Male Antifertility Agent Research Articles

Effect of Mucuna urens (horse eye bean) on the gonads of male guinea-pigs.

The effect of Mucuna urens (seeds) on the gonads and sex accessory glands of male guinea-pigs was investigated. Sexually mature guinea-pigs of proven fertility were administered orally with 70 mg/kg and 140 mg/kg body weight of crude extract daily for 8 weeks respectively. Phytochemical screening of the seeds revealed the presence of alkaloids. No death or weight loss were observed during the duration of treatment. No pregnancy occurred in females mated with the treated males. Histological observations at high dose (140 mg/kg) showed complete degeneration of sperm in the testicular tubules. In some tubules, the acrosomal cap of the sperm cells was separated from the nuclei which underwent colour changes. In some tubules only the tails were left in the lumen. The spermatids, primary and secondary spermatocytes showed pycnosis while the morphology of spermatogonia and germinal epithelium appeared normal. Some epididymides were devoid of sperm while others contained degenerated spermatozoa and cell debris. In the prostate gland there was collapse of the villi and reduction of secretion in both the prostate and seminal vesicles. At low doses (70 mg/kg), there was spermatogenic arrest at spermatid stage. These observations have shown that M. urens is a potential male antifertility agent.

Gossypol-induced DNA breaks in rat lymphocytes are secondary to cytotoxicity

Gossypol, a male antifertility and potential anticancer agent, was found to induce DNA strand breaks in rat lymphocytes. DNA breaks were measured with the single-cell gel electrophoresis (SCGE) or ‘comet’ assay. A significant increase in DNA breaks was observed after 1 h incubation at concentrations of 2 μg/ml or greater. The inclusion of 10% fetal bovine serum in the media reduced the toxicity of gossypol, and DNA breaks were only observed at a concentration of 80 μg/ml. However, the increase in DNA strand breaks, for incubations with and without serum, only occurred when cell viability was reduced to less than 70%. Examination of cell morphology and DNA fragmentation at incubations up to 5 h yielded no evidence that DNA strand breaks were occurring due to apoptosis. We conclude that gossypol is not primarily genotoxic in this cell type, and that the DNA breaks observed arose secondary to cytotoxicity.

Metabolism of the putative antifertility agents 3-(36)chloro-1-hydroxypropanone and its dimethyl ketal in the male rat.

3-(36)Chloro-1-hydroxypropanone (CHOP), a specific inhibitor of sperm glycolysis in vitro, is rapidly metabolized by the male rat to alpha-chlorohydrin, 3-chlorolactate and the inhibitory sperm metabolite, 3-chlorolactaldehyde, presumably all being of the (S)-configuration. The dimethyl ketal of (36)Cl-CHOP [3-(36)Cl-dimethyl-CHOP] is rapidly metabolized producing identical metabolites and excreted radioactivity in urine at a similar rate. As neither compound produced diuresis, glucosuria or induced the formation of spermatocoeles, conditions associated with related male antifertility and antiglycolytic agents, they could represent an alternative means of producing (S)-3-chlorolactaldehyde within spermatozoa in vivo.

Effect of ornidazole on fertility of male rats: inhibition of a glycolysis-related motility pattern and zona binding required for fertilization in vitro.

The effects of the male antifertility agent ornidazole on glycolysis as a prerequisite for fertilization were investigated in rats. Antifertility doses of ornidazole inhibited glycolysis within mature spermatozoa as determined from the lack of glucose utilization, reduced acidosis under anaerobic conditions and reduced glycolytic enzyme activity. As a consequence, cauda epididymidal spermatozoa from ornidazole-fed rats were unable to fertilize rat oocytes in vitro, with or without cumulus cells, which was not due to transfer of an inhibitor in epididymal fluid with the spermatozoa. Under IVF conditions, binding to the zona pellucida was reduced in spermatozoa from ornidazole-fed males and the spermatozoa did not undergo a change in swimming pattern, which was observed in controls. The block to fertilization could be explained by the disruption of glycolysis-dependent events, since reduced binding to the zona pellucida and a lack of kinematic changes were demonstrated by control spermatozoa in glucose-free media in the presence of respiratory substrates. The importance of glycolysis for binding to, and penetration of, the zona pellucida, and hyperactivation in rats is discussed in relation to the glycolytic production of ATP in the principal piece in which local deprivation of energy may explain the reduced force of spermatozoa from ornidazole-fed males.

Effect of ornidazole on fertility of male rats: inhibition of a glycolysis-related motility pattern and zona binding required for fertilization in vitro

The effects of the male antifertility agent ornidazole on glycolysis as a prerequisite for fertilization were investigated in rats. Antifertility doses of ornidazole inhibited glycolysis within mature spermatozoa as determined from the lack of glucose utilization, reduced acidosis under anaerobic conditions and reduced glycolytic enzyme activity. As a consequence, cauda epididymidal spermatozoa from ornidazole-fed rats were unable to fertilize rat oocytes in vitro, with or without cumulus cells, which was not due to transfer of an inhibitor in epididymal fluid with the spermatozoa. Under IVF conditions, binding to the zona pellucida was reduced in spermatozoa from ornidazole-fed males and the spermatozoa did not undergo a change in swimming pattern, which was observed in controls. The block to fertilization could be explained by the disruption of glycolysis-dependent events, since reduced binding to the zona pellucida and a lack of kinematic changes were demonstrated by control spermatozoa in glucose-free media in the presence of respiratory substrates. The importance of glycolysis for binding to, and penetration of, the zona pellucida, and hyperactivation in rats is discussed in relation to the glycolytic production of ATP in the principal piece in which local deprivation of energy may explain the reduced force of spermatozoa from ornidazole-fed males.

Gossypol as a male antifertility agent--why studies should have been continued.

It has been nearly three decades since the beginning of nationwide research into gossypol as a male antifertility agent in China. Several lines of data implicating some side effects of gossypol were published which led to the termination of this area of research in China. The present review re-examines these data and points out the confusion surrounding issues such as hypokalaemia, irreversible azoospermia and other side effects. The prospects and related difficulties for gossypol as a potential male contraceptive are also discussed.

Alpha-Glucosidase activity in the rat epididymis under different physiological conditions.

The estimation of alpha-glucosidase activity in semen is widely used as a marker of epididymal function. In the present studies, glucosidase activity was evaluated in the different segments of the rat epididymis under various physiological conditions. In addition, the effect of two known male antifertility agents, gossypol and alpha-chlorohydrin, on enzyme activity was evaluated. Enzyme activity was absent from the epididymis of rats aged 10 and 20 days but became detectable at 30 days of age when the adult pattern of distribution (highest activity in the caput epididymis) was established. Enzyme activity was reduced significantly in all segments of the epididymis at 7 days after castration and a significant decrease in activity was also observed following the administration of either gossypol or alpha-chlorohydrin. These findings are consistent with a role for alpha-glucosidase in sperm maturation in the epididymis.

Metabolism of 36Cl-ornidazole after oral application to the male rat in relation to its antifertility activity

1. The antimycotic ornidazole (a male antifertility agent in rats) was synthesized incorporating 36Cl in the chloropropyl sidechain and its metabolism was investigatedin the male rat after oral ingestion. 2. Blood levels of radioactivity were low over the first 24 h and there was no tissue accumulation of radioactivity over 48 h. 3. Most of the excreted radioactivity (20% of the ingested dose) appeared in the urine within the first 24 h. 4. Three major compounds were detected in 0-24-h urine samples and were characterized as ornidazole (13% of total radioactivity), Cl− (22%) and 3-chlorolactate (30%), the oxidation product of 3-chlorolactaldehyde. 5. No polyuria or glucosuria was observed following the oral administration of ornidazole,suggestingthat any (R)-3-chlorolactate producedwas insufficient toaffect renal metabolism. 6. Conversion of ornidazole initially to (R,S)-α-chlorohydrin or ultimately to the glycolytic inhibitor (S)-3-chlorolactaldehyde could explain its antifertility action in the male rat.

Suppressive effect of Gossypol, Gossypolone and serum from Gossypol treated rats on steroidogenesis in cultured porcine granulosa cells

Gossypol (GP), a male antifertility agent, and Gossypolone (GN), a major metabolite of GP, have been shown to have a suppressive effect on steroidogenesis in bovine luteal cells. We examined the effect of GP, GN and serum obtained from rats given GP (GP-serum) on steroidogenesis in porcine granulosa cells (PGC). To obtain GP-serum, 50-60 day old Copenhagen rats received GP (12.5 mg/kg B.W./day, sc) for 14 days. PGC were incubated with GP, GN and GP-serum at different doses for 24 hours. GP and GN significantly suppressed Progesterone (P4) and Estradiol (E2) release in a dose-dependent manner, respectively. GP-serum also significantly suppressed P4 release in a dose-dependent manner. The magnitude of suppression of P4 obtained by GP-serum could not be explained by the amount of GP remaining in the serum. Therefore, the inhibition of P4 release by GP-serum could not be solely attributed to GP.

Effects of the male antifertility agent ornidazole on sperm function in vitro and in the female genital tract.

Infertility is induced in male rats by oral administration of 400 mg ornidazole kg-1 body mass day-1 within 10 days, without drastic effect on the motility of epididymal spermatozoa obtained after 15 days of treatment. Spermatozoa were recovered from the female tract 9 h after mating with males treated with ornidazole for 10 days to identify the cause of infertility. The number and motility of spermatozoa in the uterus indicated normal seminal deposition. Spermatozoa could pass the utero-tubal junction but total numbers of spermatozoa and their velocities in the oviductal isthmus were significantly lower than those recovered from females mated to fertile, control males fed vehicle alone. However, the number of spermatozoa recovered from the ampulla was not different from control and the number of spermatozoa that had penetrated the cumulus mass was lower and none of the eggs was fertilized by spermatozoa from rats treated with ornidazole. The ability of cauda epididymal spermatozoa from ornidazole-fed rats to penetrate viscous media in vitro was lower than that of the controls. When incubated in media containing different concentrations of substrates and ions, a reduction in movement of spermatozoa was observed when glucose was the only exogenous substrate or after preincubation in substrate-free medium. These results indicate that the antifertility action of orally administered ornidazole occurs via damage to spermatozoa in the upper regions of the female tract, possibly reflecting a failure in capacitation as a result of reduced energy production.

Identification and Localization of a Novel Cathepsin S-like Proteinase in Guinea Pig Spermatozoa

Guinea pig spermatozoa were found to contain a novel cysteine proteinase that most closely resembled lysosomal cathepsin S. The responsible enzyme was shown to have an acrosomal localization. Like the cathepsin S of lymphocyte-rich tissues, e.g., lymph nodes and spleen, the spermatozoal enzyme digested protein substrates such as hemoglobin and Azocoll optimally at about pH 3.5 in the presence of pepstatin. No appreciable action was manifested in the range of pH 5-6 on protein substrates nor on the 7-(4-methyl)coumarylamide (NMec) derivatives of peptides commonly employed for the fluorometric assay of cathepsins B (benzyloxycarbonyl (Z)-Arg-Arg-NMec), H (Arg-NMec), and L (Z-Phe-Arg-NMec). Like spleen cathepsin S, the sperm enzyme selectively hydrolyzed Z-Phe-Arg-NMec, but its activity was uncharacteristically limited to a pH range of 3.0-3.5, where it displayed a typical high sensitivity to sulfhydryl reagents (HgCl 2, mersalyl acid, iodoacetate), leupeptin, Z-Phe-Phe-CHN 2, and L- trans-epoxysuccinylleucylamido(3-methyl)butane. Gossypol (a male antifertility agent) was also inhibitory. Inhibition by the mercurial sulfhydryl reagents was completely reversible with dithiothreitol. Pepstatin, a potent inhibitor of aspartic proteinases, and serine proteinase inhibitors (phenylmethylsulfonyl fluoride, beazamidine) were ineffective. Other properties displayed by the sperm extract that were uniquely characteristic of cathepsin S included stability under alkaline conditions, and a greater rate of hydrolysis when the P 2-phenylalanine of the assay substrate was replaced by two aliphatic residues, as in Z-Leu-Leu-Arg-NMec.

Development of male‐fertility‐regulating agents

Steroidal, nonsteroidal, plant-derived, gonadotropin-related and immunological agents investigated for control of male fertility are reviewed with brief descriptions of their effects, and illustrations of their structures. The physiology of the male reproductive system is presented as an introduction: an ideal male antifertility agent would inhibit spermatogenesis at the level of the Sertoli cells, without affecting endogenous androgen production by Leydig cells, needed for libido and potency. Androgens down-regulate their own production at physiological levels, but few long-acting orally active derivatives are available. Anti-androgens with mixed androgen and progestin activity, combined with a pure androgen are potentially useful. Androgen-progestin combinations are being tested by WHO as implants. Dozens of miscellaneous nonsteroidal compounds have been discovered serendipitously to have antifertility activity in men or male animals, including alkylating agents antimetabolites, antibiotics, sulfa derivatives, fungicides, trichomonocides, amebicides, alpha blockers, antimalarials, coumarins, and carbohydrate derivatives. Various plant alkaloids have been screened. Those of Hibiscus, Vitex and Plumbago species, as well as Tripterygium wilfordii glycosides, which are being evaluated in combination with gossypol, are mentioned here. Gossypol has been thoroughly tested in China, but rejected because of its side effects, particularly hypokalemic paralysis, its low therapeutic index, and uncertain recovery of fertility. Gonadotropin-releasing hormone (GnRH) agonists and antagonists are being researched in combination with androgens with some success. The GnRH antagonists to date have low activity , or cause histamine-related side effects at higher doses; the androgens require a new route such as a long-acting implant to overcome the need for daily injections. Immunological contraception for males has not progressed beyond the research stage.

Effects of embelin, a male antifertility agent, on absorptive and digestive functions of rat intestine

Oral administration of embelin (75 mg/kg per day, daily for 15 and 30 days) to male rats caused significant elevation in the uptake of d-glucose, l-alanine, l-leucine and calcium in small intestinal segments. Embelin also produced significant increases in intestinal brush border membrane-associated enzymes (sucrase, lactase, maltase, alkaline phosphatase and leucine aminopeptidase) in both intestinal homogenates and partially purified brush border membrane preparations. Significant increases were also noted for microsomal glucose-6-phosphatase and cytosolic lactate dehydrogenase. Increase in brush border membrane-associated total lipids, phospholipids, cholesterol, triacylglycerol, unesterified fatty acids and ganglioside sialic acid were seen but not in the cholesterol/phospholipid molar ratio. All these changes returned to control or near control levels following withdrawal of the drug.

Alterations and Reversibility of the Effects of Gossypol Acetic Acid on the Intestinal Uptake of End Product Nutrients in Normal and Protein Calorie-Malnourished Male Rats

The effects of gossypol acetic acid, an experimental male antifertility agent, 20 mg/kg body weight daily for 15 days and its subsequent withdrawal have been investigated on the intestinal uptake of certain end product nutrients, namely glucose, leucine, alanine and calcium, in normal and protein-calorie-malnourished (PCM) male albino rats. Gossypol feeding caused a reduction in body weight, intestinal weight and length, protein and nucleic acid contents in both normal and PCM animals. Serum parameters, e.g. total protein, albumin and globulin, also showed a decrease in PCM rats. PCM per se considerably elevated the uptake of nutrients but gossypol feeding inhibited the uptake of nutrients both in normal-fed and malnourished animals. Gossypol caused a decrease in the Na(+)-dependent (active) glucose uptake while the Na(+)-independent (passive) process was not altered. The kinetic parameters of glucose uptake indicate that gossypol might be interfering with the transport/carrier protein of these nutrients as reduction in maximum uptake velocity (Jmax) of these systems was observed without, however, any change in the affinity constant (Kt).

Histological changes in the vas deferens of rats after injection of a new male antifertility agent “SMA” and its reversibility

A new male antifertility agent, styrene maleic anhydride (SMA) dissolved in dimethyl sulphoxide (DMSO), was injected into the vas deferens of rats. The morphological changes detected were confined only to the mucosa. After four weeks, the vas deferens lumen was flushed with the solvent DMSO, and the mucosal structures returned to normal.

Species-specific toxic effects of α-chlorohydrin and 6-chloro-6-deoxyglucose on astrocytes

In 1969 α-chlorohydrin was reported to have a reversible antifertility effect in male rats. It has since been reported to have a reversible antifertility effect in many other species but not in mice and rabbits. In chronic high-dosage regimens it was reported to be neurotoxic to mice. At high doses the 6-chloro-6-deoxysugars were found to have a reversible, complete antifertility effect. 6-Chloro-6-deoxyglucose had an antifertility effect in rats and marmosets but not in mice, hamsters, guinea-pigs or rabbits. However, repeated high doses were found to be neurotoxic in the mouse and marmoset, the most prominent pathological feature being vacuolation of astrocytes. In the studies reported here the ability of α-chlorohydrin and 6-chloro-6-deoxyglucose to induce vacuolation of mouse astrocytes has been confirmed in vitro. The failure of either agent to induce vacuolation of rat astrocytes suggests that both the antifertility effect of these agents and their neurotoxic effects are species-specific. The lack of correlation between neurotoxic and antifertility effects of such agents suggests that in vitro techniques may aid the development of a safe, reversible male antifertility agent and also aid in the design of further agents for rodent control.

ChemInform Abstract: Synthesis of Some Aromatic Aldehydes and Phenols as Potential Male Antifertility Agents.

AbstractA series of title compounds such as (IV) and (VIII), which represent analogues of the antispermatogenic agent gossypol (IX) and its precursor hemigossypol (X), are prepared in order to test their activity.

Inactivation of human immunodeficiency virus in vitro by gossypol

Gossypol, a polyphenolic aldehyde extracted from cottonseed, is a male anti-fertility agent which has been reported to have anti-viral activity. In this paper we report that gossypol inactivates human immunodeficiency virus (HIV) in an in vitro system. Following exposure of cell-free incubates of HIV to 100 uM gossypol, ultracentrifugation and inoculation of the washed pellet onto H9 cells, there is no evidence of elevated reverse transcriptase activity over 21 days. Treatment with lower concentrations of gossypol reduces the peak and lengthens the time to maximal reverse transcriptase activity compared with control cultures. These observations suggest that gossypol could be used as a vaginal spermicidal/virucidal agent. The mechanism of the in vitro anti-viral action as well as the effect of orally administered gossypol on the infectivity of semen of HIV-seropositive men warrant further study.

Reversibility of the effects of gossypol acetic acid, an antispermatogenic/antifertility agent on the intestinal structure and functions of male albino rats.

The effects of Gossypol acetic acid (10 mg/kg b. wt. daily for 15 days), an experimental male antifertility agent and its subsequent withdrawal for another 15 days, on the structure and functions of the rat small intestinal tract have been investigated. Gossypol feeding causes a reduction in body weight and intestinal weight, length, protein, and nucleic acid contents. A 27%-50% reduction in the uptake of glucose, alanine, leucine, and calcium is observed after Gossypol feeding which is found to be reversible after 15 days of withdrawal of the drug. Gossypol also causes a significant reduction in the activities of sucrase, lactase, maltase and alkaline phosphatase in the intestinal homogenates as well as in the purified brush border membrane of the microvillus. A decrease in the maximum of apparent enzyme velocity and no change in the substrate affinity constant in these digestive hydrolases are observed on Gossypol treatment. It also causes a shift in the transition temperature in these enzymes and predictably changes the energy of activation both below and above the temperature of transition, although the Arrhenius expression of the temperature dependence still shows proximity, non-linearity, and is parallel to the control group. These changes are reversed on withdrawal of the drug and during the subsequent recovery period. Recovery experiments also show near identical values in kinetic parameters (Kt and Jmax) of 14C-glucose uptake in jejunal segments both in the presence and absence of Na+ ions. Also, no difference is observed between the control and recovery groups with respect to body and intestinal weight, intestinal length, and DNA, RNA, protein, lactate dehydrogenase and glucose-6-phosphate phosphohydrolase values in the intestinal homogenates. Phospholipid, cholesterol and sialic acid levels in both the groups also show nearly identical values. Molecular mechanism of the effects of Gossypol on brush border membrane-bound enzyme/carrier molecules operation is discussed in view of the kinetic and thermodynamic data obtained.

Gossypol-induced free radical toxicity to isolated islet cells

1. 1. The cytotoxicity of the polyphenolic potential male antifertility agent gossypol was investigated on isolated mouse islets cells. 2. 2. Gossypol shared many properties with the diabetogenic agent alloxan. 3. 3. Gossypol (0.1–1.0 mmol/1) induced a concentration-dependent increase of Trypan Blue uptake by the cells, indicating an increase of membrane permeability to the dye. 4. 4. Trypan Blue uptake induced by 0.5 mmol/1 gossypol was inhibited by concommitant incubation of the cells with enzymatic (200 mg/1 Superoxide dismutase, 200 mg/1 catalase, 3 mmol/1 cytochrome- c), or low-molecular weight (50 mmol/1 d-mannitol) scavengers of oxygen radicals, and the metal chelator diethylenetriaminepentacetic acid (DTPA) (50μmol/l). 5. 5. The results support the hypothesis that gossypol is B-cytotoxic by generation of noxious free radicals and that when proposing gossypol as a male antifertility agent, studies to exclude gossypol as a diabetogenic agent should first be performed in vivo.