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1.
Am J Physiol Endocrinol Metab ; 301(3): E539-47, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21693691

ABSTRACT

In Sertoli cells, anti-Müllerian hormone (AMH) expression is upregulated by FSH via cyclic AMP (cAMP), although no classical cAMP response elements exist in the AMH promoter. The response to cAMP involves NF-κB and AP2; however, targeted mutagenesis of their binding sites in the AMH promoter do not completely abolish the response. In this work we assessed whether SOX9, SF1, GATA4, and AP1 might represent alternative pathways involved in cAMP-mediated AMH upregulation, using real-time RT-PCR (qPCR), targeted mutagenesis, luciferase assays, and immunocytochemistry in the Sertoli cell line SMAT1. We also explored the signaling cascades potentially involved. In qPCR experiments, Amh, Sox9, Sf1, and Gata4 mRNA levels increased after SMAT1 cells were incubated with cAMP. Blocking PKA abolished the effect of cAMP on Sox9, Sf1, and Gata4 expression, inhibiting PI3K/PKB impaired the effect on Sf1 and Gata4, and reducing MEK1/2 and p38 MAPK activities curtailed Gata4 increase. SOX9 and SF1 translocated to the nucleus after incubation with cAMP. Mutations of the SOX9 or SF1 sites, but not of GAT4 or AP1 sites, precluded the response of a 3,063-bp AMH promoter to cAMP. In conclusion, in the Sertoli cell line SMAT1 cAMP upregulates SOX9, SF1, and GATA4 expression and induces SOX9 and SF1 nuclear translocation mainly through PKA, although other kinases may also participate. SOX9 and SF1 binding to the AMH promoter is essential to increase the activity of the AMH promoter in response to cAMP.


Subject(s)
Anti-Mullerian Hormone/metabolism , Cyclic AMP/metabolism , SOX9 Transcription Factor/metabolism , Sertoli Cells/metabolism , Steroidogenic Factor 1/metabolism , Anti-Mullerian Hormone/genetics , Cell Line , Cyclic AMP/genetics , DNA-Binding Proteins , GATA4 Transcription Factor/genetics , GATA4 Transcription Factor/metabolism , Gene Expression , Humans , Male , Promoter Regions, Genetic , RNA Splicing Factors , SOX9 Transcription Factor/genetics , Signal Transduction/physiology , Steroidogenic Factor 1/genetics , Transcription Factors , Up-Regulation
2.
DNA Cell Biol ; 23(9): 572-85, 2004 Sep.
Article in English | MEDLINE | ID: mdl-15383177

ABSTRACT

Anti-Müllerian hormone (AMH) is best known for its role as an inhibitor of the development of female internal genitalia primordia during fetal life. In the testis, AMH is highly expressed by Sertoli cells of the testis from early fetal life to puberty, when it is downregulated by the action of testosterone, acting through the androgen receptor, and meiotic spermatocytes, probably acting through TNFalpha. Basal expression of AMH is induced by SOX9; GATA4, SF1, and WT1 enhance SOX9-activated expression. When the hypothalamic-pituitary axis is active and the negative effect of androgens and germ cells is absent, for example, in the fetal and neonatal periods or in disorders like androgen insensitivity, FSH upregulates AMH expression through a nonclassical cAMP-PKA pathway involving transcription factors AP2 and NFkappaB. The maintenance and hormonal regulation of AMH expression in late fetal and postnatal life requires distal AMH promoter sequences. In the ovary, granulosa cells express AMH from late fetal life at low levels; DAX1 and FOG2 seem to be responsible for negatively modulating AMH expression. Particular features are observed in AMH expression in nonmammalian species. In birds, AMH is expressed both in the male and female fetal gonads, and, like in reptiles, its expression is not preceded by that of SOX9.


Subject(s)
Glycoproteins/genetics , Testicular Hormones/genetics , Androgens/pharmacology , Animals , Anti-Mullerian Hormone , Birds , Female , Fishes , Follicle Stimulating Hormone/metabolism , Gene Expression , Gene Expression Regulation, Developmental , Humans , Male , Mammals , Promoter Regions, Genetic , Reptiles , Sertoli Cells/metabolism , Testis/embryology , Testis/metabolism
3.
Mol Cell Endocrinol ; 211(1-2): 21-31, 2003 Dec 15.
Article in English | MEDLINE | ID: mdl-14656472

ABSTRACT

(AMH/MIS) was first suggested by Jost, more than Four decades before this gonadal glycoprotein was purified and its gene and promoter sequenced. In mammals, AMH expression is triggered by SOX9 in Sertoli cells at the onset of testicular differentiation, and regulated by SF1, GATA factors, WT1, DAX1 and FSH. Ovarian granulosa cells also secrete AMH from late foetal life. In males, AMH is secreted into the bloodstream at high levels until puberty when it is down-regulated by androgens and meiotic germ cells and its directional secretion switches from the basal compartment to the seminiferous tubule lumen. In birds and reptiles, AMH expression shows particular features. Serum AMH determination is useful to study testicular function in boys and in patients with gonadal tumours. AMH levels in seminal and follicular fluid may also be of clinical use.


Subject(s)
Glycoproteins/physiology , Testicular Hormones/physiology , Androgens/physiology , Animals , Anti-Mullerian Hormone , Birds/embryology , Birds/metabolism , Female , Follicle Stimulating Hormone/physiology , Gene Components/genetics , Gene Expression , Gene Expression Regulation, Developmental , Germ Cells/physiology , Glycoproteins/genetics , Glycoproteins/metabolism , Humans , Male , Ovary/embryology , Ovary/growth & development , Ovary/metabolism , Reptiles/embryology , Reptiles/metabolism , Sex Differentiation/genetics , Sex Differentiation/physiology , Testicular Hormones/genetics , Testicular Hormones/metabolism , Testis/embryology , Testis/growth & development , Testis/metabolism
4.
Mol Endocrinol ; 17(4): 550-61, 2003 Apr.
Article in English | MEDLINE | ID: mdl-12554789

ABSTRACT

Anti-Müllerian hormone (AMH) production by testicular Sertoli cells is high before puberty and can be further induced by FSH. Our objective was to delineate the mechanisms by which FSH stimulates AMH production. Assay of serum AMH levels and histological morphometric analysis in prepubertal FSH-deficient transgenic mice showed that serum AMH and testicular mass were decreased owing to reduced Sertoli cell number. All parameters resumed normal values in mice treated with recombinant FSH. We also analyzed the ability of FSH and the factors involved in its signaling pathway to activate AMH transcription by transfecting AMH promoter-luc reporter constructs of different lengths in a prepubertal Sertoli cell line. Our results showed that FSH activates AMH transcription via adenylate cyclase, cAMP, and protein kinase A but involving a nonclassical cAMP-response pathway requiring nuclear factor-kappaB and activating protein 2 binding sites, which lie more than 1.9 kb upstream of the AMH transcription start site. This is the first report showing the importance of distant sequences in the regulation of AMH expression. We conclude that prepubertal testicular AMH production is increased by FSH stimulation through Sertoli cell proliferation and an enhancement of AMH gene transcription.


Subject(s)
Cyclic AMP/metabolism , Follicle Stimulating Hormone/metabolism , Glycoproteins/genetics , Glycoproteins/metabolism , Sertoli Cells/cytology , Testicular Hormones/genetics , Testicular Hormones/metabolism , Testis/physiology , Animals , Anti-Mullerian Hormone , Binding Sites , Cell Division/physiology , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Cyclic AMP-Dependent Protein Kinases/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Enzyme Inhibitors/pharmacology , Follicle Stimulating Hormone/genetics , Follicle Stimulating Hormone/pharmacology , Gene Expression Regulation , Male , Mice , Mice, Transgenic , NF-kappa B/genetics , NF-kappa B/metabolism , Promoter Regions, Genetic , Sertoli Cells/drug effects , Sertoli Cells/metabolism , Signal Transduction , Transcription Factor AP-2 , Transcription Factors/genetics , Transcription Factors/metabolism , Transcription Initiation Site , Transcription, Genetic
5.
Bol. Acad. Nac. Med. B.Aires ; 79(2): 249-261, jul.-dic. 2001. tab
Article in Spanish | BINACIS | ID: bin-6814

ABSTRACT

La infección por Helicobacter pylori (H. pylori) está asociada a enfermedades inflamatorias gastroduodenales y a neoplasias gástricas como adenocarcinomas y linfomas MALT. El objetivo del presente trabajo fue evaluar las distintas metodologías diagnósticas invasivas de esta infección en relación con los resultados obtenidos por biología molecular. Se estudiaron biopsias de mucosa antral de 39 pacientes con patologías gastroduodenales endoscópica e histológicamente confirmadas, sin antibiotecoterapia en curso. Del directo de biopsia se realizaron estudios citológicos, histológicos, coloración de Gram y test rápido de ureasa. El estudio microbiológico incluyó: cultivo en Agar Base Columbia, en Agar Skirrow (AS), coloración de Gram y tipificación bioquímica (test de ureasa, catalasa y oxidasa). Se realizó técnica de la reacción en cadena de la polimerasa (PCR) para diagnóstico de H. pylori a partir de material de biopsia y cultivo. Los resultados positivos para H. pylori, en los directos de biopsias, fueron: a) citología 76,9 por ciento, b) histología 74,3 por ciento, c) coloración de Gram 87,1 por ciento, d) test de ureasa 38,5 por ciento, e) cultivo en ABC 35,9 por ciento y f) cultivo en AS 74,3 por ciento. En todos los casos el diagnóstico de H. pylori por PCR fue positivo. Los métodos directos (citología, histología y coloración de Gram) mostraron ser económicos, sensibles y con altos resultados coincidentes (94,9 por ciento). De los métodos indirectos, el test de ureasa mostró baja sensibilidad; se obtuvo un alto porcentaje de recuperación de colonias con el medio de cultivo selectivo de AS. El diagnóstico positivo de infección por H. pylori con 2 o más métodos fue posible en el 97,2 por ciento de los casos. (AU)


Subject(s)
Humans , Helicobacter Infections/diagnosis , Helicobacter pylori/cytology , Minimally Invasive Surgical Procedures/methods , Molecular Biology/methods , Polymerase Chain Reaction/methods , Gastric Mucosa/microbiology , Biopsy/statistics & numerical data , Urease/diagnosis , Reference Standards
6.
Bol. Acad. Nac. Med. B.Aires ; 79(2): 249-261, jul.-dic. 2001. tab
Article in Spanish | LILACS | ID: lil-331241

ABSTRACT

La infección por Helicobacter pylori (H. pylori) está asociada a enfermedades inflamatorias gastroduodenales y a neoplasias gástricas como adenocarcinomas y linfomas MALT. El objetivo del presente trabajo fue evaluar las distintas metodologías diagnósticas invasivas de esta infección en relación con los resultados obtenidos por biología molecular. Se estudiaron biopsias de mucosa antral de 39 pacientes con patologías gastroduodenales endoscópica e histológicamente confirmadas, sin antibiotecoterapia en curso. Del directo de biopsia se realizaron estudios citológicos, histológicos, coloración de Gram y test rápido de ureasa. El estudio microbiológico incluyó: cultivo en Agar Base Columbia, en Agar Skirrow (AS), coloración de Gram y tipificación bioquímica (test de ureasa, catalasa y oxidasa). Se realizó técnica de la reacción en cadena de la polimerasa (PCR) para diagnóstico de H. pylori a partir de material de biopsia y cultivo. Los resultados positivos para H. pylori, en los directos de biopsias, fueron: a) citología 76,9 por ciento, b) histología 74,3 por ciento, c) coloración de Gram 87,1 por ciento, d) test de ureasa 38,5 por ciento, e) cultivo en ABC 35,9 por ciento y f) cultivo en AS 74,3 por ciento. En todos los casos el diagnóstico de H. pylori por PCR fue positivo. Los métodos directos (citología, histología y coloración de Gram) mostraron ser económicos, sensibles y con altos resultados coincidentes (94,9 por ciento). De los métodos indirectos, el test de ureasa mostró baja sensibilidad; se obtuvo un alto porcentaje de recuperación de colonias con el medio de cultivo selectivo de AS. El diagnóstico positivo de infección por H. pylori con 2 o más métodos fue posible en el 97,2 por ciento de los casos.


Subject(s)
Humans , Helicobacter Infections , Helicobacter pylori , Molecular Biology , Gastric Mucosa/microbiology , Polymerase Chain Reaction , Minimally Invasive Surgical Procedures/methods , Biopsy , Reference Standards , Urease
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