RESUMEN
Acrosome exocytosis (AE), in which the sperm's single exocytotic vesicle fuses with the plasma membrane, is a complex, calcium-dependent process essential for fertilization. However, our understanding of how calcium signaling regulates AE is still incomplete. In particular, the interplay between intra-acrosomal calcium dynamics and the intermediate steps leading to AE is not well-defined. Here, we describe a method that provides spatial and temporal insights into acrosomal calcium dynamics and their relationship to membrane fusion and subsequent exocytosis of the acrosome vesicle. The method utilizes a novel transgenic mouse expressing an Acrosome-targeted Sensor for Exocytosis (AcroSensE). The sensor combines a genetically encoded calcium indicator (GCaMP) fused with mCherry. This fusion protein was specifically designed to enable the concurrent observation of acrosomal calcium dynamics and membrane fusion events. Real-time monitoring of acrosomal calcium dynamics and AE in live AcroSensE sperm is achieved using a combination of high frame-rate imaging and a stimulant delivery system that can target single sperm. This protocol also provides several examples of basic methods to quantify and analyze the raw data. Because the AcroSensE model is genetically encoded, its scientific significance can be augmented by using readily available genetic tools, such as crossbreeding with other mouse genetic models or gene-editing (CRISPR) based methods. With this strategy, the roles of additional signaling pathways in sperm capacitation and fertilization can be resolved. In summary, the method described here provides a convenient and effective tool to study calcium dynamics in a specific subcellular compartment-the sperm acrosome-and how those dynamics regulate the intermediate steps leading to membrane fusion and acrosome exocytosis.
Asunto(s)
Calcio , Semen , Masculino , Ratones , Animales , Calcio/metabolismo , Semen/metabolismo , Espermatozoides , Exocitosis/fisiología , Ratones Transgénicos , Señalización del CalcioRESUMEN
The murine epididymis has 10 distinct segments that provide the opportunity to identify compartmentalized cell physiological mechanisms underlying sperm maturation. However, despite the essential role of the epididymis in reproduction, remarkably little is known about segment-specific functions of this organ. Here, we investigate the dramatic segmental localization of the ganglioside GM1, a glycosphingolipid already known to play key roles in sperm capacitation and acrosome exocytosis. Frozen tissue sections of epididymides from adult mice were treated with the binding subunit of cholera toxin conjugated to AlexaFluor 488 to label GM1. We report that GM1-enriched vesicles were found exclusively in principal and clear cells of segment 2. These vesicles were also restricted to the lumen of segment 2 and did not appear to flow with the sperm into segment 3, within the limits of detection by confocal microscopy. Interestingly, this segment-specific presence was altered in several azoospermic mouse models and in wild-type mice after efferent duct ligation. These findings indicate that a lumicrine factor, itself dependent on spermatogenesis, controls this segmental differentiation. The RNA sequencing results confirmed global de-differentiation of the proximal epididymal segments in response to efferent duct ligation. Additionally, GM1 localization on the surface of the sperm head increased as sperm transit through segment 2 and have contact with the GM1-enriched vesicles. This is the first report of segment-specific vesicles and their role in enriching sperm with GM1, a glycosphingolipid known to be critical for sperm function, providing key insights into the segment-specific physiology and function of the epididymis.
Asunto(s)
Epidídimo , Gangliósido G(M1) , Ratones , Masculino , Animales , Epidídimo/metabolismo , Gangliósido G(M1)/metabolismo , Semen , Espermatozoides/metabolismo , EspermatogénesisRESUMEN
Secretion of the acrosome, a single vesicle located rostrally in the head of a mammalian sperm, through a process known as "acrosome exocytosis" (AE), is essential for fertilization. However, the mechanisms leading to and regulating this complex process are controversial. In particular, poor understanding of Ca2+ dynamics between sperm subcellular compartments and regulation of membrane fusion mechanisms have led to competing models of AE. Here, we developed a transgenic mouse expressing an Acrosome-targeted Sensor for Exocytosis (AcroSensE) to investigate the spatial and temporal Ca2+ dynamics in AE in live sperm. AcroSensE combines a genetically encoded Ca2+ indicator (GCaMP) fused with an mCherry indicator to spatiotemporally resolve acrosomal Ca2+ rise (ACR) and membrane fusion events, enabling real-time study of AE. We found that ACR is dependent on extracellular Ca2+ and that ACR precedes AE. In addition, we show that there are intermediate steps in ACR and that AE correlates better with the ACR rate rather than absolute Ca2+ amount. Finally, we demonstrate that ACR and membrane fusion progression kinetics and spatial patterns differ with different stimuli and that sites of initiation of ACR and sites of membrane fusion do not always correspond. These findings support a model involving functionally redundant pathways that enable a highly regulated, multistep AE in heterogeneous sperm populations, unlike the previously proposed "acrosome reaction" model.
Asunto(s)
Acrosoma , Calcio , Acrosoma/metabolismo , Reacción Acrosómica/fisiología , Animales , Calcio/metabolismo , Exocitosis/fisiología , Masculino , Mamíferos/metabolismo , Ratones , Espermatozoides/metabolismoRESUMEN
Previously, we reported the first live births of dogs using in vitro fertilization (IVF), embryo cryopreservation, and transfer. These techniques have potential applications in the conservation of endangered canids, and development of gene editing/repair technologies that could improve animal welfare by restoring normal gene function and removing predisposition to disease. Here, we used IVF as a springboard for initial attempts at genetic modification through gene editing/repair using the Clustered Regularly-Interspaced Short Palindromic Repeat (CRISPR)-CRISPR-associated endonuclease (Cas9) system. We showed previously that timing is critical for successful IVF in that the canine oocyte must be exposed to the oviductal environment beyond simply reaching metaphase II. Others have shown that timing of injection of CRISPR-Cas9 constructs is critical in gene editing, influencing the extent of genetic mosaicism. Therefore, we investigated whether timing of injection of the gene editing/repair constructs might influence the success of embryo production and gene editing in the dog. We achieved similar IVF success to our prior report in generating 2-cell control embryos, and found equally reduced embryo production whether injection was performed in oocytes prior to fertilization, or in presumptive single-cell zygotes already exposed to sperm. We had no success at generating offspring with precise single-nucleotide changes in KRT71 via homology-directed repair (HDR), but did identify mutation of FGF5 using non-homologous end joining (NHEJ). These findings underscore the difficulties inherent to gene repair, but represent important progress on reproducibility of canine IVF, improved techniques of oocyte/embryo handling, and impact of timing of injections on embryo development.
Asunto(s)
Perros/fisiología , Fertilización In Vitro/veterinaria , Edición Génica/veterinaria , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Oocitos/fisiología , Cigoto/fisiología , Animales , Sistemas CRISPR-Cas , Reparación del ADN por Unión de Extremidades/fisiología , Transferencia de Embrión , Factor 5 de Crecimiento de Fibroblastos/genética , Factor 5 de Crecimiento de Fibroblastos/metabolismo , Edición Génica/métodos , Regulación de la Expresión Génica , Genotipo , Queratinas Específicas del Pelo/genética , Queratinas Específicas del Pelo/metabolismo , Factores de TiempoRESUMEN
Sperm entering the epididymis are immotile and cannot respond to stimuli that will enable them to fertilize. The epididymis is a highly complex organ, with multiple histological zones and cell types that together change the composition and functional abilities of sperm through poorly understood mechanisms. Sperm take up taurine during epididymal transit, which may play antioxidant or osmoregulatory roles. Cysteine dioxygenase (CDO) is a critical enzyme for taurine synthesis. A previous study reported that male CDO-/- mice exhibit idiopathic infertility, prompting us to investigate the functions of CDO in male fertility. Immunoblotting and quantitative reverse transcription-polymerase chain reaction analysis of epididymal segments showed that androgen-dependent CDO expression was highest in the caput epididymidis. CDO-/- mouse sperm demonstrated a severe lack of in vitro fertilization ability. Acrosome exocytosis and tyrosine phosphorylation profiles in response to stimuli were normal, suggesting normal functioning of pathways associated with capacitation. CDO-/- sperm had a slight increase in head abnormalities. Taurine and hypotaurine concentrations in CDO-/- sperm decreased in the epididymal intraluminal fluid and sperm cytosol. We found no evidence of antioxidant protection against lipid peroxidation. However, CDO-/- sperm exhibited severe defects in volume regulation, swelling in response to the relatively hypo-osmotic conditions found in the female reproductive tract. Our findings suggest that epididymal CDO plays a key role in post-testicular sperm maturation, enabling sperm to osmoregulate as they transition from the male to the female reproductive tract, and provide new understanding of the compartmentalized functions of the epididymis.
Asunto(s)
Cisteína-Dioxigenasa/metabolismo , Fertilidad , Osmorregulación , Espermatozoides/metabolismo , Reacción Acrosómica , Animales , Antioxidantes/metabolismo , Western Blotting , Cromatografía Líquida de Alta Presión , Cisteína-Dioxigenasa/genética , Epidídimo/enzimología , Exocitosis , Femenino , Peroxidación de Lípido , Masculino , Ratones , Ratones Noqueados , Fosforilación , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Maduración del Esperma , Espermatozoides/fisiología , Taurina/análogos & derivados , Taurina/metabolismoRESUMEN
For nanobiotechnology to achieve its potential, complex organic-inorganic systems must grow to utilize the sequential functions of multiple biological components. Critical challenges exist: immobilizing enzymes can block substrate-binding sites or prohibit conformational changes, substrate composition can interfere with activity, and multistep reactions risk diffusion of intermediates. As a result, the most complex tethered reaction reported involves only 3 enzymes. Inspired by the oriented immobilization of glycolytic enzymes on the fibrous sheath of mammalian sperm, here we show a complex reaction of 10 enzymes tethered to nanoparticles. Although individual enzyme efficiency was higher in solution, the efficacy of the 10-step pathway measured by conversion of glucose to lactate was significantly higher when tethered. To our knowledge, this is the most complex organic-inorganic system described, and it shows that tethered, multi-step biological pathways can be reconstituted in hybrid systems to carry out functions such as energy production or delivery of molecular cargo.
Asunto(s)
Enzimas/metabolismo , Glucosa/metabolismo , Ácido Láctico/metabolismo , Nanopartículas/metabolismo , Animales , Mimetismo Biológico , Biotecnología , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/metabolismo , Glucosa/química , Humanos , Ácido Láctico/química , Nanopartículas/química , NanotecnologíaRESUMEN
Development of assisted reproductive technologies (ART) in the dog has resisted progress for decades, due to their unique reproductive physiology. This lack of progress is remarkable given the critical role ART could play in conserving endangered canid species or eradicating heritable disease through gene-editing technologies-an approach that would also advance the dog as a biomedical model. Over 350 heritable disorders/traits in dogs are homologous with human conditions, almost twice the number of any other species. Here we report the first live births from in vitro fertilized embryos in the dog. Adding to the practical significance, these embryos had also been cryopreserved. Changes in handling of both gametes enabled this progress. The medium previously used to capacitate sperm excluded magnesium because it delayed spontaneous acrosome exocytosis. We found that magnesium significantly enhanced sperm hyperactivation and ability to undergo physiologically-induced acrosome exocytosis, two functions essential to fertilize an egg. Unlike other mammals, dogs ovulate a primary oocyte, which reaches metaphase II on Days 4-5 after the luteinizing hormone (LH) surge. We found that only on Day 6 are oocytes consistently able to be fertilized. In vitro fertilization of Day 6 oocytes with sperm capacitated in medium supplemented with magnesium resulted in high rates of embryo development (78.8%, n = 146). Intra-oviductal transfer of nineteen cryopreserved, in vitro fertilization (IVF)-derived embryos resulted in seven live, healthy puppies. Development of IVF enables modern genetic approaches to be applied more efficiently in dogs, and for gamete rescue to conserve endangered canid species.
Asunto(s)
Transferencia de Embrión/veterinaria , Fertilización In Vitro/veterinaria , Animales , Canidae , Perros , Desarrollo Embrionario , Especies en Peligro de Extinción , Femenino , Nacimiento Vivo , Oocitos , EmbarazoRESUMEN
BACKGROUND: Rapid diagnosis for time-sensitive illnesses such as stroke, cardiac arrest, and septic shock is essential for successful treatment. Much attention has therefore focused on new strategies for rapid and objective diagnosis, such as Point-of-Care Tests (PoCT) for blood biomarkers. Here we use a biomimicry-based approach to demonstrate a new diagnostic platform, based on enzymes tethered to nanoparticles (NPs). As proof of principle, we use oriented immobilization of pyruvate kinase (PK) and luciferase (Luc) on silica NPs to achieve rapid and sensitive detection of neuron-specific enolase (NSE), a clinically relevant biomarker for multiple diseases ranging from acute brain injuries to lung cancer. We hypothesize that an approach capitalizing on the speed and catalytic nature of enzymatic reactions would enable fast and sensitive biomarker detection, suitable for PoCT devices. METHODS AND FINDINGS: We performed in-vitro, animal model, and human subject studies. First, the efficiency of coupled enzyme activities when tethered to NPs versus when in solution was tested, demonstrating a highly sensitive and rapid detection of physiological and pathological concentrations of NSE. Next, in rat stroke models the enzyme-based assay was able in minutes to show a statistically significant increase in NSE levels in samples taken 1 hour before and 0, 1, 3 and 6 hours after occlusion of the distal middle cerebral artery. Finally, using the tethered enzyme assay for detection of NSE in samples from 20 geriatric human patients, we show that our data match well (r = 0.815) with the current gold standard for biomarker detection, ELISA-with a major difference being that we achieve detection in 10 minutes as opposed to the several hours required for traditional ELISA. CONCLUSIONS: Oriented enzyme immobilization conferred more efficient coupled activity, and thus higher assay sensitivity, than non-tethered enzymes. Together, our findings provide proof of concept for using oriented immobilization of active enzymes on NPs as the basis for a highly rapid and sensitive biomarker detection platform. This addresses a key challenge in developing a PoCT platform for time sensitive and difficult to diagnose pathologies.
Asunto(s)
Envejecimiento/sangre , Bioensayo/normas , Enzimas Inmovilizadas/química , Infarto de la Arteria Cerebral Media/sangre , Fosfopiruvato Hidratasa/sangre , Accidente Cerebrovascular/sangre , Animales , Biomarcadores/sangre , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Enzimas Inmovilizadas/genética , Enzimas Inmovilizadas/metabolismo , Femenino , Genes Reporteros , Humanos , Infarto de la Arteria Cerebral Media/diagnóstico , Infarto de la Arteria Cerebral Media/fisiopatología , Luciferasas/química , Luciferasas/genética , Luciferasas/metabolismo , Masculino , Nanopartículas/química , Sistemas de Atención de Punto , Piruvato Quinasa/química , Piruvato Quinasa/genética , Piruvato Quinasa/metabolismo , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Dióxido de Silicio/química , Accidente Cerebrovascular/diagnóstico , Accidente Cerebrovascular/fisiopatología , Factores de TiempoRESUMEN
Despite numerous applications, we lack fundamental understanding of how variables such as nanoparticle (NP) size influence the activity of tethered enzymes. Previously, we showed that biomimetic oriented immobilization yielded higher specific activities versus nonoriented adsorption or carboxyl-amine binding. Here, we standardize NP attachment strategy (oriented immobilization via hexahistidine tags) and composition (Ni-NTA coated gold NPs), to test the impact of NP size (â5, 10, 20, and 50 nm) on multilayer formation, activity, and kinetic parameters (kcat, KM, kcat/KM) of enzymes representing three different classes: glucose-6-phosphate isomerase (GPI), an isomerase; Glyceraldehyde-3-phosphate dehydrogenase S (GAPDHS), an oxidoreductase; and pyruvate kinase (PK), a transferase. Contrary to other reports, we observed no trend in kinetic parameters for individual enzymes when found in monolayers (<100% enzyme coverage), suggesting an advantage for oriented immobilization versus other attachment strategies. Saturating the NPs to maximize activity per NP resulted in enzyme multilayer formation. Under these conditions, total activity per NP increased with increasing NP size. Conversely, specific activity for all three enzymes was highest when tethered to the smallest NPs, retaining a remarkable 73-94% of the activity of free/untethered enzymes. Multilayer formations caused a clear trend of kcat decreasing with increasing NP size, yet negligible change in KM. Understanding the fundamental relationships between NP size and tethered enzyme activity enables optimized design of various applications, maximizing activity per NP or activity per enzyme molecule.
Asunto(s)
Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/metabolismo , Oro/química , Nanopartículas del Metal/química , Tamaño de la Partícula , Adsorción , Histidina/química , Cinética , Modelos Moleculares , Oligopéptidos/química , Conformación ProteicaRESUMEN
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 Ca(2+) 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 Ca(2+) responses, reduced AE, and a strong subfertility phenotype. Surprisingly, AE depended on spatiotemporal information encoded by flux through CaV2.3, not merely the presence/amplitude of Ca(2+) waves. Using studies in both 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 a mechanistic understanding of membrane lipid regulation of Ca(2+) flux and therefore Ca(2+)-dependent cellular and developmental processes such as exocytosis and fertilization.
Asunto(s)
Acrosoma/metabolismo , Canales de Calcio Tipo R/fisiología , Calcio/metabolismo , Proteínas de Transporte de Catión/fisiología , Exocitosis/fisiología , Fertilización/fisiología , Gangliósido G(M1)/farmacología , Espermatozoides/metabolismo , Acrosoma/efectos de los fármacos , Animales , Células Cultivadas , Exocitosis/efectos de los fármacos , Fertilización/efectos de los fármacos , Masculino , Ratones , Oocitos/citología , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Espermatozoides/citología , Xenopus laevis/crecimiento & desarrollo , Xenopus laevis/metabolismoRESUMEN
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 into the protective, organic matrix. Here we show new applications for ferromagnetic nanoparticles (Fe3O4, MNPs) with peroxidase-like activity in potentiating the efficacy of H2O2 in biofilm degradation and prevention. Our data show that MNPs 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 the existing biofilm and prevented new biofilms from forming, killing both planktonic bacteria and those within the 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.
Asunto(s)
Bacterias/metabolismo , Materiales Biocompatibles/metabolismo , Biopelículas/efectos de los fármacos , Peróxido de Hidrógeno/química , Nanopartículas de Magnetita/química , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/farmacología , Materiales Biocompatibles/química , ADN/química , ADN/metabolismo , División del ADN/efectos de los fármacos , Óxido Ferrosoférrico/química , Oxidación-Reducción , Peroxidasas/metabolismo , Polisacáridos/química , Polisacáridos/metabolismo , Proteínas/química , Proteínas/metabolismo , Pseudomonas aeruginosa/fisiologíaRESUMEN
Maintaining activity of enzymes tethered to solid interfaces remains a major challenge in developing hybrid organic-inorganic devices. In nature, mammalian spermatozoa have overcome this design challenge by having glycolytic enzymes with specialized targeting domains that enable them to function while tethered to a cytoskeletal element. As a step toward designing a hybrid organic-inorganic ATP-generating system, we implemented a biomimetic site-specific immobilization strategy to tether two glycolytic enzymes representing different functional enzyme families: triose phosphoisomerase (TPI; an isomerase) and glyceraldehyde 3-phosphate dehydrogenase (GAPDHS; an oxidoreductase). We then evaluated the activities of these enzymes in comparison to when they were tethered via classical carboxyl-amine crosslinking. Both enzymes show similar surface binding regardless of immobilization method. Remarkably, specific activities for both enzymes were significantly higher when tethered using the biomimetic, site-specific immobilization approach. Using this biomimetic approach, we tethered both enzymes to a single surface and demonstrated their function in series in both forward and reverse directions. Again, the activities in series were significantly higher in both directions when the enzymes were coupled using this biomimetic approach versus carboxyl-amine binding. Our results suggest that biomimetic, site-specific immobilization can provide important functional advantages over chemically specific, but non-oriented attachment, an important strategic insight given the growing interest in recapitulating entire biological pathways on hybrid organic-inorganic devices.
Asunto(s)
Materiales Biomiméticos/química , Enzimas Inmovilizadas/química , Gliceraldehído-3-Fosfato Deshidrogenasas/química , Ingeniería de Proteínas/métodos , Triosa-Fosfato Isomerasa/química , Animales , Materiales Biomiméticos/metabolismo , Reactivos de Enlaces Cruzados/química , Pruebas de Enzimas , Enzimas Inmovilizadas/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Cinética , Masculino , Ratones , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Testículo/química , Testículo/enzimología , Triosa-Fosfato Isomerasa/metabolismoRESUMEN
The male germ cell-specific fatty acid-binding protein 9 (FABP9/PERF15) is the major component of the murine sperm perforatorium and perinuclear theca. Based on its cytoskeletal association and sequence homology to myelin P2 (FABP8), it has been suggested that FABP9 tethers sperm membranes to the underlying cytoskeleton. Furthermore, its upregulation in apoptotic testicular germ cells and its increased phosphorylation status during capacitation suggested multiple important functions for FABP9. Therefore, we investigated specific functions for FABP9 by means of targeted gene disruption in mice. FABP9(-/-) mice were viable and fertile. Phenotypic analysis showed that FABP9(-/-) mice had significant increases in sperm head abnormalities (~8% greater than their WT cohorts); in particular, we observed the reduction or absence of the characteristic structural element known as the "ventral spur" in ~10% of FABP9(-/-) sperm. However, deficiency of FABP9 affected neither membrane tethering to the perinuclear theca nor the fatty acid composition of sperm. Moreover, epididymal sperm numbers were not affected in FABP9(-/-) mice. Therefore, we conclude that FABP9 plays only a minor role in providing the murine sperm head its characteristic shape and is not absolutely required for spermatogenesis or sperm function.
Asunto(s)
Proteínas de Unión a Ácidos Grasos/genética , Cabeza del Espermatozoide/ultraestructura , Secuencia de Aminoácidos , Animales , Embrión de Mamíferos/metabolismo , Fertilidad/genética , Humanos , Masculino , Ratones , Microscopía Electrónica de Rastreo , Datos de Secuencia Molecular , Cabeza del Espermatozoide/fisiología , Espermatogénesis/genéticaRESUMEN
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 three 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 two different sterol-interacting proteins, apolipoprotein-A1 (apoA1) and prominin-1 (prom1) in sperm from different zones. We found that apoA1 was gradually added to the plasma membrane overlying the acrosome, whereas prom1 was not, suggesting different mechanisms for raft protein acquisition. Our results define raft-associating proteins, demonstrate functional similarities and differences among raft sub-types, and provide insights into raft-mediated epididymal protein acquisition.
Asunto(s)
Microdominios de Membrana/química , Proteoma/análisis , Espermatozoides/citología , Animales , Epidídimo/metabolismo , Masculino , Espectrometría de Masas , Microdominios de Membrana/metabolismo , Ratones , Proteoma/aislamiento & purificación , Proteoma/metabolismo , Espermatozoides/química , Espermatozoides/metabolismoRESUMEN
The development of complex hybrid organic-inorganic devices faces several challenges, including how they can generate energy. Cells face similar challenges regarding local energy production. Mammalian sperm solve this problem by generating ATP down the flagellar principal piece by means of glycolytic enzymes, several of which are tethered to a cytoskeletal support via germ-cell-specific targeting domains. Inspired by this design, we have produced recombinant hexokinase type 1 and glucose-6-phosphate isomerase capable of oriented immobilization on a nickel-nitrilotriacetic acid modified surface. Specific activities of enzymes tethered via this strategy were substantially higher than when randomly adsorbed. Furthermore, these enzymes showed sequential activities when tethered onto the same surface. This is the first demonstration of surface-tethered pathway components showing sequential enzymatic activities, and it provides a first step toward reconstitution of glycolysis on engineered hybrid devices.
Asunto(s)
Enzimas Inmovilizadas/metabolismo , Glucosa-6-Fosfato Isomerasa/metabolismo , Hexoquinasa/metabolismo , Adenosina Trifosfato , Animales , Glucosa/metabolismo , Glucosa-6-Fosfato Isomerasa/química , Hexoquinasa/química , Ratones , Níquel/química , Ácido Nitrilotriacético/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
Despite enormous interest in membrane raft micro-domains, 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 sub-types 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 micro-domain 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.
Asunto(s)
Microdominios de Membrana/metabolismo , Espermatozoides/citología , Espermatozoides/metabolismo , Animales , Fraccionamiento Celular , Detergentes/farmacología , Gangliósido G(M1)/metabolismo , Masculino , Espectrometría de Masas , Microdominios de Membrana/efectos de los fármacos , Microdominios de Membrana/ultraestructura , Proteínas de la Membrana/metabolismo , Ratones , Proteómica , Solubilidad/efectos de los fármacos , Espermatozoides/efectos de los fármacos , Espermatozoides/ultraestructura , Esteroles/metabolismoRESUMEN
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 >95% depletion of endogenous spermatogenesis, but retention of normal testis architecture. Then, 5-month-old recipient dogs (n=5) were focally irradiated on their testes prior to transplantation with mixed seminiferous tubule cells (fresh (n=2) or after 2 weeks of culture (n=3)). 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.
Asunto(s)
Espermatogénesis/fisiología , Espermatogonias/trasplante , Trasplante de Células Madre/métodos , Animales , Perros , Genotipo , Masculino , Repeticiones de Microsatélite , Modelos Animales , Espermatogénesis/efectos de la radiación , Espermatozoides/fisiología , Testículo/efectos de la radiación , Acondicionamiento Pretrasplante/métodos , Trasplante HomólogoRESUMEN
Male infertility is one possible consequence of a group of disorders arising from dysfunction of cilia. Ciliopathies include primary ciliary dyskinesia, polycystic kidney disease, Usher syndrome, nephronophthisis, Bardet-Biedl syndrome, Alstrom syndrome, and Meckel-Gruber syndrome as well as some forms of retinal degenerations. Mutations in the retinitis pigmentosa GTPase regulator gene (RPGR) are best known for leading to retinal degeneration but have also been associated with ciliary dysfunctions affecting other tissues. To further study the involvement of RPGR in ciliopathies, transgenic mouse lines overexpressing RPGR were generated. Animals carrying the transgene in varying copy numbers were investigated. We found that infertility due to aberrant spermatozoa correlated with increased copy numbers. In animals with moderately increased gene copies of Rpgr, structural disorganization in the flagellar midpiece, outer dense fibers, and fibrous sheath was apparent. In contrast, in animals with high copy numbers, condensed sperm heads were present, but the flagellum was absent in the vast majority of spermatozoa, although early steps of flagellar biogenesis were observed. This complexity of defects in flagellar assembly suggests a role of RPGR in intraflagellar transport processes.
Asunto(s)
Proteínas Portadoras/genética , Proteínas del Ojo/genética , Infertilidad Masculina/genética , Cola del Espermatozoide/metabolismo , Espermatogénesis/genética , Espermatozoides/anomalías , Animales , Transporte Biológico/genética , Proteínas Portadoras/metabolismo , Proteínas Portadoras/fisiología , Proteínas del Ojo/metabolismo , Proteínas del Ojo/fisiología , Dosificación de Gen/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Biológicos , Cola del Espermatozoide/fisiología , Cola del Espermatozoide/ultraestructura , Testículo/citología , Regulación hacia Arriba/fisiologíaRESUMEN
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.
Asunto(s)
Gangliósido G(M1)/análisis , Capacitación Espermática/fisiología , Espermatozoides/citología , Espermatozoides/fisiología , Animales , Biomarcadores/análisis , Bovinos , Procesamiento de Imagen Asistido por Computador , Masculino , Ratones , Microscopía Electrónica de Rastreo , Espermatozoides/ultraestructuraRESUMEN
PURPOSE: To map the canine rcd2 retinal degeneration locus. Rod-cone dysplasia type 2 (rcd2), an early-onset autosomal recessive form of progressive retinal atrophy (PRA), is phenotypically similar to early-onset forms of retinitis pigmentosa collectively termed Leber congenital amaurosis and segregates naturally in the collie breed of dog. Multiple genes have previously been evaluated as candidates for rcd2, but all have been excluded. METHODS: A set of informative experimental pedigrees segregating the rcd2 phenotype was produced. A genome-wide scan of these pedigrees using a set of 241 markers was undertaken. To refine the localized homology between canine and human maps, an RH map of the identified rcd2 region was built using a 3000 cR panel. A positional candidate gene strategy was then undertaken to begin to evaluate potentially causative genes. RESULTS: A locus responsible for the rcd2 phenotype was mapped to CFA7 in a region corresponding to human chromosome 1, region q32.1-q32.2. Maximum linkage was observed between rcd2 and marker FH3972 (theta = 0.02; lod = 25.53), and the critical region was flanked by markers FH2226 and FH3972. As CRB1 is the closest gene on human chromosome 1q known to cause retinal degeneration, canine gene-specific markers for CRB1 were developed, and this gene was excluded as a positional candidate for rcd2. CONCLUSIONS: The rcd2 locus represents a novel retinal degeneration gene. It is anticipated that when identified, this gene will offer new insights into retinal developmental and degenerative processes, and new opportunities for exploring experimental therapies.