RESUMEN

BACKGROUND: The formation of the male urethra depends to enzyme-mediated testosterone (T) conversion into 5α-dihydrotestosterone (DHT). Two metabolic pathways could be operating in the fetal testis to synthesize androgens: 1) the "classic" route (T→DHT) mediated by SRD5A2 and 2) a "backdoor" pathway in which DHT is synthesized by aldo-keto reductase family 1, member C2 (AKR1C2), AKR1C3, and AKR1C4 enzymes without formation of a T intermediate. OBJECTIVE: We studied four genes of the "backdoor" pathway in karyotypic males with hypospadias to ascertain whether gene defects in AKRs impair urethral DHT formation that result in hypospadias. DESIGN AND PATIENTS: The coding regions of the AKR1C2-4 and HSD17B6 genes were analyzed by PCR-SSCP and sequencing in a cohort of 25 Mexican patients (0.3-9 year-old-children) with 46,XY-hypospadias. Chi-squared tests was performed to evaluate the distribution of genotypes, alleles, and the Hardy-Weinberg (H-W) equilibrium. The effect of the genetic variants was investigated by in silico studies. RESULTS: Screening studies revealed distinct genotypic patterns at different exons of AKR1C2-4 whereas HSD17B6 presented a wild-type sequence. The DNA analyses detected two synonymous variants (c.327C>T, c.666T>C/unreported) in AKR1C2. The AKR1C3 had two variants (c.15C>G, c.230A>G), two unreported variants (c.538T>C, c.596G>A), and one silent variant (c.312G>A). Two variants (c.434C>G, c.931C>G) were identified in AKR1C4. All variants were in H-W equilibrium without structural changes. DISCUSSION: Hypospadias have been associated with defects that alter androgen biosynthesis in the human fetal testis, specifically 5α-DHT. We selected four candidate genes involved in the "backdoor" pathway for the formation of 5α-DHT. Molecular assays of the AKR1C2, AKR1C3, and AKR1C4 genes revealed a total of nine genetic single nucleotide variants. Several variants in the AKR1C genes have been associated with a variety of human pathologies. However, our studies suggest that active steroid biosynthesis via AKR1C might not be involved in hypospadias. Additionally, genetic research suggests a low involvement in the "backdoor" 5α-DHT pathway during human sexual development, specifically, the differentiation of male external genitalia. CONCLUSION: These results indicate that substitutions in AKR1C2-4 are polymorphisms and all genetic variants lacks deleterious significant association with hypospadias. The data suggest that inactivating mutations in the AKR1C2-4 and HSD17B6 genes are an infrequent cause of hypospadias, which might weaken the contribution of the "backdoor" pathway to embryonic urethral masculinization.

Asunto(s)

Hipospadias , 3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/genética , Miembro C3 de la Familia 1 de las Aldo-Ceto Reductasas , Andrógenos , Niño , Preescolar , Dihidrotestosterona , Femenino , Humanos , Hidroxiesteroide Deshidrogenasas/genética , Hipospadias/genética , Lactante , Masculino , Proteínas de la Membrana , Biología Molecular , Oxidorreductasas , Racemasas y Epimerasas , Testosterona

RESUMEN

Human steroid 5α-reductase 2 (SRD5A2) plays a determinative role in the masculinization of external genitalia. To date, approximately 114 different mutations of the SRD5A2 gene have been reported; however, little information is available about their impact on catalytic function or their three-dimensional (3D) structures. We determined the effect of point mutations on the testosterone-depend kinetic constants (Km,app and Vmax,app) and structural characteristics of SRD5A2 from Mexican patients with 46,XY-steroid 5α-reductase 2 deficiency. PCR-SSCP assays identified ten distinct gene variants and sequencing analysis identified missense mutations [p.V3I, p.S14R, p.A52T, p.F118L, p.R145W, p.R171S, p.L226P, p.F229S, p.S245Y, and p.A248V]. Mutations were re-created by site-directed mutagenesis and expressed in HEK293 cells. Functional studies demonstrated that 8 variants led to partial (Km,app = 0.16-2.6 µM; Vmax,app = 224-2640 pmol/mg P/min) or complete losses of activity compared to the wild-type enzyme (Km,app = 0.7 µM; Vmax,app = 4044 pmol/mg P/min). All the mutations were assessed using multiple software tools and the results predicted that all of the mutations were associated with disease or damage. Mapping mutations on the model of a 3D structure of SRD5A2 demonstrated alterations in contact sites with their proximal amino acids. Our data show that mutations affect the catalytic efficiency (Vmax/Km) or result in residual enzymatic activity, which could be due to erroneous interactions between amino acid residues, the substrate testosterone, or NADPH.

Asunto(s)

3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/genética , Proteínas de la Membrana/genética , 3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/química , 3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/deficiencia , 3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/metabolismo , Análisis Mutacional de ADN , Células HEK293 , Humanos , Cinética , Proteínas de la Membrana/química , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/metabolismo , Simulación del Acoplamiento Molecular , Mutagénesis Sitio-Dirigida

RESUMEN

ATP-Binding Cassette, subfamily B, member 6 (ABCB6) is a transporter that is upregulated by elevated intracellular porphyrin concentrations. In the Harderian gland (HG), the synthesis of porphyrins appears to be under the influence of gonadal steroids and to exhibit a dimorphic pattern. To explore whether ABCB6 is also influenced by sex steroids, we isolated its specific cDNA sequence and investigated its mRNA levels in the HGs of hamsters. ABCB6's cDNA sequence presents an open reading frame (ORF) of 2529 bp that encodes a predicted 842-amino acid (aa) protein with a molecular weight of 93 kDa. Multiple sequence alignments showed that ABCB6's aa sequence is highly conserved and shares the highest homology (93%) with mouse ABCB6. RT-qPCR analysis indicated that ABCB6 is expressed in all the tissues examined, exhibiting high expression levels in the liver, adrenal glands, and testis. The mRNA concentrations of ABCB6 in HGs were very similar between males and in females; similarly, gonadectomy and treatment with sex steroids appear to scarcely affect ABCB6 mRNA levels. The intraglandular content of ABCB6 mRNA showed discrete, though non-significant, variations through the estrous cycle. The results provide evidence that gonadal steroids have a minimal physiological role on the regulation of ABCB6 expression and might indicate that this transporter has a small effect on porphyrin trafficking in the HGs of hamsters. The authors would like to apologise for any inconvenience caused.

RESUMEN

ATP-Binding Cassette, subfamily B, member 6 (ABCB6) is a transporter that is upregulated by elevated intracellular porphyrin concentrations. In the Harderian gland (HG), the synthesis of porphyrins appears to be under the influence of gonadal steroids and to exhibit a dimorphic pattern. To explore whether ABCB6 is also influenced by sex steroids, we isolated its specific cDNA sequence and investigated its mRNA levels in the HGs of hamsters. ABCB6's cDNA sequence presents an open reading frame (ORF) of 2529 bp that encodes a predicted 842-amino acid (aa) protein with a molecular weight of 93 kDa. Multiple sequence alignments showed that ABCB6's aa sequence is highly conserved and shares the highest homology (93%) with mouse ABCB6. RT-qPCR analysis indicated that ABCB6 is expressed in all the tissues examined, exhibiting high expression levels in the liver, adrenal glands, and testis. The mRNA concentrations of ABCB6 in HGs were very similar between males and in females; similarly, gonadectomy and treatment with sex steroids appear to scarcely affect ABCB6 mRNA levels. The intraglandular content of ABCB6 mRNA showed discrete, though non-significant, variations through the estrous cycle. The results provide evidence that gonadal steroids have a minimal physiological role on the regulation of ABCB6 expression and might indicate that this transporter has a small effect on porphyrin trafficking in the HGs of hamsters.

Asunto(s)

Transportadoras de Casetes de Unión a ATP/metabolismo , Hormonas Esteroides Gonadales/metabolismo , Glándula de Harder/metabolismo , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Cricetinae , Femenino , Hormonas Esteroides Gonadales/aislamiento & purificación , Masculino , Mesocricetus , Alineación de Secuencia

RESUMEN

Androgen insensitivity syndrome (AIS) is an X-linked disorder caused by mutations in the NR3C4 gene, which encodes the androgen receptor (AR). In this study, we performed mutational analyses to identify AR molecular defects, in individuals with 46,XY disorders of sex development (46,XY DSD) and a presumptive diagnosis of AIS. Eighteen different gene mutations, including seven previously unreported new variants, were detected in 26 unrelated cases. These included two deletion mutations (P49fs*185 and E308f*320) in exon 1 and five substitution mutations (p.S792P, p.D829G, p.R832P, p.L839F, and p.K906E) located in the steroid-binding domain. Expression analyses of mutants generated by site-directed mutagenesis indicated that these new gene variants impaired AR function by affecting its binding activity. Seventeen of 18 mutations likely lead to reduced or absent responses to androgens, which may in turn account for the different degrees of undermasculinization observed. Our study provides insight into the functional consequences of these mutations.

Asunto(s)

Síndrome de Resistencia Androgénica/genética , Trastornos del Desarrollo Sexual/genética , Receptores Androgénicos/genética , Diferenciación Sexual/genética , Adolescente , Adulto , Niño , Preescolar , Análisis Mutacional de ADN , Femenino , Humanos , Lactante , Masculino , México , Mutación/genética , Adulto Joven

RESUMEN

According to current knowledge, two steroid 5α-reductases, designated type 1 (SRD5A1) and type 2 (SRD5A2), are present in all species examined to date. These isozymes play a central role in steroid hormone physiology by catalyzing the reduction of 3-keto-4-ene-steroids into more active 5α-reduced derivatives, including the conversion of testosterone (T) to dihydrotestosterone (DHT). A third 5α-reductase (SRD5A3, -type 3), which is overexpressed in hormone-refractory prostate cancer cells, has been identified; however, its enzymatic characteristics are practically unknown. Here, we isolated a cDNA encoding hamster Srd5a3 (hSrd5a3) and performed functional metabolic assays to investigate its biochemical properties. The cloned cDNA encodes a 330 amino acid protein that is 87% identical to the homologous protein in mice and 78% to that in humans. However, hSrd5a3 exhibits low sequence homology with its counterparts hSrd5a1 (19%) and hSrd5a2 (17%). A fusion protein consisting of hSrd5a3 and green fluorescent protein provided evidence for cytoplasmic localization in transfected mammalian cells. Real-time PCR analysis revealed that, Srd5a3 mRNA was present in nearly all hamster tissues, with high expression in the cerebellum, Harderian gland and testis. Functional assays expressing hSrd5a3 cDNA in HEK-293 cells revealed that this isozyme is unable to reduce T into DHT. Further expression assays confirmed that similar to testosterone, progesterone, androstenedione and corticosterone are not reduced by hSrd5a3 or human SRD5A3. Together, these results indicate that hSrd5a3 lacks the catalytic activity to transform 3-keto-4-ene-compounds; therefore 5α-reductase type 3 may not be involved in 5α-reduction of steroids.

Asunto(s)

3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/fisiología , 3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Biocatálisis , Secuencia Conservada , Cricetinae , Dihidrotestosterona/química , Femenino , Células HEK293 , Humanos , Masculino , Mesocricetus , Datos de Secuencia Molecular , Especificidad de Órganos , Oxidación-Reducción

RESUMEN

Ferrochelatase (protohaem ferrolyase, EC 4.99.1.1), the terminal enzyme of the haem biosynthetic pathway, catalyses the insertion of ferrous iron into protoporphyrin IX to form protohaem. The Syrian hamster Harderian gland (HG) is known for its ability to produce and accumulate large amounts of protoporphyrins. In this species, the female gland contains up to 120 times more porphyrin than the male gland. Data from biochemical studies suggest that this gland possesses the enzymatic complex for haem biosynthesis but lacks ferrochelatase activity. The abundance of intraglandular haem proteins does not support this idea. To gain more insight into this process, we isolated cDNA for ferrochelatase from hamster liver, using the 5'- and 3'- rapid amplification of complementary DNA ends (RACE), and investigated its expression in HG from males and females. The full-length cDNA comprises an open reading frame of 1269 bp encoding a polypeptide of 422 amino-acid residues. Hamster DNA sequence exhibits 92% identity to mouse and 87% identity to human sequences. The predicted hamster enzyme was shown to have structural features of mammalian ferrochelatase, including a putative NH2- terminal presequence, a central core of about 330 amino-acid residues and an extra 30-50-amino-acid stretch at the carboxyl-terminus. RNA blotting experiments indicated that this cDNA hybridized to a liver mRNA of about 2.1 kb, while a weak hybridization signal was observed with mRNA from HG preparations. RT-PCR assays confirmed the expression of specific transcripts in both tissues. Male glands contained approximately twofold more enzyme mRNA than female glands. Likewise, the intraglandular content of mRNA varied during the oestrous cycle, with the highest levels found in the oestrous phase. These cyclic variations were less evident in liver. Ovariectomy plus treatment with progesterone or 17beta-oestradiol plus progesterone increased ferrochelatase mRNA of the gland. In HG of short- or long-term castrated males, the administration of testosterone did not affect the ferrochelatase mRNA concentration. Based on mRNA expression levels, we conclude that Harderian ferrochelatase may play an active role in maintaining the physiological pool of haem required for processing cytochromes and other glandular haem proteins. Likewise, the sex-steroid hormones appear to have only a modest influence upon Harderian ferrochelatase.

Asunto(s)

Ferroquelatasa/genética , Hormonas Esteroides Gonadales/fisiología , Glándula de Harder/enzimología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting/métodos , Castración , Clonación Molecular/métodos , Cricetinae , ADN Circular/genética , Estradiol/administración & dosificación , Estradiol/fisiología , Estro/fisiología , Femenino , Expresión Génica/genética , Hígado/enzimología , Masculino , Mesocricetus , Progesterona/administración & dosificación , Progesterona/fisiología , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Testosterona/administración & dosificación , Testosterona/fisiología

RESUMEN

Aetiology of mixed gonadal dysgenesis (MGD) has not been completely elucidated. Molecular analyses have failed to demonstrate the presence of mutations in sex-determining region on Y chromosome (SRY); it has been suggested that these individuals may bear mutations in other genes involved in the testis-determining pathway. Desert hedgehog's (DHH) importance regarding male sex differentiation has been demonstrated in various studies we describe here, for the first time, two cases of MGD in which a monoallelic single base deletion in DHH is associated with the disorder. Genomic DNA was isolated from paraffin-embedded gonad tissue from 10 unrelated patients with MGD and three controls; in addition to, DNA from peripheral blood leukocytes in 100 controls. Coding sequence abnormalities in DHH were assessed by exon-specific PCR, single-stranded conformation polymorphism (SSCP) and direct sequencing. In two patients, a heterozygous 1086delG in exon 3 was found. Comparing previously described mutations in DHH to the one observed in this study, we can affirm that the phenotypic spectrum of patients with gonadal dysgenesis due to mutations in DHH is variable. This study continues to demonstrate the importance that DHH has in mammalian male sexual differentiation, providing extended evidence that DHH constitutes a key gene in gonadal differentiation.

Asunto(s)

Disgenesia Gonadal/genética , Mutación , Transactivadores/genética , Adolescente , Adulto , Preescolar , Exones , Tamización de Portadores Genéticos , Proteínas Hedgehog , Humanos , Lactante , Cariotipificación , Masculino , Valores de Referencia

RESUMEN

The KAL1 gene has a closely related nonfunctional pseudogene on the Y chromosome; a high degree of X-Y sequence similarity is observed. Some individuals present a T to C substitution at position 1833 (exon 12). Because this nucleotide differs in the X (thymine) and in the Y (cytosine) chromosome, we investigated if this was truly a polymorphism, or if in some cases the Y sequence had been amplified. The complete sequence of exon 12 of KAL1 was analyzed in 11 Kallmann Syndrome (KS) males, in 50 normal males, in 50 normal females, and in 16 patients with Ullrich-Turner Syndrome (UTS). Nucleotide 1833 was found in a heterozygous or a homozygous state in KS, normal males and normal females; UTS patients were always homozygous. Of the 61 males, 17 were heterozygous, while 11 were TT and 33 were CC. With these observations we can not assure whether these patients present a "real" polymorphism. Besides, all males were heterozygous in nucleotides 1678, 1694, 1699, 1708 and 1825, whilst females were homozygous; and in these positions, KAL1 also differs from its pseudogene. These results indicate that we are identifying the X and the Y nucleotide and these variants are not polymorphisms. Sequence variations may be pseudogene products rather than true polymorphisms, so we should always determine if the position where the variation is located differs between KAL1 and its pseudogene, because it has been suggested that the presence of various polymorphisms in affected individuals could be the cause of KS.

Asunto(s)

Proteínas de la Matriz Extracelular/genética , Proteínas del Tejido Nervioso/genética , Adulto , Cromosomas Humanos X , Cromosomas Humanos Y , Exones/genética , Femenino , Hormonas/sangre , Humanos , Síndrome de Kallmann/genética , Masculino , Datos de Secuencia Molecular , Polimorfismo Genético/genética , Sondas ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Síndrome de Turner/genética

RESUMEN

To investigate the pituitary-testicular function in nephrotic rats, a sequence of experiments was undertaken in adult male rats after a single dose of puromycin aminonucleoside (PAN). Endocrine modifications were evaluated chronologically throughout the experimental disease in order to determine the appearance of hormone alterations which lead to the axis dysfunction. Serum concentration of LH, FSH, androstenedione, total and free testosterone, estradiol as well as urine testosterone were measured by specific RIAs on days 3, 7 and 10 after treatment on nephrotic and control groups. Prolactin was also evaluated on day 10. Likewise, total weight of various androgen responsive tissues from both groups was recorded, and the number of androgen receptor (AR) binding sites were determined. To know the functional status of the hipophyseal-testicular unit, groups of nephrotic and control rats were stimulated with LHRH (300 ng/100 g b.w.) or with one or four doses of hCG (8 UI), respectively. Additionally, the relative in vitro biological activity of FSH from nephrotic and control rats before and after LHRH stimulus was determined. The results from the hormonal profile revealed clear endocrine disorders characterized by a progressive diminution of all serum hormones except prolactin and urine testosterone, which remained unmodified. The weight of the main androgen responsive tissues, the ventral prostate and the seminal vesicle, decreased parallelly to androgen diminution. The binding analysis of AR shows a significant elevation of the available androgen sites in all analyzed tissues except kidney and hypothalamus. The secretion of LH and FSH from nephrotic animals after LHRH administration was lower than that from intact animals at the registered times. Interestingly, the biological activity of FSH from nephrotic rats was not detectable at both, before and after LHRH administration. Testicular response to hCG stimuli, in terms of testosterone synthesis was not significantly different in the two groups analyzed with respect to the intact animals. By contrast, no response was observed in terms of estradiol production at either one or four doses of hCG. On the whole, the results presented herein allow us to conclude that experimental nephrosis has a harmful effect on the pituitary-testicular axis, and strongly suggests that the endocrine dysfunction is initiated at the hypophyseal level; even though a specific testicular damage is also present.

Asunto(s)

Nefrosis/fisiopatología , Hipófisis/fisiopatología , Testículo/fisiopatología , Androstenodiona/sangre , Animales , Gonadotropina Coriónica/administración & dosificación , Gonadotropina Coriónica/farmacología , AMP Cíclico/inmunología , AMP Cíclico/metabolismo , Estradiol/sangre , Hormona Folículo Estimulante/sangre , Hormona Liberadora de Gonadotropina/administración & dosificación , Hormona Liberadora de Gonadotropina/farmacología , Hipotálamo/fisiología , Hormona Luteinizante/sangre , Masculino , Nefrosis/sangre , Nefrosis/inducido químicamente , Prolactina/sangre , Puromicina Aminonucleósido/toxicidad , Radioinmunoensayo , Ratas , Ratas Wistar , Receptores Androgénicos/metabolismo , Testosterona/sangre , Testosterona/orina

RESUMEN

In this study we report the cloning and sequencing of a cDNA for cholesterol side chain cleavage cytochrome P450scc from Syrian hamster adrenal glands. Isolation of P450scc mRNA was carried out with degenerate primer PCR together with 5' and 3' RACE protocol. The full-length cDNA comprises an open reading frame of 1563 bp encoding a polypeptide of 520 amino acid residues. The predicted protein sequence exhibits well-preserved heme- and steroid-binding domains and shares 89% amino acid sequence identity with rat and mouse enzymes. Transient transfection of HEK-293 cells with the cloned cDNA leads to the formation of pregnenolone from 25-hydroxycholesterol. Northern blot analysis showed expression of mRNAs for P450scc in the major steroidogenic tissues, namely, the adrenal cortex, testis, and ovary. In addition, tissue distribution analysis using the coupled reaction of RT-PCR and Southern blotting revealed that the mRNA of the enzyme is also expressed in various nonendocrine tissues, including the epididymis, Harderian gland, and lungs. The relative abundance of specific transcripts at these novel sites suggests that P450scc could potentially play an important role in regulating local steroid hormone synthesis.

Asunto(s)

Glándulas Suprarrenales/enzimología , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Clonación Molecular , ADN Complementario/genética , Expresión Génica , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Southern Blotting , Línea Celular , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/química , Cricetinae , Embrión de Mamíferos , Humanos , Riñón , Mesocricetus/genética , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , ARN Mensajero/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección

RESUMEN

Androgen insensitivity syndrome (AIS) is an X-linked genetic disorder of male sexual differentiation caused by mutations in the androgen receptor (AR) gene. A reliable genotype-phenotype correlation in these patients does not exist as yet. Here we report the molecular studies performed on eight individuals with AIS. Exon-specific polymerase chain reaction (PCR), single-strand conformation polymorphism, and sequencing analyses, were performed in exons 2 to 8 of the AR gene. In one case, total cellular RNA was extracted from genital skin fibroblasts and reverse transcriptase-PCR was performed. Six different point mutations leading to amino acid substitutions (P682T, Q711E, G743E, F827V, H874R, D879Y), one splice-junction mutation (g-->c at +5, exon 6/intron 6), and a missense mutation without amino acid substitution (S888S) were identified. All mutations, including a de novo mutation, were previously undescribed on the steroid binding domain. Of the eight mutations identified, four led to a complete female phenotype (codons 743, 827, 874 and the donor splice site +5), two were detected in phenotypic females with partial virilization (codons 682 and 711), and two were present in phenotypic male subjects with undervirilized external genitalia, thus indicating that all of these sites determine AR functional activity.

Asunto(s)

Síndrome de Resistencia Androgénica/genética , Mutación , Receptores Androgénicos/genética , Adolescente , Niño , Preescolar , Exones , Femenino , Fibroblastos/metabolismo , Genotipo , Humanos , Lactante , Intrones , Masculino , Mutación Missense , Fenotipo , Mutación Puntual , Reacción en Cadena de la Polimerasa , Polimorfismo Conformacional Retorcido-Simple , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Procesos de Determinación del Sexo

Asunto(s)

Genes MHC Clase II , Genes MHC Clase I , Indígenas Norteamericanos/genética , Genotipo , Humanos , México/etnología

RESUMEN

The androgen insensitivity syndrome (AIS) is an X-linked form of male pseudohermaphroditism caused by mutations in the androgen receptor (AR) gene. In the present study, we analyzed the AR gene in 8 patients, 4 sporadic and 2 familial cases with the syndrome, using exon-specific polymerase chain reaction, single-stranded conformational polymorphism and sequencing analysis and identified six new single base mutations, including one nonsense mutation at the hinge region of the receptor. These molecular lesions occurred in the steroid-binding domain (SBD) and all but one affected the first nucleotide of their respective codons. A nonsense mutation in exon 4, which converts a glutamine into a premature termination signal (Q657stop), a missense mutation changing arginine instead of glycine (G743R) and a conservative substitution of leucine with valine at amino acid 830 (L830V) were detected in patients with CAIS. Three other missense mutations located in exons 4 (L701I), 5 (A765S), and 6 (Q802R) were present in individuals bearing a partial form of AIS. These data allow us to reaffirm the view that nonsense mutations in the AR results almost invariably in a CAIS phenotype and underly the importance of the SBD for the AR functional activity.

Asunto(s)

Síndrome de Resistencia Androgénica/genética , Receptores Androgénicos/genética , Adulto , Sustitución de Aminoácidos , Sitios de Unión/genética , Codón sin Sentido , ADN/análisis , ADN/genética , Exones , Humanos , Masculino , México , Mutación Missense

RESUMEN

This investigation addresses the potential regulation of enzymes involved in the biosynthesis of steroid hormones during early stages of gonadal development by follicle-stimulating hormone (FSH). Gonadal cells of 10-day-old chick embryo and cells of the left ovary of 18-day-old chick embryo were cultured for 60 h in a defined medium with or without the addition of FSH (2.0 IU/ml). At the end of the culture, cells were recovered and evaluated by biotransformation of tritiated steroid precursors and mRNA levels were evaluated by RT-PCR. The production of estrone from androstenedione was increased in the FSH-treated cells, both human FSH (hFSH) and recombinant human FSH (rhFSH), indicating a stimulatory effect on aromatase (P450arom). Similarly, the intensity of the band corresponding to P450arom mRNA was higher in hFSH and rhFSH than in control and chorionic gonadotropin (hCG) groups. The P450arom stimulation was observed in the ovary of 10- and 18-day-old chick embryo. The transformation of dehydroepiandrosterone to androstenedione was taken as evidence of 3beta-hydroxysteroid dehydrogenase function. This enzyme was stimulated in the cultured ovarian cells of 18-day-old chick embryos treated with hFSH and rhFSH compared with controls. The production of pregnenolone in the mitocondrial fraction of 18-day-old chick embryo ovary was increased when cultured with hFSH and rhFSH. This observation together with the increase in the band intensity corresponding to mRNA of P450 cholesterol side-chain cleavage indicates stimulation by FSH treatment; hCG produced a similar effect. Somatic cells of the medullary cords are proposed to be FSH target cells in the ovary of the chick embryo.

Asunto(s)

Hormona Folículo Estimulante/farmacología , Ovario/embriología , Ovario/enzimología , Esteroides/biosíntesis , 3-Hidroxiesteroide Deshidrogenasas/metabolismo , Androstenodiona/biosíntesis , Androstenodiona/metabolismo , Animales , Aromatasa/genética , Aromatasa/metabolismo , Células Cultivadas , Embrión de Pollo , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/metabolismo , Gonadotropina Coriónica/farmacología , Deshidroepiandrosterona/metabolismo , Estrona/biosíntesis , Femenino , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Ovario/efectos de los fármacos , Pregnenolona/biosíntesis , Progesterona/metabolismo , ARN Mensajero/análisis , Proteínas Recombinantes/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Testosterona/metabolismo , Factores de Tiempo , Tritio

RESUMEN

Steroid 5alpha-reductase 2 deficiency is an autosomal recessive form of male pseudohermaphroditism caused by mutations in the SRD5A2 gene. In this study, we performed DNA analyses in two unrelated subjects bearing the enzyme deficiency and found differences in the mode of transmission for the disease. The data showed that in both families the fathers were carriers for an E197D mutation, whereas the mothers were carriers for a P212R mutation. Patient 1 was identified as compound heterozygote because he had both alterations (E197D/P212R). On the contrary, patient 2 was found to be homozygous, but only for the paternal mutation. Because this finding could not be explained on the basis ofnonpaternity or a chromosomal abnormality, the presence of uniparental disomy was suggested. The reduction to homozygosity for the E197D mutation, as confirmed by restriction analysis, supported this view. The results of our study give evidence of the first case of 5alpha-reductase deficiency resulting from uniparental disomy and also disclose an alternate mechanism whereby this enzymatic disorder can derive from a single parent.

Asunto(s)

3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/deficiencia , 3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/genética , Adulto , Autorradiografía , Southern Blotting , ADN/genética , Exones/genética , Genoma , Disgenesia Gonadal 46 XY/genética , Humanos , Masculino , Mutación , Polimorfismo Conformacional Retorcido-Simple , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

Although accumulating data show that placenta is able to synthesize 1,25-dihydroxyvitamin D3, the presence of cytochrome P(450) enzyme capable of converting 25-hydroxyvitamin D3 (250HD(3)) to the biologically active form of vitamin D in this tissue, has not been yet clearly established. In this study, we have investigated the presence of 25-hydroxyvitamin D3 1alpha-hydroxylase (1alpha-(OH)ase) gene expression products in cultured human syncytiotrophoblast. Total RNA was isolated from cultured placental cells and subjected to Northern blots or RT-PCR by using 1alpha-(OH)ase-specific primers. The amplified complementary DNA fragments were analyzed by gel electrophoresis and nucleotide sequencing. Total RNA from kidney HEK 293 cells was subjected to reverse transcriptase reaction, and a 298-bp complementary DNA 1alpha-(OH)ase probe was generated by PCR. Primary cultures of human syncytiotrophoblasts exhibited 1alpha-(OH)ase activity, and a transcript for this gene could be demonstrated in these cells. Northern blot analysis revealed the presence of a 2.5-kb product, similar in size to that previously reported in kidney. RT-PCR analysis demonstrated the presence of a single transcript with nucleotide sequence identical to that previously reported for human 1alpha-(OH)ase complementary DNA clones. In addition, data are presented which suggest that differentiation of cytotrophoblast to the syncytial state was not necessary for this gene to be expressed, which may indicate a role of this enzyme all through pregnancy. The overall results of this study provide evidence for the presence of 1alpha-(OH)ase in the human placenta, suggesting that conversion of 25OHD(3) to 1,25-dihydroxyvitamin D3 in the trophoblast is most probably attributed to an enzymatic 1alpha-hydroxylation reaction.

Asunto(s)

25-Hidroxivitamina D3 1-alfa-Hidroxilasa/biosíntesis , Células Gigantes/enzimología , Trofoblastos/enzimología , 25-Hidroxivitamina D3 1-alfa-Hidroxilasa/genética , Adulto , Secuencia de Bases , Células Cultivadas , ADN Complementario/biosíntesis , ADN Complementario/genética , Femenino , Regulación Enzimológica de la Expresión Génica/genética , Células Gigantes/citología , Humanos , Riñón/metabolismo , Datos de Secuencia Molecular , Placenta/metabolismo , Embarazo , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

In Ullrich-Turner syndrome (UTS) patients, the presence of a Y-chromosome or Y-derived material has been documented in frequencies ranging from 4-61%. Mutations of SRY (testis-determining gene) constitute the cause of XY sex reversal in approximately 10-15% of females with pure gonadal dysgenesis. Most of these mutations have been described in the HMG (high mobility group) box of the gene, which is the region responsible for DNA binding and bending; however, various mutations outside the HMG box have been reported. We carried out molecular studies of the SRY gene in three patients with a UTS phenotype and bilateral streaks; two presented a 45,X/46,XY mosaic, and the third a Y marker chromosome. In two patients a missense mutation, S18N, was identified in the 5' non-HMG box region in DNA from blood and both streaks; this mutation was not identified in 75 normal males. Sequencing of the DNA region of interest was normal in the father and older brother of patient 1, demonstrating that in this patient the mutation was de novo. A previous report of a 46,XY patient with partial gonadal dysgenesis who presented the same mutation as our patients indicates the probable existence of a hot spot in this region of the SRY gene and strengthens the possibility that all gonadal dysgeneses constitute part of a spectrum of the same disorder. It also demonstrates that a single genetic abnormality can result in a wide range of phenotypic expression.

Asunto(s)

Proteínas de Unión al ADN/genética , Mosaicismo , Mutación Missense , Proteínas Nucleares , Factores de Transcripción , Síndrome de Turner/genética , Cromosoma Y , Adolescente , Sustitución de Aminoácidos , ADN/sangre , Femenino , Humanos , Hibridación Fluorescente in Situ , Cariotipificación , Masculino , Núcleo Familiar , Procesos de Determinación del Sexo , Proteína de la Región Y Determinante del Sexo

RESUMEN

BACKGROUND AND OBJECTIVE: Mutations of the steroid 5alpha-reductase type 2 (SRD5A2) gene in karyotypic males result in a spectrum of external genitalia phenotypes ranging from complete female to nearly complete male. Here we performed genomic DNA analyses from individuals bearing the enzyme deficiency in order to detect the molecular abnormalities. PATIENTS: Four unrelated 46,XY patients of Mexican origin with ambiguous external genitalia were studied. A fertile, phenotypically normal male was also included. MEASUREMENTS: Coding sequence abnormalities of the SRD5A2 gene were assessed by exon-specific polymerase chain reaction, single-stranded conformational polymorphism and sequencing analysis. RESULTS: Five different missense mutations (two of them novel mutations) were identified. Three subjects presented homozygous single base mutations. These were located at exon 2 (G115D), exon 4 (P212R) and exon 5 (R246Q), and such changes have been described previously. The fourth patient was a compound heterozygote who presented two mutations located in exons 1 and 2. We found a hitherto unreported G --> A transition at the second nucleotide of codon 85 in exon 1 (GGC --> GAC), substituting glycine for aspartic acid (G85D). This patient also presented an identical alteration at codon 115 of exon 2, which was carried by his father (G115D). Finally, in another subject who was included originally as a control, we found a C --> A transversion (yet undescribed) at codon 245 in exon 5 (S245Y). CONCLUSIONS: Four different single base mutations that cause amino acid substitutions were detected in the steroid 5alpha-reductase type 2 gene of affected individuals. One patient and a normal control had two previously undescribed mutations. Although in the latter individual we cannot exclude the possibility that the base change is a genetic polymorphism, the molecular screening of 100 chromosomes suggests strongly that the change at codon 245 does represent a heterozygous mutation. Further studies, including the recreation of the mutations, will help to reveal the biochemical consequences resulting from these changes.

Asunto(s)

3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/deficiencia , 3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/genética , Mutación Missense , Adulto , Niño , Trastornos del Desarrollo Sexual/sangre , Trastornos del Desarrollo Sexual/genética , Heterocigoto , Humanos , Lactante , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Polimorfismo Conformacional Retorcido-Simple , Análisis de Secuencia de ADN , Testosterona/análogos & derivados , Testosterona/sangre

RESUMEN

Estrogens are involved in the gonadal morphogenesis of vertebrates, and almost all hormonal effects of 17beta-estradiol are mediated through specific receptors. At the time of sexual differentiation in the chicken, or even before, there is evidence of the presence of estrogen receptors and the secretion of 17beta-estradiol. However, no information is available regarding the cellular types that express the estrogen receptor in the immature chick ovary. The present study analyzes estrogen receptor expression in germ and somatic cells of the ovary in the newly hatched chicken. Highly purified cell subpopulations of germ and somatic cells were evaluated for specific 17beta-estradiol nuclear binding. In addition, the estrogen receptor was localized at the ultrastructural level by the immunogold technique. Finally, reverse transcription and polymerase chain reaction procedures detected a steady-state level of mRNA for the estrogen receptor. Somatic cells including typical steroidogenic cells showed specific 17beta-estradiol nuclear binding, displayed the estrogen receptor, and possessed estrogen receptor transcripts. The same result was observed in primary oocytes, together with the ultrastructural localization of estrogen receptor in extended chromatin filaments. Our experimental data support the hypothesis that estrogens are involved in the function of somatic and germ cells subpopulations in the immature chicken ovary.

Asunto(s)

Ovario/citología , Ovario/metabolismo , Receptores de Estrógenos/metabolismo , Animales , Secuencia de Bases , Pollos , Cartilla de ADN/genética , Estradiol/metabolismo , Femenino , Expresión Génica , Inmunohistoquímica , Microscopía Inmunoelectrónica , Oocitos/metabolismo , Oocitos/ultraestructura , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Estrógenos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa