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1.
FASEB J ; 30(4): 1453-63, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26671998

RESUMO

Type 2 cannabinoid receptor (CB2) has been proposed to play a pivotal role in meiotic entry of male germ cells, similar to retinoic acid (RA). In this study, we showed that activation of CB2with the specific agonist JWH133 [3-(1',1'-dimethylbutyl)-1-deoxy-8-THC] (IC5010(-6)M) mimics epigenetic events induced by RA (IC5010(-7)M) in spermatogonia. Both JWH133 and RA treatments stimulate the expression of the meiotic genes c-KitandStra8, by up-regulating H3K4me3 and down-regulating H3K9me2 levels in genomic regions flanking the transcription start site. Moreover, both agents increase the expression ofPrdm9, the gene encoding a meiosis-specific histone, H3K4me3 methyltransferase, which marks hotspots of recombination in prophase I, thus resulting in a global increase in H3K4me3. Notably, prolonged administration of JWH133 to immature 7 dpp CD-1 mice induced an acceleration of the onset of spermatogenesis, whereas the specific CB2antagonist delayed germ cell differentiation. Thus, both hyper- and hypostimulation of CB2disrupted the temporal dynamics of the spermatogenic cycle. These findings highlight the importance of proper CB2signaling for the maintenance of a correct temporal progression of spermatogenesis and suggest a possible adverse effect of cannabis in deregulating this process.-Di Giacomo, D., De Domenico, E., Sette, C., Geremia, R., Grimaldi, P. Type 2 cannabinoid receptor contributes to the physiological regulation of spermatogenesis.


Assuntos
Diferenciação Celular/fisiologia , Receptor CB2 de Canabinoide/metabolismo , Transdução de Sinais/fisiologia , Espermatogênese/fisiologia , Espermatogônias/fisiologia , Animais , Western Blotting , Canabinoides/farmacologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Células Cultivadas , Expressão Gênica/efeitos dos fármacos , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/genética , Histonas/metabolismo , Indóis/farmacologia , Lisina/metabolismo , Masculino , Meiose/efeitos dos fármacos , Meiose/genética , Metilação/efeitos dos fármacos , Camundongos , Microscopia de Fluorescência , Regiões Promotoras Genéticas/genética , Receptor CB2 de Canabinoide/agonistas , Receptor CB2 de Canabinoide/antagonistas & inibidores , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Espermatogênese/efeitos dos fármacos , Espermatogênese/genética , Espermatogônias/citologia , Espermatogônias/efeitos dos fármacos , Tretinoína/farmacologia
2.
Methods Mol Biol ; 2770: 37-52, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38351445

RESUMO

Transcriptomic analyses of germ cells at different stages of differentiation have shed light on the transcriptional and post-transcriptional mechanisms regulating gene expression that ensure the correct progression of spermatogenesis and male fertility. In this chapter, we describe a method to isolate meiotic and post-meiotic germ cells, based on gravimetric sedimentation, starting from a testicular germ cell suspension isolated from a single adult mouse. We also describe how to assess the purity and quality of the collected fractions of germ cells and how to optimize the extraction from these samples of RNA for subsequent RNA-sequencing experiment. In our experience, this protocol is suitable for germ cell isolation and transcriptomic analysis for mouse models with spermatogenic defects, overcoming the limits that reduced fertility poses to the obtaining of experimental animals.


Assuntos
Espermatogênese , Testículo , Camundongos , Masculino , Animais , Espermatogênese/genética , Células Germinativas , Perfilação da Expressão Gênica , RNA/genética
3.
J Cell Sci ; 124(Pt 1): 91-9, 2011 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-21147852

RESUMO

Meiosis is a crucial process for the production of functional gametes. However, the biological significance of many genes expressed during the meiotic phase remains poorly understood, mainly because of the lethal phenotypes of the knockout mice. Functional analysis of such genes using the conditional knockout approach is hindered by the lack of suitable Cre transgenic lines. We describe here the generation of transgenic mice expressing Cre recombinase under the control of the meiotic Spo11 gene. Using LacZ-R26(loxP) and EYFP-R26(loxP) reporter mice, we show the specific expression and activity of Cre during meiosis in males and females. Spo11(Cre) mice were then crossed with floxed Nbs1 and JAM-C mice to produce conditional knockouts. A strong reduction of Nbs1 and JAM-C protein levels was found in the testis. Although Nbs1-deleted mice developed minor gonadal abnormalities, JAM-C-knockout mice showed a spermiogenetic arrest, as previously described for the null mice. These results provide strong evidence that Spo11(Cre) transgenic mice represent a powerful tool for deleting genes of interest specifically in meiotic and/or in postmeiotic germ cells.


Assuntos
Endodesoxirribonucleases/genética , Deleção de Genes , Marcação de Genes/métodos , Células Germinativas/enzimologia , Integrases/metabolismo , Animais , Endodesoxirribonucleases/metabolismo , Feminino , Células Germinativas/citologia , Integrases/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Miose , Regiões Promotoras Genéticas
4.
Cell Mol Life Sci ; 69(24): 4177-90, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22802127

RESUMO

Estrogen (E(2)) regulates spermatogenesis, yet its direct target genes have not been identified in the testis. Here, we cloned the proximal 5' flanking region of the mouse fatty acid amide hydrolase (faah) gene upstream of the luciferase reporter gene, and demonstrated its promoter activity and E(2) inducibility in primary mouse Sertoli cells. Specific mutations in the E(2) response elements (ERE) of the faah gene showed that two proximal ERE sequences (ERE2/3) are essential for E(2)-induced transcription, and chromatin immunoprecipitation experiments showed that E(2) induced estrogen receptor ß binding at ERE2/3 sites in the faah promoter in vivo. Moreover, the histone demethylase LSD1 was found to be associated with ERE2/3 sites and to play a role in mediating E(2) induction of FAAH expression. E(2) induced epigenetic modifications at the faah proximal promoter compatible with transcriptional activation by remarkably decreasing methylation of both DNA at CpG site and histone H3 at lysine 9. Finally, FAAH silencing abolished E(2) protection against apoptosis induced by the FAAH substrate anandamide. Taken together, our results identify FAAH as the first direct target of E(2).


Assuntos
Amidoidrolases/genética , Estrogênios/farmacologia , Regulação da Expressão Gênica , Oxirredutases N-Desmetilantes/fisiologia , Células de Sertoli/metabolismo , Amidoidrolases/química , Amidoidrolases/fisiologia , Animais , Apoptose , Sequência de Bases , Metilação de DNA/efeitos dos fármacos , Receptor beta de Estrogênio/metabolismo , Receptor beta de Estrogênio/fisiologia , Histona Desmetilases , Histonas/metabolismo , Masculino , Metilação , Camundongos , Dados de Sequência Molecular , Oxirredutases N-Desmetilantes/genética , Oxirredutases N-Desmetilantes/metabolismo , Regiões Promotoras Genéticas , Células de Sertoli/efeitos dos fármacos
5.
Nucleic Acids Res ; 39(12): 4961-74, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21355037

RESUMO

Sam68 plays an essential role in mouse spermatogenesis and male fertility. Herein, we report an interaction between Sam68 and the phosphorylated forms of the RNA polymerase II (RNAPII) in meiotic spermatocytes. RNase treatment decreased but did not abolish the interaction, consistently with in vitro binding of RNAPII to the Sam68 carboxyl-terminal region. Sam68 retention in the spermatocyte nucleus was dependent on the integrity of cellular RNAs, suggesting that the protein is recruited to transcriptionally active chromatin. Mouse knockout models characterized by stage-specific arrest of spermatogenesis and staining with the phosphorylated form of RNAPII documented that Sam68 expression is confined to the transcriptionally active stages of spermatogenesis. Furthermore, Sam68 associates with splicing regulators in germ cells and we report that alternative splicing of Sgce exon 8 is regulated in a Sam68-dependent manner during spermatogenesis. RNA and chromatin crosslink immunoprecipitation experiments showed that Sam68 binds in vivo to sequences surrounding the intron 7/exon 8 boundary, thereby affecting the recruitment of the phosphorylated RNAPII and of the general splicing factor U2AF65. These results suggest that Sam68 regulates alternative splicing at transcriptionally active sites in differentiating germ cells and provide new insights into the regulation of Sam68 expression during spermatogenesis.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Processamento Alternativo , Proteínas de Ligação a RNA/metabolismo , Espermatogênese/genética , Espermatozoides/metabolismo , Transcrição Gênica , Animais , Masculino , Prófase Meiótica I/genética , Camundongos , Camundongos Knockout , RNA Polimerase II/metabolismo , Sarcoglicanas/genética , Sarcoglicanas/metabolismo , Espermatócitos/enzimologia , Espermatócitos/metabolismo
6.
Hum Mol Genet ; 19(24): 4886-94, 2010 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-20881015

RESUMO

Sam68 is a multifunctional RNA-binding protein highly expressed in the gonads, whose ablation causes male infertility. Herein, we have investigated Sam68 expression in the adult ovary and its function in female fertility. Immunohistochemistry showed that Sam68 was localized in the nucleus of oocytes and follicular cells at all stages of folliculogenesis. Sam68(-/-) females were severely subfertile, and they showed a delay in the age of first pregnancy, increased breeding time for successful pregnancy and yielded smaller litters. Morphological analyses indicated a significant reduction in the number of secondary and pre-antral follicles in the ovary. These defects were associated with alteration of oestrous cycles and a reduced number of ovulated oocytes, which were only partially restored by the administration of exogenous gonadotropins. Crosslinking/immunoprecipitation experiments showed that Sam68 directly binds the mRNAs for the follicle-stimulating hormone (FSH) and the luteinizing hormone receptors (Fshr and Lhcgr), which were downregulated in ovaries of adult knockout females. Stimulation of immature females with FSH-like pregnant mare serum gonadotropin (PMSG), or of follicular cells with the FSH second messenger analogue 8Br-cAMP, caused the upregulation of Sam68. The increase in Sam68 levels paralleled that of the Fshr and Lhcgr mRNAs in the pre-ovulatory follicle and was required to allow accumulation of these transcripts in follicular cells. These studies identify a new crucial function for Sam68 in the regulation of female fertility and indicate that this protein is required to insure proper expression of the gonadotropin receptor transcripts in pre-ovulatory follicles in adult ovary.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Deleção de Genes , Gonadotropinas Equinas/farmacologia , Infertilidade Feminina/patologia , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/crescimento & desenvolvimento , Proteínas de Ligação a RNA/genética , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Ciclo Estral/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Infertilidade Feminina/genética , Masculino , Camundongos , Folículo Ovariano/patologia , Folículo Ovariano/fisiopatologia , Gravidez , Ligação Proteica/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Receptores da Gonadotropina/genética , Receptores da Gonadotropina/metabolismo , Regulação para Cima/efeitos dos fármacos
7.
J Cell Sci ; 123(Pt 6): 871-80, 2010 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-20159962

RESUMO

In the mouse, three genes that are homologous to the Drosophila Nanos (Nos) gene have been identified. Deletion of one of these genes, Nanos2, results in male sterility, owing to loss of germ cells during fetal life. Before apoptosis, Nanos2-null gonocytes enter meiosis, suggesting that Nanos2 functions as a meiotic repressor. Here, we show that Nanos2 is continuously expressed in male germ cells from fetal gonocytes to postnatal spermatogonial stem cells. We observed that the promeiotic factor AtRA, an analog of retinoic acid (RA), downregulates NANOS2 levels, in both fetal and postnatal gonocytes, while promoting meiosis. Interestingly, FGF9, a growth factor crucial for sex differentiation and survival of fetal gonocytes, upregulates levels of NANOS2 in both male and female primordial germ cells (PGCs) and in premeiotic spermatogonia. This effect was paralleled by an impairment of meiotic entry, suggesting that FGF9 acts as an inhibitor of meiosis through the upregulation of Nanos2. We found that NANOS2 interacts with PUM2, and that these two proteins colocalize in the ribonucleoparticle and polysomal fractions on sucrose gradients, supporting the notion that they bind RNA. Finally, we found that recombinant NANOS2 binds to two spermatogonial mRNAs, Gata2 and Taf7l, which are involved in germ-cell differentiation.


Assuntos
Proteínas de Transporte/genética , Fator 9 de Crescimento de Fibroblastos/farmacologia , Células Germinativas/citologia , Células Germinativas/metabolismo , Meiose/efeitos dos fármacos , Tretinoína/farmacologia , Animais , Animais Recém-Nascidos , Proteínas de Transporte/metabolismo , Regulação para Baixo/efeitos dos fármacos , Feminino , Feto/citologia , Feto/efeitos dos fármacos , Feto/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Células Germinativas/efeitos dos fármacos , Masculino , Camundongos , Óvulo/citologia , Óvulo/efeitos dos fármacos , Óvulo/metabolismo , Polirribossomos/efeitos dos fármacos , Polirribossomos/metabolismo , Ligação Proteica/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA , Ribonucleoproteínas/metabolismo , Espermatozoides/citologia , Espermatozoides/efeitos dos fármacos , Espermatozoides/metabolismo , Testículo/citologia , Testículo/efeitos dos fármacos , Testículo/metabolismo , Regulação para Cima/efeitos dos fármacos
8.
Nucleic Acids Res ; 38(9): 3005-18, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20110258

RESUMO

DNA-damaging agents cause a multifaceted cellular stress response. Cells set in motion either repair mechanisms or programmed cell death pathways, depending on the extent of the damage and on their ability to withstand it. The RNA-binding protein (RBP) Sam68, which is up-regulated in prostate carcinoma, promotes prostate cancer cell survival to genotoxic stress. Herein, we have investigated the function of Sam68 in this cellular response. Mitoxantrone (MTX), a topoisomerase II inhibitor, induced relocalization of Sam68 from the nucleoplasm to nuclear granules, together with several other RBPs involved in alternative splicing, such as TIA-1, hnRNP A1 and the SR proteins SC35 and ASF/SF2. Sam68 accumulation in nuclear stress granules was independent of signal transduction pathways activated by DNA damage. Using BrU labelling and immunofluorescence, we demonstrate that MTX-induced nuclear stress granules are transcriptionally active foci where Sam68 and the phosphorylated form of RNA polymerase II accumulate. Finally, we show that MTX-induced relocalization of Sam68 correlates with changes in alternative splicing of its mRNA target CD44, and that MTX-induced CD44 splicing depends on Sam68 expression. These results strongly suggest that Sam68 is part of a RNA-mediated stress response of the cell that modulates alternative splicing in response to DNA damage.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/análise , Processamento Alternativo , Antineoplásicos/toxicidade , Cromatina/genética , Dano ao DNA , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a RNA/análise , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linhagem Celular , Núcleo Celular/química , Citoplasma/química , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Humanos , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Masculino , Mitoxantrona/toxicidade , Mutagênicos/toxicidade , Neoplasias da Próstata/química , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Transcrição Gênica
9.
Proc Natl Acad Sci U S A ; 106(27): 11131-6, 2009 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-19541620

RESUMO

The exact role of the endocannabinoid system (ECS) during spermatogenesis has not been clarified. We used purified germ cell fractions representative of all phases of spermatogenesis and primary cultures of spermatogonia. This approach allowed the precise quantification of the cannabinoid receptor ligands, anandamide and 2-arachidonoylglycerol, and of the expression at transcriptional and transductional levels of their metabolic enzymes and receptors. Our data indicate that male mouse germ cells possess an active and complete ECS, which is modulated during meiosis, and suggest the presence of an autocrine endocannabinoid signal during spermatogenesis. Mitotic cells possess higher levels of 2-arachidonoylglycerol, which decrease in spermatocytes and spermatids. Accordingly, spermatogonia express higher and lower levels of 2-arachidonoylglycerol biosynthetic and degrading enzymes, respectively, as compared to meiotic and postmeiotic cells. This endocannabinoid likely plays a pivotal role in promoting the meiotic progression of germ cells by activating CB(2) receptors. In fact, we found that the selective CB(2) receptor agonist, JWH133, induced the Erk 1/2 MAPK phosphorylation cascade in spermatogonia and their progression toward meiosis, because it increased the number of cells positive for SCP3, a marker of meiotic prophase, and the expression of early meiotic prophase genes.


Assuntos
Moduladores de Receptores de Canabinoides/metabolismo , Endocanabinoides , Receptor CB2 de Canabinoide/metabolismo , Espermatogênese , Animais , Ácidos Araquidônicos/biossíntese , Moduladores de Receptores de Canabinoides/biossíntese , Canabinoides/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Imunofluorescência , Glicerídeos/biossíntese , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Prófase Meiótica I/efeitos dos fármacos , Camundongos , Alcamidas Poli-Insaturadas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor CB1 de Canabinoide/genética , Receptor CB1 de Canabinoide/metabolismo , Receptor CB2 de Canabinoide/agonistas , Espermatogênese/efeitos dos fármacos , Espermatogônias/citologia , Espermatogônias/efeitos dos fármacos , Espermatogônias/metabolismo , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo
10.
Biol Reprod ; 83(4): 607-15, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20574055

RESUMO

Translation of stored mRNAs accounts for protein synthesis during the transcriptionally inactive stages of spermatogenesis. A key step in mRNA translation is the assembly of the initiation complex EIF4F, which is regulated by the MTOR (mammalian target of rapamycin) and MNK1/2 (MAP kinase-interacting kinase 1 and 2) pathways. We investigated the expression and activity of regulatory proteins of these pathways in male germ cells at different stages of differentiation. All translation factors analyzed were expressed in germ cells throughout spermatogenesis. However, while EIF4G and PABP1 (poly[A]-binding protein 1) were more abundant in postmeiotic cells, MTOR and its target EIF4EBP1 (4E-BP1) decreased steadily during spermatogenesis. In vivo labeling showed that pachytene spermatocytes display higher rates of protein synthesis, which are partially dependent on MTOR and MNK activity. By contrast, haploid spermatids are characterized by lower levels of protein synthesis, which are independent of the activity of these pathways. Accordingly, MTOR and MNK activity enhanced formation of the EIF4F complex in pachytene spermatocytes but not in round spermatids. Moreover, external cues differentially modulated the activity of these pathways in meiotic and haploid cells. Heat shock decreased MTOR and MNK activity in pachytene spermatocytes, whereas round spermatids were much less sensitive. On the other hand, treatment with the phosphatase inhibitor okadaic acid activated MTOR and MNK in both cell types. These results indicate that translational regulation is differentially dependent on the MTOR and MNK pathways in mouse spermatocytes and spermatids and suggest that the late stages of germ cell differentiation display constitutive assembly of the translation initiation complex.


Assuntos
Regulação Enzimológica da Expressão Gênica/fisiologia , Meiose/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/genética , Espermatócitos/fisiologia , Serina-Treonina Quinases TOR/metabolismo , Animais , Western Blotting , Diferenciação Celular/fisiologia , Inibidores Enzimáticos/farmacologia , Fator de Iniciação 4F em Eucariotos/metabolismo , Imuno-Histoquímica , Masculino , Camundongos , Ácido Okadáico/farmacologia , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Transdução de Sinais , Espermatócitos/citologia , Espermatócitos/metabolismo , Espermatogênese/fisiologia
11.
Hum Reprod ; 25(9): 2188-202, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20601678

RESUMO

BACKGROUND: TR-KIT, a truncated form of KIT (the KITL receptor), corresponding to the c-terminal half of the intracellular split tyrosine kinase domain, is expressed during the haploid stages of mouse spermatogenesis, and is one of the candidate sperm factors possibly involved in egg activation at fertilization. METHODS: Immunocytochemistry of adult human testis, and studies of human semen samples from volunteer donors through immunofluorescence, confocal microscopy, flow cytometry, western blot and RT-PCR analyses were performed. RESULTS: We show that the TR-KIT is expressed during spermiogenesis in the human testis, and that it is maintained in human ejaculated spermatozoa. TR-KIT is localized both in the equatorial segment and in the sub-acrosomal region of the human sperm head. The equatorial localization of the TR-KIT persists after the spontaneous acrosome reaction. Cytometric analysis of several sperm samples from volunteer donors, showed variable degrees of the TR-KIT-specific immunolabeling, and a significant inverse correlation (Pearson's coefficient, r = -0.76, P < 0.0001, n = 23) of the TR-KIT positivity with markers of sperm damage, i.e. DNA fragmentation, as revealed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-nick end labeling (TUNEL) analysis and the intense clusterin positivity. We also found less significant inverse correlation with altered head morphology (r = -0.47, P < 0.05, n = 23) and direct correlation with sperm forward motility parameters (r = 0.59, P < 0.01, n = 23). CONCLUSIONS: The TR-KIT is present in the equatorial region of human spermatozoa, which is the first sperm component entering into the oocyte cytoplasm after fusion with the egg. This localization is consistent with the function previously proposed for this protein in mice. In addition, the TR-KIT represents a potential predictive parameter of human sperm quality.


Assuntos
Fragmentação do DNA , Expressão Gênica , Proteínas Proto-Oncogênicas c-kit/metabolismo , Espermatozoides/química , Espermatozoides/metabolismo , Reação Acrossômica , Adulto , Idoso , Biomarcadores/metabolismo , Forma Celular , Clusterina/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Especificidade de Órgãos , Isoformas de Proteínas/metabolismo , Transporte Proteico , Proteínas Proto-Oncogênicas c-kit/genética , RNA Mensageiro , Análise do Sêmen , Cabeça do Espermatozoide/metabolismo , Cabeça do Espermatozoide/patologia , Espermatozoides/patologia , Testículo/citologia , Testículo/metabolismo , Adulto Jovem
12.
J Pathol ; 217(3): 431-41, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19023884

RESUMO

Protein kinases that regulate the centrosome cycle are often aberrantly controlled in neoplastic cells. Changes in their expression or activity can lead to perturbations in centrosome duplication, potentially leading to chromosome segregation errors and aneuploidy. Testicular germ cell tumours (TGCTs) are characterized by amplification of centrosomes through unknown mechanisms. Herein, we report that Nek2, a centrosomal kinase required for centrosome disjunction and formation of the mitotic spindle, is up-regulated in human testicular seminomas as compared to control testes or other types of testicular germ cell tumours. In addition, Nek2 activity is also increased in human seminomas, as demonstrated by immunokinase assays. Analysis by immunohistochemistry indicated that Nek2 is prevalently localized in the nucleus of neoplastic cells of primary human seminomas. Such nuclear localization and the up-regulation of Nek2 protein were also observed in the Tcam-2 seminoma cell line. We demonstrate that nuclear localization of Nek2 is a feature of the more undifferentiated germ cells of mouse testis and correlates with expression of the stemness markers OCT4 and PLZF. These studies suggest that up-regulation of Nek2 is a frequent event in human seminomas and that this may participate in the onset or progression of neoplastic transformation through deregulation of centrosome duplication and/or nuclear events in germ cells.


Assuntos
Regulação Neoplásica da Expressão Gênica , Proteínas Serina-Treonina Quinases/genética , Seminoma/genética , Neoplasias Testiculares/genética , Regulação para Cima , Animais , Linhagem Celular Tumoral , Núcleo Celular/química , Humanos , Imunoprecipitação , Masculino , Camundongos , Microscopia de Fluorescência , Quinases Relacionadas a NIMA , Proteínas Serina-Treonina Quinases/análise
13.
Dev Biol ; 313(2): 725-38, 2008 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-18089289

RESUMO

Nanos gene encodes for zinc-finger protein with putative RNA-binding activity which shows an evolutionary conserved function in germ cell development. In the mouse, three Nanos homologs have been identified: Nanos1, Nanos2 and Nanos3. The Nanos3 ortholog is expressed in both male and female gonads of early embryo and, after birth, it is found only in the testis. Nanos3 targeted disruption results in the complete loss of germ cells in both sexes; however the role of Nanos3 in the testis during the postnatal period has not been explored yet. In this study, we show that, in prepuberal testis, Nanos3 is expressed in undifferentiated spermatogonia and that its up-regulation causes accumulation of cells in the G1 phase, indicating that this protein is able to delay the cell cycle progression of spermatogonial cells. This is in line with the observation that the cell cycle length of the undifferentiated germ cells is longer than in differentiating spermatogonia. We also demonstrate a conserved mechanism of action of Nanos3, involving the interaction with the murine RNA-binding protein Pumilio2 and consisting of a potential translational repressor activity. According to the possible role of Nanos3 in inhibiting spermatogonia cell differentiation, we show that treatment with the differentiating factor all-trans retinoic acid induces a dramatic down-regulation of its expression. These results allow to conclude that, in the prepuberal testis, Nanos3 is important to maintain undifferentiated spermatogonia via the regulation of their cell cycle.


Assuntos
Proteínas de Ligação a RNA/fisiologia , Espermatogônias/fisiologia , Animais , Linhagem Celular , Células Cultivadas , DNA Complementar/genética , Regulação para Baixo/efeitos dos fármacos , Embrião de Mamíferos , Escherichia coli/genética , Glutationa Transferase/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Humanos , Separação Imunomagnética , Hibridização In Situ , Rim/citologia , Masculino , Camundongos , Camundongos Endogâmicos , Fator 3 de Transcrição de Octâmero/metabolismo , Fases de Leitura Aberta , Plasmídeos , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/metabolismo , Espermatogênese , Espermatogônias/citologia , Testículo/anatomia & histologia , Testículo/citologia , Testículo/embriologia , Testículo/crescimento & desenvolvimento , Testículo/metabolismo , Fatores de Tempo , Transfecção , Tretinoína/farmacologia
14.
Methods Mol Biol ; 558: 299-321, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19685332

RESUMO

The studies of molecular events that occur in single cell types within a tissue often require the disaggregation of the tissue into a single cell suspension, followed by isolation of distinct cell populations. The germinal epithelium of mammals is composed of several cell types, which divide mitotically, before entering meiosis. In this chapter, we describe the isolation of five mouse germ-cell fractions by centrifugal elutriation, and characterize them by their DNA content (flow cytometry), cell morphology (DAPI staining of nuclei, Giemsa staining of squashed cells) and deposition of stage-specific meiotic markers (SYCP3, H1t, SAM68) on chromosome spreads and whole cells. Within 2 h it is possible to obtain enriched populations of elongated spermatids (up to approximately 50% of the fraction), round spermatids (up to approximately 80%), primary spermatocytes (up to approximately 89%), and secondary spermatocytes (up to approximately 17%). Furthermore, most of the collected spermatocytes of the primary spermatocyte fraction are in early-mid pachytene stage as judged by chromosome spreads, enriched up to approximately 89%. Elutriation and techniques used for characterization of germ cell fractions are described.


Assuntos
Separação Celular/métodos , Fracionamento por Campo e Fluxo/métodos , Células Germinativas/citologia , Animais , Centrifugação/instrumentação , Centrifugação/métodos , Fracionamento por Campo e Fluxo/instrumentação , Masculino , Camundongos , Modelos Biológicos
15.
Mol Biol Cell ; 17(1): 14-24, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16221888

RESUMO

Translational control plays a crucial role during gametogenesis in organisms as different as worms and mammals. Mouse knockout models have highlighted the essential function of many RNA-binding proteins during spermatogenesis. Herein we have investigated the expression and function during mammalian male meiosis of Sam68, an RNA-binding protein implicated in several aspects of RNA metabolism. Sam68 expression and localization within the cells is stage specific: it is expressed in the nucleus of spermatogonia, it disappears at the onset of meiosis (leptotene/zygotene stages), and it accumulates again in the nucleus of pachytene spermatocytes and round spermatids. During the meiotic divisions, Sam68 translocates to the cytoplasm where it is found associated with the polysomes. Translocation correlates with serine/threonine phosphorylation and it is blocked by inhibitors of the mitogen activated protein kinases ERK1/2 and of the maturation promoting factor cyclinB-cdc2 complex. Both kinases associate with Sam68 in pachytene spermatocytes and phosphorylate the regulatory regions upstream and downstream of the Sam68 RNA-binding motif. Molecular cloning of the mRNAs associated with Sam68 in mouse spermatocytes reveals a subset of genes that might be posttranscriptionally regulated by this RNA-binding protein during spermatogenesis. We also demonstrate that Sam68 shuttles between the nucleus and the cytoplasm in secondary spermatocytes, suggesting that it may promote translation of specific RNA targets during the meiotic divisions.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fosfoproteínas/metabolismo , Polirribossomos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Espermatócitos/citologia , Espermatócitos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proliferação de Células , Células Cultivadas , Proteínas de Ligação a DNA/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas Ligadas por GPI , Regulação da Expressão Gênica , Masculino , Glicoproteínas de Membrana/metabolismo , Mesotelina , Camundongos , Fosfoproteínas/genética , Fosforilação , Transporte Proteico , RNA/síntese química , RNA/química , RNA/metabolismo , Proteínas de Ligação a RNA/genética
16.
Carcinogenesis ; 29(12): 2279-88, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18809972

RESUMO

Deregulation of the phosphatidyl inositol trisphosphate kinase/AKT/mammalian target of rapamycin (mTOR) and RAS/mitogen-activated protein kinase (MAPK)/MNK pathways frequently occurs in human prostate carcinomas (PCas) and leads to aberrant modulation of messenger RNA (mRNA) translation. We have investigated the relative contribution of these pathways to translational regulation and proliferation of PCa cells. MNK-dependent phosphorylation of eIF4E is elevated in DU145 cells, which have low basal levels of AKT/mTOR activity due to the expression of the tumor suppressor PTEN. In contrast, eIF4E phosphorylation is low in PC3 and LNCaP cells with mutated PTEN and constitutively active AKT/mTOR pathway, but it can be strongly induced through inhibition of mTOR activity by rapamycin or serum depletion. Remarkably, we found that inhibition of MNKs strongly reduced the polysomal recruitment of terminal oligopyrimidine messenger RNAs (TOP mRNAs), which are known targets of mTOR-dependent translational control. Pull-down assays of the eIF4F complex indicated that translation initiation was differently affected by inhibition of MNKs and mTOR. In addition, concomitant treatment with MNK inhibitor and rapamycin exerted additive effects on polysomal recruitment of TOP mRNAs and protein synthesis. The MNK inhibitor was more effective than rapamycin in blocking proliferation of PTEN-expressing cells, whereas combination of the two inhibitors suppressed cell cycle progression in both cell lines. Microarray analysis showed that MNK affected translation of mRNAs involved in cell cycle progression. Thus, our results indicate that a balance between the activity of the AKT/mTOR and the MAPK/MNK pathway in PCa cells maintains a defined translational level of specific mRNAs required for ribosome biogenesis, cell proliferation and stress response and might confer to these cells the ability to overcome negative insults.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Ciclo Celular/fisiologia , Fator de Iniciação 4E em Eucariotos/metabolismo , Neoplasias da Próstata/metabolismo , Biossíntese de Proteínas/fisiologia , Transdução de Sinais/fisiologia , Biomarcadores Tumorais/análise , Western Blotting , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , ATPases Transportadoras de Cobre , Inibidores Enzimáticos/farmacologia , Humanos , Masculino , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Polirribossomos/efeitos dos fármacos , Polirribossomos/fisiologia , Análise Serial de Proteínas , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologia , Serina-Treonina Quinases TOR
17.
Gene Expr Patterns ; 8(5): 311-22, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18321792

RESUMO

The STAR protein Sam68 (KHDRBS1) is involved in several aspects of post-transcriptional mRNA metabolism. Herein, we have investigated the expression and subcellular localization of Sam68 during early mouse embryogenesis. We found that mouse oocytes express high levels of Sam68 mRNA, low levels of the transcript for Khdrbs2 (current symbol for Slm-1) and no Khdrbs3 (current symbol for Slm-2), two highly homologous STAR genes. Sam68 protein is expressed throughout oocyte meiotic maturation and early embryogenesis. It is released in the cytoplasm upon meiotic resumption and it slowly accumulates in the nucleus after fertilization. Unlike what was observed for other RNA-binding proteins, nuclear accumulation of Sam68 was independent of de novo mRNA transcription. However, we found that inhibition of mRNA translation by either cycloheximide or puromycin in one-cell embryos caused the accumulation of Sam68 in cytoplasmic granules. Analysis of these granules by deconvolution microscopy demonstrated that they are sites of accumulation for proteins involved in the initiation of mRNA translation, such as eIF4A1, eIF4E and eIF4G. These granules contained RNA and were dissolved by treatment with RNase A. Other proteins expressed by the zygote, like the splicing factor SC35 or the cytoplasmic kinase ERK2, did not accumulate in such structures after treatment with inhibitors of mRNA translation, indicating that the localization of Sam68 and of the translation initiation factors in these granules is a specific event. These results indicate that Sam68 is involved in translational regulation of maternal mRNAs in the zygote and in the early signaling events triggered by fertilization.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Implantação do Embrião , Proteínas de Ligação a RNA/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Alfa-Amanitina/farmacologia , Animais , Núcleo Celular/metabolismo , Células Cultivadas , Gonadotropina Coriônica/farmacologia , Cicloeximida/farmacologia , Grânulos Citoplasmáticos/metabolismo , Dactinomicina/farmacologia , Diclororribofuranosilbenzimidazol/farmacologia , Embrião de Mamíferos/efeitos dos fármacos , Embrião de Mamíferos/metabolismo , Feminino , Humanos , Camundongos , Camundongos Endogâmicos , Inibidores da Síntese de Ácido Nucleico/farmacologia , Oócitos/metabolismo , Ovário/citologia , Gravidez , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Puromicina/farmacologia , Proteínas de Ligação a RNA/genética , Substâncias para o Controle da Reprodução/farmacologia , Ribonuclease Pancreático/farmacologia , Técnicas de Cultura de Tecidos , Zigoto/citologia , Zigoto/efeitos dos fármacos
18.
Gene Expr Patterns ; 8(2): 58-70, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18036996

RESUMO

Kit ligand (KL) is a survival factor and a mitogenic stimulus for differentiating spermatogonia. However, it is not known whether KL also plays a role in the differentiative events that lead to meiotic entry of these cells. We performed a wide genome analysis of difference in gene expression induced by treatment with KL of spermatogonia from 7-day-old mice, using gene chips spanning the whole mouse genome. The analysis revealed that the pattern of RNA expression induced by KL is compatible with the qualitative changes of the cell cycle that occur during the subsequent cell divisions in type A and B spermatogonia, i.e. the progressive lengthening of the S phase and the shortening of the G2/M transition. Moreover, KL up-regulates in differentiating spermatogonia the expression of early meiotic genes (for instance: Lhx8, Nek1, Rnf141, Xrcc3, Tpo1, Tbca, Xrcc2, Mesp1, Phf7, Rtel1), whereas it down-regulates typical spermatogonial markers (for instance: Pole, Ptgs2, Zfpm2, Egr2, Egr3, Gsk3b, Hnrpa1, Fst, Ptch2). Since KL modifies the expression of several genes known to be up-regulated or down-regulated in spermatogonia during the transition from the mitotic to the meiotic cell cycle, these results are consistent with a role of the KL/kit interaction in the induction of their meiotic differentiation.


Assuntos
Proteínas Proto-Oncogênicas c-kit/fisiologia , Espermatogônias/fisiologia , Fator de Células-Tronco/fisiologia , Transcrição Gênica , Animais , Ciclo Celular , Diferenciação Celular , Células Cultivadas , DNA Complementar , Regulação da Expressão Gênica no Desenvolvimento , Genoma , Masculino , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , RNA/genética , RNA/isolamento & purificação , RNA Complementar , Espermatogônias/citologia
19.
Endocr Relat Cancer ; 14(1): 111-24, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17395980

RESUMO

Pancreatic endocrine tumours (PETs) are rare and 'indolent' neoplasms that usually develop metastatic lesions and exhibit poor response to standard medical treatments. Few studies have investigated pathways responsible for PET cell growth and invasion and no alternative therapeutic strategies have been proposed. In a recent microarray analysis for genes up-regulated in PETs, we have described the up-regulation of soluble Src family tyrosine kinases in this neoplasia, which may represent potentially promising candidates for therapy. Herein, we have investigated the expression and function of Src family kinases in PETS and PET cell lines. Western blot analysis indicated that Src is highly abundant in the PET cell lines CM and QGP-1. Immunohistochemistry and Western blot analyses showed that Src is up-regulated also in human PET lesions. Pharmacological inhibition of Src family kinases by the specific inhibitor PP2 strongly interfered with adhesion, spreading and migration of PET cell lines. Accordingly, the actin cytoskeleton was profoundly altered after inhibition of Src kinases, whereas even prolonged incubation with PP2 exerted no effect on cell cycle progression and/or apoptosis of PET cells. A transient increase in tyrosine phosphorylation of a subset of proteins was observed in QGP-1 cells adhering to the plate, with a peak at 75 min after seeding, when approximately 80% of cells were attached. Inhibition of Src kinases caused a dramatic reduction in the phosphorylation of proteins with different molecular weight that were isolated from the cell extracts by anti-phosphotyrosine immunoprecipitation or pull-down with the SH2 domain of Src. Among them, the docking protein p130Cas interacted with Src and is a major substrate of the Src kinases in QGP-1 cells undergoing adhesion. Our results suggest that Src kinases play a specific role during adhesion, spreading and migration of PET cells and may indicate therapeutical approaches directed to limiting the metastatic potential of these cells.


Assuntos
Insulinoma/metabolismo , Neoplasias Pancreáticas/metabolismo , Somatostatinoma/metabolismo , Quinases da Família src/metabolismo , Adulto , Idoso , Adesão Celular/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pirimidinas/farmacologia , Cicatrização/efeitos dos fármacos , Quinases da Família src/antagonistas & inibidores
20.
Mol Biol Cell ; 15(3): 1224-32, 2004 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-14668482

RESUMO

The mitogen-activated protein kinase (MAPK) pathway is required for maintaining the chromatin condensed during the two meiotic divisions and to avoid a second round of DNA duplication. However, molecular targets of the MAPK pathway on chromatin have not yet been identified. Here, we show that the architectural chromatin protein HMGA2 is highly expressed in male meiotic cells. Furthermore, Nek2, a serine-threonine kinase activated by the MAPK pathway in mouse pachytene spermatocytes, directly interacts with HMGA2 in vitro and in mouse spermatocytes. The interaction does not depend on the activity of Nek2 and seems constitutive. On progression from pachytene to metaphase, Nek2 is activated and HMGA2 is phosphorylated in an MAPK-dependent manner. We also show that Nek2 phosphorylates in vitro HMGA2 and that this phosphorylation decreases the affinity of HMGA2 for DNA and might favor its release from the chromatin. Indeed, we find that most HMGA2 associates with chromatin in mouse pachytene spermatocytes, whereas it is excluded from the chromatin upon the G2/M progression. Because hmga2-/- mice are sterile and show a dramatic impairment of spermatogenesis, it is possible that the functional interaction between HMGA2 and Nek2 plays a crucial role in the correct process of chromatin condensation in meiosis.


Assuntos
Proteína HMGA2/metabolismo , Meiose/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Espermatócitos/metabolismo , Animais , Células Cultivadas , Cromatina/metabolismo , Clonagem Molecular , Proteínas de Ligação a DNA/metabolismo , Masculino , Camundongos , Quinases Relacionadas a NIMA , Fosforilação , Ligação Proteica
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