mg/kg/day) for 10 weeks, on certain enzymes, which may be taken as markers for the different stages of spermatogenesis, was studied in male albino rats. Gossypol produced a significant decrease in hyaluronidase and sorbitol dehydrogenase, while no change was observed in beta-glucuronidase and acid phosphatase. A significant increase in the total lactate dehydrogenase activity was observed in the testis. The possible significance of these findings is discussed.

Ultrastructural and biochemical changes in epididymis and vas deferens of gossypol treated rats.

Adult male Wister rats when administered with 15 mg/kg body weight/day of gossypol acetic acid proved to be sterile by 10 weeks of treatment. The weight of the whole epididymis did not deviate from the controls but when the caput, corpus and cauda epididymidis were considered separately, the cauda epididymidis weight was significantly reduced. The major changes were observed in the motor apparatus of the sperm. The most common defects in the sperm were the vacuolization and complete degeneration of the midpiece mitochondria and plasma membrane. The total LDH activity of caput and cauda epididymidis were within the range of control values. Sialic acid levels of the epididymis were not affected after the treatment. These results suggest a more proximal site of action of the drug than at the epididymal level.

Gossypol affects the responsiveness of isolated rat myometrium and vas deferens

The effects of gossypol on responsiveness of both rat myometrium and vas deferens were analyzed. In myometrial strips, gossypol (1-30 micronM) produced rightward displacemtns of the cumulative concentration-response curves to acetylcholine, bradykinin and oxytocin, accompanied by reductions in maximal responses. Gossypol (30 micronM) also completely abolished the contractions induced by field stimulation of the rat vas deferens. The IC50 values for gossypol against agonist-uinduced myometrial contractions and field-stimulated vas deferens contractions were similar, ranging between 13 and 18 micronM. These results provide additional evidence that gossypol exerts a direct and irreversible inhibition of the contractility of both male and female reproductive organs

Gossipol: mecanismo de açäo sobre o sistema reprodutor masculino / Gossypol: mechanism of action on the male reproductive system

Procedeu-se a uma revisäo bibliográfica sobre o efeito do gossipol no sistema reprodutor masculino, analisando-se células da linhagem espermatogênica, espermatozóide, células de Leydig, células de Sertoli, sistema de condutos, glândulas acessórias e eixo hipotálamo-hipofisário. Em conclusäo, quanto ao efeito do gossipol: 1) Sobre as células da linhagem espermatogênica - as espermatogônias seriam pouco ou näo afetadas pelo gossipol; os espermatócitos teriam o processo de citocinese inibido; as espermátides sofreriam alteraçöes morfológicas diversas. Quanto ao espermatozóide, parece ser a célula-alvo do gossipol. Os efeitos observados referem-se à inibiçäo da motilidade e da capacidade de fertilizaçäo. Ambas as alteraçöes estäo relacionadas a modificaçöes morfológicas e enzimáticas, notando-se, entre as últimas, a inibiçäo da LDH-X e da acrosina; 2) Os efeitos sobre a célula de Leydig e de Sertoli säo controversos; 3) Estudos realizados sobre o efeito no epidídimo näo foram conclusivos; o mesmo quanto a ducto deferente e glândulas sexuais acessórias; 4) Com relaçäo ao hipotálamo, as duas referências bibliográficas encontradas referem-se a alteraçöes morfológicas de neurônios do núcleo paraventricular; 5) Näo foram encontrados resultados conclusivos quanto à hipófise e gonadotrofinas hipofisárias

Efeitos do gossipol sobre a morfofisiologia testicular / Effects of gossypol on testicular morphology and physiology

Os autores analisam as informaçöes disponíveis sobre os efeitos de gossipol na morfologia e fisiologia dos principais componentes testiculares: células de Leydig, células de Sertoli e células de linhagem espermatogênica

Gossypol injected in the rat epididymal fat pad inhibits epididymal sperm motility

El sitio primario de la acción antifertilidad del gossypol en la rata ocurruría a nivel del testículo y no en el epidídimo (Dai and Dong, 1978). Los experimentos realizados mediante ligaduras del epidídimo y administración oral de gossypol parecen demostrar la ausencia de efectos sobre el espermatozoide en el epidídimo. Para explorar hasta donde el gossypol es capaz de actuar sobre el espermatozoide contenido en el epidídimo, infiltramos la grasa adyacente al caput epidídimo con 100 ug de gossypol disueltos en 0,1 ml de aceite de sésamo. Se ligaron los conductos deferentes derechos de ambos lotes de ratas, control y tratadas. A las 24 horas de examinó bajo microscopía de contraste de fases y de campo oscuro la motilidad de los espermatozoides extraídos de los epidídimos controles y tratados. Los resultados indicaron una reducción significativa de la motilidad en los tratados con gossypol. La motidad de los tratados y ligados fue aún más baja que los tratados no ligados. Los controles ligados y no ligados mostraron una motilidad similar. El examen morfológico con TEM reveló una aparente hinchazón de la pieza media de los espermatozoides en los animales tratados. Concluimos que la introducción de gossypol dentro de la grasa epididimaria inhibe la motilidad de los espermatozoides contenidos en el epidídimo dentro de las 24 horas y que la morfotología de los espermatozoides parece sufrir alguna alteración

Efeito de gossipol sobre a isoenzima lactata desidrogenase-x / Effect of gossypol on isoenzyme lactate dehudrogenase-x

O gossipol inativa a LDH-X, isoenzima encontrada exclusivamente no testículo e espermatozóide e que participa do processo de produçäo de energia, desempenhando um importante papel no metabolismo das células germinativas, principalmente na manutençäo da mortalidade espermática.Sugere-se que o mecanismo pelo qual o gossipol exerce seu efeito anti-fertilidade seja através da inibiçäo de enzimas específicas do trato reprodutivo masculino, especialmente da LDH-X, cujos sítio(s) ativo(s) seriam inativados por produtos ativos da decomposiçäo do gossipol

Efeitos do gossypol sobre o aparelho reprodutor feminino / Effects of gossypol on the female reproductive system

Sobre o sistema reprodutor feminino observaram-se os seguintes efeitos do Gossypol: 1) alteraçöes discretas sobre a secreçäo hormonal ovariana; 2) alteraçöes do muco cervical e espermicida efetivo, quando usado por via intravaginal; 3) inibiçäo da implantaçäo, e da reaçäo decidual, em ratas