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1.
J Vis Exp ; (167)2021 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-33522502

RESUMO

Isolation of meiotic spermatocytes is essential to investigate molecular mechanisms underlying meiosis and spermatogenesis. Although there are established cell isolation protocols using Hoechst 33342 staining in combination with fluorescence-activated cell sorting, it requires cell sorters equipped with an ultraviolet laser. Here we describe a cell isolation protocol using the DyeCycle Violet (DCV) stain, a low cytotoxicity DNA binding dye structurally similar to Hoechst 33342. DCV can be excited by both ultraviolet and violet lasers, which improves the flexibility of equipment choice, including a cell sorter not equipped with an ultraviolet laser. Using this protocol, one can isolate three live-cell subpopulations in meiotic prophase I, including leptotene/zygotene, pachytene, and diplotene spermatocytes, as well as post-meiotic round spermatids. We also describe a protocol to prepare single-cell suspension from mouse testes. Overall, the procedure requires a short time to complete (4-5 hours depending on the number of needed cells), which facilitates many downstream applications.


Assuntos
Permeabilidade da Membrana Celular , Separação Celular/métodos , DNA/metabolismo , Espermatócitos/citologia , Espermatogênese , Animais , Benzimidazóis/metabolismo , Sobrevivência Celular , Dissecação , Citometria de Fluxo , Fluorescência , Masculino , Meiose , Camundongos , Estágio Paquíteno , Espalhamento de Radiação , Espermátides/citologia , Coloração e Rotulagem , Testículo/citologia
2.
Nat Commun ; 12(1): 892, 2021 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-33563972

RESUMO

Given their copy number differences and unique modes of inheritance, the evolved gene content and expression of sex chromosomes is unusual. In many organisms the X and Y chromosomes are inactivated in spermatocytes, possibly as a defense mechanism against insertions into unpaired chromatin. In addition to current sex chromosomes, Drosophila has a small gene-poor X-chromosome relic (4th) that re-acquired autosomal status. Here we use single cell RNA-Seq on fly larvae to demonstrate that the single X and pair of 4th chromosomes are specifically inactivated in primary spermatocytes, based on measuring all genes or a set of broadly expressed genes in testis we identified. In contrast, genes on the single Y chromosome become maximally active in primary spermatocytes. Reduced X transcript levels are due to failed activation of RNA-Polymerase-II by phosphorylation of Serine 2 and 5.


Assuntos
Drosophila/genética , Cromossomos Sexuais/genética , Espermatócitos/metabolismo , Animais , Drosophila/crescimento & desenvolvimento , Regulação da Expressão Gênica , Genes Ligados ao Cromossomo X/genética , Genes Ligados ao Cromossomo Y/genética , Larva/genética , Larva/crescimento & desenvolvimento , Masculino , Especificidade de Órgãos , RNA Polimerase II/metabolismo , Cromossomos Sexuais/metabolismo , Espermatogênese/genética , Testículo/citologia , Testículo/metabolismo , Transcrição Genética
3.
Ultraschall Med ; 42(1): 10-38, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33530122

RESUMO

Acute testicular pain in childhood can be caused by testicular torsion, torsion of the appendix testis, or epididymo-orchitis. Quick and reliable diagnosis is essential for determining the further course of action (surgery or conservative approach). The diagnostic tool of choice is high-resolution sonography with a linear transducer (> 10 MHz) combined with color and spectral Doppler sonography. The Doppler device settings should include a low pulse repetition frequency (< 4 cm/s), a low wall filter (< 100 Hz), and adequate gain. Comparison with the unaffected healthy testis is essential. The most important of the three diseases is torsion of the spermatic cord because it requires immediate surgical intervention and detorsion. The affected testis is enlarged and has an inhomogeneous echotexture with hypoechoic and hyperechoic areas as well as an associated hydrocele. In testicular torsion, color Doppler shows reduced or absent intratesticular vessels in comparison with the healthy contralateral testis. Spectral Doppler shows decreased flow velocities especially during diastole in intratesticular arteries and an increased resistance index. The investigation should always include imaging of the spermatic cord from the outer inguinal ring to the upper pole of the testis. In contrast to a normal finding, the vessels and the ductus deferens are not displayed as linear tubular structures but in form of a spiral twist. Ultrasound shows a target-like structure with multiple concentric rings. Color Doppler sonography shows the typical whirlpool sign. In torsion of the appendix testis, the appendix testis is enlarged in the groove between the testis and epididymis. The longitudinal diameter of the appendix testis can be greater than 5 mm. The echogenicity of the torsed appendage can vary between hypoechoic (acute torsion) and hyperechoic (prior torsion). An associated hydrocele of varying size is usually seen. Color Doppler sonography reveals a lack of perfusion of the enlarged appendix testis and increased vascularity of the testis and primarily the epididymis. Epididymo-orchitis is characterized by an enlarged epididymis and/or testis with inhomogeneous echogenicity (hypoechoic - hyperechoic). Color Doppler sonography shows increased vascularity in comparison with the unaffected testis. Spectral Doppler reveals increased diastolic flow velocities and a decreased resistance index. Idiopathic scrotal edema and an incarcerated inguinal hernia must be ruled out in the differential diagnosis.


Assuntos
Escroto , Torção do Cordão Espermático , Doenças Testiculares , Doença Aguda , Adolescente , Criança , Diagnóstico Diferencial , Epididimo , Humanos , Masculino , Escroto/diagnóstico por imagem , Torção do Cordão Espermático/diagnóstico por imagem , Testículo/diagnóstico por imagem
4.
J Vis Exp ; (167)2021 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-33522506

RESUMO

There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally been restricted to be used only on fixed samples or live cells outside of tissue labeled with strong fluorescent signal. Current super-resolution live cell imaging techniques require the use of special fluorescence probes, high illumination, multiple image acquisitions with post-acquisition processing, or often a combination of these processes. These prerequisites significantly limit the biological samples and contexts that this technique can be applied to. Here we describe a method to perform super-resolution (~140 nm XY-resolution) time-lapse fluorescence live cell imaging in situ. This technique is also compatible with low fluorescent intensity, for example, EGFP or mCherry endogenously tagged at lowly expressed genes. As a proof-of-principle, we have used this method to visualize multiple subcellular structures in the Drosophila testis. During tissue preparation, both the cellular structure and tissue morphology are maintained within the dissected testis. Here, we use this technique to image microtubule dynamics, the interactions between microtubules and the nuclear membrane, as well as the attachment of microtubules to centromeres. This technique requires special procedures in sample preparation, sample mounting and immobilizing of specimens. Additionally, the specimens must be maintained for several hours after dissection without compromising cellular function and activity. While we have optimized the conditions for live super-resolution imaging specifically in Drosophila male germline stem cells (GSCs) and progenitor germ cells in dissected testis tissue, this technique is broadly applicable to a variety of different cell types. The ability to observe cells under their physiological conditions without sacrificing either spatial or temporal resolution will serve as an invaluable tool to researchers seeking to address crucial questions in cell biology.


Assuntos
Drosophila melanogaster/citologia , Imageamento Tridimensional , Microscopia de Fluorescência/métodos , Animais , Técnicas de Cultura de Células , Sobrevivência Celular , Corantes Fluorescentes/química , Proteínas de Fluorescência Verde/metabolismo , Masculino , Microtúbulos/metabolismo , Células-Tronco/citologia , Frações Subcelulares/metabolismo , Testículo/citologia , Imagem com Lapso de Tempo , Tubulina (Proteína)/metabolismo
5.
Gan To Kagaku Ryoho ; 48(2): 303-305, 2021 Feb.
Artigo em Japonês | MEDLINE | ID: mdl-33597389

RESUMO

A man aged 65 years had undergone high orchidectomy of the right testis for diffuse large B-cell lymphoma(DLBCL) occurring primarily in the testis 11 months before. Although he was referred to another hospital for postoperative chemotherapy, he refused the treatment by self-judgement. For 1 month, he had been experiencing melena and anal pain, so he visited our department in June. Rectal palpation revealed a sub-circumference tumor palpable from the anal margin, in which a part protruded outside the anus. CT revealed a sub-circumference hypertrophic wall from the rectal Ra to the anus and intramural enlarged lymph nodes, without metastases to the other organs. Systemic gallium scintigraphy detected a strong concentration in the rectum. The endoscopic examination of the inferior region revealed a circumference type 2 tumor at Rb, and biopsy revealed DLBCL. Clinically, this case was considered a testoid DLBCL with rectal metastasis. Therefore, we performed laparoscopic rectal amputation in July, XX. sT3N1b, cM0. The postoperative course was uneventful. After the patient was discharged from our department, he received chemotherapy at another hospital. At present, 4 years 0 month postoperatively, the patient condition is favorable without recurrence. When perforation occurs in gastrointestinal DLBCL, the start of chemotherapy is delayed and the primary lesion worsen. Therefore, we performed surgical therapy first. Such cases must be evaluated for metastases or new lesions carefully.


Assuntos
Linfoma Difuso de Grandes Células B , Neoplasias Retais , Idoso , Humanos , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/cirurgia , Masculino , Recidiva Local de Neoplasia , Neoplasias Retais/tratamento farmacológico , Neoplasias Retais/cirurgia , Reto , Testículo
6.
Ecotoxicol Environ Saf ; 211: 111946, 2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-33493718

RESUMO

Increased applications of quantum dots (QDs) in the biomedical field have aroused attention for their potential toxicological effects. Although numerous studies have been carried out on the toxicity of QDs, their effects on reproductive and development are still unclear. In this study, we systematically evaluated the male reproductive toxicity and developmental toxicity of CdSe/ZnS QDs in BALB/c mice. The male mice were injected intravenously with CdSe/ZnS QDs at the dosage of 2.5 mg/kg BW or 25 mg/kg BW, respectively, and the survival status, biodistribution of QDs in testes, serum sex hormone levels, histopathology, sperm motility and acrosome integrity was measured on Day 1, 7, 14, 28 and 42 after injection. On Day 35 after treatment, male mice were housed with non-exposed female mice, and then offspring number, body weight, organ index and histopathology of major organs, blood routine and biochemical tests of offspring were measured to evaluate the fertility and offspring health. The results showed that CdSe/ZnS QDs could rapidly distribute in the testis, and the fluorescence of QDs could still be detected on Day 42 post-injection. QDs had no adverse effect on the structure of testis and epididymis, but high-dose QDs could induce apoptosis of Leydig cells in testis at an early stage. No significant differences in survival of state, body weight organ index of testis and epididymis, sex hormones levels, sperm quality, sperm acrosome integrity and fertility of male mice were observed in QDs exposed groups. However, the development of offspring was obviously influenced, which was mainly manifested in the slow growth of offspring, changes in organ index of main organs, and the abnormality of liver and kidney function parameters. Our findings revealed that CdSe/ZnS QDs were able to cross the blood-testis barrier (BTB), produce no discernible toxic effects on the male reproductive system, but could affect the healthy growth of future generations to some extent. In view of the broad application prospect of QDs in biomedical fields, our findings might provide insight into the biological safety evaluation of the reproductive health of QDs.


Assuntos
Pontos Quânticos/toxicidade , Acrossomo , Animais , Compostos de Cádmio/química , Compostos de Cádmio/toxicidade , Epididimo , Feminino , Fertilidade , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pontos Quânticos/química , Reprodução , Compostos de Selênio/farmacologia , Motilidade Espermática , Espermatozoides , Sulfetos/toxicidade , Testículo , Distribuição Tecidual , Testes de Toxicidade , Compostos de Zinco/toxicidade
7.
Pediatr Emerg Care ; 37(2): 123-125, 2021 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-33512891

RESUMO

OBJECTIVES: To determine if boys with acute testicular torsion, a surgical emergency requiring prompt diagnosis and treatment to optimize salvage of the testicle, delayed presentation to a medical facility and experienced an extended duration of symptoms (DoS), and secondarily, a higher rate of orchiectomy, during the coronavirus disease 2019 (COVID-19) pandemic. METHODS: Single-center, descriptive retrospective chart review of boys presenting with acute testicular torsion from March 15, to May 4, 2020 ("during COVID-19" or group 2), as well as for the same time window in the 5-year period from 2015 to 2019 ("pre-COVID-19" or group 1). RESULTS: A total of 78 boys met inclusion criteria, group 1 (n = 57) and group 2 (n = 21). The mean age was 12.86 ± 2.63 (group 1) and 12.86 ± 2.13 (group 2). Mean DoS before presentation at a medical facility was 23.2 ± 35.0 hours in group 1 compared with 21.3 ± 29.7 hours in group 2 (P < 0.37). When DoS was broken down into acute (<24 hours) versus delayed (≥24 hours), 41 (71.9%) of 57 boys in group 1 and 16 (76.2%) of 21 boys in group 2 presented within less than 24 hours of symptom onset (P < 0.78). There was no difference in rate of orchiectomy between group 1 and group 2 (44.7% vs 25%, P < 0.17), respectively. CONCLUSIONS: Boys with acute testicular torsion in our catchment area did not delay presentation to a medical facility from March 15, to May 4, 2020, and did not subsequently undergo a higher rate of orchiectomy.


Assuntos
/epidemiologia , Torção do Cordão Espermático/cirurgia , Adolescente , Criança , Serviço Hospitalar de Emergência , Humanos , Masculino , Orquiectomia/estatística & dados numéricos , Estudos Retrospectivos , Torção do Cordão Espermático/diagnóstico , Torção do Cordão Espermático/epidemiologia , Testículo/cirurgia , Fatores de Tempo , Tempo para o Tratamento
8.
Ecotoxicol Environ Saf ; 208: 111601, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33396121

RESUMO

Atrazine (ATZ) is among the most widely used herbicides in the world, and yet it has a potential to contaminate aquatic environments due to pesticide leaching from agricultural areas. In the Neotropical region, studies about the effects of this herbicide in native aquatic wildlife is scarce.Our study aimed at investigating the effects of a 30-day exposure to a commercial atrazine formulation on oxidative stress parameters, histopathology in testis and liver, and hormone levels in males and female of yellow-tailed tetra fish (Astyanax altiparanae). Adults were exposed to low but environmentally relevant concentrations of atrazine as follows: 0 (CTL-control), 0.5 (ATZ0.5), 1 (ATZ1), 2 (ATZ2) and 10 (ATZ10) µg/L. Our results showed decreased GST activity in gills in all groups of exposed animals and increased CAT activity in gills from the ATZ10 group. In the liver, there was an increase in lipid peroxidation in fish from ATZ1 and ATZ2 groups. Histological analysis of the liver showed increased percentage of sinusoid capillaries in ATZ2 fish, increased vascular congestion in ATZ1 and increased leukocyte infiltration in the ATZ10 group. Hepatocyte diameter analysis revealed a decrease in cell size in all groups exposed to ATZ, and a decrease in hepatocyte nucleus diameter in ATZ1, ATZ2 and ATZ10 groups. Endocrine parameters did not show significant changes following ATZ exposure, although an increase of triiodothyronine/thyroxine (T3/T4) ratio was observed in ATZ2 fish. Our results provide evidence that even low, environmentally relevant concentrations of ATZ produced oxidative damage and histological alterations in adult yellow-tailed tetra.


Assuntos
Atrazina/toxicidade , Characidae/metabolismo , Herbicidas/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Animais , Atrazina/metabolismo , Relação Dose-Resposta a Droga , Feminino , Brânquias/efeitos dos fármacos , Brânquias/metabolismo , Herbicidas/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Testículo/efeitos dos fármacos , Testículo/metabolismo , Testículo/patologia , Poluentes Químicos da Água/metabolismo
9.
Reproduction ; 161(2): R37-R44, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33434886

RESUMO

Invasion or damage of the male reproductive system is one of the reported outcomes of viral infection. Current studies have documented that SARS-CoV-2, which causes COVID-19, can damage the male reproductive system in large part by inflammatory damage caused by a cytokine storm. However, whether SARS-CoV-2 can infect the human testis directly and enter semen is controversial. Other adverse effects of SARS-CoV-2 on male reproduction are also of concern and require comprehensive evaluation. Here, we analyze the invasiveness of SARS-CoV-2 in the testis and examine reported mechanisms by which SARS-CoV-2 interferes with male reproduction. Long-term implications of SARS-CoV-2 infection on male reproduction are also discussed. It should be emphasized that although COVID-19 may induce testicular damage, a substantial decrease in male reproductive capacity awaits clinical evidence. We propose that there is an urgent need to track male COVID-19 patients during their recovery. The development of suitable experimental models, including human reproductive organoids, will be valuable to further investigate the viral impact on reproduction for current and future pandemics.


Assuntos
/complicações , Reprodução , Testículo/virologia , /análise , /fisiopatologia , Citocinas/sangue , Humanos , Sistema Hipotálamo-Hipofisário/fisiopatologia , Infertilidade Masculina/virologia , Masculino , Orquite/virologia , /fisiologia , Espermatogênese , Espermatozoides/virologia , Testículo/química , Testículo/fisiopatologia
10.
Ecotoxicol Environ Saf ; 208: 111731, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33396062

RESUMO

Cadmium (Cd) is an environmental toxicant and a nonessential metal. Cd can attack a wide range of organs, such as the liver, kidney, lung, ovary, testis, brain, and muscle in vertebrates. Among these organs, the testis might be the most sensitive organ to Cd toxicity. Metallothionein (MT) is a cysteine-rich protein with a low molecular weight, that can bind with Cd and eliminate reactive oxygen species (ROSs). Hydrogen peroxide, which as a crucial type of ROS that is induced by Cd, can be eliminated by catalase (CAT) in the self-protection of cells and to realize Cd toxicity resistance. To investigate the functions of MT and CAT in the testis of Cynops orientalis, we cloned the full-length MT and CAT genes of C. orientalis for the first time. Immunofluorescence results demonstrated that MT and CAT were expressed in Sertoli cells and all spermatogenic cells in the testis of C. orientalis. The results of the ultrastructural damage assay demonstrated that there were various impairments, which included organelle vacuolization, abnormal chromatin distribution, and apoptotic bodies, in somatic cells that were exposed to Cd. However, the anomalies of spermatozoa were located mainly in the mid-piece and head, many of which showed severely impaired structures. The results demonstrated that MT and CAT expression had distinct patterns in response to various Cd concentrations: an increase in MT mRNA levels with elevated Cd levels and a persistent increase in CAT mRNA levels with elevated Cd levels. These results suggested that MT and CAT play roles in Cd toxicity resistance in the testis and that the expression of CAT may be a better biomarker than the expression of MT for assessing Cd pollution.


Assuntos
Cádmio/toxicidade , Catalase/metabolismo , Clonagem Molecular , Substâncias Perigosas/toxicidade , Metalotioneína/metabolismo , Salamandridae/fisiologia , Testículo/efeitos dos fármacos , Animais , Sequência de Bases , Humanos , Fígado/metabolismo , Masculino , RNA Mensageiro/metabolismo , Salamandridae/genética , Salamandridae/metabolismo , Células de Sertoli/metabolismo , Espermatozoides/metabolismo , Testículo/metabolismo
11.
Ecotoxicol Environ Saf ; 208: 111714, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33396045

RESUMO

Studies on the effects of unintentional intake of pyrethroid pesticides that are akin to actual human exposure settings are very rare. Such an exposure is primarily by consuming the food products as routine diet that contain residual levels of pyrethroids. In this study, rats were orally administered for 15 months with a mixture of pyrethroids at a dose that is one-fifth (high dose; HD) or one-twenty fifth (low dose; LD) of the residual levels commonly present in the average amount of rice and vegetables consumed by Indian population. Lipid profile, kidney and liver function were assessed. Lipid peroxidation, nitric oxide, antioxidant enzyme activities and histopathological changes were analyzed in the liver, lung, kidney, pancreas, testes, caput, cauda and prostate. The effect on the male reproductive system as a function of sperm count, enzyme activity of 3ß-HSD and 17ß-HSD and the expression profile of genes involved in spermatogenesis, steroidogenesis, genetic reprogramming and apoptosis of male gametes were evaluated. Significant increase in the relative organ weight, perturbations in the activities of antioxidant enzymes, lipid profile and liver function were observed in both LD and HD groups. Damage to the anatomical architecture was evident in all the tissues due to pyrethroid toxicity. Exposure to LD and HD of pyrethroid mixture resulted in decreased sperm count, activities of 3ß-HSD and 17ß-HSD, impaired capacitation and acrosome reaction and perturbations in the expression of genes that govern male gamete production. Results of our study indicate that exposure to pyrethroids for longer durations even at doses that are far below the residual levels present in the food consumed will result in severe damage to general physiological processes as well as reproductive function.


Assuntos
Exposição Dietética/efeitos adversos , Praguicidas/toxicidade , Piretrinas/toxicidade , Animais , Antioxidantes/metabolismo , Fertilização/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Tamanho do Órgão/efeitos dos fármacos , Resíduos de Praguicidas/toxicidade , Ratos , Espermatogênese/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Espermatozoides/metabolismo , Espermatozoides/patologia , Testículo/efeitos dos fármacos , Testículo/metabolismo , Testículo/patologia
13.
Ecotoxicol Environ Saf ; 211: 111938, 2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-33476844

RESUMO

Melamine cyanuric acid (MCA) is a flame retardant linked by hydrogen bonds between melamine and cyanuric acid. MCA is used in an excellent series of phosphorus and nitrogen flame retardants. MCA can harm the kidney, liver, testis, and spleen cells. However, the effects of MCA on the emotions and behaviour of adolescent mice have not yet been investigated. In this article, male mice were exposed to MCA at 10, 20, and 40 mg/kg for four weeks. MCA exposure resulted in enhanced mouse locomotor and nocturnal activity. We also observed anxiety-like and depression-like behaviours. Moreover, after MCA exposure, the serum concentrations of thyroid-related hormones were changed, and the mRNA levels were affected. In short, MCA exposure can cause behavioural and emotion disorders.


Assuntos
Comportamento Animal/efeitos dos fármacos , Poluentes Ambientais/toxicidade , Triazinas/toxicidade , Animais , Retardadores de Chama , Rim , Masculino , Camundongos , Baço , Testículo
14.
J Zoo Wildl Med ; 51(4): 848-855, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33480565

RESUMO

Evaluation of sex ratios is a critical component of chelonian captive breeding programs and may become increasingly useful to assess the demographics of free-living populations. In many reptile species, the sex of immature animals cannot be determined based on external features. Endoscopic sex identification is an accurate and safe method to identify the sex of immature individuals of some chelonian species. A number of studies describe this technique in controlled, hospital settings and report significant interspecies variations in gonad morphology; however, there are few reports describing this technique in field conditions. In the current study, the gonadal morphology of 40 immature Western Santa Cruz tortoises (Chelonoidis porteri) on Santa Cruz Island in Galapagos, Ecuador, was assessed. A previously described endoscopic protocol was used to perform sex identification under field conditions. Tortoises were anesthetized using an intramuscular injection of ketamine (10 mg/kg) and medetomidine (0.1 mg/kg), which provided an adequate plane of anesthesia. The medetomidine was reversed with atipamezole (0.5 mg/kg). Field conditions presented challenges such as limited control over lighting, suboptimal patient positioning, and restricted power supply for endoscopy equipment. The immature testicle in Western Santa Cruz tortoises was oval, reddish pink, and tightly adhered to the coelomic membrane ventral to the kidney. The surface of the gonads resembled other species with the notable exception that the ovaries lacked a significant number of primordial follicles. These gonadal characteristics were consistent, with only one individual identified as undetermined sex of the 40 samples. This field-based endoscopic gonadal evaluation was a safe and sensitive technique for determining the sex of free-living immature Western Santa Cruz Galapagos tortoises.


Assuntos
Anestesia/veterinária , Ovário/anatomia & histologia , Maturidade Sexual , Testículo/anatomia & histologia , Analgésicos/administração & dosagem , Analgésicos/farmacologia , Animais , Feminino , Hipnóticos e Sedativos/administração & dosagem , Hipnóticos e Sedativos/farmacologia , Ketamina/administração & dosagem , Ketamina/farmacologia , Masculino , Medetomidina/administração & dosagem , Medetomidina/farmacologia , Ovário/crescimento & desenvolvimento , Testículo/crescimento & desenvolvimento , Tartarugas/fisiologia
16.
Nat Commun ; 12(1): 73, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397987

RESUMO

In the male germ cells of placental mammals, 26-30-nt-long PIWI-interacting RNAs (piRNAs) emerge when spermatocytes enter the pachytene phase of meiosis. In mice, pachytene piRNAs derive from ~100 discrete autosomal loci that produce canonical RNA polymerase II transcripts. These piRNA clusters bear 5' caps and 3' poly(A) tails, and often contain introns that are removed before nuclear export and processing into piRNAs. What marks pachytene piRNA clusters to produce piRNAs, and what confines their expression to the germline? We report that an unusually long first exon (≥ 10 kb) or a long, unspliced transcript correlates with germline-specific transcription and piRNA production. Our integrative analysis of transcriptome, piRNA, and epigenome datasets across multiple species reveals that a long first exon is an evolutionarily conserved feature of pachytene piRNA clusters. Furthermore, a highly methylated promoter, often containing a low or intermediate level of CG dinucleotides, correlates with germline expression and somatic silencing of pachytene piRNA clusters. Pachytene piRNA precursor transcripts bind THOC1 and THOC2, THO complex subunits known to promote transcriptional elongation and mRNA nuclear export. Together, these features may explain why the major sources of pachytene piRNA clusters specifically generate these unique small RNAs in the male germline of placental mammals.


Assuntos
Epigênese Genética , Éxons/genética , Mamíferos/genética , Estágio Paquíteno/genética , RNA Interferente Pequeno/metabolismo , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Acetilação , Animais , Metilação de DNA/genética , Proteínas de Ligação a DNA/metabolismo , Evolução Molecular , Histonas/metabolismo , Íntrons/genética , Masculino , Camundongos Endogâmicos C57BL , Proteínas Nucleares/metabolismo , Especificidade de Órgãos/genética , Regiões Promotoras Genéticas/genética , Processamento de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais/genética , Testículo/metabolismo , Transcrição Genética
17.
Environ Pollut ; 271: 116265, 2021 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-33385891

RESUMO

One of the causes of the global decline of amphibians is agricultural activity, responsible for causing habitat fragmentation and bringing a range of agrochemicals and fertilizers in the environment, compounds with a potential disrupting effect on non-target organisms, such as frogs. Exposure to these compounds has numerous harmful effects on the testes of these animals, which can compromise reproduction and, consequently, the maintenance of their communities. In this context, we compared the morphology and morphometry of the testes of three species of neotropical anurans (Physalaemus cuvieri, Dendropsophus minutus, and Boana albopunctata) from an agricultural area and a conservation unit. Histologically, the testicular morphology of the species was similar for both environments; however, morphometrically, there was a difference in the measured testicular parameters (locular area and area of spermatogenic cysts). Physalaemus cuvieri presented higher averages of locular and spermatogonial area in the agricultural environment, whereas the area occupied by the spermatozoa was smaller. Additionally, the testicular pigmentation, which is only present in this species, was greater in animal from the agricultural area. In D. minutus, the locular, spermatogonial, and sperm areas showed lower values in the agricultural area, whereas in B. albopunctata, the opposite pattern was found, with the area of the locule, spermatocytes, and spermatozoids being higher. Agricultural activities influence the testicular metric parameters in different species, and our results suggest that D. minutus is most sensitive to anthropic pressures. The least sensitive species is B. albopunctata. We highlight the importance of evaluating different species, since each species responds differently to agricultural activities.


Assuntos
Anuros , Testículo , Animais , Ecossistema , Masculino , Espermatogênese , Espermatogônias
18.
Environ Pollut ; 270: 116241, 2021 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-33321432

RESUMO

Cadmium (Cd) was an environmental pollutant, which could result in germ cell apoptosis in testes. Sertoli-germ cell communication was vital for germ cell development and maturity. However, little was known about the effect of Sertoli cell autophagy on Cd-induced germ cell apoptosis. Here, we used male Amh-Cre+/Atg5flox/flox (Atg5-/-) mice, loss of autophagy-related gene 5 (Atg5) in testicular Sertoli cells, to explore the obscure effects. Atg5-/- and Wild-type (WT) mice were given with cadmium chloride (CdCl2, 2.0 mg/kg) for 0-24 h. Our results showed that Cd triggered testicular germ cell apoptosis, as evidenced by the increment of TUNEL-labeled germ cells, cleaved caspase3 and cleaved poly (ADP-ribose) polymerase protein level. Additionally, Cd induced testicular autophagy, as determined by elevating the level of autophagy-related proteins, including Atg5, Atg7, LC3B-II, and the gathering of LC3 puncta. 3-methyladenine, a specific autophagy inhibitor, exacerbated Cd-caused germ cell apoptosis. Inversely, rapamycin, an autophagy inducer, relieved Cd-stimulated germ cell apoptosis. Interestingly, we found that autophagy in Sertoli cells was activated in Cd-treated WT mouse testes as evidenced by the increment of LC3 puncta surrounding SOX9, a specific Sertoli cell marker. More importantly, loss of autophagy in Sertoli cells aggravated Cd-triggered germ cell apoptosis. Taken together, these data indicate that autophagy in Sertoli cells alleviates Cd-triggered germ cell apoptosis in mouse testes.


Assuntos
Cádmio , Células de Sertoli , Animais , Apoptose , Autofagia , Cádmio/toxicidade , Células Germinativas , Masculino , Camundongos , Testículo
19.
Chemosphere ; 267: 129296, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33348264

RESUMO

The aim of this study was to: (i) determine and compare the capacity of bis (2 -ethylhexyl) phthalate (DEHP), dibutyl phthalate (DBP), bisphenol A (BPA), and their mixture to produce testicular toxicity after the subacute exposure; (ii) explore the mechanisms behind the observed changes using in silico toxicogenomic approach. Male rats were randomly split into groups (n = 6): (1) Control (corn oil); (2) DEHP (50 mg/kg b.w./day); (3) DBP (50 mg/kg b.w./day); (4) BPA (25 mg/kg b.w./day); and (5) MIX (50 mg/kg b.w./day DEHP + 50 mg/kg b.w/day DBP + 25 mg/kg b.w./day BPA). Animals were sacrificed after 28 days of oral exposure, testes were extracted and prepared for histological assessments under the light microscope (haematoxylin and eosin staining) and redox status analysis. The Comparative Toxicogenomics Database (CTD; http://CTD.mdibl.org), Cytoscape software (https://cytoscape.org) and ToppGene Suite (https://toppgene.cchmc.org) were used for data-mining. Present pathohistological study has demonstrated more pronounced testicular toxicity of the MIX group (desquamated germinal epithelium cells, enlarged cells with hyperchromatic nuclei, multinucleated cell forms and intracytoplasmic vacuoles) in comparison with the single substances, while effects on redox status parameters were either more prominent, or present only in the MIX group. In silico investigation revealed 20 genes linked to male reproductive disorders, affected by all three investigated substances. Effects on metabolism, AhR pathway, apoptosis and oxidative stress could be singled out as the most probable mechanisms involved in the subacute DEHP, DBP and BPA mixture testicular toxicity, while the effect on oxidative stress parameters was confirmed by in vivo experiment.


Assuntos
Dietilexilftalato , Testículo , Animais , Compostos Benzidrílicos , Simulação por Computador , Dietilexilftalato/metabolismo , Dietilexilftalato/toxicidade , Masculino , Oxirredução , Fenóis , Ácidos Ftálicos , Ratos , Testículo/metabolismo , Toxicogenética
20.
Gene ; 773: 145364, 2021 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-33359122

RESUMO

Vitamin E is generally believed to promote the production of ovine sperm mainly through its antioxidant effect. Our previous studies have shown that some non-antioxidant genes may also be key in mediating this process. The objective of this study was to identify key candidate proteins that were differentially expressed in response to a treatment with Vitamin E. Prepubertal ovine testicular cells were isolated and divided into two groups. They were either treated with 800 µM Vitamin E (based on our previous results) or used as a non-treated control. After 24 h, all the cells were harvested for proteomic analysis. We found 115 differentially expressed proteins, 4 of which were up-regulated and 111 were down-regulated. A GO term enrichment analysis identified 127 Biological Process, 63 Cell Component and 26 Molecular Function terms that were enriched. Within those terms, 13, 11 and 26 terms were significantly enriched, respectively. Terms related to membrane and enzyme activity including the inner acrosomal membrane, signal peptidase complex, cysteine-type endopeptidase activity, etc., were also markedly enriched, while none of the KEGG pathways were enriched. We found that many of the differentially expressed proteins, such as CD46 (membrane cofactor protein), FLNA (Filamin A), DYSF (Dysferlin), IFT20 (Intraflagellar transport 20), SPCS1 (Signal peptidase complex subunit 1) and SPCS3 (Signal peptidase complex subunit 3) were related to the acrosomal and plasma membranes. A parallel reaction monitoring (PRM) analysis verified that Vitamin E improved spermatogenesis by regulating the expression of FLNA, SPCS3, YBX3 and RARS, proteins that are associated with the plasma membranes and protamine biosynthesis of the spermatozoa.


Assuntos
Protaminas/genética , Ovinos/crescimento & desenvolvimento , Espermatogênese/genética , Vitamina E/genética , Acrossomo/metabolismo , Animais , Membrana Celular , Regulação da Expressão Gênica no Desenvolvimento/genética , Masculino , Proteômica , Ovinos/genética , Espermatozoides/metabolismo , Testículo/crescimento & desenvolvimento , Testículo/metabolismo , Vitamina E/metabolismo
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