Publications by authors named "Esperanza Berensztein"

21 Publications

  • Page 1 of 1

Testis formation in XX individuals resulting from novel pathogenic variants in Wilms' tumor 1 () gene.

Proc Natl Acad Sci U S A 2020 06 3;117(24):13680-13688. Epub 2020 Jun 3.

Human Developmental Genetics Unit, Institut Pasteur, 75724 Paris, France;

Sex determination in mammals is governed by antagonistic interactions of two genetic pathways, imbalance in which may lead to disorders/differences of sex development (DSD) in human. Among 46,XX individuals with testicular DSD (TDSD) or ovotesticular DSD (OTDSD), testicular tissue is present in the gonad. Although the testis-determining gene is present in many cases, the etiology is unknown in most -negative patients. We performed exome sequencing on 78 individuals with 46,XX TDSD/OTDSD of unknown genetic etiology and identified seven (8.97%) with heterozygous variants affecting the fourth zinc finger (ZF4) of Wilms' tumor 1 (WT1) (p.Ser478Thrfs*17, p.Pro481Leufs*15, p.Lys491Glu, p.Arg495Gln [x3], p.Arg495Gly). The variants were de novo in six families ( = 4.4 × 10), and the incidence of WT1 variants in 46,XX DSD is enriched compared to control populations ( < 1.8 × 10). The introduction of ZF4 mutants into a human granulosa cell line resulted in up-regulation of endogenous Sertoli cell transcripts and XX mice display masculinization of the fetal gonads. The phenotype could be explained by the ability of the mutated proteins to physically interact with and sequester a key pro-ovary factor β-CATENIN, which may lead to up-regulation of testis-specific pathway. Our data show that unlike previous association of WT1 and 46,XY DSD, ZF4 variants of WT1 are a relatively common cause of 46,XX TDSD/OTDSD. This expands the spectrum of phenotypes associated with WT1 variants and shows that the WT1 protein affecting ZF4 can function as a protestis factor in an XX chromosomal context.
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http://dx.doi.org/10.1073/pnas.1921676117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7306989PMC
June 2020

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

Front Endocrinol (Lausanne) 2020 25;11:72. Epub 2020 Feb 25.

Endocrinology Department, Hospital de Pediatría "Prof. Dr. Juan P. Garrahan", Buenos Aires, Argentina.

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 (ERβ) 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. ERβ expression was detected mostly in spermatogonia, primary spermatocytes, and immature spermatids. In Sertoli cells ERβ 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, ERKOβ, and ERKOαβ) 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.
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http://dx.doi.org/10.3389/fendo.2020.00072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7051936PMC
March 2021

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

Front Immunol 2019 8;10:2907. Epub 2020 Jan 8.

CONICET, Laboratorio de Inmunofarmacología, Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales (IQUIBICEN), Universidad de Buenos Aires, Buenos Aires, Argentina.

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)β 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.
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http://dx.doi.org/10.3389/fimmu.2019.02907DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6960177PMC
November 2020

Androgen Insensitivity Syndrome: Clinical Phenotype and Molecular Analysis in a Single Tertiary Center Cohort

J Clin Res Pediatr Endocrinol 2019 02 25;11(1):24-33. Epub 2018 Sep 25.

Hospital de Pediatria Garrahan, Endocrinology Service, Buenos Aires, Argentina

Objective: The aim of this study was the molecular characterization of the gene as the cause of 46,XY disorder in our population.

Methods: We studied 41, non related, 46,XY disorder of sexual differentiation index cases, having characteristics consistent with androgen insensivity syndrome (AIS). Genomic DNA was isolated from peripheral blood leukocytes of all patients and 25 family members from 17 non-related families.

Results: The gene analysis revealed an abnormal sequence in 58.5% of the index patients. All of the complete AIS (CAIS) cases were genetically confirmed, while in the partial form (PAIS) a mutation in was detected in only 13 (43.3%). Molecular studies revealed other affected or carrier relatives in 87% of the index cases. The mutations were found spread along the whole coding sequence, with a higher prevalence in the ligand binding domain. Nine out of 23 (39%) mutations were novel. In 17% of patients with detected mutations, somatic mosaicism was detected in leucocyte DNA. In our cohort, long-term follow up gender dysphoria, raised as male or female, was not found. Finally, in suspected PAIS, the identification of mutation occurred significantly less than in CAIS patients.

Conclusion: Improved knowledge of the components of the complex and signaling network might contribute to long term outcome and genetic counseling in AIS patients.
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http://dx.doi.org/10.4274/jcrpe.galenos.2018.2018.0185DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6398199PMC
February 2019

Androgen Insensitivity Syndrome at Prepuberty: Marked Loss of Spermatogonial Cells at Early Childhood and Presence of Gonocytes up to Puberty.

Sex Dev 2017 24;11(5-6):225-237. Epub 2018 Jan 24.

Servicio de Endocrinología, Hospital de Pediatría 'Prof. Dr. Juan Pedro Garrahan', Buenos Aires, Argentina.

Androgen insensitivity syndrome (AIS) is a hereditary condition in patients with a 46,XY karyotype in which loss-of-function mutations of the androgen receptor (AR) gene are responsible for defects in virilization. The aim of this study was to investigate the consequences of the lack of AR activity on germ cell survival and the degree of testicular development reached by these patients by analyzing gonadal tissue from patients with AIS prior to Sertoli cell maturation at puberty. Twenty-three gonads from 13 patients with AIS were assessed and compared to 18 testes from 17 subjects without endocrine disorders. The study of the gonadal structure using conventional microscopy and the ultrastructural characteristics of remnant germ cells using electron microscopy, combined with the immunohistochemical analysis of specific germ cell markers (MAGE-A4 for premeiotic germ cells and of OCT3/4 for gonocytes), enabled us to carry out a thorough investigation of germ cell life in an androgen-insensitive microenvironment throughout prepuberty until young adulthood. Here, we show that germ cell degeneration starts very early, with a marked decrease in number after only 2 years of life, and we demonstrate the permanence of gonocytes in AIS testis samples until puberty, describing 2 different populations. Additionally, our results provide further evidence for the importance of AR signaling in peritubular myoid cells during prepuberty to maintain Sertoli and spermatogonial cell health and survival.
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http://dx.doi.org/10.1159/000486089DOI Listing
October 2018

Histamine H4 receptor as a novel therapeutic target for the treatment of Leydig-cell tumours in prepubertal boys.

Eur J Cancer 2018 03;91:125-135

Laboratorio de Endocrinología Molecular y Transducción de Señales, Instituto de Biología y Medicina Experimental (IBYME-CONICET), Vuelta de Obligado 2490, C1428ADN, Buenos Aires, Argentina. Electronic address:

Leydig-cell tumours (LCTs) are rare endocrine tumours of the testicular interstitium, with recent increased incidence. Symptoms include precocious puberty in children; and erectile dysfunction, infertility and/or gynaecomastia, in adults. So far, scientific evidence points to aromatase (CYP19) overexpression and excessive oestrogen and insulin-like growth factor (IGF) -1 production as responsible for Leydig-cell tumourigenesis. LCTs are usually benign; however, malignant LCTs respond poorly to chemo/radiotherapy, highlighting the need to identify novel targets for treatment. Herein, we investigated the potential role of the histamine receptor H4 (HRH4) as a therapeutic target for LCTs using R2C rat Leydig tumour cells, a well-documented in vitro model for Leydigioma. Also, we studied for the first time the expression of CYP19, IGF-1R, oestrogen receptor (ER) α, ERβ, androgen receptor (AR) and HRH4 in human prepubertal LCTs versus normal prepubertal testes (NPTs). HRH4 agonist treatment inhibited steroidogenesis and proliferation in R2C cells and also negatively affected their pro-angiogenic capacity in vitro and in vivo, as assessed by evaluating the proliferative activity of human umbilical vein endothelial cells and by means of the quail chorioallantoic membrane assay, respectively. Moreover, E2 and IGF-1 inhibited HRH4 mRNA and protein levels. In human prepubertal LCTs, CYP19, IGF-1R, ERα and ERβ were overexpressed compared with NPTs. In contrast, HRH4 staining was weak in LCTs, but moderate/strong and confined to the interstitium in NPTs. Importantly, HRH4 was absent or barely detectable in seminiferous tubules or germ cells. Overall, our results point to HRH4 as a novel therapeutic target in LCTs.
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http://dx.doi.org/10.1016/j.ejca.2017.12.003DOI Listing
March 2018

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

Mol Cell Endocrinol 2017 02 23;441:46-54. Epub 2016 Sep 23.

Endocrine Service-CONICET, Hospital de Pediatria Garrahan, Buenos Aires, Argentina; National Research Council of Argentina (CONICET), Argentina.

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.
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http://dx.doi.org/10.1016/j.mce.2016.09.024DOI Listing
February 2017

H4 histamine receptors inhibit steroidogenesis and proliferation in Leydig cells.

J Endocrinol 2014 Dec 24;223(3):241-53. Epub 2014 Sep 24.

Laboratory of Molecular Endocrinology and Signal TransductionInstitute of Biology and Experimental Medicine, National Scientific and Technical Research Council (IByME-CONICET), CP 1428 Buenos Aires, ArgentinaResearch LaboratoryEndocrinology Service, Garrahan Pediatric Hospital, CP 1245 Buenos Aires, ArgentinaCell Biology UnitInstitut Pasteur de Montevideo, CP 11400 Montevideo, UruguayLaboratory of RadioisotopesSchool of Pharmacy and Biochemistry, University of Buenos Aires, CP 1113 Buenos Aires, ArgentinaInstitute for Biomedical Research (BIOMED)School of Medical Sciences, Pontifical Catholic University of Argentina, National Scientific and Technical Research Council (UCA-CONICET), CP1107 Buenos Aires, ArgentinaDepartment of Biological ChemistrySchool of Sciences, University of Buenos Aires, CP 1428 Buenos Aires, Argentina

The histamine H4 receptor (HRH4), discovered only 13 years ago, is considered a promising drug target for allergy, inflammation, autoimmune disorders and cancer, as reflected by a steadily growing number of scientific publications and patent applications. Although the presence of HRH4 has been evidenced in the testis, its specific localization or its role has not been established. Herein, we sought to identify the possible involvement of HRH4 in the regulation of Leydig cell function. We first evaluated its expression in MA-10 Leydig tumor cells and then assessed the effects of two HRH4 agonists on steroidogenesis and proliferation. We found that HRH4 is functionally expressed in MA-10 cells, and that its activation leads to the inhibition of LH/human chorionic gonadotropin-induced cAMP production and StAR protein expression. Furthermore, we observed decreased cell proliferation after a 24-h HRH4 agonist treatment. We then detected for the sites of HRH4 expression in the normal rat testis, and detected HRH4 immunostaining in the Leydig cells of rats aged 7-240 days, while 21-day-old rats also presented HRH4 expression in male gametes. Finally, we evaluated the effect of HRH4 activation on the proliferation of normal progenitor and immature rat Leydig cell culture, and both proved to be susceptible to the anti-proliferative effect of HRH4 agonists. Given the importance of histamine (2-(1H-imidazol-4-yl)ethanamine) in human (patho)physiology, continued efforts are directed at elucidating the emerging properties of HRH4 and its ligands. This study reveals new sites of HRH4 expression, and should be considered in the design of selective HRH4 agonists for therapeutic purposes.
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http://dx.doi.org/10.1530/JOE-14-0401DOI Listing
December 2014

Three novel IGF1R mutations in microcephalic patients with prenatal and postnatal growth impairment.

Clin Endocrinol (Oxf) 2015 May 7;82(5):704-11. Epub 2014 Aug 7.

Endocrinology Service, Hospital de Pediatría Garrahan, Buenos Aires, Argentina.

Background: IGF1R gene mutations have been associated with varying degrees of intrauterine and postnatal growth retardation, and microcephaly.

Objective: To identify and characterize IGF1R gene variations in a cohort of 28 Argentinean children suspected of having IGF-1 insensitivity, who were selected on the basis of the association of pre/postnatal growth failure and microcephaly.

Methods: The coding sequence and flanking intronic regions of IGF1R gene were amplified and directly sequenced. Functional characterization was performed by two in vitro assays: 1) [Methyl-(3) H] thymidine incorporation into DNA in fibroblast cell primary cultures from patients and controls treated with IGF-1 for 16-24 h. 2) PI3K/Akt pathway was evaluated with phospho-Akt (Ser473) STAR ELISA Kit (Millipore) in fibroblast cultures from patients and controls stimulated with IGF-1 for 10 min. Prepubertal clinical and GH-IGF-1 axis evaluation was followed up.

Results: We identified three novel heterozygous missense mutations in three unrelated patients, de novo p.Arg1256Ser, de novo p.Asn359Tyr and p.Tyr865Cys. In control cells, proliferation assay showed that IGF-1 significantly induced DNA synthesis at 20 h and Akt phosphorylation assay that it significantly stimulated phosphorylation after 10 min (P < 0·05 by anova and Bonferroni Tests). However, no significant increase was observed in any of the three patient fibroblasts in both functional studies. GH therapy growth response in two patients was inconsistent.

Conclusion: These variations led to failure of the IGF1R function causing pre- and postnatal growth retardation and microcephaly. Microcephaly should be considered in the evaluation of SGA patients, because it seems to favour the frequency of detection of IGF1R mutations.
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http://dx.doi.org/10.1111/cen.12555DOI Listing
May 2015

[Novel heterozygous mutation in the steroidogenic acute regulatory protein gene in a 46,XY patient with congenital lipoid adrenal hyperplasia].

Medicina (B Aires) 2013 ;73(4):297-302

Servicio de Endocrinología, Hospital Nacional de Pediatría Juan P. Garrahan, Buenos Aires, Argentina.

StAR facilitates cholesterol entry into the mitochondria as part of the transduceosome complex. Recessive mutations in the gen STAR cause classic and nonclassic congenital lipoid adrenal hyperplasia. The aim of the study was to analyze the molecular consequences of a novel heterozygous STAR mutation in a 46,XY patient with ambiguous genitalia and adrenal insufficiency. We found a de novo heterozygous IVS-2A>G STAR mutation and the reported heterozygous p.G146A SF1 polymorphism with normal CYP11A1, FDXR, FDX1, VDAC1 and TSPO genes. RT-PCR and sequencing from patient's testicular RNA showed a -exon2 transcript and the wild-type (WT) transcript. Both 37 kDa precursor and 30 kDa mature protein were detected in COS-7 cell transfected with mutant and WT plasmids. Immunofluorescence showed almost no co-localization of mitochondria and mutant protein (delta22-59StAR). Delta22-59StAR activity was 65±13% of WT. Cotransfection with WT and delta22-59StAR plasmids reduced WT activity by 62.0% ± 13.9. Novel splice-junction heterozygous STAR mutation (IVS-2A>G) resulted in the in-frame loss of amino acids 22 to 59 in the N-terminal mitochondrial targeting signal. A misfolded p.G22_L59delStAR might interfere with WT StAR activity by blocking the transduceosome complex, causing an autosomal dominant form of StAR deficiency, explaining the clinical phenotype.
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June 2014

Unique dominant negative mutation in the N-terminal mitochondrial targeting sequence of StAR, causing a variant form of congenital lipoid adrenal hyperplasia.

J Clin Endocrinol Metab 2013 Jan 21;98(1):E153-61. Epub 2012 Nov 21.

Endocrine Service, Hospital de Pediatria Garrahan, Buenos Aires C1245AAM, Argentina.

Context: Steroid acute regulatory (StAR) protein is a mitochondria-targeted protein that is part of the transduceosome complex crucial for transport of cholesterol to mitochondria. Recessive mutations cause classic and nonclassic congenital lipoid adrenal hyperplasia.

Objective: The aim of this study was to report the clinical, hormonal, genetic, and functional data of a novel heterozygous mutation in the StAR gene found in a 46,XY patient with ambiguous genitalia and neonatal severe steroidogenic deficiency.

Patient: Undetectable serum steroids with high ACTH and plasma renin activity but normal acute GnRH response were found in infancy. After gonadectomy (at 3 yr of age), serum LH and testosterone were undetectable, whereas FSH was normal but increased slowly afterward. Estrogen replacement therapy, started at 10.2 yr of age, suppressed gonadotropins (for 2 yr). However, after 1 month off estrogens, the patient showed castrated levels. At 11.9 yr old, after fludrocortisone withdrawal because of hypertension, plasma renin activity and aldosterone remained normal, suggesting mineralocorticoid recovery by a StAR-independent mechanism.

Results: We found a de novo heterozygous IVS-2A>G StAR mutation and the reported heterozygous p.G146A SF1 polymorphism with normal CYP11A1, FDXR, FDX1, VDAC1, and TSPO genes. The mutant StAR transcript lacked exon 2, resulting in the in-frame loss of amino acids 22 to 59 in the N-terminal mitochondrial targeting signal. In vitro, the mutant protein exhibited reduced StAR activity in a dominant-negative manner and almost no mitochondria localization.

Conclusions: A misfolded p.G22_L59del StAR might interfere with wild-type StAR activity by blocking the transduceosome complex, causing an autosomal dominant form of StAR deficiency, explaining the clinical phenotype. We speculated that estrogen might have modulated mineralocorticoid function and pubertal maturation in a human natural model lacking endogenous steroid production.
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http://dx.doi.org/10.1210/jc.2012-2865DOI Listing
January 2013

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.

Horm Res Paediatr 2012 14;78(2):119-26. Epub 2012 Aug 14.

Endocrinology Service, Hospital de Pediatría Garrahan, Buenos Aires, Argentina.

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.
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http://dx.doi.org/10.1159/000338346DOI Listing
February 2013

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.

Horm Res Paediatr 2011 22;75(1):70-7. Epub 2010 Sep 22.

Endocrinology Service, Hospital de Pediatria Garrahan, Buenos Aires, Argentina.

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.
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http://dx.doi.org/10.1159/000320029DOI Listing
May 2011

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

Pediatr Res 2008 Jun;63(6):662-6

Endocrine Service, Hospital de Pediatria Garrahan, Buenos Aires C1245 AAM, Argentina.

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.
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http://dx.doi.org/10.1203/PDR.0b013e31816c8ffcDOI Listing
June 2008

[Exon 5 alternative splicing of the cytochrome P450 aromatase could be a regulatory mechanism for estrogen production in humans].

Medicina (B Aires) 2007 ;67(4):369-73

Laboratorio de Investigación, Hospital Garrahan, Buenos Aires, Argentina.

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.
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September 2008

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

J Clin Endocrinol Metab 2007 Jun 3;92(6):2215-22. Epub 2007 Apr 3.

Endocrinology Service, Hospital de Pediatria Garrahan, C de los Pozos 1881, Buenos Aires, Argentina.

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.
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http://dx.doi.org/10.1210/jc.2006-2329DOI Listing
June 2007

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

Pediatr Res 2006 Dec 25;60(6):740-4. Epub 2006 Oct 25.

Research Laboratory, Hospital de Pediatria Garrahan, Buenos Aires C124 5AAM, Argentina.

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.
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http://dx.doi.org/10.1203/01.pdr.0000246072.04663.bbDOI Listing
December 2006

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

Pediatr Dev Pathol 2006 May-Jun;9(3):181-9

Laboratorio de Investigación, Hospital de Pediatria Garrahan, Buenos Aires, Argentina.

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.
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http://dx.doi.org/10.2350/06-04-0074.1DOI Listing
October 2006

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

Pediatr Res 2005 Sep;58(3):451-8

Laboratorio de Investigacion, Hospital de Pediatria Garrahan, Buenos Aires Argentina.

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.
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http://dx.doi.org/10.1203/01.PDR.0000179392.59060.93DOI Listing
September 2005

Apoptosis and proliferation of human testicular somatic and germ cells during prepuberty: high rate of testicular growth in newborns mediated by decreased apoptosis.

J Clin Endocrinol Metab 2002 Nov;87(11):5113-8

Research Laboratory, Garrahan Pediatric Hospital, C. de los Pozos 1881, Buenos Aires 1245, Argentina.

Programmed cell death and proliferation are evolutionary conserved processes that play a major role during normal development and homeostasis. In the testis, during the fetal and newborn periods, they might determine final adult size and fertility potential. In the present study, we have measured the relative number of testicular cells in apoptosis and in active proliferation in the seminiferous cords and in the interstitium, at different age periods of prepubertal testicular development in humans. Testes from 44 prepubertal subjects without endocrine and metabolic abnormalities were collected at necropsy. They were divided in three age groups (Gr): Gr 1, newborn (1- to 21-d-old neonates), n = 18, mean (+/-SD) age 0.3 +/- 0.23 months; Gr 2, post natal activation (1- to 6-month-old infants), n = 13, mean age 3.93 +/- 1.90 months; and Gr 3, early childhood period (1- to <6-yr-old boys), n = 13, mean age 31.5 +/- 18.9 months. Apoptosis was detected in 5- microm tissue sections using a modified terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay and cell proliferation was assessed by Ki-67 immunohistochemistry. Evaluation of apoptosis was confirmed by estimation of active caspase-3. Mean (+/-SD) testicular weight was 0.38 +/- 0.20, 0.54 +/- 0.35, and 0.51 +/- 0.11 g in Gr 1, Gr 2, and Gr 3, respectively. In Gr 1, there was a significant positive correlation between age and testis weight (P = 0.02). Mean (+/-SD) germ cell apoptotic index, AI, (% of apoptotic cells out of total cell number) was 15.0 +/- 6.60, 27.0 +/- 8.80 and 33.4 +/- 11.4 in Gr 1, Gr 2, and Gr 3, respectively. In Sertoli cells, it was 6.60 +/- 4.07, 22.0 +/- 14.0 and 27.5 +/- 19.8, respectively. In interstitial cells, it was 10.2 +/- 6.38, 18.0 +/- 6.70 and 25.7 +/- 15.5, respectively. In the three types of cells, AI in Gr 1 was significantly lower than in Gr 2 or Gr 3 (P < 0.05). Mean (+/-SD) germ cell proliferation index, PI, was 18.6 +/- 13.0, 10.0 +/- 6.50 and 10.9 +/- 6.24% in Gr 1, Gr 2, and Gr 3, respectively. In Sertoli cells and in interstitial cells PI was similar in the three age groups. The PI/AI ratio was used to compare relative differences among age groups. The PI/AI ratio of germ cells, Sertoli cells and interstitial cells in Gr 1 was significantly higher than in Gr 2 or Gr 3 (P < 0.05). It is concluded that, in normal subjects, there is a vigorous growth of the testis during the newborn period with subsequent stabilization during the first years of prepuberty. This cell growth seems to be mainly mediated by decreased apoptosis. The factors that modulate apoptosis of testicular cells are not known, but it is remarkable that this change takes place before the testosterone peak of the post natal gonadal activation of the first trimester of life. These changes taking place during the newborn period might be important to define testicular function in adults.
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http://dx.doi.org/10.1210/jc.2002-020032DOI Listing
November 2002