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2.
Nat Biotechnol ; 38(3): 303-308, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31959954

RESUMO

Monitoring drug-target interactions with methods such as the cellular thermal-shift assay (CETSA) is well established for simple cell systems but remains challenging in vivo. Here we introduce tissue thermal proteome profiling (tissue-TPP), which measures binding of small-molecule drugs to proteins in tissue samples from drug-treated animals by detecting changes in protein thermal stability using quantitative mass spectrometry. We report organ-specific, proteome-wide thermal stability maps and derive target profiles of the non-covalent histone deacetylase inhibitor panobinostat in rat liver, lung, kidney and spleen and of the B-Raf inhibitor vemurafenib in mouse testis. In addition, we devised blood-CETSA and blood-TPP and applied it to measure target and off-target engagement of panobinostat and the BET family inhibitor JQ1 directly in whole blood. Blood-TPP analysis of panobinostat confirmed its binding to known targets and also revealed thermal stabilization of the zinc-finger transcription factor ZNF512. These methods will help to elucidate the mechanisms of drug action in vivo.


Assuntos
Sangue/metabolismo , Proteoma/química , Proteoma/metabolismo , Bibliotecas de Moléculas Pequenas/administração & dosagem , Animais , Azepinas/administração & dosagem , Azepinas/farmacologia , Células Hep G2 , Humanos , Rim/química , Rim/metabolismo , Fígado/química , Fígado/metabolismo , Pulmão/química , Pulmão/metabolismo , Masculino , Espectrometria de Massas , Camundongos , Especificidade de Órgãos , Panobinostat/administração & dosagem , Panobinostat/farmacologia , Estabilidade Proteica , Ratos , Bibliotecas de Moléculas Pequenas/farmacologia , Baço/química , Baço/metabolismo , Testículo/química , Testículo/metabolismo , Termodinâmica , Triazóis/administração & dosagem , Triazóis/farmacologia , Vemurafenib/administração & dosagem , Vemurafenib/farmacologia
3.
J Infect Chemother ; 26(1): 51-57, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31353201

RESUMO

INTRODUCTION: Cisplatin (CIS), which is used as a therapeutic antineoplastic agent may produce gonadotoxicity in a process linked to chemotherapy. Taurine, through its potential antioxidant effect, has a protective role against cisplatin-induced oxidative stress and apoptosis. OBJECTIVE: To investigate whether taurine intake can ameliorate testicular damage induced by cisplatin and to study the possible mechanism that mediates this action, either through its antioxidant action alone or in addition to its anti-apoptotic effects. PATIENTS AND METHODS: Fifty healthy adult white male albino rats were randomly distributed into five groups, each involving ten animals. The first group represents the negative control group. The other four groups received three equal doses (3 mg/kg body weight) intraperitoneal injections of cisplatin on alternate days. In the positive control group (group 2), saline only was given. Groups 3, 4 &5 received taurine in distilled water at oral doses of 50, 150, 250 mg/kg, respectively, on alternate days followed by cisplatin (each injection of cisplatin was given 1 day after taurine). On the 28th day after the first dose of normal saline, cisplatin or taurine, blood samples were examined for testosterone levels. All rats were killed and their testes were examined. RESULTS: Rats treated with cisplatin alone showed reduced body weight in addition to reduced testicular weight, impaired sperm counts, and oxidative stress (reduced GSH, increased MDA level), decreased plasma testosterone, apoptotic marker (increased Bax, decreased bcl2). However following taurine induction, the figures for GSH and MDA changed significantly (P < 0.005) referring to the effect of taurine as a potent antioxidant. CONCLUSIONS: Cisplatin-induced germ cell apoptosis may result in decreasing spermatogenesis. However, taurine could effectively prevent nearly all of these cisplatin-induced testicular abnormalities, thereby proving to be an effective cytoprotectant.


Assuntos
Apoptose/efeitos dos fármacos , Cisplatino/toxicidade , Substâncias Protetoras/farmacologia , Taurina/farmacologia , Testículo/efeitos dos fármacos , Animais , Masculino , Estresse Oxidativo/efeitos dos fármacos , Ratos , Testículo/química , Testículo/citologia , Testículo/patologia
4.
Ecotoxicol Environ Saf ; 190: 110044, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31869717

RESUMO

Low-energy shock waves (LESWs) have been widely used in the intervention of a subset of diseased tissues and organs with good results. However, it is unclear whether they can be used directly to intervene in the testes. Therefore, the aim of this study was to determine a relatively safe energy density and impulse number for rat testes. A total of 176 male rats were randomly and equally assigned to different intervention groups. Among them, 144 rats were assigned to 18 shock subgroups with different energy densities (0.02, 0.04 and 0.06 mJ/mm2), different impulse numbers (500, 1000 and 1500 impulses) and different shock periods (2 and 8 weeks). The remaining 32 rats were divided into the sham intervention (S) groups and the blank control (N) groups with observation periods of 2 weeks and 8 weeks. One day after the last LESWs intervention, all the rats were weighed, and the concentrations of reproductive endocrine hormones were measured, the semen quality and testicular tissue oxidative stress levels were analyzed, and histomorphology and ultrastructures were observed. We found that there were no significant differences in the whole-body physiological state, testicular tissue morphology, oxidative stress state and sperm quality between the L1 shock group and the corresponding S group and N group (all p˃0.05, respectively). However, the other parameters of the shock groups caused different degrees of damage to the structure and function of rat testes, and the whole-body physiological state was also adversely affected. This study demonstrated that LESWs with an energy density of 0.02 mJ/mm2 and 500 impulses had no adverse effects on the rat testes.


Assuntos
Testículo/efeitos da radiação , Animais , Masculino , Estresse Oxidativo , Ratos , Análise do Sêmen , Espermatozoides/efeitos da radiação , Testículo/anatomia & histologia , Testículo/química , Testículo/ultraestrutura
5.
Molecules ; 25(1)2019 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-31877782

RESUMO

Bisphenol A (BPA), an endocrine disruptor, may affect in situ steroidogenesis and alter steroids levels. The present work proposes a liquid chromatography tandem mass spectrometry method to simultaneously quantify BPA, 17ß-Estradiol and testosterone in two target tissues: testis and visceral fat mass. Analytes were isolated and lipophilic impurities removed by two serial steps: liquid-liquid and solid phase extraction. All compounds were separated in a single gradient run by Kinetex F5 column and detected via multiple reaction monitoring using a triple quadrupole with a TurboIon electrospray source in both negative and positive modes. The method is selective and very sensitive. In the investigated concentration range, the linearity of the detector response is verified in both tissues. The use of specific SPE cartridges for affinity chromatography purification allows obtaining high percentages of process efficiency (68.0-83.3% for testicular tissue; 63.7-70.7% for visceral fat mass). Good repeatability and reproducibility was observed. The validated method can be efficiently applied for direct biological monitoring in testis and visceral fat mass from mice exposed to BPA. The quantification of compounds in a single assay could be achieved without a loss of sensitivity.


Assuntos
Compostos Benzidrílicos/análise , Gordura Intra-Abdominal/química , Fenóis/análise , Esteroides/análise , Testículo/química , Animais , Cromatografia Líquida , Estradiol/análise , Masculino , Camundongos , Extração em Fase Sólida , Espectrometria de Massas em Tandem , Testosterona/análise
6.
PLoS One ; 14(12): e0226560, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31887141

RESUMO

Chronic Wasting Disease (CWD) is a prion disease affecting several cervid species. Among them, white-tailed deer (WTD) are of relevance due to their value in farming and game hunting. The exact events involved in CWD transmission in captive and wild animals are still unclear. An unexplored mechanism of CWD spread involves transmissions through germplasm, such as semen. Surprisingly, the presence and load of CWD prions in semen and male sexual tissues from WTD has not been explored. Here, we described the detection of CWD prions in semen and sexual tissues of WTD bucks utilizing the Protein Misfolding Cyclic Amplification (PMCA) technology. Samples were obtained post-mortem from farmed pre-clinical, CWD positive WTD bucks possessing polymorphisms at position 96 of the PRNP gene. Our results show that overall CWD detection in these samples had a sensitivity of 59.3%, with a specificity of 97.2%. The data indicate that the presence of CWD prions in male sexual organs and fluids is prevalent in late stage, pre-clinical, CWD-infected WTD (80%-100% of the animals depending on the sample type analyzed). Our findings reveal the presence of CWD prions in semen and sexual tissues of prion infected WTD bucks. Future studies will be necessary to determine whether sexual contact and/or artificial inseminations are plausible means of CWD transmission in susceptible animal species.


Assuntos
Genitália Masculina/química , Polimorfismo Genético , Proteínas Priônicas/genética , Sêmen/química , Doença de Emaciação Crônica/diagnóstico , Animais , Autopsia , Cervos , Diagnóstico Precoce , Epididimo/química , Masculino , Técnicas de Amplificação de Ácido Nucleico , Sensibilidade e Especificidade , Testículo/química , Doença de Emaciação Crônica/transmissão
7.
BMC Complement Altern Med ; 19(1): 333, 2019 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-31771569

RESUMO

BACKGROUND: Monotropein, astragalin, and spiraeoside (MAS) are active compounds extracted from medicinal herbs; monotropein from Morinda officinalis How (Rubiaceae), astragalin (kaempferol 3-O-glucoside) from Cuscuta chinensis Lamark (Convolvulaceae) and spiraeoside from the outer scales of Allium cepa L. (Liliceae) in a ratio of 6.69:0.41:3.61. Monotropein, astragalin, and spiraeoside are well-known antioxidants, anti-inflammatory, and antinociceptive agents. The current investigation aims to study the molecular mechanism of varicocele-induced male infertility and the underlying pharmacological mechanisms of MAS. METHODS: Four groups were included: control (CTR), MAS 200 group (MAS 200 mg/kg), varicocele group (VC), and VC + MAS 200 group (MAS 200 mg/kg). Sprague-Dawley (SD) rats were treated with 200 mg/kg MAS or vehicle once daily for 28 days. The possible signaling mechanism and effects of MAS were measured via histological staining, immunohistochemistry, western blot, and biochemical assays. RESULTS: Parameters such as sperm motility and count, Johnsen's scores, spermatogenic cell density, serum testosterone, testicular superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx) and expression of the steroidogenic acute regulatory protein (StAR) improved significantly in the VC + MAS 200 group compared with the VC group. MAS treatment of varicocele-induced group significantly decreased the levels of serum luteinizing hormone (LH) and follicle-stimulating hormone (FSH), as well as testicular interleukin-6 (IL6), tumor necrosis factor-α (TNF-α), ROS/RNS, and malondialdehyde (MDA). It also decreased the apoptotic index and reduced the expression of endoplasmic reticulum (ER) protein levels (Grp78, p-IRE1α, and p-JNK) and apoptotic markers such as cleaved caspase-3 and Bax/Bcl2 ratio. CONCLUSION: This study suggests that the crosstalk between oxidative stress, ER stress, and mitochondrial pathway mediates varicocele-induced testicular germ cell apoptosis. MAS promotes spermatogenesis in varicocele-induced SD rat, probably by decreasing cytokines (IL-6, TNF-α) levels, regulating abnormal sex hormones, and decreasing oxidative stress, ER stress, and apoptosis.


Assuntos
Iridoides/farmacologia , Quempferóis/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Quercetina/análogos & derivados , Varicocele/metabolismo , Animais , Antioxidantes/farmacologia , Peso Corporal/efeitos dos fármacos , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Masculino , Mitocôndrias/efeitos dos fármacos , Tamanho do Órgão/efeitos dos fármacos , Quercetina/farmacologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Testículo/química , Testículo/efeitos dos fármacos , Testículo/patologia , Varicocele/patologia
8.
Mar Pollut Bull ; 145: 325-333, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31590794

RESUMO

Silver (Ag) is a non-essential metal known to bioaccumulate in aquatic organisms. We determined Ag concentrations in five false killer whales stranded in South America. Silver concentrations (in dry weight basis) range as 6.62-10.78 µg g-1 in liver, 0.008-7.41 µg g-1 in spleen, 0.004-5.71 µg g-1 in testis, 0.757-1.69 µg g-1 in kidney, 0.011-0.078 µg g-1 in lung and < 0.01-0.038 µg g-1 in muscle, whereas in the single samples of uterus and ovary were 0.051 and 0.023 µg g-1; respectively. Overall, Ag concentration in liver and kidney exceeded the cetacean toxic thresholds, proposed as "unhealthy concentrations" and "critically dangerous" in liver and kidney. These results warrant further eco-toxicological studies, to examine biological effects of elevated silver levels for individuals and to assess the species' conservation status with respect to marine pollution.


Assuntos
Golfinhos , Prata/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Feminino , Rim/química , Fígado/química , Masculino , Ovário/química , Prata/análise , América do Sul , Testículo/química , Poluentes Químicos da Água/análise
9.
Am J Surg Pathol ; 43(8): 1123-1128, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31094926

RESUMO

Evaluation of testicular biopsies from azoospermic men requires recognition of phases of germ cell maturation as organized architecturally within the seminiferous tubule, as well as distinguishing the inability to generate mature spermatozoa (germ cell aplasia or maturation arrest) from normal spermatogenesis, which may be associated with a reversible obstruction. While traditional fixatives (eg, Bouin solution) provide exquisite nuclear detail and preserve the architectural integrity of the seminiferous tubule, formalin fixation yields biopsies with relatively poor nuclear detail and frequent luminal sloughing of cells, making it difficult to assess sperm maturation. One clone of the anti-DOG1 antibody was recently found to be expressed in late (postspermatogonial) germ cells. We developed a dual stain including DOG1 and SF-1 to mark late germ cells and Sertoli cells, respectively, in both sloughed and intact cells. Consecutive testicular biopsies (N=28) from men with azoospermia were classified by hematoxylin and eosin morphology and stained with a dual SF-1 (Perseus)/DOG1 (Cell Marque) immunohistochemical stain. Histologic patterns included normal spermatogenesis (5 cases), hypospermatogenesis (5 cases), late maturation arrest (2 cases), Sertoli cell only pattern (15 cases), and extensive tubular hyalinization (1 case). Architectural disruption of seminiferous tubules with sloughing of cells into the lumens was noted in all biopsies, at least focally. SF-1 (nuclear) was expressed in sloughed Sertoli cells; DOG1 (cytoplasmic) in sloughed postspermatogonial germ cells (spermatocytes and spermatids). This resulted in two distinct immunophenotypes: SF-1(+)/DOG1(-) sloughed cells in cases with the Sertoli cell only pattern and SF-1(+)/DOG1(+) sloughed cells in all other histologic patterns (normal spermatogenesis, hypospermatogenesis, and maturation arrest). Because the rate of sperm retrieval is lower in men with the Sertoli cell only pattern, this immunohistochemical stain may assist pathologists in the proper interpretation of testicular biopsies, allowing better-informed decision making by patients and clinicians regarding the subsequent use of assisted reproductive technologies.


Assuntos
Anoctamina-1/análise , Azoospermia/metabolismo , Imuno-Histoquímica , Proteínas de Neoplasias/análise , Células de Sertoli/química , Espermatozoides/química , Fator Esteroidogênico 1/análise , Testículo/química , Adulto , Azoospermia/patologia , Azoospermia/fisiopatologia , Biomarcadores/análise , Biópsia , Humanos , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Prognóstico , Células de Sertoli/patologia , Espermatogênese , Espermatozoides/patologia , Testículo/patologia , Testículo/fisiopatologia
10.
Dev Dyn ; 248(6): 488-500, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30939211

RESUMO

BACKGROUND: Male germ cells are unique because they express a substantial number of variants of the general DNA binding proteins, known as histones, yet the biological significance of these variants is still unknown. In the present study, we aimed to address the expression pattern of the testis-specific histone H2B variant (TH2B) and the testis-specific histone H2A variant (TH2A) within the neonatal mouse testis. RESULTS: We demonstrate that TH2B and TH2A are present in a testis-enriched for undifferentiated spermatogonia. Co-localization studies with an undifferentiated marker, ZBTB16, revealed that TH2B and ZBTB16 co-localize in the neonatal testis. Upon the appearance of the primary spermatocytes, TH2B no longer co-localized with the ZBTB16 positive spermatogonia but were instead detected within the differentiating spermatogonia. This pattern of expression where TH2B and ZBTB16 no longer co-localize was maintained in the adult testis. CONCLUSION: These findings are in contrast to previous studies, which demonstrated that TH2B and TH2A were found only in adult spermatocytes. Our data are in support of a switch in the expression of these variants following the first round of spermatogonial differentiation. These studies reinforce current understandings that spermatogonia within the neonatal mouse testis are inherently different from those residing within the adult testis.


Assuntos
Variação Genética , Histonas/genética , Espermatogênese , Testículo/química , Animais , Animais Recém-Nascidos , Histonas/análise , Masculino , Camundongos , Espermatócitos/química
11.
Genes (Basel) ; 10(4)2019 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-31013951

RESUMO

Avian genomes have perplexed researchers by being conservative in both size and rearrangements, while simultaneously holding the blueprints for a massive species radiation during the last 65 million years (My). Transposable elements (TEs) in bird genomes are relatively scarce but have been implicated as important hotspots for chromosomal inversions. In zebra finch (Taeniopygia guttata), long terminal repeat (LTR) retrotransposons have proliferated and are positively associated with chromosomal breakpoint regions. Here, we present the genome, karyotype and transposons of blue-capped cordon-bleu (Uraeginthus cyanocephalus), an African songbird that diverged from zebra finch at the root of estrildid finches 10 million years ago (Mya). This constitutes the third linked-read sequenced genome assembly and fourth in-depth curated TE library of any bird. Exploration of TE diversity on this brief evolutionary timescale constitutes a considerable increase in resolution for avian TE biology and allowed us to uncover 4.5 Mb more LTR retrotransposons in the zebra finch genome. In blue-capped cordon-bleu, we likewise observed a recent LTR accumulation indicating that this is a shared feature of Estrildidae. Curiously, we discovered 25 new endogenous retrovirus-like LTR retrotransposon families of which at least 21 are present in zebra finch but were previously undiscovered. This highlights the importance of studying close relatives of model organisms.


Assuntos
Retroelementos , Aves Canoras/genética , Sequências Repetidas Terminais , Sequenciamento Completo do Genoma/veterinária , Animais , Evolução Molecular , Tentilhões/genética , Genoma , Sequenciamento de Nucleotídeos em Larga Escala/veterinária , Cariótipo , Masculino , Miocárdio/química , Filogenia , Testículo/química
12.
Toxicol Appl Pharmacol ; 370: 117-130, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30878504

RESUMO

The exponentially increasing use of electromagnetic field (EMF)-emitting devices imposes substantial health burden on modern societies with particular concerns of male infertility. Limited studies have addressed the modulation of this risk by protective agents. We investigated the hazardous effects of rat exposure to EMF (900 MHz, 2 h/day for 8 weeks) on male fertility and evaluated the possible protective effect of the polyamine, spermine, against EMF-induced alterations. Exposure to EMF significantly decreased sperm count, viability and motility, and increased sperm deformities. EMF-exposed rats exhibited significant reductions in serum inhibin B and testosterone along with elevated activin A, follicle-stimulating hormone, luteinizing hormone and estradiol concentrations. Testicular steroidogenic acute regulatory protein (StAR), c-kit mRNA expression and testicular activities of the key androgenic enzymes 3ß- and 17ß-hydroxysteroid dehydrogenases were significantly attenuated following exposure to EMF. Exposure led to testicular lipid peroxidation, decreased catalase and glutathione peroxidase activities and triggered nuclear factor-kappa B p65, inducible nitric oxide synthase, cyclooxygenase-2 and caspase-3 overexpression. EMF-exposed rats showed testicular DNA damage as indicated by elevated comet parameters. Spermine administration (2.5 mg/Kg/day intraperitoneally for 8 weeks) prevented EMF-induced alterations in the sperm and hormone profiles, StAR and c-kit expression and androgenic enzyme activities. Spermine hampered EMF-induced oxidative, inflammatory, apoptotic and DNA perturbations. Histological and histomorphometric analysis of the testes supported all biochemical findings. In conclusion, rat exposure to EMF disrupts sperm and hormone profiles with underlying impairment of steroidogenesis and spermatogenesis. Spermine confers protection against EMF-associated testicular and reproductive aberrations, at least in part, via antioxidant, anti-inflammatory and anti-apoptotic mechanisms.


Assuntos
Campos Eletromagnéticos/efeitos adversos , Infertilidade Masculina/etiologia , Infertilidade Masculina/prevenção & controle , Espermina/administração & dosagem , 17-Hidroxiesteroide Desidrogenases/metabolismo , Animais , Apoptose/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Hormônios Esteroides Gonadais/biossíntese , Hormônios Esteroides Gonadais/sangue , Infertilidade Masculina/patologia , Masculino , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Wistar , Contagem de Espermatozoides , Motilidade Espermática/efeitos dos fármacos , Espermatogênese/efeitos dos fármacos , Espermatozoides/anormalidades , Espermatozoides/efeitos dos fármacos , Espermatozoides/fisiologia , Testículo/química , Testículo/metabolismo , Testículo/patologia
13.
PLoS One ; 14(3): e0210537, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30865634

RESUMO

We previously found relationships between body condition and physiological function affecting health and welfare of female tourist camp elephants in Thailand, and used that approach to conduct a similar study of bull elephants in the same camps (n = 13). A body condition score (BCS) was done every other month, and fecal glucocorticoid metabolite (FGM) concentrations were measured twice monthly for 1 year. Effects of season, camp management and tourist activity on lipid profiles [total cholesterol (TC), low density lipoproteins (LDL), high density lipoproteins (HDL), triglycerides (TG)] and metabolic factors [insulin, glucose, fructosamine, glucose to insulin ratio (G:I)] were determined and correlated to measures of body condition, testosterone and FGM. Positive correlations were found between BCS and TG, between FGM and TG, HDL and glucose, and between testosterone and HDL, whereas BCS and testosterone were negatively associated with the G:I. There was a significant positive relationship between FGM and testosterone. Elevated FGM concentrations were associated with altered lipid and metabolic profiles and were higher in winter compared to summer and rainy seasons. Insulin and glucose levels were higher, while the G:I was lowest in the winter season. Strong positive associations were found between TC and HDL, LDL and HDL and glucose, and glucose and insulin. By contrast, negative relationships were found between the G:I and HDL and glucose, and between insulin and G:I. Differences also were found between High and Low tourist season months for FGM, insulin, and G:I. Last, there was notable variation among the camps in measured parameters, which together with tourist season effects suggests camp management may affect physiological function and welfare; some negatively like feeding high calorie treats, others positively, like exercise. Last, compared to females, bull elephants appear to be in better physical health based on normal BCSs, lower insulin levels and higher G:I ratios.


Assuntos
Animais de Zoológico/fisiologia , Glucocorticoides/metabolismo , Glucose/metabolismo , Insulina/metabolismo , Lipídeos/análise , Testículo/química , Testosterona/análise , Bem-Estar do Animal , Animais , Elefantes , Fezes/química , Nível de Saúde , Atividades Humanas , Masculino , Metabolômica , Estações do Ano , Tailândia
14.
Biomater Sci ; 7(4): 1422-1436, 2019 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-30666997

RESUMO

Extracellular matrix-derived scaffolds provide an efficient platform for the generation of organ-like structures. Successful development of testicular organoids (TOs) with the capability of supporting complete spermatogenesis has not been reported yet. Here, we have developed an optimized method for the decellularization of ram testicular tissue fragments. Our findings showed that testicular fragments treated with a serial combination of Triton X-100 and SDS in PBS for 48 h resulted in the efficient removal of cellular materials and retention of the extracellular matrix (ECM) components. In order to fabricate testis-derived scaffolds (TDSs), the testicular ECM (T-ECM) was digested in acid/pepsin, followed by neutralization of pre-gel solution to form a hydrogel. Then, the hydrogels were freeze-dried and cross-linked using a chemical method. To reach the optimal concentration for the T-ECM in the fabrication of TDSs, the scaffold properties including porosity, pore size, swelling behavior, and degradation were evaluated. Our study suggested that 25 mg ml-1 of the T-ECM is the best concentration for the fabrication of macroporous TDSs for demonstrating lower pore size, homogeneously distributed pores, and a higher swelling ratio. Furthermore, inoculation of neonatal mouse testicular cells onto TDSs resulted in the generation of multicellular TOs in which the differentiation of spermatogonial cells into post-meiotic cells was confirmed. Hormonal analysis of TDSs revealed the functionality of TOs in the secretion of testosterone and inhibin B. The current study also demonstrated that macroporous TDSs could provide a novel platform for testicular tissue engineering and in vitro spermatogenesis.


Assuntos
Organoides/química , Testículo/química , Animais , Matriz Extracelular/química , Masculino , Camundongos , Tamanho da Partícula , Porosidade , Propriedades de Superfície , Engenharia Tecidual
15.
Nat Commun ; 10(1): 433, 2019 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-30683861

RESUMO

Aberrant sperm flagella impair sperm motility and cause male infertility, yet the genes which have been identified in multiple morphological abnormalities of the flagella (MMAF) can only explain the pathogenic mechanisms of MMAF in a small number of cases. Here, we identify and functionally characterize homozygous loss-of-function mutations of QRICH2 in two infertile males with MMAF from two consanguineous families. Remarkably, Qrich2 knock-out (KO) male mice constructed by CRISPR-Cas9 technology present MMAF phenotypes and sterility. To elucidate the mechanisms of Qrich2 functioning in sperm flagellar formation, we perform proteomic analysis on the testes of KO and wild-type mice. Furthermore, in vitro experiments indicate that QRICH2 is involved in sperm flagellar development through stabilizing and enhancing the expression of proteins related to flagellar development. Our findings strongly suggest that the genetic mutations of human QRICH2 can lead to male infertility with MMAF and that QRICH2 is essential for sperm flagellar formation.


Assuntos
Infertilidade Masculina/genética , Mutação com Perda de Função , Proteínas dos Microtúbulos/genética , Cauda do Espermatozoide/metabolismo , Proteínas de Ancoragem à Quinase A/deficiência , Proteínas de Ancoragem à Quinase A/genética , Adulto , Animais , Proteínas de Ligação ao Cálcio/deficiência , Proteínas de Ligação ao Cálcio/genética , Consanguinidade , Expressão Gênica , Perfilação da Expressão Gênica , Proteínas de Choque Térmico/deficiência , Proteínas de Choque Térmico/genética , Humanos , Infertilidade Masculina/metabolismo , Infertilidade Masculina/patologia , Masculino , Camundongos , Camundongos Knockout , Linhagem , Fosfoproteínas/deficiência , Fosfoproteínas/genética , Motilidade Espermática , Cauda do Espermatozoide/patologia , Cauda do Espermatozoide/ultraestrutura , Testículo/química , Testículo/metabolismo , Sequenciamento Completo do Genoma
16.
Sci Rep ; 8(1): 16577, 2018 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-30409985

RESUMO

The aim of this study was to investigate the effect of early calf-hood nutrition on the transcriptomic profile of the arcuate nucleus of the hypothalamus, anterior pituitary and testes in Holstein-Friesian bulls. Holstein-Friesian bull calves with a mean (±S.D.) age and bodyweight of 19 (±8.2) days and 47.5 (±5.3) kg, respectively, were offered a high (n = 10) or low (n = 10) plane of nutrition in order to achieve an overall growth rate of 1.2 and 0.5 kg/day. At 126 (±3) days of age, calves were euthanized, hypothalamus (arcuate region), anterior pituitary and testicular parenchyma samples were harvested and RNAseq analysis was performed. There were 0, 49 and 1,346 genes differentially expressed in the arcuate nucleus, anterior pituitary and testicular tissue of bull calves on the low relative to the high plane of nutrition, respectively (P < 0.05; False Discovery Rate <0.05). Cell cycle processes in the anterior pituitary were down regulated in the low relative to the high plane of nutrition; there was no differential expression of genes related to reproductive processes. Gene expression involved in cholesterol and androgen biosynthesis in the testes were down regulated in animals on the low plane of nutrition. This study provides insight into the effect of early life plane of nutrition on the regulation of the HPT axis.


Assuntos
Núcleo Arqueado do Hipotálamo/química , Perfilação da Expressão Gênica/veterinária , Adeno-Hipófise/química , Testículo/química , Androgênios/biossíntese , Animais , Núcleo Arqueado do Hipotálamo/metabolismo , Peso Corporal , Bovinos , Colesterol/biossíntese , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Estado Nutricional , Adeno-Hipófise/metabolismo , Análise de Sequência de RNA/veterinária , Testículo/metabolismo
17.
J Proteome Res ; 17(12): 4178-4185, 2018 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-30277781

RESUMO

In 2012, the Chromosome-centric Human Proteome Project (C-HPP) launched an investigation for missing proteins (MPs) to complete the Human Proteome Project (HPP). The majority of the MPs were distributed in low-molecular-weight (LMW) ranges, especially from 0 to 40 kDa. LMW protein identification is challenging, owing to their short length, low abundance, and hydrophobicity. Furthermore, many sequences from trypsin digestion are unlikely to yield detectable peptides or a reasonable quality of MS2 spectrum. Therefore, we focused on small MPs by combining LMW protein enrichment and a pair of complementary proteases strategy with trypsin and LysargiNase for human testis samples. In-depth testis LMW protein profiling resulted in the identification of 4063 proteins, of which 2565 were LMW proteins and 1130 had pairs of peptides generated from both trypsin and LysargiNase. This provided additional mass spectral evidence of further verification of small MPs. Finally, two MPs were verified from the seven MP candidates. One of them, Q8N688 , was verified with two series of continuous and complementary b/y-product ions from the pairs of spectra for tryptic and LysargiNase digested peptides after the "mirror spectrum" matching. This make the confident identification of the representative peptides for the target MPs. On the contrary, the two verified peptides for Q86WR6 were identified with the same strategy from the gel-separation and gel-elution samples, respectively. Although the other five MP candidates showed high-quality spectra, they could not be sufficiently distinguished as PE1s and require further verification. All MS data sets have been deposited in the ProteomeXchange with identifier PXD010093.


Assuntos
Peptídeos/análise , Testículo/química , Humanos , Masculino , Espectrometria de Massas/métodos , Peso Molecular , Peptídeo Hidrolases/metabolismo
18.
Anat Rec (Hoboken) ; 301(12): 2080-2085, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30312540

RESUMO

Sertoli cells of the mammalian seminiferous epithelium form unique subcellular actin-related structures at intercellular junctions. The appearance of these so called "tubulobulbar complexes" (TBCs) precedes both sperm release at the apex of the epithelium and the movement of early spermatogenic cells out of the spermatogonial stem cell niche at the base of the epithelium. TBCs are considered to be part of the mechanism of junction endocytosis by Sertoli cells. The structures contain junction proteins and morphologically identifiable junctions, and are associated with markers of endocytosis. Here we review the current state of knowledge about the structure and function of TBCs. As the complexes form, they morphologically resemble and have the molecular signature of clathrin-coated pits with extremely long necks. As they mature, the actin filament networks around the "necks" of the structures progressively disassemble and the membrane cores expand or swell into distinct "bulbs". These bulbs acquire extensive membrane contact sites with associated cisternae of endoplasmic reticulum. Eventually the bulbs undergo scission and continue through endosomal compartments of the Sertoli cells. The morphology and composition of TBC indicates to us that the structures likely evolved from the basic clathrin-mediated endocytosis mechanism common to cells generally, and along the way they incorporated unique features to accommodate the cyclic turnover of massive and "intact" intercellular junctions that occurs during spermatogenesis. Anat Rec, 301:2080-2085, 2018. © 2018 Wiley Periodicals, Inc.


Assuntos
Actinas/metabolismo , Clatrina/metabolismo , Endocitose/fisiologia , Junções Intercelulares/metabolismo , Testículo/metabolismo , Actinas/análise , Animais , Clatrina/análise , Humanos , Junções Intercelulares/química , Masculino , Epitélio Seminífero/química , Epitélio Seminífero/citologia , Epitélio Seminífero/metabolismo , Células de Sertoli/química , Células de Sertoli/metabolismo , Testículo/química , Testículo/citologia
19.
Anal Chem ; 90(22): 13306-13314, 2018 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-30350618

RESUMO

We have previously demonstrated liquid extraction surface analysis (LESA) high field asymmetric waveform ion mobility spectrometry (FAIMS) mass spectrometry imaging of proteins in thin tissue sections of brain and liver. Here, we present an improved approach that makes use of multiple static FAIMS parameters at each sampled location and allows a significant improvement in the number of proteins, lipids, and drugs that can be imaged simultaneously. The approach is applied to the mass spectrometry imaging of control and cassette-dosed rat kidneys. Mass spectrometry imaging of kidneys typically requires washing to remove excess hemoglobin; however, that is not necessary with this approach. Multistep static FAIMS mass spectrometry resulted in a 6- to 16-fold increase in the number of proteins detected in the absence of FAIMS, in addition to smaller increases over single step static FAIMS (chosen for optimum transmission of total protein ions). The benefits of multistep static FAIMS mass spectrometry for protein detection are also shown for sections of testes. The numbers of proteins detected following multistep FAIMS increased between 2- and 3-fold over single step FAIMS and between 2- and 14-fold over LESA alone. Finally, to date, LESA mass spectrometry of proteins in tissue has been undertaken solely on fresh frozen samples. In this work, we demonstrate that heat-preserved tissues are also suitable for these analyses. Heat preservation of tissue improved the number of proteins detected by LESA MS for both kidney and testes tissue (by between 2- and 4-fold). For both tissue types, the majority of the proteins additionally detected in the heat-treated samples were subsequently detected in the frozen samples when FAIMS was incorporated. Improvements in the numbers of proteins detected were observed for LESA FAIMS MS for the kidney tissue; for testes tissue, fewer total proteins were detected following heat preservation; however, approximately one-third were unique to the heat-preserved samples.


Assuntos
Espectrometria de Mobilidade Iônica/métodos , Extração Líquido-Líquido/métodos , Proteínas/análise , Espectrometria de Massas em Tandem/métodos , Animais , Química Encefálica , Temperatura Baixa , Temperatura Alta , Rim/química , Masculino , Ratos Wistar , Propriedades de Superfície , Testículo/química
20.
J Proteome Res ; 17(12): 4171-4177, 2018 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-30280576

RESUMO

Subsequent to conducting the Chromosome-Centric Human Proteome Project, we have focused on human testis-enriched missing proteins (MPs) since 2015. For protein coverage to be enhanced, a multiprotease strategy was used for separation of samples by 10% SDS-PAGE. For the separating efficiency to be improved, a high-pH reverse phase (RP) separation strategy was applied to fractionate complex samples in this study. A total of 11,558 proteins was identified, which is the largest proteome data set for single human tissue sample so far. On the basis of this large-scale data set, we verified 14 MPs (PE2) in neXtProt (2018-01) after spectrum quality analysis, isobaric post-translational modification, and single amino acid variant filtering, and synthesized peptide matching. Tissue expression analysis showed that 3 of 14 MPs were testis-specific proteins. Functional analysis showed that 10 of 14 MPs were closely related to liver tumor, liver carcinoma, and hepatocellular carcinoma. Another 100 MPs were listed as candidates but required additional verification information. All MS data sets have been deposited into the ProteomeXchange with the identifier PXD009737.


Assuntos
Proteoma/análise , Testículo/química , Eletroforese em Gel de Poliacrilamida , Variação Genética , Humanos , Neoplasias Hepáticas/química , Masculino , Espectrometria de Massas , Peptídeo Hidrolases/metabolismo , Processamento de Proteína Pós-Traducional , Proteômica/métodos
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