RESUMO
The thyroid gland produces thyroid hormones (TH), which are essential regulators for growth, development and metabolism. The thyroid is mainly controlled by the thyroid-stimulating hormone (TSH) that binds to its receptor (TSHR) on thyrocytes and mediates its action via different G protein-mediated signaling pathways. TSH primarily activates the Gs-pathway, and at higher concentrations also the Gq/11-pathway, leading to an increase of intracellular cAMP and Ca2+, respectively. To date, the physiological importance of other G protein-mediated signaling pathways in thyrocytes is unclear. Congenital hypothyroidism (CH) is defined as the lack of TH at birth. In familial cases, high-throughput sequencing methods have facilitated the identification of novel mutations. Nevertheless, the precise etiology of CH yet remains unraveled in a proportion of cases. Genetically modified mouse models can reveal new pathophysiological mechanisms of thyroid diseases. Here, we will present an overview of genetic mouse models for thyroid diseases, which have provided crucial insights into thyroid gland development, function, and growth with a special focus on TSHR and microRNA signaling.
Assuntos
Camundongos Transgênicos , Glândula Tireoide/embriologia , Glândula Tireoide/crescimento & desenvolvimento , Glândula Tireoide/fisiologia , Animais , Hipotireoidismo Congênito/genética , Hipotireoidismo Congênito/patologia , Humanos , Camundongos , Receptores da Tireotropina/genética , Receptores da Tireotropina/fisiologia , Transdução de Sinais/fisiologia , Doenças da Glândula Tireoide/genética , Doenças da Glândula Tireoide/patologia , Hormônios Tireóideos/fisiologia , Tireotropina/genética , Tireotropina/fisiologiaRESUMO
Transgenic (TG) female mice expressing bLHbeta-CTP (a chimeric protein derived from the beta-subunit of bovine luteinizing hormone [LH] and a fragment of the beta-subunit of human chorionic gonadotropin [hCG]) exhibit elevated serum LH, infertility, polycystic ovaries, and ovarian tumors. In humans, increased LH secretion also occurs in infertility and polycystic ovarian syndrome, often concomitant with adrenocortical dysfunction. We therefore investigated adrenal function in LH overexpressing bLHbeta-CTP female mice. The size of their adrenals was increased by 80% with histological signs of cortical stimulation. Furthermore, adrenal steroid production was increased, with up to 14-fold elevated serum corticosterone. Primary adrenal cells from TG and control females responded similarly to ACTH stimulation, but, surprisingly, the TG adrenals responded to hCG with significantly increased cAMP, progesterone, and corticosterone production. LH receptor (LHR) expression and activity were also elevated in adrenals from female TG mice, but gonadectomized TG females showed no increase in corticosterone, suggesting that the dysfunctional ovaries of the intact TG females promote adrenocortical hyperfunction. We suggest that, in intact TG females, enhanced ovarian estrogen synthesis causes increased secretion of prolactin (PRL), which elevates LHR expression. Chronically elevated serum LH, augmented by enhanced PRL production, induces functional LHR expression in mouse adrenal cortex, leading to elevated, LH-dependent, corticosterone production. Thus, besides polycystic ovaries, the bLHbeta-CTP mice provide a useful model for studying human disorders related to elevated LH secretion and adrenocortical hyperfunction.
Assuntos
Córtex Suprarrenal/metabolismo , Hormônio Luteinizante/sangue , Receptores do LH/metabolismo , Esteroides/biossíntese , Hormônio Adrenocorticotrópico/farmacologia , Fatores Etários , Animais , Bovinos , Células Cultivadas , Gonadotropina Coriônica/genética , Gonadotropina Coriônica/farmacologia , Corticosterona/sangue , Feminino , Histocitoquímica , Hibridização In Situ , Hormônio Luteinizante/genética , Masculino , Camundongos , Camundongos Transgênicos , Progesterona/metabolismo , Prolactina/sangue , RNA Mensageiro/metabolismo , Receptores do LH/genética , Proteínas Recombinantes de Fusão/farmacologiaRESUMO
Normal spermatogenesis is highly dependent on well-balanced germ cell proliferation, differentiation, and apoptosis. However, the molecular mechanisms that govern these processes are largely unknown. Retinoblastoma family proteins (pRb, p107 and p130) are potentially important regulators of cell growth, differentiation and apoptosis. pRb has been shown to be expressed in the rat testis and involved in the regulation of spermatogenesis. In the present study, the expression and localization of the other two pRb family members, p107 and p130, were analysed at both mRNA and protein levels during testicular development and spermatogenesis using Northern, Western blotting, immunohistochemistry, and in situ hybridization. Furthermore, changes of levels and phosphorylation status of pRb family proteins in response to growth suppression and/or apoptosis induction were investigated using a seminiferous tubule culture system and three animal models. Our data suggest that: (1) pRb family proteins are differentially expressed in the rat testis and they function in a cell-type-specific manner during testicular development and spermatogenesis; (2) they participate in the control of germ cell cycle and act in a cell cycle-phase-specific fashion during germ cell proliferation, and (3) they are also involved in the regulation of apoptosis of germ cells and Leydig cells.
Assuntos
Proteínas Nucleares/genética , Fosfoproteínas/genética , Proteínas , Proteína do Retinoblastoma/genética , Espermatogênese/genética , Testículo/citologia , Acetatos/farmacologia , Animais , Apoptose/genética , Diferenciação Celular/genética , Divisão Celular/genética , Regulação da Expressão Gênica , Masculino , Mesilatos/farmacologia , Família Multigênica , Proteínas Nucleares/biossíntese , Fosfoproteínas/biossíntese , Fosforilação , Ratos , Ratos Sprague-Dawley , Proteína do Retinoblastoma/biossíntese , Proteína p130 Retinoblastoma-Like , Túbulos Seminíferos/citologia , Espermatócitos/efeitos dos fármacosRESUMO
Transgenic (TG) mice, expressing the Simian Virus 40 T-antigen (Tag) under a 6-kb fragment of the murine inhibin alpha-subunit promoter (inh alpha p), develop gonadal tumors of granulosa/theca or Leydig cell origin. We showed previously that adrenocortical tumors develop if the TG mice are gonadectomized but never develop in intact animals. However, if functional gonadectomy was induced by GnRH antagonist treatment or by cross-breeding the TG mice into the hypogonadotropic hpg genetic background, neither gonadal nor adrenal tumors appeared. Since the most obvious difference between the gonadectomized and GnRH-antagonist-treated or Tag/hpg double mutant mice is the elevated gonadotropin secretion in the first group, we examined whether the adrenal tumorigenesis would be gonadotropin-dependent. Surprisingly, both the adrenal tumors and a cell line (C alpha 1) derived from one of them expressed highly functional LH receptors (LHR), as assessed by Northern hybridization, immunocytochemistry, ligand binding, and human CG (hCG)-stimulated cAMP and steroid production. No FSH receptor expression was found in the adrenal tumors by RT-PCR. hCG treatment of the C alpha 1 cells stimulated their proliferation, as measured by [3H]thymidine incorporation. This effect was related to hCG-stimulated steroidogenesis since progesterone, testosterone, and estradiol, at physiological concentrations, also stimulated the C alpha 1 cell proliferation. Different adrenocortical cells expressed initially LHR and Tag, whereas both were highly expressed in the tumor cells. In conclusion, the high level of functional LHR in the adrenal tumors indicates that this receptor can function as tumor promoter when ectopically expressed and stimulated by the ligand hormone.
Assuntos
Neoplasias do Córtex Suprarrenal/genética , Antígenos Transformantes de Poliomavirus/fisiologia , Tumor de Células da Granulosa/genética , Inibinas , Tumor de Células de Leydig/genética , Hormônio Luteinizante , Hormônio Luteinizante/farmacologia , Neoplasias Hormônio-Dependentes/genética , Neoplasias Ovarianas/genética , Peptídeos/fisiologia , Regiões Promotoras Genéticas , Neoplasias Testiculares/genética , Tumor da Célula Tecal/genética , Neoplasias do Córtex Suprarrenal/fisiopatologia , Animais , Antígenos Transformantes de Poliomavirus/genética , Castração , Transformação Celular Neoplásica/genética , Gonadotropina Coriônica/farmacologia , Cruzamentos Genéticos , Replicação do DNA/efeitos dos fármacos , Feminino , Hormônios Esteroides Gonadais/farmacologia , Hormônio Liberador de Gonadotropina/análogos & derivados , Hormônio Liberador de Gonadotropina/toxicidade , Gonadotropinas Hipofisárias/deficiência , Tumor de Células da Granulosa/fisiopatologia , Humanos , Tumor de Células de Leydig/fisiopatologia , Hormônio Luteinizante/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Mutantes , Camundongos Transgênicos , Neoplasias Hormônio-Dependentes/fisiopatologia , Especificidade de Órgãos , Neoplasias Ovarianas/fisiopatologia , Peptídeos/genética , Receptores do FSH/análise , Receptores do LH/biossíntese , Receptores do LH/fisiologia , Proteínas Recombinantes de Fusão/fisiologia , Vírus 40 dos Símios/fisiologia , Neoplasias Testiculares/fisiopatologia , Tumor da Célula Tecal/fisiopatologia , Células Tumorais CultivadasRESUMO
MicroRNAs (miRNAs) are important regulators of posttranscriptional gene expression and involved in embryonic development, regulation of cell differentiation, and growth. Dicer1 is a key enzyme in the maturation process of functional miRNAs. However, miRNA-mediated regulation of normal thyroid function and growth is largely unknown. To understand the role of miRNAs in the thyroid, we generated constitutive and tamoxifen-inducible, thyrocyte-specific Dicer1 knockout mice. The mice with perinatal Dicer1 deletion (cTgDcrKO) showed impaired follicular organization, increased fibrosis, and accumulation of adipocytes in the thyroid. Similar histological changes were observed in tamoxifen-induced adult Dicer1-deficient mice (iTgDcrKO). The thyroid phenotype in both knockout (KO) lines was associated with significantly down-regulated mRNA expression of thyroid transcription factor 1 (Ttf-1/Nkx2-1), thyroid peroxidase, and thyroglobulin (Tg) and up-regulated expression of genes involved in Tgf-ß signaling. Furthermore, in cTgDcrKO mice, which developed mild hypothyroidism, the protein expression of Nkx2-1, thyroglobulin, Paired box 8, and TSH receptor were clearly down-regulated compared with controls. Despite similar down-regulation of Dicer1 in cTgDcrKO and iTgDcrKO compared with controls, Dicer1 deletion in adult mice thyrocytes did not lead to acute hypothyroidism. No significant differences in thyroid weights between cTgDcrKO, iTgDcrKO, and controls were observed. However, a goitrogenic diet induced a 4-fold increase in thyroid weight in control animals, whereas it had no effect on iTgDcrKO thyroids. In summary, Dicer1 deficiency in thyrocytes is associated with intrathyroid fibrosis, adipogenesis, and enhanced expression of Tgf-ß signaling genes. Furthermore, our data indicate that Dicer1 is required for thyroid follicular organization, thyrocyte differentiation, and goiter development.
Assuntos
RNA Helicases DEAD-box/genética , Bócio/prevenção & controle , Ribonuclease III/genética , Glândula Tireoide/metabolismo , Animais , RNA Helicases DEAD-box/metabolismo , Regulação para Baixo , Bócio/genética , Bócio/metabolismo , Camundongos , Camundongos Knockout , MicroRNAs , Receptores da Tireotropina/genética , Receptores da Tireotropina/metabolismo , Ribonuclease III/metabolismo , Tireoglobulina/genética , Tireoglobulina/metabolismo , Tireotropina/sangue , Tiroxina/sangue , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
We investigated in this study the effects of ovine PRL on endocrine functions of cultured murine Leydig tumor cells (mLTC-1). The parameters studied were the activation of signal transduction systems involving cAMP and intracellular free Ca(2+), the expression of Janus kinase 2 (JAK2), expression and function of LH and PRL receptors (R), expression of the steroidogenic acute regulatory (StAR) protein, and stimulation of steroidogenesis. Very similar biphasic dose- and time-dependent responses of all the parameters studied were found upon PRL stimulation, comprising a fast inhibition within 24 h in response to high PRL doses (>/=30 microgram/liter), and a slow stimulation, between 48-72 h, in response to lower PRL doses (1-10 microgram/liter). In addition, extracellular Ca(2+) (1.5 mmol/liter) increased the effect of PRL on human CG (hCG)-stimulated StAR messenger RNA expression and progesterone (P) production. Importantly, the biphasic effects of PRL on LHR gene expression and hCG-mediated P production were abolished in the presence of anti-PRL antiserum, demonstrating specificity of PRL action. The PRL effects on StAR expression, and steroid and cAMP production, apparently reflect its effects on LHR function. The relevance of the PRL effects observed in mLTC-1 cells was supported by demonstration of similar PRL responses in hCG-stimulated testosterone production of isolated mouse Leydig cells. Collectively, these findings clearly demonstrate the biphasic regulatory actions of PRL, and clarify some facets of the controversial role of this hormone in Leydig cell function.
Assuntos
Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Tumor de Células de Leydig/fisiopatologia , Prolactina/farmacologia , Proteínas Proto-Oncogênicas , Neoplasias Testiculares/fisiopatologia , Animais , Cálcio/metabolismo , Gonadotropina Coriônica/metabolismo , Humanos , Janus Quinase 2 , Cinética , Masculino , Camundongos , Fosfoproteínas/genética , Proteínas Tirosina Quinases/genética , Receptores do LH/efeitos dos fármacos , Receptores do LH/genética , Receptores do LH/fisiologia , Receptores da Prolactina/genética , Ovinos , Células Tumorais CultivadasRESUMO
To further investigate the regulatory mechanisms responsible for the control of testicular inhibin/activin subunit gene expression, inhibin-alpha, -betaA, and -betaB messenger RNA (mRNA) levels were assessed after ethylene dimethane sulfonate (EDS)-induced destruction of Leydig cells (LC) in different animal models: the intact rat, the rat treated with high doses of testosterone, and the unilaterally cryptorchid rat. In intact rats, EDS selectively eliminates the mature adult-type LCs, activating the proliferation and differentiation of preexisting LC precursors into a new population of functionally active LCs. In this model, a single dose of EDS (75 mg/kg BW, ip) induced a significant increase in testicular inhibin-alpha and -betaB mRNA levels 5 days after treatment (5.0- and 5.5-fold increases, respectively), whereas inhibin-betaA mRNA remained undetectable upon Northern hybridization in control and EDS-treated testes. Moreover, in situ hybridization analysis demonstrated that the increased expression of inhibin-alpha and -betaB mRNAs observed 5 days after EDS takes place mainly in Sertoli cells. Along with LC repopulation, the expression level of inhibin-alpha and -betaB messages declined, and inhibin-alpha mRNA returned to control values on day 40 after EDS. This treatment, however, failed to alter the pattern of testicular expression of FSH receptor and androgen-binding protein mRNAs, thus suggesting selectivity for the above effects. In EDS-treated rats supplemented with high doses of testosterone, the preexisting mature LCs are destroyed, but, due to elevated testosterone concentrations, disruption of spermatogenesis is attenuated, and the post-EDS rise in serum gonadotropins is blocked; the latter prevents LC regeneration. In this model, a 5.0-fold increase in inhibin-alpha mRNA levels, similar to that found in intact animals, was detected 5 days after EDS administration, but the rise in inhibin-betaB levels was partially delayed. In addition, the blockade of LC repopulation resulted in permanent elevation of inhibin-alpha and -betaB messages throughout the study period. In unilaterally cryptorchid rats, the abdominal testis shows disrupted spermatogenesis and altered paracrine environment that expedites LC repopulation after EDS treatment. In this model, the abdominal testes showed a significant 2.5-fold increase in inhibin-alpha mRNA levels 5 days after EDS, but no effect was found in those of inhibin-betaB. Further, the faster rate of LC repopulation resulted in precocious decline of inhibin-alpha mRNA levels. Finally, the expression of inhibin/activin subunit mRNAs was monitored during postnatal testicular development, specifically at the time of regression of fetal-type LCs and appearance of those of the adult type. High levels of expression of inhibin-alpha and -betaB mRNAs were detected in neonatal and infantile testes. A sharp decline in both messages took place between days 15-20, i.e. at the time when fetal-type Leydig cells are replaced by adult-type cells. From this time point onward, inhibin-alpha and -betaB mRNA levels remained low, ranging between 15-30% of the maximum. In conclusion, our results suggest that the adult-type LCs differentially modulate the expression of inhibin/activin subunit genes and point to a major inhibitory role in this cell type on expression of the inhibin-alpha gene.
Assuntos
Expressão Gênica , Inibinas/genética , Células Intersticiais do Testículo/fisiologia , Mesilatos/farmacologia , Testículo/metabolismo , Ativinas , Envelhecimento , Animais , Northern Blotting , Criptorquidismo/metabolismo , Hibridização In Situ , Cinética , Células Intersticiais do Testículo/efeitos dos fármacos , Masculino , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Testículo/crescimento & desenvolvimentoRESUMO
Recently, we demonstrated that triiodothyronine (T(3)) stimulated steroid hormone biosynthesis and steroidogenic acute regulatory (StAR) protein expression in mLTC-1 mouse Leydig tumor cells through the mediation of steroidogenic factor 1 (SF-1). We now report a dual response mechanism of T(3) on steroidogenesis and StAR expression, and on LH receptor (LHR) expression and binding in mLTC-1 cells. T(3) acutely (8 h), induced a 260% increase in StAR messenger RNA (mRNA) expression over the basal level which was coincident with an increase in progesterone (P) production. In contrast, chronic stimulation with T(3) (beyond 8 h), resulted in an attenuation of StAR expression and P production. This attenuation was most likely caused by a decrease in cholesterol delivery to the inner mitochondrial membrane as demonstrated by incubations with the hydrophilic steroid precursors, 22R hydroxycholesterol and pregnenolone, which restored P synthesis. In similar studies, chronic treatment with T(3) increased the levels of cytochrome P450scc mRNA by 83%, whereas those of cytochrome P450 17alpha-hydroxylase and 3ss-hydroxysteroid dehydrogenase decreased. The diminished response in steroidogenesis following chronic T(3) exposure was not a result of alterations in StAR mRNA stability, but rather was due to inhibition of transcription of the StAR gene. Similar acute stimulatory and chronic inhibitory responses to T(3) were found when LHR mRNA expression and LHR ligand binding were examined. Transfections with an LHR or StAR promoter/luciferase reporter construct demonstrated that a 173-bp fragment of the LHR promoter containing an SF-1 binding motif was involved in T(3) response, as was the SF-1 recognition site at -135 bp in the StAR promoter. Furthermore, the importance of SF-1 in T(3) function was also verified employing mutation in the bases of SF-1 sequences using electrophoretic mobility shift assays. The potential physiological relevance of these findings was demonstrated when similar responses were obtained in mice rendered hypo and hyperthyroid. Collectively, these observations further characterize the thyroid-gonadal connection and provide insights into the mechanisms for a dual regulatory role of thyroid hormone in Leydig cell functions.
Assuntos
Células Intersticiais do Testículo/fisiologia , Mitocôndrias/metabolismo , Fosfoproteínas/genética , Progesterona/metabolismo , Receptores do LH/genética , Transcrição Gênica/efeitos dos fármacos , Tri-Iodotironina/farmacologia , 3-Hidroxiesteroide Desidrogenases/genética , 3-Hidroxiesteroide Desidrogenases/metabolismo , Animais , Células Cultivadas , Colesterol/metabolismo , Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , Cicloeximida/farmacologia , Genes Reporter , Membranas Intracelulares/metabolismo , Células Intersticiais do Testículo/efeitos dos fármacos , Luciferases/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Fosfoproteínas/metabolismo , Receptores do LH/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esteroide 17-alfa-Hidroxilase/genética , Esteroide 17-alfa-Hidroxilase/metabolismo , Tiroxina/farmacologia , TransfecçãoRESUMO
Considering the major role of LH in the control of Leydig cell (LC) development and function, we aimed to characterize further the pattern of LH receptor (LHR) expression in two experimental paradigms: the rat treated with ethylene dimethane sulfonate (EDS), in which the selective destruction of preexisting mature LCs induces the proliferation and differentiation of newly formed LCs, a process that takes place in the presence of high levels of gonadotropins; and the EDS-rat treated with a high dose of testosterone (EDS + T), in which the LH secretion is suppressed, and consequently LC development after EDS arrested. In EDS rats, serum T was suppressed and testicular LHR binding became undetectable on days 5 and 15 after treatment. The pattern of LHR messenger RNA (mRNA) expression was profoundly modified: only one of the splice variants [1.8-kilobase (kb)] persisted, whereas the others disappeared. On days 20 and 45 after EDS, along with LC repopulation, serum T and LHR binding recovered, and the pattern of LHR mRNA expression gradually returned to that resembling controls. In EDS + T rats, a similar drop in testicular LHR binding and change in the pattern of LHR mRNA expression was detected on days 5 and 15 after treatment. However, on days 20 and 45, no recovery either in LHR binding or in expression of the longer LHR mRNA splice variants was observed, showing that LH is needed to induce LHR expression in repopulating LCs, at least to a quantitatively significant level. To gain further insight into the mechanism(s) by which LH acts on LC precursors, the translational status of the 1.8-kb LHR transcript, persistently expressed after EDS, was analyzed and compared with that of the 6.8-kb message. In polysome distribution analysis of total testicular RNA, the 6.8-kb LHR message was highly associated with polysomes, whereas the 1.8-kb variant was mainly localized to prepolysomal fractions, both in control and EDS testes, thus predicting lower translational efficiency. In addition, considering that only LCs express LHRs in the testis, the time course of the reappearance of functional receptors was mapped by evaluating testicular responsiveness to human recombinant LH in vitro. No response to LH stimulation was detected 5 days after EDS. However, cAMP response to LH was observed on days 15 and 20, regardless of the presence of high (EDS) or suppressed (EDS + T) LH in the donor animal. Hence, the appearance of functional LHRs, qualitatively, can take place in the absence of measurable LH levels. In EDS-treated rats, the appearance of the cAMP response coincided with those of pregnenolone, progesterone, and T. In contrast, no LH-induced steroid release was observed in EDS + T rats, indicating that steroidogenic response in developing LC requires LH priming. In conclusion, the appearance of functional LHRs, at a low level of expression, in LC precursors is an LH-independent developmental event, essential for the subsequent LH-dependent maturational steps, including the onset of steroidogenesis and increased LHR expression. In addition, our results cast doubt on a major functional role of the truncated (1.8-kb) form of LHR mRNA, which persists after EDS at a high level of expression, in the early Leydig cell precursors.
Assuntos
Células Intersticiais do Testículo/citologia , Células Intersticiais do Testículo/efeitos dos fármacos , Mesilatos/farmacologia , Receptores do LH/biossíntese , Receptores do LH/fisiologia , Testículo/metabolismo , Análise de Variância , Animais , Northern Blotting , Morte Celular/efeitos dos fármacos , AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Hormônio Foliculoestimulante/sangue , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Hormônio Luteinizante/sangue , Hormônio Luteinizante/farmacologia , Hormônio Luteinizante/fisiologia , Masculino , Pregnenolona/metabolismo , Progesterona/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores do LH/genética , Proteínas Recombinantes/farmacologia , Células-Tronco/química , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Testículo/citologia , Testosterona/sangue , Testosterona/metabolismoRESUMO
We have developed a transgenic (TG) mouse model for tumorigenesis of gonadal somatic cells using a 6 kb fragment of the mouse inhibin-alpha subunit promoter (Inh-alpha) fused with the simian virus 40 T-antigen (Tag) coding sequence. Gonadal tumors, of Leydig or granulosa cell origin, develop in the TG mice with 100% penetrance by the age of 5-8 months. Conspicuously, if the mice are gonadectomized, they develop adrenal tumors. Gonadal and adrenal tumorigenesis in these mice seem to be gonadotropin dependent. On the other hand, testosterone stimulates the proliferation of a cell line (C alpha 1) established from one of the adrenal tumors. The purpose of the present study was therefore to investigate further whether testosterone affects the growth of these gonadal and adrenal tumors in vivo. Two experimental models were used: (1) Tag TG/hypogonadotropic (hpg) double mutant mice and (2) castrated Tag TG mice. Both were treated between 1-2 and 7-8 months of age with Silastic rods (length 2 cm) containing testosterone. None of the control or testosterone-treated Tag/hpg mice developed gonadal or adrenal tumors. The castrated Tag TG mice displayed, upon microscopical examination, early stages of adrenal tumors, whereas those receiving testosterone did not show such changes. Testosterone increased the weights of gonads in the Tag/hpg mice, and those of uteri and seminal vesicles in both groups. In contrast, the adrenal weights were significantly reduced in both groups by testosterone treatment. Gonadal histology of the testosterone-treated mice showed hyperplasia of testicular Leydig cells and ovarian stroma. Spermatogenesis was induced by testosterone in the Tag/hpg mice. Adrenal histology of the testosterone-treated animals demonstrated the disappearance of the X-zone. Serum levels of FSH in testosterone-treated Tag/hpg mice were significantly increased, while those of serum LH were decreased. In conclusion, the present result indicate that the suppression of gonadotropins by testosterone implants in castrated Inh-alpha/Tag TG mice prevents the tumorigenesis of their adrenals. In intact Tag/hpg mice, testosterone implants were not able to induce gonadal or adrenal tumorigenesis. Although testosterone treatment was able to induce interstitial cell hyperplasia in gonads of the Inh-alpha/Tag mice, direct gonadotropin action is responsible for gonadal and adrenal tumorigenesis.
Assuntos
Neoplasias das Glândulas Suprarrenais/prevenção & controle , Inibinas , Neoplasias Ovarianas/prevenção & controle , Peptídeos/genética , Neoplasias Testiculares/prevenção & controle , Testosterona/uso terapêutico , Análise de Variância , Animais , Implantes de Medicamento , Feminino , Hormônio Foliculoestimulante/sangue , Hormônio Luteinizante/sangue , Masculino , Camundongos , Camundongos Transgênicos , Orquiectomia , OvariectomiaRESUMO
In the rat, the cytotoxic drug ethylene dimethane sulfonate (EDS) selectively eliminates mature Leydig cells (LCs) from testicular interstitium, activating a complex process of proliferation and differentiation of pre-existing LC precursors. We observed previously that after EDS treatment, the early LC precursors persistently express a truncated 1.8 kb form of LH receptor (LHR) mRNA. This prompted us to study whether experimental cryptorchidism, known to alter the process of LC repopulation, can influence the pattern of testicular LHR mRNA expression after EDS administration. EDS treatment completely eliminated mature LCs both in control and unilaterally cryptorchid (UC) rats. This response was followed by gradual reappearance of newly formed, functionally active LCs, as evidenced by the recovery in testicular LHR content and plasma testosterone levels in both experimental groups. Noteworthy, the rate of LC repopulation was higher in the abdominal testes of UC rats, in keeping with previous findings. Interestingly, the 1.8 kb LHR transcript was persistently expressed in scrotal testes at all time-points, but undetectable upon Northern hybridization in abdominal testes at early stages after EDS administration, when low levels of expression of truncated LHR transcripts could only be detected by semi-quantitative RT-PCR analysis. In addition, the faster LC repopulation in cryptorchid testes was associated with precocious recovery of the complete array of LHR mRNA transcripts, including the 1.8 kb species. These changes appeared acutely and irreversibly, as unilateral positioning of scrotal testes into the abdomen resulted in a rapid loss of expression of the 1.8 kb LHR transcript, whereas scrotal relocation of the UC testes failed to alter the pattern of LHR gene expression. In conclusion, experimental cryptorchidism changes the pattern of LHR mRNA expression in rat testis after selective LC destruction by EDS. This change, i.e. repression of the 1.8 kb LHR transcript after EDS administration, is acute and irreversible, and likely related to the impairment of testicular microenvironment following cryptorchidism. However, even though at low levels, the expression of truncated forms of LHR mRNA appears to be a universal feature of proliferating LC precursors. The UC testis may represent a good model for analysis of the regulatory signals involved in the control of LHR gene expression.
Assuntos
Criptorquidismo/metabolismo , RNA Mensageiro/metabolismo , Receptores do LH/genética , Testículo/metabolismo , Análise de Variância , Animais , Northern Blotting , Southern Blotting , Expressão Gênica , Células Intersticiais do Testículo/efeitos dos fármacos , Masculino , Mesilatos , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismoRESUMO
The LH receptor (LHR) is a member of the family of G protein-coupled seven-times plasma membrane transversing receptors. Its gene consists of 11 exons, the last one encoding the transmembrane and intracellular domains of the receptor. The FSHR, and its gene, resemble structurally those of the LHR, with the exception that the sequences corresponding to exon 10 in LHR are missing in FSHR, which is thus encoded by a total of ten exons. Our recent studies on the marmoset monkey testis LHR cDNA indicated that an 81 bp nucleotide sequence, encoding the complete exon 10 of the LHR gene in other mammalian species, is absent in this species without affecting the LHR function. To study further the role of the exon 10 encoded sequences of the LHR in the gonadotropin receptor function, a deletion of exon 10 from the human LHR (hLHdeltaexon10R), and a chimeric hFSHR with exon 10 from hLHR inserted (hFSHLHexon10R), were constructed in expression vectors. The results presented here demonstrate that 293 cells transfected with the hLHdeltaexon10R display a decrease in the proportion of the receptor binding at the cell surface, compared with cells transfected with wild-type hLHR. However, the cells expressing hLHdeltaexon10R showed similar high affinity binding of [125I]iodo-hCG as those transfected with wild-type hLHR, in either intact cells or their detergent extracts. In addition, cells expressing the hLHdeltaexon10R and wild-type hLHR displayed similar dose-response of cAMP production to hCG stimulation. Cells transfected with chimeric hFSHLHexon10R showed barely detectable [125I]iodo-FSH binding in intact cells compared with those transfected with wild-type hFSHR. The FSH binding detected in cellular detergent extracts displayed 10-fold lower binding activity than wild-type receptors, in spite of similar level of immunoreactive FSHR protein expression in the transfected cells. The hFSHLHexon10R had a modest 5-fold lower binding affinity for FSH as compared with wild-type hFSHR. In conclusion, the present study indicates that the sequences encoding exon 10 of the hLHR are essential for the LHR expression at the plasma membrane, but deleterious for function if inserted into the hFSHR.
Assuntos
Membrana Celular/metabolismo , Éxons , Expressão Gênica , Receptores do FSH/genética , Receptores do LH/genética , Animais , Callithrix , Linhagem Celular , Embrião de Mamíferos , Humanos , Rim , Masculino , Ratos , Proteínas Recombinantes de Fusão , Testículo , TransfecçãoRESUMO
Minority peoples like the Romanies have divergent cultures. Typical cultural aspects for medical personnel to consider would include greetings and other communication, family and social support, dressing and habits of cleanliness, marriage and sexuality, honor, and other issues of importance to any human being. Some minority cultures have no geographic boundaries but they still may adopt the lifestyles of the country they live in. Physicians have to reckon with these different cultural patterns when dealing with patients. Patients must be treated equally at the same time when their personal needs require individual consideration. This consideration is reflected in both verbal and non-verbal communication with the other. Both the sender and the receiver of a message would need to know of the other. Minority groups tend to know more about the majority groups than vice versa. Most health care providers belong to the majority group and would be expected to learn more about the other. Problem-based learning can help students to understand attitudes of minority patients (like the Romanies) and handle the situation. In this instance, the students collected theory base from existing legal, cultural, and other resources and interviewed a Romany woman to verify that the information pertaining to the female case was correct. This combination of theory and experience was considered useful in preparing a case presented to a seminar with 116 medical and dental students in 1994.
Assuntos
Características Culturais , Grupos Minoritários , Educação de Pacientes como Assunto/métodos , Aprendizagem Baseada em Problemas , Roma (Grupo Étnico)/psicologia , Neoplasias da Mama/etnologia , Comunicação , Feminino , Finlândia , Humanos , Pessoa de Meia-IdadeRESUMO
Transgenic (TG) mice, bearing the Simian Virus 40 T-antigen (Tag) under a 6-kb fragment of the murine inhibin alpha-subunit promoter (inhalpha), develop gonadal tumors of granulosa or Leydig cell origin with 100% penetrance by the age of 5-7 months. When these TG mice were gonadectomized prepubertally, between 21-25 days of life, adrenal gland tumors were observed in each mouse by the age of 5-7 months. No adrenal tumors were detected in any intact TG, gonadectomized or intact or control non-TG littermates. The adrenocortical tumors appeared to originate from the X-zone of the adrenal cortex. If functional gonadectomy was induced by GnRH antagonist treatment or by cross-breeding of the TG mice into hypogonadotropic hpg genetic background, neither gonadal nor adrenal tumorigenesis appeared. This prompted a hypothesis that adrenal tumor development in inhalpha/Tag TG mice is related to elevated gonadotropin secretion, which is the most obvious difference between the surgical and functional gonadectomy models. The adrenal tumors and a cell line (Calpha1) derived from them, was found to express luteinizing hormone receptor (LHR), but no FSHR, and hCG treatment stimulated their proliferation. No FSHR was found in the adrenal glands. On the basis of this it was suggested that expression of the potent oncogene T-antigen, allow LH in adrenocortical cells to function as a tumor promoter, and induction of high level functional LHR expression in adrenal tumors. Given the induction of expression and regulation of the GATA-4 and GATA-6 zinc finger family of transcription factors in the gonads by gonadotropins, it was in our interest to explore their expression in the adrenals. We utilized the inalpha/Tag TG mouse model and pathological human adrenal samples to explore the role of GATA-4 and GATA-6 in adrenocortical tumorigenesis. Abundant GATA-6 mRNA expression was found in normal control adrenal cortex during mouse development, whereas GATA-4 mRNA was undetectable. In striking contrast to this, GATA-6 was absent from murine adrenocortical tumors, while GATA-4 mRNA expression was dramatically upregulated in the murine adrenal tumors as well as in human adrenocortical carcinomas. Taken together, these results suggest different roles for GATA-4 and GATA-6 in the adrenal gland, and implicate GATA-4 in adrenal LHR expression and tumorigenesis. Immunohistochemical detection of GATA-4 may serve as a useful marker in differential diagnosis of human adrenal tumors. In addition, the inhalpha/Tag TG model will be helpful for exploring the molecular mechanisms underlying adrenocortical tumorigenesis, ectopic LHR expression in adrenals and the GATA-4/LHR interaction that is related to adrenal tumorigenesis in TG mice.
Assuntos
Neoplasias do Córtex Suprarrenal/genética , Proteínas de Ligação a DNA/genética , Receptores do LH/genética , Fatores de Transcrição/genética , Neoplasias do Córtex Suprarrenal/terapia , Animais , Antígenos Transformantes de Poliomavirus/genética , Modelos Animais de Doenças , Fator de Transcrição GATA4 , Fator de Transcrição GATA6 , Terapia Genética , Inibinas/genética , Camundongos , Camundongos Transgênicos , Regiões Promotoras Genéticas , Proteínas Recombinantes de FusãoRESUMO
The shedding of human rhinovirus (HRV) after an acute, naturally acquired infection has not been described in detail. We determined the duration of HRV shedding in immunocompetent children and adults, and in patients with primary hypogammaglobulinaemia. Subjects with symptoms of respiratory tract infection, and their household contacts, were screened for HRV by reverse transcription PCR. They were followed by serial, self-collected nasal swab specimens until negative for HRV or infected by another HRV type. We followed 62 HRV infections in 54 subjects. The mean (95% CI) duration of HRV shedding was 11.4 (8.2-14.7) days in children, 10.1 (7.4-12.9) days in adults, and 40.9 (26.4-55.4) days in patients with hypogammaglobulinaemia (p <0.001). The duration of respiratory tract symptoms correlated with the duration of virus shedding (p 0.002). A new infection by another HRV type soon after the first episode was common. We conclude that the shedding times of HRV are relatively short in otherwise healthy individuals. In contrast, prolonged shedding over 28 days is frequent in patients with hypogammaglobulinaemia despite immunoglobulin replacement therapy.
Assuntos
Imunodeficiência de Variável Comum/complicações , Infecções por Picornaviridae/virologia , Infecções Respiratórias/virologia , Rhinovirus/isolamento & purificação , Eliminação de Partículas Virais , Adulto , Criança , Pré-Escolar , Imunodeficiência de Variável Comum/virologia , Seguimentos , Humanos , Lactente , Pessoa de Meia-Idade , Mucosa Nasal/virologia , Infecções por Picornaviridae/imunologia , RNA Viral/genética , RNA Viral/isolamento & purificação , Infecções Respiratórias/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de TempoRESUMO
The significance of the interaction between Sertoli cell-produced stem cell factor (SCF) and its receptor, c-kit, on Leydig cells (LCs) during LC development and differentiation is unknown. In the present study, we investigated the potential role of the SCF/c-kit system in LC apoptosis and precursor LC proliferation after ethylene dimethane sulfonate (EDS) treatment in rats. A function-blocking anti-c-kit antibody, ACK-2, was used to block SCF/c-kit interaction at four time points, corresponding to the peak of LC apoptosis and three waves of proliferation of precursor LCs. Blockade of SCF/c-kit interaction by ACK-2 accelerated LC apoptosis and inhibited proliferation of precursor LCs during the first two waves of precursor LC proliferation around days 3-4 and day 10, but not the third wave of precursor LC proliferation around day 20 after EDS treatment. The data suggest that the soluble SCF might act as a survival factor for mature LCs and a growth factor for precursor LCs after EDS-induced LC depletion. This is also supported by a close correlation between the oscillating levels of soluble SCF mRNA and the profiles of LC apoptosis and regeneration. Since regeneration of the LC population after EDS treatment resembles the development of adult-type LCs during prepubertal life, the present findings imply that soluble SCF might participate in regulation of the formation of the LC population during testicular development. Our data also support a model in which delicate and reciprocal regulation exists between soluble SCF production by Sertoli cells, testosterone production by LCs, and pituitary gonadotropins.
Assuntos
Células Intersticiais do Testículo/citologia , Células Intersticiais do Testículo/efeitos dos fármacos , Mesilatos/farmacologia , Proteínas Proto-Oncogênicas c-kit/metabolismo , Fator de Células-Tronco/metabolismo , Células-Tronco/citologia , Animais , Anticorpos/imunologia , Anticorpos/farmacologia , Apoptose/efeitos dos fármacos , Bromodesoxiuridina/metabolismo , Diferenciação Celular , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , DNA/biossíntese , Hormônio Foliculoestimulante/sangue , Imuno-Histoquímica , Hibridização In Situ , Marcação In Situ das Extremidades Cortadas , Células Intersticiais do Testículo/metabolismo , Hormônio Luteinizante/sangue , Masculino , Mitógenos/metabolismo , Proteínas Proto-Oncogênicas c-kit/imunologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Fator de Células-Tronco/genética , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Testosterona/sangue , Fatores de TempoRESUMO
BACKGROUND: Asthma is generally regarded as a disease with strong T(H)2-type cytokine expression, whereas in autoimmune disorders, such as coeliac disease (CD), insulin-dependent diabetes mellitus (IDDM), and rheumatoid arthritis (RA), T(H)1-type expression is seen. According to the cross-regulatory properties of T(H)1 and T(H)2 cells, one would assume that these diseases exist in different patient populations. OBJECTIVE: We sought to test the hypothesis that asthma could exist in children with T(H)1-type diseases, such as CD, IDDM, and RA. METHODS: Comparison was made of the cumulative incidence of asthma in children with CD, IDDM, or RA by linking Finnish Medical Birth Register data on the whole 1987 birth cohort (n = 60,254 births) with the data of several national health registers to obtain information on the incidences of these diseases during the first 7 years of life. RESULTS: The cumulative incidence of asthma in children with CD (24.6%) or RA (10.0%) was significantly higher than in children without CD (3.4%) or RA (3.4%; P < .001 and P = .016, respectively). Asthma tended to be more common in children with IDDM than in children without IDDM. CONCLUSION: These data indicate that the T(H)1 and T(H)2 diseases can coexist, indicating a common environmental denominator behind the disease processes.