RESUMEN

Aging leads to a general decline in protective immunity. The most common age-associated effects are in seen T-cell mediated immune function. Adult mice whose immune systems show only moderate changes in T-cell subsets tend to live longer than age-matched siblings that display extensive T-cell subset aging. Importantly, at the time of reproductive decline, the increase in disease risks in women significantly outpace those of men. In female mice, there is a significant decline in central and peripheral naïve T-cell subsets at the time of reproductive failure. Available evidence indicates that this naïve T-cell decline is sensitive to ovarian function and can be reversed in post-reproductive females by transplantation of young ovaries. The restoration of naïve T-cell subsets due to ovarian transplantation was impressive compared with post-reproductive control mice, but represented only a partial recovery of what was lost from 6 months of age. Apparently, the influence of ovarian function on immune function may be an indirect effect, likely moderated by other physiological functions. Estradiol is significantly reduced in post-reproductive females, but was not increased in post-reproductive females that received new ovaries, suggesting an estradiol-independent, but ovarian-dependent influence on immune function. Further evidence for an estradiol-independent influence includes the restoration of immune function through the transplantation of young ovaries depleted of follicles and through the injection of isolated ovarian somatic cells into the senescent ovaries of old mice. While the restoration of naïve T-cell populations represents only a small part of the immune system, the ability to reverse this important functional parameter independent of estradiol may hold promise for the improvement of post-reproductive female immune health. Further studies of the non-reproductive influence of the ovary will be needed to elucidate the mechanisms of the relationship between the ovary and health.

Asunto(s)

Estradiol , Linfocitos T , Femenino , Ratones , Animales , Ovario/fisiología , Reproducción/fisiología , Envejecimiento/fisiología

RESUMEN

PURPOSE: Successful identification of transcriptomic biomarkers within human IVF embryos may enhance implantation prediction and provide insights not available through conventional embryo biopsy genomic analysis. We demonstrate proof-of-concept for a methodology to assess overall embryo gene expression using qPCR with blastocoel fluid-conditioned media by examining the comparative presence of apoptotic genes. METHODS: Blastocoel fluid-conditioned media were collected from 19 embryos (11 euploid) following trophectoderm biopsy of day-5 ICSI-IVF blastocysts. Media were assessed for apoptotic gene expression via qPCR. Statistical analysis of gene expression was conducted via Wilcoxon Signed-Ranks test (overall expression), multivariate ANOVA (functional gene groups), and chi-square test of independence (gene level). RESULTS: A significantly higher overall apoptotic gene expression within euploid versus aneuploid embryos (p = 0.001) was observed. There was significantly (p = 0.045) higher expression of pro-apoptotic genes between implanted and not implanted embryos. Pro- vs. anti-apoptotic gene expression from all euploid embryos approached significance (p = 0.053). The ploidy status-based claim is further substantiated at the gene level with significantly higher expression of BBC3 (p = 0.012) and BCL2L13 (p = 0.003) in euploid embryos compared to aneuploid embryos. CONCLUSIONS: In this preliminary study, we demonstrate that (1) qualitative analysis of blastocoel fluid-conditioned media gene expression is possible, (2) global trends of expression are potentially related to clinical outcomes, and (3) gene-level expression trends exist and may be another viable metric for comparative expression between samples. The presence of statistical significance within analyses conducted with this sample size warrants a larger investigation of blastocoel fluid-conditioned media as an additional beneficial predictive tool for future IVF cases.

Asunto(s)

Diagnóstico Preimplantación , Aneuploidia , Blastocisto , Medios de Cultivo Condicionados , Femenino , Fertilización In Vitro/métodos , Expresión Génica , Humanos , Embarazo , Diagnóstico Preimplantación/métodos

RESUMEN

Male factors account for roughly half of infertility cases, with most male infertility diagnosed as idiopathic. Researchers predicting intrauterine insemination success rates have identified multiple prognostic factors, including semen parameters and seminal fluid composition. Seminal plasma contains extracellular exosomes, which contain RNAs and proteins involved in spermatogenesis. The contents of seminal plasma exosomes may be an indicator of overall sperm quality or fertility potential; therefore, analysis of exosomes may provide a measure for sperm viability and fertilization potential. In our study, exosomes were isolated and purified from seminal plasma obtained from IUI treatments with known pregnancy outcomes. We used a unique method to isolate the exosomes which made use of the hydrophobic interaction chromatography method. RNASeq was performed on RNAs from the purified exosomes. This analysis revealed holistic trends, including increased expression associated with RNA originating from chromosomes 1, 10, 12, 16 and 21. Overall, total RNA was significantly decreased and rRNA was significantly increased in successful IUI attempts. Furthermore, we found specific mRNAs and lincRNAs associated with positive versus negative pregnancy outcomes. Our study isolated and purified seminal plasma exosomes without ultracentrifugation, and it provides further evidence for differences in seminal plasma exosome molecular contents associated with pregnancy status.

Asunto(s)

Exosomas , Infertilidad Masculina , Cromatografía , Femenino , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Inseminación , Inseminación Artificial , Masculino , Embarazo , Resultado del Embarazo , ARN , Semen , Espermatozoides

RESUMEN

The threat of diminished antibiotic discovery has global health care in crisis. In the United States, it is estimated each year that over 2 million bacterial infections are resistant to first-line antibiotic treatments and cost in excess of 20 billion dollars. Many of these cases result from infection with the ESKAPE pathogens ( Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species), which are multidrug-resistant bacteria that often cause community- and hospital-acquired infections in both healthy and immunocompromised patients. Physicians have turned to last-resort antibiotics like polymyxins to tackle these pathogens, and as a consequence, polymyxin resistance has emerged and is spreading. Barring the discovery of new antibiotics, another route to successfully mitigate polymyxin resistance is to identify compounds that can complement the existing arsenal of antibiotics. We recently designed and performed a large-scale robotic screen to identify 43 bioactive compounds that act synergistically with polymyxin B to inhibit the growth of polymyxin-resistant Escherichia coli Of these 43 compounds, 5 lead compounds were identified and characterized using various Gram-negative bacterial organisms to better assess their synergistic activity with polymyxin. Several of these compounds reduce polymyxin to an MIC of <2 µg/ml against polymyxin-resistant and polymyxin-heteroresistant Gram-negative pathogens. Likewise, four of these compounds exhibit antimicrobial activity against Gram-positive bacteria, one of which rapidly eradicated methicillin-resistant Staphylococcus aureus We present multiple first-generation (i.e., not yet optimized) compounds that warrant further investigation and optimization, since they can act both synergistically with polymyxin and also as lone antimicrobials for combating ESKAPE pathogens.

Asunto(s)

Antibacterianos/farmacología , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Acinetobacter baumannii/efectos de los fármacos , Colistina/farmacología , Enterococcus faecium/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Klebsiella pneumoniae/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Polimixina B/farmacología , Polimixinas/farmacología , Pseudomonas aeruginosa/efectos de los fármacos , Staphylococcus aureus/efectos de los fármacos

RESUMEN

Burkholderia mallei causes the highly contagious and debilitating zoonosis glanders, which infects via inhalation or percutaneous inoculation and often culminates in life-threatening pneumonia and sepsis. In humans, glanders is difficult to diagnose and requires prolonged antibiotic therapy with low success rates. No vaccine exists to protect against B. mallei, and there is concern regarding its use as a bioweapon. The authors previously identified the protein BpaB as a potential target for devising therapies due to its role in adherence to host cells and the formation of biofilms in vitro and its contribution to pathogenicity in a mouse model of glanders. In the present study, the authors developed an immunostaining approach to probe tissues of experimentally infected animals and demonstrated that BpaB is produced exclusively in vivo by wild-type B. mallei in target organs from mice and marmosets. They detected the expression of BpaB by B. mallei both extracellularly and within macrophages, neutrophils, and epithelial cells in respiratory tissues (7/10 marmoset; 2/2 mouse). The authors also noted the intracellular expression of BpaB by B. mallei in macrophages in the regional lymph nodes of mice (2/2 tissues) and MALT of marmosets (4/5 tissues). It is interesting that B. mallei bacteria infecting distal organs did not express BpaB (2/2 mice; 3/3 marmosets), suggesting that the protein is not necessary for bacterial fitness in these anatomic locations. These findings underscore the value of BpaB as a target for developing medical countermeasures and provide insight into its role in pathogenesis.

Asunto(s)

Burkholderia mallei/patogenicidad , Muermo/microbiología , Factores de Virulencia/metabolismo , Animales , Anticuerpos Antibacterianos/inmunología , Antígenos Bacterianos/inmunología , Burkholderia mallei/inmunología , Burkholderia mallei/metabolismo , Callithrix/microbiología , Muermo/metabolismo , Macrófagos/microbiología , Ratones , Ratones Endogámicos BALB C , Factores de Virulencia/inmunología

RESUMEN

Burkholderia mallei, a facultative intracellular bacterium and tier 1 biothreat, causes the fatal zoonotic disease glanders. The organism possesses multiple genes encoding autotransporter proteins, which represent important virulence factors and targets for developing countermeasures in pathogenic Gram-negative bacteria. In the present study, we investigated one of these autotransporters, BatA, and demonstrate that it displays lipolytic activity, aids in intracellular survival, is expressed in vivo, elicits production of antibodies during infection, and contributes to pathogenicity in a mouse aerosol challenge model. A mutation in the batA gene of wild-type strain ATCC 23344 was found to be particularly attenuating, as BALB/c mice infected with the equivalent of 80 median lethal doses cleared the organism. This finding prompted us to test the hypothesis that vaccination with the batA mutant strain elicits protective immunity against subsequent infection with wild-type bacteria. We discovered that not only does vaccination provide high levels of protection against lethal aerosol challenge with B. mallei ATCC 23344, it also protects against infection with multiple isolates of the closely related organism and causative agent of melioidosis, Burkholderia pseudomallei Passive-transfer experiments also revealed that the protective immunity afforded by vaccination with the batA mutant strain is predominantly mediated by IgG antibodies binding to antigens expressed exclusively in vivo Collectively, our data demonstrate that BatA is a target for developing medical countermeasures and that vaccination with a mutant lacking expression of the protein provides a platform to gain insights regarding mechanisms of protective immunity against B. mallei and B. pseudomallei, including antigen discovery.

Asunto(s)

Anticuerpos Antibacterianos/inmunología , Burkholderia mallei/inmunología , Burkholderia pseudomallei/inmunología , Melioidosis/prevención & control , Animales , Proteínas Bacterianas/genética , Burkholderia mallei/genética , Burkholderia mallei/crecimiento & desarrollo , Burkholderia mallei/patogenicidad , Burkholderia pseudomallei/patogenicidad , Modelos Animales de Enfermedad , Muermo/inmunología , Muermo/microbiología , Muermo/prevención & control , Inmunoglobulina G/inmunología , Melioidosis/inmunología , Melioidosis/microbiología , Ratones , Ratones Endogámicos BALB C , Mutación , Vacunación , Factores de Virulencia/genética

RESUMEN

Determining the chemical composition of biological materials is paramount to the study of natural phenomena. Here, we describe the composition of model gram-negative outer membranes, focusing on the predominant assembly, an asymmetrical bilayer of lipid molecules. We also give an overview of lipid biosynthetic pathways and molecular mechanisms that organize this material into the outer membrane bilayer. An emphasis is placed on the potential of these pathways as targets for antibiotic development. We discuss deviations in composition, through bacterial cell surface remodeling, and alternative modalities to the asymmetric lipid bilayer. Outer membrane lipid alterations of current microbiological interest, such as lipid structures found in commensal bacteria, are emphasized. Additionally, outer membrane components could potentially be engineered to develop vaccine platforms. Observations related to composition and assembly of gram-negative outer membranes will continue to generate novel discoveries, broaden biotechnologies, and reveal profound mysteries to compel future research.

Asunto(s)

Membrana Celular/metabolismo , Bacterias Gramnegativas/metabolismo , Membrana Dobles de Lípidos/química , Membrana Celular/química , Membrana Celular/genética , Bacterias Gramnegativas/química , Bacterias Gramnegativas/genética , Membrana Dobles de Lípidos/metabolismo

RESUMEN

Burkholderia mallei is a highly pathogenic bacterium that causes the zoonosis glanders. Previous studies indicated that the genome of the organism contains eight genes specifying autotransporter proteins, which are important virulence factors of Gram-negative bacteria. In the present study, we report the characterization of one of these autotransporters, BpaB. Database searches identified the bpaB gene in ten B. mallei isolates and the predicted proteins were 99-100% identical. Comparative sequence analyses indicate that the gene product is a trimeric autotransporter of 1,090 amino acids with a predicted molecular weight of 105-kDa. Consistent with this finding, we discovered that recombinant bacteria expressing bpaB produce a protein of ≥ 300-kDa on their surface that is reactive with a BpaB-specific monoclonal antibody. Analysis of sera from mice infected with B. mallei indicated that animals produce antibodies against BpaB during the course of disease, thus establishing production of the autotransporter in vivo. To gain insight on its role in virulence, we inactivated the bpaB gene of B. mallei strain ATCC 23344 and determined the median lethal dose of the mutant in a mouse model of aerosol infection. These experiments revealed that the bpaB mutation attenuates virulence 8-14 fold. Using a crystal violet-based assay, we also discovered that constitutive production of BpaB on the surface of B. mallei promotes biofilm formation. To our knowledge, this is the first report of a biofilm factor for this organism.

Asunto(s)

Proteínas Bacterianas/genética , Biopelículas/crecimiento & desarrollo , Burkholderia mallei/genética , Burkholderia mallei/patogenicidad , Muermo/microbiología , Sistemas de Secreción Tipo V/genética , Aerosoles , Animales , Anticuerpos Antibacterianos/química , Anticuerpos Monoclonales/química , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Burkholderia mallei/metabolismo , Línea Celular , Clonación Molecular , Células Epiteliales/microbiología , Células Epiteliales/patología , Escherichia coli/genética , Escherichia coli/metabolismo , Femenino , Eliminación de Gen , Expresión Génica , Muermo/mortalidad , Muermo/patología , Muermo/transmisión , Humanos , Macrófagos/microbiología , Macrófagos/patología , Ratones , Ratones Endogámicos BALB C , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análisis de Supervivencia , Sistemas de Secreción Tipo V/química , Sistemas de Secreción Tipo V/metabolismo , Virulencia

RESUMEN

Burkholderia mallei is a host-adapted bacterium that does not persist outside of its equine reservoir. The organism causes the zoonosis glanders, which is endemic in Asia, Africa, the Middle East and South America. Infection by B. mallei typically occurs via the respiratory or percutaneous route, and the most common manifestations are life-threatening pneumonia and bacteremia. Glanders is difficult to diagnose and requires prolonged antibiotic therapy with low success rates. There is no vaccine to protect against B. mallei and there is concern regarding its use as a biothreat agent. Thus, experiments were performed to establish a non-human primate model of intranasal infection to study the organism and develop countermeasures. Groups of marmosets (Callithrix jacchus) were inoculated intranasally with B. mallei strain ATCC 23344 and monitored for clinical signs of illness for up to 13 days. We discovered that 83% of marmosets inoculated with doses of 2.5 X 10(4) to 2.5 X 10(5) bacteria developed acute lethal infection within 3-4 days. Signs of disease were severe and included lethargy, inappetence, conjunctivitis, mucopurulent and hemorrhagic nasal discharges, and increased respiratory effort with abdominal lifts. Burkholderia mallei was cultured from the lungs, spleen and liver of these animals, and pathologic examination of tissues revealed lesions characteristic of glanders. Challenge experiments also revealed that 91% of animals infected with doses ranging from 25 to 2.5 X 10(3) bacteria exhibited mild non-specific signs of illness and were culture negative. One marmoset inoculated with 2.5 X 10(3) organisms developed moderate signs of disease and reached humane end-points 8 days post-infection. The liver and spleen of this animal were colonized with the agent and pathological analysis of tissues showed nasal, splenic and hepatic lesions. Taken together, these data indicate that the marmoset is a suitable model to study respiratory infection by B. mallei.

Asunto(s)

Burkholderia mallei/patogenicidad , Callithrix/microbiología , Muermo/etiología , Administración Intranasal , Animales , Carga Bacteriana , Modelos Animales de Enfermedad , Femenino , Muermo/patología , Muermo/transmisión , Caballos , Humanos , Hígado/microbiología , Hígado/patología , Pulmón/microbiología , Pulmón/patología , Masculino , Especificidad de la Especie , Bazo/microbiología , Bazo/patología , Zoonosis/etiología , Zoonosis/patología , Zoonosis/transmisión

RESUMEN

The ubiquitin-proteasome system (UPS) controls intracellular protein turnover in a substrate-specific manner via E3-type ubiquitin ligases. Mammalian fertilization and particularly sperm penetration through the oocyte vitelline coat, the zona pellucida (ZP), is regulated by UPS. We use an extrinsic substrate of the proteasome-dependent ubiquitin-fusion degradation pathway, the mutant ubiquitin UBB(+1), to provide evidence that an E3-type ligase activity exists in sperm-acrosomal fractions. Protein electrophoresis gels from such de novo ubiquitination experiments contained a unique protein band identified by tandem mass spectrometry as being similar to ubiquitin ligase UBR7 (alternative name: C14ORF130). Corresponding mRNA was amplified from boar testis and several variants of the UBR7 protein were detected in boar, mouse and human sperm extracts by Western blotting. Genomic analysis indicated a high degree of evolutionary conservation, remarkably constant purifying selection and conserved testis expression of the UBR7 gene. By immunofluorescence, UBR7 was localized to the spermatid acrosomal cap and sperm acrosome, in addition to hotspots of proteasomal activity in spermatids, such as the cytoplasmic lobe, caudal manchette, nucleus and centrosome. During fertilization, UBR7 remained with the ZP-bound acrosomal shroud following acrosomal exocytosis. Thus, UBR7 is present in the acrosomal cap of round spermatids and within the acrosomal matrix of mature boar spermatozoa. These data provide the first evidence of ubiquitin ligase activity in mammalian spermatozoa and indicate UBR7 involvement in spermiogenesis.

Asunto(s)

Espermatozoides/enzimología , Ubiquitina-Proteína Ligasas/metabolismo , Acrosoma/efectos de los fármacos , Acrosoma/metabolismo , Animales , Anticuerpos Bloqueadores/farmacología , Western Blotting , Fertilización In Vitro , Técnica del Anticuerpo Fluorescente , Humanos , Masculino , Ratones , Filogenia , Transporte de Proteínas/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Espermátides/citología , Espermátides/efectos de los fármacos , Espermátides/metabolismo , Espermatozoides/citología , Espermatozoides/efectos de los fármacos , Porcinos , Testículo/efectos de los fármacos , Testículo/metabolismo , Ubiquitina-Proteína Ligasas/genética

RESUMEN

Burkholderia pseudomallei, the etiologic agent of melioidosis, is a saprophytic bacterium readily isolated from wet soils of countries bordering the equator. Burkholderia mallei is a host-adapted clone of B. pseudomallei that does not persist outside of its equine reservoir and causes the zoonosis glanders, which is endemic in Asia, Africa, the Middle East and South America. Infection by these organisms typically occurs via percutaneous inoculation or inhalation of aerosols, and the most common manifestation is severe pneumonia leading to fatal bacteremia. Glanders and melioidosis are difficult to diagnose and require prolonged antibiotic therapy with low success rates. There are no vaccines available to protect against either Burkholderia species, and there is concern regarding their use as biological warfare agents given that B. mallei has previously been utilized in this manner. Hence, experiments were performed to establish a mouse model of aerosol infection to study the organisms and develop countermeasures. Using a hand-held aerosolizer, BALB/c mice were inoculated intratracheally with strains B. pseudomallei 1026b and B. mallei ATCC23344 and growth of the agents in the lungs, as well as dissemination to the spleen, were examined. Mice infected with 10(2), 10(3) and 10(4) organisms were unable to control growth of B. mallei in the lungs and bacteria rapidly disseminated to the spleen. Though similar results were observed in mice inoculated with 10(3) and 10(4) B. pseudomallei cells, animals infected with 10(2) organisms controlled bacterial replication in the lungs, dissemination to the spleen, and the extent of bacteremia. Analysis of sera from mice surviving acute infection revealed that animals produced antibodies against antigens known to be targets of the immune response in humans. Taken together, these data show that small volume aerosol inoculation of mice results in acute disease, dose-dependent chronic infection, and immune responses that correlate with those seen in human infections.

Asunto(s)

Aerosoles/administración & dosificación , Anticuerpos Antibacterianos/sangre , Bacteriemia/inmunología , Burkholderia mallei/inmunología , Burkholderia pseudomallei/inmunología , Muermo/inmunología , Melioidosis/inmunología , Administración por Inhalación , Animales , Bacteriemia/sangre , Bacteriemia/microbiología , Bacteriemia/patología , Armas Biológicas , Burkholderia mallei/patogenicidad , Burkholderia pseudomallei/patogenicidad , Modelos Animales de Enfermedad , Femenino , Muermo/sangre , Muermo/microbiología , Muermo/patología , Caballos , Humanos , Pulmón/inmunología , Pulmón/microbiología , Pulmón/patología , Melioidosis/sangre , Melioidosis/microbiología , Melioidosis/patología , Ratones , Ratones Endogámicos BALB C , Bazo/inmunología , Bazo/microbiología , Bazo/patología

RESUMEN

CASE DESCRIPTION: A 1.5-year-old mixed-breed dog was examined because of a 1-month history of anorexia, vomiting, diarrhea, and weight loss. CLINICAL FINDINGS: The dog was very thin on physical examination (body condition score, 3/9). Results of all diagnostic tests were within reference limits except intestinal thickening and lymphadenopathy were identified on abdominal ultrasound examination. During exploratory laparotomy, thickening at the ileocecal-colic junction and within the transverse colon and mesenteric lymphadenopathy were identified, and the ileocecal-colic junction was resected. Histopathologic evaluation of the ileocecal-colic junction and full-thickness biopsy specimens from other sites as well as results of a serum ELISA were diagnostic for gastrointestinal Pythium insidiosum infection. TREATMENT AND OUTCOME: Pythiosis was initially treated medically with administration of itraconazole and terbinafine by mouth, but the colonic lesion was progressive with this regimen. Two months after diagnosis, a subtotal colectomy was performed; marginal excision (0.6 cm) was obtained at the aboral margin. The dog was treated with 3 doses of a pythiosis vaccine beginning approximately 2 weeks after surgery and was continued on itraconazole and terbinafine for 5 months. Parenteral and enteral nutrition as well as considerable general supportive care were required postoperatively. Six months after treatment, the dog had a normal serum ELISA titer. Two years after treatment, the dog had returned to preoperative weight and was clinically normal. CLINICAL RELEVANCE: This patient had an unusually positive therapeutic response to chronic, extensive, marginally excised gastrointestinal pythiosis.

Asunto(s)

Enfermedades de los Perros/microbiología , Enfermedades Intestinales/veterinaria , Pitiosis/veterinaria , Vacunas/inmunología , Animales , Antiinfecciosos/administración & dosificación , Antiinfecciosos/uso terapéutico , Enfermedades de los Perros/patología , Enfermedades de los Perros/terapia , Perros , Enrofloxacina , Fluoroquinolonas/uso terapéutico , Enfermedades Intestinales/microbiología , Enfermedades Intestinales/terapia , Itraconazol/administración & dosificación , Itraconazol/uso terapéutico , Masculino , Naftalenos/administración & dosificación , Naftalenos/uso terapéutico , Pitiosis/terapia , Pythium/inmunología , Terbinafina

Asunto(s)

Ascomicetos/aislamiento & purificación , Diarrea/veterinaria , Enfermedades de los Perros/orina , Giardia/aislamiento & purificación , Urinálisis/veterinaria , Animales , Diarrea/microbiología , Diarrea/parasitología , Enfermedades de los Perros/microbiología , Enfermedades de los Perros/parasitología , Perros , Heces/parasitología , Femenino , Urinálisis/normas

RESUMEN

Despite decades of research, the mechanism by which the fertilizing spermatozoon penetrates the mammalian vitelline membrane, the zona pellucida (ZP) remains one of the unexplained fundamental events of human/mammalian development. Evidence has been accumulating in support of the 26S proteasome as a candidate for echinoderm, ascidian and mammalian egg coat lysin. Monitoring ZP protein degradation by sperm during fertilization is nearly impossible because those few spermatozoa that penetrate the ZP leave behind a virtually untraceable residue of degraded proteins. We have overcome this hurdle by designing an experimentally consistent in vitro system in which live boar spermatozoa are co-incubated with ZP-proteins (ZPP) solubilized from porcine oocytes. Using this assay, mimicking sperm-egg interactions, we demonstrate that the sperm-borne proteasomes can degrade the sperm receptor protein ZPC. Upon coincubation with motile spermatozoa, the solubilized ZPP, which appear to be ubiquitinated, adhered to sperm acrosomal caps and induced acrosomal exocytosis/formation of the acrosomal shroud. The degradation of the sperm receptor protein ZPC was assessed by Western blotting band-densitometry and proteomics. A nearly identical pattern of sperm receptor degradation, evident already within the first 5 min of coincubation, was observed when the spermatozoa were replaced with the isolated, enzymatically active, sperm-derived proteasomes. ZPC degradation was blocked by proteasomal inhibitors and accelerated by ubiquitin-aldehyde(UBAL), a modified ubiquitin protein that stimulates proteasomal proteolysis. Such a degradation pattern of ZPC is consistent with in vitro fertilization studies, in which proteasomal inhibitors completely blocked fertilization, and UBAL increased fertilization and polyspermy rates. Preincubation of intact zona-enclosed ova with isolated active sperm proteasomes caused digestion, abrasions and loosening of the exposed zonae, and significantly reduced the fertilization/polyspermy rates after IVF, accompanied by en-mass detachment of zona bound sperm. Thus, the sperm borne 26S proteasome is a candidate zona lysin in mammals. This new paradigm has implications for contraception and assisted reproductive technologies in humans, as well as animals.

Asunto(s)

Fertilización/fisiología , Complejo de la Endopetidasa Proteasomal/metabolismo , Receptores de Superficie Celular/metabolismo , Espermatozoides/metabolismo , Zona Pelúcida/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas del Huevo/química , Proteínas del Huevo/metabolismo , Femenino , Masculino , Mamíferos/metabolismo , Mamíferos/fisiología , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Modelos Biológicos , Datos de Secuencia Molecular , Complejo de la Endopetidasa Proteasomal/fisiología , Procesamiento Proteico-Postraduccional/fisiología , Receptores de Superficie Celular/química , Interacciones Espermatozoide-Óvulo/fisiología , Porcinos , Glicoproteínas de la Zona Pelúcida

RESUMEN

PURPOSE: Identification of the biomarkers of oocyte quality, and developmental and reprogramming potential is of importance to assisted reproductive technology in humans and animals. EXPERIMENTAL DESIGN: PerkinElmer ExacTag™ Kit was used to label differentially proteins in pig oocyte extracts (oocyte proteome) and pig oocyte-conditioned in vitro maturation media (oocyte secretome) obtained with high- and low-quality oocytes. RESULTS: We identified 16 major proteins in the oocyte proteome that were expressed differentially in high- versus low-quality oocytes. More abundant proteins in the high-quality oocyte proteome included kelch-like ECH-associated protein 1 (an adaptor for ubiquitin-ligase CUL3), nuclear export factor CRM1 and ataxia-telangiectasia mutated protein kinase. Dystrophin (DMD) was more abundant in low-quality oocytes. In the secretome, we identified 110 proteins, including DMD and cystic fibrosis transmembrane conductance regulator, two proteins implicated in muscular dystrophy and cystic fibrosis, respectively. Monoubiquitin was identified in the low-quality-oocyte secretome. CONCLUSIONS AND CLINICAL IMPLICATIONS: A direct, quantitative proteomic analysis of small oocyte protein samples can identify potential markers of oocyte quality without the need for a large amount of total protein. This approach will be applied to discovery of non-invasive biomarkers of oocyte quality in assisted human reproduction and in large animal embryo transfer programs.

Asunto(s)

Oocitos/metabolismo , Proteómica/métodos , Animales , Biomarcadores/metabolismo , Femenino , Regulación de la Expresión Génica , Humanos , Proteoma/metabolismo , Porcinos

RESUMEN

The molecular basis underlying the binding of spermatozoa to their homologous eggs and the subsequent induction of acrosomal exocytosis remain a major unresolved issue in mammalian fertilization. Novel cell adhesion systems are now being explored to advance this research. Triantennary and tetraantennary N-glycans have previously been implicated as the major carbohydrate sequences that mediate the initial binding of spermatozoa to the specialized egg coat (zona pellucida) in the murine and porcine models. Mouse spermatozoa also undergo binding to rabbit erythrocytes (rRBCs), presumably via the interaction of their lectin-like egg-binding proteins with branched polylactosamine sequences present on these somatic cells. Experiments presented in this study confirm that boar spermatozoa also bind to rRBCs. However, unlike mouse spermatozoa, boar spermatozoa also undergo acrosomal exocytosis within 30 min after binding to rRBCs. Both binding and induction of acrosomal exocytosis in this system did not require the participation of terminal Galalpha1-3Gal sequences that are found on rRBCs. Pronase glycopeptides derived from rRBCs inhibited the binding of boar sperm to porcine oocytes by 91% at a final concentration of 0.3 mg/ml under standard IVF conditions. Binding in this porcine cell adhesion model was also completely blocked at this concentration of glycopeptide. Thus, adhesion results from the interaction of the egg-binding protein expressed on the surface of boar spermatozoa with the glycans presented on rRBCs. This cell adhesion model will be useful for investigating the molecular basis of gamete binding and the induction of acrosomal exocytosis in the pig.

Asunto(s)

Acrosoma/fisiología , Metabolismo de los Hidratos de Carbono , Adhesión Celular/fisiología , Exocitosis/fisiología , Oocitos/fisiología , Espermatozoides/fisiología , Porcinos/fisiología , Acrosoma/metabolismo , Acrosoma/ultraestructura , Animales , Eritrocitos/metabolismo , Eritrocitos/ultraestructura , Citometría de Flujo/veterinaria , Pruebas de Hemaglutinación/veterinaria , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica de Transmisión/veterinaria , Oocitos/citología , Oocitos/metabolismo , Oocitos/ultraestructura , Espermatozoides/citología , Espermatozoides/metabolismo , Espermatozoides/ultraestructura

RESUMEN

Proteolysis of ubiquitinated sperm and oocyte proteins by the 26S proteasome is necessary for the success of mammalian fertilization, including but not limited to acrosomal exocytosis and sperm-zona pellucida (ZP) penetration. The present study examined the role of PSMD4, an essential non-ATPase subunit of the proteasomal 19S regulatory complex responsible for proteasome-substrate recognition, in sperm-ZP penetration during porcine fertilization in vitro (IVF). Porcine sperm-ZP penetration, but not sperm-ZP binding, was blocked in the presence of a monoclonal anti-PSMD4 antibody during IVF. Inclusion in the fertilization medium of mutant ubiquitins (Ub+1 and Ub5+1), which are refractory to processing by the 19S regulatory complex and associated with Alzheimer's disease, also inhibited fertilization. This observation suggested that subunit PSMD4 is exposed on the sperm acrosomal surface, a notion that was further supported by the binding of non-cell permeant, biotinylated proteasomal inhibitor ZL3VS to the sperm acrosome. Immunofluorescence localized PSMD4 in the sperm acrosome. Immunoprecipitation and proteomic analysis revealed that PSMD4 co-precipitated with porcine sperm-associated acrosin inhibitor (AI). Ubiquitinated species of AI were isolated from boar sperm extracts by affinity purification of ubiquitinated proteins using the recombinant UBA domain of p62 protein. Some proteasomes appeared to be anchored to the sperm head inner acrosomal membrane, as documented by co-fractionation studies. In conclusion, the 19S regulatory complex subunit PSMD4 is involved in the sperm-ZP penetration during fertilization. The recognition of substrates on the ZP by the 19S proteasomal regulatory complex is essential for the success of porcine/mammalian fertilization in vitro.

Asunto(s)

Inhibidores de Proteasoma , Interacciones Espermatozoide-Óvulo , Espermatozoides/enzimología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/farmacología , Western Blotting , Proteínas Portadoras/inmunología , Fertilización In Vitro/efectos de los fármacos , Masculino , Datos de Secuencia Molecular , Complejo de la Endopetidasa Proteasomal/inmunología , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteómica , Proteínas de Secreción de la Vesícula Seminal/inmunología , Interacciones Espermatozoide-Óvulo/efectos de los fármacos , Porcinos , Inhibidor de Tripsina Pancreática de Kazal/inmunología , Proteínas Ubiquitinadas/metabolismo

RESUMEN

The 26S proteasome is a multi-subunit protease specifically targeting ubiquitinated proteins. A consensus has emerged from studies by multiple laboratories on the role of sperm-borne proteasomes in human, mouse, pig, bovine, ascidian and echinoderm fertilization. Major findings from the studies in various mammalian and non-mammalian fertilization systems are (1) proteasomes are present in the mammalian sperm acrosome and on the acrosomal surface; (2) ubiquitinated proteins are present on the mammalian, ascidian and echinoderm egg coat; (3) proteasomal proteolytic and ubiquitin-deconjugating (deubiquitinating) activities can be detected in viable, motile mammalian spermatozoa; (4) proteasomes remain associated with the sperm head following ZP-induced acrosomal exocytosis; (5) inhibition of ubiquitination and proteasomal proteolysis blocks fertilization in mammals, ascidians and echinoderms; (6) inhibition of proteasomal proteolysis alters the course of mammalian sperm capacitation and acrosomal exocytosis induced by sperm binding to the egg coat, zona pellucida (ZP); (7) depletion of the sperm surface-associated ATP blocks porcine and echinoderm fertilization, most likely by affecting the integrity of sperm proteasomes, of which several subunits are ATPases; (8) inhibition of proteasomal proteolysis blocks sperm-ZP penetration, but does not alter the rate of sperm-ZP binding in mammals, and (9) experimental modification of sperm-associated deubiquitinating activities shifts the balance of monospermic fertilization to polyspermic fertilization in vitro. Altogether, these studies provide evidence for the involvement of the 26S proteasome in multiple steps of animal and human fertilization, offering a novel model of sperm-egg coat interactions, and identifying a range of potential new sperm quality markers and contraceptive targets.

Asunto(s)

Acrosoma/fisiología , Fertilización , Complejo de la Endopetidasa Proteasomal/fisiología , Capacitación Espermática , Espermatozoides/fisiología , Acrosoma/ultraestructura , Animales , Humanos , Masculino , Modelos Biológicos , Espermatozoides/ultraestructura , Ubiquitinación

RESUMEN

Peroxiredoxin 2 (PRDX2) is a highly efficient redox protein that neutralizes hydrogen peroxide, resulting in protection of cells from oxidative damage and in regulation of peroxide-mediated signal transduction events. The oxidized form of PRDX2 is reverted back to the reduced form by the thioredoxin system. In the present study, we investigated the presence of PRDX2 in mouse and boar spermatozoa and in mouse spermatids using proteomic techniques and immunocytochemistry. Sperm and spermatid extracts displayed a 20-kDa PRDX2 band on Western blotting. PRDX2 occurred as a Triton-soluble form in spermatids and as a Triton-insoluble form in mature spermatozoa. Boar seminiferous tubule extracts were immunoprecipitated with PRDX2 antibody and separated by SDS-PAGE. Peptide mass fingerprinting by matrix-assisted laser desorption ionization-time of flight (TOF) and microsequencing by nanospray quadrupole-quadrupole TOF tandem mass spectrometry revealed the presence of PRDX2 ions in the immunoprecipitated band, along with sperm mitochondria-associated cysteine-rich protein, cellular nucleic acid-binding protein, and glutathione peroxidase 4. In mouse spermatocytes and spermatids, diffuse labeling of PRDX2 was observed in the cytoplasm and residual bodies. After spermiation, PRDX2 localization became confined to the mitochondrial sheath of the sperm tail midpiece. Boar spermatozoa displayed similar PRDX2 localization as in mouse spermatozoa. Boar spermatozoa with disrupted acrosomes expressed PRDX2 in the postacrosomal sheath region. Peroxidase enzyme activity of boar sperm extracts was evaluated by estimating the rate of NADPH oxidation in the presence or absence of a glutathione depletor (diethyl maleate) or a glutathione reductase inhibitor (carmustine). Diethyl maleate partially inhibited peroxidase activity, whereas carmustine showed an insignificant effect. These observations suggest that glutathione and glutathione reductase activity contribute only partially to the total peroxidase activity of the sperm extract. While the specific role of PRDX2 in the total peroxidase activity of sperm extract is still an open question, the present study for the first time (to our knowledge) shows the presence of PRDX2 in mammalian spermatozoa. Peroxidase activity in sperm extracts is not due to the glutathione system and therefore possibly involves PRDX2 and other peroxiredoxins.

Asunto(s)

Peroxidasas/metabolismo , Peroxirredoxinas/metabolismo , Espermátides/enzimología , Animales , Antineoplásicos Alquilantes , Carmustina , Citoplasma/metabolismo , Peróxido de Hidrógeno/metabolismo , Inmunohistoquímica , Masculino , Ratones , NADP/metabolismo , Peroxirredoxinas/química , Solubilidad , Cabeza del Espermatozoide/enzimología , Porcinos , Testículo/enzimología

RESUMEN

Cyanobacteria and similar organisms produced most of the oxygen found in Earth's atmosphere, which implies that early photosynthetic organisms would have lived in an atmosphere that was rich in CO2 and poor in O2. We investigated the tolerance of several cyanobacteria to very high (>20 kPa) concentrations of atmospheric CO2. Cultures of Synechococcus PCC7942, Synechocystis PCC7942, Plectonema boryanum, and Anabaena sp. were grown in liquid culture sparged with CO2-enriched air. All four strains grew when transferred from ambient CO2 to 20 kPa partial pressure of CO2 (pCO2), but none of them tolerated direct transfer to 40 kPa pCO2. Synechococcus and Anabaena survived 101 kPa (100%) pCO2 when pressure was gradually increased by 15 kPa per day, and Plectonema actively grew under these conditions. All four strains grew in an anoxic atmosphere of 5 kPa pCO2 in N2. Strains that were sensitive to high CO2 were also sensitive to low initial pH (pH 5-6). However, low pH in itself was not sufficient to prevent growth. Although mechanisms of damage and survival are still under investigation, we have shown that modern cyanobacteria can survive under Earth's primordial conditions and that cyanobacteria-like organisms could have flourished under conditions on early Mars, which probably had an atmosphere similar to early Earth's.

Asunto(s)

Dióxido de Carbono/metabolismo , Cianobacterias/crecimiento & desarrollo , Cianobacterias/metabolismo , Anabaena/crecimiento & desarrollo , Anabaena/metabolismo , Planeta Tierra , Exobiología , Agua Dulce/microbiología , Concentración de Iones de Hidrógeno , Marte , Origen de la Vida , Plectonema/crecimiento & desarrollo , Plectonema/metabolismo , Synechococcus/crecimiento & desarrollo , Synechococcus/metabolismo , Synechocystis/crecimiento & desarrollo , Synechocystis/metabolismo