RESUMEN
Spermatogenesis is a highly coordinated process. Signaling from nuclear hormone receptors, like those for retinoic acid (RA), is important for normal spermatogenesis. However, the mechanisms regulating these signals are poorly understood. Mediator complex subunit 1 (MED1) is a transcriptional enhancer that directly modulates transcription from nuclear hormone receptors. MED1 is present in male germ cells throughout mammalian development, but its function during spermatogenesis is unknown. To determine its role, we generated mice lacking Med1 specifically in their germ cells beginning just before birth. Conditional Med1 knockout males are fertile, exhibiting normal testis weights and siring ordinary numbers of offspring. RA-responsive gene products stimulated by RA gene 8 (Stra8) and synaptonemal complex protein 3 (Sycp3) are first detected in knockout spermatogonia at the expected time points during the first wave of spermatogenesis, and persist with normal patterns of cellular distribution in adult knockout testes. Meiotic progression, however, is altered in the absence of Med1. At postnatal day 7 (P7), zygotene-stage knockout spermatocytes are already detected, unlike in control testes, with fewer pre-leptotene-stage cells and more leptotene spermatocytes observed in the knockouts. At P9, Med1 knockout spermatocytes prematurely enter pachynema. Once formed, greater numbers of knockout spermatocytes remain in pachynema relative to the other stages of meiosis throughout testis development and its maintenance in the adult. Meiotic exit is not inhibited. We conclude that MED1 regulates the temporal progression of primary spermatocytes through meiosis, with its absence resulting in abbreviated pre-leptotene, leptotene, and zygotene stages, and a prolonged pachytene stage.
Asunto(s)
Subunidad 1 del Complejo Mediador/metabolismo , Meiosis/genética , Espermatogénesis/genética , Animales , Masculino , Subunidad 1 del Complejo Mediador/genética , Ratones , Ratones Noqueados , Fase Paquiteno/genética , Testículo/metabolismoRESUMEN
Chromatin remodeling is important for cell differentiation. Histone methyltransferase EZH2 and histone demethylase JMJD3 (KDM6B) modulate levels of histone H3 lysine 27 trimethylation (H3K27me3). Interplay between the two modulators influence lineage specification in stem cells. Here, we identified microRNA MIR146A to be a negative regulator of JMJD3. In the osteogenic differentiation of human mesenchymal stem cells (hMSCs), we observed an upregulation of JMJD3 and a downregulation of MIR146A. Blocking JMJD3 activity in differentiating hMSCs reduced transcript levels of osteogenic gene RUNX2. H3K27me3 levels decreased at the RUNX2 promoter during cell differentiation. Modulation of MIR146A levels in hMSCs altered JMJD3 and RUNX2 expression and affected osteogenic differentiation. We conclude that JMJD3 promotes osteogenesis in differentiating hMSCs, with MIR146A regulating JMJD3.
Asunto(s)
Diferenciación Celular , Histona Demetilasas con Dominio de Jumonji/genética , Células Madre Mesenquimatosas/fisiología , MicroARNs/genética , Interferencia de ARN , Regiones no Traducidas 3' , Secuencia de Bases , Células Cultivadas , Regulación hacia Abajo , Represión Enzimática , Histonas/metabolismo , Humanos , Histona Demetilasas con Dominio de Jumonji/metabolismo , Osteogénesis , Unión ProteicaRESUMEN
The differentiation of adult stem cells involves extensive chromatin remodeling, mediated in part by the gene products of histone deacetylase (HDAC) family members. While the transcriptional downregulation of HDACs can impede stem cell self-renewal in certain contexts, it may also promote stem cell maintenance under other circumstances. In self-renewing, differentiating, and aging spermatogonial stem cells (SSCs), the gene expression dynamics of HDACs have not yet been characterized. To gain further insight with these studies, we analyzed the transcriptional profiles of six HDAC family members, previously identified to be the most highly expressed in self-renewing SSCs, during stem cell differentiation and aging. Here we discovered that in both differentiating and aging SSCs the expression of Sirt4 increases, while the expression of Hdac2, Hdac6, and Sirt1 decreases. When SSCs are exposed to the lifespan-enhancing drug rapamycin in vivo, the resultant HDAC gene expression patterns are opposite of those seen in the differentiating and aging SSCs, with increased Hdac2, Hdac6, and Sirt1 and decreased Hdac8, Hdac9, and Sirt4. Our findings suggest that HDACs important for stem cell maintenance and oxidative capacity are downregulated as adult stem cells differentiate or age. These results provide important insights into the epigenetic regulation of stem cell differentiation and aging in mammals.
Asunto(s)
Senescencia Celular/genética , Histona Desacetilasas/metabolismo , Espermatogonias/metabolismo , Células Madre/metabolismo , Animales , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Regulación hacia Abajo , Expresión Génica , Perfilación de la Expresión Génica , Histona Desacetilasa 2/biosíntesis , Histona Desacetilasa 6 , Histona Desacetilasas/biosíntesis , Histona Desacetilasas/genética , Masculino , Ratones , Proteínas Mitocondriales/biosíntesis , Proteínas Represoras/biosíntesis , Sirolimus/farmacología , Sirtuina 1/biosíntesis , Sirtuinas/biosíntesis , Espermatogonias/citología , Espermatogonias/efectos de los fármacos , Células Madre/citología , Células Madre/efectos de los fármacos , Transcripción GenéticaRESUMEN
Chromatin modifier Swi-independent 3a (SIN3A), together with associated histone deacetylases, influences gene expression during development and differentiation through a variety of transcription factors in a cell-specific manner. Sin3a is essential for the maintenance of inner cell mass cells of mouse blastocysts, embryonic fibroblasts, and myoblasts, but is not required for the survival of trophectoderm or Sertoli cells. To better understand how this transcriptional regulator modulates cells at different developmental stages within a single lineage, we used conditional gene targeting in mice to ablate Sin3a from perinatal quiescent male gonocytes and from postnatal differentiating spermatogonia. Mitotic germ cells expressing stimulated by retinoic acid gene 8 (Stra8) that lacked Sin3a exhibited increased DNA damage and apoptosis, yet collectively progressed through meiosis and spermiogenesis and generated epididymal sperm at approximately 50% of control levels, sufficient for normal fertility. In contrast, perinatal gonocytes lacking Sin3a underwent rapid depletion that coincided with cell cycle reentry, exhibiting 2.5-fold increased histone H3 phosphorylation upon cycling that suggested a prophase/metaphase block; germ cells were almost entirely absent two weeks after birth, resulting in sterility. Gene expression profiling of neonatal testes containing Sin3a-deleted gonocytes identified upregulated transcripts highly associated with developmental processes and pattern formation, and downregulated transcripts involved in nuclear receptor activity, including Nr4a1 (Nur77). Interestingly, Nr4a1 levels were elevated in testes containing Stra8-expressing, Sin3a-deleted spermatogonia. SIN3A directly binds to the Nr4a1 promoter, and Nr4a1 expression is diminished upon spermatogonial differentiation in vitro. We conclude that within the male germline, Sin3a is required for the mitotic reentry of gonocytes, but is dispensable for the maintenance of differentiating spermatogonia and subsequent spermatogenic processes.
Asunto(s)
Diferenciación Celular/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Células Germinativas/crecimiento & desarrollo , Proteínas Represoras/metabolismo , Espermatogonias/crecimiento & desarrollo , Proteínas Adaptadoras Transductoras de Señales , Animales , Apoptosis/genética , Linaje de la Célula/fisiología , Inmunoprecipitación de Cromatina , Daño del ADN/genética , Cartilla de ADN/genética , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica/genética , Marcación de Gen/métodos , Células Germinativas/citología , Histonas/metabolismo , Inmunohistoquímica , Masculino , Ratones , Análisis por Micromatrices , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Fosforilación , Proteínas/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Represoras/deficiencia , Proteínas Represoras/genética , Complejo Correpresor Histona Desacetilasa y Sin3 , Testículo/citología , Testículo/metabolismoRESUMEN
Impaired biogenesis of microRNAs disrupts spermatogenesis and leads to infertility in male mice. Spermatogonial differentiation is a key step in spermatogenesis, yet the mechanisms that control this event remain poorly defined. In this study, we discovered microRNA 146 (Mir146) to be highly regulated during spermatogonial differentiation, a process dependent on retinoic acid (RA) signaling. Mir146 transcript levels were diminished nearly 180-fold in differentiating spermatogonia when compared with undifferentiated spermatogonia. Luciferase assays revealed the direct binding of Mir146 to the 3' untranslated region of the mediator complex subunit 1 (Med1), a coregulator of retinoid receptors (RARs and RXRs). Overexpression of Mir146 in cultured undifferentiated spermatogonia reduced Med1 transcript levels, as well as those of differentiation marker kit oncogene (Kit). MED1 protein was also diminished. Conversely, inhibition of Mir146 increased the levels of Kit. When undifferentiated spermatogonia were exposed to RA, Mir146 was downregulated along with a marker for undifferentiated germ cells, zinc finger and BTB domain containing 16 (Zbtb16; Plzf); Kit was upregulated. Overexpression of Mir146 in RA-treated spermatogonia inhibited the upregulation of Kit, stimulated by retinoic acid gene 8 (Stra8), and spermatogenesis- and oogenesis-specific basic helix-loop-helix 2 (Sohlh2). Inhibition of Mir146 in RA-treated spermatogonia greatly enhanced the upregulation of these genes. We conclude that Mir146 modulates the effects of RA on spermatogonial differentiation.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , MicroARNs/metabolismo , Espermatogonias/citología , Tretinoina/farmacología , Animales , Línea Celular , Regulación de la Expresión Génica , Masculino , Subunidad 1 del Complejo Mediador/genética , Subunidad 1 del Complejo Mediador/metabolismo , Ratones , MicroARNs/genética , Espermatogonias/efectos de los fármacosRESUMEN
Insulin resistance is a central feature of the PCOS and may increase cardiovascular risk. Owing to insulin resistance, the metabolic syndrome is more prevalent in women with PCOS compared with unaffected women. Metformin improves the metabolic profile in PCOS in short-term studies. In this study, we evaluated the long-term effect of metformin on metabolic parameters in women with PCOS during routine care without a controlled diet.We performed a retrospective medical chart review of 70 women with PCOS receiving metformin from an academic endocrine clinic. Metabolic risk factors were compared before and after metformin treatment. Time trends of these metabolic parameters were also analysed. After a mean follow-up of 36.1 months with metformin treatment, improvements were observed for BMI (-1.09 +/- 3.48 kg/m2, p = 0.0117), diastolic blood pressure (-2.69 +/- 10.35 mmHg, p = 0.0378), and HDL cholesterol (+5.82 +/- 11.02 mg/dL, p <0.0001).The prevalence of metabolic syndrome decreased from 34.3% at baseline to 21.4% (p = 0.0495). The course of BMI reduction during metformin treatment was significantly more pronounced in women with PCOS with metabolic syndrome at baseline, compared with those without the metabolic syndrome (p = 0.0369 for interaction). In conclusion, metformin improved the metabolic profile of women with PCOS over 36.1 months, particularly in HDL cholesterol, diastolic blood pressure and BMI.