Sperm impairing microbial factor: potential candidate for male contraception.

BACKGROUND: Despite significant advances in contraceptive options for women, vasectomy and condoms are the only options available for male contraception. Due to this limitation, the burden of contraception resides on the shoulders of females only. Therefore, there is an urgent need to develop a safe, effective and reversible method of contraception for men. Amongst the alternative approaches, microbial derived products are gaining attention of the scientific world to combat unintended pregnancies. Earlier in our laboratory, sperm impairing microbial factor (Sperm immobilization factor) isolated from Staphylococcus aureus has shown excellent contraceptive efficacy in female mice. Keeping this in mind, the present study was carried out to exploit the sperm immobilization factor (SIF) as potential male contraceptive using vas deferens for administration in mouse model. METHODS: SIF (10, 50, 100 or 200 µg) was inoculated in the lumen of right vas deferens whereas the left vas deferens served as control. The mice were sacrificed at Day 3, 7, 14, 21, 30, 45, 60 and 90 after inoculation and the results in terms of change in body weight, seminal parameters, Tissue somatic indices (TSI), haematological parameters, serum level of testosterone, lipid peroxidation and histology were studied. In order to ratify the SIF induced azoospermia SIF (200 µg) was administered with different doses viz. 100, 200, 300, 400 or 500 µg of SIF binding receptor extracted from mouse spermatozoa. RESULTS: The weight profile studies of all the experimental groups showed no significant change in the initial and final body weight. In case of seminal parameters, the results revealed that right vas deferens treated with SIF showed azoospermia and with 200 µg of SIF it persisted up to 90 days. TSI of reproductive organs and non-reproductive organs showed no significant change in all the experimental groups. The haematological indices were found to be unaltered throughout the course of investigation however significant decrease in testosterone level was observed in the treated mice. The treatment also affected the oxidative status of the testis. Further, histological studies revealed hypospermatogenesis and late maturation arrest on treated side whereas the left side which served as control showed normal tissue histology. SIF induced azoospermia was ameliorated when administered with 400 µg of SIF binding receptor from mouse spermatozoa. CONCLUSION: SIF, when administered via intra vas deferens route, could lead to complete azoospermia. Therefore, it could be considered as a potential male contraceptive.

Escherichia coli recombinant sperm immobilizing factor RecX as a potential vaginal contraceptive.

BACKGROUND: To control the overpopulation and unintended pregnancies, vaginal contraceptives have gained recent surge of interest because of its topical application with possible avoidance of systemic effects. However non-specific cytotoxicity associated with detergent-based synthetic vaginal contraceptive agents limits their use and generates considerable interest in the development of vaginal contraceptives of biological origin for controlling reproduction and ultimately growing population. In this study, we have cloned, over-expressed an Escherichia coli gene encoding a sperm immobilizing factor (SIF) that inhibits sperm motility for the development of vaginal contraceptive from a biological source i.e. E. coli. The contraceptive efficacy of the Escherichia coli recombinant sperm immobilizing factor (r-SIF) was also determined. METHODS: Genomic DNA library of an E. coli strain isolated from semen sample of an infertile male was constructed for the identification and cloning of E. coli SIF coding gene. This gene was sub-cloned in pBADmycHisB for over-expression and the r-SIF was purified using Ni-NTA affinity chromatography. Effect of r-SIF on mouse sperm motility, viability and on morphology was evaluated. Binding of r-SIF to mouse sperm was demonstrated by fluorescent labeling. Contraceptive efficacy of r-SIF was checked in murine model. RESULTS: Genomic library resulted in five hundred transformants; five clones were found positive for sperm immobilizing activity. The protein product of the insert DNA sequence in one of the transformants showed maximum sperm immobilizing activity. Sequence analysis of ORFs in the insert revealed homology to recX on both nucleotide and protein level. 40 µg of the purified r-SIF showed immediate spermicidal activity in vitro for mouse sperm. Scanning electron micrograph of the r-SIF treated sperm showed intense morphological damage to sperm. FITC labeled r-SIF showed highest fluorescence at the head region of the sperm. 5 µg of purified r-SIF exhibited a complete contraceptive effect in mouse model. CONCLUSION: r-SIF could be seen as potential target to be developed as potent and safe vaginal contraceptive in future.

Isolation of a spermatozoal immobilization factor from Staphylococcus aureus filtrates.

Staphylococcus aureus isolated from the cervix of an infertile woman was found to cause complete immobilization of human spermatozoa in vitro. Only the cell culture and cell-free supernatant showed immobilization activity, indicating that the sperm immobilization factor might be released extracellularly by the organism because no activity was observed with the washed cells. Heat treatment of the supernatant at 60 degrees C for 10 min waived its immobilizing activity, indicating that the active component may be a protein. The bioactive molecule from the supernatant was purified to homogeneity by ammonium sulfate precipitation, gel permeation chromatography, and ion exchange chromatography. Sperm immobilization factor (SIF) was found to be an approximately 20 kDa protein. SIF at a concentration of 10 microg/mL was required to cause 100% immobilization of human spermatozoa after 30 min of incubation at 37 degrees C, whereas a concentration of 150 microg/mL caused immediate immobilization, and a concentration of 200 microg/mL resulted in instant loss of viability of human spermatozoa, observed by eosin-nigrosin staining. Scanning electron microscopy showed that the treatment of human spermatozoa with SIF caused multiple defects in the head, midpiece, neck, and tail region of human spermatozoa.

Immobilization effect of Ruta graveolens L. on human sperm: a new hope for male contraception.

AIM OF THE STUDY: Contraceptive plants which were introduced by folk in traditional remedies are investigated worldwide. In this study, the contraceptive effects of Ruta graveolens L., which has been mentioned for male contraceptive in Iranian traditional folk medicine, was experimented on human sperm. MATERIALS AND METHODS: Different doses of lyophilized aqueous extract of Ruta graveolens L. were added to an amount of fresh semen, containing 10(6) cells in a 1:1 volumic ratio. Motility and viability of cells, DNA status, mitochondrial activity and sperm revival tests were carried out. RESULTS: The sperm immobilization effects of the extract appeared immediately in a does-dependent manner and 100% of the sperms became immotile at a concentration of 100mg/ml but other parameters were intact. After washing the sperms, motility was returned in 30.8+/-3.2% of the sperms, besides coiled tails in 38.6+/-5.5% of the treated cells, in comparison to 12.5+/-2.0% of the control group (p=0.001). The part of the extract, responsible for immobilization of the sperms was stable upon boiling. CONCLUSIONS: As the cells were alive and immotile, probably some ionic currents are blocked by a thermostable component of the plant which can be promising as a new male channel blocker contraceptive.

The zoapatle. IX. In vitro effect of Montanoa tomentosa and Montanoa frutescens upon human sperm and red cells.

The effect of zoapatle aqueous crude extract (ZACE) was further studied and partially characterized upon human and rabbit spermatozoa. ZACE prepared from Montanoa tomentosa s.s.p tomentosa did not influence sperm motility or viability in a wide range of ZACE concentrations tested; on the other hand, ZACE prepared from Montanoa frutescens had immediate and constant inhibitory effect upon motility and decreased cell viability. Red cell lysis was readily observed with ZACE-frutescens, but not with ZACE-tomentosa. The effect of time on ZACE-frutescens potency for inducing red cell lysis was observed.

A human seminal plasma protein blocks the motility of human spermatozoa.

An inhibitor of the motility of demembranated spermatozoa has been shown to be present in human seminal plasma. This seminal plasma motility inhibitor (SPMI) was purified to apparent homogeneity and tested on intact human spermatozoa. Motility parameters of spermatozoa incubated with the sperm motility inhibitor were evaluated with the video automated Cell Soft system. SPMI decreased the percentage of motile spermatozoa in a dose-dependent manner and motility was completely blocked in the presence of 1600 units/ml. Sperm velocity and beat/cross frequency showed a similar progressive decrease as the inhibitor was augmented. However, linearity was essentially not affected. The effects of SPMI on the percentage of motile spermatozoa increased with the time of contact between the inhibitor and spermatozoa. After 120 min., the IC50 was 35% lower than that observed at five min. The presence of seminal plasma did not prevent the inhibitory effects of the seminal plasma factor on sperm motility parameters. On the contrary, a potentiating effects was observed. The data suggest that the SPMI could play a significant role in cases of infertility caused by asthenospermia.

Immunoglobulins from human follicular fluid induce the acrosome reaction in human sperm.

The ability of human follicular fluid (hFF), retrieved from women undergoing IVF to induce the acrosome reaction (AR) in human sperm has been documented by several laboratories. However, the nature of the active factors in the hFF and the physiological meaning of the AR induction are highly controversial. We performed a three step purification scheme for hFF and all the fractions were screened for the AR-inducing activity. AR activity was associated with a protein fraction of M(r) > 180 kD that on further analysis under PAGE was found to be composed by subunits of apparent M(r) 50,000 and 29,000. The N-terminal sequences of these bands showed a 100% homology with the heavy and light chains of human IgG. A polyclonal antibody raised against the purified protein and anti-human IgG were both able to suppress the acrosome reaction-inducing activity of crude hFF. However, neither normal human serum nor a purified preparation of human IgG were able to mimic the AR-inducing activity of hFF. We concluded that the AR-inducing activity of hFF is, at least in part, due to the presence of antisperm antibodies.

Nature's motility blockers: controlling human sperm motility machinery from the outside. Chemical characterization of a peritoneal fluid lipid that induces sperm immobilization.

A molecule isolated from the peritoneal fluids of women undergoing laparoscopy for in-vitro fertilization techniques has been chemically characterized and identified as 1-palmitic-3-phosphorylcholine (lysophosphatidylcholine, LPC). This lipid is able, at physiological concentrations, to completely inhibit sperm motility in vitro in a dose-dependent way. Synthetic LPC induced rapid and complete arrest of sperm motility when added to sperm suspensions at physiological concentrations without any damage to cell membranes. Taken together, these results suggest that LPC may represent a previously unrecognized in-vivo modulator of human sperm motility.