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Despite its great scientific and technological importance, wall-bounded turbulence is an unresolved problem in classical physics that requires new perspectives to be tackled. One of the key strategies has been to study interactions among the energy-containing coherent structures in the flow. Such interactions are explored in this study using an explainable deep-learning method. The instantaneous velocity field obtained from a turbulent channel flow simulation is used to predict the velocity field in time through a U-net architecture. Based on the predicted flow, we assess the importance of each structure for this prediction using the game-theoretic algorithm of SHapley Additive exPlanations (SHAP). This work provides results in agreement with previous observations in the literature and extends them by revealing that the most important structures in the flow are not necessarily the ones with the highest contribution to the Reynolds shear stress. We also apply the method to an experimental database, where we can identify structures based on their importance score. This framework has the potential to shed light on numerous fundamental phenomena of wall-bounded turbulence, including novel strategies for flow control.
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BACKGROUND: Numerous conventional magnetic resonance imaging (cMRI) parameters were previously found to differentiate parkinsonian disorders with statistical significance, but effect size has not been considered. OBJECTIVES: To quantify effect size of previously identified cMRI parameters that differentiated parkinsonian disorders with statistical significance. METHOD: A PubMed search limited to studies assessing cMRI parameters in at least 2 of Parkinson's disease, progressive supranuclear palsy, multiple system atrophy, and corticobasal degeneration/syndrome were selected. Either Cohen's d or positive and negative likelihood (LR+/-) as well as diagnostic odds ratios (DORs) were calculated as appropriate. cMRI parameter was considered useful if Cohen's d > 1.94 (<20% overlap) or if LR+ > 10, LR- < 0.1, or DOR > 20. RESULTS: Literature search identified 8848 publications and 36 were included for analysis. Putaminal (Cohen's d 2.07; DOR 23-infinity), pontine (DOR 32-infinity), and middle cerebellar peduncle (Cohen's d 2.24; DOR infinity) abnormalities were most useful in differentiating multiple system atrophy while reduced midbrain (Cohen's d 2.33-8.69; DOR infinity) and superior cerebellar peduncle (Cohen's d 2.47; DOR 51-infinity) diameters separated progressive supranuclear palsy. Corticobasal degeneration/syndrome does not have any distinguishing cMRI features, but reduced midbrain diameter may help differentiate corticobasal degeneration/syndrome from Parkinson's disease (DOR infinity). When LR- was calculated, all of these features carried a value of <0.1. CONCLUSION: A number of cMRI features consistently demonstrated large effect size in separating parkinsonian disorders. However, it is the presence and not absence of these cMRI features that is most useful in patients with low to moderate pretest probability.
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BACKGROUND: While numerous validated questionnaires measuring psychiatric symptoms in Parkinson's disease (PD) are available, a quick multifaceted screening tool is lacking. OBJECTIVE: To generate the Brief Psychiatric Questionnaire (BPQ) that quickly screens for depression, anxiety, and apathy and to evaluate its content validity against three reference scales. METHODS: Forty-seven questions were drafted and measured against the Geriatric Depression Scale (GDS), State Trait Anxiety Inventory (Form Y2) (STAI-Y2), and Apathy Evaluation Scale (AES). Data were reduced by principal component analysis and linear regression. Content validity and repeatability were assessed in a second cohort. RESULTS: Data from ninety-five patients were used for BPQ development. Variation explained by the final linear regression models was 52% for GDS (R 2 = 0.521, F(2,94) = 49.97, p < 0.0001), 65% for STAI-Y2 (R 2 = 0.652, F(4,94) = 42.08, p < 0.0001), and 14% for AES (R 2 = 0.135, F(1,94) = 14.51, p < 0.0001). From the initial pool, only five questions remained for further testing. BPQ questions correctly identified 88% in the second cohort of 33 patients scoring more than five on GDS and 91% who scored in the highest decile of STAI-Y2, but only 51% who scored in the highest decile of AES. Moderate to strong correlation (r = 0.464 - 0.733, p < 0.004) between predicted scores based on BPQ questions and actual scores of three validated questionnaires was demonstrated. Good repeatability of BPQ questions was demonstrated by moderate to high intraclass correlation coefficients (0.47-0.772, p < 0.01). CONCLUSIONS: BPQ questions were able to accurately identify patients at risk of depression and anxiety but not apathy. It is brief and multifaceted and can act as a preconsultation tool to prompt further psychiatric assessment.
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The biology of transport of spermatids and spermatid adhesion across the seminiferous epithelium during the epithelial cycle remains largely unexplored. Nonetheless, studies have implicated the role of motor proteins in these cellular events. In this article, we report findings to unravel the role of myosin VIIa, an F-actin-based barbed (+)-end-directed motor protein, to support cellular transport and adhesion in the testis. Using RNA interference to knock down myosin VIIa in Sertoli cells cultured in vitro as a study model was shown to perturb the Sertoli cell tight junction permeability barrier, mediated through disorganization of actin- or microtubule (MT)-based cytoskeletons owing to disruptive changes on the spatiotemporal expression of F-actin or MT-regulatory proteins. Consistent with these in vitro findings, knockdown of myosin VIIa in the testis in vivo also induced disorganization of the actin- and MT-based cytoskeletons across the seminiferous epithelium, mediated by disruptive changes in the spatiotemporal expression of actin- and MT-based regulatory proteins. More important, the transport of spermatids and organelles across the epithelium, as well as cell adhesion, was grossly disrupted. For instance, step 19 spermatids failed to be transported to the adluminal compartment near the tubule lumen to undergo spermiation; in this manner, step 19 spermatids were persistently detected in stage IX and XII tubules, intermingling with step 9 and 12 spermatids, respectively. Also, phagosomes were detected near the tubule lumen in stage I to III tubules when they should have been degraded near the base of the seminiferous epithelium via the lysosomal pathway. In summary, myosin VIIa motor protein was crucial to support cellular transport and adhesion during spermatogenesis.
Assuntos
Junções Aderentes/metabolismo , Miosina VIIa/fisiologia , Epitélio Seminífero/fisiologia , Células de Sertoli/fisiologia , Espermatogênese , Actinas/metabolismo , Animais , Adesão Celular , Citoesqueleto/metabolismo , Masculino , Fagossomos/metabolismo , Interferência de RNA , Ratos Sprague-Dawley , Espermátides , Proteínas de Junções Íntimas/metabolismoRESUMO
Notwithstanding the huge demand in bio-imaging and optoelectronics, the construction of highly emissive deep red/near infrared (DR/NIR) organic luminogens is still a big challenge because a narrow energy gap generally leads to low photoluminescence quantum yield. It is even more difficult to afford DR/NIR emitters in the solid state due to the aggregation caused quenching (ACQ) effect. In this work, we found that the direct attachment of a tetraphenylethylene substituted arylamine to the electron accepting 2,1,3-benzothiadiazole produces DR/NIR AIE luminogens with bright emission facilely and efficiently. And the emission wavelengths could be tuned from the red to the DR/NIR region by regulating the variety of the substituents. The long emission wavelength and high photoluminescence quantum yield of these AIEgens are ascribed to the effective intramolecular charge transfer and the suppressed intramolecular motion. Furthermore, non-doped OLEDs based on one of the AIEgens showed an EL emission at 684 nm with a large radiance of 5772 mW Sr-1 m-2 and an impressive external quantum efficiency (EQE) of 1.73%.
RESUMO
In the mammalian testis, spermatogenesis is dependent on the microtubule (MT)-specific motor proteins, such as dynein 1, that serve as the engine to support germ cell and organelle transport across the seminiferous epithelium at different stages of the epithelial cycle. Yet the underlying molecular mechanism(s) that support this series of cellular events remain unknown. Herein, we used RNAi to knockdown cytoplasmic dynein 1 heavy chain (Dync1h1) and an inhibitor ciliobrevin D to inactivate dynein in Sertoli cells in vitro and the testis in vivo, thereby probing the role of dynein 1 in spermatogenesis. Both treatments were shown to extensively induce disruption of MT organization across Sertoli cells in vitro and the testis in vivo. These changes also perturbed the transport of spermatids and other organelles (such as phagosomes) across the epithelium. These changes thus led to disruption of spermatogenesis. Interestingly, the knockdown of dynein 1 or its inactivation by ciliobrevin D also perturbed gross disruption of F-actin across the Sertoli cells in vitro and the seminiferous epithelium in vivo, illustrating there are cross talks between the two cytoskeletons in the testis. In summary, these findings confirm the role of cytoplasmic dynein 1 to support the transport of spermatids and organelles across the seminiferous epithelium during the epithelial cycle of spermatogenesis.
Assuntos
Dineínas/metabolismo , Espermátides/metabolismo , Espermatogênese/fisiologia , Testículo/metabolismo , Animais , Transporte Biológico/fisiologia , Dineínas/genética , Masculino , Quinazolinonas/farmacologia , Interferência de RNA , Ratos , Células de Sertoli/efeitos dos fármacos , Células de Sertoli/metabolismo , Espermátides/efeitos dos fármacos , Espermatogênese/efeitos dos fármacos , Testículo/efeitos dos fármacosRESUMO
BACKGROUND: Interference refers to learned associations and established behaviors "interfering" with response to new material. It forms a core pillar of executive functions, which are commonly affected in Parkinson's disease (PD). Cognitive interference test (CIT) forms part of a smartphone application designed for ambulatory assessment in PD. OBJECTIVE: The aims of this study were to establish that CIT could effectively demonstrate interference and would perform comparably to the Stroop Color-Word Test Victoria version (VST) despite PD-related motor impairment. METHODS: Ninety-nine patients with PD were recruited. Initial evaluation included CIT, VST, Montreal cognitive assessment (MOCA), and Movement Disorders Society-sponsored revision of the -Unified Parkinson's Disease Rating Scale (MDS-UPDRS-III). A group of patients underwent repeat assessment within 2 weeks. Thirty-four healthy controls were recruited for comparison. RESULTS: Patients' mean age was 66.2 years, disease duration was 8.7 years, on-state MDS-UPDRS-III was 22, and MOCA total score was 27. CIT effectively generated interference, whereby the total time taken to complete the incongruent task was 20% longer compared to that of the baseline task. CIT key test items demonstrated convergent validity to VST (r = 0.478-0.644, p < 0.0001) and satisfactory repeatability (intraclass correlation coefficient 0.46-0.808, p ≤ 0.0002). Performance on key CIT test parameters deteriorated with increasing age (r = 0.225-0.478, p < 0.01) and MDS-UPDRS-III total score (r = 0.354-0.481, p < 0.0001). When compared to controls and patients with less motor impairment, patients MDS-UPDRS-III > 30 took longer to complete CIT and VST and had lower MOCA-attention sub-score, implying that the degree of motor impairment could not be the sole explanation for reduced CIT performance. CONCLUSIONS: We established that despite motor impairment, the novel approach of using smartphone technology to test interference in PD patients is feasible.
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Função Executiva/fisiologia , Doença de Parkinson/psicologia , Smartphone , Análise e Desempenho de Tarefas , Adulto , Idoso , Idoso de 80 Anos ou mais , Cognição/fisiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Índice de Gravidade de DoençaRESUMO
Levodopa-carbidopa intestinal gel (LCIG) is an effective treatment for Parkinson disease. Initiating therapy involves an initial naso-jejunal (NJ) titration phase. The NJ phase is prolonged with significant morbidity. The aim of this study is to assess the impact of proceeding without the NJ phase on resource utilisation and the outcomes of patients. Twenty-five patients were started on LCIG using the patients existing levodopa equivalent dose (LED). We recorded change in LED, length of hospital stay, readmission rates and use of outpatient services and clinical outcomes within 6 months. The median length of stay was 4.5 days. Patients had four outpatient clinic reviews and 2.5 community nurse contacts within 6 months. There was no significant change in daily LED on discharge (P = 0.56). There were significant improvements in all Unified Parkinson Disease Rating Scale subscores (P < 0.05), the Freezing of Gait scale (P < 0.01) and Parkinson Disease Quality Of Life 39 score (P < 0.01). Initiating LCIG without the NJ phase resulted in short admissions, a minimal outpatient burden and no significant requirement for dose titration while producing good clinical outcomes.
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Antiparkinsonianos/administração & dosagem , Carbidopa/administração & dosagem , Jejuno/efeitos dos fármacos , Tempo de Internação/tendências , Levodopa/administração & dosagem , Doença de Parkinson/diagnóstico , Doença de Parkinson/tratamento farmacológico , Administração Intranasal , Idoso , Antiparkinsonianos/sangue , Carbidopa/sangue , Combinação de Medicamentos , Feminino , Gastrostomia/métodos , Géis , Humanos , Bombas de Infusão Implantáveis , Jejunostomia/métodos , Jejuno/metabolismo , Levodopa/sangue , Masculino , Pessoa de Meia-Idade , Doença de Parkinson/sangueRESUMO
During spermatogenesis, developing elongating/elongated spermatids are highly polarized cells, displaying unique apico-basal polarity. For instance, the heads of spermatids align perpendicular to the basement membrane with their tails pointing to the tubule lumen. Thus, the maximal number of spermatids are packed within the limited space of the seminiferous epithelium to support spermatogenesis. Herein, we reported findings that elongating/elongated spermatids displayed planar cell polarity (PCP) in adult rat testes in which the proximal end of polarized spermatid heads were aligned uniformly across the plane of the seminiferous epithelium based on studies using confocal microscopy and 3-dimensional (D) reconstruction of the seminiferous tubules. We also discovered that spermatid PCP was regulated by PCP protein Vangl2 (Van Gogh-like protein 2) since Vangl2 knockdown by RNAi was found to perturb spermatid PCP. More important, Vangl2 exerted its regulatory effects through changes in the organization of the microtubule (MT)-based cytoskeleton in the seminiferous epithelium. These changes were mediated via the downstream signaling proteins atypical protein kinase C ξ (PKCζ) and MT-associated protein (MAP)/microtubule affinity-regulating kinase 2 (MARK2). These findings thus provide new insights regarding the biology of spermatid PCP during spermiogenesis.
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Polaridade Celular , Citoesqueleto/metabolismo , Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Espermátides/citologia , Espermátides/metabolismo , Testículo/metabolismo , Animais , Citoesqueleto/genética , Masculino , Microtúbulos/genética , Proteínas do Tecido Nervoso/genética , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Testículo/citologiaRESUMO
The blood-testis barrier is a unique ultrastructure in the mammalian testis, located near the basement membrane of the seminiferous tubule that segregates the seminiferous epithelium into the basal and the adluminal (apical) compartment. Besides restricting paracellular and transcellular passage of biomolecules (e.g., paracrine factors, hormones), water, electrolytes, and other substances including toxicants and/or drugs to enter the adluminal compartment of the epithelium, the BTB is an important ultrastructure that supports spermatogenesis. As such, a sensitive and reliable assay to monitor its integrity in vivo is helpful for studying testis biology. This assay is based on the ability of an intact BTB to exclude the diffusion of a small molecule such as sulfo-NHS-LC-biotin (C20H29N4NaO9S2, Mr. 556.59, a water-soluble and membrane-impermeable biotinylation reagent) from the basal to the apical compartment of the seminiferous epithelium. Herein, we summarize the detailed procedures on performing the assay and to obtain semiquantitative data to assess the extent of BTB damage when compared to positive controls, such as treatment of rats with cadmium chloride (CdCl2) which is known to compromise the BTB integrity.
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Biotina/metabolismo , Barreira Hematotesticular/fisiologia , Epitélio Seminífero/metabolismo , Células de Sertoli/metabolismo , Junções Íntimas/metabolismo , Animais , Barreira Hematotesticular/efeitos dos fármacos , Células Cultivadas , Masculino , Ratos , Ratos Sprague-Dawley , Epitélio Seminífero/citologia , Células de Sertoli/citologiaRESUMO
The blood-testis barrier (BTB) is an important ultrastructure in the testis that supports meiosis and postmeiotic spermatid development since a delay in the establishment of a functional Sertoli cell barrier during postnatal development in rats or mice by 17-20 day postpartum (dpp) would lead to a delay of the first wave of meiosis. Furthermore, irreversible disruption of the BTB by toxicants also induces infertility in rodents. Herein, we summarize recent findings that BTB dynamics (i.e., disassembly, reassembly, and stabilization) are supported by the concerted efforts of the actin- and microtubule (MT)-based cytoskeletons. We focus on the role of two actin nucleation protein complexes, namely, the Arp2/3 (actin-related protein 2/3) complex and formin 1 (or the formin 1/spire 1 complex) known to induce actin nucleation, respectively, by conferring plasticity to actin cytoskeleton. We also focus on the MT plus (+)-end tracking protein (+TIP) EB1 (end-binding protein 1) which is known to confer MT stabilization. Furthermore, we discuss in particular how the interactions of these proteins modulate BTB dynamics during spermatogenesis. These findings also yield a novel hypothetical concept regarding the molecular mechanism that modulates BTB function.
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Actinas/metabolismo , Barreira Hematotesticular/fisiologia , Citoesqueleto/metabolismo , Microtúbulos/metabolismo , Células de Sertoli/metabolismo , Espermatogênese , Animais , Células Cultivadas , Humanos , Masculino , Camundongos , Ratos , Células de Sertoli/citologiaRESUMO
Germ cell differentiation during the epithelial cycle of spermatogenesis is accompanied by extensive remodeling at the Sertoli cell-cell and Sertoli cell-spermatid interface to accommodate the transport of preleptotene spermatocytes and developing spermatids across the blood-testis barrier (BTB) and the adluminal compartment of the seminiferous epithelium, respectively. The unique cell junction in the testis is the actin-rich ectoplasmic specialization (ES) designated basal ES at the Sertoli cell-cell interface, and the apical ES at the Sertoli-spermatid interface. Since ES dynamics (i.e., disassembly, reassembly and stabilization) are supported by actin microfilaments, which rapidly converts between their bundled and unbundled/branched configuration to confer plasticity to the ES, it is logical to speculate that actin nucleation proteins play a crucial role to ES dynamics. Herein, we reported findings that Spire 1, an actin nucleator known to polymerize actins into long stretches of linear microfilaments in cells, is an important regulator of ES dynamics. Its knockdown by RNAi in Sertoli cells cultured in vitro was found to impede the Sertoli cell tight junction (TJ)-permeability barrier through changes in the organization of F-actin across Sertoli cell cytosol. Unexpectedly, Spire 1 knockdown also perturbed microtubule (MT) organization in Sertoli cells cultured in vitro. Biochemical studies using cultured Sertoli cells and specific F-actin vs. MT polymerization assays supported the notion that a transient loss of Spire 1 by RNAi disrupted Sertoli cell actin and MT polymerization and bundling activities. These findings in vitro were reproduced in studies in vivo by RNAi using Spire 1-specific siRNA duplexes to transfect testes with Polyplus in vivo-jetPEI as a transfection medium with high transfection efficiency. Spire 1 knockdown in the testis led to gross disruption of F-actin and MT organization across the seminiferous epithelium, thereby impeding the transport of spermatids and phagosomes across the epithelium and perturbing spermatogenesis. In summary, Spire 1 is an ES regulator to support germ cell development during spermatogenesis.
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Citoplasma/metabolismo , Proteínas dos Microfilamentos/metabolismo , Epitélio Seminífero/metabolismo , Células de Sertoli/metabolismo , Espermátides/metabolismo , Espermatogênese/fisiologia , Animais , Masculino , Ratos , Ratos Sprague-Dawley , Epitélio Seminífero/citologia , Células de Sertoli/citologia , Espermátides/citologia , Junções Íntimas/metabolismoRESUMO
In adult mammalian testes, spermatids, most notably step 17-19 spermatids in stage IV-VIII tubules, are aligned with their heads pointing toward the basement membrane and their tails toward the tubule lumen. On the other hand, these polarized spermatids also align across the plane of seminiferous epithelium, mimicking planar cell polarity (PCP) found in other hair cells in cochlea (inner ear). This orderly alignment of developing spermatids during spermiogenesis is important to support spermatogenesis, such that the maximal number of developing spermatids can be packed and supported by a fixed population of differentiated Sertoli cells in the limited space of the seminiferous epithelium in adult testes. In this review, we provide emerging evidence to demonstrate spermatid PCP in the seminiferous epithelium to support spermatogenesis. We also review findings in the field regarding the biology of spermatid cellular polarity (e.g., head-tail polarity and apico-basal polarity) and its inter-relationship to spermatid PCP. Furthermore, we also provide a hypothetical concept on the importance of PCP proteins in endocytic vesicle-mediated protein trafficking events to support spermatogenesis through protein endocytosis and recycling.
Assuntos
Polaridade Celular/fisiologia , Transdução de Sinais/fisiologia , Espermátides/fisiologia , Espermatogênese/fisiologia , Animais , Humanos , Masculino , Células de Sertoli/citologia , Células de Sertoli/metabolismo , Espermátides/citologia , Testículo/citologia , Testículo/metabolismoRESUMO
Signaling pathways that regulate blood-tissue barriers are important for studying the biology of various blood-tissue barriers. This information, if deciphered and better understood, will provide better therapeutic management of diseases particularly in organs that are sealed by the corresponding blood-tissue barriers from systemic circulation, such as the brain and the testis. These barriers block the access of antibiotics and/or chemotherapeutical agents across the corresponding barriers. Studies in the last decade using the blood-testis barrier (BTB) in rats have demonstrated the presence of several signaling pathways that are crucial to modulate BTB function. Herein, we critically evaluate these findings and provide hypothetical models regarding the underlying mechanisms by which these signaling molecules/pathways modulate BTB dynamics. This information should be carefully evaluated to examine their applicability in other tissue barriers which shall benefit future functional studies in the field. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.
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Barreira Hematotesticular/metabolismo , Conexinas/metabolismo , Junções Comunicantes/metabolismo , Modelos Cardiovasculares , Transdução de Sinais/fisiologia , Animais , Humanos , MasculinoRESUMO
Cell polarity in the adult mammalian testis refers to the polarized alignment of developing spermatids during spermiogenesis and the polarized organization of organelles (e.g., phagosomes, endocytic vesicles, Sertoli cell nuclei, Golgi apparatus) in Sertoli cells and germ cells to support spermatogenesis. Without these distinctive features of cell polarity in the seminiferous epithelium, it is not possible to support the daily production of millions of sperm in the limited space provided by the seminiferous tubules in either rodent or human males through the adulthood. In short, cell polarity provides a novel mean to align spermatids and the supporting organelles (e.g., phagosomes, Golgi apparatus, endocytic vesicles) in a highly organized fashion spatially in the seminiferous epithelium during the epithelial cycle of spermatogenesis. This is analogous to different assembling units in a manufacturing plant such that as developing spermatids move along the "assembly line" conferred by Sertoli cells, different structural/functional components can be added to (or removed from) the developing spermatids during spermiogenesis, so that functional spermatozoa are produced at the end of the assembly line. Herein, we briefly review findings regarding the regulation of cell polarity in the testis with specific emphasis on developing spermatids, supported by an intriguing network of regulatory proteins along a local functional axis. Emerging evidence has suggested that cell cytoskeletons provide the tracks which in turn confer the unique assembly lines in the seminiferous epithelium. We also provide some thought-provoking concepts based on which functional experiments can be designed in future studies.
Assuntos
Polaridade Celular , Citoesqueleto/metabolismo , Células de Sertoli/metabolismo , Testículo/metabolismo , Animais , Humanos , Masculino , Microtúbulos/metabolismo , Células de Sertoli/citologia , Espermátides/citologia , Espermatogênese , Testículo/citologiaRESUMO
The blood-testis barrier (BTB), conferred by Sertoli cells in the mammalian testis, is an important ultrastructure that supports spermatogenesis. Studies using animal models have shown that a disruption of the BTB leads to meiotic arrest, causing defects in spermatogenesis and male infertility. To better understand the regulation of BTB dynamics, we report findings herein to understand the role of ribosomal protein S6 (rpS6), a downstream signaling protein of mammalian target of rapamycin complex 1 (mTORC1), in promoting BTB disruption in the testis in vivo, making the barrier "leaky." Overexpression of wild-type rpS6 (rpS6-WT, the full-length cDNA cloned into the mammalian expression vector pCI-neo) and a constitutively active quadruple phosphomimetic mutant cloned into pCI-neo (p-rpS6-MT) vs. control (empty pCI-neo vector) was achieved by transfecting adult rat testes with the corresponding plasmid DNA using a Polyplus in vivo-jetPEI transfection reagent. On the basis of an in vivo functional BTB integrity assay, p-rpS6-MT was found to induce BTB disruption better than rpS6-WT did (and no effects in empty vector control), leading to defects in spermatogenesis, including loss of spermatid polarity and failure in the transport of cells (e.g., spermatids) and organelles (e.g., phagosomes), to be followed by germ exfoliation. More important, rpS6-WT and p-rpS6-MT exert their disruptive effects through changes in the organization of actin- and microtubule (MT)-based cytoskeletons, which are mediated by changes in the spatiotemporal expression of actin- and MT-based binding and regulatory proteins. In short, mTORC1/rpS6 signaling complex is a regulator of spermatogenesis and BTB by modulating the organization of the actin- and MT-based cytoskeletons.
Assuntos
Barreira Hematotesticular/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/fisiologia , Proteína S6 Ribossômica/fisiologia , Espermatogênese/fisiologia , Testículo/metabolismo , Animais , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Permeabilidade , Ratos , Ratos Sprague-Dawley , Ratos Transgênicos , Proteína S6 Ribossômica/genética , Transdução de Sinais/genética , Espermatogênese/genética , Testículo/fisiologia , Regulação para Cima/genéticaRESUMO
PFOS induces Sertoli cell injury using testicular cells isolated from rodent testes, but it remains unknown if PFOS has similar effects in humans. Herein, we maintained human Sertoli cells in a mitotically active state in vitro, thus enabling transfection experiments that altered gene expression to explore the molecular mechanism(s) underlying toxicant-induced cell injury. Human Sertoli cells obtained from men at ages 15, 23, 36 and 40 were cultured in vitro. These differentiated Sertoli cells remained mitotically active when cultured in the presence of 10% FBS (fetal bovine serum), with a replication time of ~1-3 weeks. At ~80% confluency, they were used for studies including toxicant exposure, immunoblotting, immunofluorescence analysis, tight junction (TJ)-permeability assessment, and overexpression of BTB (blood-testis barrier) regulatory genes such as FAK and its phosphomimetic mutants. PFOS was found to induce Sertoli cell injury through disruptive effects on actin microfilaments and microtubule (MT) organization across the cell cytosol. As a consequence, these cytoskeletal networks failed to support cell adhesion at the BTB. Overexpression of a FAK phosphomimetic and constitutively active mutant p-FAK-Y407E in these cells was capable of rescuing the PFOS-induced injury through corrective cellular organization of cytoskeletal elements. SUMMARY: PFOS induces human Sertoli cell injury which can be rescued by overexpressing p-FAK-Y407E mutant.
Assuntos
Ácidos Alcanossulfônicos/toxicidade , Fluorocarbonos/toxicidade , Proteína-Tirosina Quinases de Adesão Focal/genética , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Mutação/genética , Células de Sertoli/patologia , Actinas/metabolismo , Adolescente , Adulto , Células Cultivadas , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Humanos , Masculino , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Permeabilidade , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Transporte Proteico/efeitos dos fármacos , Células de Sertoli/efeitos dos fármacos , Células de Sertoli/metabolismo , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismo , Adulto JovemRESUMO
During spermatogenesis, immature spermatocytes traverse the blood-testis barrier (BTB) and enter the apical apartment of seminiferous epithelium for further development. This course involves extensive junction disassembly and reassembly at the BTB. P-glycoprotein is known to be coded by two genes in rodents, namely Abcb1a and Abcb1b. Our previous studies showed that simultaneously silencing Abcb1a and Abcb1b genes in Sertoli cells impeded BTB integrity. However, the individual role of Abcb1a and Abcb1b in regulating BTB dynamics remains uninvestigated. Here, single knockdown of Abcb1a by RNAi impeded the in vitro Sertoli cell permeability barrier via redistributing TJ proteins, accelerating endocytosis, and affecting endocytic vesicle-mediated protein transportation that undermined Sertoli cell barrier. F5-peptide model was used to induce cell junction disruption and subsequent restructuring in primary Sertoli cells. F5-peptide perturbed this barrier, but its removal allowed barrier 'resealing'. Abcb1b knockdown was found to inhibit barrier resealing following F5-peptide removal by suppressing the restore of the expression and distribution of junction proteins at BTB, and reducing the migration of internalized junction proteins back to Sertoli cell interface. In summary, Abcb1a is critical in maintaining BTB integrity, while Abcb1b is crucial for junction reassembly at the BTB.