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Auclair S.,CNRS Physiology of Reproduction and Behaviors | Auclair S.,University of Tours | Rossetti R.,University of Milan | Meslin C.,CNRS Physiology of Reproduction and Behaviors | And 10 more authors.
PLoS ONE | Year: 2013

Bone Morphogenetic Protein 15 (BMP15) is a TGFβ-like oocyte-derived growth factor involved in ovarian folliculogenesis as a critical regulator of many granulosa cell processes. Alterations of the BMP15 gene have been found associated with different ovarian phenotypic effects depending on the species, from sterility to increased prolificacy in sheep, slight subfertility in mouse or associated with primary ovarian insufficiency (POI) in women. To investigate the evolving role of BMP15, a phylogenetic analysis of this particular TGFβ family member was performed. A maximum likelihood phylogenetic tree of several TGFβ/BMP family members expressed by the ovary showed that BMP15 has a very strong divergence and a rapid evolution compared to others. Moreover, among 24 mammalian species, we detected signals of positive selection in the hominidae clade corresponding to F146, L189 and Y235 residues in human BMP15. The biological importance of these residues was tested functionally after site directed-mutagenesis in a COV434 cells luciferase assay. By replacing the positively selected amino acid either by alanine or the most represented residue in other studied species, only L189A, Y235A and Y235C mutants showed a significant increase of BMP15 signaling when compared to wild type. Additionally, the Y235C mutant was more potent than wild type in inhibiting progesterone secretion of ovine granulosa cells in primary culture. Interestingly, the Y235C mutation was previously identified in association with POI in women. In conclusion, this study evidences that the BMP15 gene has evolved faster than other members of the TGFß family and was submitted to a positive selection pressure in the hominidae clade. Some residues under positive selection are of great importance for the normal function of the protein and thus for female fertility. Y235 represents a critical residue in the determination of BMP15 biological activity, thus indirectly confirming its role in the onset of POI in women. © 2013 Auclair et al. Source


Persani L.,Laboratorio Of Ricerche Endocrino Metaboliche | Persani L.,University of Milan | Bonomi M.,Laboratorio Of Ricerche Endocrino Metaboliche | Lleo A.,Center for Autoimmune Liver Diseases | And 14 more authors.
Journal of Autoimmunity | Year: 2012

Multiple mechanisms have been proposed to explain the peculiar distribution of autoimmune thyroiditis (AIT) among women and men. Most attention has been focused on the detection of the role of estrogens and the X chromosome. Specifically, a potential role for X haploinsufficiency has been proposed in the female patient population and an association with the disease has been confirmed. Our knowledge of the etiopathogenesis of autoimmunity in male patients remains, however, limited. Next to the possible role of androgens and their imbalances, the Y chromosome appears as a potential candidate for influence of the immune function in men. Herein we analyzed a population of male patients with AIT (n=31) and healthy controls (n=88) to define a potential association of disease and the loss of the Y chromosome. Y chromosome loss increases in AIT compared to unaffected subjects; these phenomenon increases with aging as expected, however, the degree of loss is significantly increased in the patient population compared to the healthy controls. We were, thus, able to confirm the existence of an analogous mechanism in the male population to previously identified X haploinsufficiency in female patients with AIT. We propose that this commonality might represent a relevant feature in the etiopathogenesis of AIT that should be further investigated. © 2011 Elsevier Ltd. Source


Libri D.V.,Laboratorio Of Ricerche Endocrino Metaboliche | Kleinau G.,Charite - Medical University of Berlin | Vezzoli V.,University of Milan | Busnelli M.,CNR Institute of Neuroscience | And 12 more authors.
Journal of Clinical Endocrinology and Metabolism | Year: 2014

Introduction: Defects of prokineticin pathway affect the neuroendocrine control of reproduction, but their role in the pathogenesis of central hypogonadism remains undefined, and the functional impact of the missense PROKR2 variants has been incompletely characterized. Material and Methods: In a series of 246 idiopathic central hypogonadism patients,wefound three novel (p.V158I, p.V334M, and p.N15TfsX30) and six already known (p.L173R, p.T260M, p.R268C, p.V274D, p.V331M, and p.H20MfsX23) germline variants in the PROKR2 gene. We evaluated the effects of seven missense alterationsontwodifferent prokineticin receptor 2 (PROKR2)-dependent pathways: inositol phosphate-Ca2+ (Gq coupling) and cAMP (Gs coupling). Results: PROKR2 variants were found in 16 patients (6.5%). Expression levels of variants p.V158I and p.V331M were moderately reduced, whereas they were markedly impaired in the remaining cases, except p.V334M, which was significantly overexpressed. The variants p.T260M, p.R268C, and p.V331M showed no remarkable changes in cAMP response (EC50) whereas the IP signaling appeared more profoundly affected. In contrast, cAMP accumulation cannot be stimulated through the p.L173R and p.V274D, but IP EC50 was similar to wt inp.L173R and increased by 10-fold in p.V274D. The variant p.V334M led to a 3-fold increase of EC50 for both cAMP and IP. Conclusion: Our study shows that single PROKR2 missense allelic variants can either affect both signaling pathways differently or selectively. Thus, the integrity of both PROKR2-dependent cAMP and IP signals should be evaluated for a complete functional testing of novel identified allelic variants. Copyright © 2014 by the Endocrine Society. Source


De Filippis T.,Laboratorio Of Ricerche Endocrino Metaboliche | Marelli F.,Laboratorio Of Ricerche Endocrino Metaboliche | Nebbia G.,Clinica Pediatrica De Marchi | Porazzi P.,Laboratorio Of Ricerche Endocrino Metaboliche | And 17 more authors.
Journal of Clinical Endocrinology and Metabolism | Year: 2016

Context: The pathogenesis of congenital hypothyroidism (CH) is still largely unexplained. We previously reported that perturbations of the Notch pathway and knockdown of the ligand jagged1 cause a hypothyroid phenotype in the zebrafish. Heterozygous JAG1 variants are known to account for Alagille syndrome type 1 (ALGS1), a rare multisystemic developmental disorder characterized by variable expressivity and penetrance. Objective: Verify the involvement of JAG1 variants in the pathogenesis of congenital thyroid defects and the frequency of unexplained hypothyroidism in a series of ALGS1 patients. Design, Settings, and Patients: A total of 21 young ALGS1 and 100 CH unrelated patients were recruited in academicandpublic hospitals. TheJAG1variantswerestudied in vitroandin the zebrafish. Results:Wereport a previously unknown nonautoimmune hypothyroidism in 6/21 ALGS1 patients, 2 of them with thyroid hypoplasia. We found 2 JAG1 variants in the heterozygous state in 4/100 CH cases (3 with thyroid dysgenesis, 2 with cardiac malformations). Five out 7 JAG1 variants are new. Different bioassays demonstrate that the identified variants exhibit a variable loss of function. In zebrafish, the knock-down of jag1a/b expression causes a primary thyroid defect, and rescue experiments of the hypothyroid phenotype with wild-type or variant JAG1 transcripts support a role for JAG1 variations in the pathogenesis of the hypothyroid phenotype seen in CH and ALGS1 patients. Conclusions: clinical and experimental data indicate that ALGS1 patients have an increased risk of nonautoimmune hypothyroidism, and that variations in JAG1 gene can contribute to the pathogenesis of variable congenital thyroid defects, including CH. © 2016 by the Endocrine Society. Source


Porazzi P.,University of Milan | Marelli F.,University of Milan | Benato F.,University of Padua | De Filippis T.,Laboratorio Of Ricerche Endocrino Metaboliche | And 5 more authors.
Endocrinology | Year: 2012

The mechanisms underlying the early steps of thyroid development are largely unknown. In search for novel candidate genes implicated in thyroid function, we performed a gene expression analysis on thyroid cells revealing that TSH regulates the expression of several elements of the Notch pathway, including the ligand Jagged1. Because the Notch pathway is involved in cell-fate determination of several foregut-derived endocrine tissues, we tested its contribution in thyroid development using the zebrafish, a teleost model recapitulating the mammalian molecular events during thyroid development. Perturbing the Notch signaling (e.g. mib mutants, γ-secretase inhibition, or Notch intracellular domain overexpression), we obtained evidence that this pathway has a biological role during the earlier phases of thyroid primordium induction, limiting the number of cells that proceed to a specialized fate and probably involving actions from surrounding tissues. Moreover, we were able to confirm the expression of Jagged1 during different phases of zebrafish thyroid development, as well as in mouse and human thyroid tissues. The two orthologues to the single jagged1 gene (JAG1) in humans, jag1a and jag1b, are expressed with different spatiotemporal patterns in the developing zebrafish thyroid. Both jag1a and jag1b morphants, as well as jag1b mutant fish line, display thyroid hypoplasia and impaired T4 production; this thyroid phenotype was rescued by coinjection of human JAG1 mRNA. In conclusion, Notch pathway is involved in the early steps of thyroid morphogenesis, and Jagged1-Notch signal is required for zebrafish thyroid development and function. Thus, genetic alterations affecting the Notch pathway may confer susceptibility for thyroid dysgenesis. Copyright © 2012 by The Endocrine Society. Source

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