Sperm Immobilization Factor Research Articles

An experimental study to decipher the implications of antigenic sharing between Proteus mirabilis and mouse spermatozoa in eliciting an antisperm immune response: A potential culprit in immune infertility

The present study aims to investigate the antigenic cross reactivity between the receptor from Proteus mirabilis and spermatozoa against a common sperm immobilization factor, SIF, by calorimetric and competitive inhibition studies, and the immunogenicity of this receptor to evoke the formation of antisperm antibodies and their subsequent role in fertility outcome. The sperm binding receptor from Proteus mirabilis (PM-SBR) was extracted from ultrasonicated cell debris by treating it for 12 h at 37°C with 1 M NaCl. After being purified by gel permeation chromatography, its molecular weight as determined by SDS-PAGE was observed to be ≈ 47 kDa. The detrimental impacts of Sperm immobilizing factor (SIF) on spermatozoa viz. motility, viability, and morphology were mitigated when SIF was preincubated with various concentrations of PM-SBR. Using isothermal titration calorimetry, the entropy of the SIF-PM-SBR interaction was found to be -18.31 kJ/mol, whereas the free energy was 28.4 J/mol K. FTIR analysis was used to evaluate the binding interactions between PM-SBR and SIF. In addition, mice that were administered antibodies against PM-SBR were unable to conceive, in contrast to mice that were administered Phosphate buffer saline (PBS) or pre-immunization serum as controls. In light of this, we may conclude that anti-PM-SBR antibodies act as anti-sperm antibodies. Our work found that molecular mimicry between Proteus mirabilis and spermatozoa may cause antisperm immune reactivity. As a result of an immunological response to PM-SBR, infected individuals may produce antibodies against an epitope similar to one found on spermatozoa which helps in developing new strategies for managing autoimmune responses and infertility.

Evaluation of Antifertility Effects of Sperm Immobilization Factor from Escherichia coli on Male Mice

Earlier in our laboratory, sperm immobilization factor (SIF) isolated from Escherichia coli has shown admirable contraceptive efficacy in female mice and thus generated interest in evaluating the same as a potential candidate for male contraception. Keeping this in mind, the present study was aimed at exploitation of SIF as contraceptive agent in male mouse model after intra testis administration. For this, Balb/c mice were administered with different concentrations (10, 50, 100, 200, or 400µg) of SIF and sacrificed on days 3,7,14,21,30,45,60, and 90. After sacrifice, results in terms of change in body weight, tissue somatic indices, seminal parameters, histology, hematological parameters, lipid peroxidation, testosterone level, and cytokines level were evaluated. The weight profile and TSI of all experimental groups showed no significant change. In case of seminal parameters, 400μg of SIF led to azoospermia up to 90 days and tissue histology also revealed hypo-spermatogenesis. No significant change was observed in haematological parameters after SIF (400µg) administration however, a significant decrease was observed in testosterone levels. The oxidative status and cytokines profile was also affected after SIF (400µg) inoculation. Thus, when administered via intra testis route, SIF can lead to azoospermia and poses to be a potent and safe antifertility agent.

Sperm impairing microbial factor: potential candidate for male contraception

BackgroundDespite 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.MethodsSIF (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.ResultsThe 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.ConclusionSIF, when administered via intra vas deferens route, could lead to complete azoospermia. Therefore, it could be considered as a potential male contraceptive.

Sperm immobilization factor of Candida albicans: A proposed mechanism of infertility in female mice

Candida species are the most frequently isolated fungi, responsible for blood stream and urinary tract infections. Various studies have shown Candida albicans to be associated with impediment of sperm parameters. Therefore to determine the underlying mechanism, the standard strain of Candida albicans (MTCC 1637) was employed and it was found to cause complete immobilization of mouse spermatozoa in vitro. Further, sperm immobilization activity was shown by only cell-free supernatant, indicating that the sperm immobilization factor might be released extracellularly, as no activity was observed with the washed cells. The sperm immobilization factor (SIF) from supernatant was precipitated out with ammonium sulphate at the saturation of 60% to 80% and was purified by gel permeation chromatography followed by ion exchange chromatography.

Shared Antigenic Determinants Between Spermatozoa and Bacteria: An Experimental Study

Sperm immobilization factor (SIF), the secretory protein of Staphylococcus aureus, is known to cause complete immobilization, death and morphological alterations in mouse spermatozoa in vitro. However, the present study aims to explore a newer dimension of SIF i.e., to bind to motile and non-motile bacteria and its ability to induce immobilization of motile bacteria in vitro. The results showed that 800µg of SIF caused complete immobilization of motile bacteria, however, death and morphological alterations could not be observed even with 1000µg of SIF. Furthermore, this SIF-mediated bacterial immobilization was reversed when each of the SIF-binding receptor from mouse spermatozoa and bacteria (Escherichia coli and Streptococcus pyogenes) was incubated with bacteria, thereby, providing an experimental evidence of similarity between the antigenic determinants present on spermatozoa and bacteria against a common ligand, SIF.

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.

Characterization of Sperm Immobilization Factor from Escherichia coli and Its Receptor to Study the Underlying Mechanism of Sperm Immobilization

Sperm immobilization factor (SIF) was extracted and purified from Escherichia coli filtrate. Characterization of SIF using Liquid chromatography –mass spectrometry (LC-MS) revealed its sequence similarity to chaperone protein HchA of E. coli O157:H7. Further the effect of SIF on sperm functions such as acrosome reaction was studied and the results showed that SIF (500µg/ml) significantly inhibits calcium ionophore induced acrosome reaction. SIF was also found to decrease Mg ++ ATPase activity of spermatozoa from 869.5 (control) to zero at 25µg/ml. The corresponding SIF binding receptor was also isolated and purified. The tryptic peptide analysis of SIF binding receptor by MALDI-TOF (Matrix assisted laser desorption ionization- time of flight) showed its sequence similarity to chain A structure of human apolactoferrin. Further specificity of SIF and SIF binding receptor was studied by calorimetric studies that showed binding constant (K), enthalpy of binding (ΔH°), free energy (ΔG) and entropy (ΔS) to be 1460/M and -11.7kJ/mole, -18.76 kJ/mole and 22.77 J/moleK, respectively.

Human Sperm Interaction withStaphylococcus aureus: A Molecular Approach

Sperm immobilization factor (SIF) causing 100% immobilization of spermatozoa isolated from Staphylococcus aureus when characterized using LC-MS (Liquid chromatography-mass spectrometry) showed that this 20 kDa protein had peptide sequence similarity with hsp-70 protein. It was found to completely (100%) inhibit Mg++ ATPase activity of spermatozoa at concentration of 100 μg mL−1. Sperm samples treated with SIF also showed reduction in calcium ionophore-induced acrosome reaction as compared to control samples (treated with calcium ionophore alone). Binding studies of FITC labelled SIF with spermatozoa using fluorescent microscopy showed binding of SIF to the surface of spermatozoa indicating the presence of SIF binding receptor. The receptor was extracted by 3M NaCl and purified by gel permeation chromatography. Characterization of the receptor by MALDI-TOF (Matrix-assisted laser desorption ionization-time of flight) indicated that the receptor shared sequence similarity with MHC class II antigen. A calorimetric study showed that the receptor moiety on spermatozoa was specific for the purified ligand as binding of the receptor to ligand was enthalpically (−11.9 kJ mole−1) as well as entropically (21.53 J mole−1 K−1) favored resulting in the Gibb's free energy of −18.57 kJ mole−1.

Molecular Mimicry Between Spermatozoa and Bacteria

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. Sperm immobilization factor was isolated from S. aureus. Sperm immobilization factor binding receptors were isolated from spermatozoa and bacteria. 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. Results provide evidence for molecular similarity between bacteria and spermatozoa.

Editorial Comment to Receptor dependent immobilization of spermatozoa by sperm immobilization factor isolated from Escherichia coli: Proof of evidence

International Journal of UrologyVolume 18, Issue 8 p. 603-604 Editorial Comment to Receptor dependent immobilization of spermatozoa by sperm immobilization factor isolated from Escherichia coli: Proof of evidence Decio Armanini M.D., Decio Armanini M.D. Department of Medical and Surgical Sciences, Endocrinology, University of PaduaSearch for more papers by this authorFrancesco L Giorgino M.D., Francesco L Giorgino M.D. A.G.E.O., Padua, Italy. decio.armanini@unipd.itSearch for more papers by this authorCristina Fiore M.Sc., Cristina Fiore M.Sc. Department of Medical and Surgical Sciences, Endocrinology, University of PaduaSearch for more papers by this author Decio Armanini M.D., Decio Armanini M.D. Department of Medical and Surgical Sciences, Endocrinology, University of PaduaSearch for more papers by this authorFrancesco L Giorgino M.D., Francesco L Giorgino M.D. A.G.E.O., Padua, Italy. decio.armanini@unipd.itSearch for more papers by this authorCristina Fiore M.Sc., Cristina Fiore M.Sc. Department of Medical and Surgical Sciences, Endocrinology, University of PaduaSearch for more papers by this author First published: 22 June 2011 https://doi.org/10.1111/j.1442-2042.2011.02801.xCitations: 1Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article.Citing Literature Volume18, Issue8August 2011Pages 603-604 RelatedInformation

Receptor dependent immobilization of spermatozoa by sperm immobilization factor isolated from Escherichia coli: Proof of evidence

To examine the receptor-ligand interaction between E. coli and spermatozoa resulting in sperm immobilization. Sperm immobilization factor (SIF) was isolated and purified from filtrate of E. coli suspension. Receptor on human spermatozoa for SIF was made to isolated and purified by salt extraction, gel permeation and ion exchange chromatography. Receptor dependent immobilization of spermatozoa by SIF was confirmed by competitive inhibition by addition of the purified receptor and binding to sperm receptor by Fluorescin Isothiocynate (FITC) labelled SIF using fluorescence microscopy. Heat labile, non-dialyzable, sperm immobilization factor of ∼56 kDa was isolated and purified from E. coli. Using SIF as a tool, receptor of 113 kDa could be isolated and purified from human spermatozoa. Addition of purified receptor completely inhibited sperm immobilization and death induced by SIF suggesting receptor-dependent immobilization of spermatozoa. Further incubation of sperm with FITC labelled SIF resulted in staining of whole sperm. The study provides evidence of receptor-ligand interaction between E. coli and spermatozoa.

Mechanism of Sperm Immobilization byEscherichia coli

Aim. To explore the influence of Escherichia coli on the motility of human spermatozoa and its possible mechanism. Methods. Highly motile preparations of spermatozoa from normozoospermic patients were coincubated with Escherichia coli for 4 hours. At 1, 2 and 4 hours of incubation, sperm motility was determined. The factor responsible for sperm immobilization without agglutination was isolated and purified from filtrates. Results. This report confirms the immobilization of spermatozoa by E. coli and demonstrates sperm immobilization factor (SIF) excreted by E. coli. Further this factor was purified by ammonium sulfate precipitation, gel permeation chromatography, and ion-exchange chromatography. Purified SIF (56 kDa) caused instant immobilization without agglutination of human spermatozoa at 800 μg/mL and death at 2.1 mg/mL. Spermatozoa incubated with SIF revealed multiple and profound alterations involving all superficial structures of spermatozoa as observed by scanning electron microscopy. Conclusion. In conclusion, these results have shown immobilization of spermatozoa by E. coli and demonstrate a factor (SIF) produced and secreted by E. coli which causes variable structural damage as probable morphological correlates of immobilization.

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.