Studies on the male antifertility agent gossypol acetic acid: in vitro studies on the effect of gossypol acetic acid on human spermatozoa

The hypothesis that gossypol (an active ingredient associated with cottonseed oil) affects enzymes and other constituents of spermatozoa, resulting in alterations in motility and sperm viability after in vitro treatment, was tested using human semen samples. At a gossypol acetic acid concentration (and a 30 minute incubation) of .01 mcg, the percent motility was 71+ or -2.5 (P .01); at .1 mcg concentration, percent motility was 69+ or -4 (P .01); at 1 mcg motility was 66+ or -5 (P .005); at 10 cg, motility was 38.5+ or -3.5 (P .001). Control values for sperm motility were 77+ or -3. These investigations suggested that treatment with gossypol depletes production of adenosine triphosphate in the sperms and thus their metabolism does not proceed normally, rendering them immotile. It appears that the high vulnerability of the testis to gossypol and the uncoupling of oxidative phosphorylation in the respiratory chain of mitochondria of germ cells may be the mechanism responsible for infertility induction.     

Antifertility and ultrastructural effects of optical isomers of gossypol administered intratesticularly in rats

This study reports on the effects of optical isomers of gossypol administered intratesticularly on testicular ultrastructure and fertility in rats. Gossypool isomers were administered to adult male rats by daily intratesticular injections for up to two days at a dose of 1200 ugm/testis. The results of this study have demonstrated that the intratesticular injection of racemic and (-) gossypol at a dose of 1200 ugm for 1 or 2 days mimic the effect of gossypol on rat testis after oral administration of 20 to 30 mg dose for 5-7 weeks; (-)-gossypol was found to be 81% effective in inducing mitochondrial sheath damage in the late spermatids of stage VI, VII and VIII tubules compared to 67% for racemic and 7% for (+)-gossypol, the intratesticular injection of (+/-) and (-)-gossypol (1200 ugm/testis, daily for 1 or 2 days) induces complete infertility three weeks after the last injection.     

Antiproliferative effect of gossypol and its optical isomers on human reproductive cancer cell lines

The antiproliferative effect of gossypol and its optical isomers on various human cell lines of reproductive and nonreproductive tissue origin was studied. Various reproductive cancer cell lines of ovarian, gestational, and testicular origin were highly sensitive (IC50 values of 0.86-1.98) to gossypol. The antiproliferative action of gossypol was not restricted to reproductive cancers, as non-reproductive cancer cell lines were also equally sensitive (IC50 values of 0.69-3.55). In addition, actively proliferating untransformed cells such as fibroblasts and PHA-activated lymphocytes were also sensitive (IC50 values of 0.87-2.51). (-)-Gossypol was 3.6-12.4 times more potent than (+)-gossypol and 1.48-2.65 times more potent than (+/-)-gossypol. The most sensitive indicator of gossypol action was a decrease in DNA synthesis followed by inhibition of protein synthesis and uptake of rhodamine-123 by mitochondria as tested in an ovarian cancer cell line (OVCA 433) and a fibroblast line (Hs27). These results indicate that gossypol possesses a general nonselective antiproliferative action toward human cells in vitro. Further, the pharmacologic activity of gossypol as an antiproliferative agent is primarily attributable to its (-) isomer, which is also the active isomer as a contraceptive.     

Spermatogenic events in rat and mice following intratesticular administration of optical isomers of gossypol

Male rats and mice were administered racemic and dextrorotatory gossypol intratesticularly at the dose of 200 micrograms/testis. In a separate experiment, 100 micrograms of dextrorotatory and levorotatory gossypol was administered to male mice. Animals were sacrificed 72 hours after drug treatment. In another experiment, mice were sacrificed 3, 10, 20, 30 and 60 days after racemic gossypol (200 micrograms/testis) administration. Marked degenerative changes in rat and mice tests were observed in the animals receiving racemic gossypol. Dextrorotatory gossypol had less pronounced effect, both in rat and mice. Stage 7 and 16 spermatids were most susceptible to the deleterious effects of racemic gossypol. In mice, 57.52% and 85.40% decrease in stage 7 and 16 spermatids were observed 72 hrs after drug administration. A progressive recovery was observed after drug treatment; the damage (stage 7 & 16) was reduced to 7.92% and 21.30% after 60 days. Histopathological changes in mice testis following 100 micrograms of levorotatory gossypol were distinctly different from those of the dextrorotatory (100 micrograms/testis) gossypol. On the basis of observations made by us, it can be suggested that mice equally respond to the antifertility effect of gossypol following intratesticular administration. The dextrorotatory gossypol, both in rat and mice, had less pronounced effect on the histoarchitecture of the testis in comparison to racemic and levorotatory gossypol. Our observations further suggest that this animal model provides meaningful information on the mechanism of action of gossypol, and recovery of spermatogenesis is possible after termination of drug treatment.     

Variations of gossypol susceptibility in rat spermatozoa during spermatogenesis

Sperm collected at two epididymal regions from various rat groups force-fed with gossypol were assayed for their ATP levels and motilities to examine their response to the antifertility effect of gossypol, a yellowish polyphenolic pigment from cottonseed oil. Rats receiving gossypol administration for 2 weeks began to show a proportional decrease in spermatozoal ATP content and motility and this antifertility effect deepened with time. Recovery from the gossypol-induced ATP decrease also developed at 2 weeks after the removal of gossypol administration. This rapid development of both the inhibitory and the recovery effects in a normal 53-day spermatogenic process might have stemmed from a change of susceptibility to gossypol inhibition during spermatogenesis. A model showing a more susceptible middle stage in spermatogenesis is proposed.     

Effect of gossypol on immature male rats

In immature male rats the body growth rate, the testis and epididymis weight were not affected by gossypol treatment (4, 8 and 40 mg/kg body weight/24 hrs. for 30 days). There was however a marked reduction in the weight of the prostate after high dose gossypol administration. Furthermore gossypol treatment did not show any effect on the histoarchitecture of the testis nor did the drug treatment have any effect on sperm motility. The plasma levels of testosterone, LH and FSH in gossypol treated animals were no different from those on the controls.     

Efficacy and safety of papaverine as an in vitro motility enhancer on human spermatozoa

Purpose

The aim of this study was to examine the ability and safety of papaverine supplementation for in vitro sperm motility enhancement. In addition, sperm motility enhancement of papaverine was compared to pentoxifylline and theophylline. The post-thaw spermatozoa were used as an asthenozoospermia model.

Methods

Post thaw sperm suspensions were divided into two groups: papaverine (100 μmol/L) and control, and each was investigated in two subgroups of 30- and 60-min exposure times. Detailed motility parameters were detected using a computerized sperm motility analyzer. Acrosomal status, viability, apoptosis, and DNA fragmentation were evaluated by flow cytometry. Furthermore, the motility-enhancing capacity of papaverine, pentoxifylline, and theophylline was compared.

Results

Cryopreservation impaired sperm parameters dramatically but no significant changes occurred in acrosomal status and apoptosis. Supplementation of papaverine enhanced motility parameters consistently at all exposure intervals, significantly. However, viability was lower at the 60th minute compared to the 30th minute (p=0.019). Papaverine did not alter any acrosomal or apoptotic markers at any time points. All of the compounds compared in this study increased the motility parameters, where theophylline supplementation provided significantly better improvement in total motility compared to papaverine and pentoxifylline.

Conclusion

Our results suggest that in vitro papaverine treatment for 30 min adequately improves motility of post-thaw sperm, without leading to acrosome reaction, DNA damage, and viability loss. Theophylline’s potency on increasing the ratio of total motile spermatozoa was found significantly superior than the two tested compounds. Prospective clinical studies with embryo production, pregnancy, and live birth data should be undertaken.

     

Quantified ultrastructural study of spermatozoa in unexplained failure of in vitro fertilization

Failure of in vitro fertilization or very low cleavage rates may occur even though oocyte and semen parameters seem satisfactory. Quantified ultrastructural study of spermatozoa was performed in such cases of failure (n =6) or low cleavage rate (<20%; n =4). Through 1 to 11 retrievals, the number of inseminated oocytes ranged from 14 to 145. The results were compared to those of six fertile men. Quantification was achieved by cataloguing cell defects of the spermatozoon heads and mid-/principal pieces of the flagella. Using the data from each specimen, the percentages of total cellular abnormalities in the head/mid-/principal pieces were established. At the level of the head overall percentages for six groups of defects were determined. The overall percentage of combined head abnormalities, defined as the presence of at least three of these six defects on the same spermatozoon head, was established. Statistical differences among control and patient groups were analyzed by nonparametric Mann—Whitney Utest. The percentages of anomalies of the midpiece and of the principal piece were not significantly different between patients and controls. Motility assessed by spermogram was considered functionally uncompromised. In eight patients the percentage of cell alterations of the head (93–100 vs 77.3 ± 6.4%) and the percentage of combined anomalies of the head (78.1–100 vs 60.8 ± 8.5%) were significantly different between patients and controls. In two cases, the percentages established for all head parameters considered were not globally different from those observed in controls. Thus in 8 cases of 10, electron microscopy with quantified analysis supplied valuable evidence about the poor quality of these sperm samples judged as normal under light microscopy and may provide an explanation for their impaired fertilizability. In the two other cases the fact that the sperm appeared to be ultrastructurally normal does not rule out functional sperm pathology. Alternatively, defects in the oocyte may also account for unexplained failures of in vitro fertilization.     

Albumin and the acrosome change in mammalian spermatozoa

Samples of follicular fluid were collected from ovaries of women, rabbits, cats, pigs, and cows, and examined for their effects on the acrosome of rabbits' spermatozoa. The occurrence of the acrosome reaction was assessed by staining. All follicular fluids were effective in inducing the reaction. Those taken from humans, cow, or pig follicles were more effective than those from cats or rabbits. The factor in follicular fluid was a protein of large molecular weight, and its properties were dependent on the presence of calcium ions. The reactive protein in bovine follicular fluid evoked the acrosome reaction in hamster spermatozoa. The protein may have a role in vivo although other studies in vitro have indicated that it can be replaced by the use of other compounds in culture media used for capacitation and fertilization.     

Effect of fructose on motility, acrosome reaction and in vitro fertilization capability of boar spermatozoa

Aim:  The present study was carried out to investigate the effects of fructose supplementation in glucose containing mTALP medium on motility, acrosome reaction and in vitro fertilization capability of boar spermatozoa.
Methods:  Boar spermatozoa were preincubated, swum-up, resuspended and then incubated for 6 h in mTALP medium supplemented with 0, 0.5, or 1.0 mmol fructose in the presence of 5.0 mmol glucose. After completion of the specified incubation period, motility was determined subjectively on the basis of speed of progression and on the type of forward movement of spermatozoa; acrosome status was evaluated by applying a triple staining technique; and in vitro fertilization capability was assessed by acetic–orcein staining.
Results:  The combination of fructose and glucose (0.5 + 5.0 mmol) supplements in mTALP medium improved sperm motility significantly ( P <  0.05), more than glucose alone (5.0 mmol) at 2–6 h of incubation. Acrosome reaction (live spermatozoa) and the sperm penetration rate was increased significantly ( P <  0.05) when the spermatozoa were treated with the combination of fructose and glucose compared with glucose alone, but the incidence of polyspermy was not significantly different between the treatments.
Conclusion:  These results suggest that the combination of glucose and fructose as supplements in mTALP medium improve the progressive motility, acrosome reaction and fertilization capability of boar spermatozoa. (Reprod Med Biol 2006; 5: 255–261)     

Effect of gossypol on domestic fowl, Gallus domesticus

Domestic fowls (Gallus domesticus) were administered gossypol (10 mg/kg body weight/day) by oral intubation for 15 weeks. Drug treatment did not have any effect on body growth rate. The drug treatment, however, caused a marked decrease in the weights of testis and epididymis. Predominant changes in the histoarchitecture of testis (desquamation of germinal epithelium and inhibition of spermatogenesis) were observed following gossypol treatment. Epididymal tubules in gossypol treated animals were devoid of spermatozoa. Gossypol treatment had no effect on hematological parameters (total erythrocyte count, total leucocyte count, hematocrit and hemoglobin) studied in the present investigations.     

Effect of relaxin on human spermatozoa

To study its effect on the motility of human spermatozoa, relaxin was added at different concentrations to human semen samples of various qualities as well as to washed spermatozoa. Relaxin in physiologic concentrations (10-100 ng/mL) had no significant effect on sperm motility in normal semen samples. However, the addition of relaxin to semen samples with low sperm motility significantly increased the motility. Addition of relaxin similarly increased the motility of spermatozoa from normal semen samples that were either aged or washed; the treatment resulted in a decrease in motility. When sperm motility was optimal, as in normal samples, addition of relaxin did not increase motility. However, in some situations of decreased motility, addition of relaxin resulted in improvement of spermatozoan motility. Relaxin may have clinical value in the treatment of male infertility.     

Effect of gossypol on the fertility of male rats.

Thirty male rats were grouped into 5 groups of 6 animals each. Animals in groups II-V were given gossypol at a dose of 5 mg/kg, 10 mg/kg, 20 mg/kg and 40 mg/kg body weight per day for 45 days respectively. Animals of group I served as control. A significant decrease in body weight after administration of 40 mg/kg body weight of gossypol was observed; low doses of gossypol, however did not affect the body weight. Testis, epididymis, prostate and seminal vesicles weights decreased gradually with the increasing doses of gossypol. With the increasing doses of gossypol, a marked decrease in the vas deferens sperm motility was observed. At 40 mg/kg dose there was a total inhibition of sperm motility. Histological studies after 5 mg/kg revealed no apparent sign of degeneration, while after 10 mg/kg dose the changes in the individual cell types were accompanied by overall disorganisation of the germinal epithelium involving displacement of the spermatocytes. The rats treated with 20-40 mg/kg gossypol showed a pronounced deleterious effect on the histological structure of the testis. The drug effect was dose dependent developing sequentially; from the uppermost layer of elongated spermatids affecting round spermatids and finally spermatocytes. Quantitatively the ratios of pachytene spermatocytes: resting spermatocytes, stage 7 spermatids: pachytene spermatocytes, and stage 19 spermatids: stage 7 spermatids and tubular diameter and germinal height decreased significantly. The activities of glucose-6-phosphatase, fructose 1, 6-diphosphatase, glucose-6-phosphate isomerase in testis decreased significantly at high dose (40 mg/kg), while the activity of amylase and glycogen content increased significantly with the increasing doses of gossypol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phage infection in vaginal lactobacilli: an in vitro study

Objective: During bacterial vaginosis, an unexplained decrease of vaginal lactobacilli occurs. To identify whether these lactobacilli could be infected by phages, we isolated phages from vaginal lactobacilli and analyzed their potential virulence in attacking vaginal lactobacilli in vitro.Methods: Vaginal samples were obtained from 39 reproductive-aged women. The selective Rogosa SL agar was used to isolate lactobacilli, from which phages induced by mitomycin C or released spontaneoulsy were analyzed by the agar spot method.Results: Of 20 samples from women with vaginal infections, 12 did not have lactobacilli. From the remaining 8 infection samples and the 19 samples from healthy women, 37 Lactobacillus strains were isolated, from which 7 temperate phages were identified. Upon analysis, all 7 phages infected vaginal lactobacilli from the same and/or different women in vitro. Two phages, Phikc005 and Phikc007, had a broad host range, infecting 7 of 8 species tested. A control intestinal Lactobacillus phage also lysed several vaginal strains. One vaginal phage, Phikc039, was apparently lytic against vaginal lactobacilli from 7 other women. This phage was characterized as follows: plaque morphology, small and clear; burst size, 300 phages per cell; spontaneous induction rate, 1 per 10(6) cells; DNA, double-stranded and linear, 41 kb; and shape, a hexogonal head and a non-contractile tail.Conclusions: Bacteriophages were isolated from vaginal lactobacilli of some women and were shown in vitro to lyse vaginal Lactobacillus strains from the same and/or different women. It was suggested that vaginal lactobacilli might be suppressed by phages.     

Investigation of mammalian ovulation with an in vitro perfused rabbit ovary preparation.

An in vitro perfused rabbit ovary preparation has been developed to study local factors responsible for the ovulatory process. This preparation enables: (1) isolation of the ovary from systemic influences, (2) direct serial observations of follicle development and ovulation, (3) addition to the perfusing medium of substances that may influence ovulation acting at the level of the ovary, and (4) correlation of the times of individual follicle rupture with ovarian contractile patterns. With this model the temporal requirements for human chorionic gonadotropin in the process of ovulation have been studied. The direct effects on ovulation of prostaglandin F3 alpha (PGF 2 alpha), the antihistamine chlorpheniramine, and calcium deprivation were also investigated. Chlorpheniramine and EDTA each led to inhibition of ovulation and depressed ovarian contractility. The use of the isolated in vitro ovary preparation permits characterization of the local requirements for ovulation.     

Effect of pentoxifylline on immotile testicular spermatozoa

Purpose: A model for differentiating live and dead sperm cells during intracytoplasmic sperm injection (ICSI) is proposed. Methods: We used pentoxifylline, a phosphodiesterase inhibitor known to enhance sperm motility, to initiate motility in testicular spermatozoa. Ten immotile testicular sperm samples were divided into two parts for examination of sperm motility with and without pentoxifylline treatment at 30, 60, and 90 min. Results: The samples without pentoxifylline remained immotile even after 90 min of incubation: the addition of pentoxifylline initiated sperm motility in all samples: 51.8±10.2, 64.4±9.4, and 70.8±8.9% (mean±SD) at 30, 60, and 90 min, respectively. Conclusions: That pentoxifylline may be used to differentiate live testicular spermatozoa during ICSI, which may improve fertilization and pregnancy rates, is suggested.