RESUMO
The high mobility group factor NUPR1, also known as p8 and com1, plays a role in temporal expression of the beta subunit of luteinizing hormone, LHB, during gonadotroph development. At Embryonic Day (e) 16.5, LHB is detectable in wild-type (Nupr1(+/+)) but not Nupr1 knockout (Nupr1(-/-)) mice. LHB is initiated by e17.5 in Nupr1(-/-) mice, and expression is fully recovered by Postnatal Day (p) 2. Factors indicative of pituitary maturation, GATA2, CGA, and TSH, are not differentially expressed in Nupr1(-/-) and Nupr1(+/+) embryos at e17.5. Therefore, the delay in LHB expression does not appear to result from delayed pituitary development. In addition, the role of NUPR1 in gonadotropin expression appears specific for LHB, as no difference in FSHB is observed in Nupr1(-/-) and Nupr1(+/+) embryos. The gonads are also impacted by the absence of NUPR1. Ovaries of female Nupr1(-/-) mice lack corpora lutea (CL) at 8 wk, an age at which CL are present in all Nupr1(+/+) littermates. Sexual maturity is recovered by 11 wk in Nupr1(-/-) mice. Conversely, the testes of Nupr1(-/-) males appear normal through 8 mo of age. By 10 mo, however, these mice develop a condition in which a significant number of seminiferous tubules lack germ cells, an abnormality reminiscent of human Sertoli-cell-only syndrome. NUPR1 is undetectable in Nupr1(+/+) gonadotrophs by p2 and remains absent in adulthood, but quantitative PCR analysis indicates Nupr1(+/+) adult ovaries and testes express Nupr1 mRNA. Therefore, the ovarian and testicular phenotypes may be due to the loss of NUPR1 directly at the gonads.
Assuntos
Proteínas de Ligação a DNA/genética , Hormônio Luteinizante Subunidade beta/genética , Proteínas de Neoplasias/genética , Ovário/embriologia , Síndrome de Células de Sertoli/genética , Testículo/patologia , Animais , Diferenciação Celular/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Embrião de Mamíferos , Feminino , Subunidade beta do Hormônio Folículoestimulante/genética , Subunidade beta do Hormônio Folículoestimulante/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Gonadotrofos/metabolismo , Gonadotrofos/fisiologia , Masculino , Camundongos , Camundongos Knockout , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/fisiologia , Ovário/crescimento & desenvolvimento , Ovário/metabolismo , Hipófise/embriologia , Hipófise/crescimento & desenvolvimento , Hipófise/metabolismo , Síndrome de Células de Sertoli/patologia , Maturidade Sexual/genética , Maturidade Sexual/fisiologia , Testículo/embriologia , Testículo/crescimento & desenvolvimento , Distribuição TecidualRESUMO
The 5-hydroxytryptamine type 3A receptor (5-HT3AR) is a ligand-gated cation channel activated by serotonin. This receptor is expressed throughout the nervous system as well as in the pituitary gland. Although it has been documented that the 5-HT3AR modulates exocytosis in neurons, its role in the pituitary gland has not been determined. Previous research has shown that the 5-HT3AR modulates circulating gonadotropin levels in vivo. It is unclear, however, if its activation in the pituitary gland mediates these effects or if receptors elsewhere in the hypothalamus-pituitary-gondal axis are responsible. To investigate the potential for the 5-HT3AR to modulate gonadotropin release from pituitary gonadotropes, the gonadotrope-derived LbetaT2 cell line was used as a model system and radioimmunoassays were employed to investigate how 5-HT3AR activation influences luteinizing hormone (LH) release. Our studies demonstrate that gonadotropin releasing hormone (GnRH)-stimulated LH release was decreased by the 5-HT3AR-specific antagonist MDL 72222 in a concentration-dependent manner. In addition, it was found that overexpressing the 5-HT3AR in LbetaT2 cells enhanced both basal and GnRH-stimulated LH release and also increased LHbeta gene promoter activity. These results suggest that the 5-HT3AR may participate in the hypothalamus-pituitary-gonadal axis at the level of the pituitary gonadotrope to mediate pituitary hormone release.
Assuntos
Hormônio Luteinizante Subunidade beta/genética , Hormônio Luteinizante/genética , Hormônio Luteinizante/metabolismo , Hipófise/metabolismo , Receptores 5-HT3 de Serotonina/biossíntese , Animais , Biguanidas/farmacologia , Northern Blotting , Linhagem Celular , Luciferases/biossíntese , Luciferases/genética , Hipófise/citologia , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Radioimunoensaio , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Antagonistas da Serotonina/farmacologia , Agonistas do Receptor de Serotonina/farmacologia , Tropanos/farmacologiaRESUMO
The serotonin type 3(A) receptor (5-HT3(A)R) is a ligand-gated ion channel (LGIC) that modulates a diverse set of cognitive and physiological functions. The 5-HT3(A)R, as with other LGICs, is a pentameric ion channel comprising five glycoprotein subunits. Although the N-terminal of the 5-HT3(A)R contains three putative N-linked glycosylation sites, the importance of each glycosylation site has not yet been established. To address this question, we used tunicamycin treatment and site-directed mutagenesis to inhibit selectively N-linked glycosylation at each site and then examined the effects of these treatments on receptor expression and function in transiently transfected heterologous cells. We show that the murine 5-HT3(A)R is glycosylated and that each N-linked glycosylation site plays a role in receptor regulation. Our findings suggest that N109 is necessary for receptor assembly, whereas N174 and N190 are important for plasma membrane targeting and ligand binding. Furthermore, we demonstrate that each site is necessary for 5-HT3(A)R-mediated Ca(2+) influx. We conclude that N-glycosylation is a critical step in the maturation, trafficking, and function of the murine 5-HT3(A)R.