ERRγ1 and Aromatase Expression in Human Placental Tissues from Term Deliveries of Large for Gestational Age (LGA) Newborns.

INTRODUCTION: Being born either large (LGA) or small for gestational age (SGA) has been associated with an increased risk of developing metabolic syndrome in adulthood. However, the mechanism underlying this early programming remained unclear. Estrogen-related receptor gamma (ERRγ) is an orphan nuclear receptor with a high expression in human placenta, particularly ERRγ1. ERRγ has been proposed to play a central role in controlling genes involved in energy metabolism. In placenta, ERRγ1 acts as an oxygen-responsive transcription factor regulating aromatase (Aro) expression during trophoblast differentiation. Aromatase is an enzyme that catalyzes the synthesis of estrogens from androgens and is located in the syncytiotrophoblast. An adequate estrogen-androgen balance is required for normal pregnancy progression. Our aim was to analyze ERRγ1 and Aro mRNA in human placenta from term LGA newborns. We propose that ERRγ1 and CYP19A1 expressions in human placenta from LGA newborns are impaired, which would modify fetal programming of LGA newborns, since an imbalance in intrauterine estrogen-androgen ratio would be occurred Methods: Total RNA was obtained from placental tissues of LGA (GA: 39-41 weeks, n=8) and adequate for gestational age (AGA; 39-40 weeks, n=10) newborns. ERRγ1 and Aro mRNA variants were analyzed by RT2-PCR. Primers for Aro analysis were specific for Total aromatase (TotalAro) binding in exons 2-3 and for Active aromatase (ActAro) in exons 9-10. Aro protein was analyzed by Western-blot. RESULTS: ERRγ1 mRNA was significantly higher in LGA compare to AGA. TotalAro mRNA was significantly lower in LGA in comparison with AGA control. Similar results with Aro protein. In contrast ActAro/TotalAro ratio was higher in LGA compared to the AGA control. CONCLUSIONS: High expression of ERRγ1 as well as ActAro/TotalAro ratio in LGA suggests that ERRγ1 is involved in ActAro variant expression and hence disrupted estrogen-androgen balance in the intrauterine environment. We propose that dysregulation of ERRγ1 in placenta might modify the estrogen-androgen balance in the intrauterine environment in LGA newborns, possibly representing one of the key factors in the regulation of fetal programming.

Endocannabinoid signaling impairs syncytialization: Using flow cytometry to evaluate forskolin-induced cell fusion.

Cytotrophoblast cells fuse to form the syncytiotrophoblast, the main structure responsible for the placenta's specialized functions. This complex process denominated syncytialization is fundamental for a correct pregnancy outcome. We observed that the endocannabinoid anandamide disrupts syncytialization employing traditional techniques and flow cytometry in BeWo cell line.

Estrogens in Human Male Gonadotropin Secretion and Testicular Physiology From Infancy to Late Puberty.

Several reports in humans as well as transgenic mouse models have shown that estrogens play an important role in male reproduction and fertility. Estrogen receptor alpha (ERα) and beta (ERß) are expressed in different male tissues including the brain. The estradiol-binding protein GPER1 also mediates estrogen action in target tissues. In human testes a minimal ERα expression during prepuberty along with a marked pubertal up-regulation in germ cells has been reported. ERß expression was detected mostly in spermatogonia, primary spermatocytes, and immature spermatids. In Sertoli cells ERß expression increases with age. The aromatase enzyme (cP450arom), which converts androgens to estrogens, is widely expressed in human tissues (including gonads and hypothalamus), even during fetal life, suggesting that estrogens are also involved in human fetal physiology. Moreover, cP450arom is expressed in the early postnatal testicular Leydig cells and spermatogonia. Even though the aromatase complex is required for estrogen synthesis, its biological relevance is also related to the regulation of the balance between androgens and estrogens in different tissues. Knockout mouse models of aromatase (ArKO) and estrogen receptors (ERKOα, ERKOß, and ERKOαß) provide an important tool to study the effects of estrogens on the male reproductive physiology including the gonadal axis. High basal serum FSH levels were reported in adult aromatase-deficient men, suggesting that estrogens are involved in the negative regulatory gonadotropin feedback. However, normal serum gonadotropin levels were observed in an aromatase-deficient boy, suggesting a maturational pattern role of estrogen in the regulation of gonadotropin secretion. Nevertheless, the role of estrogens in primate testis development and function is controversial and poorly understood. This review addresses the role of estrogens in gonadotropin secretion and testicular physiology in male humans especially during childhood and puberty.

VIP Promotes Recruitment of Tregs to the Uterine-Placental Interface During the Peri-Implantation Period to Sustain a Tolerogenic Microenvironment.

Uterine receptivity and embryo implantation are two main processes that need a finely regulated balance between pro-inflammatory and tolerogenic mediators to allow a successful pregnancy. The neuroimmune peptide vasoactive intestinal peptide (VIP) is a key regulator, and it is involved in the induction of regulatory T cells (Tregs), which are crucial in both processes. Here, we analyzed the ability of endogenous and exogenous VIP to sustain a tolerogenic microenvironment during the peri-implantation period, particularly focusing on Treg recruitment. Wild-type (WT) and VIP-deficient mice [heterozygous (HT, +/-), knockout (KO, -/-)], and FOXP3-knock-in-GFP mice either pregnant or in estrus were used. During the day of estrus, we found significant histological differences between the uterus of WT mice vs. VIP-deficient mice, with the latter exhibiting undetectable levels of FOXP3 expression, decreased expression of interleukin (IL)-10, and vascular endothelial growth factor (VEGF)c, and increased gene expression of the Th17 proinflammatory transcription factor RORγt. To study the implantation window, we mated WT and VIP (+/-) females with WT males and observed altered FOXP3, VEGFc, IL-10, and transforming growth factor (TGF)ß gene expression at the implantation sites at day 5.5 (d5.5), demonstrating a more inflammatory environment in VIP (+/-) vs. VIP (+/+) females. A similar molecular profile was observed at implantation sites of WT × WT mice treated with VIP antagonist at d3.5. We then examined the ability GFP-sorted CD4+ cells from FOXP3-GFP females to migrate toward conditioned media (CM) obtained from d5.5 implantation sites cultured in the absence/presence of VIP or VIP antagonist. VIP treatment increased CD4+FOXP3+ and decreased CD4+ total cell migration towards implantation sites, and VIP antagonist prevented these effects. Finally, we performed adoptive cell transfer of Tregs (sorted from FOXP3-GFP females) in VIP-deficient-mice, and we observed that FOXP3-GFP cells were mainly recruited into the uterus/implantation sites compared to all other tested tissues. In addition, after Treg transfer, we found an increase in IL-10 expression and VEGFc in HT females and allowed embryo implantation in KO females. In conclusion, VIP contributes to a local tolerogenic response necessary for successful pregnancy, preventing the development of a hostile uterine microenvironment for implantation by the selective recruitment of Tregs during the peri-implantation period.

Accelerated Pubertal Tempo in a 46,XY Aromatase-Deficient Patient.

BACKGROUND: Aromatase deficiency is a rare autosomal recessive disorder. 46,XY-affected patients often remain undiagnosed until late puberty. Only 2 pediatric cases have been reported. Data on pubertal development in affected males are scarce. AIM: To report the clinical phenotype and hormonal studies of an aromatase-deficient boy during the prepubertal and early pubertal period. RESULTS: The patient was the older brother of a 46,XX girl with aromatase deficiency. Molecular analysis revealed a previously reported homozygous mutation (Arg192Cys) in the CYP19A1 gene. Pubertal onset was at 9.8 years. At 11.3 years of age, signs of rapidly progressive puberty were seen. Laboratory tests revealed normal pubertal basal and GnRH-stimulated gonadotropin levels, normal Sertoli cell markers, and increased testosterone. The prepubertal lumbar spine bone mineral density (BMD) was normal but pubertal bone mineral accrual was incomplete, leading to osteopenia. CONCLUSION: Estrogen restraint on gonadotropin secretion has been demonstrated in animal and human models. Interestingly, our patient presented with accelerated puberty and apparently normal pituitary gonadal function. These findings suggest that aromatase activity may be required to define pubertal progression in boys. Estrogen deficiency due to aromatase deficiency is responsible for insufficient bone mineral accrual during puberty.

DNA methylation is not involved in specific down-regulation of HSD3B2, NR4A1 and RARB genes in androgen-secreting cells of human adrenal cortex.

We hypothesized that DNA methylation is involved in human adrenal functional zonation. mRNAs expression and methylation pattern of RARB, NR4A1 and HSD3B2 genes in human adrenal tissues (HAT) and in pediatric virilizing adrenocortical tumors (VAT) were analyzed. For analysis of the results samples were divided into 3 age groups according to FeZ involution, pre and post-adrenarche ages. In all HAT, similar RARB mRNA was found including microdissected zona reticularis (ZR) and zona fasciculata, but HSD3B2 and NR4A1 mRNAs were lower in ZR (p < 0.05). NR4A1 and RARB promoters remained unmethylated in HAT and VAT. No adrenal zone-specific differences in NR4A1 methylation were observed. In summary, RARB was not associated with ZR-specific downregulation of HSD3B2 in postnatal human adrenocotical zonation. DNA methylation would not be involved in NR4A1 adrenocortical cell-type specific downregulation. Lack of CpG islands in HSD3B2 suggested that HSD3B2 ZR-specific downregulation would not be directly mediated by DNA methylation.

An Intron 9 CYP19 Gene Variant (IVS9+5G>A), Present in an Aromatase-Deficient Girl, Affects Normal Splicing and Is Also Present in Normal Human Steroidogenic Tissues.

BACKGROUND/AIMS: Splicing CYP19 gene variants causing aromatase deficiency in 46,XX disorder of sexual development (DSD) patients have been reported in a few cases. A misbalance between normal and aberrant splicing variants was proposed to explain spontaneous pubertal breast development but an incomplete sex maturation progress. The aim of this study was to functionally characterize a novel CYP19A1 intronic homozygote mutation (IVS9+5G>A) in a 46,XX DSD girl presenting spontaneous breast development and primary amenorrhea, and to evaluate similar splicing variant expression in normal steroidogenic tissues. METHODS: Genomic DNA analysis, splicing prediction programs, splicing assays, and in vitro protein expression and enzyme activity analyses were carried out. CYP19A1 mRNA expression in human steroidogenic tissues was also studied. RESULTS: A novel IVS9+5G>A homozygote mutation was found. In silico analysis predicts the disappearance of the splicing donor site in intron 9, confirmed by patient peripheral leukocyte cP450arom and in vitro studies. Protein analysis showed a shorter and inactive protein. The intron 9 transcript variant was also found in human steroidogenic tissues. CONCLUSIONS: The mutation IVS9+5G>A generates a splicing variant that includes intron 9 which is also present in normal human steroidogenic tissues, suggesting that a misbalance between normal and aberrant splicing variants might occur in target tissues, explaining the clinical phenotype in the affected patient.

Five new cases of 46,XX aromatase deficiency: clinical follow-up from birth to puberty, a novel mutation, and a founder effect.

CONTEXT: Aromatase is the key enzyme for estrogen biosynthesis and is encoded by the CYP19A1 gene. Since 1991, several molecular CYP19A1 gene alterations associated with aromatase deficiency have been described in both sexes. OBJECTIVE: The objective of the study was to detect CYP19A1 mutations in five aromatase-deficient 46,XX patients, to describe the clinical follow-up from birth to puberty and to perform haplotype analysis associated with the high-frequency c.628G>A splice mutation in Argentinean patients. DESIGN: The design of the study was the sequencing of the coding and flanking intronic regions of the CYP19A1 gene in all patients and parents. Haplotype analysis of patients carrying the c.628G>A mutation was also performed. PATIENTS: Clinical and biochemical findings in five new cases and one previously reported female aromatase-deficient patient (46,XX) are described. All patients presented with ambiguous genitalia at birth. Congenital adrenal hyperplasia due to 21-hydroxylase deficiency as well as other steroidogenic defects were ruled out. RESULTS: Phenotypic variability among the affected patients was found during follow-up. Direct sequencing of the CYP19A1 gene from genomic DNA revealed one novel mutation (c.574C>T) in two patients. In silico analysis predicted the c.574C>T mutation to be probably damaging. Four of six nonrelated patients presented with the c.628G>A splice mutation. Haplotype analysis showed that the c.628G>A splice mutation is associated with the same haplotype in our population. CONCLUSIONS: Increased knowledge on phenotypical variability found in female aromatase-deficient patients is useful to improve the detection rate in this disorder. In our population, a genetic founder defect has probably contributed to an increase in the incidence of the c.628G>A splice mutation.

Preserved fertility in a patient with a 46,XY disorder of sex development due to a new heterozygous mutation in the NR5A1/SF-1 gene: evidence of 46,XY and 46,XX gonadal dysgenesis phenotype variability in multiple members of an affected kindred.

In humans, steroidogenic factor 1 (NR5A1/SF-1) mutations have been reported to cause gonadal dysgenesis, with or without adrenal failure, in both 46,XY and 46,XX individuals. We have previously reported extreme within-family variability in affected 46,XY patients. Even though low ovarian reserve with preserved fertility has been reported in females harboring NR5A1 gene mutations, fertility has only been observed in one reported case in affected 46,XY individuals. A kindred with multiple affected members presenting gonadal dysgenesis was studied. Four 46,XY individuals presented severe hypospadias at birth, one of them associated with micropenis and cryptorchidism. The other 3 developed spontaneous male puberty, and 1 has fathered 5 children. Four 46,XX patients presented premature ovarian failure (one of them was not available for the study) or high follicle-stimulating hormone levels. Mutational analysis of the NR5A1 gene revealed a novel heterozygous mutation, c.938G→A, predicted to cause a p.Arg313Hys amino acid change. A highly conserved amino acid of the ligand-binding domain of the mature protein is affected, predicting abnormal protein function. We confirm that preserved fertility can be observed in patients with a 46,XY disorder of sex development due to heterozygous mutations in the NR5A1 gene.

Three new SF-1 (NR5A1) gene mutations in two unrelated families with multiple affected members: within-family variability in 46,XY subjects and low ovarian reserve in fertile 46,XX subjects.

BACKGROUND: Three novel heterozygous SF-1 gene mutations affecting multiple members of two unrelated families with a history of 46,XY disorders of sex development (DSD) and 46,XX ovarian insufficiency are described. METHODS: clinical and mutational analysis of the SF-1 gene in 9 subjects of two families. RESULTS: family 1 had 2 affected 46,XY DSD subjects. One, born with severe perineal hypospadias, was raised as a male, and presented normal adolescence. The other, born with ambiguous genitalia, uterus, and mild testicular dysgenesis, was raised as a female. A W279X heterozygous mutation and an intronic deletion (g3314-3317delTCTC (IVS 4 + 8) was found in the SF-1 gene. In family 2, 4/6 affected siblings had 46,XY DSD or hypospadias. An affected 46,XX sister had normal sexual development but increased FSH levels. The 37-year-old affected mother had entered menopause. An Y183X heterozygous mutation was detected. CONCLUSION: an extreme within-family phenotypic variability, ranging from severe prenatal undervirilization to normal pubertal development, was observed in 46,XY-affected siblings, indicating that other unknown factors might be involved in the phenotype. Low ovarian reserve and preserved fertility in 46,XX subjects can be observed in heterozygous SF-1 gene mutations.

Metformin, estrogen replacement therapy and gonadotropin inhibition fail to improve insulin sensitivity in a girl with aromatase deficiency.

BACKGROUND: Insulin resistance (IR), abnormal lipid profile, and other features of the metabolic syndrome have been described in CYP19 gene knockout mice and in aromatase-deficient adult men but not in prepubertal affected girls. AIMS: To study insulin sensitivity, as well as the effects of estrogen, metformin and GnRHa treatment on glucose homeostasis, in an aromatase-deficient girl. METHODS: Clinical, metabolic and hormonal follow-up data, from 8 to 12 years of age, is presented. RESULTS: At 9 years of age, IR (HOMA 5.6) and glucose intolerance was detected, along with high serum testosterone (2.28 nmol/l), androstenedione (4.92 nmol/l) and FSH (13.4 mIU/ml) levels. Estrogen replacement was ineffective to suppress gonadotropin and androgen levels, as well as IR. Under metformin therapy, she developed type 2 diabetes and acanthosis nigricans. GnRHa administration for 1 year resulted in marked decreases in gonadotropin and serum androgens, but severe IR persisted. CONCLUSION: Postnatal estrogen replacement and a marked decrease of endogenous androgens failed to improve IR and glucose tolerance. We propose that, in females, the increment of androgens and/or lack of estrogens during fetal life might alter the mechanism of fetal programming of insulin sensitivity.

Role of IGFs and insulin in the human testis during postnatal activation: differentiation of steroidogenic cells.

Immunoexpression of IGF-I, IGF-II, type 1 IGF receptor (IGFR), insulin receptor (IR), and GH receptor (GHR) was analyzed in human testis, in three age groups (Gr): Gr1 (neonates), Gr2 (postnatal testicular activation), and Gr3 (early prepuberty). In interstitial cells, low IGF-I and GHR, but moderate IR immunoexpression was observed in all Grs. However, high expression of IGF-II in Gr1, and moderate expression of IGFR in Gr1 and Gr2 were found. In Leydig cell (LC), high expression of IGF-II, moderate expression of IGFR and GHR, and undetectable IGF-I was found. Moreover, IR was highly expressed in Gr2. The effect of IGF-I on cell proliferation (PI) and apoptosis (AI), induction of cytochrome P450 side chain cleavage (cP450scc) immunoexpression, 3beta-hydroxysteroid dehydrogenase mRNA and testosterone (T) secretion was evaluated in human testis cell cultures. IGF-I increased P450scc immunoexpression, 3beta-hydroxysteroid dehydrogenase mRNA, T secretion, and PI, but decreased AI. We propose that IGF-II, mainly through IR, is involved in functional LC differentiation. In some interstitial cells, probably in LC precursors, IGF-II/IR could be involved, among other factors, in the stimulation of PI and/or inhibition of AI, and in LC differentiation.

[Exon 5 alternative splicing of the cytochrome P450 aromatase could be a regulatory mechanism for estrogen production in humans]. / El splicing alternativo del exón 5 de la citocromo p450 aromatasa podría ser un mecanismo de regulación de la producción de estrógenos en humanos.

P450 aromatase (P450Aro), involved in androgen to estrogen conversion, is encoded by the CYP19 gene. P450Aro c655G>A mutation described in heterozygous form in a girl and in homozygous form in an adult male with P450Aro deficiency results in an aberrant splicing due to disruption of a donor splice site. A truncated inactive protein would be expected if intron5 is retained. Surprisingly, the girl described with this mutation showed spontaneous breast development and pubertal estradiol (E2) levels suggesting residual P450Aro activity (AA). Formerly, we postulate the in frame E5 skipping as a consequence of this mutation generating a protein with some degree of activity. When P450Aro mRNA expression was analysed from patient's lymphocytes, an aberrant spliced mRNA lacking E5 (-E5mRNA) was detected, suggesting an association between E5 skipping and the presence of the mutation. Splicing assays in Y1 cells confirmed this association. -Ex5 cDNA expression in Y1 cells resulted in an inactive protein that could not explain patient's phenotype. Exon 5 might be predicted as a poorly defined exon suggesting a susceptibility to splicing mutations and physiological alternative splicing (AS) events. Therefore, -Ex5mRNA was assessed as a natural occurring alternative transcript in normal human steroidogenic tissues. As P450Aro -E5mRNA expression was detected in human term placenta, prepubertal testis and prepubertal adrenal, we might speculate that AS of P450Aro coding region would occur in humans and would be involved in the complex AA regulation. Furthermore, tissue specific regulation of AS might suggest low expression of +E5mRNA from the c655G>A allele explaining residual AA evidenced in the affected girl.

El splicing alternativo del exón 5 de la citocromo p450 aromatasa podría ser un mecanismo de regulación de la producción de estrógenos en humanos / Exon 5 alternative splicing of the cytochrome P450 aromatase could be a regulatory mechanism for estrogen production in humans

La enzima P450 aromatasa (P450Aro) participa en la síntesis de estrógenos a partir de andrógenos. La mutación c655G>A, descripta en forma heterocigota en una niña y en forma homocigota en un hombre adulto, ambos con déficit de aromatasa, genera la disrupción del sitio dador de splicing exón5-intrón5. Se ha postulado que la retención del intrón5 y la generación de una proteína truncada inactiva serían las consecuencias de esta mutación. Sorpresivamente, la paciente presentó desarrollo espontáneo de mamas y niveles puberales de estradiol, sugiriendo una actividad aromatasa (AA) residual. En principio postulamos que la mutación c655G>A generaría la pérdida del exón5 con conservación del marco de lectura, generándose una proteína con menor actividad que podría explicar el déficit parcial. La expresión del ARNm sin exón5 (ARNm- E5) en linfocitos de la paciente sugiere una asociación entre la pérdida del exón y la presencia de la mutación; posteriormente confirmada realizando ensayos de splicing en células Y1. Sin embargo, la expresión del cDNAE5 en células Y1 presentó una AA nula que no explicaría un déficit parcial. La expresión del ARNm-E5 fue detectada en placenta, testículo y adrenal humanos como una variante de splicing normal. Estos resultados indicarían la ocurrencia de splicing alternativo (SA) en la zona codificante de P450Aro como un posible mecanismo regulador de la producción de estrógenos en tejidos esteroidogénicos humanos. La mutación c655G>A podría alterar los mecanismos fisiológicos reguladores del SA del exón5 favoreciendo su exclusión. De esta forma, bajos niveles de ARNm+E5 podrían expresarse aun en presencia de la mutación explicando el fenotipo de déficit parcial observado en la paciente.

P450 aromatase (P450Aro), involved in androgen to estrogen conversion, is encoded by the CYP19 gene. P450Aro c655G>A mutation described in heterozygous form in a girl and in homozygous form in an adult male with P450Aro deficiency results in an aberrant splicing due to disruption of a donor splice site. A truncated inactive protein would be expected if intron5 is retained. Surprisingly, the girl described with this mutation showed spontaneous breast development and pubertal estradiol (E2) levels suggesting residual P450Aro activity (AA). Formerly, we postulate the in frame E5 skipping as a consequence of this mutation generating a protein with some degree of activity. When P450Aro mRNA expression was analysed from patient's lymphocytes, an aberrant spliced mRNA lacking E5 (-E5mRNA) was detected, suggesting an association between E5 skipping and the presence of the mutation. Splicing assays in Y1 cells confirmed this association. -Ex5 cDNA expression in Y1 cells resulted in an inactive protein that could not explain patient's phenotype. Exon 5 might be predicted as a poorly defined exon suggesting a susceptibility to splicing mutations and physiological alternative splicing (AS) events. Therefore, -Ex5mRNA was assessed as a natural occurring alternative transcript in normal human steroidogenic tissues. As P450Aro -E5mRNA expression was detected in human term placenta, prepubertal testis and prepubertal adrenal, we might speculate that AS of P450Aro coding region would occur in humans and would be involved in the complex AA regulation. Furthermore, tissue specific regulation of AS might suggest low expression of +E5mRNA from the c655G>A allele explaining residual AA evidenced in the affected girl.

El splicing alternativo del exón 5 de la citocromo p450 aromatasa podría ser un mecanismo de regulación de la producción de estrógenos en humanos / Exon 5 alternative splicing of the cytochrome P450 aromatase could be a regulatory mechanism for estrogen production in humans

La enzima P450 aromatasa (P450Aro) participa en la síntesis de estrógenos a partir de andrógenos. La mutación c655G>A, descripta en forma heterocigota en una niña y en forma homocigota en un hombre adulto, ambos con déficit de aromatasa, genera la disrupción del sitio dador de splicing exón5-intrón5. Se ha postulado que la retención del intrón5 y la generación de una proteína truncada inactiva serían las consecuencias de esta mutación. Sorpresivamente, la paciente presentó desarrollo espontáneo de mamas y niveles puberales de estradiol, sugiriendo una actividad aromatasa (AA) residual. En principio postulamos que la mutación c655G>A generaría la pérdida del exón5 con conservación del marco de lectura, generándose una proteína con menor actividad que podría explicar el déficit parcial. La expresión del ARNm sin exón5 (ARNm- E5) en linfocitos de la paciente sugiere una asociación entre la pérdida del exón y la presencia de la mutación; posteriormente confirmada realizando ensayos de splicing en células Y1. Sin embargo, la expresión del cDNAE5 en células Y1 presentó una AA nula que no explicaría un déficit parcial. La expresión del ARNm-E5 fue detectada en placenta, testículo y adrenal humanos como una variante de splicing normal. Estos resultados indicarían la ocurrencia de splicing alternativo (SA) en la zona codificante de P450Aro como un posible mecanismo regulador de la producción de estrógenos en tejidos esteroidogénicos humanos. La mutación c655G>A podría alterar los mecanismos fisiológicos reguladores del SA del exón5 favoreciendo su exclusión. De esta forma, bajos niveles de ARNm+E5 podrían expresarse aun en presencia de la mutación explicando el fenotipo de déficit parcial observado en la paciente.(AU)

P450 aromatase (P450Aro), involved in androgen to estrogen conversion, is encoded by the CYP19 gene. P450Aro c655G>A mutation described in heterozygous form in a girl and in homozygous form in an adult male with P450Aro deficiency results in an aberrant splicing due to disruption of a donor splice site. A truncated inactive protein would be expected if intron5 is retained. Surprisingly, the girl described with this mutation showed spontaneous breast development and pubertal estradiol (E2) levels suggesting residual P450Aro activity (AA). Formerly, we postulate the in frame E5 skipping as a consequence of this mutation generating a protein with some degree of activity. When P450Aro mRNA expression was analysed from patients lymphocytes, an aberrant spliced mRNA lacking E5 (-E5mRNA) was detected, suggesting an association between E5 skipping and the presence of the mutation. Splicing assays in Y1 cells confirmed this association. -Ex5 cDNA expression in Y1 cells resulted in an inactive protein that could not explain patients phenotype. Exon 5 might be predicted as a poorly defined exon suggesting a susceptibility to splicing mutations and physiological alternative splicing (AS) events. Therefore, -Ex5mRNA was assessed as a natural occurring alternative transcript in normal human steroidogenic tissues. As P450Aro -E5mRNA expression was detected in human term placenta, prepubertal testis and prepubertal adrenal, we might speculate that AS of P450Aro coding region would occur in humans and would be involved in the complex AA regulation. Furthermore, tissue specific regulation of AS might suggest low expression of +E5mRNA from the c655G>A allele explaining residual AA evidenced in the affected girl.(AU)

The cytochrome P450 aromatase lacking exon 5 is associated with a phenotype of nonclassic aromatase deficiency and is also present in normal human steroidogenic tissues.

OBJECTIVE: The previously described c655G>A mutation of the human cytochrome P450 aromatase gene (P450aro, CYP19) results in aberrant splicing due to disruption of a donor splice site. To explain the phenotype of partial aromatase deficiency observed in a female patient described with this mutation, molecular consequences of the c655G>A mutation were investigated. DESIGN: To investigate whether the c655G>A mutation causes an aberrant spliced mRNA lacking exon 5 (-Ex5), P450aro RNA was analysed from the patient's lymphocytes by reverse transcription polymerase chain reaction (RT-PCR) and by splicing assays performed in Y1 cells transfected with a P450aro -Ex5 expression vector. Aromatase activity of the c655G>A mutant was predicted by three dimensional (3D) protein modelling studies and analysed in transiently transfected Y1 cells. Exon 5 might be predicted as a poorly defined exon suggesting a susceptibility to both splicing mutations and physiological alternative splicing events. Therefore, expression of the -Ex5 mRNA was also assessed as a possibly naturally occurring alternative splicing transcript in normal human steroidogenic tissues. PATIENTS: An aromatase deficient girl was born with ambiguous genitalia. Elevated serum LH, FSH and androgens, as well as cystic ovaries, were found during prepuberty. At the age of 8.4 years, spontaneous breast development and a 194.6 pmol/l serum oestradiol level was observed. RESULTS: The -Ex5 mRNA was found in lymphocytes of the P450aro deficient girl and her father, who was a carrier of the mutation. Mutant minigene expression resulted in complete exon 5 skipping. As expected from 3D protein modelling, -Ex5 cDNA expression in Y1 cells resulted in loss of P450aro activity. In addition, the -Ex5 mRNA was present in placenta, prepubertal testis and adrenal tissues. CONCLUSIONS: Alternative splicing of exon 5 of the CYP19 gene occurs in the wild type (WT) as well as in the c655G>A mutant. We speculate that for the WT it might function as a regulatory mechanism for aromatization, whereas for the mutant a relative prevalence of the shorter over the full-length protein might explain the phenotype of partial aromatase deficiency.

Identification and developmental changes of aromatase and estrogen receptor expression in prepubertal and pubertal human adrenal tissues.

CONTEXT: The mechanisms of postnatal adrenal zonation remain unclear. OBJECTIVE: To provide a clue for a possible role of estrogens in adrenarche, we studied the expression of estrogen receptor (ER)alpha, ERbeta, G protein-coupled receptor (GPR)30, and cP450aromatase (cP450arom) in human adrenal tissue. DESIGN: Human adrenal tissue was collected from three postnatal age groups (Grs): Gr 1, younger than 3 months (n = 12), fetal zone involution; Gr 2, 3 months to 6 yr (n = 17), pre-adrenarche; and Gr 3, older than 6-20 yr (n = 12), post-adrenarche period. RESULTS: ERbeta mRNA in Grs 1 and 3 was higher than in Gr 2 (P < 0.05). By immunohistochemistry and laser capture microdissection followed by RT-PCR, ERbeta was expressed in zona reticularis and fetal zone, GPR30 in zona glomerulosa (ZG) and adrenal medulla, while ERalpha mRNA and protein were undetectable. cP450arom mRNA in Gr 3 was higher than in Grs 1 and 2 (P < 0.05), and localized to ZG and adrenal medulla by laser capture microdissection. cP450arom Immunoreactivity was observed in adrenal medulla in the three Grs and in subcapsular ZG of Gr 3. Double-immunofluorescence studies revealed that cP450arom and chromogranin A only colocalize in adrenal medulla of subjects younger than 18 months. In these samples, exon 1.b-derived transcript was 3.5-fold higher, while exon 1.a-, 1.c-, and 1.d-derived transcripts were 3.3-, 1.9-, and 1.7-fold lower, respectively, than in subjects older than 6 yr. CONCLUSIONS: Our results suggest that estrogens produced locally in adrenal medulla would play a role in zona reticularis functional differentiation through ERbeta. The cP450arom and GPR30 expression in subcapsular ZG, colocalizing with a high-cell proliferation index, previously reported, suggests a local GPR30-dependent estrogen action in proliferation and migration of progenitor adrenal cells.

Expression of aromatase, estrogen receptor alpha and beta, androgen receptor, and cytochrome P-450scc in the human early prepubertal testis.

The expression of aromatase, estrogen receptor alpha (ERalpha) and beta (ERbeta), androgen receptor (AR), and cytochrome P-450 side chain cleavage enzyme (cP450scc) was studied in prepubertal testis. Samples were divided in three age groups (GRs): GR1, newborns (1- to 21-d-old neonates, n = 5); GR2, postnatal activation stage (1- to 7-mo-old infants, n = 6); GR3, childhood (12- to 60-mo-old boys, n = 4). Absent or very poor detection of ERalpha by immunohistochemistry in all cells and by mRNA expression was observed. Leydig cells (LCs) of GR1 and GR2 showed strong immunostaining of aromatase and cP450scc but weak staining of ERbeta and AR. Interstitial cells (ICs) and Sertoli cells (SCs) expressed ERbeta, particularly in GR1 and GR2. Strong expression of AR was found in peritubular cells (PCs). For all markers, expression in GR3 was the weakest. In germ cells (GCs), i.e. gonocytes and spermatogonia, aromatase and ERbeta were immunoexpressed strongly whereas no expression of ERalpha, AR, or cP450scc was detected. It is proposed that in newborn and infantile testis, testosterone acting on PCs might modulate infant LC differentiation, whereas the absence of AR in SCs prevents development of spermatogenesis. The role of estrogen is less clear, but it could modulate the preservation of an adequate pool of precursor LCs and GCs.

High TGFbeta1, estrogen receptor, and aromatase gene expression in a large cell calcifying sertoli cell tumor (LCCSCT): implications for the mechanism of oncogenesis.

Large cell calcifying Sertoli cell tumors (LCCSCT) are associated with Carney complex and Peutz-Jeghers syndrome. The mechanisms linking these 2 genetic defects to the genesis of this tumor are obscure. Studies of CYP19 (aromatase) and transforming growth factor (TGF)-beta1 messenger RNA (mRNA) abundance, estrogen receptor (ER), TGFbeta1, and TGFbeta type II receptor (R) immunochemistry were carried out in the testis of a patient with this tumor to gain information on possible mechanisms of cell tumor development. Testicular tissue of a prepubertal patient, collected at gonadectomy, was separated into 2 macroscopically distinct fractions: tumoral nodules (Tu) and extratumoral, normal-looking testicular tissue (ExTu). The patient was a 9.5-year-old boy with a 5-year history of bilateral gynecomastia (Tanner stage 4), no pubic hair, incipient genital development, and bilateral testicular nodules. Multiple pigmented lesions of the skin were present. Bilateral mammectomy and gonadectomy was performed. RNA was extracted from Tu and ExTu for semiquantitative reverse transcriptase-polymerase chain reaction of CYP19 and TGFbeta1. Protein expression of ER, TGFbeta1, and TGFbeta type II R in Tu and ExTu was detected by immunohistochemistry. Cell proliferation was estimated by Ki-67 antigen immunochemistry and apoptosis using a modified TUNEL assay. Mean expression of aromatase and TGFbeta1 mRNAs in Tu was 6- and 2.3-fold higher than in ExTu, respectively (P<0.05). Tumoral cells exhibited ER staining with a predominant extranuclear localization. Positive staining of Sertoli cells in Tu was higher than in ExTu. TGFbeta1 immunostaining of the interstitial cells in Tu was higher than in ExTu. TGFbeta type II R immunostaining was detected in most Sertoli and interstitial cells, but intensity in ExTu was lower than in Tu. No significant difference was detected in the proliferation index, but in Tu, the percentage of Sertoli cells in apoptosis (1.4%) was significantly lower (P<0.01) than in ExTu (14.0%). The following hypothesis is proposed. The congenital gene defects of Carney complex or of Peutz-Jeghers syndrome might trigger a cascade of intracellular events that leads to overexpression of aromatase in Sertoli cells, favoring the development of a LCCSCT. At some point in the evolution of the disease, a mutational event might induce a higher expression of the ER. Also, TGFbeta1 protein expression is increased in neighboring cells. In this environment, TGFbeta1 might switch from tumor suppressor to oncogenic factor and, along with estrogen-ER complexes, might favor tumor progression by inhibiting apoptosis.

Expression of the IGF system in human adrenal tissues from early infancy to late puberty: implications for the development of adrenarche.

IGF-1, IGF-2, and type 1 IGF receptor (IGF-R1) mRNA expression and immunolocalization and cell proliferation index were studied in human adrenals from early infancy to late puberty. Adrenals were obtained from transplantation donors or from necropsies of endocrinologically normal subjects. Subjects were divided into three age groups: group 1, <3 mo of age, involution of fetal adrenals; group 2, 3 mo to 6 y of age, preadrenarche; and group 3, older than 6 y up to 20 y of age, postadrenarche. Cell proliferation index (Ki-67) in the outer, subcapsular, zona glomerulosa was significantly higher than in zona fasciculata of all groups and in zona reticularis or fetal zone. IGF-1 mRNA (semiquantitative reverse transcriptase-PCR and Northern blot) in group 2 was significantly higher than in group 1 and group 3 (p < 0.05). IGF2 mRNA in group 1 was significantly higher than in the other groups. IGF-R1 mRNA in group 3 was significantly higher than in group 2 but not different from group 1. Strong IGF-1, IGF-2, and IGF-R1 immunostaining signal was observed in the outer, subcapsular, zona glomerulosa and in zona fasciculata in the three groups, whereas a very weak IGF-1 and IGF-R1 immunostaining signal was found in fetal zone cells of group 1 and in zona reticularis of group 3. We propose that IGF-1 could be a factor involved in the postnatal mechanism of progenitor adrenal cell proliferation and migration. Our data also suggest that IGF-1 is not a direct regulatory factor of adrenal androgen production by zona reticularis cells.