RESUMO
The published online version contains mistakes in Table 1, Table 2 and Fig. 2. See below for the corrected Tables and Figure.
RESUMO
The gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), are important hormones in vertebrate reproduction. The isolation of gonadotropins from the pituitary gland is sub-optimal, as the cross-contamination of one hormone with another is common and often results in the variation in the measured activity of LH and FSH. The production of recombinant hormones is, therefore, a viable approach to solve this problem. This study aimed to express recombinant rat, mouse, and mastomys FSH and LH in Chinese hamster ovary (CHO) cells. Their common α-subunits along with their hormone-specific ß-subunits were encoded in a single mammalian expression vector. FSH from all three species was expressed, whereas expression was achieved only for the mouse LH. Immunohistochemistry for rat alpha subunit of glycoprotein hormone (αGSU) and LHß and FSHß subunits confirmed the production of the dimeric hormone in CHO cells. The recombinant rodent gonadotropins were confirmed to be biologically active; estradiol production was increased by recombinant FSH in granulosa cells, while recombinant LH increased testosterone production in Leydig cells.
Assuntos
Hormônio Foliculoestimulante/biossíntese , Vetores Genéticos , Hormônio Luteinizante/biossíntese , Animais , Células CHO , Cricetulus , Hormônio Foliculoestimulante/genética , Hormônio Luteinizante/genética , Masculino , Camundongos Endogâmicos C57BL , Murinae , Ratos Wistar , Proteínas Recombinantes/biossínteseRESUMO
Sox2-expressing stem/progenitor cells in the anterior lobe of the pituitary gland form two types of micro-environments (niches): the marginal cell layer and dense cell clusters in the parenchyma. In relation to the mechanism of regulation of niches, juxtacrine signaling via ephrin and its receptor Eph is known to play important roles in various niches. The ephrin and Eph families are divided into two subclasses to create ephrin/Eph signaling in co-operation with confined partners. Recently, we reported that ephrin-B2 localizes specifically to both pituitary niches. However, the Ephs interacting with ephrin-B2 in these pituitary niches have not yet been identified. Therefore, the present study aims to identify the Ephs interacting with ephrin-B2 and the cells that produce them in the rat pituitary gland. In situ hybridization and immunohistochemistry demonstrated cell type-specific localization of candidate interacting partners for ephrin-B2, including EphA4 in cells located in the posterior lobe, EphB1 in gonadotropes, EphB2 in corticotropes, EphB3 in stem/progenitor cells and EphB4 in endothelial cells in the adult pituitary gland. In particular, double-immunohistochemistry showed cis-interactions between EphB3 and ephrin-B2 in the apical cell membranes of stem/progenitor cell niches throughout life and trans-interactions between EphB2 produced by corticotropes and ephrin-B2 located in the basolateral cell membranes of stem/progenitor cells in the early postnatal pituitary gland. These data indicate that ephrin-B2 plays a role in pituitary stem/progenitor cell niches by selective interaction with EphB3 in cis and EphB2 in trans.
Assuntos
Efrina-B2/metabolismo , Hipófise/metabolismo , Ratos/metabolismo , Receptores da Família Eph/metabolismo , Animais , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Efrina-B2/análise , Hipófise/citologia , Hipófise/crescimento & desenvolvimento , Hipófise/ultraestrutura , Mapas de Interação de Proteínas , Ratos/crescimento & desenvolvimento , Ratos Wistar , Receptor EphB3/análise , Receptor EphB3/metabolismo , Receptores da Família Eph/análise , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
PROP1 is a pituitary specific transcription factor that plays a crucial role in pituitary organogenesis. The Prop1 shows varied expression patterns that promptly emerge and then fade during the early embryonic period. However, the regulatory mechanisms governing Prop1 expression remain unclear. Here, we investigated whether Prop1 was under epigenetic regulation by DNA methylation. Bisulfite sequencing was performed on DNA obtained from the pituitary glands and livers of rats on embryonic days (E) 13.5 and E14.5, and postnatal days (P) 4 and P30. The methylation of CpG sites in seven regions from 3-kb upstream of the Prop1 transcription start site through to its second intron were examined. Certain differences in CpG-methylation levels were observed in Region-1 (-2772 b to -2355 b), Region-4 (-198 b to +286 b), Region-5 (+671 b to +990 b), and Region-6 (+1113 b to +1273 b) based on comparisons between pituitary and liver DNA on E13.5. DNA methylation in pituitary glands on E14.5, P4, and P30 was generally similar to that observed in in the pituitary gland on E13.5, whereas the anterior and intermediate lobes of the pituitary gland on P4 and P30 showed only small differences. These results indicate that Prop1 is under regulation by CpG methylation during the early period of pituitary primordium development around E13.5.
Assuntos
Metilação de DNA , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Hipófise/embriologia , Hipófise/fisiologia , Animais , Biologia Computacional , Ilhas de CpG , DNA/metabolismo , Epigênese Genética , Feminino , Perfilação da Expressão Gênica , Fígado/metabolismo , Masculino , Ratos , Ratos Wistar , TemperaturaRESUMO
The anterior and intermediate lobes of the pituitary gland develop through invagination of the oral ectoderm and as they are endocrine tissues, they participate in the maintenance of vital functions via the synthesis and secretion of numerous hormones. We recently observed that several extrapituitary cells invade the anterior lobe of the developing pituitary gland. This raised the question of the origin(s) of these S100ß-positive cells, which are not classic endocrine cells but instead comprise a heterogeneous cell population with plural roles, especially as stem/progenitor cells. To better understand the roles of these S100ß-positive cells, we performed immunohistochemical analysis using several markers in S100ß/GFP-TG rats, which express GFP in S100ß-expressing cells under control of the S100ß promoter. GFP-positive cells were present as mesenchymal cells surrounding the developing pituitary gland and at Atwell's recess but were not present in the anterior lobe on embryonic day 15.5. These cells were negative for SOX2, a pituitary stem/progenitor marker, and PRRX1, a mesenchyme and pituitary stem/progenitor marker. However, three days later, GFP-positive and PRRX1-positive (but SOX2-negative) cells were observed in the parenchyma of the anterior lobe. Furthermore, some GFP-positive cells were positive for vimentin, p75, isolectin B4, DESMIN, and Ki67. These data suggest that S100ß-positive cells of extrapituitary origin invade the anterior lobe, undergoing proliferation and diverse transformation during pituitary organogenesis.
Assuntos
Células-Tronco Mesenquimais/metabolismo , Adeno-Hipófise/citologia , Adeno-Hipófise/metabolismo , Subunidade beta da Proteína Ligante de Cálcio S100/metabolismo , Animais , Biomarcadores , Feto , Imunofluorescência , Expressão Gênica , Genes Reporter , Masculino , Organogênese/genética , Hormônios Hipofisários/metabolismo , Regiões Promotoras Genéticas , Ratos , Subunidade beta da Proteína Ligante de Cálcio S100/genéticaRESUMO
The LIM-homeobox transcription factors LHX2 and LHX3s (LHX3a and LHX3b) are thought to be involved in regulating the pituitary glycoprotein hormone subunit genes Cga and Fshß. These two factors show considerable differences in their amino acid sequences for DNA binding and protein-protein interactions and in their vital function in pituitary development. Hence, we compared the DNA binding properties and transcriptional activities of Cga and Fshß between LHX2 and LHX3s. A gel mobility shift assay for approximately 1.1 kb upstream of Cga and 2.0 kb upstream of Fshß varied in binding profiles between LHX2 and LHX3s. DNase I footprinting revealed DNA binding sites in 8 regions of the Cga promoter for LHX2 and LHX3s with small differences in the binding range and strength. In the Fshß promoter, 14 binding sites were identified for LHX2 and LHX3, respectively. There were alternative binding sites to either gene in addition to similar differences observed in the Cga promoter. The transcriptional activities of LHX2 and LHX3s according to a reporter assay showed cell-type dependent activity with repression in the pituitary gonadotrope lineage LßT2 cells and stimulation in Chinese hamster ovary lineage CHO cells. Reactivity of LHX2 and LHX3s was observed in all regions, and differences were observed in the 5'-upstream region of Fshß. However, immunohistochemistry showed that LHX2 resides in a small number of gonadotropes in contrast to LHX3. Thus, LHX3 mainly controls Cga and Fshß expression.
Assuntos
Subunidade beta do Hormônio Folículoestimulante/metabolismo , Subunidade alfa de Hormônios Glicoproteicos/metabolismo , Proteínas com Homeodomínio LIM/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Animais , Sítios de Ligação , Células CHO , Linhagem Celular , Cricetinae , Cricetulus , Desoxirribonuclease I/metabolismo , Subunidade beta do Hormônio Folículoestimulante/genética , Subunidade alfa de Hormônios Glicoproteicos/genética , Imuno-Histoquímica , Camundongos , Hipófise/metabolismo , Regiões Promotoras Genéticas , Domínios Proteicos , SuínosRESUMO
Pituitary-specific transcription factor PROP1, a factor important for pituitary organogenesis, appears on rat embryonic day 11.5 (E11.5) in SOX2-expressing stem/progenitor cells and always coexists with SOX2 throughout life. PROP1-positive cells at one point occupy all cells in Rathke's pouch, followed by a rapid decrease in their number. Their regulatory factors, except for RBP-J, have not yet been clarified. This study aimed to use the 3 kb upstream region and 1st intron of mouse prop1 to pinpoint a group of factors selected on the basis of expression in the early pituitary gland for expression of Prop1. Reporter assays for SOX2 and RBP-J showed that the stem/progenitor marker SOX2 has cell type-dependent inhibitory and activating functions through the proximal and distal upstream regions of Prop1, respectively, while RBP-J had small regulatory activity in some cell lines. Reporter assays for another 39 factors using the 3 kb upstream regions in CHO cells ultimately revealed that 8 factors, MSX2, PAX6, PIT1, PITX1, PITX2, RPF1, SOX8 and SOX11, but not RBP-J, regulate Prop1 expression. Furthermore, a synergy effect with SOX2 was observed for an additional 10 factors, FOXJ1, HES1, HEY1, HEY2, KLF6, MSX1, RUNX1, TEAD2, YBX2 and ZFP36Ll, which did not show substantial independent action. Thus, we demonstrated 19 candidates, including SOX2, to be regulatory factors of Prop1 expression.