RESUMEN
This study aimed at the efficacy of sequential treatment of bone marrow-derived mesenchymal stem cell secretion for busulfan-treated azoospermia in mice. The conditioned media (CM) was obtained from bone marrow mesenchymal stem cells (MSCs) or 293 cells. Chemically induced azoospermia mice received 200 µl MSC-CM or 293-CM twice a week intravenously for three consecutive weeks. The histological assessment of spermatogenic recovery quantifying the expression of meiosis-associated genes, and Sertoli cell barrier functional factors were assessed. The characteristics of TM4 cells (Sertoli cell line) after pre-incubation of MSC-CM in vitro were also obtained. The MSC-CM group had the most spermatogenic colonies among the three groups (p < .05), but no spermatids were seen. Expressions of the meiosis-associated genes Dazl, Vasa, Miwi, Stra8, CyclinA1, Pgk2 and Scp3 in MSC-CM testis were remarkably higher compared with 293-CM and busulfan groups respectively (p < .05). The levels of Sertoli cell barrier functional factors, for example ICAM-1 and N-cadherin, were significantly increased during MSC-CM treatment (p < .05). Moreover, pre-incubation of MSC-CM particularly accelerated the CD54 (ICAM-1) and CD44 expressions of TM4 cells and promoted cell inherent adhesion. MSC-CM treatment can significantly improve the short-term restoration of spermatogonial structures of chemically induced azoospermia related to facilitating Sertoli cell adhesion integrity.
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Azoospermia , Células Madre Mesenquimatosas , Animales , Azoospermia/inducido químicamente , Azoospermia/terapia , Busulfano/toxicidad , Humanos , Masculino , Ratones , Células de Sertoli , EspermatogénesisRESUMEN
BACKGROUND: The current study aimed to determine the impact of SARS-CoV-2 infection on male fertility. METHODS: This is a single-center, hospital-based observational study that included autopsied testicular and epididymal specimens of deceased COVID-19 male patients (n=6) and recruited recovering COVID-19 inpatients (n=23) with an equal number of age-matched controls, respectively. We performed histopathological examinations on testicular and epididymal specimens, and also performed TUNEL assay and immunohistochemistry. Whereas, we investigated the semen specimen for sperm parameters and immune factors. FINDINGS: Autopsied testicular and epididymal specimens of COVID-19 showed the presence of interstitial edema, congestion, red blood cell exudation in testes, and epididymides. Thinning of seminiferous tubules was observed. The number of apoptotic cells within seminiferous tubules was significantly higher in COVID-19 compared to control cases. It also showed an increased concentration of CD3+ and CD68+ in the interstitial cells of testicular tissue and the presence of IgG within seminiferous tubules. Semen from COVID-19 inpatients showed that 39.1% (n=9) of them have oligozoospermia, and 60.9% (n=14) showed a significant increase in leucocytes in semen. Decreased sperm concentration, and increased seminal levels of IL-6, TNF-α, and MCP-1 compared to control males were observed. INTERPRETATION: Impairment of spermatogenesis was observed in COVID-19 patients, which could be partially explained as a result of an elevated immune response in testis. Additionally, autoimmune orchitis occurred in some COVID-19 patients. Further research on the reversibility of impairment and developing treatment are warranted. FUNDING: This study was supported by Ministry of Science and Technology of China Plan, Hubei Science and Technology Plan, National Key Research and Development Program of China, HUST COVID-19 Rapid Response Call, China and National Natural Science Foundation of China; these funding bodies are public institutions, and they had no role in study conception, design, interpretation of results, and manuscript preparation.
RESUMEN
The present study was designed to investigate the therapeutic effect of bone marrow MSC-derived factors on gonadotropic toxicity induced by busulfan in vivo. The conditioned media (CM) was obtained from MSCs in serum-free incubation for 48 hr and concentrated ~25-fold by ultrafiltration. The CM of HEK 293 cells was treated as control (293-CM). MSC-CM was injected into busulfan mice via caudal veins after 1 day of busulfan treatment for 2 weeks (200 µl per dose/twice weeklyï¼. Compared to the 293-CM group, testicular injury was delayed in MSC-CM group, including reduced vacuolations of cells in the basal compartment of the seminiferous epithelium and detachment of cells from basement membrane. Apoptotic spermatogenic cells were significantly decreased in MSC-CM group (p ï¼ 0.05). Interesting N-cadherinï¼ICAM-1 and P-cadherin expressions significantly increased in MSC-CM group, while occludin, ZO-1 and connexin 43 expressions showed no difference among MSC-CM, 293-CM and busulfan groups. Present results suggest MSC-secreted factors protect spermatogenesis impairment after busulfan treatment by reducing the apoptosis of spermatogenic cells and enhancing intercellular adhesion molecule expressions.
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Barrera Hematotesticular/efectos de los fármacos , Busulfano/toxicidad , Medios de Cultivo Condicionados/farmacología , Infertilidad Masculina/tratamiento farmacológico , Células Madre Mesenquimatosas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Barrera Hematotesticular/citología , Barrera Hematotesticular/patología , Cadherinas/metabolismo , Adhesión Celular/efectos de los fármacos , Medios de Cultivo Condicionados/metabolismo , Modelos Animales de Enfermedad , Células HEK293 , Humanos , Infertilidad Masculina/inducido químicamente , Infertilidad Masculina/patología , Molécula 1 de Adhesión Intercelular/metabolismo , Masculino , Ratones , Espermatogénesis/efectos de los fármacosRESUMEN
Human fibroblasts were isolated from foreskin of a clinically diagnosed 40-year old patient with idiopathic infertility. The fibroblasts were reprogrammed with the Yamanaka KOSM transcriptional factors using the retroviral vectors. The obtained induced pluripotent stem cell (iPSC) line showed pluripotency verified by the expression of pluripotency markers, NANOG, SOX2, OCT4, TRA-1-60, and SSEA-4. And the iPSC line was demonstrated to have the three germ layers differentiation capacity in vivo by teratoma assay. The iPSC line also showed normal karyotype. This patient-specific iPSC line can be used to explore the mechanism for idiopathic male infertility.
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Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Infertilidad/metabolismo , Adulto , Diferenciación Celular/fisiología , Reprogramación Celular/genética , Reprogramación Celular/fisiología , Fibroblastos/metabolismo , Estratos Germinativos/citología , Estratos Germinativos/metabolismo , Humanos , Infertilidad/patología , Masculino , Factores de Transcripción/metabolismoRESUMEN
Busulfan is an alkane sulphonate currently used as an anticancer drug and to prepare azoospermic animal models, because it selectively destroys differentiated spermatogonia in the testes. However, few studies have focussed on the exact effects of busulfan treatment on the epididymis currently. The present study assessed the effect of busulfan on epididymal morphology and the blood-epididymis barrier in mice. We treated mice with a single injection of busulfan and detected the effect at different time points. We showed that busulfan was toxic to the morphological structure and function of the epididymis. Furthermore, busulfan treatment down-regulated the epididymal expression of vimentin and zonula occludens-1 (ZO-1) at the mRNA and protein levels. In addition, there was an increase in total androgen receptor (AR) levels, whereas the estrogen receptor-α (ER-α) levels were reduced, both in the caput and cauda regions after busulfan treatment, which may be secondary to the testicular damage. In conclusion, our study describes the effects of busulfan administration on the mouse epididymis and also provides a potential understanding of male infertility arising from chemotherapy-related defects in the epididymis.
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Busulfano/efectos adversos , Epidídimo/efectos de los fármacos , Vimentina/metabolismo , Proteína de la Zonula Occludens-1/metabolismo , Animales , Regulación hacia Abajo/efectos de los fármacos , Epidídimo/metabolismo , Receptor alfa de Estrógeno/metabolismo , Masculino , Ratones , Ratones Endogámicos ICR , Receptores Androgénicos/metabolismo , Testículo/efectos de los fármacos , Testículo/metabolismoRESUMEN
Dermal fibroblasts play a vital role in maintaining skin function. They not only synthesize and secrete extracellular matrix molecules, but also produce a complex mixture of bioactive factors, which both contribute to immune regulation and wound healing. Fibroblasts isolated from skin tissue exhibit wide range of potentials, especially in regenerative medicine. The use of fibroblast cultures for medical purposes requires standardization of cell preparations. To achieve this, we isolated and characterized dermal fibroblasts from human foreskin with a standardized method. The obtained cells grew as typical morphology of fibroblasts, and expressed intermediate filament protein vimentin and nestin. Immunophenotypic analysis indicated that the isolated fibroblasts expressed mesenchymal surface markers CD73, CD90, CD44 and CD105, and were negative for haematopoietic markers CD45 and CD34. Growth kinetics analysis of the cells showed high proliferative properties. Furthermore, cryopreservation had no influence on cell morphology and growth properties. Here, we describe a standardized, repeatable method for isolation of fibroblasts from human foreskin tissues and identify their biological characters according to morphologic, immunohistologic and proliferative criteria, which would be meaningful for future clinical trials and regenerative medicine purposes.
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Diferenciación Celular/genética , Fibroblastos/metabolismo , Prepucio/crecimiento & desarrollo , Infertilidad Masculina/metabolismo , Adipogénesis/genética , Linaje de la Célula/genética , Proliferación Celular/genética , Fibroblastos/patología , Prepucio/patología , Regulación del Desarrollo de la Expresión Génica , Humanos , Infertilidad Masculina/patología , Masculino , Células Madre Mesenquimatosas/citología , Nestina/biosíntesis , Vimentina/biosíntesisRESUMEN
This paper proposes a method to monitor atmospheric HCHO and CHOCHO with high temporal resolution based on differential optical absorption spectroscopy (DOAS) in Shanghai urban area. Based on the characteristic absorbing structure of HCHO and CHOCHO, different fitting intervals were chosen for spectral analysis in order to avoid the absorption of interfering gases and reduce the residuals of spectral analysis. The resulting optical thickness of the target gas is used to obtain HCHO and CHOCHO concentrations, which were averaged at (4.0±1.6) and (3.4±1.2) µg·m-3 in October 2013, respectively. The averaged concentrations of HCHO in workdays were higher than those in holidays due to the impacts of its anthropogenic emission sources, while no obvious differences of averaged CHOCHO concentrations between workdays and holidays were observed. Diurnal patterns of HCHO and CHOCHO were alike. In the early morning, both the HCHO and CHOCHO concentrations peaked at 06:0007:00, and then decreased rapidly to the minimum around 09:00. Afterwards, the concentrations increased continuously until sunset and kept in a relatively stable level in the evening. To explore the possible emission source and formation mechanism of atmospheric HCHO, four typical periods, i.e. steady-state stage at night, morning rush hours, photochemical reaction stage and evening rush hours, were classified for source apportionment.NO2 is regarded as the indicator for primary source of ambient HCHO.As the intermediate products of photochemical reactions, HCHO has a similar formation mechanism in common with CHOCHO. Therefore, it is reasonable to use CHOCHO as an indicator for secondary source of ambient HCHO. The linear regression analysis showed a good agreement between modeled and observed HCHO concentrations, the correlation coefficients R2 ranged from 0.60 to 0.81. Secondary sources of HCHO were estimated to contribute to one third of ambient HCHO concentrations in Shanghai urban area.
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The purification of mouse bone marrow mesenchymal stem cells (BMSCs) by using the standard method of whole bone marrow adherence to plastic still remains ineffective. An increasing number of studies have indicated compact bone as an alternative source of BMSCs. We isolated BMSCs from cultured compact bone fragments and investigated the proliferative capacity, surface immunophenotypes, and osteogenic and adipogenic differentiations of the cells after the first trypsinization. The fragment culture was based on the fact that BMSCs were assembled in compact bones. Thus, the procedure included flushing bone marrow out of bone cavity and culturing the fragments without any collagenase digestion. The cell yield from cultured fragments was slightly less than that from cultured bone marrow using the same bone quantity. However, the trypsinized cells from cultured fragments exhibited significantly higher proliferation and were accompanied with more CD90 and CD44 expressions and less CD45 expression. The osteogenic and adipogenic differentiation capacity of cells from cultured fragments were better than those of cells from bone marrow. The directly adherent culture of compact bone is suitable for mouse BMSC isolation, and more BMSCs with potentially improved proliferation capacity can be obtained in the primary culture.
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Mast cells (MC) and basophils share expression of the high-affinity receptor for IgE (FcεRI) but can be distinguished by their divergent expression of KIT and CD49b. In BALB/c mice, MC lineage cells expressing high levels of FcεRI by flow cytometry were seen only in bone marrow whereas those expressing intermediate levels of FcεRI were present in bone marrow and spleen of naive mice and in mesenteric lymph nodes (mLN) of Trichinella spiralis-infected mice. These FcεRI(+)KIT(+)CD49b(-) cells had a membrane phenotype similar to i.p. connective tissue-type MC, but were smaller and hypogranular by flow cytometry forward and side scatter profiles, respectively. Consistent with this, they lacked the prominent secretory granules identified by histochemistry and immunodetection for the MC-specific granule proteases that are readily seen in mature jejunal mucosal MC that also are induced by the infection and present at the same time. The concentration of these MC lineage cells in mLN determined by flow cytometry was comparable to that of MC progenitors (MCp) measured by limiting dilution and clonal expansion with maturation. We observed upregulation of IL-4 transcription by MCp in mLN and spleens of helminth-infected 4get mice, and we demonstrated by intracellular cytokine staining production of IL-4 and IL-6 by the mLN MCp in helminth-infected mice. Furthermore, treatment of helminth-infected mice with anti-FcεRI mAb, a protocol known to deplete basophils, also depleted mLN MCp. Thus, this study identifies a hypogranular subset of MCp recruited to mLN by helminth infection that may be an important unrecognized source of cytokines.
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Gránulos Citoplasmáticos/inmunología , Interleucina-4/biosíntesis , Interleucina-6/biosíntesis , Ganglios Linfáticos/inmunología , Mastocitos/inmunología , Triquinelosis/inmunología , Animales , Movimiento Celular/genética , Movimiento Celular/inmunología , Células Cultivadas , Gránulos Citoplasmáticos/parasitología , Gránulos Citoplasmáticos/patología , Regulación hacia Abajo/inmunología , Genes Reporteros , Interleucina-4/genética , Ganglios Linfáticos/parasitología , Ganglios Linfáticos/patología , Mastocitos/parasitología , Mastocitos/patología , Mesenterio/inmunología , Mesenterio/patología , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones Transgénicos , ARN Mensajero/biosíntesis , Células Madre/inmunología , Células Madre/parasitología , Células Madre/patología , Trichinella spiralis , Triquinelosis/parasitología , Triquinelosis/patología , Regulación hacia Arriba/genética , Regulación hacia Arriba/inmunologíaRESUMEN
Prostaglandin E(2) (PGE(2)) is an abundant lipid inflammatory mediator with potent but incompletely understood anti-inflammatory actions in the lung. Deficient PGE(2) generation in the lung predisposes to airway hyperresponsiveness and aspirin intolerance in asthmatic individuals. PGE(2)-deficient ptges(-/-) mice develop exaggerated pulmonary eosinophilia and pulmonary arteriolar smooth-muscle hyperplasia compared with PGE(2)-sufficient controls when challenged intranasally with a house dust mite extract. We now demonstrate that both pulmonary eosinophilia and vascular remodeling in the setting of PGE(2) deficiency depend on thromboxane A(2) and signaling through the T prostanoid (TP) receptor. Deletion of TP receptors from ptges(-/-) mice reduces inflammation, vascular remodeling, cytokine generation, and airway reactivity to wild-type levels, with contributions from TP receptors localized to both hematopoietic cells and tissue. TP receptor signaling ex vivo is controlled heterologously by E prostanoid (EP)(1) and EP(2) receptor-dependent signaling pathways coupling to protein kinases C and A, respectively. TP-dependent up-regulation of intracellular adhesion molecule-1 expression is essential for the effects of PGE(2) deficiency. Thus, PGE(2) controls the strength of TP receptor signaling as a major bronchoprotective mechanism, carrying implications for the pathobiology and therapy of asthma.
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Alérgenos/toxicidad , Antígenos Dermatofagoides/toxicidad , Asma/inmunología , Dinoprostona/inmunología , Neumonía/inmunología , Eosinofilia Pulmonar/inmunología , Tromboxano A2/inmunología , Animales , Asma/inducido químicamente , Asma/genética , Dinoprostona/genética , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/inmunología , Oxidorreductasas Intramoleculares/genética , Oxidorreductasas Intramoleculares/inmunología , Masculino , Ratones , Ratones Noqueados , Neumonía/inducido químicamente , Neumonía/genética , Prostaglandina-E Sintasas , Eosinofilia Pulmonar/inducido químicamente , Eosinofilia Pulmonar/genética , Subtipo EP1 de Receptores de Prostaglandina E/genética , Subtipo EP1 de Receptores de Prostaglandina E/inmunología , Subtipo EP2 de Receptores de Prostaglandina E/genética , Subtipo EP2 de Receptores de Prostaglandina E/inmunología , Receptores de Tromboxanos/genética , Receptores de Tromboxanos/inmunología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transducción de Señal/inmunología , Tromboxano A2/genética , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genética , Regulación hacia Arriba/inmunologíaRESUMEN
Cysteinyl leukotriene (cysLT) overproduction is a hallmark of aspirin-exacerbated respiratory disease (AERD), but its mechanism is poorly understood. Because adherent platelets can convert the leukocyte-derived precursor leukotriene (LT)A(4) to LTC(4), the parent cysLT, through the terminal enzyme LTC(4) synthase, we investigated the contribution of platelet-dependent transcellular cysLT production in AERD. Nasal polyps from subjects with AERD contained many extravascular platelets that colocalized with leukocytes, and the percentages of circulating neutrophils, eosinophils, and monocytes with adherent platelets were markedly higher in the blood of subjects with AERD than in aspirin-tolerant controls. Platelet-adherent subsets of leukocytes had higher expression of several adhesion markers than did platelet nonadherent subsets. Adherent platelets contributed more than half of the total LTC(4) synthase activity of peripheral blood granulocytes, and they accounted for the higher level of LTC(4) generation by activated granulocytes from subjects with AERD compared with aspirin-tolerant controls. Urinary LTE(4) levels, a measure of systemic cysLT production, correlated strongly with percentages of circulating platelet-adherent granulocytes. Because platelet adherence to leukocytes allows for both firm adhesion to endothelial cells and augmented transcellular conversion of leukotrienes, a disturbance in platelet-leukocyte interactions may be partly responsible for the respiratory tissue inflammation and the overproduction of cysLTs that characterize AERD.
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Aspirina/efectos adversos , Asma Inducida por Aspirina/inmunología , Plaquetas/inmunología , Cisteína/inmunología , Leucocitos/inmunología , Leucotrienos/inmunología , Pólipos Nasales/inducido químicamente , Adulto , Anciano , Araquidonato 5-Lipooxigenasa/inmunología , Araquidonato 5-Lipooxigenasa/metabolismo , Aspirina/inmunología , Plaquetas/efectos de los fármacos , Broncoconstricción/inmunología , Cisteína/metabolismo , Femenino , Granulocitos/efectos de los fármacos , Granulocitos/inmunología , Humanos , Integrinas/inmunología , Leucotrieno E4/inmunología , Leucotrienos/metabolismo , Masculino , Persona de Mediana Edad , Pólipos Nasales/inmunología , Inhibidores de Agregación Plaquetaria/efectos adversos , Inhibidores de Agregación Plaquetaria/inmunología , Adulto JovenRESUMEN
The effect of loss-of-function of Attractin (Atrn) on the male mouse reproduction system was examined in the study. The weights and pathological changes of testes and epididymes were compared between Atrn mutant (Atrn(mg-3J)) mice and wild-type mice (C3HeB/FeJ) at different months of age. The number and motility of sperms were measured in the mutant and control mice. Furthermore, the testicular lactate dehydrogenase (LDH) and succinate dehydrogenase (SDH) in these animals were detected. The fertility potential of the sperms was observed in vivo and in vitro. The results showed that the testes of 3-month-old Atrn (mg-3J) mice experienced no significantly different pathological changes from the control mice at the same month of age but the SDH activity was substantially reduced. In the 5-month-old mutant mice, as compared with the control mice, mild vacuolation was found in the testes, the density and motility of sperms were decreased in the epididymes, the sperm fertility was impaired and the testicular enzyme activity was reduced. It is concluded that the age-related Atrn gene progressively loses its function and can cause testis vacuolation and impaired sperm function, which may be responsible for the impairment of male reproductive ability.
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Infertilidad Masculina/genética , Proteínas de la Membrana/genética , Mutación , Motilidad Espermática , Testículo/patología , Animales , Epidídimo/patología , L-Lactato Deshidrogenasa/metabolismo , Masculino , Ratones , Ratones Endogámicos C3H , Recuento de Espermatozoides , Succinato Deshidrogenasa/metabolismo , Testículo/enzimología , Testículo/metabolismoRESUMEN
Attractin (ATRN) and Attractin-like 1 (ATRNL1) are highly similar type I transmembrane proteins. Atrn null mutant mice have a pleiotropic phenotype including dark fur, juvenile-onset spongiform neurodegeneration, hypomyelination, tremor, and reduced body weight and adiposity, implicating ATRN in numerous biological processes. Bioinformatic analysis indicated that Atrn and Atrnl1 arose from a common ancestral gene early in vertebrate evolution. To investigate the genetics of the ATRN system and explore potential redundancy between Atrn and Atrnl1, we generated and characterized Atrnl1 loss- and gain-of-function mutations in mice. Atrnl1 mutant mice were grossly normal with no alterations of pigmentation, central nervous system pathology or body weight. Atrn null mutant mice carrying a beta-actin promoter-driven Atrnl1 transgene had normal, agouti-banded hairs and significantly delayed onset of spongiform neurodegeneration, indicating that over-expression of ATRNL1 compensates for loss of ATRN. Thus, the two genes are redundant from the perspective of gain-of-function but not loss-of-function mutations.
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Proteínas Adaptadoras Transductoras de Señales/clasificación , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas de la Membrana/clasificación , Proteínas de la Membrana/genética , Pigmentación/genética , Secuencia de Aminoácidos , Animales , Evolución Molecular , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Fenotipo , FilogeniaAsunto(s)
Proteínas de la Membrana , Proteína de Señalización Agouti , Animales , Sistema Nervioso Central/anomalías , Sistema Nervioso Central/metabolismo , Sistema Nervioso Central/patología , Metabolismo Energético , Color del Cabello/genética , Color del Cabello/fisiología , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/fisiología , Mutación , Obesidad/genética , Obesidad/metabolismoRESUMEN
OBJECTIVE: To localize the Mahoganoid protein and Mahoganoid mRNA in the testes and epididymides of mature male rats. METHODS: Testes and epididymides obtained from mature male SD rats (n = 20) were fixed by 4% poly formaldehyde and sliced for immunohistochemical (IHC) test and in situ hybridization (ISH) test, respectively, for detecting Mahoganoid protein and Mahoganoid mRNA. RESULTS: In both the 2 tests, clear brown staining was observed in Leydig cells, spermatogonia, primary spermatocytes, spermatids, Sertoli cells, and peritubular myoid cells. Both the Mahoganoid protein and its mRNA were mainly located on cell membrane and cytoplasm. And in the epididymis tissues, the Mahoganoid protein and Mahoganoid mRNA were respectively expressed within the membrane and cytoplasm of principal cells, basic cells and epithelial cells. CONCLUSION: Both Mahoganoid protein and Mahoganoid mRNA are expressed in the male rat reproductive system and localized in Leydig cells, Sertoli cells, spermatogenic cells and epithelial cells. They play an important role in spermatogenesis but their physiological significance remains to be clarified.
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Epidídimo/metabolismo , Testículo/metabolismo , Ubiquitina-Proteína Ligasas/biosíntesis , Animales , Inmunohistoquímica , Hibridación in Situ , Masculino , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
OBJECTIVE: Given the fact that the Attractin protein express widely in the testis of mature male rat, this paper aims at studying the distribution of the Attractin protein and Attractin mRNA in the testicular tissue of rats of different ages. METHODS: Testes and epididymides were obtained from newborn (8 hours after birth), prepubertal (5 days), pubertal (20 days), postpubertal (50 days) and mature (70 days) Wistar rats; the tissues were fixed and the Attractin protein and mRNA detected respectively by immunohistochemical and in situ hybridization techniques. RESULTS: The Attractin protein and mRNA expressed respectively within Leydig cells, primitive spermatogonia, primary spermatocytes, spermatids, Sertoli cells and peritubular myoid cells. The Attractin protein stain was mainly located on the cell membrane and cytoplasm, and Attractin mRNA distributed in the nucleus and cytoplasm. There was no immunopositive staining in spermatozoa and epididymides. CONCLUSION: The Attractin protein and Attractin mRNA are both expressed widely in the testis of rats of different ages. It suggests that the rat testis has the ability of synthesizing the Attractin protein throughout sexual development, but its physiological function and biochemical mechanism yet remain to be further studied.
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Proteínas de la Membrana/biosíntesis , Testículo/metabolismo , Animales , Animales Recién Nacidos , Células Germinativas/metabolismo , Inmunohistoquímica , Hibridación in Situ , Masculino , Proteínas de la Membrana/genética , ARN Mensajero/genética , Ratas , Ratas WistarRESUMEN
Hepatitis C virus (HCV) is an RNA virus infecting 1 in every 40 people worldwide. Development of new therapeutics for treating HCV has been hampered by the lack of small-animal models. We have adapted existing hydrodynamic transfection methods to optimize the delivery of RNAs to the cytoplasm of mouse liver cells in vivo. Transfected HCV genomic RNA failed to replicate in mouse liver, suggesting a post-entry block to viral replication. Real-time imaging of HCV internal ribosome entry site (IRES) firefly luciferase reporter mRNA translation in living mice demonstrated that the HCV IRES was functional in mouse liver. We then used this system as a model for studying HCV RNA translation in mice. We compared translation by several mutant HCV IRES variants in cell lysates, cultured cells, and mouse liver. We measured the contribution to translation of a cap, HCV 3'-untranslated region (UTR), poly(A) tail, domains II, IIIb, IIIabc, IIIabcd, IIId, and the initiator codon. Efficient translation required a 3'-UTR in mice and HeLa cells, but not in rabbit reticulocyte lysates. Translational regulation of transfected RNAs was stringent in mice. The method we describe could be useful for studies in mice of antisense or ribozyme inhibitors targeting the IRES as well as other RNA biochemical studies in vivo.