RESUMEN
The receptor for the pituitary glycoprotein hormone FSH (FSHR) and the nuclear hormone receptor steroidogenic factor 1 (SF-1) play important roles in control of the hypothalamic-pituitary- gonadal axis. FSHR is essential for integrating the pituitary FSH signal to gonadal response, while SF-1 is an important transcriptional regulator of many genes that function within this axis and is essential for the development of gonads and adrenal glands. Given the critical role of SF-1 in regulation of the gonads and the coexpression of FSHR and SF-1 in Sertoli and granulosa cells, we examined the ability of SF-1 to regulate transcription of the FSHR gene. We found that SF-1 stimulated rat FSHR promoter activity in a dose-dependent and promoter-specific manner. Examination of various promoter deletion mutants indicated that SF-1 acts through the proximal promoter region and upstream promoter sequences. An E box element within the proximal promoter is essential for activation of the FSHR promoter by SF-1. This element binds the transcriptional regulators USF1 and USF2 (upstream stimulatory factors 1 and 2) but not SF-1, as shown by electrophoretic mobility shift assays. In addition, functional studies identified a requirement for the USF proteins in SF-1 activation of FSHR and mapped an important regulatory domain within exons 4 and 5 of USF2. Cotransfection studies revealed that activation of protein kinase A leads to inhibition of SF-1-stimulated transcription of FSHR, while it synergized with SF-1 to activate the equine LH beta-promoter (ebeta). Thus, stimulation of the cAMP pathway differentially regulates SF-1 activation of the FSHR and ebeta-promoters.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Proteínas de Unión al ADN/fisiología , Regiones Promotoras Genéticas/fisiología , Receptores de HFE/fisiología , Factores de Transcripción/fisiología , Animales , Secuencia de Bases , AMP Cíclico/metabolismo , Sondas de ADN/química , Proteínas de Unión al ADN/antagonistas & inhibidores , Electroforesis en Gel de Poliacrilamida , Factores de Transcripción Fushi Tarazu , Regulación de la Expresión Génica/fisiología , Proteínas de Homeodominio , Humanos , Ratones , Datos de Secuencia Molecular , Mutación , Ratas , Receptores Citoplasmáticos y Nucleares , Receptores de HFE/biosíntesis , Receptores de HFE/genética , Factor Esteroidogénico 1 , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismo , Activación Transcripcional/fisiología , Transfección , Células Tumorales Cultivadas , Factores Estimuladores hacia 5'RESUMEN
Dmrt1 is a recently described gene that is expressed exclusively in the testis and is required for postnatal testis differentiation. Here we describe the expression of Dmrt1 in postnatal rat testis and Sertoli cells. RNase protection analysis was used to examine Dmrt1 messenger RNA (mRNA) levels in intact testis during postnatal development and in primary cultures of Sertoli cells under various culture conditions. We show that Dmrt1 mRNA levels rise significantly beginning approximately 10 days after birth and remain elevated until after the third postnatal week. Thereafter, mRNA levels drop coincident with the proliferation of germ cells in the testis. In freshly isolated Sertoli cells, Dmrt1 mRNA levels were robust but decreased significantly when the cells were placed in culture for 24 h. Treatment of Sertoli cells with either FSH or 8-bromo-cAMP resulted in a significant rise in Dmrt1 mRNA levels. This cAMP response was sensitive to treatment with the transcriptional inhibitor actinomycin D but not to the translational inhibitor cycloheximide. The cAMP-dependent rise in Dmrt1 mRNA also required activation of protein kinase A, as mRNA induction was sensitive to the inhibitor H89. Studies also show that Dmrt1 expression was inhibited by phorbol esters (PMA) but only modestly effected by serum.
Asunto(s)
Animales Recién Nacidos/fisiología , Hormona Folículo Estimulante/farmacología , Expresión Génica/efectos de los fármacos , Células de Sertoli/fisiología , Factores de Transcripción/genética , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Envejecimiento/metabolismo , Secuencia de Aminoácidos/genética , Animales , Animales Recién Nacidos/genética , Secuencia de Bases/genética , Células Cultivadas , Clonación Molecular , AMP Cíclico/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , ADN Complementario/genética , Activación Enzimática/fisiología , Inducción Enzimática/efectos de los fármacos , Masculino , Datos de Secuencia Molecular , ARN Mensajero/metabolismo , Ratas , Proteínas Recombinantes/farmacología , Células de Sertoli/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología , Transcripción GenéticaRESUMEN
Expression of the FSH receptor (FSHR) is limited to granulosa cells of the ovary and Sertoli cells of the testis. Previous studies showed that an E box in the proximal promoter of the FSHR gene is required for transcription and that the predominant E box binding proteins are the ubiquitous transcription factors, upstream stimulatory factor 1 (USF1) and USF2. Through cotransfection analysis, we have shown that both wild-type and dominant negative forms of the USF proteins regulate the rat FSHR promoter and that transcriptional activation of FSHR required several domains within the amino-terminal portion of the USF proteins. Analysis of the FSHR promoter region using in vivo genomic footprinting indicated that the E box is occupied by proteins in Sertoli cells but not in cells that fail to express the receptor, despite the presence of the USF proteins. To help delineate the regions of the rat FSHR gene required for correct spatial and temporal expression, transgenic mice harboring two constructs containing variable amounts of 5'-flanking sequence (5,000 bp and 100 bp) were generated. Examination of 16 different transgenic lines revealed varied transgene expression profiles with multiple lines having different amounts of ectopic expression and two lines failing to express the transgene. In addition, little or no expression was observed in Sertoli cells. These studies indicate that additional regulatory sequences outside the region from -5,000 to +123 bp are needed for proper expression in Sertoli cells.
Asunto(s)
Proteínas de Unión al ADN , Receptores de HFE/genética , Células de Sertoli/fisiología , Factores de Transcripción/metabolismo , Transcripción Genética , Proteínas Virales , Animales , Animales Recién Nacidos , Secuencia de Bases , Femenino , Regulación de la Expresión Génica , Integrasas/genética , Masculino , Ratones , Ratones Endogámicos , Ratones Transgénicos , Datos de Secuencia Molecular , Mutación , Especificidad de Órganos , Regiones Promotoras Genéticas , Estructura Terciaria de Proteína , Ratas , Receptores de HFE/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Testículo/fisiología , Factores de Transcripción/genética , Activación Transcripcional , Factores Estimuladores hacia 5'RESUMEN
Steroidogenic factor 1 (SF-1), also known as adrenal 4-binding protein, is a member of the nuclear hormone receptor family that regulates transcription of genes encoding hormones and steroidogenic enzymes important to the function of the hypothalamic-pituitary-gonadal axis. The mammalian Ftz-F1 gene encodes SF-1 and is required for development of adrenal glands and gonads. To better understand the mechanisms regulating this gene in the gonads, we have examined its expression in the testis and characterized the promoter region for SF-1 in two testicular cell types. SF-1 promoter activity was examined in primary cultures of Sertoli cells and cell lines representative of Sertoli and Leydig cells. Deletion mutagenesis of the promoter identified several regions: both 5' and 3' to the transcriptional start sites that are important for transcriptional activity. Two elements, an E box and a CCAAT box, were found to be important for SF-1 transcription in the testis. An oligodeoxynucleotide containing both of these elements bound three specific protein complexes. The binding of one complex required only sequences within the E box and cross-reacted with antibodies against the basic helix-loop-helix ZIP proteins USF1 and USF2. A second specific complex required sequences within both the E box and CCAAT box for efficient binding, while a third complex predominantly interacted with sequences within the CCAAT motif. The presence of multiple protein complexes binding these sites suggests that regulation through these elements may involve interactions with different factors that depend on the state of the cell and its environment.
Asunto(s)
Proteínas de Unión al ADN/genética , Secuencias Reguladoras de Ácidos Nucleicos , Testículo/fisiología , Factores de Transcripción/genética , Animales , Secuencia de Bases , Reacciones Cruzadas , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción Fushi Tarazu , Regulación de la Expresión Génica , Proteínas de Homeodominio , Células Intersticiales del Testículo/metabolismo , Masculino , Ratones , Datos de Secuencia Molecular , Mutación , Regiones Promotoras Genéticas , Ratas , Ratas Sprague-Dawley , Receptores Citoplasmáticos y Nucleares , Elementos de Respuesta/genética , Células de Sertoli/metabolismo , Factor Esteroidogénico 1 , Testículo/citología , Factores de Transcripción/inmunología , Factores de Transcripción/metabolismo , Transcripción Genética , Factores Estimuladores hacia 5' , Dedos de Zinc/genéticaRESUMEN
The FSH receptor (FSHR) is expressed only in granulosa cells of the ovary and Sertoli cells of the testis. This highly specific pattern of gene expression asserts that transcriptional events unique to these two cell types are responsible for activation of the FSHR gene. We have characterized the promoter elements required for activity of the rat FSHR gene in a Sertoli cell line MSC-1, primary cultures of rat Sertoli cells, and two non-Sertoli cell lines. Transient transfection analysis of deletion and block replacement mutants identified several elements, both 5' and 3' to the transcriptional start sites, that are essential for full promoter activity in Sertoli cells. These studies confirmed the use of an important E box element (CACGTG), which had the single greatest impact on promoter function. Bases within the core CACGTG of the E box, as well as flanking sequences, were shown to be essential for its function. Electrophoretic mobility shift assays identified both upstream stimulatory factor 1 (USF1) and USF2 as primary components of the complexes binding the E box. Sequence requirements for USF binding in vitro modestly diverged from the sequence requirements for in vivo function of the element. Comparison of the E box binding proteins in different cell types revealed that similar proteins bind the E box in Sertoli and non-Sertoli cell lines. Extracts from primary cultures of rat and mouse Sertoli cells have a second E box-binding complex that cross-reacts with USF antibodies that is not present in the cell lines.
Asunto(s)
Regiones Promotoras Genéticas , Receptores de HFE/genética , Células de Sertoli/metabolismo , Animales , Emparejamiento Base , Secuencia de Bases , Células Cultivadas , Reacciones Cruzadas , Proteínas de Unión al ADN/inmunología , Proteínas de Unión al ADN/metabolismo , Masculino , Ratones , Datos de Secuencia Molecular , Mutación , Ratas , Receptores de HFE/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Factores de Transcripción/inmunología , Factores de Transcripción/metabolismo , Transcripción Genética , Factores Estimuladores hacia 5'RESUMEN
The pituitary glycoprotein hormones LH and FSH regulate the reproductive cycle and are sensitive to feedback by gonadal steroids. The common alpha-subunit shared by these hormones is transcriptionally repressed by androgen receptor (AR) in the presence of its ligand dihydrotestosterone. This identifies at least one mechanism that contributes to AR-dependent suppression of gonadotropin synthesis. Repression of alpha-subunit transcription by AR requires only the sequences within the first 480 bp of the promoter. While this region contains a high-affinity binding site for AR, this element does not mediate the suppressive effects of androgens. Instead, two other elements within the promoter-regulatory region (alpha-basal element and cAMP-regulatory element), which are important for expression of the alpha-subunit gene in gonadotropes, mediate the effects of AR. This suggests that AR inhibits activity of the alpha-subunit promoter by interfering with the transcriptional properties of the proteins that bind to alpha-basal element and the cAMP-regulatory elements. Furthermore, transfection analysis of various mutant ARs identified both the DNA-binding and ligand-binding domains of the receptor as critical for repression. Comparisons with the MMTV promoter revealed distinct structural requirements that underlie the transactivation and transrepression properties of AR.
Asunto(s)
ADN/metabolismo , Hormona Folículo Estimulante/metabolismo , Hormona Luteinizante/metabolismo , Hipófisis/metabolismo , Receptores Androgénicos/metabolismo , Transducción de Señal , Activación Transcripcional , Animales , Sitios de Unión , Proteínas de Unión al ADN/metabolismo , Hormona Folículo Estimulante/genética , Ligandos , Hormona Luteinizante/genética , Ratones , Receptores Androgénicos/genéticaRESUMEN
The alpha subunit gene encodes a common subunit shared by all glycoprotein hormones. This single copy gene is expressed in pituitary gonadotropes and thyrotropes of all mammals and in placental trophoblasts of primates and horses. Tandem cAMP response elements (CREs) in the promoter of the human gene are key mediators of this pattern of cell-specific expression. Replacing the palindromic CREs with non-primate variant CREs significantly attenuated activity in trophoblasts but not in gonadotropes. Furthermore, proteins binding the palindromic CRE cross-reacted with antibodies for CREB, CREM, ATF1, ATF2, and c-Jun, while proteins binding the variant CRE cross-reacted only with ATF2 and c-Jun antibodies. The data suggest that ATF2 and c-Jun can activate transcription through the CREs in gonadotropes but not in trophoblasts. Additional analyses indicated that while promoters with either palindromic or variant CREs have similar overall activity in gonadotropes, the variant CREs make a much smaller contribution to promoter activity than their palindromic counterparts. The weaker contribution of the variant CREs is compensated by the activity of two upstream elements present in the promoter. This compensation probably occurs through an indirect mechanism, as the binding affinity of proteins to the CRE is not influenced by the presence of these upstream elements.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas de Unión al ADN , Hormonas Glicoproteicas de Subunidad alfa/genética , Gónadas/metabolismo , Trofoblastos/metabolismo , Factor de Transcripción Activador 1 , Factor de Transcripción Activador 2 , Electroforesis en Gel de Poliacrilamida , Humanos , Leucina Zippers , Mutagénesis Sitio-Dirigida , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-jun/metabolismo , Factores de Transcripción/metabolismoRESUMEN
To identify elements of the human alpha subunit gene necessary for cell-specific expression, we generated an array of block mutations spanning approximately 400 base pairs (bp) of promoter proximal region and examined them using transient transfection analysis in pituitary (alpha T3) and placental (BeWo) cell lines. Comparison of promoter activity in the two cell types revealed both common and unique elements required for transcription in pituitary and placenta. Two strong elements, the cyclic AMP response element (CRE) and the upstream regulatory element (URE), regulate expression of the alpha subunit gene in BeWo cells. In contrast, promoter activity in alpha T3 cells requires an array of weaker elements. These include the CREs, the URE, as well as two previously described elements, pituitary glycoprotein hormone basal element (PGBE) and gonadotrope-specific element (GSE), and two new elements we designated as the alpha basal elements 1 and 2 (alpha BE1 and alpha BE2). These new elements reside between -316 and -302 bp (alpha BE1) and -296 and -285 bp (alpha BE2) of the human alpha subunit promoter and bind distinct proteins designated alpha BP1 and alpha BP2, respectively. Southwestern blot analysis revealed that alpha BE1 specifically binds 54- and 56-kDa proteins. Additional studies disclosed several potential interactions between proteins that bind the CRE and proteins that occupy PGBE, alpha BE1, and alpha BE2, suggesting that gonadotrope-specific expression occurs through a unique composite regulatory element that includes components of the placenta-specific enhancer.
Asunto(s)
Regulación de la Expresión Génica , Hormonas Glicoproteicas de Subunidad alfa/biosíntesis , Hominidae/genética , Hipófisis/metabolismo , Placenta/metabolismo , Regiones Promotoras Genéticas , Secuencias Reguladoras de Ácidos Nucleicos , Animales , Secuencia de Bases , Línea Celular , Sistema Libre de Células , Clonación Molecular , Cartilla de ADN , Femenino , Humanos , Luciferasas/biosíntesis , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Reacción en Cadena de la Polimerasa , Embarazo , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes/biosíntesis , Transfección , beta-Galactosidasa/biosíntesisRESUMEN
Recently, a mouse Sertoli cell line (MSC-1) was established from transgenic mice carrying a fusion gene composed of human Müllerian inhibitory substance transcriptional regulatory sequences linked to the SV40 T-antigen gene. This cell line contained a morphologically heterogeneous population of cells that expressed characteristic Sertoli cell mRNAs such as transferrin, the beta-subunit for inhibin, and sulfated glycoprotein-2 (SGP-2). In this study, we compared various characteristics of MSC-1 cells to primary cultures of Sertoli cells from immature rats or adult mice. Our observations indicated that: 1) These cells were ultrastructurally similar to mouse Sertoli cells in culture; 2) MSC-1 cells expressed mRNA for androgen-binding protein (ABP) and SGP-1, but not the receptor for FSH; 3) The expression of SGP-2 mRNA and secretion of SGP-2 increased approximately 2-fold when cells were cultured at 41 degrees C, the nonpermissive temperature for the SV40 virus, while SGP-1 and transferrin mRNA levels and secretion were unaffected; 4) Proliferation of these cells was serum-dose dependent and temperature dependent and could be inhibited by incubating MSC-1 cells at 41 degrees C; 5) Proliferation was also significantly reduced after cells were incubated in the presence of dibutyryl cAMP (dbcAMP) for 6 days; 6) Fifteen subcultures produced from single MSC-1 cells displayed similar levels of mRNA expression for transferrin or SGP-1. Together these data indicate that the MSC-1 cell line is composed of a single cell type displaying numerous characteristics of Sertoli cells. This cell line may provide a unique source of tissue for studying various aspects of Sertoli cell behavior.
Asunto(s)
Chaperonas Moleculares , Células de Sertoli/metabolismo , Proteína de Unión a Andrógenos/genética , Animales , Hormona Antimülleriana , Antígenos Transformadores de Poliomavirus/genética , División Celular , Línea Celular , Clusterina , Glicoproteínas/genética , Glicoproteínas/metabolismo , Inhibidores de Crecimiento/genética , Masculino , Ratones , Ratones Transgénicos , ARN Mensajero/metabolismo , Ratas , Receptores de HFE/genética , Proteínas Recombinantes de Fusión , Secuencias Reguladoras de Ácidos Nucleicos , Saposinas , Células de Sertoli/ultraestructura , Hormonas Testiculares/genética , Transferrina/genética , Transferrina/metabolismoRESUMEN
Testicular androgens suppress the synthesis and secretion of the pituitary gonadotropins, in particular, luteinizing hormone. This suppressive effect includes transcription of both the common alpha subunit gene and the unique beta subunit genes. Herein, we demonstrate that 1500 base pairs (bp) of proximal 5'-flanking region derived from the human alpha subunit gene and a shorter 315-bp segment of the bovine alpha subunit gene confer negative regulation by androgen to the gene encoding bacterial chloramphenicol acetyltransferase in transgenic mice. Cotransfection assays with human androgen receptor indicated that the 1500-bp promoter region of the human alpha subunit gene also confers androgen regulation (transcriptional suppression) to reporter genes in both pituitary and placental cell lines. This raises the possibility of a role for DNA binding in suppression of alpha subunit transcription by activated androgen receptor. Consistent with this possibility, we have used a gel-mobility shift assay to detect several high affinity binding sites for androgen receptor located in the proximal promoter of the human alpha subunit gene. The strongest androgen receptor binding site is located at approximately -101 in the proximal 5'-flanking region. This steroid receptor binding site overlaps another binding site that defines one of several contiguous cis-acting regulatory elements required for basal transcriptional activity. Thus, binding of activated androgen receptor to this region may block the binding of a requisite trans-acting factor and lead to an attenuation in transcription. We conclude that this interaction, which occurs directly at the level of the pituitary, represents one of several physiological avenues through which androgens regulate gonadotropin gene expression.
Asunto(s)
Andrógenos/fisiología , Hormonas Glicoproteicas de Subunidad alfa/genética , Regiones Promotoras Genéticas , Receptores Androgénicos/metabolismo , Transcripción Genética , Animales , Secuencia de Bases , Cloranfenicol O-Acetiltransferasa/antagonistas & inhibidores , Cloranfenicol O-Acetiltransferasa/genética , Cloranfenicol O-Acetiltransferasa/metabolismo , ADN/metabolismo , Estrógenos/fisiología , Regulación de la Expresión Génica , Humanos , Masculino , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Orquiectomía , Receptores Androgénicos/genética , Secuencias Reguladoras de Ácidos NucleicosRESUMEN
We have characterized a series of rat genomic clones that code for the FSH receptor (FSHR) gene and approximately 14.8 kilobases of DNA up-stream of the transcriptional start sites. Southern blot analysis indicated that there was only a single gene for the FSHR. Primer extension and S1 nuclease experiments revealed the presence of two major transcriptional start sites at positions -80 and -98 relative to the translational start site. Transient expression studies of a fusion gene containing 830 basepairs of DNA 5' to the translational start site linked to the reporter gene chloramphenicol acyltransferase have shown that this portion of the gene is capable of acting as a transcriptional promoter in rat Sertoli cells. The FSHR gene contained 10 exons and nine introns. The first nine exons encoded the extensive amino-terminal domain of the receptor, while the last exon encoded the transmembrane-spanning and cytoplasmic domains. A repeated motif similar to that observed in the leucine-rich glycoprotein family was delineated within exons 2-9. Comparison of the FSHR gene to the LH receptor gene revealed a number of striking similarities which clearly indicate that these receptors evolved through gene duplication. The ancestral gene for these receptors presumably arose from a series of tandem duplications of the leucine-rich motif, which when combined with the common ancestral gene of the G-protein-coupled receptor family led to the current gene structure of the glycoprotein hormone receptors.
Asunto(s)
Receptores de HFE/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Evolución Biológica , Clonación Molecular/métodos , Exones/fisiología , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/fisiología , Ratas , Receptores de HL/genética , Homología de Secuencia de Ácido NucleicoRESUMEN
In order to better understand the role of FSH in reproduction, we have studied the expression of its receptor in the male rat. A DNA probe for the rat FSH receptor (FSHR) was generated by the polymerase chain reaction, and a partial genomic clone was isolated. Northern blot analysis revealed two transcripts for the FSHR, a 2.6-kilobase (kb) transcript, which is the predominant mRNA, and a 4.5-kb transcript. The two transcripts were specific to the testis and ovary, and in the testis, the expression of the mRNA appeared to be primarily in the Sertoli cells. Northern blot analysis showed the presence of FSHR mRNA in testes from rats ranging in age from 10-60 days as well as in Sertoli cells cultured from 20-day-old and adult animals. FSHR mRNA levels in testes synchronized to specific stages of the seminiferous epithelium were shown by Northern blot analysis to be highest in stages XIII-II and to decrease to a minimum at stages VII-VIII. The levels of FSHR mRNA underwent more than a 3-fold change during the cycle of the seminiferous epithelium.
Asunto(s)
ARN Mensajero/metabolismo , Receptores de HFE/genética , Células de Sertoli/metabolismo , Testículo/metabolismo , Envejecimiento , Animales , Secuencia de Bases , Northern Blotting , Epitelio/metabolismo , Expresión Génica , Masculino , Datos de Secuencia Molecular , Ratas , Ratas Endogámicas , Receptores de HFE/biosíntesis , Túbulos Seminíferos/metabolismoRESUMEN
The data presented in this manuscript are all based on some inferences drawn from past experimental observations. The first is that the synchronized testis model is representative of the normal testis. Support for this premise comes from the studies on SGP-2, SGP-1 and transferrin where results using in situ hybridization and Northern blots are similar for normal and for synchronized testes. The second inference is that normalizing all of the data to the relative levels of SGP-1 mRNA adjusts for the possible differential loading of mRNA samples. The logic of this practice is based on the observation obtained using in situ hybridization that SGP-1 mRNA levels did not change across the cycle. The third assumption was that total mRNA levels do not change greatly across the cycle. Wholesale changes in testicular mRNA such as the doubling of all of the mRNA transcripts per testis would not be accounted for by these studies. We feel that this is an unlikely complication because of the strong correlation between much of the data and the known biology. In addition, there is a strong correlation between our data on the FSHR mRNA and the binding data for FSH obtained by another laboratory and different techniques. The available data in the literature reveals that most of the Sertoli cell products which change in relative concentrations during the cycle of the seminiferous epithelium appear to have a maximum in either stages VII or IX or in stages XIII to III. Thus, the Sertoli cells in the cycle could be described has having two different functional modes. In mode A maximal levels of mRNA for a specific Sertoli cell product are roughly found in stages VII-IX and in mode B the maximal levels are found in stages XIII to III (Fig. 5). The distribution of the receptor mRNA and ABP mRNA can also be described in terms of these two modes of Sertoli cell function. Transferrin receptors, retinoic acid receptors, and androgen-binding protein appear to fall into mode A while FSH and androgen receptors fall into mode B (Table 1). Products which have antithetical functions such as FSH and inhibin or cystatin and cathepsin L are found in different modes. We propose that most of the actions of the Sertoli cell during the cycle can be specified by the dual modes described above rather than by an infinite number of operational modes.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Receptores de Superficie Celular/biosíntesis , Epitelio Seminífero/metabolismo , Animales , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Expresión Génica , Masculino , Receptores Androgénicos/biosíntesis , Receptores Androgénicos/genética , Receptores de HFE/biosíntesis , Receptores de HFE/genética , Receptores de Ácido Retinoico , Receptores de Transferrina/biosíntesis , Receptores de Transferrina/genética , Tretinoina/metabolismoRESUMEN
The 2-oxoglutarate dehydrogenase complex was isolated from the cellular slime mould, Dictyostelium discoideum, and purified 113-fold. The enzyme exhibited Michaelis-Menten kinetics and the Km values for 2-oxoglutarate, CoA, and NAD were 1.0 mM, 0.002 mM, and 0.07 mM, respectively. The Ki value for succinyl-CoA was determined to be 0.004 mM and the Ki for NADH was 0.018 mM. AMP had positive effects whereas ATP had negative effects on the enzyme activity. The kinetic constants determined in this study and the reaction mechanism suggested can now be incorporated into a transition model of the tricarboxylic acid cycle during differentiation of D. discoideum.