RESUMEN
KRAS plays critical roles in regulating a range of normal cellular events as well as pathological processes in many tissues mediated through a variety of signaling pathways, including ERK1/2 and AKT signaling, in a cell-, context- and development-dependent manner. The in vivo function of KRAS and its downstream targets in gonadal steroidogenic cells for the development and homeostasis of reproductive functions remain to be determined. To understand the functions of KRAS signaling in gonadal theca and interstitial cells, we generated a Kras mutant (tKrasMT) mouse line that selectively expressed a constitutively active Kras G12D in these cells. Kras G12D expression in ovarian theca cells did not block follicle development to the preovulatory stage. However, tKrasMT females failed to ovulate and thus were infertile. The phosphorylated ERK1/2 and forkhead box O1 (FOXO1) and total FOXO1 protein levels were markedly reduced in tKrasMT theca cells. Kras G12D expression in theca cells also curtailed the phosphorylation of ERK1/2 and altered the expression of several ovulation-related genes in gonadotropin-primed granulosa cells. To uncover downstream targets of KRAS/FOXO1 signaling in theca cells, we found that the expression of bone morphogenic protein 7 (Bmp7), a theca-specific factor involved in ovulation, was significantly elevated in tKrasMT theca cells. Chromosome immunoprecipitation assays demonstrated that FOXO1 interacted with the Bmp7 promoter containing forkhead response elements and that the binding activity was attenuated in tKrasMT theca cells. Moreover, Foxo1 knockdown caused an elevation, whereas Foxo1 overexpression resulted in an inhibition of Bmp7 expression, suggesting that KRAS signaling regulates FOXO1 protein levels to control Bmp7 expression in theca cells. Thus, the anovulation phenotype observed in tKrasMT mice may be attributed to aberrant KRAS/FOXO1/BMP7 signaling in theca cells. Our work provides the first in vivo evidence that maintaining normal KRAS activity in ovarian theca cells is crucial for ovulation and female fertility.
RESUMEN
Numb (Nb) and Numb-like (Nbl) are functionally redundant adaptor proteins that critically regulate cell fate and morphogenesis in a variety of organs. We selectively deleted Nb and Nbl in testicular germ cells by breeding Nb/Nbl floxed mice with a transgenic mouse line Tex101-Cre. The mutant mice developed unilateral or bilateral cystic dilation in the rete testis (RT). Dye trace indicated partial blockages in the testicular hilum. Morphological and immunohistochemical evaluations revealed that the lining epithelium of the cysts possessed similar characteristics of RT epithelium, suggesting that the cyst originated from dilation of the RT lumen. Spermatogenesis and the efferent ducts were unaffected. In comparisons of isolated germ cells from mutants to control mice, the Notch activity considerably increased and the expression of Notch target gene Hey1 significantly elevated. Further studies identified that germ cell Fgf4 expression negatively correlated the Notch activity and demonstrated that blockade of FGF receptors mediated FGF4 signaling induced enlargement of the RT lumen in vitro. The crucial role of the FGF4 signaling in modulation of RT development was verified by the selective germ cell Fgf4 ablation, which displayed a phenotype similar to that of germ cell Nb/Nbl null mutant males. These findings indicate that aberrant over-activation of the Notch signaling in germ cells due to Nb/Nbl abrogation impairs the RT development, which is through the suppressing germ cell Fgf4 expression. The present study uncovers the presence of a lumicrine signal pathway in which secreted/diffusible protein FGF4 produced by germ cells is essential for normal RT development.
RESUMEN
INTRODUCTION: Adenosine, 5'-Se-methyl-5'-seleno-,2',3'-diacetate (NPC43) is a recently identified small, non-peptidyl molecule which restores normal insulin signaling in a mouse model of type 2 diabetes (Lan et al). The present study investigated the ability of NPC43 as an oral and injectable insulin-replacing agent to activate insulin receptor (INSR) and counter hyperglycemia in streptozotocin (STZ)-induced type 1 diabetic (T1D) mice. RESEARCH DESIGN AND METHODS: In this study, STZ was intraperitoneally injected into wild-type mice to induce hyperglycemia and hypoinsulinemia, the main features of T1D. These STZ-induced T1D mice were given NPC43 orally or intraperitoneally and blood glucose levels were measured using a glucometer. Protein levels of phosphorylated and total Insrß, protein kinase B (Akt) and AS160 (critical for glucose uptake) in the skeletal muscle and liver of STZ-induced T1D mice following oral NPC43 treatment were determined by western blot analysis. In addition, hepatic expression of activated Insr in STZ-induced T1D mice after intraperitoneal NPC43 treatment was measured by ELISA. Student's t-test was used for statistical analysis. RESULTS: Oral administration of NPC43 at a dose of 5.4 or 10.8 mg/kg body weight (mpk) effectively lowered blood glucose levels in STZ-induced T1D mice at ≥1 hour post-treatment and the glucose-lowering activity of oral NPC43 persisted for 5 hours. Blood glucose levels were also reduced in STZ-induced T1D mice following intraperitoneal NPC43 (5.4 mpk) treatment. Protein levels of phosphorylated Insrß, Akt and AS160 were significantly increased in the skeletal muscle and liver of STZ-induced T1D mice after oral NPC43 (5.4 mpk) treatment. In addition, activation of hepatic Insr was observed in STZ-induced T1D mice following intraperitoneal NPC43 (5.4 mpk) treatment. CONCLUSIONS: We conclude that NPC43 is a de facto fast-acting oral and injectable insulin mimetic which activates Insr and mitigates hyperglycemia in a mouse model of T1D.
Asunto(s)
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 1 , Diabetes Mellitus Tipo 2 , Hiperglucemia , Administración Oral , Animales , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Hiperglucemia/tratamiento farmacológico , Ratones , Receptor de Insulina/uso terapéutico , Estreptozocina/uso terapéuticoRESUMEN
The pathophysiology of type 2 diabetes mellitus (T2D) is characterized by reduced or absent insulin receptor (INSR) responsiveness to its ligand, elevated hepatic glucose output and impaired glucose uptake in peripheral tissues, particularly skeletal muscle. Treatments to reduce hyperglycemia and reestablish normal insulin signaling are much sought after. Any agent which could be orally administered to restore INSR function, in an insulin-independent manner, would have major implications for the management of this global disease. We have discovered a non-peptidyl small molecule, adenosine, 5'-Se-methyl-5'-seleno-, 2',3'-diacetate [referred to as non-peptidyl compound #43 (NPC43)], which restores INSR signaling in the complete absence of insulin. Initial screening of numerous compounds in human HepG2 liver cells revealed that NPC43 significantly inhibited glucose production. The compound was potently anti-hyperglycemic and anti-hyperinsulinemic in vivo, in insulin-resistant T2D Leprdb/db mice, following either acute or chronic treatment by oral gavage and intraperitoneal injection, respectively. The compound acted at the level of INSR and activated it in both liver and skeletal muscle of Leprdb/db mice. In cell culture, the compound activated INSR in both liver and skeletal muscle cells; furthermore, it cooperated with insulin to depress glucose-6-phosphatase catalytic subunit (G6pc) expression and stimulate glucose uptake, respectively. Our results indicated that the compound directly interacted with INSRα, triggering appropriate phosphorylation and activation of the receptor and its downstream targets. Unlike insulin, NPC43 did not activate insulin-like growth factor 1 receptor in either liver or skeletal muscle. We believe this compound represents a potential oral and/or injectable insulin replacement therapy for diabetes and diseases associated with insulin resistance.
Asunto(s)
Adenosina/uso terapéutico , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Hiperglucemia/tratamiento farmacológico , Hipoglucemiantes/uso terapéutico , Receptor de Insulina/metabolismo , Adenosina/análogos & derivados , Animales , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/metabolismo , Glucosa/metabolismo , Células Hep G2 , Humanos , Hiperglucemia/complicaciones , Hiperglucemia/metabolismo , Hipoglucemiantes/química , Insulina/metabolismo , Resistencia a la Insulina , Masculino , Metilación , Ratones , Ratones Endogámicos C57BL , Compuestos de Organoselenio/química , Compuestos de Organoselenio/uso terapéuticoRESUMEN
Gametogenetin (GGN) binding protein 2 (GGNBP2) is a zinc finger protein expressed abundantly in spermatocytes and spermatids. We previously discovered that Ggnbp2 resection caused metamorphotic defects during spermatid differentiation and resulted in an absence of mature spermatozoa in mice. However, whether GGNBP2 affects meiotic progression of spermatocytes remains to be established. In this study, flow cytometric analyses showed a decrease in haploid, while an increase in tetraploid spermatogenic cells in both 30- and 60-day-old Ggnbp2 knockout testes. In spread spermatocyte nuclei, Ggnbp2 loss increased DNA double-strand breaks (DSB), compromised DSB repair and reduced crossovers. Further investigations demonstrated that GGNBP2 co-immunoprecipitated with a testis-enriched protein GGN1. Immunofluorescent staining revealed that both GGNBP2 and GGN1 had the same subcellular localizations in spermatocyte, spermatid and spermatozoa. Ggnbp2 loss suppressed Ggn expression and nuclear accumulation. Furthermore, deletion of either Ggnbp2 or Ggn in GC-2spd cells inhibited their differentiation into haploid cells in vitro. Overexpression of Ggnbp2 in Ggnbp2 null but not in Ggn null GC-2spd cells partially rescued the defect coinciding with a restoration of Ggn expression. Together, these data suggest that GGNBP2, likely mediated by its interaction with GGN1, plays a role in DSB repair during meiotic progression of spermatocytes.
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Proteínas Portadoras/genética , Meiosis/genética , Espermatogénesis/genética , Hormonas Testiculares/genética , Proteínas Adaptadoras Transductoras de Señales , Animales , Roturas del ADN de Doble Cadena , Reparación del ADN/genética , Regulación del Desarrollo de la Expresión Génica/genética , Masculino , Ratones , Espermatocitos/crecimiento & desarrollo , Espermatocitos/metabolismo , Espermatozoides/crecimiento & desarrollo , Espermatozoides/metabolismoRESUMEN
Numerous studies have been conducted to understand the molecular mechanisms controlling mammalian secondary palate development such as growth, reorientation and fusion. However, little is known about the signaling factors regulating palate initiation. Mouse fibroblast growth factor (FGF) receptor 2 gene (Fgfr2) is expressed on E11.5 in the palate outgrowth within the maxillary process, in a region that is responsible for palate cell specification and shelf initiation. Fgfr2 continues to express in palate on E12.5 and E13.5 in both epithelial and mesenchymal cells, and inactivation of Fgfr2 expression in mesenchymal cells using floxed Fgfr2 allele and Osr2-Cre leads to cleft palate at various stages including reorientation, horizontal growth and fusion. Notably, some mutant embryos displayed no sign of palate shelf formation suggesting that FGF receptor 2 mediated FGF signaling may play an important role in palate initiation.
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Hueso Paladar/crecimiento & desarrollo , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Animales , Fisura del Paladar/genética , Femenino , Mutación con Pérdida de Función , Masculino , Células Madre Mesenquimatosas/metabolismo , Ratones , Hueso Paladar/citología , Hueso Paladar/metabolismoRESUMEN
Gametogenetin binding protein 2 (GGNBP2) is an evolutionarily conserved zinc finger protein. Although Ggnbp2-null embryos in the B6 background died because of a defective placenta, 6.8% of Ggnbp2-null mice in the B6/129 mixed background were viable and continued to adulthood. Adult Ggnbp2-null males were sterile, with smaller testes and an azoospermic phenotype, whereas mutant females were fertile. Histopathological analysis of 2-month-old Ggnbp2-null testes revealed absence of mature spermatozoa in the seminiferous tubules and epididymides and reduction of the number of spermatids. Ultrastructural analysis indicated dramatic morphological defects of developing spermatids in the Ggnbp2-null testes, including irregularly shaped acrosomes, acrosome detachment, cytoplasmic remnant, ectopic manchette, and ill-formed head shape in both elongating and elongated spermatids. However, the numbers of spermatogonia, spermatocytes, Leydig cells, and Sertoli cells in Ggnbp2-null testes did not significantly differ from the wild-type siblings. Gonadotropins, testosterone, and the blood-testis barrier were essentially unaffected. Western blot analyses showed increases in α-E-catenin, ß-catenin, and N-cadherin, decreases in E-cadherin, afadin, and nectin-3, and no changes in vinculin, nectin-2, focal adhesion kinase, and integrin-ß1 protein levels in Ggnbp2-null testes compared to wild-type siblings. Together, this study demonstrates that GGNBP2 is critically required for maintenance of the adhesion integrity of the adlumenal germ epithelium and is indispensable for normal spermatid transformation into mature spermatozoa in mice.
Asunto(s)
Proteínas Portadoras/genética , Infertilidad Masculina/genética , Mutación/genética , Espermatogénesis/genética , Proteínas Adaptadoras Transductoras de Señales , Animales , Barrera Hematotesticular/metabolismo , Cadherinas/metabolismo , Femenino , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Espermátides/metabolismo , Espermátides/patología , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
Theca cell-selective Pten mutation (tPtenMT) in mice resulted in increases in PDK1 and Akt phosphorylation, indicating an over-activation of PI3K signaling in the ovaries. These mice displayed elevated androgen levels, ovary enlargement, antral follicle accumulation, early fertility loss and increased expression of Lhcgr and genes that are crucial to androgenesis. These abnormalities were partially reversed by treatments of PI3K or Akt inhibitor. LH actions in Pten deficient theca cells were potentiated. The phosphorylation of Foxo1 was increased, while the binding of Foxo1 to forkhead response elements in the Lhcgr promoter was reduced in tPtenMT theca cells, implying a mechanism by which PI3K/Akt-induced upregulation of Lhcgr in theca cells might be mediated by reducing the inhibitory effect of Foxo1 on the Lhcgr promoter. The phenotype of tPtenMT females is reminiscent of human PCOS and suggests that dysregulated PI3K cascade in theca cells may be involved in certain types of PCOS pathogenesis.
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Andrógenos/metabolismo , Eliminación de Gen , Ovario/fisiopatología , Fosfohidrolasa PTEN/genética , Células Tecales/metabolismo , Envejecimiento/metabolismo , Animales , Clorpropamida/análogos & derivados , Clorpropamida/farmacología , Femenino , Fertilidad , Proteína Forkhead Box O1/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Hormona Luteinizante/farmacología , Masculino , Ratones Endogámicos C57BL , Ratones Mutantes , Modelos Biológicos , Ovario/efectos de los fármacos , Fosfohidrolasa PTEN/metabolismo , Fosforilación/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Unión Proteica/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora , Receptores de HL/genética , Receptores de HL/metabolismo , Esteroides/biosíntesis , Testosterona/sangre , Células Tecales/efectos de los fármacosRESUMEN
Gametogenetin-binding protein 2 (GGNBP2) is encoded in human chromosome 17q12-q23, a region known as a breast and ovarian cancer susceptibility locus. GGNBP2, also referred to ZFP403, has a single C2H2 zinc finger and a consensus LxxLL nuclear receptor-binding motif. Here, we demonstrate that GGNBP2 expression is reduced in primary human breast tumors and in breast cancer cell lines, including T47D, MCF-7, LCC9, LY2, and MDA-MB-231 compared with normal, immortalized estrogen receptor α (ERα) negative MCF-10A and MCF10F breast epithelial cells. Overexpression of GGNBP2 inhibits the proliferation of T47D and MCF-7 ERα positive breast cancer cells without affecting MCF-10A and MCF10F. Stable GGNBP2 overexpression in T47D cells inhibits 17ß-estradiol (E2)-stimulated proliferation as well as migration, invasion, anchorage-independent growth in vitro, and xenograft tumor growth in mice. We further demonstrate that GGNBP2 protein physically interacts with ERα, inhibits E2-induced activation of estrogen response element-driven reporter activity, and attenuates ER target gene expression in T47D cells. In summary, our in vitro and in vivo findings suggest that GGNBP2 is a novel breast cancer tumor suppressor functioning as a nuclear receptor corepressor to inhibit ERα activity and tumorigenesis.
Asunto(s)
Neoplasias de la Mama/metabolismo , Regulación hacia Abajo , Receptor alfa de Estrógeno/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Neoplasias de la Mama/patología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Estradiol/farmacología , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Células MCF-7 , Ratones , Trasplante de Neoplasias , Elementos de Respuesta/efectos de los fármacosRESUMEN
BACKGROUND: Fibroblast growth factor (FGF) signaling is thought to play diverse roles in the male reproductive system. However, its role in testicular cells for spermatogenesis and fertility remains unclear. METHODS: In this study, the expression and localization of Fgfr 1 (FGF Receptor) and Fgfr 2 in the postnatal mouse testes were examined by RT-PCR, Western blotting and immunohistochemistry. The in vivo function of each receptor in testicular germ cells was determined using germ cell-specific Fgfr mutant animals, Tex101-iCre;Fgfr (flox/flox) and Tex101-iCre;Fgfr (flox/flox) mice. The results were analyzed by Kruskal-Wallis test and Dunn's Post-test. RESULTS: Both Fgfr1 and Fgfr2 were expressed in the testis throughout the entire postnatal development. Prominent immunostaining of these FGFRs was observed in interstitial and peritubular cells with little or no changes in all phases during postnatal development. Positive staining of these receptors was also detected in germ cells including elongated spermatids and spermatozoa. Germ cell-specific Fgfr1 or Fgfr2 mutant mice were viable with no developmental abnormalities in the testes and accessory sex organs. Fertility studies showed that the fecundity of both mutant mouse lines did not significantly differ from wild-type siblings (n=4, p>0.05). Further analysis indicated the presence of other Fgfrs in testicular germ cells including Fgfr 3, 4 and 5. CONCLUSION: The results demonstrated that Fgfr1 and 2 are expressed in all testicular cell types and that neither Fgfr1 nor Fgfr2 in testicular germ cells is essential for spermatogenesis and fertility. Future studies are needed to investigate the potential functional redundancy among five Fgfrs in male germ cells for spermatogenesis and fertility.
RESUMEN
We have generated a transgenic mouse line that expresses improved Cre recombinase (iCre) under the control of the testis-expressed gene 101 (Tex101) promoter. This transgenic mouse line was named Tex101-iCre. Using the floxed ROSA reporter mice, we found that robust Cre recombinase activity was detected in postnatal testes with weak or no activity in other tissues. Within the testis, Cre recombinase was active in spermatogenic cells as early as the prospermatogonia stage at day 1 after birth. In 30- and 60-day-old mice, positive Cre recombinase activity was detected not only in prospermatogonia but also in spermatogenic cells at later stages of spermatogenesis. There was little or no Cre activity in interstitial cells. Breeding wild-type females with homozygous floxed fibroblast growth factor receptor 2 (Fgfr2) males carrying the Tex101-iCre transgene did not produce any progeny with the floxed Fgfr2 allele. All the progeny inherited a recombined Fgfr2 allele, indicating that complete deletion of the floxed Fgfr2 allele by Tex101-iCre can be achieved in the male germline. Furthermore, FGFR2 protein was not detected in spermatocytes and spermatids of adult Fgfr2(fl/fl) ;Tex101-iCre mice. Taken together, our results suggest that the Tex101-iCre mouse line allows the inactivation of a floxed gene in spermatogenic cells in adult mice, which will facilitate the functional characterization of genes in normal spermatogenesis and male fertility.
Asunto(s)
Antígenos de Superficie/genética , Regulación Enzimológica de la Expresión Génica , Células Germinativas/enzimología , Integrasas/genética , Integrasas/metabolismo , Animales , Antígenos de Superficie/metabolismo , Femenino , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Regiones Promotoras Genéticas , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/metabolismoRESUMEN
Using an in silico approach, we identified a putative zinc finger domain-containing transcription factor (zinc finger protein 105, ZFP105) enriched in the adult mouse testis. RT-PCR analyses showed that Zfp105 was indeed highly expressed in adult mouse testis and that its expression was regulated during postnatal development. To further characterize Zfp105 expression, we generated a Zfp105:beta-galactosidase (LacZ) knock-in reporter mouse line (Zfp105(LacZ/+)) in which a Zfp105:LacZ fusion gene was expressed. Whole-mount LacZ analyses of adult Zfp105(LacZ/+) tissues showed robust LacZ staining in the testis, very weak staining in the ovary, and no staining in the spleen, liver, kidney, heart, lung, thymus, adrenal gland, uterus, or oviduct. Sectional LacZ staining showed that ZFP105 was highly expressed in pachytene spermatocytes. ZNF35, the human ortholog of Zfp105, was also highly expressed in human testis. Immunofluorescence analysis showed that ZNF35 was located primarily in the cytoplasm of male germ cells. More importantly, reduced male fertility was observed in adult Zfp105(LacZ/LacZ) mice. Histological studies showed the presence of undifferentiated spermatogenic cells in the lumen of seminiferous tubules at stage VII and in the epididymal lumen of adult Zfp105(LacZ/LacZ) mice. Taken together, our results suggest that ZFP105 is a male germ-cell factor and plays a role in male reproduction.
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Proteínas de Unión al ADN/metabolismo , Fertilidad/fisiología , Testículo/fisiología , Factores de Transcripción/metabolismo , Animales , Proteínas de Unión al ADN/genética , Femenino , Técnicas de Sustitución del Gen , Genes Reporteros , Células Germinativas/citología , Células Germinativas/metabolismo , Humanos , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Análisis por Micromatrices , Espermatogénesis/fisiología , Testículo/química , Testículo/citología , Distribución Tisular , Factores de Transcripción/genéticaRESUMEN
To maintain the female reproductive lifespan, the majority of ovarian primordial follicles are preserved in a quiescent state in order to provide ova for later reproductive life. However, the molecular mechanism that maintains the long quiescence of primordial follicles is poorly understood. Here we provide genetic evidence to show that the tumor suppressor tuberous sclerosis complex 1 (Tsc1), which negatively regulates mammalian target of rapamycin complex 1 (mTORC1), functions in oocytes to maintain the quiescence of primordial follicles. In mutant mice lacking the Tsc1 gene in oocytes, the entire pool of primordial follicles is activated prematurely due to elevated mTORC1 activity in the oocyte, ending up with follicular depletion in early adulthood and causing premature ovarian failure (POF). We further show that maintenance of the quiescence of primordial follicles requires synergistic, collaborative functioning of both Tsc and PTEN (phosphatase and tensin homolog deleted on chromosome 10) and that these two molecules suppress follicular activation through distinct ways. Our results suggest that Tsc/mTORC1 signaling and PTEN/PI3K (phosphatidylinositol 3 kinase) signaling synergistically regulate the dormancy and activation of primordial follicles, and together ensure the proper length of female reproductive life. Deregulation of these signaling pathways in oocytes results in pathological conditions of the ovary, including POF and infertility.
Asunto(s)
Oocitos/metabolismo , Folículo Ovárico/citología , Insuficiencia Ovárica Primaria/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Modelos Animales de Enfermedad , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Oocitos/citología , Folículo Ovárico/metabolismo , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Insuficiencia Ovárica Primaria/fisiopatología , Factores de Transcripción/genética , Proteína 1 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/genéticaRESUMEN
Nuclear receptor subfamily 6, group A, member 1 (NR6A1) is an orphan member of the nuclear receptor superfamily and is required for normal mouse embryonic development. In adult mice, NR6A1 is predominantly expressed in spermatogenic cells and growing oocytes of the gonads and has a role in female reproduction by modulating the transcription of the oocyte-specific genes bone morphogenetic protein 15 (Bmp15) and growth differentiation factor 9 (Gdf9). In our goal to further understand the functional role of NR6A1 during postnatal development, we generated a Nr6a1:beta-galactosidase (LacZ) knockin reporter (Nr6a1(LacZ/+)) mouse line in which the Nr6a1:LacZ fusion gene was expressed and then characterized Nr6a1 expression in these reporter mice by performing LacZ staining. Our RT-PCR analyses showed that Nr6a1 was expressed in a variety of somatic tissues (e.g., oviduct and lung) other than gonads of normal adult mice. In adult Nr6a1(LacZ/+) mice, robust LacZ staining was observed in the gametes of gonads. Strong positive LacZ staining was also observed in the sperm of the epididymis, epithelial cells of the oviduct, and bronchioles within the lung. In adult Nr6a1(LacZ/+) mice, positive LacZ staining was observed in other somatic tissues, including hippocampus, cerebral cortex, cerebellum, and thalamus of brain; pars intermedia and pars anterior of pituitary; parathyroid; and islet of pancreas. NR6A1 expression in sperm within the epididymis, epithelial cells in the oviduct, and bronchioles of the lung was further confirmed by immunohistochemical studies. Nr6a1 is expressed not only in the germ cells of mouse gonads but also in a variety of somatic tissues, including epididymis, oviduct, brain, and pituitary. The extra-germ cell expression of NR6A1 makes it a less attractive contraceptive.
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Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Secuencia de Bases , Encéfalo/metabolismo , Cartilla de ADN/genética , Epidídimo/metabolismo , Femenino , Expresión Génica , Genes Reporteros , Operón Lac , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Miembro 1 del Grupo A de la Subfamilia 6 de Receptores Nucleares , Oocitos/metabolismo , Oviductos/metabolismo , Hipófisis/metabolismo , Embarazo , Espermatozoides/metabolismo , Distribución TisularRESUMEN
A transgenic mouse line that expresses iCre under regulation of the Cytochrome P(450) 17alpha-hydroxylase/17, 20-lyase (Cyp17) promoter was developed as a novel transgenic mouse model for the conditional deletion of genes specifically in the theca/interstitial cells of the ovary and Leydig cells of the testis. In this report, we describe the development of Cyp17iCre mice and the application of these mice for conditional deletion of the estrogen receptor alpha (Esr1) gene in the theca/interstitial and Leydig cells of the female and male gonad, respectively. These mice will prove a powerful tool to inactivate genes in the gonad in a cell-specific manner.
Asunto(s)
Receptor alfa de Estrógeno/deficiencia , Eliminación de Gen , Ingeniería Genética/métodos , Integrasas/metabolismo , Ovario/metabolismo , Esteroide 17-alfa-Hidroxilasa/genética , Testículo/metabolismo , Animales , Embrión de Mamíferos/embriología , Embrión de Mamíferos/enzimología , Receptor alfa de Estrógeno/genética , Femenino , Vectores Genéticos/genética , Integrasas/genética , Masculino , Ratones , Ratones Transgénicos , Especificidad de Órganos , Esteroide 17-alfa-Hidroxilasa/metabolismoRESUMEN
In the mammalian ovary, progressive activation of primordial follicles from the dormant pool serves as the source of fertilizable ova. Menopause, or the end of female reproductive life, occurs when the primordial follicle pool is exhausted. However, the molecular mechanisms underlying follicle activation are poorly understood. We provide genetic evidence that in mice lacking PTEN (phosphatase and tensin homolog deleted on chromosome 10) in oocytes, a major negative regulator of phosphatidylinositol 3-kinase (PI3K), the entire primordial follicle pool becomes activated. Subsequently, all primordial follicles become depleted in early adulthood, causing premature ovarian failure (POF). Our results show that the mammalian oocyte serves as the headquarters of programming of follicle activation and that the oocyte PTEN-PI3K pathway governs follicle activation through control of initiation of oocyte growth.
Asunto(s)
Oocitos/fisiología , Folículo Ovárico/fisiología , Fosfohidrolasa PTEN/fisiología , Animales , Femenino , Atresia Folicular , Ratones , Ratones Transgénicos , Oocitos/citología , Oocitos/crecimiento & desarrollo , Tamaño de los Órganos , Folículo Ovárico/citología , Ovario/anatomía & histología , Ovario/fisiología , Ovulación , Fosfohidrolasa PTEN/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Insuficiencia Ovárica Primaria/fisiopatología , Proteínas Quinasas/metabolismo , Proteína S6 Ribosómica/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TORRESUMEN
Hedgehog (Hh) signaling emerges as a potential pathway contributing to fat formation during postnatal development. In this report, we found that Patched 1 (Ptc1), a negative regulator of Hh signaling, was expressed in the epididymal fat pad of adult mice. Reduced total white fat mass and epididymal adipocyte cell size were observed in naturally occurring spontaneous mesenchymal dysplasia (mes) adult mice (Ptc1(mes/mes)), which carry a deletion of Ptc1 at the carboxyl-terminal cytoplasmic region. Increased expression of truncated Ptc1, Ptc2 and Gli1, the indicators of ectopic activation of Hh signaling, was observed in epididymal fat pads of adult Ptc1(mes/mes) mice. In contrast, expression of peroxisome proliferator-activated receptor gamma, CCAAT/enhancer binding protein alpha, adipocyte P2 and adipsin were reduced in epididymal fat pads of adult Ptc1(mes/mes) mice. Taken together, our results indicate that deletion of carboxyl-terminal tail of Ptc1 can lead to the reduction of white fat mass during postnatal development.
Asunto(s)
Adipogénesis/genética , Tejido Adiposo Blanco/fisiología , Tejido Adiposo/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Transducción de Señal/genética , Adipogénesis/fisiología , Animales , Composición Corporal , Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Factor D del Complemento/metabolismo , Cartilla de ADN/genética , Eliminación de Gen , Proteínas Hedgehog/metabolismo , Ratones , PPAR gamma/metabolismo , Receptores Patched , Receptor Patched-1 , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Using an in silico approach, a putative nuclear receptor-interacting protein 0610009K11Rik was identified in mouse testis. We named this gene testis-specific nuclear receptor-interacting protein-1 (Tnrip-1). Tnrip-1 was predominantly expressed in the testis of adult mouse tissues. Expression of Tnrip-1 in the testis was regulated during postnatal development, with robust expression in 14-day-old or older testes. In situ hybridization analyses showed that Tnrip-1 is highly expressed in pachytene spermatocytes and spermatids. Consistent with its mRNA expression, Tnrip-1 protein was detected in adult mouse testes. Immunohistochemical studies showed that Tnrip-1 is a nuclear protein and mainly expressed in pachytene spermatocytes and round spermatids. Moreover, co-immunoprecipitation analyses showed that endogenous Tnrip-1 protein can interact with germ cell nuclear receptor (GCNF) in adult mouse testes. Our results suggest that Tnrip-1 is a testis-specific and GCNF-interacting protein which may be involved in the modulation of GCNF-mediated gene transcription in spermatogenic cells within the testis.
Asunto(s)
Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Núcleo Celular/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Espermatozoides/citología , Espermatozoides/metabolismo , Testículo/metabolismo , Animales , Animales Recién Nacidos , Proteínas de Unión al ADN/metabolismo , Regulación del Desarrollo de la Expresión Génica , Masculino , Ratones , Ratones Endogámicos C57BL , Miembro 1 del Grupo A de la Subfamilia 6 de Receptores Nucleares , Especificidad de Órganos , Fase Paquiteno , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Espermátides/citología , Espermátides/metabolismo , Espermatocitos/citología , Espermatocitos/metabolismo , Testículo/citologíaRESUMEN
Orphan nuclear receptors such as germ cell nuclear factor (GCNF), steroidogenic factor 1 (SF-1) and liver receptor homolog-1 (LRH-1), are emerging as important ovarian factors in regulating female reproduction. Within the ovary, GCNF (NR6A1) expression is restricted to the oocyte, while SF-1 (NR5A1) is expressed only in the somatic cells, such as granulosa, thecal and luteal cells, and interstitial cells. LRH-1 (NR5A2), an orphan receptor closely related to SF-1, is expressed only in the granulosa cells of the follicles and luteal cells within the ovary. Recent studies using conditional knockout strategies to bypass the embryonic lethality of GCNF and SF-1 null mice have uncovered important roles of GCNF and SF-1 in the oocyte and granulosa cells, respectively. In this review, we will summarize the major findings of GCNF and SF-1 in the ovary from the studies of conditional GCNF and SF-1 knockout mice. The potential role of LRH-1 in the ovary is also briefly discussed. Understanding the ovarian functions of these orphan nuclear receptors may lead to the development of new agents for regulation of female fertility and new medicines for the treatment of female idiopathic infertility, premature ovarian failure, polycystic ovarian syndrome and ovarian cancer.
Asunto(s)
Proteínas Nucleares/fisiología , Ovario/metabolismo , Animales , Femenino , Humanos , Ratones , Enfermedades del Ovario/diagnóstico , Enfermedades del Ovario/metabolismo , Enfermedades del Ovario/fisiopatología , Enfermedades del Ovario/terapiaRESUMEN
Oocyte-specific deletion of ovarian genes using Cre/loxP technology provides an excellent tool to understand their physiological roles during folliculogenesis, oogenesis, and preimplantation embryonic development. We have generated a transgenic mouse line expressing improved Cre recombinase (iCre) driven by the mouse growth differentiation factor-9 (GDF-9) promoter. The resulting transgenic mouse line was named GDF-9-iCre mice. Using the floxed ROSA reporter mice, we found that Cre recombinase was expressed in postnatal ovaries, but not in heart, liver, spleen, kidney, and brain. Within the ovary, the Cre recombinase was exclusively expressed in the oocytes of primordial follicles and follicles at later developmental stages. The expression of iCre of GDF-9-iCre mice was shown to be earlier than the Cre expression of Zp3Cre and Msx2Cre mice, in which the Cre gene is driven by zona pellucida protein 3 (Zp3) promoter and a homeobox gene Msx2 promoter, respectively, in the postnatal ovary. Breeding wild-type males with heterozygous floxed germ cell nuclear factor (GCNF) females carrying the GDF-9-iCre transgene did not produce any progeny having the floxed GCNF allele, indicating that complete deletion of the floxed GCNF allele can be achieved in the female germline by GDF-9-iCre mice. These results suggest that GDF-9-iCre mouse line provides an excellent genetic tool for understanding functions of oocyte-expressing genes involved in folliculogenesis, oogenesis, and early embryonic development. Comparison of the ontogeny of the Cre activities of GDF-9-iCre, Zp3Cre, and Msx2Cre transgenic mice shows there is sequential Cre activity of the three transgenes that will allow inactivation of a target gene at different points in folliculogenesis.