RESUMO
Nonsense-mediated RNA decay (NMD) is a highly conserved and selective RNA turnover pathway that depends on the endonuclease SMG6. Here, we show that SMG6 is essential for male germ cell differentiation in mice. Germ-cell conditional knockout (cKO) of Smg6 induces extensive transcriptome misregulation, including a failure to eliminate meiotically expressed transcripts in early haploid cells, and accumulation of NMD target mRNAs with long 3' untranslated regions (UTRs). Loss of SMG6 in the male germline results in complete arrest of spermatogenesis at the early haploid cell stage. We find that SMG6 is strikingly enriched in the chromatoid body (CB), a specialized cytoplasmic granule in male germ cells also harboring PIWI-interacting RNAs (piRNAs) and the piRNA-binding protein PIWIL1. This raises the possibility that SMG6 and the piRNA pathway function together, which is supported by several findings, including that Piwil1-KO mice phenocopy Smg6-cKO mice and that SMG6 and PIWIL1 co-regulate many genes in round spermatids. Together, our results demonstrate that SMG6 is an essential regulator of the male germline transcriptome, and highlight the CB as a molecular platform coordinating RNA regulatory pathways to control sperm production and fertility.
Assuntos
Endorribonucleases , Grânulos de Ribonucleoproteínas de Células Germinativas , Espermatogênese , Transcriptoma , Animais , Masculino , Camundongos , Células Germinativas/metabolismo , RNA Interferente Pequeno/genética , Espermátides/metabolismo , Espermatogênese/genética , Endorribonucleases/metabolismoRESUMO
Presynaptic increase in striatal dopamine is the primary dopaminergic abnormality in schizophrenia, but the underlying mechanisms are not understood. Here, we hypothesized that increased expression of endogenous GDNF could induce dopaminergic abnormalities that resemble those seen in schizophrenia. To test the impact of GDNF elevation, without inducing adverse effects caused by ectopic overexpression, we developed a novel in vivo approach to conditionally increase endogenous GDNF expression. We found that a 2-3-fold increase in endogenous GDNF in the brain was sufficient to induce molecular, cellular, and functional changes in dopamine signalling in the striatum and prefrontal cortex, including increased striatal presynaptic dopamine levels and reduction of dopamine in prefrontal cortex. Mechanistically, we identified adenosine A2a receptor (A2AR), a G-protein coupled receptor that modulates dopaminergic signalling, as a possible mediator of GDNF-driven dopaminergic abnormalities. We further showed that pharmacological inhibition of A2AR with istradefylline partially normalised striatal GDNF and striatal and cortical dopamine levels in mice. Lastly, we found that GDNF levels are increased in the cerebrospinal fluid of first episode psychosis patients, and in post-mortem striatum of schizophrenia patients. Our results reveal a possible contributor for increased striatal dopamine signalling in a subgroup of schizophrenia patients and suggest that GDNF-A2AR crosstalk may regulate dopamine function in a therapeutically targetable manner.
Assuntos
Dopamina , Esquizofrenia , Animais , Camundongos , Dopamina/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/farmacologia , Esquizofrenia/metabolismo , Corpo Estriado/metabolismo , Transdução de SinaisRESUMO
Hydroxysteroid 17ß dehydrogenase 12 (HSD17B12) is suggested to be involved in the elongation of very long chain fatty acids. Previously, we have shown a pivotal role for the enzyme during mouse development. In the present study we generated a conditional Hsd17b12 knockout (HSD17B12cKO) mouse model by breeding mice homozygous for a floxed Hsd17b12 allele with mice expressing the tamoxifen-inducible Cre recombinase at the ROSA26 locus. Gene inactivation was induced by administering tamoxifen to adult mice. The gene inactivation led to a 20% loss of body weight within 6 days, associated with drastic reduction in both white (83% males, 75% females) and brown (65% males, 60% females) fat, likely due to markedly reduced food and water intake. Furthermore, the knockout mice showed sickness behavior and signs of liver toxicity, specifically microvesicular hepatic steatosis and increased serum alanine aminotransferase (4.6-fold in males, 7.7-fold in females). The hepatic changes were more pronounced in females than males. Proinflammatory cytokines, such as interleukin-6 (IL-6), IL-17, and granulocyte colony-stimulating factor, were increased in the HSD17B12cKO mice indicating an inflammatory response. Serum lipidomics study showed an increase in the amount of dihydroceramides, despite the dramatic overall loss of lipids. In line with the proposed role for HSD17B12 in fatty acid elongation, we observed accumulation of ceramides, dihydroceramides, hexosylceramides, and lactosylceramides with shorter than 18-carbon fatty acid side chains in the serum. The results indicate that HSD17B12 is essential for proper lipid homeostasis and HSD17B12 deficiency rapidly results in fatal systemic inflammation and lipolysis in adult mice.
Assuntos
17-Hidroxiesteroide Desidrogenases/fisiologia , Homeostase/fisiologia , 17-Hidroxiesteroide Desidrogenases/genética , Tecido Adiposo Marrom/efeitos dos fármacos , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/efeitos dos fármacos , Tecido Adiposo Branco/metabolismo , Animais , Comportamento Animal , Peso Corporal/genética , Citocinas/metabolismo , Ácidos Graxos/metabolismo , Comportamento Alimentar , Feminino , Homeostase/genética , Metabolismo dos Lipídeos/genética , Metabolismo dos Lipídeos/fisiologia , Lipidômica , Hepatopatias/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Caracteres Sexuais , Tamoxifeno/farmacologiaRESUMO
Sperm differentiation requires specific protein transport for correct sperm tail formation and head shaping. A transient microtubular structure, the manchette, appears around the differentiating spermatid head and serves as a platform for protein transport to the growing tail. Sperm flagellar 2 (SPEF2) is known to be essential for sperm tail development. In this study we investigated the function of SPEF2 during spermatogenesis using a male germ cell-specific Spef2 knockout mouse model. In addition to defects in sperm tail development, we observed a duplication of the basal body and failure in manchette migration resulting in an abnormal head shape. We identified cytoplasmic dynein 1 and GOLGA3 as novel interaction partners for SPEF2. SPEF2 and dynein 1 colocalize in the manchette and the inhibition of dynein 1 disrupts the localization of SPEF2 to the manchette. Furthermore, the transport of a known SPEF2-binding protein, IFT20, from the Golgi complex to the manchette was delayed in the absence of SPEF2. These data indicate a possible novel role of SPEF2 as a linker protein for dynein 1-mediated cargo transport along microtubules.
Assuntos
Proteínas/fisiologia , Espermátides/crescimento & desenvolvimento , Espermátides/fisiologia , Espermatogênese/fisiologia , Animais , Proteínas de Transporte/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Dineínas do Citoplasma/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microtúbulos/fisiologia , Transporte Proteico/genética , Transporte Proteico/fisiologia , Proteínas/genética , Cauda do Espermatozoide/fisiologia , Cauda do Espermatozoide/ultraestrutura , Espermátides/citologia , Espermatogênese/genéticaRESUMO
Accuracy of protein synthesis is enabled by the selection of amino acids for tRNA charging by aminoacyl-tRNA synthetases (ARSs), and further enhanced by the proofreading functions of some of these enzymes for eliminating tRNAs mischarged with noncognate amino acids. Mouse models of editing-defective cytoplasmic alanyl-tRNA synthetase (AlaRS) have previously demonstrated the importance of proofreading for cytoplasmic protein synthesis, with embryonic lethal and progressive neurodegeneration phenotypes. Mammalian mitochondria import their own set of nuclear-encoded ARSs for translating critical polypeptides of the oxidative phosphorylation system, but the importance of editing by the mitochondrial ARSs for mitochondrial proteostasis has not been known. We demonstrate here that the human mitochondrial AlaRS is capable of editing mischarged tRNAs in vitro, and that loss of the proofreading activity causes embryonic lethality in mice. These results indicate that tRNA proofreading is essential in mammalian mitochondria, and cannot be overcome by other quality control mechanisms.
Assuntos
Alanina-tRNA Ligase/genética , Mitocôndrias/genética , Edição de RNA , RNA de Transferência/genética , Aminoacilação de RNA de Transferência/genética , Alanina-tRNA Ligase/metabolismo , Sequência de Aminoácidos , Animais , Células Cultivadas , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Mamíferos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Mutação , Biossíntese de Proteínas/genética , RNA de Transferência/metabolismo , Homologia de Sequência de AminoácidosRESUMO
The pituitary gonadotrophins and testosterone are the main hormonal regulators of spermatogenesis, but estradiol is also known to play a role in the process. The hormonal responses in the testis are partially mediated by somatic Sertoli cells that provide nutritional and physical support for differentiating male germ cells. Hydroxysteroid (17ß) dehydrogenase 1 (HSD17B1) is a steroidogenic enzyme that especially catalyzes the conversion of low potent 17keto-steroids to highly potent 17ß-hydroxysteroids. In this study, we show that Hsd17b1 is highly expressed in Sertoli cells of fetal and newborn mice, and HSD17B1 knockout males present with disrupted spermatogenesis with major defects, particularly in the head shape of elongating spermatids. The cell-cell junctions between Sertoli cells and germ cells were disrupted in the HSD17B1 knockout mice. This resulted in complications in the orientation of elongating spermatids in the seminiferous epithelium, reduced sperm production, and morphologically abnormal spermatozoa. We also showed that the Sertoli cell-expressed HSD17B1 participates in testicular steroid synthesis, evidenced by a compensatory up-regulation of HSD17B3 in Leydig cells. These results revealed a novel role for HSD17B1 in the control of spermatogenesis and male fertility, and that Sertoli cells significantly contribute to steroid synthesis in the testis.-Hakkarainen, J., Zhang, F.-P., Jokela, H., Mayerhofer, A., Behr, R., Cisneros-Montalvo, S., Nurmio, M., Toppari, J., Ohlsson, C., Kotaja, N., Sipilä, P., Poutanen, M. Hydroxysteroid (17ß) dehydrogenase 1 expressed by Sertoli cells contributes to steroid synthesis and is required for male fertility.
Assuntos
17-Hidroxiesteroide Desidrogenases/biossíntese , Fertilidade/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Células de Sertoli/enzimologia , Espermatogênese/fisiologia , Esteroides/biossíntese , 17-Hidroxiesteroide Desidrogenases/genética , Animais , Masculino , Camundongos , Camundongos Knockout , Epitélio Seminífero/citologia , Epitélio Seminífero/enzimologia , Células de Sertoli/citologia , Espermátides/citologia , Espermátides/enzimologiaRESUMO
Hydroxysteroid (17ß) dehydrogenases (HSD17Bs) form an enzyme family characterized by their ability to catalyze reactions in steroid and lipid metabolism. In the present study, we characterized the phenotype of HSD17B13-knockout (HSD17B13KO) mice deficient in Hsd17b13. In these studies, hepatic steatosis was detected in HSD17B13KO male mice, indicated by histologic analysis and by the increased triglyceride concentration in the liver, whereas reproductive performance and serum steroid concentrations were normal in HSD17B13KO mice. In line with these changes, the expression of key proteins in fatty acid synthesis, such as FAS, acetyl-CoA carboxylase 1, and SCD1, was increased in the HSD17B13KO liver. Furthermore, the knockout liver showed an increase in 2 acylcarnitines, suggesting impaired mitochondrial ß-oxidation in the presence of unaltered malonyl CoA and AMPK expression. The glucose tolerance did not differ between wild-type and HSD17B13KO mice in the presence of lower levels of glucose 6-phosphatase in HSD17B13KO liver compared with wild-type liver. Furthermore, microgranulomas and increased portal inflammation together with up-regulation of immune response genes were observed in HSD17B13KO mice. Our data indicate that disruption of Hsd17b13 impairs hepatic-lipid metabolism in mice, resulting in liver steatosis and inflammation, but the enzyme does not play a major role in the regulation of reproductive functions.-Adam, M., Heikelä, H., Sobolewski, C., Portius, D., Mäki-Jouppila, J., Mehmood, A., Adhikari, P., Esposito, I., Elo, L. L., Zhang, F.-P., Ruohonen, S. T., Strauss, L., Foti, M., Poutanen, M. Hydroxysteroid (17ß) dehydrogenase 13 deficiency triggers hepatic steatosis and inflammation in mice.
Assuntos
17-Hidroxiesteroide Desidrogenases/deficiência , Fígado Gorduroso/enzimologia , Metabolismo dos Lipídeos , Acetil-CoA Carboxilase/genética , Acetil-CoA Carboxilase/metabolismo , Animais , Ácido Graxo Sintase Tipo I/genética , Ácido Graxo Sintase Tipo I/metabolismo , Fígado Gorduroso/genética , Fígado Gorduroso/patologia , Glucose-6-Fosfatase/genética , Glucose-6-Fosfatase/metabolismo , Inflamação/enzimologia , Inflamação/genética , Inflamação/patologia , Camundongos , Camundongos Knockout , Mitocôndrias Hepáticas/enzimologia , Mitocôndrias Hepáticas/genética , Mitocôndrias Hepáticas/patologia , Oxirredução , Estearoil-CoA Dessaturase/genética , Estearoil-CoA Dessaturase/metabolismoRESUMO
miRNAs are important regulators of gene expression and are often deregulated in cancer. We have previously shown that miR-32 is an androgen receptor-regulated miRNA overexpressed in castration-resistant prostate cancer and that miR-32 can improve prostate cancer cell growth in vitro. To assess the effects of miR-32 in vivo, we developed transgenic mice overexpressing miR-32 in the prostate. The study indicated that transgenic miR-32 expression increases replicative activity in the prostate epithelium. We further observed an aging-associated increase in the incidence of goblet cell metaplasia in the prostate epithelium. Furthermore, aged miR-32 transgenic mice exhibited metaplasia-associated prostatic intraepithelial neoplasia at a low frequency. When crossbred with mice lacking the other allele of tumor-suppressor Pten (miR-32xPten+/- mice), miR-32 expression increased both the incidence and the replicative activity of prostatic intraepithelial neoplasia lesions in the dorsal prostate. The miR-32xPten+/- mice also demonstrated increased goblet cell metaplasia compared with Pten+/- mice. By performing a microarray analysis of mouse prostate tissue to screen downstream targets and effectors of miR-32, we identified RAC2 as a potential, and clinically relevant, target of miR-32. We also demonstrate down-regulation of several interesting, potentially prostate cancer-relevant genes (Spink1, Spink5, and Casp1) by miR-32 in the prostate tissue. The results demonstrate that miR-32 increases proliferation and promotes metaplastic transformation in mouse prostate epithelium, which may promote neoplastic alterations in the prostate.
Assuntos
Regulação Neoplásica da Expressão Gênica/genética , MicroRNAs/genética , Próstata/patologia , Neoplasias da Próstata/genética , Animais , Proliferação de Células/genética , Transformação Celular Neoplásica/patologia , Epitélio/patologia , Masculino , Camundongos , Neoplasias da Próstata/patologia , Receptores Androgênicos/metabolismoRESUMO
Wingless-type MMTV integration site family (WNT)16 is a key regulator of bone mass with high expression in cortical bone, and Wnt16(-/-) mice have reduced cortical bone mass. As Wnt16 expression is enhanced by estradiol treatment, we hypothesized that the bone-sparing effect of estrogen in females is WNT16-dependent. This hypothesis was tested in mechanistic studies using two genetically modified mouse models with either constantly high osteoblastic Wnt16 expression or no Wnt16 expression. We developed a mouse model with osteoblast-specific Wnt16 overexpression (Obl-Wnt16). These mice had several-fold elevated Wnt16 expression in both trabecular and cortical bone compared with wild type (WT) mice. Obl-Wnt16 mice displayed increased total body bone mineral density (BMD), surprisingly caused mainly by a substantial increase in trabecular bone mass, resulting in improved bone strength of vertebrae L3. Ovariectomy (ovx) reduced the total body BMD and the trabecular bone mass to the same degree in Obl-Wnt16 mice and WT mice, suggesting that the bone-sparing effect of estrogen is WNT16-independent. However, these bone parameters were similar in ovx Obl-Wnt16 mice and sham operated WT mice. The role of WNT16 for the bone-sparing effect of estrogen was also evaluated in Wnt16(-/-) mice. Treatment with estradiol increased the trabecular and cortical bone mass to a similar extent in both Wnt16(-/-) and WT mice. In conclusion, the bone-sparing effects of estrogen and WNT16 are independent of each other. Furthermore, loss of endogenous WNT16 results specifically in cortical bone loss, whereas overexpression of WNT16 surprisingly increases mainly trabecular bone mass. WNT16-targeted therapies might be useful for treatment of postmenopausal trabecular bone loss.
Assuntos
Densidade Óssea/fisiologia , Osteoblastos/metabolismo , Coluna Vertebral/metabolismo , Proteínas Wnt/biossíntese , Animais , Estrogênios , Feminino , Camundongos , Camundongos Knockout , Osteoblastos/citologia , Proteínas Wnt/genéticaRESUMO
Females are, in general, more insulin sensitive than males. To investigate whether this is a direct effect of sex-steroids (SS) in white adipose tissue (WAT), we developed a male mouse model overexpressing the aromatase enzyme, converting testosterone (T) to estradiol (E2), specifically in WAT (Ap2-arom mice). Adipose tissue E2 levels were increased while circulating SS levels were unaffected in male Ap2-arom mice. Importantly, male Ap2-arom mice were more insulin sensitive compared with WT mice and exhibited increased serum adiponectin levels and upregulated expression of Glut4 and Irs1 in WAT. The expression of markers of macrophages and immune cell infiltration was markedly decreased in WAT of male Ap2-arom mice. The adipogenesis was enhanced in male Ap2-arom mice, supported by elevated Pparg expression in WAT and enhanced differentiation of preadipocyte into mature adipocytes. In summary, increased adipose tissue aromatase activity reduces adipose tissue inflammation and improves insulin sensitivity in male mice. We propose that estrogen increases insulin sensitivity via a local effect in WAT on adiponectin expression, adipose tissue inflammation, and adipogenesis.
Assuntos
Tecido Adiposo Branco/metabolismo , Aromatase/genética , Estradiol/metabolismo , Resistência à Insulina/genética , Testosterona/metabolismo , Adipócitos , Adipogenia/genética , Adiponectina/metabolismo , Tecido Adiposo Branco/imunologia , Animais , Técnicas de Introdução de Genes , Transportador de Glucose Tipo 4/metabolismo , Inflamação , Proteínas Substratos do Receptor de Insulina/metabolismo , Macrófagos/imunologia , Masculino , Camundongos , PPAR gama/metabolismo , Regulação para CimaRESUMO
OBJECTIVE: miR-21 is an oncomir highly upregulated in hepatocellular carcinoma and in early stages of liver diseases characterised by the presence of steatosis. Whether upregulation of miR-21 contributes to hepatic metabolic disorders and their progression towards cancer is unknown. This study aims at investigating the role of miR-21/miR-21* in early stages of metabolic liver disorders associated with diet-induced obesity (DIO). DESIGN: Constitutive miR-21/miR-21* knockout (miR21KO) and liver-specific miR-21/miR-21* knockout (LImiR21KO) mice were generated. Mice were then fed with high-fat diet (HFD) and alterations of the lipid and glucose metabolism were investigated. Serum and ex vivo explanted liver tissue were analysed. RESULTS: Under normal breeding conditions and standard diet, miR-21/miR-21* deletion in mice was not associated with any detectable phenotypic alterations. However, when mice were challenged with an obesogenic diet, glucose intolerance, steatosis and adiposity were improved in mice lacking miR-21/miR-21*. Deletion of miR-21/miR-21* specifically in hepatocytes led to similar improvements in mice fed an HFD, indicating a crucial role for hepatic miR-21/miR-21* in metabolic disorders associated with DIO. Further molecular analyses demonstrated that miR-21/miR-21* deletion in hepatocytes increases insulin sensitivity and modulates the expression of multiple key metabolic transcription factors involved in fatty acid uptake, de novo lipogenesis, gluconeogenesis and glucose output. CONCLUSIONS: Hepatic miR-21/miR-21* deficiency prevents glucose intolerance and steatosis in mice fed an obesogenic diet by altering the expression of several master metabolic regulators. This study points out miR-21/miR-21* as a potential therapeutic target for non-alcoholic fatty liver disease and the metabolic syndrome.
Assuntos
Dieta Hiperlipídica , Fígado Gorduroso , Transtornos do Metabolismo de Glucose/metabolismo , Glucose/metabolismo , Hepatócitos , MicroRNAs/metabolismo , Obesidade/metabolismo , Animais , Gorduras na Dieta/efeitos adversos , Modelos Animais de Doenças , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Intolerância à Glucose/metabolismo , Hepatócitos/metabolismo , Hepatócitos/patologia , Camundongos , Camundongos KnockoutRESUMO
Hydroxysteroid (17ß)-dehydrogenase type 1 (HSD17B1) catalyzes the conversion of low active 17-ketosteroids, androstenedione (A-dione) and estrone (E1) to highly active 17-hydroxysteroids, testosterone (T) and E2, respectively. In this study, the importance of HSD17B1 in ovarian estrogen production was determined using Hsd17b1 knockout (HSD17B1KO) mice. In these mice, the ovarian HSD17B enzyme activity was markedly reduced, indicating a central role of HSD17B1 in ovarian physiology. The lack of Hsd17b activity resulted in increased ovarian E1:E2 and A-dione:T ratios, but we also observed reduced progesterone concentration in HSD17B1KO ovaries. Accordingly with the altered steroid production, altered expression of Star, Cyp11a1, Lhcgr, Hsd17b7, and especially Cyp17a1 was observed. The ovaries of HSD17B1KO mice presented with all stages of folliculogenesis, while the corpus luteum structure was less defined and number reduced. Surprisingly, bundles of large granular cells of unknown origin appeared in the stroma of the KO ovaries. The HSD17B1KO mice presented with severe subfertility and failed to initiate pseudopregnancy. However, the HSD17B1KO females presented with normal estrous cycle defined by vaginal smears and normal puberty appearance. This study indicates that HSD17B1 is a key enzyme in ovarian steroidogenesis and has a novel function in initiation and stabilization of pregnancy.
Assuntos
17-Hidroxiesteroide Desidrogenases/deficiência , Ciclo Estral , Infertilidade Feminina/enzimologia , Luteinização , Ovário/metabolismo , Progesterona/biossíntese , 17-Hidroxiesteroide Desidrogenases/biossíntese , 17-Hidroxiesteroide Desidrogenases/genética , 17-Hidroxiesteroide Desidrogenases/metabolismo , Animais , Enzima de Clivagem da Cadeia Lateral do Colesterol/biossíntese , Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , Feminino , Infertilidade Feminina/genética , Masculino , Camundongos , Camundongos Knockout , Ovário/patologia , Fosfoproteínas/biossíntese , Fosfoproteínas/genética , Gravidez , Progesterona/genética , Maturidade Sexual/genética , Esteroide 17-alfa-Hidroxilase/biossíntese , Esteroide 17-alfa-Hidroxilase/genéticaRESUMO
Embigin (Gp70), a receptor for fibronectin and an ancillary protein for monocarboxylate transporters, is known to regulate stem cell niches in sebaceous gland and bone marrow. Here, we show that embigin expression is at high level during early mouse embryogenesis and that embigin is essential for lung development. Markedly increased neonatal mortality of Emb-/- mice can be explained by the compromised lung maturation: in Emb-/- mice (E17.5) the number and the size of the small airways and distal airspace are significantly smaller, there are fewer ATI and ATII cells, and the alkaline phosphatase activity in amniotic fluid is lower. Emb-/- lungs show less peripheral branching already at E12.5, and embigin is highly expressed in lung primordium. Thus, embigin function is essential at early pseudoglandular stage or even earlier. Furthermore, our RNA-seq analysis and Ki67 staining results support the idea that the development of Emb-/- lungs is rather delayed than defected.
RESUMO
Hydroxysteroid (17ß) dehydrogenase (HSD17B) enzymes convert 17-ketosteroids to 17beta-hydroxysteroids, an essential step in testosterone biosynthesis. Human XY individuals with inactivating HSD17B3 mutations are born with female-appearing external genitalia due to testosterone deficiency. However, at puberty their testosterone production reactivates, indicating HSD17B3-independent testosterone synthesis. We have recently shown that Hsd17b3 knockout (3-KO) male mice display a similar endocrine imbalance, with high serum androstenedione and testosterone in adulthood, but milder undermasculinization than humans. Here, we studied whether HSD17B1 is responsible for the remaining HSD17B activity in the 3-KO male mice by generating a Ser134Ala point mutation that disrupted the enzymatic activity of HSD17B1 (1-KO) followed by breeding Hsd17b1/Hsd17b3 double-KO (DKO) mice. In contrast to 3-KO, inactivation of both HSD17B3 and HSD17B1 in mice results in a dramatic drop in testosterone synthesis during the fetal period. This resulted in a female-like anogenital distance at birth, and adult DKO males displayed more severe undermasculinization than 3-KO, including more strongly reduced weight of seminal vesicles, levator ani, epididymis, and testis. However, qualitatively normal spermatogenesis was detected in adult DKO males. Furthermore, similar to 3-KO mice, high serum testosterone was still detected in adult DKO mice, accompanied by upregulation of various steroidogenic enzymes. The data show that HSD17B1 compensates for HSD17B3 deficiency in fetal mouse testis but is not the enzyme responsible for testosterone synthesis in adult mice with inactivated HSD17B3. Therefore, other enzymes are able to convert androstenedione to testosterone in the adult mouse testis and presumably also in the human testis.
Assuntos
17-Hidroxiesteroide Desidrogenases , Camundongos Knockout , Testículo , Testosterona , Animais , Masculino , Testículo/metabolismo , Testículo/embriologia , Camundongos , 17-Hidroxiesteroide Desidrogenases/metabolismo , 17-Hidroxiesteroide Desidrogenases/genética , 17-Hidroxiesteroide Desidrogenases/deficiência , Feminino , Testosterona/sangue , Testosterona/metabolismo , Feto/metabolismo , Estradiol Desidrogenases/metabolismo , Estradiol Desidrogenases/genéticaRESUMO
BACKGROUND: Kiss1 neurons in the hypothalamic arcuate-nucleus (ARC) play key roles in the control of GnRH pulsatility and fertility. A fraction of ARC Kiss1 neurons, termed KNDy, co-express neurokinin B (NKB; encoded by Tac2). Yet, NKB- and Kiss1-only neurons are also found in the ARC, while a second major Kiss1-neuronal population is present in the rostral hypothalamus. The specific contribution of different Kiss1 neuron sub-sets and kisspeptins originating from them to the control of reproduction and eventually other bodily functions remains to be fully determined. METHODS: To tease apart the physiological roles of KNDy-born kisspeptins, conditional ablation of Kiss1 in Tac2-expressing cells was implemented in vivo. To this end, mice with Tac2 cell-specific Kiss1 KO (TaKKO) were generated and subjected to extensive reproductive and metabolic characterization. RESULTS: TaKKO mice displayed reduced ARC kisspeptin content and Kiss1 expression, with greater suppression in females, which was detectable at infantile-pubertal age. In contrast, Tac2/NKB levels were fully preserved. Despite the drop of ARC Kiss1/kisspeptin, pubertal timing was normal in TaKKO mice of both sexes. However, young-adult TaKKO females displayed disturbed LH pulsatility and sex steroid levels, with suppressed basal LH and pre-ovulatory LH surges, early-onset subfertility and premature ovarian insufficiency. Conversely, testicular histology and fertility were grossly conserved in TaKKO males. Ablation of Kiss1 in Tac2-cells led also to sex-dependent alterations in body composition, glucose homeostasis, especially in males, and locomotor activity, specifically in females. CONCLUSIONS: Our data document that KNDy-born kisspeptins are dispensable/compensable for puberty in both sexes, but required for maintenance of female gonadotropin pulsatility and fertility, as well as for adult metabolic homeostasis. SIGNIFICANCE STATEMENT: Neurons in the hypothalamic arcuate nucleus (ARC) co-expressing kisspeptins and NKB, named KNDy, have been recently suggested to play a key role in pulsatile secretion of gonadotropins, and hence reproduction. However, the relative contribution of this Kiss1 neuronal-subset, vs. ARC Kiss1-only and NKB-only neurons, as well as other Kiss1 neuronal populations, has not been assessed in physiological settings. We report here findings in a novel mouse-model with elimination of KNDy-born kisspeptins, without altering other kisspeptin compartments. Our data highlights the heterogeneity of ARC Kiss1 populations and document that, while dispensable/compensable for puberty, KNDy-born kisspeptins are required for proper gonadotropin pulsatility and fertility, specifically in females, and adult metabolic homeostasis. Characterization of this functional diversity is especially relevant, considering the potential of kisspeptin-based therapies for management of human reproductive disorders.
Assuntos
Gonadotropinas , Kisspeptinas , Masculino , Feminino , Camundongos , Humanos , Animais , Kisspeptinas/genética , Neurônios/metabolismo , Puberdade , Hormônio Liberador de Gonadotropina/metabolismo , Núcleo Arqueado do Hipotálamo/metabolismo , FertilidadeRESUMO
Background: The human adrenal cortex undergoes several rapid remodeling steps during its lifetime. In rodents, similar remodeling occurs postnatally in the "X-zone" layer through unknown mechanisms. Furthermore, little is known regarding the impact of thyroid hormone (TH) on adrenal glands in humans. Methods: To investigate the impact of TH on adrenal pathophysiology, we created two genetic murine models mimicking human nonautoimmune hypothyroidism and hyperthyroidism. Moreover, we analyzed serum thyrotropin (TSH) and steroid hormone concentrations in patients diagnosed with congenital hypothyroidism and premature adrenarche (PA). Results: We found that TH receptor beta-mediated hypertrophy of the X-zone significantly elevated the adrenal weights of hyperthyroid women. In the hypothyroid model, the X-zone was poorly developed in both sexes. Moreover, large reciprocal changes in the expression levels of genes that regulate adrenal cortical function were observed with both models. Unexpectedly, up- and downregulation of several genes involved in catecholamine synthesis were detected in the adrenal glands of the hypothyroid and hyperthyroid models, respectively. Furthermore, TSH and adrenal steroid concentrations correlated positively in pediatric patients with congenital hypothyroidism and PA. Conclusions: Our results revealed that congenital hypothyroidism and hyperthyroidism functionally affect adrenal gland development and related steroidogenic activity, as well as the adrenal medulla.
Assuntos
Hipotireoidismo Congênito , Hipertireoidismo , Animais , Criança , Hipotireoidismo Congênito/genética , Feminino , Expressão Gênica , Humanos , Masculino , Camundongos , Hormônios Tireóideos , TireotropinaRESUMO
During the last two decades a large number of genetically modified mouse lines with altered gonadotropin action have been generated. These mouse lines fall into three categories: the lack-of-function mice, gain-of-function mice, and the mice generated by breeding the abovementioned lines with other disease model lines. The mouse strains lacking gonadotropin action have elucidated the necessity of the pituitary hormones in pubertal development and function of gonads, and revealed the processes from the original genetic defect to the pathological phenotype such as hypo- or hypergonadotropic hypogonadism. Conversely, the strains of the second group depict consequences of chronic gonadotropin action. The lines vary from those expressing constitutively active receptors and those secreting follicle-stimulating hormone (FSH) with slowly increasing amounts to those producing human choriogonadotropin (hCG), amount of which corresponds to 2000-fold luteinizing hormone (LH)/hCG biological activity. Accordingly, the phenotypes diverge from mild anomalies and enhanced fertility to disrupted gametogenesis, but eventually chronic, enhanced and non-pulsatile action of both FSH and LH leads to female and male infertility and/or hyper- and neoplasias in most of the gonadotropin gain-of-function mice. Elevated gonadotropin levels also alter the function of several extra-gonadal tissues either directly or indirectly via increased sex steroid production. These effects include promotion of tumorigenesis in tissues such as the pituitary, mammary and adrenal glands. Finally, the crossbreedings of the current mouse strains with other disease models are likely to uncover the contribution of gonadotropins in novel biological systems, as exemplified by the recent crossbreed of LHCG receptor deficient mice with Alzheimer disease mice.
Assuntos
Modelos Animais de Doenças , Doenças do Sistema Endócrino/genética , Gonadotropinas/genética , Gonadotropinas/fisiologia , Camundongos Transgênicos , Animais , Doenças do Sistema Endócrino/metabolismo , Doenças do Sistema Endócrino/patologia , Doenças do Sistema Endócrino/fisiopatologia , Feminino , Gonadotropinas/deficiência , Gonadotropinas/metabolismo , Humanos , Masculino , Camundongos , Ovário/metabolismo , Ovário/patologia , Ovário/fisiologia , Hipófise/metabolismo , Hipófise/patologia , Hipófise/fisiologia , Testículo/metabolismo , Testículo/patologia , Testículo/fisiologiaRESUMO
miR-22 is one of the most abundant miRNAs in the liver and alterations of its hepatic expression have been associated with the development of hepatic steatosis and insulin resistance, as well as cancer. However, the pathophysiological roles of miR-22-3p in the deregulated hepatic metabolism with obesity and cancer remains poorly characterized. Herein, we observed that alterations of hepatic miR-22-3p expression with non-alcoholic fatty liver disease (NAFLD) in the context of obesity are not consistent in various human cohorts and animal models in contrast to the well-characterized miR-22-3p downregulation observed in hepatic cancers. To unravel the role of miR-22 in obesity-associated NAFLD, we generated constitutive Mir22 knockout (miR-22KO) mice, which were subsequently rendered obese by feeding with fat-enriched diet. Functional NAFLD- and obesity-associated metabolic parameters were then analyzed. Insights about the role of miR-22 in NAFLD associated with obesity were further obtained through an unbiased proteomic analysis of miR-22KO livers from obese mice. Metabolic processes governed by miR-22 were finally investigated in hepatic transformed cancer cells. Deletion of Mir22 was asymptomatic when mice were bred under standard conditions, except for an onset of glucose intolerance. However, when challenged with a high fat-containing diet, Mir22 deficiency dramatically exacerbated fat mass gain, hepatomegaly, and liver steatosis in mice. Analyses of explanted white adipose tissue revealed increased lipid synthesis, whereas mass spectrometry analysis of the liver proteome indicated that Mir22 deletion promotes hepatic upregulation of key enzymes in glycolysis and lipid uptake. Surprisingly, expression of miR-22-3p in Huh7 hepatic cancer cells triggers, in contrast to our in vivo observations, a clear induction of a Warburg effect with an increased glycolysis and an inhibited mitochondrial respiration. Together, our study indicates that miR-22-3p is a master regulator of the lipid and glucose metabolism with differential effects in specific organs and in transformed hepatic cancer cells, as compared to non-tumoral tissue.
RESUMO
Human genetic evidence demonstrates that WNT1 mutations cause osteogenesis imperfecta (OI) and early-onset osteoporosis, implicating WNT1 as a major regulator of bone metabolism. However, its main cellular source and mechanisms of action in bone remain elusive. We generated global and limb bud mesenchymal cell-targeted deletion of Wnt1 in mice. Heterozygous deletion of Wnt1 resulted in mild trabecular osteopenia due to decreased osteoblast function. Targeted deletion of Wnt1 in mesenchymal progenitors led to spontaneous fractures due to impaired osteoblast function and increased bone resorption, mimicking the severe OI phenotype in humans with homozygous WNT1 mutations. Importantly, we showed for the first time that Wnt1 signals strictly in a juxtacrine manner to induce osteoblast differentiation and to suppress osteoclastogenesis, in part via canonical Wnt signaling. In conclusion, mesenchymal cell-derived Wnt1, acting in short range, is an essential regulator of bone homeostasis and an intriguing target for therapeutic interventions for bone diseases. © 2019 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals, Inc.
Assuntos
Diferenciação Celular , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/citologia , Osteoclastos/citologia , Via de Sinalização Wnt , Proteína Wnt1/metabolismo , Animais , Doenças Ósseas Metabólicas/patologia , Linhagem Celular , Núcleo Celular/metabolismo , Fraturas Ósseas/patologia , Deleção de Genes , Heterozigoto , Camundongos Knockout , Osteoblastos/metabolismo , Osteoclastos/metabolismo , OsteogêneseRESUMO
The crucial effects of androgens on the male skeleton are at least partly mediated via the androgen receptor (AR). In addition to hormone binding, the AR activity is regulated by post-translational modifications, including SUMOylation. SUMOylation is a reversible modification in which Small Ubiquitin-related MOdifier proteins (SUMOs) are attached to the AR and thereby regulate the activity of the AR and change its interactions with other proteins. To elucidate the importance of SUMOylation of AR for male bone metabolism, we used a mouse model devoid of the two AR SUMOylation sites (ARSUM-; K381R and K500R are substituted). Six-month-old male ARSUM- mice displayed significantly reduced trabecular bone volume fraction in the distal metaphyseal region of femur compared with wild type (WT) mice (BV/TV, -19.1⯱â¯4.9%, Pâ¯<â¯0.05). The number of osteoblasts per bone perimeter was substantially reduced (-60.5⯱â¯7.2%, Pâ¯<â¯0.001) while no significant effect was observed on the number of osteoclasts in the trabecular bone of male ARSUM- mice. Dynamic histomorphometric analysis of trabecular bone revealed a reduced bone formation rate (-32.6⯱â¯7.4%, Pâ¯<â¯0.05) as a result of reduced mineralizing surface per bone surface in ARSUM- mice compared with WT mice (-24.3⯱â¯3.6%, Pâ¯<â¯0.001). Furthermore, cortical bone thickness in the diaphyseal region of femur was reduced in male ARSUM- mice compared with WT mice (-7.3⯱â¯2.0%, Pâ¯<â¯0.05). In conclusion, mice devoid of AR SUMOylation have reduced trabecular bone mass as a result of reduced bone formation. We propose that therapies enhancing AR SUMOylation might result in bone-specific anabolic effects with minimal adverse effects in other tissues.