RESUMO
Small Kinetochore-Associated Protein (SKAP)/Kinastrin is a multifunctional protein with proposed roles in mitosis, apoptosis and cell migration. Exact mechanisms underlying its activities in these cellular processes are not completely understood. SKAP is predicted to have different isoforms, however, previous studies did not differentiate between them. Since distinct molecular architectures of protein isoforms often influence their localization and functions, this study aimed to examine the expression profile and functional differences between SKAP isoforms in human and mouse. Analyses of various human tissues and cells of different origin by RT-PCR, and by Western blotting and immunocytochemistry applying newly generated anti-SKAP monoclonal antibodies revealed that human SKAP exists in two protein isoforms: ubiquitously expressed SKAP16 and testis/sperm-specific SKAP1. In mouse, SKAP1 expression is detectable in testis at 4 weeks postnatally, when the first wave of spermatogenesis in mice is complete and the elongated spermatids are present in the testes. Furthermore, we identified Pontin as a new SKAP1 interaction partner. SKAP1 and Pontin co-localized in the flagellar region of human sperm suggesting a functional relevance for SKAP1-Pontin interaction in sperm motility. Since most previous studies on SKAP were performed with the testis-specific isoform SKAP1, our findings provide a new basis for future studies on the role of SKAP in both human somatic cells and male germ cells, including studies on male fertility.
Assuntos
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Animais , Apoptose , Humanos , Masculino , Camundongos , Mitose , Especificidade de Órgãos/genética , Domínios Proteicos/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Motilidade dos Espermatozoides , Espermatogênese , Espermatozoides/metabolismo , Testículo/metabolismoRESUMO
The Adhesion family forms a large branch of the pharmacologically important superfamily of G protein-coupled receptors (GPCRs). As Adhesion GPCRs increasingly receive attention from a wide spectrum of biomedical fields, the Adhesion GPCR Consortium, together with the International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification, proposes a unified nomenclature for Adhesion GPCRs. The new names have ADGR as common dominator followed by a letter and a number to denote each subfamily and subtype, respectively. The new names, with old and alternative names within parentheses, are: ADGRA1 (GPR123), ADGRA2 (GPR124), ADGRA3 (GPR125), ADGRB1 (BAI1), ADGRB2 (BAI2), ADGRB3 (BAI3), ADGRC1 (CELSR1), ADGRC2 (CELSR2), ADGRC3 (CELSR3), ADGRD1 (GPR133), ADGRD2 (GPR144), ADGRE1 (EMR1, F4/80), ADGRE2 (EMR2), ADGRE3 (EMR3), ADGRE4 (EMR4), ADGRE5 (CD97), ADGRF1 (GPR110), ADGRF2 (GPR111), ADGRF3 (GPR113), ADGRF4 (GPR115), ADGRF5 (GPR116, Ig-Hepta), ADGRG1 (GPR56), ADGRG2 (GPR64, HE6), ADGRG3 (GPR97), ADGRG4 (GPR112), ADGRG5 (GPR114), ADGRG6 (GPR126), ADGRG7 (GPR128), ADGRL1 (latrophilin-1, CIRL-1, CL1), ADGRL2 (latrophilin-2, CIRL-2, CL2), ADGRL3 (latrophilin-3, CIRL-3, CL3), ADGRL4 (ELTD1, ETL), and ADGRV1 (VLGR1, GPR98). This review covers all major biologic aspects of Adhesion GPCRs, including evolutionary origins, interaction partners, signaling, expression, physiologic functions, and therapeutic potential.
Assuntos
Moléculas de Adesão Celular/metabolismo , AMP Cíclico/fisiologia , Modelos Moleculares , Receptores Acoplados a Proteínas G/metabolismo , Sistemas do Segundo Mensageiro , Animais , Adesão Celular , Moléculas de Adesão Celular/química , Membrana Celular/enzimologia , Membrana Celular/metabolismo , Movimento Celular , Humanos , Agências Internacionais , Ligantes , Farmacologia/tendências , Farmacologia Clínica/tendências , Isoformas de Proteínas/agonistas , Isoformas de Proteínas/química , Isoformas de Proteínas/classificação , Isoformas de Proteínas/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/classificação , Transdução de Sinais , Sociedades Científicas , Terminologia como AssuntoRESUMO
Human spermatogonia (Spg) and their fetal precursors express fibroblast growth factor receptor 3 (FGFR3). To further elucidate the role of FGFR3 in the control of Spg self-renewal, proliferation, and/or differentiation, and to narrow down the FGFR3-positive cell type(s) in the normal adult human testis, tissue sections and whole mount preparations of seminiferous tubules were analyzed combining immunofluorescence and confocal fluorescence microscopy. FGFR3 protein was chiefly observed in cellular membranes and cytoplasmic vesicles of a subpopulation of type A Spg, which comprised the chromatin rarefaction zone-containing type A(dark). Cytoplasmic expression of FGFR3 and nuclear expression of proliferation-associated antigen KI-67 were mutually exclusive. Similarly, FGFR3-positive Spg were negative for Doublesex and Mab-3 related transcription factor 1 (DMRT1). By contrast, undifferentiated embryonic cell transcription factor 1 (UTF1) and survival time-associated PHD finger in ovarian cancer 1 protein (SPOC1) were co-expressed in the nuclei of FGFR3-positive Spg. Whole mounted seminiferous tubules illustrated the clonogenic arrangement of the FGFR3/UTF1 double-positive Spg, which mainly occurred as pairs or quadruplets and, different from the KIT-positive Spg, showed no overlap with KI-67 labeled clusters. Taken together, in the adult human testis, FGFR3 expression is a feature of small clones of rarely dividing type A Spg which resemble "undifferentiated" Spg, including the spermatogonial stem cells.
Assuntos
Divisão Celular , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/biossíntese , Espermatogônias/metabolismo , Membrana Celular/metabolismo , Vesículas Citoplasmáticas/metabolismo , Proteínas de Ligação a DNA/biossíntese , Imunofluorescência , Humanos , Antígeno Ki-67/biossíntese , Masculino , Microscopia Confocal , Proteínas Nucleares/biossíntese , Proteínas Proto-Oncogênicas c-kit/análise , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/análise , Túbulos Seminíferos/citologia , Túbulos Seminíferos/metabolismo , Espermatogênese/fisiologia , Testículo/citologia , Testículo/metabolismo , Transativadores/biossíntese , Fatores de Transcrição/análise , Fatores de Transcrição/biossínteseRESUMO
BACKGROUND: Mammalian spermatogenesis is a process that involves a complex expression program in both somatic and germ cells present in the male gonad. A number of studies have attempted to define the transcriptome of male meiosis and gametogenesis in rodents and primates. Few human transcripts, however, have been associated with testicular somatic cells and germ cells at different post-natal developmental stages and little is known about their level of germline-specificity compared with non-testicular tissues. METHODS: We quantified human transcripts using GeneChips and a total of 47 biopsies from prepubertal children diagnosed with undescended testis, infertile adult patients whose spermatogenesis is arrested at consecutive stages and fertile control individuals. These results were integrated with data from enriched normal germ cells, non-testicular expression data, phenotype information, predicted regulatory DNA-binding motifs and interactome data. RESULTS: Among 3580 genes for which we found differential transcript concentrations in somatic and germ cells present in human testis, 933 were undetectable in 45 embryonic and adult non-testicular tissues, including many that were corroborated at protein level by published gene annotation data and histological high-throughput protein immunodetection assays. Using motif enrichment analyses, we identified regulatory promoter elements likely involved in germline development. Finally, we constructed a regulatory disease network for human fertility by integrating expression signals, interactome information, phenotypes and functional annotation data. CONCLUSIONS: Our results provide broad insight into the post-natal human testicular transcriptome at the level of cell populations and in a global somatic tissular context. Furthermore, they yield clues for genetic causes of male infertility and will facilitate the identification of novel cancer/testis genes as targets for cancer immunotherapies.
Assuntos
Criptorquidismo/metabolismo , Regulação da Expressão Gênica , Infertilidade Masculina/metabolismo , Proteoma/metabolismo , Espermatogênese , Testículo/metabolismo , Adulto , Motivos de Aminoácidos , Animais , Criança , Criptorquidismo/patologia , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Perfilação da Expressão Gênica , Humanos , Infertilidade Masculina/patologia , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Especificidade de Órgãos , Regiões Promotoras Genéticas , Proteoma/química , Proteoma/genética , RNA Mensageiro/metabolismo , Ratos , Especificidade da Espécie , Testículo/crescimento & desenvolvimento , Testículo/patologiaRESUMO
It is unclear whether the distinct nuclear morphologies of human A(dark) (Ad) and A(pale) (Ap) spermatogonia are manifestations of different stages of germ cell development or phases of the mitotic cycle, or whether they may reflect still unknown molecular differences. According to the classical description by Clermont, human dark type A spermatogonium (Ad) may contain one, sometimes two or three nuclear 'vacuolar spaces' representing chromatin rarefaction zones. These structures were readily discerned in paraffin sections of human testis tissue during immunohistochemical and immunofluorescence analyses and thus represented robust morphological markers for our study. While a majority of the marker proteins tested did not discriminate between spermatogonia with and without chromatin rarefaction zones, doublesex- and mab-3-related transcription factor (DMRT1), tyrosine kinase receptor c-Kit/CD117 (KIT) and proliferation-associated antigen Ki-67 (KI-67) appeared to be restricted to subtypes which lacked the rarefaction zones. Conversely, exosome component 10 (EXOSC10) was found to accumulate within the rarefaction zones, which points to a possible role of this nuclear domain in RNA processing.
Assuntos
Espermatogônias/citologia , Espermatogônias/metabolismo , Biomarcadores/metabolismo , Diferenciação Celular , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Cromatina/metabolismo , Cromatina/ultraestrutura , Proteínas de Ligação a DNA/metabolismo , Exorribonucleases/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Humanos , Imuno-Histoquímica , Antígeno Ki-67/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Masculino , Microscopia Confocal , Microscopia de Fluorescência , Proteínas Nucleares/metabolismo , Proteína com Dedos de Zinco da Leucemia Promielocítica , Proteínas Proto-Oncogênicas c-kit/metabolismo , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Espermatogênese , Espermatogônias/classificação , Transativadores/metabolismo , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: A key step in studying the biology of spermatogonia is to determine their global gene expression profile. However, disassociation of these cells from the testis may alter their profile to a considerable degree. To characterize the molecular phenotype of human spermatogonia, including spermatogonial stem cells (SSCs), within their cognate microenvironment, a rare subtype of human defective spermatogenesis was exploited in which spermatogonia were the only germ cell type. METHODS: The global expression profile of these samples was assessed on the Affymetrix microarray platform and compared with tissues showing homogeneous Sertoli-cell-only appearance; selected genes were validated by quantitative real-time PCR and immunohistochemistry on disparate sample sets. RESULTS: Highly significant differences in gene expression levels correlated with the appearance of spermatogonia, including 239 best candidates of human spermatogonially expressed genes. Specifically, fibroblast growth factor receptor 3 (FGFR3), desmoglein 2 (DSG2), E3 ubiquitin ligase c-CBL (casitas B-cell lymphoma), cancer/testis antigen NY-ESO-1 (CTAG1A/B), undifferentiated embryonic cell transcription factor 1 (UTF1) and synaptosomal-associated protein, 91 kDa homolog (SNAP91) were shown to represent specific biomarkers of human spermatogonia. CONCLUSIONS: These biomarkers, specifically the surface markers FGFR3 and DSG2, may facilitate the isolation and enrichment of human stem and/or progenitor spermatogonia and thus lay a foundation for studies of long-term maintenance of human SSCs/progenitor cells, spermatogonial self-renewal, clonal expansion and differentiation.
Assuntos
Espermatogônias/citologia , Espermatogônias/metabolismo , Testículo/citologia , Testículo/metabolismo , Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Biomarcadores/metabolismo , Diferenciação Celular , Desmogleína 2/genética , Desmogleína 2/metabolismo , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Imuno-Histoquímica , Infertilidade Masculina/genética , Infertilidade Masculina/metabolismo , Infertilidade Masculina/patologia , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Mapeamento de Interação de Proteínas , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Testículo/embriologiaRESUMO
The male reproductive tract expresses a diverse array of adhesion-GPCRs, many in a highly specific and regulated manner. Despite this specificity of expression, little is known about the function of this receptor family in male reproductive physiology. Insights into function are beginning to emerge with the increasing availability of genetically modified mice harbouring mutations in these genes. Gpr64 is the best characterised of the adhesion-GPCRs in the male reproductive system and the phenotype of Gpr64 knock-out mice implicates this receptor in the regulation of fluid absorption in the efferent ducts and proximal epididymis. This chapter summarizes recent data concerning this receptor and other family members in the male reproductive system.
Assuntos
Receptores Acoplados a Proteínas G/metabolismo , Sistema Urogenital/fisiologia , Animais , Caderinas/genética , Caderinas/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Masculino , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores Acoplados a Proteínas G/genética , Sistema Urogenital/anatomia & histologiaRESUMO
BACKGROUND: Multiple profilin isoforms exist in mammals; at least four are expressed in the mammalian testis. The testis-specific isoforms profilin-3 (PFN3) and profilin-4 (PFN4) may have specialized roles in spermatogenic cells which are distinct from known functions fulfilled by the "somatic" profilins, profilin-1 (PFN1) and profilin-2 (PFN2). RESULTS: Ligand interactions and spatial distributions of PFN3 and PFN4 were compared by biochemical, molecular and immunological methods; PFN1 and PFN2 were employed as controls. beta-actin, phosphoinositides, poly-L-proline and mDia3, but not VASP, were confirmed as in vitro interaction partners of PFN3. In parallel experiments, PFN4 bound to selected phosphoinositides but not to poly-L-proline, proline-rich proteins, or actin. Immunofluorescence microscopy of PFN3 and PFN4 revealed distinct subcellular locations in differentiating spermatids. Both were associated first with the acroplaxome and later with the transient manchette. Predicted 3D structures indicated that PFN3 has the actin-binding site conserved, but retains only approximately half of the common poly-L-proline binding site. PFN4, in comparison, has lost both, polyproline and actin binding sites completely, which is well in line with the experimental data. CONCLUSION: The testis-specific isoform PFN3 showed major hallmarks of the well characterized "somatic" profilin isoforms, albeit with distinct binding affinities. PFN4, on the other hand, did not interact with actin or polyproline in vitro. Rather, it seemed to be specialized for phospholipid binding, possibly providing cellular functions which are distinct from actin dynamics regulation.
Assuntos
Profilinas/metabolismo , Espermátides/metabolismo , Testículo/metabolismo , Actinas/metabolismo , Animais , Sítios de Ligação , Simulação por Computador , Humanos , Cinética , Masculino , Peptídeos/metabolismo , Fosfolipídeos/metabolismo , Estrutura Terciária de Proteína , Ratos , EspermatogêneseRESUMO
BACKGROUND: Post-meiotically expressed genes in the testis are essential for the proper progression of spermatogenesis, and yet, aside from the construction of individual transgenic mice using specific promoters to drive reporter plasmids, there are only very limited possibilities for relevant and quantitative analysis of gene promoters. This is due to the special nature of post-meiotic haploid cells, which to date are not represented in any appropriate cell-lines. This article reports the development of novel methodology using isolated and cultured rat seminiferous tubules in a multiwell format, into which promoter-reporter constructs can be introduced by a combination of microinjection and electroporation. METHODS: Culture conditions were developed which allowed the continued incubation of isolated rat seminiferous tubules for up to 48 h without obvious cell death and loss of post-meiotic cells. Transfection of intact seminiferous tubules by microinjection and electroporation was optimized to achieve high expression efficiencies of control plasmids, using either fluorescent protein or luciferase as reporters, thereby allowing both morphological as well as quantitative assessment. RESULTS: Successful transfection was achieved into all cell types except for mature spermatozoa. However, there appeared to be only limited cell-type specificity for the promoters used, even though these had appeared to be specific when used in transgenic animals. CONCLUSION: We have devised a methodology which allows relatively high throughput analysis of post-meiotic gene promoters into primary cells of intact seminiferous tubules. An apparent lack of cell-type specificity suggests that the gene fragments used do not contain sufficient targeting information, or that the transient episomal expression of the constructs does not encourage appropriate expression specificity. The results also highlight the doubtful interpretation of many studies using heterologous transfection systems to analyse post-meiotically expressed genes.
Assuntos
Eletroporação/métodos , Regulação da Expressão Gênica/fisiologia , Genes Reporter/genética , Regiões Promotoras Genéticas , Túbulos Seminíferos/metabolismo , Técnicas de Cultura de Tecidos/métodos , Transfecção/métodos , Animais , Proteínas de Bactérias/genética , Linhagem Celular , Sobrevivência Celular , Luciferases/genética , Proteínas Luminescentes/genética , Masculino , Camundongos , Microinjeções/métodos , Ratos , Células de Sertoli/metabolismoRESUMO
A role for HE6/GPR64 in male excurrent ducts in the regulation of water balance was suggested from targeted gene mutation in the mouse. Results of the present immunolocalization study strengthen this hypothesis. Employing monospecific antibodies and laser confocal microscopy, we studied the localization of the receptor protein in the human and wild-type mouse ductuli efferentes and epididymis. We show that HE6/GPR64 is specifically associated with cell types and subcellular domains involved in the process of fluid reabsorption. In the mouse, dual labelling with anti-tubulin antibodies revealed that HE6/GPR64 was absent from the (kino-) cilia of ciliated cells. Instead, the receptor protein accumulated in the non-ciliated principal cells. Specifically, strong immunofluorescence was observed in the apical compartment of these cells. Dual labelling with phalloidin and anti-ezrin antibodies revealed that in the mouse the bulk amount of HE6/GPR64 protein co-localized with the F-actin-ezrin scaffold in brush border-like microvilli of ductuli efferentes and long stereocilia of the epididymis proper. In the ductuli efferentes, HE6/GPR64 also co-localized with the subapical F-actin network immediately below the microvilli. Comparable immunostaining patterns were observed in human and mouse; however, a specific feature of the human ductuli efferentes was an intense HE6/GPR64-related labelling of crypt-like grooves or furrows of hitherto unknown function.
Assuntos
Epididimo/química , Receptores Acoplados a Proteínas G/análise , Ducto Deferente/química , Citoesqueleto de Actina/química , Animais , Humanos , Técnicas Imunoenzimáticas , Masculino , Camundongos , Microscopia ConfocalRESUMO
Fibronectin type II (Fn2) module-containing proteins in the male genital tract are characterized by different numbers of Fn2 modules. Predominantly two classes exist which are distinct by having either two or four Fn2 modules. Minor variants with three Fn2 modules were also found in the human and the porcine epididymis. To reveal their relationship, mRNAs and proteins of representatives of these classes were studied in human, in Sus scrofa, and in rodents. Adult boars expressed members of both classes, i.e. ELSPBP1 and pB1, in subsequent regions of the epididymis, and both were under androgenic control. Human and rodent epididymides, on the other hand, alternatively contained only representatives of one of these two classes, i.e. ELSPBP1 in the human and two different pB1-related counterparts in rodents. ELSPBP1 and pB1-related genomic sequences were closely linked in chromosomal regions HSA 19q and SSC 6 q11-q21; conserved synteny between these regions is well established. On the other hand, in a syntenic region on mouse chromosome 7, ELSPBP1-related sequences were lacking. Tight binding to the sperm membrane via a choline-mediated mechanism was a common feature of the two classes of Fn2-module proteins, suggesting related function(s). However, differences in their regionalized expression patterns along the male genital tract as well as in association sites on the sperm surface suggested a species-specific sequential order in sperm binding.
Assuntos
Fibronectinas/genética , Fibronectinas/fisiologia , Proteínas de Plasma Seminal/genética , Sequência de Aminoácidos , Animais , Proteínas de Transporte/genética , Mapeamento Cromossômico , Clonagem Molecular , DNA Complementar/isolamento & purificação , Expressão Gênica , Ligação Genética , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Filogenia , Proteínas de Plasma Seminal/fisiologia , Homologia de Sequência de Aminoácidos , SuínosRESUMO
Human epididymal protein 6 (HE6; also known as GPR64) is an orphan member of the LNB-7TM (B(2)) subfamily of G-protein-coupled receptors. Family members are characterized by the dual presence of a secretin-like (type II) seven-transmembrane (7TM) domain and a long cell adhesion-like extracellular domain. HE6 is specifically expressed within the efferent ductules and the initial segment of the epididymis, ductal systems involved in spermatozoon maturation. Here, we report that targeted deletion of the 7TM domain of the murine HE6 gene results in male infertility. Mutant mice reveal a dysregulation of fluid reabsorbtion within the efferent ductules, leading to a backup of fluid accumulation in the testis and a subsequent stasis of spermatozoa within the efferent ducts. The fertility phenotype of HE6 knockout mice identifies this receptor as a potential nonsteroidal, nontesticular target for future male contraceptives and identifies an in vivo function for a member of this unusual gene family.
Assuntos
Epididimo/patologia , Infertilidade Masculina/genética , Receptores Acoplados a Proteínas G/genética , Animais , Northern Blotting , Epididimo/metabolismo , Deleção de Genes , Infertilidade Masculina/metabolismo , Infertilidade Masculina/patologia , Masculino , Camundongos , Camundongos Knockout , RNA Mensageiro/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Testículo/patologiaRESUMO
AIM: To determine the effectiveness of the sk11, sk9 and sk11 TNUA5 Sertoli cell lines in binding germ cells in vitro. METHODS: The immortalized Sertoli cell lines sk9, sk11 and sk11 TNUA5 were used in co-culture experiments with germ cells in media with or without reproductive hormones and incubated for 44 h at 32 degrees . The number of germ cells bound to Sertoli cells was then determined and statistically analyzed. Western blot analysis and reverse transcriptase-polymerase chain reaction (RT-PCR) studies were employed to investigate the presence of cell adhesion proteins and follicle stimulating hormone (FSH) receptor, respectively. RESULTS: No statistical difference between the number of bound step-8 spermatids and bound pre-step 8 spermatids on Sertoli cells from any of the cell lines existed. After the addition of germ cells, Sertoli cells showed more lipid accumulation in their cytoplasm, indicating active phagocytosis. Western blot analysis in the sk11 TNUA5 line indicated the expression of N-cadherin. FSH-only and testosterone-only treatments increased N-cadherin expression, regardless of germ cell addition. The addition of germ cells to the sk11 TNUA5 Sertoli cells increased the expression of espin, as did the addition of FSH with germ cells. RT-PCR studies of the sk11 TNUA5 cells indicated that the mRNA for FSH receptor decreased with successive passages. CONCLUSION: In vitro binding between isolated germ cells and sk9, sk11 or sk11 TNUA5 Sertoli cells is not feasible, and therefore these cell lines are not useful for the in vitro investigation of Sertoli-germ cell interactions and primary Sertoli cell isolates must still be used.
Assuntos
Linhagem Celular Transformada/citologia , Células de Sertoli/citologia , Espermátides/citologia , Animais , Caderinas/genética , Caderinas/metabolismo , Adesão Celular/fisiologia , Linhagem Celular Transformada/metabolismo , Técnicas de Cocultura , Expressão Gênica , Processamento de Imagem Assistida por Computador , Masculino , Camundongos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , RNA Mensageiro/metabolismo , Receptores do FSH/genética , Receptores do FSH/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células de Sertoli/metabolismo , Espermátides/metabolismoRESUMO
Seminal plasma proteins are secretory proteins originating mainly from the epididymis and the accessory sex glands. They are involved in the remodelling of the sperm surface which occurs during sperm transit through the male genital tract and continues later at ejaculation. During this process, collectively called post-testicular sperm maturation, the spermatozoa acquire the ability to fertilise an egg. Seminal plasma proteins have been shown to contribute to early and central steps of the fertilisation sequence, e.g. the establishment of the oviductal sperm reservoir, modulation of capacitation and gamete interaction. The major equine seminal plasma proteins belong to three protein classes, which contain widely occurring protein modules. Fn-2 type proteins are characterised by two or four tandemly arranged Fn-2 modules and have been implicated in the modulation of sperm capacitation. Multiple members of the cysteine-rich secretory proteins (CRISP) have been identified in the male genital tract of a number of species. CRISP proteins have been shown to be involved in various functions related to sperm-oocyte fusion, innate host defense function and ion channel blockage. Spermadhesins occur only in ungulate species. Their carbohydrate- and zona pellucida-binding properties would suggest a role of these proteins in gamete recognition. The major proteins of equine seminal plasma have been isolated and characterised regarding their expression along the male genital tract, protein structure and their functions.
Assuntos
Fertilização/fisiologia , Cavalos , Proteínas de Plasma Seminal/fisiologia , Animais , Cisteína/análise , Epididimo/metabolismo , Fibronectinas/análise , Fibronectinas/química , Genitália Masculina/metabolismo , Glicoproteínas/química , Glicoproteínas/fisiologia , Masculino , Sêmen/química , Proteínas de Plasma Seminal/análise , Proteínas de Plasma Seminal/química , Capacitação EspermáticaRESUMO
The SV40 large T-antigen has been widely used to convert various cell types to a transformed phenotype, and also to induce progressive tumours in transgenic animals. The objectives of this review are to compare and discuss three different approaches to generate epididymal epithelial cell lines using the large T-antigen. In the first approach, retroviral transfection of primary cultures was used to immortalize canine epididymal cells in vitro; the other two approaches used transgenic mice expressing the large T-antigen. In one of these in vivo approaches, a construct consisting of the coding sequence of a temperature sensitive (ts) SV40 large T-antigen was inserted in a mouse genome. When the cells are exposed to the permissive temperature of 33 degrees C, functional expression of the large T-antigen occurs and cells start to proliferate. In the second in vivo approach a tissue-specific promoter, the 5kb GPX5 promoter, was used to direct expression of the large T-antigen to the epididymal duct epithelium.
Assuntos
Antígenos Virais de Tumores/metabolismo , Transformação Celular Viral , Epididimo/metabolismo , Células Epiteliais/metabolismo , Epitélio/metabolismo , Vírus 40 dos Símios/imunologia , Animais , Linhagem Celular Transformada , Epididimo/citologia , Células Epiteliais/citologia , Masculino , Camundongos , Camundongos TransgênicosRESUMO
A human caput epididymidal cDNA, HE2C, was cloned based on its homology to the known chimpanzee counterpart, suggesting that the encoded beta-defensin-like peptide represented a conserved component of the innate epididymidal epithelial defense system in primates. An approximately 6kDa HE2- related peptide was co-purified together with other HE2 isoforms from human seminal plasma by affinity chromatography. By its antibody reactivity as shown by Western blot analysis, this peptide was distinct from the more abundant HE2 isoforms and was concluded to correspond to HE2C. Similar to other HE2-encoded isoforms, the endogenous HE2C was proteolytically processed from a larger precursor by a furin-like prohormone convertase. This was confirmed by N-terminal sequencing. In order to study the structural and functional properties of HE2C it was recombinantly expressed in insect cells. Post-translational processing also occurred within these cells, yielding the mature processed HE2C peptide. Correct disulfide bonding of the recHE2C peptide was shown by p-aminophenylarsineoxide(PAPAO)-agarose binding assay. Purified recHE2C strongly bound to Escherichia coli DH5alpha and Bacillus subtilis; however, it did not exhibit microbicidal activity when tested in a radial diffusion assay against these bacteria. Different from the previously described beta-defensins, the mature HE2C peptide has an anionic pI and an algebraic net charge of -1. Also, it lacks the amphipathic transitions, which, according to the Shai-Matzusaki-Huang model, are prerequisite for the membranolytic activity of antimicrobial peptides.
Assuntos
Antígenos de Superfície/química , DNA Complementar/química , Glicopeptídeos/química , Fragmentos de Peptídeos/química , Proteínas Recombinantes/química , beta-Defensinas/química , Animais , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Linhagem Celular , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/metabolismo , Glicopeptídeos/genética , Glicopeptídeos/metabolismo , Humanos , Masculino , Fragmentos de Peptídeos/metabolismo , RNA/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , beta-Defensinas/metabolismoRESUMO
The epithelial cells lining the epididymal duct play an important role in establishing and maintaining the luminal fluid microenvironment. A cDNA, canine epididymal cDNA 11 (CE11), was cloned from the dog epididymis predicting a transport protein involved in this process. The full-length sequence encoded a 12-transmembrane domain protein of 481 amino acids; a splice variant predicted a shorter isoform. Northern blot analysis and in situ transcript hybridization revealed that the CE11 mRNA is highly expressed by the epididymal duct epithelium. By reverse transcription-polymerase chain reaction, however, lower levels of both splice variants were detected in other tissues as well. Database searches suggested that CE11 is homologous to the previously described so-called thymic stromal cotransporter (TSCOT) with weak similarity to sugar (and other) cotransporters. We show here that the mouse and human TSCOT mRNAs, although enriched in immune tissues, have a broader distribution than previously assumed. Employing Western blot analysis, we identified the endogenous CE11 protein in membrane preparations of the canine epididymis. In accordance with the occurrence of 2 splice variants, 2 immunopositive bands were detected, most probably representing the predicted CE11 isoforms. Immunoperoxidase staining and indirect immunofluorescence localized the antigen in the apical membrane compartment of the adult organ, suggesting that CE11 functions as an apical transport protein involved in the establishment and/or maintenance of the specific luminal microenvironment of the epididymal duct.
Assuntos
Proteínas de Transporte/genética , Epididimo/citologia , Epididimo/metabolismo , Células Epiteliais/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Clonagem Molecular , DNA Complementar , Cães , Interações Hidrofóbicas e Hidrofílicas , Hibridização In Situ , Masculino , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Estrutura Terciária de Proteína , RNA Mensageiro/análiseRESUMO
AIM: Identification of the rodent counterparts of human and canine epididymal cDNAs HE3, HE4 and Ce8/Ly6G5C by sequence homology and analysis of their expression patterns and regulation level in the rat. METHODS: "Electronic screening" of Expressed Sequence Tag (EST) and genomic databases, followed by RT-PCR and Northern blot analysis. RESULTS: Rodent ESTs and genomic sequences homologous to HE3, HE4 and Ce8/Ly6G5C were identified in the public databases and the "full-length" rat cDNAs cloned. To emphasise their homology to the human and canine genes, they were named Me3/Re3, Me4/Re4 and Re8 for mouse and rat counterparts, respectively. mRNA expression patterns were analysed in rats, including rat HE1 and HE5/CD52 counterparts as controls. Re3 and Re8 mRNAs were only found in the rat epididymis, while Re4 showed a broader tissue distribution. Within the epididymis, Re3 and Re4 mRNAs were detected in all regions; Re8, on the other hand, was restricted to the caput. During postnatal development, Re3 and control mRNAs were found from the earliest stages investigated, while Re8 mRNA was observed only from day 24 postnatum, corresponding to the onset of spermatogenesis in the prepubertal testis. Castration and testosterone supplementation of adult male rats suggested that none of the cloned mRNAs was directly androgen-regulated. Efferent duct ligation, however, showed that Re8 mRNA levels depended on testicular factors other than androgens. CONCLUSION: The novel rodent cDNAs can now be used to monitor epididymal gene expression more closely and to set up various regulatory and functional studies.
Assuntos
DNA Complementar/química , Epididimo/química , Homologia de Sequência , Sequência de Aminoácidos , Animais , Northern Blotting , Proteínas de Transporte/química , Proteínas de Transporte/genética , Cães , Proteínas Secretadas pelo Epidídimo/química , Proteínas Secretadas pelo Epidídimo/genética , Glicoproteínas/química , Glicoproteínas/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Masculino , Dados de Sequência Molecular , Proteínas/química , Proteínas/genética , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Distribuição Tecidual , Proteínas de Transporte Vesicular , beta-DefensinasRESUMO
BACKGROUND: The molecular basis of human testicular dysfunction is largely unknown. Global gene expression profiling of testicular biopsies might reveal an expression signature of spermatogenic failure in azoospermic men. METHODS: Sixty-nine individual testicular biopsy samples were analysed on two microarray platforms; selected genes were validated by quantitative real-time PCR and immunohistochemistry. RESULTS: A minimum of 188 transcripts were significantly increased on both platforms. Their levels increased with the severity of spermatogenic damage and reached maximum levels in samples with Sertoli-cell-only appearance, pointing to genes expressed in somatic testicular cells. Over-represented functional annotation terms were steroid metabolism, innate defence and immune response, focal adhesion, antigen processing and presentation and mitogen-activated protein kinase K signalling pathway. For a considerable proportion of genes included in the expression signature, individual transcript levels were in keeping with the individual mast cell numbers of the biopsies. When tested on three disparate microarray data sets, the gene expression signature was able to clearly distinguish normal from defective spermatogenesis. More than 90% of biopsy samples clustered correctly into the corresponding category, emphasizing the robustness of our data. CONCLUSIONS: A gene expression signature of human spermatogenic failure was revealed which comprised well-studied examples of inflammation-related genes also increased in other pathologies, including autoimmune diseases.
Assuntos
Azoospermia/genética , Perfilação da Expressão Gênica/métodos , Espermatogênese/genética , Testículo/patologia , Azoospermia/metabolismo , Biópsia , Humanos , Imuno-Histoquímica/métodos , Inflamação , Masculino , Mastócitos/citologia , Mastócitos/metabolismo , Meiose , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células de Sertoli/metabolismo , Células de Sertoli/patologia , Transdução de SinaisRESUMO
Targeted disruption of the epididymis-specific HE6/Gpr64 receptor gene in mice led to male infertility. In order to characterize the phenotype at a molecular level, we compared the gene expression patterns of wild type (wt) versus knockout (KO) caput epididymides. The caput region of KO males, although morphologically normal, nevertheless showed an aberrant expression pattern. Combining micro array analysis, differential library screening, Northern blot analysis and quantitative RT-PCR, we found that the knockout of the HE6/Gpr64 receptor was mainly associated with the downregulation of genes specific to the initial segment. The list of KO downregulated transcripts comprised Enpp2/autotaxin, the lipocalins 8 and 9, the beta-defensin Defb42, cystatins 8 and 12, as well as the membrane proteins Adam (A Disintegrin And Metalloprotease) 28, claudin-10, EAAC1, and the novel Me9. Clusterin/ApoJ and osteopontin/Spp1 mRNAs, on the other hand, were upregulated in the KO tissues. The Me9 transcript was studied in further detail, and we report here a cluster of related epididymis-specific genes. Me9 is specifically expressed in the initial segment and is representative of a novel and highly conserved mammalian gene family. The family consists of single-exon genes only; intron-containing paralogs have not yet been ascertained. The cloned cDNA sequences predicted hydrophobic polytopic membrane proteins containing the DUF716 motif. Protein expression was shown in the rodent caput epididymidis but remained uncertain in primates.