RESUMO
STUDY QUESTION: Is there a relationship between maternal occupational exposure to endocrine-disrupting chemicals (EDCs) during pregnancy and the semen quality of their sons? SUMMARY ANSWER: Our results suggest an association between maternal occupational exposure to potential EDCs, especially to pesticides, phthalates and heavy metals, and a decrease in several semen parameters. WHAT IS KNOWN ALREADY: Sexual differentiation, development and proper functioning of the reproductive system are largely dependent on steroid hormones. Although there is some animal evidence, studies on maternal exposure to EDCs during pregnancy and its effect on the semen quality of sons are scarce and none have focused on maternal occupational exposure. STUDY DESIGN, SIZE, DURATION: A cross-sectional study aiming to evaluate semen quality was carried out among Swiss conscripts aged 18 to 22 years between 2005 and 2017. PARTICIPANTS/MATERIALS, SETTING, METHODS: Conscript and parent questionnaires were completed prior to the collection of a semen sample. Semen parameters were categorised according to the guidelines of the World Health Organization (WHO). Data on maternal employment during pregnancy were provided by the parent questionnaire. Maternal occupational exposure to potential EDC categories was defined using a job-exposure matrix (JEM). Logistic regressions were used to analyse the relationship between maternal occupational exposure to EDCs and each semen parameter adjusted for potential confounding factors. Results are presented using odds ratios and 95% confidence intervals. MAIN RESULTS AND THE ROLE OF CHANCE: In total, 1,737 conscripts provided a conscript and parent questionnaire, as well as a semen sample; among these 1,045 of their mothers worked during pregnancy. Our study suggests an association between occupational exposure of mothers during pregnancy to potential EDCs and low semen volume and total sperm count, particularly for exposure to pesticides (OR 2.07, 95% CI 1.11-3.86 and OR 2.14, 95% CI 1.05-4.35), phthalates (OR 1.92, 95% CI 1.10-3.37 and OR 1.89, 95% CI 1.01-3.55), and heavy metals (OR 2.02, 95% CI 1.14-3.60 and OR 2.29, 95% CI 1.21-4.35). Maternal occupational exposure to heavy metals was additionally associated with a low sperm concentration (OR 1.89, 95% CI 1.06-3.37). LIMITATIONS, REASONS FOR CAUTION: Several limitations should be noted, such as the indirect method for maternal occupational exposure assessment during the pregnancy (JEM) and the cross-sectional design of the study. WIDER IMPLICATIONS OF THE FINDINGS: Our observations reinforce the need to inform pregnant women of potential hazards during pregnancy that could impair their child's fertility. Additional studies are needed to confirm the involvement of EDCs. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Swiss Centre for Applied Human Toxicology: SCAHT and the 'Fondation privée des Hôpitaux Universitaires de Genève'. The collection of human biological material used for this study was supported by the FABER Foundation, the Swiss National Science Foundation (SNSF): NFP 50 'Endocrine Disruptors: Relevance to Humans, Animals and Ecosystems', the Medical Services of the Swiss Army (DDPS) and Medisupport. The authors declare they have no competing financial interests. TRIAL REGISTRATION NUMBER: N/A.
Assuntos
Disruptores Endócrinos , Exposição Ocupacional , Adulto , Criança , Estudos Transversais , Ecossistema , Disruptores Endócrinos/toxicidade , Feminino , Humanos , Masculino , Exposição Materna/efeitos adversos , Exposição Ocupacional/efeitos adversos , Gravidez , Sêmen , Análise do SêmenRESUMO
Impaired testicular descent (cryptorchidism) is one of the most frequent congenital abnormalities in humans, involving 2% of male births. Cryptorchidism can result in infertility and increases risk for development of germ-cell tumours. Testicular descent from abdomen to scrotum occurs in two distinct phases: the trans-abdominal phase and the inguino-scrotal phase. Currently, little is known about the factors that regulate the trans-abdominal phase of testicular descent. Leydig insulin-like hormone (Insl3) is a member of the insulin hormone superfamily expressed in the developing testis. We show here that mice mutant for Insl3 are viable, but exhibit bilateral cryptorchidism due to developmental abnormalities of the gubernaculum, resulting in abnormal spermatogenesis and infertility. Female homozygotes have impaired fertility associated with deregulation of the oestrus cycle. These findings reveal roles for Insl3 in the development of the urogenital tract and in female fertility. Insl3 may act as a hormone to regulate the growth and differentiation of the gubernaculum, thereby mediating intra-abdominal testicular descent.
Assuntos
Criptorquidismo/genética , Proteínas/genética , Animais , Sequência de Bases , Criptorquidismo/patologia , Primers do DNA/genética , Feminino , Fertilidade/genética , Hormônios/genética , Insulina/genética , Masculino , Camundongos , Camundongos Knockout , Fenótipo , Sistema Urogenital/patologiaRESUMO
BACKGROUND: Sperm counts have been steadily decreasing over the past five decades with regional differences in the Western world. The reasons behind these trends are complex, but numerous insights indicate that environmental and lifestyle factors are important players. OBJECTIVE: To evaluate semen quality and male reproductive health in Switzerland. MATERIALS AND METHODS: A nationwide cross-sectional study was conducted on 2523 young men coming from all regions of Switzerland, recruited during military conscription. Semen volume, sperm concentration, motility, and morphology were analyzed. Anatomy of the genital area and testicular volume was recorded. Testicular cancer incidence rates in the general population were retrieved from Swiss regional registries. RESULTS: Median sperm concentration adjusted for period of sexual abstinence was 48 million/mL. Comparing with the 5th percentile of the WHO reference values for fertile men, 17% of men had sperm concentration below 15 million/mL, 25% had less than 40% motile spermatozoa, and 43% had less than 4% normal forms. Disparities in semen quality among geographic regions, urbanization rates, and linguistic areas were limited. A larger proportion of men with poor semen quality had been exposed in utero to maternal smoking. Furthermore, testicular cancer incidence rates in the Swiss general population increased significantly between 1980 and 2014. DISCUSSION: For the first time, a systematic sampling among young men has confirmed that semen quality is affected on a national level. The median sperm concentration measured is among the lowest observed in Europe. No specific geographical differences could be identified. Further studies are needed to determine to what extent the fertility of Swiss men is compromised and to evaluate the impact of environmental and lifestyle factors. CONCLUSION: A significant proportion of Swiss young men display suboptimal semen quality with only 38% having sperm concentration, motility, and morphology values that met WHO semen reference criteria.
Assuntos
Oligospermia/epidemiologia , Análise do Sêmen , Motilidade dos Espermatozoides/fisiologia , Espermatozoides/fisiologia , Adolescente , Estudos Transversais , Feminino , Humanos , Estilo de Vida , Masculino , Exposição Materna/efeitos adversos , Contagem de Espermatozoides , Suíça/epidemiologia , Adulto JovemRESUMO
Receptors of the insulin/insulinlike growth factor (IGF) family have been implicated in the regulation of pancreatic beta-cell growth and insulin secretion. The insulin receptor-related receptor (IRR) is an orphan receptor of the insulin receptor gene (Ir) subfamily. It is expressed at considerably higher levels in beta cells than either insulin or IGF-1 receptors, and it has been shown to engage in heterodimer formation with insulin or IGF-1 receptors. To address whether IRR plays a physiologic role in beta-cell development and regulation of insulin secretion, we have characterized mice lacking IRR and generated a combined knockout of Ir and Irr. We report that islet morphology, beta-cell mass, and secretory function are not affected in IRR-deficient mice. Moreover, lack of IRR does not impair compensatory beta-cell hyperplasia in insulin-resistant Ir(+/-) mice, nor does it affect beta-cell development and function in Ir(-/-) mice. We conclude that glucose-stimulated insulin secretion and embryonic beta-cell development occur normally in mice lacking Irr.
Assuntos
Receptor de Insulina/fisiologia , Animais , Insulina/fisiologia , Fator de Crescimento Insulin-Like I/fisiologia , Ilhotas Pancreáticas/embriologia , Ilhotas Pancreáticas/fisiologia , Camundongos , Camundongos Knockout , Receptor IGF Tipo 1/fisiologiaRESUMO
Epigenetics is defined as the study of the heritable changes in gene expression that are transmitted from a mother cell to a daughter cell or a parent to its progeny, that occur without modifications in DNA sequences. The epigenetic mechanisms of gene regulation include DNA methylation and post-translational modifications of histone tails such as acetylation. Generally speaking, the more methylated the DNA is, and the less acetylated the associated histones are, the less a gene will be expressed. Herein, we introduce the reader to the field of epigenetic reprogramming and its importance during embryonic development and gametogenesis. We also summarize the current litterature on environmental influences on epigenetic processes including the epigenetic transgenerational actions of endocrine disruptors and diet.
Assuntos
Desenvolvimento Embrionário/genética , Epigênese Genética , Epigênese Genética/fisiologia , HumanosRESUMO
We have isolated nine putative odorant receptor genes from the chick, named COR1 to COR9, that belong to the large multigene family of olfactory G protein-coupled receptors found in the fish, rat, mouse, dog, and human. By combining genomic DNA blot analysis, low stringency library screenings, and several PCR analyses, we were able to detect approximately 20 COR genes in the chick genome highly related to COR1-9. By in situ hybridization of newborn and adult, COR expression was detected only in the olfactory epithelium, and exhibited a random spatial distribution. During development, COR expression was observed as early as embryonic stage E5. Different levels of gene expression were observed for the COR1-9 genes: at E5, COR1-6 expression was high compared to the expression of COR7, COR8, and COR9. Surprisingly, at E5, a row of COR1-6 positive cells probably associated with the olfactory nerve extended outside the olfactory placode, reaching the anterior pole of the developing forebrain. These results suggest that, in addition to their role as putative odorant receptors, some COR may play a role in the development of the avian olfactory system.
Assuntos
Condutos Olfatórios/embriologia , Receptores Odorantes/genética , Olfato , Sequência de Aminoácidos , Animais , Southern Blotting , Embrião de Galinha , Galinhas , Regulação da Expressão Gênica no Desenvolvimento , Hibridização In Situ , Camundongos , Dados de Sequência Molecular , Condutos Olfatórios/química , Reação em Cadeia da Polimerase , Receptores Odorantes/biossíntese , Alinhamento de SequênciaRESUMO
The mammalian trkB locus undergoes alternative splicing to produce two different types of brain-derived neurotrophic factor receptors. The first type is the full-length receptor tyrosine kinase (TrkB(Tk+); the second type is a truncated receptor lacking the intracellular tyrosine kinase domain (TrkB(Tk-)). To investigate the function of both types of TrkB receptor in vivo, we have generated knockout mice lacking all isoforms of the TrkB receptor (trkB-/-) and compared sensory neuron survival in these mice to that in the previously described TrkB kinase domain knockout mice (trkB(k)-/-). We observed that the presence of truncated TrkB receptors in trkB(k)-/- mice results in more severe sensory neuron losses. Increased neuron losses associated with the presence of truncated TrkB were most severe in regions where neuron survival is most dependent on brain-derived neurotrophic factor and neurotrophin-3. Our data suggest that truncated TrkB receptors negatively influence neuron survival by interfering with the function of catalytic TrkB receptors.
Assuntos
Diferenciação Celular/genética , Sobrevivência Celular/genética , Gânglios Sensitivos/crescimento & desenvolvimento , Gânglios Sensitivos/metabolismo , Neurônios Aferentes/metabolismo , Receptor trkB/deficiência , Animais , Animais Recém-Nascidos , Carbocianinas , Domínio Catalítico/genética , Morte Celular/genética , Cóclea/crescimento & desenvolvimento , Cóclea/inervação , Cóclea/metabolismo , Gânglios Sensitivos/citologia , Camundongos , Camundongos Knockout , Modelos Biológicos , Fatores de Crescimento Neural/metabolismo , Neurônios Aferentes/citologia , Isoformas de Proteínas/genética , Receptor trkB/genética , Receptor trkC/genética , Receptor trkC/metabolismo , Transdução de Sinais/genética , Gânglio Espiral da Cóclea/citologia , Gânglio Espiral da Cóclea/crescimento & desenvolvimento , Gânglio Espiral da Cóclea/metabolismo , Taxa de Sobrevida , Regulação para Cima/genéticaRESUMO
Brain-derived neurotrophic factor (BDNF) and its cognate receptor tyrosine kinase B (TrkB) play important roles in regulating survival, structure, and function of CNS neurons. One method of studying the functions of these molecules has utilized in vitro hippocampal slice preparations. An important caveat to using slices, however, is that slice preparation itself might alter the expression of BDNF, thereby confounding experimental results. To address this concern, BDNF immunoreactivity was examined in rodent slices using two different methods of slice preparation. Rapid and anatomically selective regulation of BDNF content followed slice preparation using both methodologies; however, different patterns of altered BDNF immunoreactivity were observed. First, in cultured slices, BDNF content decreased in the dentate molecular layer and increased in the CA3 pyramidal cell layer and the mossy fiber pathway of the hippocampus after 30 min. Furthermore, an initially "punctate" pattern of BDNF labeling observed in the mossy fiber pathway of control sections changed to homogenous labeling of the pathway in vitro. In contrast to these findings, slices prepared as for acute slice physiology exhibited no change in BDNF content in the molecular layer and mossy fiber pathway 30 min after slicing, but exhibited significant increases in the dentate granule and CA3 pyramidal cell layers. These findings demonstrate that BDNF protein content is altered following slice preparation, that different methods of slice preparation produce different patterns of BDNF regulation, and raise the possibility that BDNF release and TrkB activation may also be regulated. These consequences of hippocampal slice preparation may confound analyses of exogenous or endogenous BDNF on hippocampal neuronal structure or function.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Hipocampo/metabolismo , Análise de Variância , Animais , Animais Recém-Nascidos , Fator Neurotrófico Derivado do Encéfalo/deficiência , Fator Neurotrófico Derivado do Encéfalo/genética , Contagem de Células/métodos , Hipocampo/anatomia & histologia , Imuno-Histoquímica/métodos , Masculino , Camundongos , Camundongos Knockout , Microscopia Confocal/métodos , Técnicas de Cultura de Órgãos , Ratos , Ratos Sprague-Dawley , Sinapsinas/genética , Sinapsinas/metabolismo , Fatores de TempoRESUMO
In vivo, neuronal over-expression of the anti-apoptotic protein Bcl-2 prevents axotomy-induced motoneuron death and prolongs life in a mouse model of familial amyotrophic lateral sclerosis. The mechanism of these protective effects is still unknown. We have examined, in situ, the influence of Bcl-2 over-expression on the messenger RNA level of two pro-apoptotic, bax and cpp32, and one anti-apoptotic, bcl-xl, regulators of neuronal death. In neonates wild-type mice, cpp32 mRNA was increased in axotomized, dying motoneurons. No changes in bax and bcl-xl messenger RNAs expression were detected. A similar course was observed in protected axotomized neonate motoneurons of transgenic mice over-expressing Bcl-2. In adult wild-type mice no motoneuron death was detected one week after axotomy: bax and cpp32 messenger RNAs were increased and bcl-xl messenger RNA was decreased. Four weeks after the lesion, 60% of the lesioned facial motoneurons had disappeared. In the remaining motoneurons only cpp32 messenger RNA expression was superior to control level. In Bcl-2 transgenic mice, no axotomy-induced facial motoneurons death was detected but the course of the neosynthesis of cell death genes messenger RNAs was similar to wild-type mice. Bax, Bcl-x and CPP32 immunoreactivity were increased in facial motoneurons after axotomy. Thus, fatal axotomy induces cell death genes bax and cpp32 messenger RNAs neosynthesis which is not prevented by athanatal Bcl-2 over-expression. This suggests that the protective effect of Bcl-2 results from interactions with Bax and CPP32 at the post-translation level without repercussion at the messenger RNA level. Axotomy induces cell death messenger RNA neosynthesis potentially harmful at long-term despite Bcl-2 over-expression.
Assuntos
Caspases/genética , Nervo Facial/fisiologia , Regulação Enzimológica da Expressão Gênica , Neurônios Motores/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Transcrição Gênica , Animais , Animais Recém-Nascidos , Apoptose , Axotomia , Caspase 3 , Genes bcl-2 , Humanos , Hibridização In Situ , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/genética , Proteína X Associada a bcl-2 , Proteína bcl-XRESUMO
Spermatogenesis relies on the precise regulation of the self-renewal and differentiation of spermatogonia to provide a continuous supply of differentiating germ cells. The understanding of the cellular pathways regulating this equilibrium remains unfortunately incomplete. This investigation aimed to elucidate the testicular and ovarian functions of the glucocorticoid-induced leucine zipper protein (GILZ) encoded by the X-linked Tsc22d3 (Gilz) gene. We found that GILZ is specifically expressed in the cytoplasm of proliferating spermatogonia and preleptotene spermatocytes. While Gilz mutant female mice were fully fertile, constitutive or male germ cell-specific ablation of Gilz led to sterility due to a complete absence of post-meiotic germ cells and mature spermatozoa. Alterations were observed as early as postnatal day 5 during the first spermatogenic wave and included extensive apoptosis at the spermatogonial level and meiotic arrest in the mid-late zygotene stage. Overall, these data emphasize the essential role played by GILZ in mediating spermatogonial survival and spermatogenesis.
Assuntos
Espermatogênese/fisiologia , Espermatogônias/metabolismo , Fatores de Transcrição/metabolismo , Animais , Apoptose/genética , Apoptose/fisiologia , Proliferação de Células , Células Cultivadas , Feminino , Masculino , Camundongos , Espermatócitos/metabolismo , Espermatogênese/genética , Fatores de Transcrição/genéticaRESUMO
In mammals, insulin and insulin-like growth factors (IGFs: IGF1 and IGF2) act through 2 structurally related receptors, the insulin receptor (INSR) and the type 1 IGF receptor (IGF1R), both of which are expressed in developing oocytes. IGF1 plays an important role in female reproduction, and female Igf1 knockout mice fail to ovulate and are infertile. On the other hand, little is known about the in vivo role of the insulin signaling pathway in oocytes during follicular development, although exposure to insulin or IGF1 in vitro improves oocyte maturation. To further address the significance of insulin/IGF signaling, we used conditional mutant mice and ablated the function of the genes encoding INSR, IGF1R, or both receptors specifically in developing mouse oocytes. Our genetic evidence showed unexpectedly that the female reproductive functions are not affected when Insr, Igf1r or both Insr;Igf1r are ablated in oocytes, as the female mice are fertile and exhibit normal estrous cyclicity, oocyte development and maturation, parturition frequency, and litter size. In view of these novel observations indicating that the insulin/IGF signaling is not essential in oocytes, the IGF1-dependent female fertility is re-evaluated and discussed.
Assuntos
Diferenciação Celular , Oócitos/citologia , Oogênese/genética , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/metabolismo , Animais , Western Blotting , Feminino , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Receptor IGF Tipo 1/genética , Receptor de Insulina/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
In vertebrates, odors are thought to be detected by a multigene family encoding several hundreds of seven-transmembrane-domain G-protein-coupled receptors found in fish, rat, mouse, dog, and human. Recently, the putative odorant receptor (OR) gene family in the chicken has been characterized. Twelve members have been isolated and subdivided into six subfamilies. Herein, we have further characterized the chicken olfactory receptor subfamily 7 (COR7) composed of two highly related genes (named COR7a and COR7b) which are 98.5% identical. By in situ hybridization experiments, both COR7a and COR7b transcripts were detected in the olfactory epithelium from embryonic day 6 (E6) to the new born stage. Within the olfactory epithelium, the spatial distribution of COR7a and COR7b labeled cells was random. We also observed that every individual positive cell did not coexpress the COR7a and COR7b genes. Interestingly, the COR7b gene was found to be transiently expressed in the notochord from E2 to E6, whereas COR7a or any of the other known members of the COR gene family were not detected in this mesodermal tissue. These data suggest that, in addition to its potential role as an OR in the olfactory system, COR7b may also have a function in the notochord that is essential for the dorsoventral organization of the neural tube and of the somitic mesoderm. We also discuss the possible role(s) of a putative OR present in both the notochord and the sensory olfactory epithelium.
Assuntos
Notocorda/química , Mucosa Olfatória/química , Receptores Odorantes/genética , Olfato/genética , Sequência de Aminoácidos , Animais , Embrião de Galinha , Biblioteca Genômica , Hibridização In Situ , Dados de Sequência Molecular , Família Multigênica , Proteínas do Tecido Nervoso/genética , Mucosa Olfatória/embriologia , RNA Mensageiro/isolamento & purificação , Receptores Odorantes/isolamento & purificação , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Fatores de Tempo , Distribuição TecidualRESUMO
Classical embryology has provided a clear view of the timing and hormonal cues that govern sexual differentiation. Molecular biology has added important details to this picture. The cloning of SRY, MIS, and INSL3 provide insight into the molecular signals that provide important cues at the cellular level. Continued understanding of these pathways may provide the necessary information to one day reverse defects of sexual differentiation.
Assuntos
Genitália Masculina/crescimento & desenvolvimento , Glicoproteínas , Hormônios/fisiologia , Diferenciação Sexual/fisiologia , Animais , Hormônio Antimülleriano , Estrogênios/fisiologia , Fertilidade/genética , Genitália Masculina/embriologia , Inibidores do Crescimento/genética , Inibidores do Crescimento/metabolismo , Humanos , Insulina , Masculino , Proteínas/genética , Proteínas/metabolismo , Hormônios Testiculares/genética , Hormônios Testiculares/metabolismoRESUMO
Male sexual differentiation relies upon testicular secretion of the hormones testosterone, Mullerian inhibiting substance, and insulin-3 (Insl3). Insl3 is responsible for testicular descent through virilization and outgrowth of the embryonic gubernaculum. In mouse, prenatal exposure to 17beta-estradiol and the nonsteroidal synthetic estrogen diethylstilbestrol (DES) disturbs the endocrine balance, causing demasculinizing and feminizing effects in the male embryo, including impaired testicular descent (cryptorchidism). In the current study, we show that maternal exposure to estrogens, including 17alpha- and beta-estradiol, as well as DES, specifically down regulates Insl3 expression in embryonic Leydig cells, thereby providing a mechanism for cryptorchidism. These experiments may have implications for the widespread use of estrogenic substances in agriculture and the environment.
Assuntos
Criptorquidismo/induzido quimicamente , Estradiol/efeitos adversos , Animais , Estradiol/administração & dosagem , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Insulina , Masculino , Camundongos , Microscopia Eletrônica de Varredura , Gravidez , Proteínas/genética , Testículo/embriologia , Testículo/metabolismo , Testículo/ultraestrutura , Transcrição Gênica/efeitos dos fármacosRESUMO
Recoverin is a calcium sensor that regulates rhodopsin phosphorylation in a calcium-dependent manner. Cloning experiments indicate the presence of a numerous gene family, called the NCS family, encoding recoverin-like proteins expressed predominantly in neurons. Here, we report the cloning of three novel NCS genes, and demonstrate that at least six distinct members of the NCS family (including recoverin, S-modulin, vilip 1, NCS-1, Ce-NCS-1, and Ce-NCS-2) specifically inhibit rhodopsin phosphorylation. The presence of species homologues within the NCS family suggests that this function might be shared by at least 12 (out of 18) NCS proteins. Recent studies indicate that recoverin inhibits rhodopsin phosphorylation by directly regulating rhodopsin kinase, a G protein coupled receptor kinase (GRK). Since several NCS proteins are found in neurons throughout the entire nervous system, they may regulate other members of the GRK family. Together, our data suggest a general role for NCS proteins in the regulation of calcium-dependent phosphorylation in the nervous system.
Assuntos
Encéfalo/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Proteínas do Olho , Lipoproteínas , Família Multigênica , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Neuropeptídeos/metabolismo , Proteínas Quinases/metabolismo , Rodopsina/genética , Rodopsina/metabolismo , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/metabolismo , Bovinos , Galinhas , Clonagem Molecular , Sequência Consenso , Receptor Quinase 1 Acoplada a Proteína G , Proteínas de Ligação ao GTP/metabolismo , Hipocalcina , Homeostase , Humanos , Dados de Sequência Molecular , Proteínas Sensoras de Cálcio Neuronal , Fosforilação , Filogenia , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Recoverina , Rodopsina/biossíntese , Homologia de Sequência de AminoácidosRESUMO
Development of the nervous system relies on stringent regulation of genes that are crucial to this process. TrkA, the receptor for nerve growth factor (NGF), is tightly regulated during embryonic development and is essential for the survival and differentiation of neural crest-derived sensory and sympathetic neurons. We have previously identified a mouse TrkA enhancer and have characterized several cis regulatory elements that are important for appropriate TrkA expression in vivo. We now report the cloning of a novel gene encoding a Kruppel-like factor from a mouse dorsal root ganglion expression library. This Kruppel-like factor, named mKlf7, binds specifically to an Ikaros core binding element that is crucial for in vivo TrkA enhancer function. Using in situ hybridization, we demonstrate that mKlf7 is coexpressed with TrkA in sensory and sympathetic neurons during embryogenesis and in adulthood. These data are consistent with the idea that mKlf7 may directly regulate TrkA gene expression in the peripheral nervous system.
Assuntos
Proteínas de Ligação a DNA/genética , Neurônios Aferentes/metabolismo , Proteínas Nucleares/genética , Receptor trkA/genética , Sistema Nervoso Simpático/citologia , Fatores de Transcrição/genética , Dedos de Zinco , Sequência de Aminoácidos , Animais , Sequência de Bases , Sistema Nervoso Central/embriologia , Sistema Nervoso Central/metabolismo , Clonagem Molecular , DNA Complementar , Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos , Feminino , Expressão Gênica , Humanos , Fatores de Transcrição Kruppel-Like , Camundongos , Camundongos Endogâmicos ICR , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Neurons of the vertebrate olfactory epithelium (OE) regenerate continuously throughout life. The capacity of these neurons to regenerate and make new and precise synaptic connections in the olfactory bulb provides a useful model to study factors that may control or mediate neuronal regeneration. Expression and in vitro studies have suggested potential roles for the neurotrophins in the olfactory system. To directly examine whether neurotrophins are required for olfactory neuron development, we characterized in vivo the role of the neurotrophins in the primary olfactory system. For this, we generated mutant mice for TrkA, TrkB, TrkC, and also for BDNF and NT3 together with P2-IRES-tau-LacZ trangenic mice. Histochemical staining for beta-galactosidase at birth allowed in vivo analysis of the P2 subpopulation of olfactory neurons as well as their projections to the olfactory bulb. Our data indicate that Trk signaling is not required for normal embryonic development of the olfactory system.
Assuntos
Fatores de Crescimento Neural/metabolismo , Bulbo Olfatório/embriologia , Mucosa Olfatória/embriologia , Nervo Olfatório/embriologia , Condutos Olfatórios/embriologia , Receptores de Fator de Crescimento Neural/metabolismo , Animais , Camundongos , Camundongos Knockout , Regeneração Nervosa , Neurônios/metabolismo , Bulbo Olfatório/citologia , Mucosa Olfatória/citologia , Nervo Olfatório/citologia , Condutos Olfatórios/citologiaRESUMO
Persistent stimulation of G protein-coupled receptors by agonists leads rapidly to reduced responses, a phenomenon described as desensitization. It involves primarily the phosphorylation of receptor sites by specific kinases of the G protein-coupled receptor kinase (GRK) family. The beta-adrenergic receptor kinase 1 (GRK2) desensitizes agonist-activated beta2-adrenergic receptors, whereas rhodopsin kinase (GRK1) phosphorylates and inactivates photon-activated rhodopsin. Little is known about the role of calcium in desensitization. Here we report the characterization of a novel neuronal calcium sensor (NCS) named NCS-1 possibly involved in the regulation of receptor phosphorylation. NCS-1 is a new member of the EF-hand superfamily, which includes calmodulin, troponin C, parvalbumin, and recoverins. By Northern analysis and in situ hybridization, we discovered that NCS-1 is specifically expressed in the central and peripheral nervous systems. Chick NCS-1 has 72% of amino acid identity with Drosophila frequenin, a protein found in the nervous system and at the motor nerve terminals of neuromuscular junctions. By analogy with the reported function for two other members of the NCS family, we discuss whether G protein-coupled receptors or GRKs are the targets of neuronal calcium sensors.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Neurônios/metabolismo , Receptores de Superfície Celular/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Ligação ao Cálcio/genética , Galinhas , Primers do DNA/genética , Hibridização In Situ , Dados de Sequência Molecular , Fosforilação , Filogenia , Receptores Proteína Tirosina Quinases/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Distribuição TecidualRESUMO
VILIP and NCS-1, neural-specific, 22-kDa Ca(2+)-binding proteins possessing four EF-hands, were expressed in Escherichia coli to study their divalent cation properties. Flow dialysis (Ca2+ binding) and equilibrium gel filtration (Mg2+ binding) revealed that both recombinant proteins possess only two active metal-binding sites, which can accommodate either Ca2+ or Mg2+. VILIP binds cations without cooperativity with intrinsic affinity constants K'Ca of 1.0 x 10(6) M-1 and K'Mg of 4.8 x 10(3) M-1.Mg2+ antagonizes Ca2+ binding by shifting the isotherms to higher free Ca2+ concentrations without changing their shape. The competition equation yields a K'Mg, comp value of 180 M-1 for both sites. NCS-1 binds two Mg2+ without cooperativity with K'Mg of 8.3 x 10(4) M-1 and two Ca2+ with very strong positive cooperativity (nH = 1.96). In the absence of Mg2+ the K'Ca1 and K'Ca2 values are 8.9 x 10(4) and 1.4 x 10(8) M-1, respectively, which represent an allosteric increase of 1600-fold. Mg2+ shifts the Ca(2+)-binding isotherms to higher Ca2+ concentrations, yielding a K'Mg, comp value of 800 M-1 for both sites. Thus VILIP and NCS-1 show three remarkable differences in the Ca2+/Mg2+ binding parameters: 1) VILIP binds Ca2+ with much lower affinity than NCS-1; 2) VILIP binds Ca2+ in a noncooperative way, whereas NCS-1 shows maximal positive cooperativity; 3) in VILIP the Mg2+/Ca2+ antagonism is much weaker than in NCS-1. Conformational changes monitored by Trp fluorescence indicate that the metal-free forms already are highly structured. Ca2+ binding promotes a 20-30% increase of fluorescence in both proteins, but whereas the Mg2+ form of VILIP has the same fluorescence properties as the metal-free form, Mg(2+)-saturated NCS-1 has those of the Ca2+ form. Near UV difference spectra confirmed that in VILIP the Mg2+ form is very similar to the metal-free form; in NCS-1 it is different, especially in the Tyr region. NCS-1 possesses one unique Cys-38 in EF-hand site I. Its reactivity (kSH) toward 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) is the same for the Ca(2+)- and Mg(2+)-loaded protein, but kSH is 4-fold higher in metal-free NCS-1. VILIP possesses two additional thiols, one of which is inaccessible to DTNB in the native protein. The reactivity of the two accessible thiols is identical in the metal-free and Mg2+ forms and 5-fold higher than in the Ca2+ form.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Receptores de Detecção de Cálcio , Animais , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/química , Cátions , Galinhas , Magnésio/metabolismo , Proteínas do Tecido Nervoso/química , Neurocalcina , Ligação Proteica , Conformação Proteica , Espectrometria de Fluorescência , Compostos de Sulfidrila/química , Triptofano/químicaRESUMO
PURPOSE: Testicular descent is controlled by 2 morphological and hormonal steps. Transabdominal testicular descent is mediated by gubernacular swelling and regression of the cranial suspensory ligament. Müllerian inhibiting substance (MIS) has been proposed to stimulate the swelling but this remains controversial. Recently, a mouse mutant for Leydig insulin-like hormone (Insl3) was found to have undescended testis and deficient gubernaculum. We examine the testicular position of Insl3 mutant mice and the development of gubernacula. MATERIALS AND METHODS: Mice with Insl3 homozygotes (-/-), heterozygotes (+/-) and wild-types (+/+) were examined at embryonic day 16.5 and birth. Macroscopic dissections and measurements of the testicular position, as well as microscopic analysis (hematoxylin and eosin, and Masson's trichrome) were performed. RESULTS: Of the mice 11 Insl3 homozygote males had significantly impaired testicular descent at embryonic day 16.5 and birth (p <0.01), and the cord was thin and elongated, while 14 heterozygotes and 7 wild-types had normal testicular descent. Microscopically, the gubernaculum of Insl3 homozygotes was small with some muscle development but no central core of mesenchyme at embryonic day 16.5. On the other hand, heterozygotes and wild-types had normal gubernacular development with a swelling reaction. CONCLUSIONS: Insl3 mutants show feminized gubernaculum with deficient mesenchymal core. Insl3 appears to have some role in the gubernacular swelling reaction in mice.