Hydrogen peroxide (H2O2) is a “green chemical” that has various cleaning and disinfectant uses, including as an anti-bacterial agent for hygienic and medical treatments. However, its efficacy is limited against biofilm-producing bacteria, because of poor penetration of the protective, organic matrix. Here we show new applications for ferromagnetic nanoparticles (Fe3O4, MNP) with peroxidase-like activity in potentiating the efficacy of H2O2 in biofilm degradation and prevention. Our data show that MNP enhanced oxidative cleavage of biofilm components (model nucleic acids, proteins, and oligosaccharides) in the presence of H2O2. When challenged with live, biofilm-producing bacteria, the MNP-H2O2 system efficiently broke down existing biofilm and prevented new biofilm from forming, killing both planktonic bacteria and those within biofilm. By enhancing oxidative cleavage of various substrates, the MNP-H2O2 system provides a novel strategy for biofilm elimination, and other applications utilizing oxidative breakdown.

Lipid rafts, membrane sub-domains enriched in sterols and sphingolipids, are controversial because demonstrations of rafts have often utilized fixed cells. We showed in living sperm that the ganglioside G(M1) localized to a micron-scale membrane sub-domain in the plasma membrane overlying the acrosome. We investigated four models proposed for membrane sub-domain maintenance. G(M1) segregation was maintained in live sperm incubated under non-capacitating conditions, and after sterol efflux, a membrane alteration necessary for capacitation. The complete lack of G(M1) diffusion to the post-acrosomal plasma membrane (PAPM) in live cells argued against the transient confinement zone model. However, within seconds after cessation of sperm motility, G(M1) dramatically redistributed several microns from the acrosomal sub-domain to the post-acrosomal, non-raft sub-domain. This redistribution was not accompanied by movement of sterols, and was induced by the pentameric cholera toxin subunit B (CTB). These data argued against a lipid-lipid interaction model for sub-domain maintenance. Although impossible to rule out a lipid shell model definitively, mice lacking caveolin-1 maintained segregation of both sterols and G(M1), arguing against a role for lipid shells surrounding caveolin-1 in sub-domain maintenance. Scanning electron microscopy of sperm freeze-dried without fixation identified cytoskeletal structures at the sub-domain boundary. Although drugs used to disrupt actin and intermediate filaments had no effect on the segregation of G(M1), we found that disulfide-bonded proteins played a significant role in sub-domain segregation. Together, these data provide an example of membrane sub-domains extreme in terms of size and stability of lipid segregation, and implicate a protein-based membrane compartmentation mechanism.

No abstract

Spermatogonial stem cell transplantation (SSCT) offers unique approaches to investigate SSC and to manipulate the male germline. We report here the first successful performance of this technique in the dog, which is an important model of human diseases. First, we investigated an irradiation protocol to deplete endogenous male germ cells in recipient testes. Histologic examination confirmed O95% depletion of endogenous spermatogenesis, but retention of normal testis architecture. Then, 5-month-old recipient dogs (nZ5) were focally irradiated on their testes prior to transplantation with mixed seminiferous tubule cells (fresh (nZ2) or after 2 weeks of culture (nZ3)). The dogs receiving cultured cells showed an immediate allergic response, which subsided quickly with palliative treatment. No such response was seen in the dogs receiving fresh cells, for which a different injection medium was used. Twelve months post-injection recipients were castrated and sperm was collected from epididymides. We performed microsatellite analysis comparing DNA from the epididymal sperm with genomic DNA from both the recipients and the donors. We used six markers to demonstrate the presence of donor alleles in the sperm from one recipient of fresh mixed tubule cells. No evidence of donor alleles was detected in sperm from the other recipients. Using quantitative PCR based on single nucleotide polymorphisms (SNPs), about 19.5% of sperm were shown to be donor derived in the recipient. Our results demonstrate the first successful completion of SSCT in the dog, an important step toward transgenesis through the male germline in this valuable biomedical model. Reproduction (2008) 136 823-831

Despite enormous interest in membrane raft microdomains, no studies in any cell type have defined the relative compositions of the raft fractions on the basis of their major components-sterols, phospholipids, and proteins-or additional raft-associating lipids such as the ganglioside, G M1 . Our previous localization data in live sperm showed that the plasma membrane overlying the acrosome represents a stabilized platform enriched in G M1 and sterols. These findings, along with the physiological requirement for sterol efflux for sperm to function, prompted us to characterize sperm membrane fractions biochemically. After confirming limitations of commonly-used detergent-based approaches, we utilized a non-detergent-based method, separating membrane fractions that were reproducibly distinct based on sterol, G M1 , phospholipid and protein compositions (both mass amounts and molar ratios). Based on fraction buoyancy and biochemical composition, we identified at least three highly reproducible subtypes of membrane raft. Electron microscopy revealed that raft fractions were free of visible contaminants and were separated by buoyancy rather than morphology. Quantitative proteomic comparisons and fluorescence localization of lipids suggested that different organelles contributed differentially to individual raft sub-types, but that multiple membrane microdomain sub-types could exist within individual domains. This has important implications for scaffolding functions broadly associated with rafts. Most importantly, we show that the common practice of characterizing membrane domains as either "raft" or "non-raft" oversimplifies the actual biochemical complexity of cellular membranes.

ABSTRACT:We previously showed that in live murine and bovine sperm heads, the ganglioside G M1 localizes to the sterol-rich plasma membrane overlying the acrosome (APM). Labeling G M1 using the pentameric cholera toxin subunit B (CTB) induced a dramatic redistribution of signal from the APM to the sterol-poor postacrosomal plasma membrane (PAPM) upon sperm death. We now show a similar phenomenon in the flagellum where CTB induces G M1 redistribution to sterol-poor membrane subdomains of the annulus and flagellar zipper. Because sterol efflux from the plasma membrane is required for capacitation, we examined whether G M1 localization might be useful to detect membrane changes associated with capacitation and/or acrosomal exocytosis. First, incubation of murine and bovine sperm with their respective stimuli for capacitation did not change G M1 distribution in live cells. However, incubation of sperm of both species with specific stimuli for capacitation, followed by the use of specific fixation conditions, induced reproducible, stimulus-specific patterns of G M1 distribution. By assessing changes in G M1 distribution in response to progesterone-induced AE, we show that these patterns reflect the response of murine sperm populations to capacitating stimuli. These data suggest that G M1 localization can be used as a diagnostic tool for evaluating sperm response to stimuli for capacitation and/or AE. Such information could be useful when deciding between technologies of assisted reproduction or when screening for male fertility. Furthermore, stimulus-specific changes in G M1 distribution showed that sperm could respond to NaHCO 3 or mediators of sterol efflux independently, thereby refining existing models of capacitation.

Mammalian sperm are transcriptionally and translationally inactive. To meet changing needs in the epididymis and female tract, they rely heavily on post-translational modifications and protein acquisition/degradation. Membrane rafts are sterol and sphingolipid-enriched micro-domains that organize and regulate various pathways. Rafts have significance in sperm by transducing the stimulus of sterol efflux into changes in intracellular signaling that confer fertilization competence. We recently characterized 3 biochemically distinct sub-types of sperm rafts, and now present profiles for proteins targeting to and associating with these sub-types, along with a fraction largely comprised of "non-raft" domains. Proteomics analysis using a gel-based LC-MS/MS approach identified 190 strictly validated proteins in the raft sub-types. Interestingly, many of these are known to be expressed in the epididymis, where sperm membrane composition matures. To investigate potential roles for rafts in epididymal protein acquisition, we compared the expression and localization of 2 different sterol-interacting proteins, apolipoprotein-A1 and prominin-1 in sperm from different zones. We found that apolipoprotein-A1 was gradually added to the plasma membrane overlying the acrosome, whereas prominin-1 was not, suggesting different mechanisms for raft protein acquisition. Our results define raft-associating proteins, demonstrate functional similarities and differences among raft subtypes, and provide insights into raft-mediated epididymal protein acquisition.

Summary Membrane lipid regulation of cell function is poorly understood. In early development, sterol efflux and the ganglioside GM1 regulate sperm acrosome exocytosis (AE) and fertilization competence through unknown mechanisms. Here, we show that sterol efflux and focal enrichment of GM1 trigger Ca2+ influx necessary for AE through CaV2.3, whose activity has been highly controversial in sperm. Sperm lacking CaV2.3’s pore-forming α1E subunit showed altered Ca2+ responses, reduced AE, and a strong sub-fertility phenotype. Surprisingly, AE depended on spatio-temporal information encoded by flux through CaV2.3—not merely the presence/ amplitude of Ca2+ waves. Using both studies in sperm and voltage clamp of Xenopus oocytes, we define a molecular mechanism for GM1/CaV2.3 regulatory interaction, requiring GM1’s lipid and sugar components and CaV2.3’s α1E and α2δ subunits. Our results provide mechanistic understanding of membrane lipid regulation of Ca2+ flux and therefore Ca2+-dependent cellular and developmental processes such as exocytosis and fertilization.