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.

Amelioration of sperm immobilisation factor-induced infertility by bacterial antigenic determinants cross-reacting with spermatozoa.

A strain of Staphylococcus aureus, capable of invitro immobilisation of human and mouse spermatozoa, was already present in our laboratory. Therefore, in the present study, the factor responsible (sperm immobilisation factor, SIF) was isolated and purified. It was found to compromise not only motility, but also viability, morphology and Mg2+-ATPase activity of mouse spermatozoa. Also, SIF (250µgmL-1), when administered intravaginally in female BALB/c mice before mating, showed 100% contraceptive effect. Moreover, fluorescein isothiocyanate-labelled SIF was also found to bind mouse spermatozoa and various motile as well as non-motile bacteria, indicating the presence of common SIF-binding receptors on spermatozoa and bacteria. Further, to demonstrate molecular mimicry, the amelioration of SIF-induced impairment of sperm function by a SIF-binding bacterial receptor was compelling. For this, the SIF-binding receptor from Escherichia coli (E-SBR) was purified and evaluated for its ameliorative effect on SIF-induced sperm impairment invitro and invivo. Interestingly, upon the addition of mouse spermatozoa to SIF pre-incubated with E-SBR, an ameliorative effect against SIF-induced impairment of sperm function could be observed through analysis of normal sperm parameters (motility, viability, morphology, Mg2+-dependent ATPase levels). E-SBR also blocked binding of labelled SIF to spermatozoa and bacteria and alleviated SIF-induced infertility in female BALB/c mice. This provided evidence for molecular similarities between bacteria and spermatozoa, owing to which anti-bacterial antibodies cross-reacting with spermatozoa might be produced and infertility might follow.

Receptor from Streptococcus pyogenes as a potential antidote against sperm immobilization factor-induced sperm impairment and infertility.

The present study was undertaken to demonstrate the existence of mimicry between spermatozoa and bacteria. For this, the shared antigenic determinants between mouse spermatozoa and Streptococcus pyogenes against a common ligand, sperm immobilization factor (SIF), were isolated. The mimicry was established on the basis of their ability to ameliorate the SIF-mediated compromised sperm parameters in vitro viz. motility, viability, morphology and Mg2+-ATPase activity of spermatozoa. Further, both the receptors i.e. SIF-binding receptor from mouse spermatozoa (MS-SBR) and SIF-binding receptor from S. pyogenes (S-SBR) were able to block the binding of FITC-labelled SIF to spermatozoa and bacteria. The in vivo studies also showed that MS-SBR (10 µg)/S-SBR (25 µg) could alleviate SIF-induced infertility in female BALB/c mice, further providing evidence for molecular similarities between bacteria and spermatozoa.

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.

Evaluación de la motilidad espermática usando dos crioprotectores / Evaluation of sperm motility using two cryoprotectants

La infertilidad, es un problema que afecta a una gran cantidad de parejas. Una de sus causas es la disminución de la calidad seminal debido, por ejemplo, a tratamientos gonadotóxicos. La criopreservación seminal es la técnica que permite conservar y almacenar espermatozoides sin que pierdan su capacidad fecundante; siendo esta una herramienta fundamental en reproducción asistida. El objetivo de este trabajo ha sido optimizar la técnica de criopreservación. Para ello se llevó a cabo un estudio, sobre muestras de pacientes en estudio por problemas de fertilidad, en el que se compararon dos medios de criopreservación (SpermCryo™All-round y CryoSperm™) y la aplicación o no de un baño en nitrógeno líquido a las muestras (previo a su almacenamiento); así como el efecto del tiempo que transcurre desde la eyaculación hasta el procesado sobre la calidad de la muestra. Las posibles variaciones fueron estudiadas con un analizador automático, mediante la realización de test pre- y post-congelación para comprobarla movilidad espermática

Infertility is a problem that affects a lot of couples. One of its causes is a decreased semen quality due to, for example, gonadotoxic treatments. The cryopreservation of human semen is the technique that allows sperm preserving and storing without losing their fertilizing capacity; being a fundamental tool in assisted reproduction. The aim of this study was to optimize the cryopreservation technique. To this end, a study carried out on samples of patients under study by fertility problems, in which two cryoprotectant media (SpermCryo™ All-round and CryoSperm™) and the execution or non-execution of an immersion of the samples in liquid nitrogen (before storage) were compared; and the effect of the time between ejaculation and the processing on the quality of the sample. Variations were studied with an automatic analyzer by performing pre- and post-thaw sperm motility tests. The results show no difference between the two cryoprotectants media, but seems to have a tendency to obtain better postthaw mobility with either depending on sample characteristics. Moreover, the liquid nitrogen bath had no apparent effects on post-thaw results. However, we must highlight the importance of time in the processing of semen samples once liquefied, to avoid decreased sperm quality. To improve post-thaw outcomes the key lies in the necessity to adjust the freezing protocol to the sample characteristics and a correct implementation of the protocol cryopreservation (selection and addition of cryoprotectant media...); favoring the management of infertility and the success of assisted reproduction techniques

Evaluation of antifertility effect of gel formulation containing sperm immobilizing factor: In vitro and in vivo studies.

Sexually active women often seek protection against unplanned pregnancies. Latter can be effectively controlled by consistent use of spermicides during each coital act. However, side effects associated with the use of available synthetic spermicidal agents have directed the interest towards identifying newer and safer agents. Present studies were undertaken to formulate a vaginal contraceptive gel, containing sperm immobilizing factor (SIF) isolated from Staphylococcus aureus using 1% w/v Carbopol. SIF loaded gel formulation was characterized for various in vitro parameters i.e. pH, spreadability, texture profiling, rheological properties, and in vitro release studies. The prepared formulation was found to possess significant spreading properties, gel firmness and strength, and released about 80% of SIF within 30min. Latter can completely immobilize human spermatozoa within 20s, at a dose of 200µg/ml. SIF in the proposed gel formulation showed 100% contraceptive effect when used at amount as low as of 10µg, thus confirming the possibility to develop it as a potent vaginal contraceptive for future use.

A unique dithiocarbamate chemistry during design & synthesis of novel sperm-immobilizing agents.

1-Substituted piperazinecarbodithioates were obtained by an unusual removal of CS2 in benzyl substituted dithiocarbamate derivatives under acid and basic conditions during design and synthesis of 1,4-(disubstituted)piperazinedicarbodithioates as double edged spermicides. A plausible mechanism for CS2 removal has been proposed. All synthesized compounds were subjected to spermicidal, antitrichomonal and antifungal activities. Twenty-one compounds irreversibly immobilized 100% sperm (MEC, 0.06-31.6 mM) while seven compounds exhibited multiple activities. Benzyl 4-(2-(piperidin-1-yl)ethyl) piperazine-1-(carbodithioate) (18) and 1-benzyl 4-(2-(piperidin-1-yl)ethyl)piperazine-1,4-bis(carbodithioate) (24) exhibited appreciable spermicidal (MEC, 0.07 and 0.06 mM), antifungal (MIC, 0.069-0.14 and >0.11 mM) and antitrichomonal (MIC, 1.38 and 0.14 mM) activities. The probable mode of action of these compounds seems to be through sulfhydryl binding which was confirmed by fluorescence labeling of sperm thiols.

Estudio de los daños estructurales tras la inmovilización espermática previa a la microinyección intracitoplasmática de espermatozoides en sujetos teratozoospérmicos / Study of structural damage after sperm immobilisation prior to intracytoplasmic sperm injection in teratozoospermic males

Objetivo: Describir y comparar las alteraciones ultraestructurales que puede provocar la inmovilización espermática previa a la microinyección intracitoplasmática de espermatozoides, así como los daños en el ADN y el estado del acrosoma en espermatozoides de sujetos teratozoospérmicos y normozoospérmicos. Material y métodos: Se utilizaron 15 muestras seminales procedentes de donantes normozoospérmicos y 20 muestras de pacientes teratozoospérmicos del Instituto Bernabeu de Alicante. Para el estudio con microscopia electrónica de transmisión se utilizaron ovocitos humanos como receptáculo de los espermatozoides. La fragmentación del ADN se valoró mediante la técnica TUNEL, y el estado del acrosoma, utilizando la lectina Pisum sativum conjugada con FITC. El análisis estadístico entre los diferentes grupos se realizó mediante un test ANOVA. Resultados: Los resultados mostraron que, tras la inmovilización, los espermatozoides procedentes tanto de sujetos normozoospérmicos como de teratozoospérmicos sufrieron las mismas alteraciones ultraestructurales a nivel de la membrana plasmática y acrosomal. Tras valorar la fragmentación de ADN mediante TUNEL observamos que en los espermatozoides inmovilizados el porcentaje de espermatozoides con ADN fragmentado era similar en ambos grupos (normozoospérmicos vs. teratozoospérmicos). En cambio, el porcentaje de espermatozoides con reacción acrosómica fue significativamente más elevado en los inmovilizados que en el grupo control, tanto en los sujetos normozoospérmicos como teratozoospérmicos. Conclusión: Los resultados de este estudio evidencian que los da˜nos estructurales provocados por la inmovilización, en espermatozoides procedentes de sujetos normozoospérmicos y teratozoospérmicos, son independientes de la morfología espermática. Y además, dicho proceso no provoca fragmentación del ADN. Sin embargo, la inmovilización tanto en el grupo de los sujetos normozoospérmicos como teratozoospérmicos provoca una inducción mecánica de la reacción acrosómica (AU)

Objective: To describe and compare the ultrastructural alterations, DNA fragmentation and acrosome status that could cause sperm immobilisation prior to intracytoplasmic sperm injection, in samples from normozoospermic and teratozoospermic males. Material and methods: For this study we used 15 sperm samples from consenting normozoospermic donors and 20 samples from teratozoospermic males, submitted for assisted reproduction at Instituto Bernabeu of Alicante. To assess the ultrastructural alterations induced by immobilisation, human oocytes were used as containers for spermatozoa and then observed by transmission electron microscopy. DNA fragmentation was assessed using TUNEL and acrosome status using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin. The control group consisted of sperm without manipulation. Statistical analysis between groups was performed by ANOVA. Results: The results showed that immobilised spermatozoa presented the same damage in plasma and acrosomal membranes in the samples from both normozoospermic and teratozoospermic subjects. After assessing DNA fragmentation in the cells from normozoospermic and teratozoospermic patients by the TUNEL technique, we observed that the percentage of spermatozoa with DNA fragmentation did not increase in the immobilised group compared with the control group. However, the percentage of acrosome-reacted sperm was significantly greater in immobilised spermatozoa than in the control group in both normozoospermic and teratozoospermic males. Conclusion: The results of this study show that the structural damage caused by immobilization in spermatozoa from teratozoospermic and normozoospermic males is independent of sperm morphology. In addition, the immobilisation does not cause DNA fragmentation. However, the percentage of acrosome-reacted sperm was greater in the immobilised group than in the control group, in both normozoospermic and teratozoospermic subjects (AU)

¿A qué velocidad «muere» el ácido desoxirribonucleico del espermatozoide tras descongelar muestras seminales procedentes de donantes? / At what speed does sperm deoxyribonucleic acid “die” after donor semen samples are thawed?

Objetivo. Analizar los niveles de daño que se registran en el ADN de espermatozoides de donantes y estimar la velocidad a la que este se degrada tras la descongelación. Material y métodos. Dosis seminales procedentes de donantes (n = 50) y un grupo control formado por pacientes normozoospérmicos (n = 40). Se estudiaron los valores de fragmentación del ADN espermático (SDF) en su nivel basal, así como los valores de SDF tras incubación de las muestras a 37 °C durante 2, 6 y 24 h. Se calcularon las velocidades de degradación del ADN por tramos de incubación. Resultados. El semen criopreservado de donante presenta unos niveles basales de SDF 2 veces inferiores a los observados en los controles, y su ADN es 2,5 veces más longevo que el del grupo control. Niveles basales de SDF sobre un 8% generan una sensibilidad de un 82% y una especificidad de un 65% para discriminar entre los donantes y los controles. Los valores de incremento del daño de 1,8% por hora, analizados durante las 2 primeras horas de incubación, identifican a los donantes con un 77% de sensibilidad y un 65% de especificidad. Ambos valores no muestran ninguna correlación dentro del grupo de los controles, ni entre los donantes. Conclusiones. El establecimiento de este tipo de valores umbral se podría utilizar para identificar donantes considerados como «superdonantes» en relación con sus bajos niveles de SDF y su alta estabilidad de la cromatina. Los donantes que se seleccionaron en las diferentes clínicas presentan características equiparables para estos parámetros(AU)

Objective. The study was made to analyze the baseline levels of damage recorded in sperm DNA fragmentation (SDF) and to estimate sperm DNA longevity as observed in donors after thawing. Material and methods. Fifty donors and forty individuals attending a clinic and classified as a normo-zoospermic population were compared. The baseline SDF levels and the increasing rate of SDF (r-SDF) obtained after thawing when the sperm was incubated for a period of 24 h with different sub-sampling performed after 2, 6 and 24 h of incubation were considered as the independent variables and compared. Results. Cryopreserved donor sperm exhibited baseline SDF values approximately 2 times lower than those observed in the control group. DNA stability was 2.5 times higher than that observed in the control cohort. Baseline values of SDF of approximately 8% generates 65% sensitivity and 82% specificity to discriminate between the donors and controls. Values of increase of damage of 1.8% per hour, analyzed during the first hours of incubation, identify the donor characteristics with 77% sensibility and 65% specificity. Neither value show any correlation within the control and donor cohorts group. Conclusion. The establishment of these types of threshold values can be used to identify donors considered as “super-donors” in relation to their low levels of SDF and high chromatin stability. The donors selected from the different clinics participating in this study showed similar characteristics for these parameters(AU)

Rat sperm immobilisation effects of a protein from Ricinus communis (Linn.): an in vitro comparative study with nonoxynol-9.

Previous study conducted in our department showed that 50% ethanolic extract of the root of Ricinus communis possess reversible antifertility effect and a 62-kDa protein (Rp) from this extract is responsible for the antifertility effects. In this study, we compared the spermicidal effect of this Rp with nonoxynol-9 (N-9) in vitro. The sperm immobilisation studies showed that 100 µg ml(-1) of Rp was able to immobilise the sperms completely within 30 s. Sperm revival test revealed that the spermicidal effect was irreversible. There was also a significant reduction in sperm viability and hypo-osmotic swelling in Rp and N-9 treated groups in comparison with the control. In Rp and N-9 treated groups, the number of acrosome-reacted cells was found to be high and also caused agglutination of the spermatozoa, indicating the loss of intactness of the plasma membrane, which was further supported by the significant reduction in the activity of membrane bound 5'-nucleotidase, acrosomal acrosin. In short, the protein Rp possesses spermicidal activity in vitro and its effects are similar to that of nonoxynol 9.

Molecular mimicry between spermatozoa and bacteria.

PURPOSE: Sperm immobilization factor isolated from Staphylococcus aureus immobilizes human spermatozoa as well as motile bacteria, showing that sperm immobilization factor receptor might be shared by bacteria and human spermatozoa. Thus, we sought to identify a common sperm immobilization factor binding receptor on spermatozoa and bacteria. MATERIALS AND METHODS: Sperm immobilization factor was isolated from S. aureus. Sperm immobilization factor binding receptors were isolated from spermatozoa and bacteria. RESULTS: Antisperm antibodies in humans cross-reacted with bacteria antibodies. Thus, molecular similarities between determinants of spermatozoa and pathogenic microorganisms can be inferred. Sperm immobilization factor isolated from S. aureus immobilized spermatozoa (150 µg/ml) and motile bacteria, including Escherichia coli, Pseudomonas aeruginosa and Proteus mirabilis (500 µg/ml). This showed the presence of a common sperm immobilization factor binding conformation on spermatozoa and bacteria. Furthermore, receptors binding sperm immobilization factor were extracted and purified from spermatozoa, E. coli, P. aeruginosa and P. mirabilis. Molecular mimicry between spermatozoa and bacteria was confirmed by observing the blockage of spermatozoa immobilization by sperm immobilization factor in the presence of receptors isolated from spermatozoa, E. coli, P. aeruginosa or P. mirabilis. Also, a higher concentration of sperm immobilization factor (200 µg/ml) caused sperm death. Blocking the death of spermatozoa induced by sperm immobilization factor in the presence of these receptors provided further evidence for a common receptor. CONCLUSIONS: Results provide evidence for molecular similarity between bacteria and spermatozoa.

Spermicidal action of a protein isolated from ethanolic root extracts of Achyranthes aspera: an in vitro study.

A previous study conducted in our department, showed that 50% ethanolic extract of the roots of Achyranthes aspera possess spermatotoxic effects. Preliminary studies also revealed that the active principle may be a protein. In this study a 58 kDa Achyranthes protein (Ap) was isolated from Achyranthes aspera using standard protocols and their effects on the rat sperm was studied in vitro in comparison with nonoxynol-9 (N-9). The sperm immobilization studies showed that about 150 µg of Ap was able to immobilize sperms completely within seconds at a lower concentration than N-9 (250 µg). The sperm revival test revealed that the spermicidal effect was irreversible. There was also a significant reduction in sperm viability and hypo-osmotic swelling in the Ap-treated and N-9 treated groups in comparison to the control. In the Ap and N-9 treated groups the number of acrosome reacted cells were found to be high and it also caused agglutination of the sperms indicating the loss of intactness of the plasma membrane which was further supported by the significant reduction in the activity of membrane bound 5' nucleotidase and acrosin enzyme. Hence this study showed that the protein isolated from the roots of Achyranthes aspera possess spermicidal activity in vitro and can act as a spermicide similar to that of nonoxynol 9. Ap also possessed spermicidal activity against human sperms in vitro.

Comparative study on the spermicidal activity of organic solvent fractions from hydroethanolic extracts of Achyranthes aspera and Stephania hernandifolia in human and rat sperm.

BACKGROUND: This study was conducted to determine the most effective fraction of the hydroethanolic (water:ethanol, 1:1) extracts of Stephania hernandifolia leaves and Achyranthes aspera roots (in a composite manner at a ratio of 1:3, respectively) that will provide maximum spermicidal activity in human and rat spermatozoa out of five different ratios (1:1, 1:3, 1:7, 3:1 and 7:1) that have been studied in pilot experiments. STUDY DESIGN: n-Hexane, chloroform and ethyl acetate fractions of the hydroethanolic (1:1) extracts of S. hernandifolia and A. aspera were mixed at 1:3. Different concentrations were tested for sperm immobilization, sperm viability, acrosome status, 5'-nucleotidase activity and nuclear chromatin decondensation using human and rat spermatozoa for the selection of the most effective concentration. RESULTS: Out of three fractions of the hydroethanolic (1:1) extracts of the said plants, the n-hexane fraction was most effective, and the chloroform fraction exhibited minimum activity for this purpose. At a concentration of 0.1 g/mL hexane fraction, all sperm of the human sample were immobilized immediately (within 20 s). In case of the rat sample, all epididymal spermatozoa were immobilized immediately (within 20 s) by treatment with hexane fraction at a concentration of 0.004 g/mL. All human sperm were found to be nonviable within 20 min. The activity of acrosome enzymes was reduced, and significant release of 5'-nucleotidase (a plasma membrane marker) into the surrounding medium was noted after treatment with 0.1 g/mL hexane fraction, indicating that the hexane fraction affected the cytoarchitecture of the sperm plasma membrane. The maximum number of human sperm failed to decondense when treated with 0.1 g/mL hexane fraction, and sperm motility was also irreversible. The hexane fraction was tested in rats as vaginal contraceptive and showed 100% efficacy, indicating its potential for development as vaginal contraceptive. CONCLUSION: The findings indicate that, among the different fractions, the hexane fraction of the hydroethanolic extracts of the two plants produced the most effective spermicidal activity and can be considered as vaginal contraceptive.

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.

The effects of lead and zinc on the quality of semen of albino rats.

OBJECTIVE: To determine the effects of lead and zinc administration on the quality of semen of albino rats. STUDY DESIGN: Experimental study. PLACE AND DURATION OF STUDY: Basic Medical Sciences Institute, Jinnah Postgraduate Medical Centre, Karachi, from August 2003 to December 2005. METHODOLOGY: Sixty adult albino rats selected for the study were divided into three groups, group A received injection normal saline 1 ml intraperitoneally daily for 8 weeks. Group B received lead chloride in a dose of 10 mg/kg body weight intraperitoneally daily. Group C received lead chloride in a dose of 10 mg/kg body weight and zinc chloride in a dose of 1 mg/kg body weight intraperitoneally daily. On the day of completion of treatment, the animals were sacrificed; epididymis was used for semen analysis. Student's t-test was used to determinate significance; the p-value < 0.05 was taken as significant. RESULTS: The number of sperms was 7.3, 1.7 and 6.6 million cells/ml in groups A, B and C respectively. Sperm's count decreased by 87% in group B (p < 0.001, CI 4117082.4 - 6965747.6) as compared to group A. Compared with group C, the sperm's count was decreased to 75% (p < 0.001, CI -5417413 to -4416987). The immotility of sperms was 27%, 57% and 26% in groups A, B and C respectively. There was 30% decreased motility of sperm in group B (p < 0.001, CI -30.19425 to -19.80575) as compared to group A. Compared with group C, the immotile sperm were increased to 31% (p < 0.001, CI 19.87494 - 30.92506). CONCLUSION: Lead produced toxic effects on germinal epithelium and altered the quality of semen which was improved by zinc.

Sperm motility and viability in west African dwarf rams treated with Euphorbia hirta / Motilidad y viabilidad espermática en carneros enanos del oeste africano tratados con Euphorbia hirta

Four adult sexually matured and clinically healthy West African Dwarf (WAD) rams aged between 24 and 30 months were used for the study. The rams were first used as control and later as experimental animals upon being orally dosed with Euphorbia hirta extract at 400mg/kg body weight for 14 days. Semen samples were collected from the rams a day after the administration of the plant extra and seven days after. The objective of the study was to investigate the effect of Euphorbia hirta on the semen picture of WAD rams. There were significantly differences (P <0.05) in the semen picture as reflected in a reduction of sperm motility from 80% to 47.5% and live-dead ratio from 90.75% to 32.5% in the control and post-experimental stages of the study respectively. This indicates that the fertilization capacity and livability of spermatozoa were negatively affected. There were no significant differences in the values of body parameters measured across the stages of the study. The plant is therefore not recommended for medicinal purpose in male animals.

Cuatro carneros enanos adultos de África Occidental sexualmente maduros y clínicamente sanos, con edades comprendidas entre los 24 y 30 meses, fueron utilizados para este estudio. Los carneros fueron utilizados como control y, más tarde, como animales de experimentación al ser medicados por vía oral con extracto de Euphorbia hirta en 400mg/kg peso corporal durante 14 días. Se recogieron muestras de semen de los carneros un día después de la administración de la planta y siete días después. El objetivo del estudio fue investigar el efecto de Euphorbia hirta en las imágenes de esperma de carneros enanos África Occidental. Hubo diferencias significativas (P <0,05) en la imagen del semen como reflejo de una reducción de la motilidad espermática del 80% al 47,5% y un ratio de vivos-muertos de 90,75% a 32,5% en la etapa control y después de las fases experimentales del estudio, respectivamente. Esto indica que la capacidad de fertilización y calidad de vida de los espermatozoides fueron afectados negativamente. No hubo diferencias significativas en los valores de los parámetros corporales medidos a través de las etapas del estudio. La planta por tanto no es recomendable para fines medicinales en los animales machos.

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.

Sperm immobilizing properties of lemon juice.

The aim of this study was to evaluate the sperm-immobilizing properties of lemon juice to determine if they are consistent with its traditional contraceptive use. It was found that lemon juice supernatant (LJS) has high osmolality (550-60 mOsm) and low pH (2.2-2.6) and that addition of LJS to semen to give a final concentration of 20% v/v reduced the pH from around 8.4 to 4.1. This acidification was associated with irreversible cessation of all sperm movements within 1 minute. In conclusion, lemon juice should be further evaluated for acceptability, safety, and efficacy as a topical vaginal contraceptive agent.

Calcium (hydrogen-1-malate) hexahydrate on Echeveria gibbiflora leaves and its effect on sperm cells.

Echeveria gibbiflora is a plant widely used for its contraceptive activity in traditional Mexican medicine. Data on calcium crystals in plants are not outstanding. In the case of the Echeveria gibbiflora leaves, however, its quality, quantity, and salt type are quite surprising; one striking result of its X-ray crystallographic data shows the presence of calcium bis (hydrogen-1-malate) hexahydrate [2(C4H5O(5)1), Ca(1)2+, 6(H2O1)]. This highly soluble compound might explain the rapid shape changes of calcium crystals. Because SEM-EDS analysis shows that calcium malate crystals were obtained in a highly pure state and the immobilization and agglutination pattern that OBACE show on human and bull spermatozoa are not found even when high concentrations of calcium bis (hydrogen-1-malate) hexahydrate salt are present it is not feasible to involucrate molecules as calcium malate as part of the OBACE contraceptive activity.

The effect of oleanolic acid on sperm motion characteristics and fertility of male Wistar rats.

The study was designed to examine the effect of oleanolic acid on cauda epididymal sperm motion using a computer-aided sperm analysis system and to elucidate the relationship between sperm motion and fertility, as a tool for contraceptive studies. Oleanolic acid-polyvinylpyrrollidone suspension was orally administered to adult male Wistar rats for 30 days, followed by a 14-day drug withdrawal from half of the rats in the group. Control rats received only polyvinylpyrrollidone. All males were mated with untreated females. Treated males failed to impregnate females, whereas control and oleanolic acid withdrawn males achieved 100% pregnancies. Sperm motion analysed on the Sperm Motility Quantifier (SMQ) showed significant differences in linearity (P < 0.001) and wobble (P < 0.01) between control and treated groups. However, the curvilinear velocities were not significantly different (P > 0.05) among all the groups. Sperm motility patterns verified differences among kinematic parameters.