Evaluation of the Spermicidal and Contraceptive Activity of Platycodin D,a Saponin from Platycodon grandiflorum

Background

The extract of Platycodon grandiflorum has been reported to have effective spermicidal activity. This study was designed to evaluate the spermicidal and contraceptive activity, as well as the safety, of Platycodin D (PD), a major saponin in Platycodon grandiflorum.

Methods

Using the computer-aided sperm analysis (CASA) test criteria, the sperm-immobilizing activity of PD was studied using highly motile human sperm. The sperm viability was assessed by fluorescent staining using SYBR-14 (living sperm) and propidium iodide (dead sperm). The sperm membrane integrity was assessed by evaluating the hypo-osmotic swelling (HOS) and examinations by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The in vivo contraceptive efficacy was evaluated in rats using post-intrauterine PD application. The comet assay was employed to determine whether PD caused DNA damage in the sperm. Vaginal biopsies were also performed to determine whether the PD gel induced vaginal inflammation.

Results

A dose-dependent effect of PD on the sperm motility and viability was observed. The maximum spermicidal effect was observed with a 0.25 mM concentration of PD. More than 70% of the PD-treated sperm lost their HOS responsiveness at a concentration of 0.20 mM PD, indicating that PD caused injury to the sperm plasma membrane. TEM and SEM revealed significant damage to both the head and tail membranes of the sperm. PD decreased the fertility to zero in rats, was non-DNA damaging and was not harmful to the vaginal tissue in the rats.

Conclusion

PD has significant spermicidal activity that should be explored in further studies.     

Effets in vitro des dermaseptines sur les caractéristiques du sperme

The spermicidal efficacy of synthetic peptides, dermaseptin (DS1) and (DS4), was studied under in vitro conditions using human spermatozoa. The data showed that sperm motility was inhibited with various concentrations of dermaseptins at different intervals ranging from 2 to 240 min. The effective 100% inhibitory concentration (EC100) of DS4 sperm immobilization assay was equal to 50 mg/ml at 30 min, while the EC 100 of DS1 was equal to 100 mg/ml. The presence of 0.1% of chelating agent, EDTA, reduced the EC100 of DS4 to 5 mg/ml, while less than a twofold enhancement in DS 1 activity was observed in combination with EDTA. The action of dermaseptins on sperm motility was observed to be dose-dependent. Addition of pentoxifylline, which is known to enhance sperm motility, and Ca²⁺, which is a key element for sperm movement, did not prevent the spermicidal action of dermaseptins.     

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.     

Spermicidal and Contraceptive Potential of Desgalactotigonin: A Prospective Alternative of Nonoxynol-9

Crude decoction of Chenopodium album seed showed spermicidal effect at MIC 2 mg/ml in earlier studies. Systematic isolation, characterization and evaluation revealed that the major metabolite Desgalactotigonin (DGT) is the most effective principle in both in vitro and in vivo studies. The in vitro studies comprises (a) rat and human sperm motility and immobilizing activity by Sander-Cramer assay; (b) sperm membrane integrity was observed by HOS test and electron microscopy; (c) microbial potential was examined in Lactobacillus broth culture, and (d) the hemolytic index was determined by using rat RBCs. The in vivo contraceptive efficacy was evaluated by intra uterine application of DGT in rat. Lipid peroxidation and induction of apoptosis by DGT on human spermatozoa were also studied. The minimum effective concentration (MEC) of DGT that induced instantaneous immobilization in vitro was 24.18 µM for rat and 58.03 µM for human spermatozoa. Microbial study indicated DGT to be friendly to Lactobacillus acidophilus. Implantation was prevented in DGT treated uterine horn while no hindrance occurred in the untreated contra lateral side. At the level of EC₅₀, DGT induced apoptosis in human spermatozoa as determined by increased labeling with Annexin-V and decreased polarization of sperm mitochondria. Desgalactotigonin emerged 80 and 2×10⁴ times more potent than the decoction and Nonoxynol-9 respectively. It possesses mechanism based detrimental action on both human and rat spermatozoa and spares lactobacilli and HeLa cells at MEC which proves its potential as a superior ingredient for the formulation of a contraceptive safer/compatible to vaginal microflora.     

Structural and functional characterization of seven spermicidal vanadium(IV) complexes: potentiation of activity by methyl substitution on the cyclopentadienyl rings.

In a systematic effort to identify and develop effective vanadocene(IV) complexes as a new class of contraceptive agents, the effect of methyl substitution in the cyclopentadienyl rings of Cpx2VCl2 on their spermicidal activity has been examined. The spermicidal activities of compounds Cpx2VCl2 [Cpx = Me5Cp (Cp*) (1), Cp (3), MeCp (Cp') (5)], as well as two of their corresponding vanadium(V) oxidation products Cp*V(O)Cl2 (2) and CpV(O)Cl2 (4), were examined by computer-assisted sperm analysis (CASA). These analyses have established that penta-substitution of the Cp ring by electron-donating methyl groups augments the spermicidal activity 10-fold. The corresponding V(V) oxo compounds, Cp*V(O)Cl2 (2) and CpV(O)Cl2 (4), tested under identical conditions did not show as effective spermicidal activity even though these complexes have a pseudo-tetrahedral geometry similar to the active vanadocene(IV) dichlorides. Two pseudo-octahedral V(IV) complexes with tris-pyrazolyl borate as ligand, (HBpz3)V(O)Cl.DMF (6) and (HB(3,5-Me2pz)3)V(O)Cl.DMF (7), were also found to exhibit potent spermicidal activity. Although some vanadium(IV) complexes may immobilize sperm due to the generation of .OH radicals, the lack of spermicidal activity of VOSO4 which generates .OH radicals, and the potent spermicidal activity of [Cp2V(acac)][O3SCF3] (8), and [Cp2V(DeDtc)][O3SCF3] (9) which do not generate .OH radicals, indicate that .OH radical mediated reactions may not be essential for the spermicidal activity of vanadium(IV) complexes.     

Effects of methyl-beta-cyclodextrin-mediated cholesterol depletion in porcine sperm compared to somatic cells

In this study, the use of methyl-beta-cyclodextrin (MBCD) to support capacitation of sperm cells was studied. Sperm were incubated with MBCD or alternatively capacitated in an in vitro fertilization medium. The effects of these incubations on phospholipid scrambling (using merocyanin), cholesterol depletion, GM-1 localization (using cholera-toxin B (CTX)), and membrane deterioration were assessed. For comparison, this was also tested in MBCD-treated MDCK cells. In MDCK cells, upto 71% of cholesterol was depleted, which coincided with a more diffuse CTX staining without any obvious effects on cell viability. In sperm, a similar depletion of 53% cholesterol was found after a 10 mM MBCD treatment. However, no merocyanin response was observed in viable sperm after MBCD treatments (indicating a lack of membrane changes associated with sperm capacitation). In contrast to MDCK, cells >1 mM MBCD caused plasma membrane disintegration and rendered sperm immotile. At higher concentrations also acrosome disruption was noted. CTX staining was absent at < 0.1 mM MBCD incubations but appeared at higher MBCD levels and was found to be specific for deteriorated cells that showed morphological signs of acrosome disruption. No significant plasma membrane deterioration, acrosome disruption, and sperm immotility nor CTX staining and only a modest (< 15%) cholesterol depletion were observed in conventionally capacitated sperm, where 40% of the intact sperm showed merocyanin staining. Taken together, the results indicate that membranes of sperm are more sensitive to MBCD-mediated cholesterol depletion than MDCK cells and that the use of MBCD to support sperm capacitation cannot be recommended due to its spermicidal effects.     

Initial evaluation of fertilin as an immunocontraceptive antigen and molecular cloning of the cynomolgus monkey fertilin β subunit

Fertilin (PH-30) is a sperm surface protein that functions in sperm adhesion and fusion with the egg plasma membrane. Because of its essential function in fertilization, fertilin is a potential target for novel contraceptive approaches. In a pilot fertility trial, immunization of male guinea pigs with purified guinea pig fertilin resulted in complete infertility. The contraceptive effect was partial (two out of six animals were infertile) when female guinea pigs were immunized with the antigen. These results suggest that fertilin or domains of fertilin may be effective as immunocontraceptive antigens. As a step toward achieving this goal, we communicate the cDNA and deduced amino acid sequence of the monkey fertilin β subunit. © 1996 Wiley Liss, Inc.     

Potentiation of spermicidal activity of 2',4'-dichlorobenzamil by lidocaine

The present investigation was designed to study the spermicidal activity of lidocaine, a membrane stabilizer, and its combination with 2',4'-dichlorobenzamil hydrochloride, a Na+-Ca2+ exchange inhibitor, on human semen and spermatozoa separated from semen. Both drugs per se produced dose- and time-dependent reduction in motility of ejaculated human sperm. Lidocaine was found to potentiate the spermicidal activity of benzamil resulting in significant decrease in time for producing complete loss of ejaculated sperm motility. Sperm revival test revealed irreversible loss of sperm viability indicating a spermicidal rather than spermiostatic action by both the drugs. Furthermore, both benzamil (10-40 mM) per se and benzamil-lidocaine combination (0.5 and 16 mM) produced contraception in rabbit model.     

In vitro spermicidal activity of peptides from amphibian skin: dermaseptin S4 and derivatives

Sexually transmitted infections and unplanned pregnancies present a great risk to the reproductive health of women. Therefore, female-controlled vaginal products directed toward disease prevention and contraception are needed urgently. In the present study, efforts were made to evaluate the contraceptive potency of dermaseptin DS4, an antimicrobial peptide derived from frog skin. To assess the structure-activity relationship between the native DS4 and its derivatives, a set of chemically modified peptides was synthesized and evaluated. Normal human semen samples were used to detect the spermicidal activity of the new compounds. HeLa cultures were used to determine the safety of compounds toward their toxicity. Fluorescent-binding assays were performed to evaluate the rapidity and the irreversibility of the sperm-immobilizing activity of peptides. All DS4 derivatives elicited concentration-dependent spermicidal activity at microgram concentrations (EC(100) values: 25 microg/ml-l mg/ml). The order was K4S4=S4a>S4>K4S4(1-16)a>S4(6-28). In cytotoxicity assay, some compounds were found to be significantly safer than nonoxynol-9, the most widely used spermicide, and their activity was not accompanied by total loss of plasma membrane integrity as detected by fluorescent microscopy. Our data also show that increasing the number of positive charges of the peptide resulted in a reduced cytotoxicity without affecting the spermicidal effect. This study indicates that dermaseptins are spermicidal molecules that deserve to be tested as topical contraceptive with useful activities that can add to their prophylaxis, safety, and effectiveness.     

The spermicidal and antitrichomonas activities of SSRI antidepressants

The study investigated spermicidal and antitrichomonas activities of selective serotonin reuptake inhibitor (SSRI) antidepressants with a view to generate new lead for development of dual-function spermicidal microbicides, which is an urgent global need. Fluoxetine, Sertraline, and Fluvoxamine exhibited both spermicidal and anti-STI (antitrichomonas) activities in vitro, whereas Paroxetine and Citalopram showed only the spermicidal activity. Fluoxetine exhibited better activity profile than the other antidepressant drugs with its spermicidal and antitrichomonas activities being comparable to that of the OTC contraceptive Nonoxynol-9. The non-detergent nature of Fluoxetine and a much lower spermicidal ED50 value (than N-9) may add considerably to its merit as a candidate for microbicidal contraceptive. Thus, the antidepressants exhibiting both spermicidal and antitrichomonas activities might provide useful lead for the development of novel, dual-function spermicidal contraceptives.     

Biological effects of Trichoderma harzianum peptaibols on mammalian cells

Trichoderma species isolated from water-damaged buildings were screened for toxicity by using boar sperm cells as indicator cells. The crude methanolic cell extract from Trichoderma harzianum strain ES39 inhibited the boar sperm cell motility at a low exposure concentration (50% effective concentration, 1 to 5 microg [dry weight] ml of extended boar semen(-1)). The same exposure concentration depleted the boar sperm cells of NADH(2). Inspection of the exposed boar sperm cells by transmission electron microscopy revealed damage to the plasma membrane. By using the black lipid membrane technique, it was shown that the semipurified metabolites (eluted from a SepPak C(18) cartridge) of T. harzianum strain ES39 induced voltage-dependent conductivity. The high-performance liquid chromatography-purified metabolites of T. harzianum strain ES39 dissipated the mitochondrial membrane potential (Deltapsi(m)) of human lung epithelial carcinoma cells (cell line A549). The semipurified metabolites (eluted from a SepPak C(18) cartridge) of T. harzianum strain ES39 were analyzed by mass spectrometry (MS). Matrix-assisted laser desorption ionization and nanoflow electrospray ionization MS revealed five major peptaibols, each of which contained 18 residues and had a mass ranging from 1,719 to 1,775 Da. Their partial amino acid sequences were determined by collision-induced dissociation tandem MS.     

Maturing the sperm: unique mechanisms for modifying integral proteins in the sperm plasma membrane

The epididymis has been understudied, in part due to its cancer resistance and the development of effective technologies for sperm injection and in vitro fertilization. However, it is worthy of study because--absent advanced reproductive technology--its proper function is essential for conceiving children: sperm leaving the testis are immature and nonfertile. Epididymal functions can be divided into several general categories (1) concentration of sperm; (2) functional maturation; (3) storage in a quiescent state until ejaculation; (4) removal of degenerating sperm; (5) provision of appropriate conditions for survival; (6) transport by the myoid cells; (7) protection; (8) maintenance of the blood epididymal barrier. In the past decade investigators have focused on those maturational changes of the integral proteins of the sperm plasma membrane which are directly related to sperm-ova interactions. It has traditionally been thought that changes in the sperm plasma membrane proteins were limited to simple binding or removal of proteins or interactions with the proteases, glycosylases and glycotransferases present. However, the epididymis can also release secretory products in bulk through apical blebs and inject integral membrane proteins with epididymosomes which fuse with the plasma membrane. The epididymis also activates and cleaves enzymes present on the sperm surface (e.g., germ cell angiotensin converting enzyme), thus enabling them to modify proteins on the sperm membrane. Aside from the need to understand epididymal function relative to the sperm, basic science on epididymal physiology is warranted because it may help us understand the functioning of androgens, protection of tissues from oxidative damage, and resistance to cancer and benign hyperplasic growth.     

Assessment of spermicides by a stripping technique against human spermatozoa

Fifty-two (52) compounds were tested for spermicidal activity by titration against human spermatozoa. The gradual decrease in mean sperm size was measured against increasing concentration of spermicide and the end-point was taken as the point at which all the peripheral cytoplasm had been removed and only the sperm core of nucleus and tail fibres remained. There were 14 compounds that produced this total effect. All were detergents, of various types, and the effect was purely physical. The most potent compounds caused complete stripping at 0.5-50 pmol/cell and most are already used in spermicidal preparations. A further 11 compounds, including sodium hypochlorite and some phenols, caused partial stripping, while 4 compounds caused sperm swelling. The test was not suitable for assessment of metabolic cell poisons.     

Inhibition of fertilization of the rabbit ova in vitro by the antibody to the inner acrosomal membrane of rabbit spermatozoa

The antibody to the rabbit sperm inner acrosomal membrane, raised in guinea pig, completely inhibited the fertilization of rabbit ova in vitro. The F(ab')2 of the antibody was equally effective in inhibiting fertilization. The antibody appeared to exert its inhibitory effect by binding to the inner acrosomal membrane of acrosome-reacted sperm. The antibody-treated sperm did not attach to or penetrate the zona pellucida. Thus, anti-IAM offers a great potential as a contraceptive agent.     

Fungitoxic activity of some essential oils

Of the five essential oils screened against Helminthosporium oryzae, the oils of Cymbopogon martinii (ginger grass oil), Cymbopogon oliveri, Cymbopogon sp. (rosa sofia oil) and Trachyspermum ammi (dethymolysed oil) exhibited strong fungitoxicity and showed wide range of activity. The oils were found more active than some of the prevalent synthetic fungicides and thus might be exploited as natural fungicides if successful infield trials. Besides, these oils were found toxic to various human pathogens.     

Thermoreversible gel formulation containing sodium lauryl sulfate as a potential contraceptive device

The contraceptive properties of a gel formulation containing sodium lauryl sulfate were investigated in both in vitro and in vivo models. Results showed that sodium lauryl sulfate inhibited, in a concentration-dependent manner, the activity of sheep testicular hyaluronidase. Sodium lauryl sulfate also completely inhibited human sperm motility as evaluated by the 30-sec Sander-Cramer test. The acid-buffering capacity of gel formulations containing sodium lauryl sulfate increased with the molarity of the citrate buffers used for their preparations. Furthermore, experiments in which semen was mixed with undiluted gel formulations in different proportions confirmed their physiologically relevant buffering capacity. Intravaginal application of the gel formulation containing sodium lauryl sulfate to rabbits before their artificial insemination with freshly ejaculated semen completely prevented egg fertilization. The gel formulation containing sodium lauryl sulfate was fully compatible with nonlubricated latex condoms. Taken together, these results suggest that the gel formulation containing sodium lauryl sulfate could represent a potential candidate for use as a topical vaginal spermicidal formulation to provide fertility control in women.     

A novel approach to the generation of antibodies against phylogenetically preserved sperm antigens

Conventional methods for immunization of laboratory animals against human spermatozoa proved not to be efficient enough to identify phylogenetically conserved sperm-specific antigens. A combination of vasectomy and subcutaneous administration of autologous testis homogenates was tested in 5 New-Zealand rabbits, and in 7 Long-Evans and 8 Spraque-Dawley rats in an attempt to induce an autoimmune response against such antigens. This experimental procedure resulted in a generation of sperm autoantibodies cross-reactive with human, rabbit and rat spermatozoa, as demonstrated by sperm-agglutination, ELISA and flow cytometry (FCM). No specific binding to human seminal plasma was detected by ELISA, indicating that intrinsic sperm membrane antigens rather than sperm-coating antigens were involved in establishing cross-reactivity with human spermatozoa. This suggestion was confirmed by the finding that rabbit autoantisera reacted more strongly against epididymal than against ejaculated human spermatozoa as shown by FCM. Humoral antispermatozoal response correlated well with impaired spermatogenesis in rabbits. The autoimmunized rats revealed severe alterations in reproductive tissues, including testicular and epididymal sperm granulomas; however, they showed a lower incidence of circulating antibodies. The results indicate that the established experimental model in rabbits can be further used to identify and characterize evolutionary preserved intrinsic sperm membrane autoantigens, which are desirable candidates for contraceptive vaccine development.     

Molecular modification of Penaeus monodon sperm in female thelycum and its consequent responses

Using Penaeus monodon as the model, we demonstrated the molecular changes and the mechanism of thelycal-dependent sperm modification resulting in an enhanced acrosome reaction (AR) response. Attention was paid to the modification of the sperm plasma membrane which was mediated through an adsorption or removal of sperm peripheral and integral membrane proteins as indicated by the different profiles of these proteins in spermatophore (S) and thelycal (T) sperm. In vitro adsorption of Alexa-488 conjugated T proteins onto the entire S-sperm surface confirmed protein transfer in a time-dependent manner. Specific anchoring of 83 and 140 kDa proteins to sperm peripheral proteins as well as 53/55 and 60 kDa proteins to sperm lipids was demonstrated. Apart from membrane modification, a substantial increase in protein tyrosine phosphorylation was shown to be closely associated with T-dependent sperm modification event. The physiological significance of this sperm modification in enhancing sperm AR response, which required at least 3 days of T residence in order for the sperm to gain a complete AR response, was also elucidated.