RESUMO
Gametogenetin (Ggn) is a testicular germ cell-specific gene specifically expressed from late pachytene spermatocytes through round spermatids. The function of gametogenetin protein 1 (GGN1) remains unknown. Here, we used the yeast two-hybrid approach to look for more GGN1 interacting proteins. We found that gametogenetin binding protein 1 (GGNBP1), gametogenetin binding protein 2 (GGNBP2) and ornithine decarboxylase antizyme 3 (OAZ3) were potential GGN1 interaction partners. We determined the regions mediating the interactions and further showed the interactions between the proteins in mammalian cells by colocalization and coimmunoprecipitation experiments. Our work suggested that GGN1, GGNBP1, GGNBP2 and OAZ3 could be involved in a common process associated with spermatogenesis.
Assuntos
Proteínas de Transporte/metabolismo , Hormônios Testiculares/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Transporte/análise , Linhagem Celular , Núcleo Celular/química , Citoplasma/química , Humanos , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Camundongos , Espermatogênese , Espermatozoides/metabolismo , Hormônios Testiculares/química , Testículo/citologia , Técnicas do Sistema de Duplo-Híbrido , Leveduras/genéticaRESUMO
Anti-Müllerian hormone (AMH), a TGF-beta family member, determines whether an individual develops a uterus and Fallopian tubes. Mutations in the AMH gene lead to persistent Müllerian duct syndrome in males. The wild-type human AMH protein is synthesized as a disulfide-linked dimer of two identical 70-kDa polypeptides, which undergoes proteolytic processing to generate a 110-kDa N-terminal dimer and a bioactive 25-kDa TGF-beta-like C-terminal dimer. We have studied the biosynthesis and secretion of wild-type AMH and of seven persistent Müllerian duct syndrome proteins, containing mutations in either the N- or C-terminal domain. Mutant proteins lacking the C-terminal domain are secreted more rapidly than full-length AMH, whereas single amino acid changes in both domains can have profound effects on protein stability and folding. The addition of a cysteine in an N-terminal domain mutant, R194C, prevents proper folding, whereas the elimination of the cysteine involved in forming the interchain disulfide bond, in a C-terminal domain mutant, C525Y, leads to a truncation at the C terminus. A molecular model of the AMH C-terminal domain provides insights into how some mutations could affect biosynthesis and function.
Assuntos
Transtornos do Desenvolvimento Sexual/genética , Glicoproteínas/química , Glicoproteínas/genética , Glicoproteínas/metabolismo , Mutação , Hormônios Testiculares/química , Hormônios Testiculares/genética , Hormônios Testiculares/metabolismo , Sequência de Aminoácidos , Animais , Hormônio Antimülleriano , Células COS , Cricetinae , Cisteína/genética , Glicosilação , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Dobramento de Proteína , Processamento de Proteína Pós-Traducional , Estrutura Terciária de ProteínaRESUMO
Anti-Müllerian hormone (AMH), inhibins and activins are members of the transforming growth factor (TGFbeta) superfamily and are known to have a variety of actions concerning reproduction, hormonogenesis, development processes and differentiation. Inhibins and activins are dimeric glycoproteins that are defined by their actions on the pituitary gonadotroph cells. AMH, inhibins and activins have a vast array of actions usually exerted through paracrine and endocrine mechanisms. The recent availability of specific inhibin assays has demonstrated that inhibin B is the relevant circulating inhibin form in the human male. Inhibin B seems to be a useful marker of spermatogenesis, but serum and seminal inhibin B levels are not predictive parameters for the selection of azoospermic men as candidates for testicular sperm extraction (TESE). AMH in seminal plasma may be important for sperm production, and is a good marker for sertoli cell development. It might be the only one seminal marker of spermatogenesis in non-obstructive azoospermia. Nevertheless, many of these studies were carried out with small patient numbers, and consequently must be interpreted with caution. In women ongoing assisted reproductive therapy (ART), day 3 inhibin B and AMH levels predict the number of oocytes retrieved, but cannot predict likelihood of pregnancy. Further studies are needed to determine if AMH and inhibin predict ART outcomes better than classical parameters (age, FSH levels and follicular ultrasonography). AMH and inhibin are also specific markers of Sertoli- and granulosa-cell origin in gonadal tumors.
Assuntos
Ativinas/fisiologia , Glicoproteínas/fisiologia , Inibinas/fisiologia , Técnicas Reprodutivas , Hormônios Testiculares/fisiologia , Ativinas/química , Hormônio Antimülleriano , Feminino , Glicoproteínas/química , Humanos , Inibinas/química , Masculino , Valor Preditivo dos Testes , Gravidez , Taxa de Gravidez , Hormônios Testiculares/químicaRESUMO
In addition to its role in causing Müllerian duct regression, Müllerian inhibiting substance (MIS) is implicated in the regulation of steroidogenesis, breast and prostate growth, and ovarian follicle recruitment, all of which are processes controlled or influenced by the hypothalamic-pituitary-gonadal axis. Whereas the direct effect of MIS on gonadal, prostate, and breast cells is under investigation, the ability of MIS to modulate pituitary function, thereby affecting those tissues indirectly, has not yet been studied. Using LbetaT2 cells, a murine gonadotrope-derived cell line, we have evaluated the effects of MIS on the expression of the gonadotropin genes. We show that both LbetaT2 cells and adult rat pituitaries express MIS type II receptor (MISRII) mRNA. Within 2 h, follicle-stimulating hormone beta subunit (FSHbeta) mRNA levels are significantly induced by addition of MIS to LbetaT2 cells and remain elevated through 8 h of treatment. Transcriptional activation of both the FSHbeta and luteinizing hormone beta subunit (LHbeta) gene promoters was observed by MIS, which enhances the effect of gonadotropin-releasing hormone (GnRH) agonist on the FSHbeta gene promoter and synergizes with the GnRH agonist to stimulate LHbeta gene promoter activity. Addition of MIS to LbetaT2 cells stimulates the activity of the rat LHbeta gene promoter with as little as 1 microg/ml and in a dose-dependent manner. These studies report both MISRII expression in rat pituitary cells and a gonadotrope-derived cell line and MIS-mediated activation of LHbeta and FSHbeta gene expression, and suggest that MIS may modulate the hypothalamic-pituitary-gonadal axis at more than one level.
Assuntos
Regulação da Expressão Gênica , Glicoproteínas , Gonadotropinas/biossíntese , Gonadotropinas/genética , Inibidores do Crescimento/metabolismo , Hormônios Testiculares/metabolismo , Animais , Hormônio Antimülleriano , Northern Blotting , Western Blotting , Linhagem Celular , Relação Dose-Resposta a Droga , Inibidores do Crescimento/química , Luciferases/metabolismo , Hormônio Luteinizante/metabolismo , Camundongos , Hipófise/metabolismo , Hipófise/fisiologia , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Hormônios Testiculares/química , Fatores de Tempo , Transcrição Gênica , Ativação TranscricionalRESUMO
The YWK-II protein in human spermatozoa is structurally related to the betaA4-amyloid precursor protein of Alzheimer disease and has high similarity with amyloid precusor homologues. Antibodies to the YWK-II protein agglutinate human spermatozoa and may be a potential cause of infertility. In the present study, a yeast two-hybrid system (MATCHMAKER Two-Hybrid System 2; Clontech, Palo Alto, CA) was used to screen a rat ovary cDNA library for potential ligands capable of interacting with the YWK-II component. Müllerian-inhibiting substance was found to interact with the extracellular domain of YWK-II protein. The interaction was confirmed by a binding experiment in vitro and surface plasmon resonance assays. The recombinant Müllerian-inhibiting substance can significantly increase the viability and longevity of human spermatozoa after 5 and 22 h of incubation, presumably through binding the YWK-II component on the sperm membrane. The results of this study indicate that the YWK-II sperm membrane protein may function as a receptor for Müllerian-inhibiting substance.
Assuntos
Precursor de Proteína beta-Amiloide , Membrana Celular/química , Glicoproteínas , Inibidores do Crescimento/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso , Espermatozoides/ultraestrutura , Hormônios Testiculares/metabolismo , Sequência de Aminoácidos , Animais , Hormônio Antimülleriano , Sobrevivência Celular/efeitos dos fármacos , Feminino , Biblioteca Gênica , Inibidores do Crescimento/química , Inibidores do Crescimento/genética , Humanos , Masculino , Proteínas de Membrana/química , Proteínas de Membrana/fisiologia , Dados de Sequência Molecular , Plasmídeos/genética , Ratos , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Espermatozoides/fisiologia , Hormônios Testiculares/química , Hormônios Testiculares/genética , Transfecção , Técnicas do Sistema de Duplo-HíbridoRESUMO
Newly synthesized mammalian spermatozoa undergo critical modifications as they pass along the epididymis. The modifications endow spermatozoa with fertilizing ability and occur largely as a consequence of epididymal gene expression. With this in mind, we here employed a cDNA cloning strategy designed to identify key epididymal gene products. We describe a novel cynomolgus monkey (Macaca fascicularis) epididymal transcript designated cy-ESP13.2, of 690 nucleotides. The putative human ortholog was cloned and is highly conserved. Both cDNA sequences predict small, secretory proteins with a disulfide-stabilized core. Anti-peptide polyclonal antibodies were raised to a predicted cy-ESP13.2 surface loop. Western blotting with these antibodies revealed high-level, epididymis-specific expression of cy-ESP13.2, consistent with the pattern of cy-ESP13.2 mRNA expression assessed by Northern blotting. cy-ESP13.2 protein was of 30 kDa and was readily detectable in epithelial cells lining the efferent ductules, initial segment, and cauda regions of the epididymis, but not on spermatozoa. Similarities to members of the four-disulfide-core family suggest clues to ESP13.2 function.
Assuntos
Epididimo/química , Metaloproteínas/isolamento & purificação , Hormônios Testiculares/isolamento & purificação , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Clonagem Molecular , DNA Complementar/química , Proteínas Secretadas pelo Epidídimo , Humanos , Macaca fascicularis , Masculino , Metaloproteínas/química , Metaloproteínas/genética , Dados de Sequência Molecular , RNA Mensageiro/análise , Alinhamento de Sequência , Hormônios Testiculares/química , Hormônios Testiculares/genéticaRESUMO
Gonadal morphogenesis is very similar among mammals, birds, and reptiles. Despite this similarity, each group utilises quite different genetic triggers for sex determination. In mammals, testis development is initiated by action of the Y-chromosome gene SRY. Current evidence suggests that SRY may act together with a related gene, SOX9, to activate another gene(s) in the pathway of testicular differentiation. A downstream candidate for regulation by SRY and SOX9 is AMH. In mouse, Sox9 is expressed in the Sertoli cells of the embryonic testis and it precedes the onset of Amh expression. During mouse gonadogenesis, Amh is confined to the embryonic testis, although it later shows postnatal expression in the ovary. Reptiles such as the American alligator, which exhibit temperature-dependent sex determination (TSD) do not have dimorphic sex chromosomes and apparently no SRY orthologue. SOX9 is expressed during testis differentiation in the alligator; however, it appears to be expressed too late to cause testis determination. Here we describe the cloning and expression of the alligator AMH gene and show that AMH expression precedes SOX9 expression during testis differentiation. This is the opposite to that observed in the mouse where SOX9 precedes AMH expression. The data presented here, as well as findings from recent expression studies in the chick, suggest that AMH expression is not regulated by SOX9 in the non-mammalian vertebrates.
Assuntos
Jacarés e Crocodilos/embriologia , Jacarés e Crocodilos/genética , Regulação da Expressão Gênica no Desenvolvimento , Glicoproteínas , Inibidores do Crescimento/genética , Proteínas de Grupo de Alta Mobilidade/genética , Morfogênese/fisiologia , Processos de Determinação Sexual , Hormônios Testiculares/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Hormônio Antimülleriano , Sequência de Bases , Galinhas , Feminino , Inibidores do Crescimento/química , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Ductos Paramesonéfricos/embriologia , Fatores de Transcrição SOX9 , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Temperatura , Hormônios Testiculares/química , Testículo/embriologiaRESUMO
MAb 4E9, raised against a detergent extract of rat cauda epididymal sperm, recognizes a 26 kD glycoprotein that is found on the plasma membrane of the sperm tail in cauda, but not caput, sperm (Moore et al., 1994). It also recognizes an epididymis-secreted protein that has been shown to be protein E (Xu and Hamilton, 1996). It is felt that the secreted protein becomes associated with sperm, but there has been no biochemical evidence of molecular identity between the secreted and membrane proteins. In this report, the membrane form of the antigen has been purified by reverse phase HPLC. Cyanogen bromide cleavage of the purified protein yielded 3 peptides that were purified, also by reverse phase HPLC. One of the peptides yielded an unambiguous sequence of 34 amino acids that is identical to an internal peptide of the protein found in epididymal fluid. This is the first report showing sequence identity between an epididymis-secreted protein and a protein of the sperm plasma membrane.
Assuntos
Anticorpos Monoclonais , Epididimo/metabolismo , Proteínas de Membrana/química , Metaloproteínas/química , Proteínas Nucleares/química , Espermatozoides/química , Hormônios Testiculares/química , Sequência de Aminoácidos , Animais , Membrana Celular/química , Cromatografia Líquida de Alta Pressão , Proteínas Secretadas pelo Epidídimo , Masculino , Proteínas de Membrana/isolamento & purificação , Metaloproteínas/isolamento & purificação , Dados de Sequência Molecular , Peso Molecular , Proteínas Nucleares/isolamento & purificação , Mapeamento de Peptídeos , Ratos , Hormônios Testiculares/isolamento & purificaçãoRESUMO
We have recently cloned a novel proteoglycan initially identified in human testis and hence previously called testican. A close examination of the overall protein structure reveals three main regions: four osteonectin/SPARC-like domains encompassing the amino-terminal and central part of the deduced protein, a Kazal-like motif overlapping the third domain, and the CWCV domain in the carboxyl-terminal end region of the protein core. We propose to call it SPOCK, the acronym of SPARC/Osteonectin CWCV and Kazal-like domains proteoglycan, according to its specific multidomain structure. To get further insight into the function, a Northern blot analysis was performed in order to determine the site of expression in various adult tissues; a 5.2 kb transcript appeared only but strongly in mouse brain. The structure of the murine brain proteoglycan was determined through molecular cloning; human and mouse deduced proteins are highly homologous with 95% overall amino acid identity. Murine brain serial sections hybridized with cDNA and immunological probes revealed identical distribution in discrete cerebral regions, such as CA3 hippocampal region and cerebellum. Immunoelectron microscopy showed the antigen selectively localized in the post-synaptic density of scattered pyramidal neurons and Purkinje cells. Structural analysis, a main expression in nervous system and preliminary assignment of the human gene in a critical region for neuropathologies, suggest that SPOCK may be of importance in neural development and neurodegenerative diseases.
Assuntos
Química Encefálica , Clonagem Molecular , Proteoglicanas/genética , Hormônios Testiculares/genética , Sequência de Aminoácidos , Animais , Expressão Gênica , Humanos , Camundongos , Dados de Sequência Molecular , Doenças Neuromusculares/metabolismo , Proteoglicanas/química , Hormônios Testiculares/químicaRESUMO
A protein designated as BE-20 was purified from cauda epididymal fluid of the rabbit by preparative polyacrylamide gel electrophoresis and HPLC on a mono Q HR5/5 anion exchange column. The purified protein migrated with an estimated Mt of 20,000 when analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The amino acid sequence of the N-terminus of the BE-20 protein was determined. The initial eight amino acid residues were His-Gly-Ala-Asp-Lys-Pro-Gly-Val. The corresponding 23 mer oligonucleotide (5'-CATGGCGCTGACAAGCCTGGGGT-3') was synthesized and used as sense primer with rabbit epididymal mRNA as template in the RT-PCR system. The purified BE-20 cDNA consisted of 499 bp with an open reading frame of 285 bp encoding a deduced polypeptide composed of 95 amino acids. The BE-20 cDNA had 78.5% identity in 479 bp overlap with human epididymis-specific HE4 cDNA. The amino acid sequences of the initial 30 amino acid residues of the N-terminus of the purified protein and the deduced polypeptides were as follows: N-His-Gly-Ala-Asp-Lys-Pro-Gly-Val-Cys-Pro-Gln-Leu-Ser-Ala-Asp-Leu-Asn-Cy s- Thr-Gln-Asp-Cys-Arg-Ala-Asp-Gln-Asp-Cys-Ala-Glu. The deduced polypeptide contained 16 cysteine residues and had partial sequence homology with proteins belonging to the four-disulfide core family of extracellular proteinase inhibitors. The BE-20 protein may play a role in sperm maturation and/or capacitation.
Assuntos
Epididimo/química , Glicoproteínas/genética , Hormônios Testiculares/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Proteínas do Citoesqueleto , Primers do DNA/síntese química , DNA Complementar/química , Glicoproteínas/química , Glicoproteínas/isolamento & purificação , Humanos , Masculino , Dados de Sequência Molecular , Coelhos , Análise de Sequência , Homologia de Sequência , Hormônios Testiculares/química , Hormônios Testiculares/isolamento & purificação , ZixinaRESUMO
Sympathetic denervation of the rat cauda epididymidis by surgical removal of the inferior mesenteric ganglion (IMG) results in an excessive accumulation of sperm in the cauda epididymidis as well as significant changes in cauda sperm motility and cauda epididymal gross histology. The objective of the present study was to determine if the cauda-specific changes in sperm storage, sperm motility, and epididymal histology following the loss of sympathetic innervation were accompanied by changes in the protein composition of epididymal fluid. One and 4 weeks after surgical IMG removal or sham operations, luminal fluid obtained from the caput and cauda epididymidis and cauda epididymal sperm-associated proteins were subjected to two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and silver-stained proteins were quantitated. One week after IMG removal, two cauda epididymal fluid (CEF) proteins (2 and 13) had increased 43% and 49%, respectively, whereas four CEF proteins (5, 8, 9, and 19) had decreased between 30% and 73% compared to controls. Four weeks after IMG removal, changes in CEF proteins observed 1 week following surgery were no longer present, but the staining intensities of three additional CEF proteins (11, 12, and 18) were reduced an average of 70% compared to control CEF proteins. By obstructing the cauda epididymidis, we confirmed that the changes in CEF protein composition observed following IMG removal were not the result of sperm accumulation but were due directly to the loss of innervation; the staining intensity of CEF protein 2 increased as a result of excessive sperm accumulation in the cauda epididymidis both in the presence and absence of innervation from the IMG. No significant changes in caput epididymal fluid proteins or cauda epididymal sperm-associated proteins were detected following IMG removal. These data show that the protein composition of rat CEF is significantly affected by the loss of sympathetic innervation and suggest that neuronal input may play an important role in the maintenance of epididymal function.
Assuntos
alfa-Globulinas/química , Denervação Autônoma , Epididimo/metabolismo , Plexo Hipogástrico/cirurgia , Metaloproteínas/química , Proteínas/química , Hormônios Testiculares/química , Animais , Eletroforese em Gel de Poliacrilamida , Proteínas Secretadas pelo Epidídimo , Epididimo/inervação , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Epididymis-secreted proteins D and E have been purified to homogeneity and partially characterized, and it is shown that monoclonal antibody (MAb) 4E9 (raised against a detergent extract of rat caudal epididymal sperm [Moore et al., 1994: Mol Reprod Dev 37(2):181-194]) recognizes protein E, but not protein D. The molecular weight of protein D (approximately 30 kD) is approximately 2 kD lower than protein E (approximately 32 kD). The NH2-terminus of each protein is blocked; however, microsequencing of internal peptides confirms earlier reports of significant sequence identity between the two proteins. High performance liquid chromatography tryptic peptide mapping showed peak differences between the two proteins, but it was not possible to obtain amino acid sequence in the peaks that were different. The epitope for MAb 4E9 was localized in the blocked NH2-terminus-CNBr peptide derived from protein E. The epitope was destroyed by protease treatment of protein E. Removal of N-linked oligosaccharides did not destroy the epitope for MAb 4E9 and did not affect the molecular weight difference between the proteins.
Assuntos
Epididimo/química , Metaloproteínas/química , Hormônios Testiculares/química , Amidoidrolases/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Brometo de Cianogênio , Proteínas Secretadas pelo Epidídimo , Masculino , Metaloproteínas/imunologia , Metaloproteínas/isolamento & purificação , Dados de Sequência Molecular , Oligossacarídeos , Mapeamento de Peptídeos , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase , Ratos , Ratos Sprague-Dawley , Espermatozoides/imunologia , Hormônios Testiculares/imunologia , Hormônios Testiculares/isolamento & purificação , TripsinaRESUMO
Müllerian duct regression in male embryos is due to early production by fetal Sertoli cells of anti-Müllerian hormone, a homodimeric protein of the transforming growth factor- beta superfamily. In mammals, both female Müllerian ducts develop into the uterus and Fallopian tubes, whereas in birds, the right oviduct does not develop. To gain insight into sex differentiation in birds, we have cloned the cDNA for chick anti-Müllerian hormone using antibodies raised against the partially purified protein. Expression cloning was required because of the lack of cross-hybridization between mammalian and chick anti-Müllerian hormone DNA. The chick DNA and protein are significantly longer, due to insertions that abolish nucleotide homology, except in the cDNA coding for the C-terminal, bioactive part of the protein. Nevertheless, the general structure of the gene, sequenced from the transcription initiation to the polyadenylation site, and the main features of the protein are conserved between the chick and mammals. The chick anti-Müllerian hormone gene is expressed at high levels in Sertoli cells of the embryonic testes and in lower amounts in both ovaries, higher levels being reached on the left side after 10 days of incubation.
Assuntos
Expressão Gênica , Glicoproteínas , Inibidores do Crescimento/biossíntese , Inibidores do Crescimento/química , Hormônios Testiculares/biossíntese , Hormônios Testiculares/química , Sequência de Aminoácidos , Animais , Hormônio Antimülleriano , Sequência de Bases , Northern Blotting , Bovinos , Embrião de Galinha , Galinhas , Clonagem Molecular , Sequência Conservada , Primers do DNA , DNA Complementar , Humanos , Íntrons , Masculino , Mamíferos , Camundongos , Dados de Sequência Molecular , Ductos Paramesonéfricos/fisiologia , Técnicas de Cultura de Órgãos , Reação em Cadeia da Polimerase , Ratos , Homologia de Sequência de Aminoácidos , Testículo/embriologia , Testículo/metabolismo , Transcrição GênicaRESUMO
A high molecular-mass protein complex from the archaebacterium Thermoplasma acidophilum, referred to here as the 'thermosome', is built from two subunits (M(r) 58 and 60). The thermosome has been purified to homogeneity. The molecular mass of the native complex was determined to be 1061 +/- 30 Da by scanning transmission electron microscopy. It shows a weak ATPase activity and is able to bind denatured polypeptides. Averages obtained from electron micrographs of negatively stained molecules in the end-on and side-on orientations, respectively, were compared with those of the t-complex polypeptide 1 ring complex (TRiC), isolated from bovine testes. Both molecules consist of two stacked pseudo eightfold symmetric rings which build up a cylindrical particle with a large cavity in the center. Sequence alignments of peptides generated from both subunits of the thermosome and different subunits of TRiC reveal a high partial similarity to each other and to the archaebacterial chaperonin thermophilic factor 55 from Sulfolobus shibatae as well as to eukaryotic TCP1 proteins. These striking structural similarities confirm the proposition that all these molecules belong to a single protein family which is structurally and functionally related to the GroEL class of molecular chaperones.
Assuntos
Proteínas de Bactérias/química , Chaperoninas/química , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Associadas aos Microtúbulos , Proteínas Nucleares/química , Thermoplasma/química , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/ultraestrutura , Bovinos , Chaperoninas/genética , Chaperoninas/metabolismo , Eletroforese em Gel de Poliacrilamida , Técnicas In Vitro , Masculino , Microscopia Eletrônica , Dados de Sequência Molecular , Peso Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Desnaturação Proteica , Homologia de Sequência de Aminoácidos , Hormônios Testiculares/química , Hormônios Testiculares/genética , Hormônios Testiculares/metabolismo , Thermoplasma/genética , Thermoplasma/metabolismo , Ubiquitina-Proteína Ligases , Região do Complexo-t do GenomaRESUMO
We have cloned cDNAs involved in germ cell-specific expression. For this, a subtracted cDNA library was generated by subtracting cDNAs derived from supporting cells of mutant testis from wild-type testis cDNAs. Detailed analyses of mRNA expression revealed that the genes corresponding to the cloned cDNAs were exclusively expressed in testes and were developmentally controlled.
Assuntos
Clonagem Molecular , Regulação da Expressão Gênica no Desenvolvimento , Espermátides/metabolismo , Espermatogênese/genética , Hormônios Testiculares/genética , Testículo/metabolismo , Fatores de Despolimerização de Actina , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteína de Capeamento de Actina CapZ , Diferenciação Celular , DNA Complementar/genética , Destrina , Biblioteca Gênica , Masculino , Camundongos , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/genética , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Sondas RNA , Espermatócitos/metabolismo , Espermatogônias/metabolismo , Hormônios Testiculares/química , Testículo/citologia , Transcrição GênicaRESUMO
In Percoll purified Leydig cells from mature rat incubated in Ham F12/DME medium, we have demonstrated that the testosterone production (1 ng/10(6) Leydig cells/3 h) is increased 9 fold in presence of a saturating amount of oLH but diminished when larger doses of gonadotropin are used. However, if SCM or STM is added together with oLH to the Leydig cell incubation medium, a further increase (69%) of testosterone output is noticed. Obviously, when the testosterone production is low consecutive to a desensitizing dose of oLH, the STM or SCM improves at least 2 fold the steroid synthesis. Therefore, the paracrine factor (or factors) presents in SCM (or STM) which is testis specific and acts in synergy with dbcAMP and 22R-hydroxycholesterol, uses an alternative pathway to improve rat Leydig cell testosterone production since its biological effect is highly potent in absence and in presence of oLH. Therefore, these data bring new insights in the regulation of rat Leydig cell steroidogenesis through cAMP-independent channel suggesting either an increase of the cholesterol side chain cleavage activity and/or availability of the cholesterol to mitochondria.
Assuntos
Células Intersticiais do Testículo/metabolismo , Células de Sertoli/química , Testosterona/biossíntese , Animais , Técnicas In Vitro , Masculino , Ratos , Ratos Sprague-Dawley , Túbulos Seminíferos/química , Túbulos Seminíferos/metabolismo , Células de Sertoli/metabolismo , Hormônios Testiculares/química , Hormônios Testiculares/metabolismoAssuntos
Glicoproteínas , Inibidores do Crescimento/fisiologia , Ductos Paramesonéfricos/fisiologia , Hormônios Testiculares/fisiologia , Sequência de Aminoácidos , Animais , Hormônio Antimülleriano , Bovinos , Inibidores do Crescimento/química , Humanos , Masculino , Dados de Sequência Molecular , Ratos , Hormônios Testiculares/químicaRESUMO
A basic 54-kDa protein (pI approximately 8.8) that cross-reacts with anti-caltrin antisera has been detected and isolated by gel filtration and cation exchange chromatography from seminal vesicle content of the rat. The soluble protein spontaneously precipitated in NaHCO3-buffered solution at pH 7.8, but it was kept soluble in imidazole buffer containing EDTA and dithiothreitol at pH 7.0. In addition to the main band of 54 kDa, two faint immunoreactive fractions with molecular weights around 45,000 and 14,000 were also revealed by Western blotting. The presence of the rat caltrin sequence within the primary structure of the 54-kDa molecule has been investigated by sequencing the peptides generated by trypsin digestion. The sequence of the first 46 amino acid residues of rat caltrin has been found in one of the fragments produced by enzymatic cleavage. However, the exact location of the caltrin sequence in the whole 54-kDa protein has not been determined. The purified 54-kDa protein did not inhibit Ca2+ uptake by epididymal spermatozoa. Results indicated that this molecule represents an inactive precursor of caltrin and is enzymatically processed in the lumen of the seminal vesicle to the small and active calcium transport inhibitor protein. The immunoreactive proteins with intermediate molecular weights (45,000 and 14,000) could represent partially degraded products of the precursor. The lack of inhibitory activity of the precursor may be related to the molecule's having a size and conformation that would make it unable to interact with caltrin receptors on the sperm surface.
Assuntos
Proteínas Secretadas pela Próstata , Proteínas/isolamento & purificação , Glândulas Seminais/química , Hormônios Testiculares/isolamento & purificação , Sequência de Aminoácidos , Animais , Western Blotting , Cálcio/metabolismo , Eletroforese em Gel de Poliacrilamida , Ponto Isoelétrico , Masculino , Dados de Sequência Molecular , Peso Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Proteínas/química , Proteínas/farmacologia , Ratos , Proteínas de Plasma Seminal , Espermatozoides/efeitos dos fármacos , Espermatozoides/metabolismo , Hormônios Testiculares/química , Hormônios Testiculares/farmacologia , Tripsina/metabolismoAssuntos
Glicoproteínas , Inibidores do Crescimento/imunologia , Isoanticorpos/imunologia , Hormônios Testiculares/imunologia , Animais , Hormônio Antimülleriano , Sítios de Ligação de Anticorpos , Cromossomos Humanos Par 19 , Transtornos do Desenvolvimento Sexual , Epitopos , Feminino , Regulação da Expressão Gênica , Inibidores do Crescimento/química , Inibidores do Crescimento/genética , Humanos , Masculino , Células de Sertoli/metabolismo , Hormônios Testiculares/química , Hormônios Testiculares/genética , Cromossomo YRESUMO
Mullerian inhibiting substance (MIS) is the gonadal hormone that causes regression of the Mullerian ducts, the anlagen of the female internal reproductive structures, during male embryogenesis. MIS is a member of the large transforming growth factor-beta (TGF beta) multigene family of glycoproteins that are involved in the regulation of growth and differentiation. The proteins in this gene family are all produced as dimeric precursors and undergo posttranslational processing for activation, requiring cleavage and dissociation to release bioactive C-terminal fragments. Similarly, the 140 kilodalton (kDa) disulfide-linked homodimer of MIS is proteolytically cleaved to generate its active C-terminal fragments. The sexually dimorphic expression of MIS in Sertoli cells of the testis and granulosa cells of the ovary is critical for normal differentiation of the internal reproductive tract structures. A number of extra-Mullerian functions such as control of germ cell maturation and gonadal morphogenesis, induction of the abdominal phase of testicular descent, suppression of lung maturation, and growth inhibition of transformed cells have also been proposed for this growth-inhibitory hormone and will be discussed. This article will summarize the current understanding of the biology and multiple functions of MIS including its activation, regulation, and mechanism of action and discuss areas of interest in ongoing research.