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1.
Cell Physiol Biochem ; 54(1): 40-52, 2020 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-31942786

RESUMO

BACKGROUND/AIMS: AIRE is known for its involvement in autoreactive T-cell deletion in thymic epithelium. Though extrathymic expression of AIRE is well documented, the functional relevance of AIRE in non-thymus tissues is emerging. AIRE is expressed in neonatal and adult testis, and has been implicated in sporadic germ cell apoptosis in developing testis. In this study we examined whether AIRE has any role in inducing apoptosis in cultured spermatogonial cells. METHODS: We over-expressed AIRE or CARD domain of AIRE in GC1-spg cells and evaluated its impact on cell cycle using fluorescence activated cell sorting following Hoechst 33342 staining. Apoptosis was assayed using Annexin-V staining. Caspase-3 cleavage was assessed on western blots and caspase-3 expression was quantitated using realtime PCR. RESULTS: We report that C18-4 cells which are derived from Type A spermatogonia expressed AIRE, while GC1-spg which is closer to Type B spermatogonia was negative for AIRE expression. Overexpression of AIRE or CARD domain of AIRE induced Caspase-3 expression in GC1-spg cells. Silencing of AIRE in C18-4 cells inhibited Caspase-3 expression. When overexpressed, AIRE and CARD brought about a very negligible increase in germ cell death and resulted in altered cell cycle pattern with a reduction in G1 phase. This was not associated with any increase in activation of Caspase-3. CONCLUSION: We conclude that the CARD domain of AIRE enhances caspase-3 expression through possible direct DNA binding and triggers non-apoptotic downstream signaling in cultured spermatogonial cells.


Assuntos
Apoptose , Caspase 3/genética , Espermatogônias/citologia , Fatores de Transcrição/genética , Animais , Linhagem Celular , Humanos , Masculino , Camundongos , Espermatogônias/metabolismo , Testículo/citologia , Testículo/metabolismo , Regulação para Cima
2.
BMC Complement Altern Med ; 19(1): 362, 2019 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-31829240

RESUMO

BACKGROUND: Infertility and gonadal dysfunction are well known side-effects by cancer treatment in males. In particularly, chemotherapy and radiotherapy induced testicular damage, resulting in prolonged azoospermia. However, information regarding therapeutics to treat spermatogenesis disturbance after cancer treatment is scarce. Recently, we demonstrated that Goshajinkigan, a traditional Japanese medicine, can completely rescue severe busulfan-induced aspermatogenesis in mice. In this study, we aimed to detect the effects of Goshajinkigan on aspermatogenesis after irradiation. METHODS: This is animal research about the effects of traditional Japanese medicine on infertility after cancer treatment. C57BL/6 J male mice received total body irradiation (TBI: a single dose of 6Gy) at 4 weeks of age and after 60 days were reared a Goshajinkigan (TJ107)-containing or TJ107-free control diet from day 60 to day 120. Then, two untreated females were mated with a single male from each experimental group. On day 60, 120 and 150, respectively, the sets of testes and epididymis of the mice in each group after deep anesthetization were removed for histological and cytological examinations. RESULTS: Histological and histopathological data showed that 6Gy TBI treatment decreased the fertility rate (4/10) in the control diet group; in contrast, in the TJ107-diet group, the fertility rate was 10/10 (p < 0.05 vs. 6Gy group). Supplementation with TJ107 was found to rescue the disrupted inter-Sertoli tight junctions via the normalization of claudin11, occludin, and ZO-1 expression and reduce serum anti-germ cell autoantibodies. CONCLUSIONS: These findings show the therapeutic effect on TBI-induced aspermatogenesis and the recovering disrupted gonadal functions by supplementation with TJ107.


Assuntos
Azoospermia/patologia , Medicamentos de Ervas Chinesas/farmacologia , Lesões Experimentais por Radiação/patologia , Protetores contra Radiação/farmacologia , Espermatogênese , Animais , Epididimo/citologia , Epididimo/patologia , Epididimo/efeitos da radiação , Feminino , Japão , Masculino , Medicina Tradicional do Leste Asiático , Camundongos , Camundongos Endogâmicos C57BL , Espermatogênese/efeitos dos fármacos , Espermatogênese/efeitos da radiação , Testículo/citologia , Testículo/patologia , Testículo/efeitos da radiação
3.
Endocr Regul ; 53(2): 93-99, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-31517623

RESUMO

OBJECTIVE: Stem cell therapy, specifically, pre-induction of mesenchymal stem cells toward male germ-like cells may be useful in patients with azoospermia. The aim of this study was to evaluate in vitro differentiation of mouse bone marrow-derived mesenchymal stem cells (BMSCs) into male germ-like cells by indirect co-culture with testicular cells in the presence of bone morphogenetic protein 4 (BMP4). METHODS: Experimental groups included: control (mouse BMSCs), treatment group-1 (BMSCs treated with BMP4), treatment group-2 (indirect co-culture of BMSCs with mouse testicular cells in the presence of BMP4) and treatment group-3 (indirect co-culture of BMSCs with testicular cells). BMSCs-derived male germ-like cells were evaluated by the expression of Dazl, and Stra8 using RT-qPCR. RESULTS: Stra8 gene expression was significantly increased in the treatment group-2 and Dazl gene was significantly increased in the treatment group-1 compared to other groups. In conclusion, indirect co-culturing of BMSCs with testicular cells and BMP4 leads to the differentiation of BMSCs into male germ-like cells which express specific male germ-like genes. Testicular cells released factors that contributed to the differentiation of BMSCs into male germ progenitor cells. CONCLUSION: This study suggests that mesenchymal stem cells may be differentiated into male germ-like cells and therefore, may be a novel treatment option for men with azoospermia.


Assuntos
Proteína Morfogenética Óssea 4/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Testículo/citologia , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/fisiologia , Células Cultivadas , Técnicas de Cocultura/métodos , Células Germinativas/efeitos dos fármacos , Células Germinativas/fisiologia , Humanos , Masculino , Células-Tronco Mesenquimais/fisiologia , Camundongos , Espermatozoides/fisiologia
4.
Environ Pollut ; 255(Pt 1): 113097, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31520908

RESUMO

Decabromodiphenyl ether (BDE-209) is commonly used as a flame retardant, usually in products that were utilized in electronic equipment, plastics, furniture and textiles. To identify the impacts of BDE-209 on the male reproductive system and the underlying toxicological mechanisms, 40 male ICR mice were randomly divided into four groups, which were then exposed to BDE-209 at 0, 7.5, 25 and 75 mg kg-1 d-1 for four weeks, respectively. With regard to the in vitro study, GC-2spd cells were treated with BDE-209 at 0, 2, 8 and 32 µg mL-1 for 24 h, respectively. The results from the in vivo experiments showed that BDE-209 resulted in damage to the testis structure, led to cell apoptosis in testis and decreased sperm number and motility, while sperm malformation rates were significantly increased. Moreover, BDE-209 could induce oxidative stress with decreased testosterone levels, result in DNA damage and activate DNA damage response signaling pathways (ATM/Chk2, ATR/Chk1 and DNA-PKcs/XRCC4/DNA ligase Ⅳ). The data from the in vitro experiments showed that BDE-209 led to cytotoxicity by reducing cell viability and increasing LDH release as well. BDE-209 also induced DNA strand breaks, cell cycle arrest at G1 phase and elevated reactive oxygen species (ROS) level in GC-2 cells. These results suggested that BDE-209 could lead to male reproductive toxicity by inducing DNA damage and failure of DNA damage repair which resulted in cell cycle arrest and apoptosis of spermatogenic cell. The present study provided new evidence to elucidate the potential mechanism of male reproductive toxicity induced by BDE-209.


Assuntos
Apoptose/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Retardadores de Chama/toxicidade , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Éteres Difenil Halogenados/toxicidade , Espermatozoides/patologia , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Reparo do DNA/genética , Masculino , Camundongos , Camundongos Endogâmicos ICR , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Contagem de Espermatozoides , Motilidade Espermática/efeitos dos fármacos , Espermatozoides/citologia , Testículo/citologia , Testículo/patologia , Testosterona/sangue
5.
Arch Insect Biochem Physiol ; 102(4): e21612, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31482645

RESUMO

Zn72D encodes the Drosophila zinc finger protein Zn72D. It was first identified to be involved in phagocytosis and indicated to have a role in immunity. Then it was demonstrated to have a function in RNA splicing and dosage compensation in Drosophila melanogaster. In this study, we discovered a new function of Zn72D in male fertility. We showed that knockdown of Zn72D in fly testes caused an extremely low egg hatch rate. Immunofluorescence staining of Zn72D knockdown testes exhibited scattered spermatid nuclei and no actin cones or individualization complexes (ICs) during spermiogenesis, whereas the early-stage germ cells and the spermatocytes were observed clearly. There were no mature sperms in the seminal vesicles of Zn72D knockdown fly testes, although a few sperms could be found close to the seminal vesicle. We further showed that many cytoskeleton-related genes were significantly downregulated in fly testes due to Zn72D knockdown. Taken together these findings suggest that Zn72D may have an important function in spermatogenesis by sustaining the cytoskeleton-based morphogenesis and individualization thus ensuring the proper formation of sperm in D. melanogaster.


Assuntos
Proteínas de Transporte/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Espermatogênese/genética , Animais , Drosophila melanogaster/crescimento & desenvolvimento , Feminino , Fertilidade/genética , Expressão Gênica , Masculino , Ovário/metabolismo , Testículo/citologia , Testículo/crescimento & desenvolvimento , Testículo/metabolismo
6.
Reprod Biol Endocrinol ; 17(1): 71, 2019 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-31472681

RESUMO

BACKGROUND: Palmitic acid (PA) is a common saturated fatty acid that induces apoptosis in various types of cells, including testicular Leydig cells. There is evidence suggesting that PA is increased in patients with obesity and that PA-induced cell apoptosis may play an important role in obesity-related male infertility. Curcumin, a natural polyphenol, has been reported to exert cytoprotective effects in various cell types. However, the cytoprotective effect of curcumin against PA-induced apoptosis in Leydig cells remains unknown. Therefore, the current study was performed to investigate the protective effects of curcumin in response to PA-induced toxicity and apoptosis in murine Leydig tumor cell line 1 (MLTC-1) cells and explore the mechanism underlying its anti-apoptotic action. METHODS: MLTC-1 cells were cultured in Roswell Park Institute-1640 medium and divided into five groups. First four groups were treated with 50-400 µM PA, 400 µM PA + 5-40 µM curcumin, 400 µM PA + 500 nM 4-phenylbutyric acid (4-PBA, an endoplasmic reticulum (ER) stress inhibitor), and 500 nM thapsigargin (TG, an ER stress inducer) + 20 µM curcumin, respectively, followed by incubation for 24 h. Effects of PA and/or curcumin on viability, apoptosis, and ER stress in MLTC-1 cells were then determined by cell proliferation assay, flow cytometry, and western blot analysis. The fifth group of MLTC-1 cells was exposed to 400 µM of PA and 5 IU/mL of human chorionic gonadotropin (hCG) for 24 h in the absence and presence of curcumin, followed by measurement of testosterone levels in cell-culture supernatants by enzyme-linked immunosorbent assay (ELISA). Rats fed a high-fat diet (HFD) were treated with or without curcumin for 4 weeks, and the testosterone levels were detected by ELISA. RESULTS: Exposure to 100-400 µM PA reduced cell viability, activated caspase 3, and enhanced the expression levels of the apoptosis-related protein BCL-2-associated X protein (BAX) and ER stress markers glucose-regulated protein 78 (GRP78) and CCAAT/enhancer binding protein homologous protein (CHOP) in MLTC-1 cells. Treating cells with 500 nM 4-PBA significantly attenuated PA-induced cytotoxicity through inhibition of ER stress. Curcumin (20 µM) significantly suppressed PA- or TG-induced decrease in cell viability, caspase 3 activity, and the expression levels of BAX, CHOP, and GRP78. In addition, treating MLTC-1 cells with 20 µM curcumin effectively restored testosterone levels, which were reduced in response to PA exposure. Similarly, curcumin treatment ameliorated the HFD-induced decrease in serum testosterone level in vivo. CONCLUSIONS: The present study suggests that PA induces apoptosis via ER stress and curcumin ameliorates PA-induced apoptosis by inhibiting ER stress in MLTC-1 cells. This study suggests the application of curcumin as a potential therapeutic agent for the treatment of obesity-related male infertility.


Assuntos
Apoptose/efeitos dos fármacos , Curcumina/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Células Intersticiais do Testículo/efeitos dos fármacos , Ácido Palmítico/farmacologia , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Células Intersticiais do Testículo/citologia , Células Intersticiais do Testículo/metabolismo , Masculino , Camundongos , Fenilbutiratos/farmacologia , Substâncias Protetoras/farmacologia , Ratos Sprague-Dawley , Testículo/citologia
7.
Adv Exp Med Biol ; 1169: 225-242, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31487027

RESUMO

Germ cells transfer genetic materials from one generation to the next, which ensures the continuation of the species. Spermatogenesis, the process of male germ cell production, is one of the most productive systems in adult tissues. This high productivity depends on the well-coordinated differentiation cascade in spermatogonia, occurring via their synchronized cell division and proliferation. Spermatogonial stem cells (SSCs) are responsible for maintaining the spermatogonial population via self-renewal and the continuous generation of committed progenitor cells that differentiate into spermatozoa. Like other stem cells in the body, SSCs are defined by their self-renewal and differentiation abilities. A functional transplantation assay, in which these biological properties of SSCs can be quantitatively evaluated, was developed using mice, and the cell surface characteristics and intracellular marker gene expression of murine SSCs were successfully determined. Another approach to elucidate SSC identity is a cell lineage-tracing experiment using transgenic mice, which can track the SSC behavior in the testes. Recent studies using both these experimental approaches have revealed that the SSC identity changed depending upon the developmental, homeostatic, and regenerative circumstances. In addition, single-cell transcriptomic analyses have further indicated the instability of marker gene expression in SSCs. More studies are needed to unify the results of the determination of SSC identity based on the functional properties and accumulating transcriptomic data of SSCs, to elucidate the functional interaction between SSC behavior and gene products and illustrate the conserved features of SSCs amidst their heterogeneity. Furthermore, the deterministic roles of distinct SSC niches under different physiological conditions in the SSC heterogeneity and its causal regulators must also be clarified in future studies.


Assuntos
Células-Tronco Germinativas Adultas , Espermatogônias , Animais , Células Cultivadas , Masculino , Camundongos , Espermatogênese , Espermatogônias/citologia , Testículo/citologia
8.
Ecotoxicol Environ Saf ; 184: 109579, 2019 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-31505405

RESUMO

DNA methylation have been suggested as possible mediators of long-term health effects of environmental stressors. This study aimed to evaluate the potential therapy of methylation of S-adenosyl-l-methionine (SAM) on PFOS induced trangeneral reproductive toxicity. In this study, postnatal 5d Sprague Dawley rats were randomly divided into four groups: control, PFOS, PFOS + SAM, and PFOS + Decitabine (DAC). The F0 rats were exposed to 5 mg/kg PFOS and SAM or DAC until PND60. The development of the offsprings were monitored without PFOS exposure. The fertility in F0, F1 rats, and change in F1 testes were observed. The results were as follows. The significant increase in F0 pregnancy rate, and survival rate in F1 offspring in PFOS + SAM relative to PFOS group were observed. Changes of birth weights and physical development in F1 offspring with SAM were approached as a corresponding variation of the control after the deparation period. No pregnant in F1 maternal rats in the PFOS and DAC groups were found, but pregnant in the SAM group. Significantly decrease in the percentage of abnormal seminiferous tubules and increase in expression of promyelocytic leukemia zinc finger (PLZF+) spermatogonial stem cells in F1 testis compared with the PFOS group. Taken together, Methyl donor SAM improve PLZF + spermatogonia stem cell proliferation, attenuate damage in testicular tissue structure, which subsequently improve the transgenerational growth retard and infertility induced by PFOS chronic stress.


Assuntos
Ácidos Alcanossulfônicos/toxicidade , Fluorcarbonetos/toxicidade , Efeitos Tardios da Exposição Pré-Natal/prevenção & controle , Reprodução/efeitos dos fármacos , S-Adenosilmetionina/uso terapêutico , Animais , Peso ao Nascer , Decitabina/uso terapêutico , Feminino , Masculino , Gravidez , Taxa de Gravidez , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Efeitos Tardios da Exposição Pré-Natal/mortalidade , Efeitos Tardios da Exposição Pré-Natal/patologia , Ratos Sprague-Dawley , Espermatogônias/citologia , Espermatogônias/efeitos dos fármacos , Taxa de Sobrevida , Testículo/citologia , Testículo/efeitos dos fármacos
9.
Int J Mol Sci ; 20(18)2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31510090

RESUMO

The negative association between psychological stress and male fertility has been known for many years. This study was aimed at (i) identifying spermatogenesis impairment induced by psychological stress in rats and (ii) exploring the role of glucocorticoid receptor (GR) signaling in these adverse effects (if they exist). Male Sprague Dawley rats were exposed to a six-week period of unpredictable chronic mild stress (uCMS) along with cotreatment of GR antagonist RU486 (1 mg/kg/day). Testicular damage was assessed by testicular pathological evaluation, epididymal sperm concentration, serum testosterone levels, testicular apoptotic cell measurements, and cell cycle progression analyses. Rats in the uCMS group had decreased levels of serum testosterone and decreased epididymal sperm concentration. The uCMS-treated rats also had decreased numbers of spermatids and increased levels of apoptotic seminiferous tubules; additionally, cell cycle progression of spermatogonia was arrested at the G0/G1 phase. Furthermore, uCMS exposure caused an increase in serum corticosterone level and activated GR signaling in the testes including upregulated GR expression. RU486 treatment suppressed GR signaling and alleviated the damaging effects of stress, resulting in an increased epididymal sperm concentration. Overall, this work demonstrated for the first time that the activation of GR signaling mediates stress-induced spermatogenesis impairment and that this outcome is related to cell apoptosis and cell cycle arrest in germ cells.


Assuntos
Epididimo/metabolismo , Receptores de Glucocorticoides/metabolismo , Espermatogênese/fisiologia , Espermatozoides/metabolismo , Estresse Psicológico/fisiopatologia , Testículo/metabolismo , Animais , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Epididimo/citologia , Epididimo/efeitos dos fármacos , Masculino , Mifepristona/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores de Glucocorticoides/antagonistas & inibidores , Túbulos Seminíferos/citologia , Túbulos Seminíferos/efeitos dos fármacos , Túbulos Seminíferos/metabolismo , Contagem de Espermatozoides , Espermatogênese/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Testículo/citologia , Testículo/efeitos dos fármacos , Testosterona/sangue
10.
Anat Histol Embryol ; 48(5): 505-507, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31389074

RESUMO

The distribution of actin filaments was examined in the seminiferous epithelium of the Habu (Trimeresurus flavoviridis; snake), by transmission electron microscopy and fluorescence histochemistry. By transmission electron microscopy, actin filaments were clearly found only at the site between Sertoli cell and spermatid without a lattice-like structure. Fluorescence histochemistry showed a weak labelling of actin filaments in the seminiferous epithelium, whereas these findings seem to be common among reptiles, they are different from those in mammals. Additionally, the bundles of actin filaments adjacent to the plasma membrane of Sertoli cells, appeared in other reptiles, were not observed in the Habu.


Assuntos
Citoesqueleto de Actina/ultraestrutura , Epitélio Seminífero/citologia , Animais , Masculino , Epitélio Seminífero/ultraestrutura , Células de Sertoli/citologia , Espermátides/ultraestrutura , Testículo/citologia , Trimeresurus
11.
Nat Commun ; 10(1): 3821, 2019 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-31444359

RESUMO

Meiosis is the specialized cell division during which parental genomes recombine to create genotypically unique gametes. Despite its importance, mammalian meiosis cannot be studied in vitro, greatly limiting mechanistic studies. In vivo, meiocytes progress asynchronously through meiosis and therefore the study of specific stages of meiosis is a challenge. Here, we describe a method for isolating pure sub-populations of nuclei that allows for detailed study of meiotic substages. Interrogating the H3K4me3 landscape revealed dynamic chromatin transitions between substages of meiotic prophase I, both at sites of genetic recombination and at gene promoters. We also leveraged this method to perform the first comprehensive, genome-wide survey of histone marks in meiotic prophase, revealing a heretofore unappreciated complexity of the epigenetic landscape at meiotic recombination hotspots. Ultimately, this study presents a straightforward, scalable framework for interrogating the complexities of mammalian meiosis.


Assuntos
Núcleo Celular/metabolismo , Epigênese Genética/fisiologia , Código das Histonas/fisiologia , Histonas/genética , Meiose/fisiologia , Acetilação , Animais , Núcleo Celular/genética , Cromatina/metabolismo , Imunoprecipitação da Cromatina , Quebras de DNA de Cadeia Dupla , Metilação de DNA/fisiologia , Sequenciamento de Nucleotídeos em Larga Escala , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Masculino , Camundongos , Regiões Promotoras Genéticas/genética , Recombinação Genética/fisiologia , Testículo/citologia
12.
PLoS Genet ; 15(8): e1008316, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31437213

RESUMO

The ubiquitin proteasome system regulates meiotic recombination in yeast through its association with the synaptonemal complex, a 'zipper'-like structure that holds homologous chromosome pairs in synapsis during meiotic prophase I. In mammals, the proteasome activator subunit PA200 targets acetylated histones for degradation during somatic DNA double strand break repair and during histone replacement during spermiogenesis. We investigated the role of the testis-specific proteasomal subunit α4s (PSMA8) during spermatogenesis, and found that PSMA8 was localized to and dependent on the central region of the synaptonemal complex. Accordingly, synapsis-deficient mice show delocalization of PSMA8. Moreover, though Psma8-deficient mice are proficient in meiotic homologous recombination, there are alterations in the proteostasis of several key meiotic players that, in addition to the known substrate acetylated histones, have been shown by a proteomic approach to interact with PSMA8, such as SYCP3, SYCP1, CDK1 and TRIP13. These alterations lead to an accumulation of spermatocytes in metaphase I and II which either enter massively into apoptosis or give rise to a low number of aberrant round spermatids that apoptose before histone replacement takes place.


Assuntos
Fertilidade/genética , Infertilidade Masculina/genética , Metáfase/genética , Complexo de Endopeptidases do Proteassoma/genética , Subunidades Proteicas/genética , Animais , Apoptose/genética , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Knockout , Proteínas Nucleares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Subunidades Proteicas/metabolismo , Espermatócitos/metabolismo , Espermatogênese/genética , Complexo Sinaptonêmico/metabolismo , Testículo/citologia , Testículo/metabolismo
13.
Yi Chuan ; 41(8): 686-702, 2019 Aug 20.
Artigo em Chinês | MEDLINE | ID: mdl-31447420

RESUMO

Spermatogonial stem cells (SSCs) are male germline stem cells that reside in the basement membrane of the seminiferous tubule in the testis. SSCs are characterized by their capability of self-renewal to maintain the stem cell pool throughout the lifespan and commitments to germ line after puberty, thus transmitting the genetic information from parents to the SSC-derived progenies. SSCs can be isolated from testis, propagated in vitro, and induced to differentiate into varied germ cells. Although significant progress has been made in the field of rodent SSCs, the SSCs of large animals have advanced slowly. Studies on SSCs of large animal models can offer insights into the physiological and pathological mechanism of human reproduction. Moreover, SSCs of agricultural large animals can be used as an essential tool for multiplication of elite animal individuals, and generation of genetically modified livestock with valuable economic traits. In this review, we summarize the recent progress on SSCs of large animal models for agricultural and medical purposes, and discuss the present problems and future prospects. This review can give an overall view of large animal SSCs as respect to their applications in novel alternative reproductive technologies, generation of transgenic animals, treatment of male infertility and regenerative medicine.


Assuntos
Espermatogônias/citologia , Células-Tronco/citologia , Animais , Animais Geneticamente Modificados , Humanos , Infertilidade Masculina , Masculino , Modelos Animais , Testículo/citologia
14.
Nat Commun ; 10(1): 3858, 2019 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-31451685

RESUMO

The Polycomb group of proteins is required for the proper orchestration of gene expression due to its role in maintaining transcriptional silencing. It is composed of several chromatin modifying complexes, including Polycomb Repressive Complex 2 (PRC2), which deposits H3K27me2/3. Here, we report the identification of a cofactor of PRC2, EZHIP (EZH1/2 Inhibitory Protein), expressed predominantly in the gonads. EZHIP limits the enzymatic activity of PRC2 and lessens the interaction between the core complex and its accessory subunits, but does not interfere with PRC2 recruitment to chromatin. Deletion of Ezhip in mice leads to a global increase in H3K27me2/3 deposition both during spermatogenesis and at late stages of oocyte maturation. This does not affect the initial number of follicles but is associated with a reduction of follicles in aging. Our results suggest that mature oocytes Ezhip-/- might not be fully functional and indicate that fertility is strongly impaired in Ezhip-/- females. Altogether, our study uncovers EZHIP as a regulator of chromatin landscape in gametes.


Assuntos
Proteínas Oncogênicas/metabolismo , Óvulo/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Espermatozoides/metabolismo , Adulto , Animais , Linhagem Celular Tumoral , Cromatina/metabolismo , Feminino , Células HEK293 , Histonas/metabolismo , Humanos , Masculino , Camundongos , Camundongos Knockout , Mutação , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/isolamento & purificação , Oogênese , Ovário/citologia , Ovário/patologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Células Sf9 , Espermatogênese , Testículo/citologia , Testículo/patologia
15.
PLoS Genet ; 15(7): e1008062, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31295251

RESUMO

Stem cells rely on instructive cues from their environment. Alterations in microenvironments might contribute to tissue dysfunction and disease pathogenesis. Germline stem cells (GSCs) and cyst stem cells (CySC) in Drosophila testes are normally maintained in the apical area by the testicular hub. In this study, we found that reproduction leads to accumulation of early differentiating daughters of CySCs and GSCs in the testes of aged male flies, due to hyperactivation of Jun-N-terminal kinase (JNK) signaling to maintain self-renewal gene expression in the differentiating cyst cells. JNK activity is normally required to maintain CySCs in the apical niche. A muscle sheath surrounds the Drosophila testis to maintain its long coiled structure. Importantly, reproduction triggers accumulation of the tumor necrosis factor (TNF) Eiger in the testis muscle to activate JNK signaling via the TNF receptor Grindelwald in the cyst cells. Reducing Eiger activity in the testis muscle sheath suppressed reproduction-induced differentiation defects, but had little effect on testis homeostasis of unmated males. Our results reveal that reproduction in males provokes a dramatic shift in the testicular microenvironment, which impairs tissue homeostasis and spermatogenesis in the testes.


Assuntos
Células-Tronco Germinativas Adultas/citologia , Drosophila melanogaster/fisiologia , Reprodução , Células-Tronco Germinativas Adultas/metabolismo , Animais , Diferenciação Celular , Autorrenovação Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Feminino , Homeostase , Sistema de Sinalização das MAP Quinases , Masculino , Proteínas de Membrana/metabolismo , Comportamento Sexual Animal , Espermatogênese , Nicho de Células-Tronco , Testículo/citologia , Testículo/metabolismo
16.
BMC Evol Biol ; 19(1): 137, 2019 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-31269894

RESUMO

BACKGROUND: Previously, we have demonstrated that genes involved in ovarian function are highly conserved throughout evolution. In this study, we aimed to document the conservation of genes involved in spermatogenesis from flies to vertebrates and their expression profiles in vertebrates. RESULTS: We retrieved 379 Drosophila melanogaster genes that are functionally involved in male reproduction according to their mutant phenotypes and listed their vertebrate orthologs. 83% of the fly genes have at least one vertebrate ortholog for a total of 625 mouse orthologs. This conservation percentage is almost twice as high as the 42% rate for the whole fly genome and is similar to that previously found for genes preferentially expressed in ovaries. Of the 625 mouse orthologs, we selected 68 mouse genes of interest, 42 of which exhibited a predominant relative expression in testes and 26 were their paralogs. These 68 mouse genes exhibited 144 and 60 orthologs in chicken and zebrafish, respectively, gathered in 28 groups of paralogs. Almost two thirds of the chicken orthologs and half of the zebrafish orthologs exhibited a relative expression ≥50% in testis. Finally, our focus on functional in silico data demonstrated that most of these genes were involved in the germ cell process, primarily in structure elaboration/maintenance and in acid nucleic metabolism. CONCLUSION: Our work confirms that the genes involved in germ cell development are highly conserved across evolution in vertebrates and invertebrates and display a high rate of conservation of preferential testicular expression among vertebrates. Among the genes highlighted in this study, three mouse genes (Lrrc46, Pabpc6 and Pkd2l1) have not previously been described in the testes, neither their zebrafish nor chicken orthologs. The phylogenetic approach developed in this study finally allows considering new testicular genes for further fundamental studies in vertebrates, including model species (mouse and zebrafish).


Assuntos
Galinhas/genética , Evolução Molecular , Testículo/metabolismo , Peixe-Zebra/genética , Animais , Drosophila melanogaster/genética , Masculino , Camundongos , Filogenia , Espermatogênese/genética , Testículo/citologia
17.
Gen Comp Endocrinol ; 282: 113216, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31278920

RESUMO

The Atlantic Bluefin Tuna (ABFT, Thunnus thynnus) is one of the most intensely exploited fisheries resources in the world. In spite of the years of studies on ABFT, basic aspects of its reproductive biology remain uncertain. To gain insight regarding the seasonal changes of the reproductive characteristics of the eastern stock of ABFT, blood and tissue samples were collected from mature specimens caught in the Mediterranean basin during the reproductive (May-June) and non-reproductive season (Oct-Nov). Histological analysis of the gonads of May-June samples indicated that there were females which were actively spawning (contained post-ovulatory follicles) and females that were not actively spawning that had previtellogenic and fully vitellogenic oocytes. In males, testis were at early or late stage of spermatogenesis during the reproductive season. In Oct-Nov, ovaries contained mostly previtellogenic oocytes as well as ß and α atretic follicles while the testis predominantly contained spermatogonia and few cysts with spermatocytes and spermatozoa. Gonadosomatic index (GSI) in females was highest among the actively spawning individuals while in males GSI was higher in early and late spermatogenic individuals compared to those that were spent. Plasma sex steroids levels varied with the reproductive season. In females, estradiol (E2), was higher in May-June while testosterone (T) and progesterone (P) did not vary. In males, E2 and T were higher in May-June while P levels were similar at the two sampling points. Circulating follicle stimulating hormone (FSH) was higher in Oct-Nov than in May-June both in males and females. Vitellogenin (VTG) was detected in plasma from both males and females during the reproductive season with levels in females significantly higher than in males. VTG was undetected in Oct-Nov samples. Since choriogenesis is an important event during follicle growth, the expression of three genes involved in vitelline envelope formation and hardening was measured and results showed significantly higher levels in ovaries in fish caught in May-June with respect to those sampled in Oct-Nov. In addition, a set of genes encoding for ion channels that are responsible for oocyte hydration and buoyancy, as well as sperm viability, were characterized at the two time points, and these were found to be more highly expressed in females during the reproductive season. Finally, the expression level of three mRNAs encoding for different lipid-binding proteins was analyzed with significantly higher levels detected in males, suggesting sex-specific expression. Our findings provide additional information on the reproductive biology of ABFT, particularly on biomarkers for the assessment of the state of maturation of the gonad, highlighting gender-specific signals and seasonal differences.


Assuntos
Reprodução/fisiologia , Estações do Ano , Atum/fisiologia , Animais , Feminino , Hormônio Foliculoestimulante/sangue , Gametogênese/genética , Regulação da Expressão Gênica , Hormônios Esteroides Gonadais/metabolismo , Masculino , Folículo Ovariano/citologia , Ovário/citologia , Ovário/metabolismo , Testículo/citologia , Testículo/metabolismo , Atum/sangue , Atum/genética , Vitelogeninas/sangue
18.
Nat Commun ; 10(1): 3389, 2019 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-31358756

RESUMO

Small RNAs have important functions. However, small RNAs in primate oocytes remain unexplored. Herein, we develop CAS-seq, a single-cell small RNA sequencing method, and profile the small RNAs in human oocytes and embryos. We discover a class of ~20-nt small RNAs that are predominantly expressed in human and monkey oocytes, but not in mouse oocytes. They are specifically associated with HIWI3 (PIWIL3), whereas significantly shorter than the commonly known PIWI-interacting RNAs (piRNAs), designated as oocyte short piRNAs (os-piRNAs). Notably, the os-piRNAs in human oocytes lack 2'-O-methylation at the 3' end, a hallmark of the classic piRNAs. In addition, the os-piRNAs have a strong 1U/10 A bias and are enriched on the antisense strands of recently evolved transposable elements (TEs), indicating the potential function of silencing TEs by cleavage. Therefore, our study has identified an oocyte-specific piRNA family with distinct features and provides valuable resources for studying small RNAs in primate oocytes.


Assuntos
Oócitos/metabolismo , RNA Interferente Pequeno/genética , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Animais , Proteínas Argonauta/genética , Proteínas Argonauta/metabolismo , Células do Cúmulo/citologia , Células do Cúmulo/metabolismo , Elementos de DNA Transponíveis , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Feminino , Humanos , Masculino , Camundongos , Oócitos/citologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Testículo/citologia , Testículo/metabolismo
19.
Gen Comp Endocrinol ; 283: 113227, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31348956

RESUMO

Water temperature is a critical external factor influencing gonadal development in fish. This research aimed to study the impact of elevated temperature on testicular germ cell survival and reproductive capacity of Nile tilapia. Male Nile tilapia were exposed to high temperatures of either 36 (HT1) or 37 °C (HT2) for 3000 degree-days (DD) and thereafter returned to the control temperature of 27 °C (CT) for 2200 DD. The deleterious effects on testicular germ and somatic cells were observed histologically, characterised by vacuolisation, atrophy and the loss of spermatogenic cells in testes with a more severe impact of HT2 compared to HT1. Interestingly, serum 11-ketotestosterone (11-KT) and testosterone (T) levels tended to be higher during the heat treatments than CT. Expression levels of germline-specific genes piwil1, piwil2 and nanos2 and Bcl-2 family genes, bcl-xLb and baxa were significantly reduced during the heat treatment compared to CT, more so in the HT2, while the levels of nanos3 and gfra1 transcripts were only significantly reduced in HT2, implying a significant loss of spermatogonial stem cell (SSC) and spermatogonia in HT2. The effect of HT2 is further evidenced by the significantly reduced sperm density and fertilisation rate compared to CT and HT1 at the end of the recovery period but complete sterility was not induced by HT2. Overall, the present study showed significant effects of HT2 on germ cell survival with histological changes in testes, reduced milt quality, increased 11-KT, and decreased expression of germline-specific genes, SSC marker genes and Bcl-2 family genes in testes which could therefore be potential target genes for sterilisation by genome editing.


Assuntos
Ciclídeos/metabolismo , Espermatozoides/citologia , Temperatura Ambiente , Testículo/citologia , Testículo/metabolismo , Animais , Ciclídeos/sangue , Ciclídeos/genética , Regulação da Expressão Gênica , Hormônios Esteroides Gonadais/sangue , Masculino , Espermatozoides/ultraestrutura , Testosterona/análogos & derivados , Testosterona/sangue
20.
Theriogenology ; 138: 111-120, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31325741

RESUMO

Multilamellar bodies (MLBs) are produced and secreted by many cell types. In this study, we report the existence and ultrastructure of MLBs that are produced by Leydig cells and identification of telocytes in the testicular interstitium of naked mole rat. This study was performed on both breeder and non-breeder male naked mole rats using light microscopy, transmission electron microscopy, and morphometric approaches. In the testicular interstitium, the most prominent cells were Leydig cells, which contained numerous lipid droplets (LDs) in the cytoplasm. We found that MLBs were associated with the LDs of Leydig cells and were secreted into the extracellular or interstitial environment via exocytosis. After their release from Leydig cells, MLBs localized to the space between Leydig cells near blood vessels and attached to telocytes. We also identified telocytes in the testicular interstitium, and their cellular extensions were distributed throughout the interstitium. MLBs were aligned along the cellular extensions of telocytes, and membrane-to-membrane contact was observed between the cellular extensions of telocytes and MLBs, suggesting that telocytes may play a role in the transport of MLBs within the interstitial space. No ultrastructural differences were found in Leydig cells, telocytes, or MLBs between breeder and non-breeder testes. However, morphometric analysis revealed a significant difference in the number of MLBs between the breeder and non-breeder animals. Furthermore, both selective autophagy of LDs and non-selective autophagy were observed in Leydig cells. Typical features of macrolipophagy were also observed, as a few LDs were entirely enclosed by a limiting membrane. Remarkably, autophagy may be a key factor in the biogenesis of MLBs and steroid hormone production. The appearance of MLBs in the testicular interstitium of naked mole rats could thus be related to lipid storage and trafficking.


Assuntos
Corpos de Inclusão/ultraestrutura , Células Intersticiais do Testículo/citologia , Células Intersticiais do Testículo/ultraestrutura , Ratos-Toupeira , Telócitos/citologia , Testículo , Animais , Autofagia/fisiologia , Células Intersticiais do Testículo/fisiologia , Masculino , Microscopia , Microscopia Eletrônica de Transmissão , Telócitos/ultraestrutura , Testículo/citologia , Testículo/ultraestrutura
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