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James A Dias - One of the best experts on this subject based on the ideXlab platform.

  • single molecule analyses of fully functional fluorescent protein tagged Follitropin Receptor reveal homodimerization and specific heterodimerization with lutropin Receptor
    Biology of Reproduction, 2015
    Co-Authors: Joseph E Mazurkiewicz, Barbara Lindaushepard, Katharine Herrickdavis, Margarida Barroso, Alfredo Ulloaaguirre, Richard M Thomas, James A Dias
    Abstract:

    We have previously shown that the carboxyl terminus (cT) of human follicle-stimulating hormone (FSH, Follitropin) Receptor (FSHR) is clipped before insertion into the plasma membrane. Surprisingly, several different constructs of FSHR fluorescent fusion proteins (FSHR-FPs) failed to traffic to the plasma membrane. Subsequently, we discovered that substituting the extreme cT of luteinizing hormone (LH) Receptor (LHR) to create an FSHR-LHRcT chimera has no effect on FSHR functionality. Therefore, we used this approach to create an FSHR-LHRcT-FP fusion. We found this chimeric FSHR-LHRcT-FP was expressed in HEK293 cells at levels similar to reported values for FSHR in human granulosa cells, bound FSH with high affinity, and transduced FSH binding to produce cAMP. Quantitative fluorescence resonance energy transfer (FRET) analysis of FSHR-LHRcT-YFP/FSHR-LHRcT-mCherry pairs revealed an average FRET efficiency of 12.9 ± 5.7. Advanced methods in single-molecule analyses were applied in order to ascertain the oligomerization state of the FSHR-LHRcT. Fluorescence correlation spectroscopy coupled with photon-counting histogram analyses demonstrated that the FSHR-LHRcT-FP fusion protein exists as a freely diffusing homodimer in the plasma membrane. A central question is whether LHR could oligomerize with FSHR, because both Receptors are coexpressed in differentiated granulosa cells. Indeed, FRET analysis revealed an average FRET efficiency of 14.4 ± 7.5 when the FSHR-LHR cT-mCherry was coexpressed with LHR-YFP. In contrast, coexpression of a 5-HT2cVSV-YFP with FSHR-LHR cT-mCherry showed only 5.6 ± 3.2 average FRET efficiency, a value indistinguishable from the detection limit using intensity-based FRET methods. These data demonstrate that coexpression of FSHR and LHR can lead to heterodimerization, and we hypothesize that it is possible for this to occur during granulosa cell differentiation.

  • Trafficking of the Follitropin Receptor
    Methods in Enzymology, 2013
    Co-Authors: Alfredo Ulloa-aguirre, James A Dias, George Bousfield, I. Huhtaniemi, Eric Reiter
    Abstract:

    The Follitropin or follicle-stimulating hormone Receptor (FSHR) belongs to a highly conserved subfamily of the G protein-coupled Receptor (GPCR) superfamily and is mainly expressed in specific cells in the gonads. As any other GPCR, the newly synthesized FSHR has to be correctly folded and processed in order to traffic to the cell surface plasma membrane and interact with its cognate ligand. In this chapter, we describe in detail the conditions and procedures used to study outward trafficking of the FSHR from the endoplasmic reticulum to the plasma membrane. We also describe some methods to analyze phosphorylation, beta-arrestin recruitment, internalization, and recycling of this particular Receptor, which have proved useful in our hands for dissecting its downward trafficking and fate following agonist stimulation.

  • Trafficking of the Follitropin Receptor
    Methods in enzymology, 2013
    Co-Authors: Alfredo Ulloa-aguirre, James A Dias, George Bousfield, I. Huhtaniemi, Eric Reiter
    Abstract:

    The Follitropin or follicle-stimulating hormone Receptor (FSHR) belongs to a highly conserved subfamily of the G protein-coupled Receptor (GPCR) superfamily and is mainly expressed in specific cells in the gonads. As any other GPCR, the newly synthesized FSHR has to be correctly folded and processed in order to traffic to the cell surface plasma membrane and interact with its cognate ligand. In this chapter, we describe in detail the conditions and procedures used to study outward trafficking of the FSHR from the endoplasmic reticulum to the plasma membrane. We also describe some methods to analyze phosphorylation, β-arrestin recruitment, internalization, and recycling of this particular Receptor, which have proved useful in our hands for dissecting its downward trafficking and fate following agonist stimulation.

  • Human Follitropin Receptor (FSHR) interacts with the adapter protein 14-3-3tau.
    Molecular and cellular endocrinology, 2004
    Co-Authors: Brian D. Cohen, Cheryl A Nechamen, James A Dias
    Abstract:

    The human Follitropin (follicle stimulating hormone, FSH) Receptor (FSHR) is a G protein-coupled Receptor (GPCR). To identify cytoplasmic proteins that may regulate FSHR function, a yeast-based interaction trap was performed. A linked construct of the first and second intracellular loops (iL1-iL2 bait) of FSHR was used as bait and a human ovarian cDNA library was used as prey. Among the proteins identified that interacted with the bait was 14-3-3tau, a member of a family of homodimeric cytoplasmic adapter proteins. Human granulosa cells, the site of FSHR expression in the ovary, were found to contain 14-3-3tau. Importantly, 14-3-3tau co-immunoprecipitated with FSHR stably expressed in HEK 293 cells. Its association with FSHR was Follitropin-dependent. Over-expression of 14-3-3tau resulted in a modest decrease of Follitropin-induced cAMP accumulation. Collectively, these data support a role for 14-3-3tau in Follitropin action. The finding that 14-3-3tau interacts with FSHR is novel and should lead to new insights into the regulation of GPCR in general and FSHR specifically.

  • Human Follitropin Receptor (FSHR) interacts with the adapter protein 14-3-3τ
    Molecular and Cellular Endocrinology, 2004
    Co-Authors: Brian D. Cohen, Cheryl A Nechamen, James A Dias
    Abstract:

    The human Follitropin (follicle stimulating hormone, FSH) Receptor (FSHR) is a G protein-coupled Receptor (GPCR). To identify cytoplasmic proteins that may regulate FSHR function, a yeast-based interaction trap was performed. A linked construct of the first and second intracellular loops (iL1-iL2 bait) of FSHR was used as bait and a human ovarian cDNA library was used as prey. Among the proteins identified that interacted with the bait was 14-3-3tau, a member of a family of homodimeric cytoplasmic adapter proteins. Human granulosa cells, the site of FSHR expression in the ovary, were found to contain 14-3-3tau. Importantly, 14-3-3tau co-immunoprecipitated with FSHR stably expressed in HEK 293 cells. Its association with FSHR was Follitropin-dependent. Over-expression of 14-3-3tau resulted in a modest decrease of Follitropin-induced cAMP accumulation. Collectively, these data support a role for 14-3-3tau in Follitropin action. The finding that 14-3-3tau interacts with FSHR is novel and should lead to new insights into the regulation of GPCR in general and FSHR specifically.

M. Ram Sairam - One of the best experts on this subject based on the ideXlab platform.

  • Bone Marrow Transplantation Restores Follicular Maturation and Steroid Hormones Production in a Mouse Model for Primary Ovarian Failure
    PloS one, 2012
    Co-Authors: Mohsen Ghadami, Xinlei Chen, M. Ram Sairam, Ebtehal El-demerdash, Dong Zhang, Salama A. Salama, Awadh A. Binhazim, Anthony E. Archibong, Billy R. Ballard, Ayman Al-hendy
    Abstract:

    Recent studies suggest that bone marrow stem cells (BMSCs) are promising grafts to treat a variety of diseases, including reproductive dysfunction. Primary ovarian failure is characterized by amenorrhea and infertility in a normal karyotype female, with an elevated serum level of follicle-stimulating hormone (FSH) and a decrease level of estrogen caused by a mutation in FSH Receptor (FSHR) gene. Currently, there is no effective treatment for this condition. The phenotype of FSHR (−/−) mouse, FORKO (Follitropin Receptor knockout), is a suitable model to study ovarian failure in humans. Female FORKO mice have elevated FSH, decreased estrogen levels, are sterile because of the absence of folliculogenesis, and display thin uteri and small nonfunctional ovaries. In this study, we determined the effects of BMSC transplantation on reproductive physiology in this animal model. Twenty four hours post BMSC transplantation, treated animals showed detectable estroidogeneic changes in daily vaginal smear. Significant increase in total body weight and reproductive organs was observed in treated animals. Hemotoxylin and eosin (H&E) evaluation of the ovaries demonstrated significant increase in both the maturation and the total number of the follicles in treated animals. The FSH dropped to 40–50% and estrogen increased 4–5.5 times in the serum of treated animals compared to controls. The FSHR mRNA was detected in the ovaries of treated animals. Our results show that intravenously injected BMSCs were able to reach the ovaries of FORKO mice, differentiate and express FHSR gene, make FSHR responsive to FSH, resume estrogen hormone production, and restore folliculogenesis.

  • Potentiation of vascular oxidative stress and nitric oxide-mediated endothelial dysfunction by high-fat diet in a mouse model of estrogen deficiency and hyperandrogenemia.
    Journal of the American Society of Hypertension : JASH, 2009
    Co-Authors: Danesh Javeshghani, M. Ram Sairam, Ernesto L. Schiffrin, Rhian M. Touyz
    Abstract:

    Estrogen deficiency is associated with increased cardiovascular risk due, in part, to hypertension, endothelial dysfunction, obesity, and hypercholesterolemia. Underlying mechanisms for this remain unclear. Here, we investigated whether high-fat intake aggravates vascular dysfunction through oxidative stress and inflammation, which could predispose to cardiovascular injury in conditions of estrogen deficiency, such as menopause. We studied female homozygous Follitropin Receptor knock out (FORKO) mice, which have hormonal features of clinical menopause and hypertension and wild-type (WT) and heterozygote mice (HTZ), fed a standard or high-fat diet for 4 months. Vascular function and structure were evaluated in arterial segments by pressurized myography. Acetylcholine (ACh)-induced vasodilation was reduced in FORKO vs. WT mice (P < .001). Nϖ-nitro-l-arginine-methyl-ester inhibited Ach-induced relaxation in all groups on normal chow and in WT and HTZ on high-fat diet (FD) but had no effect in fat-fed FORKO mice. Antioxidant cocktail (superoxide dismutase, catalase, Tempol) increased ACh responses only in high-fat diet FORKO mice (P < .05). Vascular media-to-lumen ratio was increased and reactive oxygen species (ROS) generation, nitrotyrosine formation, and plasma nitrite levels were augmented in fat-fed FORKO vs. FORKO on normal chow. High-fat diet did not influence vascular inflammatory responses in any group. Our data demonstrate that FORKO mice have altered nitric oxide-sensitive vasorelaxation and vascular remodeling, which are aggravated by high-fat diet. Underlying mechanisms for this may involve decreased NO formation and increased generation of ROS and nitrotyrosine. These findings suggest that high-fat intake potentiates vascular damage in estrogen-deficient states, an effect involving increased oxidative stress.

  • Changes in Adiponectin and Inflammatory Genes in Response to Hormonal Imbalances in Female Mice and Exacerbation of Depot Selective Visceral Adiposity by High-Fat Diet: Implications for Insulin Resistance
    Endocrinology, 2007
    Co-Authors: Hui Zhang, Jayaprakash Aravindakshan, Xinlei Chen, M. Ram Sairam
    Abstract:

    Early obesity and late onset of insulin resistance associated with hormonal imbalances occur in FSH Receptor-deficient Follitropin Receptor knockout female mice. This study tests the hypothesis that chronic high-fat diet aggravates obesogenic changes in a depot-specific manner and explores some molecular links of hormone imbalances with insulin resistance. In SV 129 mice, hormonal imbalances seem obligatory for exacerbation of diet-induced obesity. Visceral adiposity, glucose intolerance, and lipid disturbances in 9-month Follitropin Receptor knockout females were associated with decrease in adiponectin signaling. High-molecular-weight plasma adiponectin and adipose tissue adiponectin mRNA were decreased. Adiponectin Receptors R1 and R2 mRNA was selectively altered in mesenteric fat but not periuterine fat. R2 decreased in the liver and R1 was higher in muscle. Whereas hepatic adenosine monophosphate T-activated protein kinase activity was down-regulated, both phosphoenolpyruvate carboxykinase and glucose...

  • Early alterations in ovarian surface epithelial cells and induction of ovarian epithelial tumors triggered by loss of FSH Receptor.
    Neoplasia (New York N.Y.), 2007
    Co-Authors: Xinlei Chen, Jayaprakash Aravindakshan, Yinzhi Yang, M. Ram Sairam
    Abstract:

    Little is known about the behavior of the ovarian surface epithelium (OSE), which plays a central role in ovarian cancer etiology. It has been suggested that incessant ovulation causes OSE changes leading to transformation and that high gonadotropin levels during postmenopause activate OSE Receptors, inducing proliferation. We examined the chronology of OSE changes, including tumor appearance, in a mouse model where ovulation never occurs due to deletion of Follitropin Receptor. Changes in epithelial cells were marked by pan-cytokeratin (CK) staining. Histologic changes and CK staining in the OSE increased from postnatal day 2. CK staining was observed inside the ovary by 24 days and increased thereafter in tumor-bearing animals. Ovaries from a third of aged (1 year) mutant mice showed CK deep inside, indicating cell migration. These tumors resembled serous papillary adenoma of human ovaries. Weak expression of GATA-4 and elevation of PCNA, cyclooxygenase-1, cyclooxygenase-2, and platelet-derived growth factor Receptors α and β in mutants indicated differences in cell proliferation, differentiation, and inflammation. Thus, we report that OSE changes occur long before epithelial tumors appear in FORKO mice. Our results suggest that neither incessant ovulation nor follicle-stimulating hormone Receptor presence in the OSE is required for inducing ovarian tumors; thus, other mechanisms must contribute to ovarian tumorigenesis.

  • The tale of Follitropin Receptor diversity: a recipe for fine tuning gonadal responses?
    Molecular and cellular endocrinology, 2006
    Co-Authors: M. Ram Sairam, P Suresh Babu
    Abstract:

    The original concept (dogma) of a single FSH Receptor entity coupling to G(s) protein to activate adenylate cyclase and producing cAMP as second messenger appears inadequate to explain pleiotropic actions of the hormone. The identification and expression of alternatively spliced gonadotropin Receptors, suggest that alternative splicing could serve as a mechanism for creating Receptor diversity. Studies focused on sheep and mouse gonadal tissues show that the single large gene of approximately 250kb is a modular structure whose pre-mRNA undergoes alternative splicing creating several subtypes (at least four FSH-R1 to R4 identified to date). With segments of the N-terminus that are identical different topographies are generated by differing carboxyl termini. The same gene thus produces Receptor types with different motifs that can display dominant positive, dominant negative, growth factor/cytokine type and potentially soluble binding protein features. Functional relevance is shown by modulation of Receptor variants during hormonal stimulation. Presence of equivalent segments of the gene in the human and bovine suggests conservation and predicts similarity in structures and function. Thus, the complex cellular biology of Follitropin Receptors that may interact differently with polymorphic forms (glycosylation variants) of FSH represents an intricate scheme to regulate hormone signaling.

M R Sairam - One of the best experts on this subject based on the ideXlab platform.

  • Molecular Genetics of the Follitropin Receptor: Structural Diversity, Mutations, Gene Knock Out and Biological Implications
    Indian National Science Academy, 2015
    Co-Authors: M R Sairam
    Abstract:

    Molecular Genetics of the Follitropin Receptor: Structural Diversity, Mutations, Gene Knock Out and Biological Implication

  • Chronology and complexities of ovarian tumorigenesis in FORKO mice: age-dependent gene alterations and progressive dysregulation of Major Histocompatibility Complex (MHC) Class I and II profiles.
    Molecular and Cellular Endocrinology, 2010
    Co-Authors: Jayaprakash Aravindakshan, Xinlei Chen, M R Sairam
    Abstract:

    Among gynecologic malignancies ovarian cancer is the deadliest and most difficult to detect at early stages. As ovarian tumors have long latency and are relatively more frequent in postmenopausal women, revealing chronological changes in model systems might help in the discovery of novel molecular targets and diagnostic biomarkers for disease detection and management. Follitropin Receptor knockout (FORKO) mice with early and sustained sex steroid hormone disharmony develop various age-dependent ovarian abnormalities including increased incidence ovarian tumors in complete absence of ovulation. These mutants show various tumor cell types including those related to ovarian surface epithelium around 12-15 months of age. To explore why the FORKO mice develop ovarian tumors later in life, we assessed global gene expression changes during the pre-tumor period (at 8 months). Age-matched wild-type and FORKO mice were compared to gain a comprehensive view of genes that are misregulated, even before overt tumors appear in mutants. Applying a conservative 2-fold change to detect changes, our study identified 476 genes (338 upregulated and 138 downregulated) to be altered between 8-month-old FORKO and wild-type ovaries. Using Ingenuity Pathway Analysis (IPA), we found highly significant alterations in five functional networks in pre-tumor stage FORKO ovaries. Notably, the top network to change in 8-month-old FORKO ovaries was associated with functions implicated in immune system development and function. We selected 9 immune related genes that are reportedly altered in Epithelial Ovarian Cancer (EOC) in women and confirmed their expression and chronology of changes in FORKO ovaries before and after tumor development. Our data indicate that immune surveillance mechanisms are compromised with in a 4-month window of tumorigenic alterations. In addition, expression of previously unrecognized genes misregulated in the dysfunctional FORKO ovaries suggests mechanisms not yet appreciated to date. We propose that a better understanding of genes that change before overt tumors develop could provide useful insights into ovarian carcinogenesis and open the door to additional new targets for treating ovarian cancers.

  • Toward gene therapy of premature ovarian failure: intraovarian injection of adenovirus expressing human FSH Receptor restores folliculogenesis in FSHR(−/−) FORKO mice
    Molecular human reproduction, 2010
    Co-Authors: Mohsen Ghadami, M R Sairam, Ebtehal El-demerdash, Salama A. Salama, Awadh A. Binhazim, Anthony E. Archibong, Billy R. Ballard, X. Chen, Ayman Al-hendy
    Abstract:

    A homozygous missense mutation, C566T, in the follicle stimulation hormone Receptor (FSHR) gene has been linked to premature ovarian failure. The disease leads to infertility in a normal karyotype female with an elevated follicle stimulating hormone (FSH) and decreased serum estrogen level. Female mice carrying mutated FSHR gene, called Follitropin Receptor knockout (FORKO), display similar phenotype and are sterile because of a folliculogenesis block at a primary stage. We investigated the effects of bilateral intra-ovarian injection of an adenovirus expressing a normal copy of human FSHR on the reproductive system of 6–10 weeks female FORKO mice. Ad-LacZ was injected directly into each ovary of the control group. Animals were sacrificed at 2, 4, 8 and 12 weeks post-injection and tissues collected for evaluation. Treated mice showed estrogenic changes in daily vaginal smear whereas control animals remained fixated in the diestrus stage. Histological evaluation showed on average 26 ± 4 follicles/ovary in treated group with 8 ± 2 follicles at the antral stage compared with only 5 ± 2 with zero follicles at antral stage in Ad-LacZ control mice. There was no significant change in serum level of progesterone, however, estrogen level increased 2–3-fold (P < 0.02) and FSH decreased by up to 50% (P < 0.04) in treated animals. FSHR mRNA was detected in the ovaries of the treated group. In conclusion, intra-ovarian injection of an adenovirus expressing human FSHR gene is able to restore FSH responsiveness and reinitiate ovarian folliculogenesis as well as resume estrogen production in female FORKO mice. Ad-LacZ injections indicate the absence of systemic viral dissemination or germ line transmission of adenovirus DNA to offspring.

  • age dependent bimodal gdnf regulation during ovarian tumorigenesis in Follitropin Receptor mutant mice
    Biochemical and Biophysical Research Communications, 2006
    Co-Authors: Jayaprakash Aravindakshan, Xinlei Chen, M R Sairam
    Abstract:

    Most ovarian tumors in women occur upon aging. Follitropin Receptor knockout (FORKO) mice are sterile and have age-dependent abnormalities including increased ovarian tumor incidence. To explore why atrophic ovaries of FORKO mice become tumorigenic later in life, we compared gene expression profiles by microarray at different ages. Here we show an unexpected ovarian expression of GDNF and its bimodal regulation. GDNF was down-regulated at a young age but up-regulated in aging FORKO mice prior to tumor appearance. Immunohistochemistry localized GDNF in the oocyte as well as somatic granulosa and stromal cells. GDNF protein also showed an age-dependent increase in the ovary, being lower in young mutants and increasing by 6 months. We found evidence for GDNF up-regulation in GC tumors and a potential role for androgen. The peripheral expression pattern and functions of this powerful neurotropic factor suggest mediation of processes involved in pathology of ovarian compartments.

  • perspectives on reproductive senescence and biological aging studies in genetically altered Follitropin Receptor knockout forko mice
    Experimental Gerontology, 2004
    Co-Authors: Natalia Danilovich, Dusica Maysinger, M R Sairam
    Abstract:

    Increased life expectancy leads to increased age-associated health issues in both sexes. For menopausal women the most important of these appear to result from the severe estrogen deficiency caused by ovarian dysfunction. The consequences among others include hot flashes, osteoporosis, obesity, impaired memory, higher incidence of Alzheimer's disease and cardiovascular disease. Ovarian function and steroidogenesis are influenced by pituitary gonadotropins, including follicle-stimulating hormone (FSH), whose actions are mediated through ovarian Receptors. This article highlights our recent data pertinent to aging as derived from a novel genetically modified animal model [the FORKO mouse (Follitropin Receptor KnockOut) lacking the FSH Receptor. FORKO female mice experience a chronic depletion of estrogen (E2) from early development, and have phenotypes similar to aging women, with ovarian failure, obesity, skeletal changes, and ovarian tumors. A variety of findings support the conclusion that E2 deficiency in FORKO mice is responsible for their neural impairments associated with glial cell hypertrophy, region-specific brain cells loss, and abnormal behavior. Findings from mice with FSH Receptor haploinsufficiency mice ('menopausal mice') are also shedding light on the molecular basis of menopausal conditions that include degeneration of the hippocampus. Many phenotypes noted in the null condition also occur in +/- females but in an age related manner. Thus, the FORKO mouse becomes an excellent model to investigate mechanisms underlying age-related changes especially when these events are accelerated, as in menopausal women. Opportunities abound to assess the potential benefits/adverse effects of hormone replacement regimen on various targets.

Ram M Sairam - One of the best experts on this subject based on the ideXlab platform.

  • Differential Expression of Claudin Family Proteins in Mouse Ovarian Serous Papillary Epithelial Adenoma in Aging
    2015
    Co-Authors: Fsh Receptor–deficient Mutants, Jayaprakash Aravindakshan, Xinlei Chen, Ram M Sairam
    Abstract:

    Ovarian cancer is a deadly disease with long latency. To understand the consequences of loss of follicle-stimulating hormone Receptor (FSH-R) signaling and to explore why the atrophic and anovulatory ovaries of Follitropin Receptor knockout (FORKO) mice develop different types of ovarian tumors, including serous papillary epithelial adenoma later in life, we usedmRNA expression profiling to gain a comprehensive view of misregulated genes. Using real-time quantitative re-verse transcription–polymerase chain reaction, pro-tein analysis, and cellular localization, we show, for the first time, in vivo evidence that, in the absence of FSH-R signaling, claudin-3, claudin-4, and claudin-11 are selectively upregulated, whereas claudin-1 de-creases in ovarian surface epithelium and tumors in comparison to wild type. In vitro experiments using a mouse ovarian surface epithelial cell line derived from wild-type females reveal direct hormonal influence on claudin proteins. Although recent studies suggest that cell junction proteins are differentially expressed in ovarian tumors in women, the etiology of such changes remains unclear. Our results suggest an altered hormonal environment resulting from FSH-R loss as a cause of early changes in tight junction pro-teins that predispose the ovary to late-onset tumors that occur with aging. More importantly, this study identifies claudin-11 overexpression in mouse ovarian serous cystadenoma

  • doi: 10.1210/en.2007-0572 Impairment of the Natriuretic Peptide System in Follitropin Receptor Knockout Mice and Reversal by Estradiol: Implications for Obesity-Associated
    2013
    Co-Authors: Hypertension In Menopause, Ram M Sairam, Najara O. Belo, Adelina Dos M. Reis
    Abstract:

    Estrogen is considered a major regulator of adipose tissue in females. Estrogen increases circulating levels of atrial natriuretic peptide (ANP), a hormone with renal and cardiovascular effects. The aim of this study was to determine the status of the natriuretic peptide system in female Follitropin-Receptor knockout (FORKO) mice that could be associated with obesity and hypertension observed in these mutants. Furthermore, estradiol treatment was used to reverse alterations observed. FORKO and wild-type (WT) mice received daily injections of estradiol for 4 d. On the fifth day, blood was collected for determination of plasma ANP levels, and selected tissues were collected for determination of ANP, natriuretic peptide Receptor type-A (NPR-A) and type-C (NPR-C) gene expression by RT-PCR and binding of [ 125 I]ANP by autoradiography. At ESTROGEN IS A MAJOR factor in regulating adipos

  • impairment of the natriuretic peptide system in Follitropin Receptor knockout mice and reversal by estradiol implications for obesity associated hypertension in menopause
    Endocrinology, 2008
    Co-Authors: Ram M Sairam, Najara O. Belo, Adelina M Reis
    Abstract:

    Estrogen is considered a major regulator of adipose tissue in females. Estrogen increases circulating levels of atrial natriuretic peptide (ANP), a hormone with renal and cardiovascular effects. The aim of this study was to determine the status of the natriuretic peptide system in female Follitropin-Receptor knockout (FORKO) mice that could be associated with obesity and hypertension observed in these mutants. Furthermore, estradiol treatment was used to reverse alterations observed. FORKO and wild-type (WT) mice received daily injections of estradiol for 4 d. On the fifth day, blood was collected for determination of plasma ANP levels, and selected tissues were collected for determination of ANP, natriuretic peptide Receptor type-A (NPR-A) and type-C (NPR-C) gene expression by RT-PCR and binding of [125I]ANP by autoradiography. At 5 months of age, FORKO mice were heavier and had more adipose tissue than WT mice. FORKO mice had lower plasma ANP levels and atrial ANP gene expression and higher renal and ad...

  • increased blood pressure vascular inflammation and endothelial dysfunction in androgen deficient Follitropin Receptor knockout male mice
    Journal of The American Society of Hypertension, 2007
    Co-Authors: Danesh Javeshghani, Ernesto L. Schiffrin, Ram M Sairam, Rhian M. Touyz
    Abstract:

    The relationship between testosterone, vascular function, and blood pressure remains unclear. Here we utilized a mouse model of andropause, Follitropin Receptor knockout (FORKO) male mice, which are testosterone-deficient, to investigate whether vascular function and structure are altered and whether this is associated with elevated blood pressure. Blood pressure was measured by radiotelemetry, and vascular function and structure were assessed in isolated pressurized mesenteric resistance arteries in wild-type (WT) and FORKO mice. Diastolic and mean arterial pressures were significantly higher in FORKO than in WT mice (P < .05). Resistance arteries of FORKO mice had greater media-to-lumen ratio (10.4 vs. 8.2; P < .05) and reduced relaxation responses to acetylcholine (Ach) (62% vs. 94% at Ach 10−4 mol/L, P < .05) in pressurized preparations. Nω-nitro-L-arginine (L-NAME) reduced Ach-induced relaxation equally in both groups (45% to 46%), and plasma nitrite was lower (P < .05) in FORKO mice. However, the L-NAME-resistant relaxation was smaller in FORKO (16% vs. 48% at Ach 10−4 mol/L, P < .05). In FORKO, expression of intracellular adhesion molecule-1 and vascular cell adhesion molecule-1 was enhanced by immunohistochemistry, and vascular estrogen Receptors (ER)α/ERβ expression ratio was decreased 5-fold by immunoblot analysis. Vasoconstrictor responses to angiotensin II were blunted, and angiotensin Receptor 1 expression was decreased in FORKO mice. Our data indicate that in androgen-deficient FORKO mice, blood pressure is elevated and resistance arteries exhibit endothelial dysfunction, structural remodeling, and vascular inflammation. These phenomena may be related to reduced expression of ERα and/or to decreased testosterone levels and indicate that androgens may play an important role in modulating vascular function and regulation of blood pressure.

  • impairments of heat shock protein expression and mapk translocation in the central nervous system of Follitropin Receptor knockout mice
    Experimental Gerontology, 2007
    Co-Authors: Lada Rumora, Ram M Sairam, Jasmina Lovric, Dusica Maysinger
    Abstract:

    The central nervous system is exposed to the chronic oxidative stress during aging when the endogenous defence weakens and the load of reactive oxygen species enhances. Sex hormones and heat shock proteins (Hsps) participate in these responses to stress. Their regulation is disturbed in aging. We assessed the expression of Hsps in hippocampus and cortex of Follitropin Receptor knockout (FORKO) mice, known to exhibit gender and age-dependent imbalance in sex steroids and gonadotropins. These imbalances could contribute to an impaired regulation of Hsps thereby increasing the risk of developing neurodegenerative disorders. Our study shows that, in the hippocampus the expression of Hsp70 and Hsp25 was reduced in 20-month-old FORKO mice. However, in the cortex both Hsps were significantly down regulated only in elderly females. There is a well-established co-regulation between Hsps and mitogen-activated protein kinases (MAPKs). Significant, gender-specific impairments in the translocation of phosphorylated ERK and JNK were found in the CNS structures in aged FORKO mice. Our results suggest that hormonal imbalances lead to a disturbed subcellular distribution of activated MAPKs which contribute to the impairments of signal transduction networks maintaining normal physiological functions in the cortex and hippocampus that are associated with neurodegenerative changes in aging.

Sabine Costagliola - One of the best experts on this subject based on the ideXlab platform.

  • Molecular mechanisms selected by evolution m primates for self-protection against human chorionic gonadotropin
    Bulletin et memoires de l'Academie royale de medecine de Belgique, 2008
    Co-Authors: Sabine Costagliola, Anne De Leener
    Abstract:

    The constitutive activation of the Follitropin Receptor (FSHR) could lead to promiscuous toxic activation by placental chorionic gonadotropin (CG) during human pregnancy. This study demonstrated that the evolution of the transmembrane region of the simian FSHR parallels the progressive accumulation of CG copies in the primate genomes. We assist to a purifying selection to keep the FSHR constitutively inactive and thus insensitive to CG.

  • identification of the first germline mutation in the extracellular domain of the Follitropin Receptor responsible for spontaneous ovarian hyperstimulation syndrome
    Human Mutation, 2008
    Co-Authors: Anne De Leener, Gilbert Vassart, Leonardo Pardo, Gianluigi Caltabiano, Sanly Erkan, Mehmet Idil, Sabine Costagliola
    Abstract:

    The Receptors for Follitropin (FSHR), thyrotropin (TSHR), and lutropin/chorionic gonadotropin (LHCGR) are the members of the glycoprotein hormone (GPH) Receptors (GPHR) family. They present a bipartite structure with a large extracellular amino-terminal domain (ECD), responsible for high-affinity hormone binding, and a carboxyl-terminal serpentine region, implicated in transduction of the activation signal. Spontaneous ovarian hyperstimulation syndrome (sOHSS) is a rare genetic condition in which human chorionic gonadotropin (hCG) promiscuously stimulates the FSHR during the first trimester of pregnancy. Surprisingly, germline FSHR mutations responsible for the disease have so far been found only in the transmembrane helices of the serpentine region of the FSHR, outside the hormone binding domain. When tested functionally, all mutants were abnormally sensitive to both hCG and thyrotropin (TSH) while displaying constitutive activity. This loss of ligand specificity was attributed to the lowering of an intramolecular barrier of activation rather than to an increase of binding affinity. Here we report the first germline mutation responsible for sOHSS (c.383C4A, p.Ser128Tyr), located in the ECD of the FSHR. Contrary to the mutations described previously, the p.Ser128Tyr FSHR mutant displayed increase in affinity and sensitivity toward hCG and did not show any constitutive activity, nor promiscuous activation by TSH. Thus, sOHSS can be achieved from different molecular mechanisms involving each functional domains of the FSHR. Based on the structure of the FSHR/ FSH complex and site-directed mutagenesis studies, we provide robust molecular models for the GPH/GPHR complexes and we propose a molecular explanation to the binding characteristics of the p.Ser128Tyr mutant. Hum Mutat 29(1), 91–98, 2008. r 2007 Wiley-Liss, Inc.

  • identification of the first germline mutation in the extracellular domain of the Follitropin Receptor responsible for spontaneous ovarian hyperstimulation syndrome
    Human Mutation, 2008
    Co-Authors: Anne De Leener, Gilbert Vassart, Leonardo Pardo, Gianluigi Caltabiano, Sanly Erkan, Mehmet Idil, Sabine Costagliola
    Abstract:

    The Receptors for Follitropin (FSHR), thyrotropin (TSHR), and lutropin/chorionic gonadotropin (LHCGR) are the members of the glycoprotein hormone (GPH) Receptors (GPHR) family. They present a bipartite structure with a large extracellular amino-terminal domain (ECD), responsible for high-affinity hormone binding, and a carboxyl-terminal serpentine region, implicated in transduction of the activation signal. Spontaneous ovarian hyperstimulation syndrome (sOHSS) is a rare genetic condition in which human chorionic gonadotropin (hCG) promiscuously stimulates the FSHR during the first trimester of pregnancy. Surprisingly, germline FSHR mutations responsible for the disease have so far been found only in the transmembrane helices of the serpentine region of the FSHR, outside the hormone binding domain. When tested functionally, all mutants were abnormally sensitive to both hCG and thyrotropin (TSH) while displaying constitutive activity. This loss of ligand specificity was attributed to the lowering of an intramolecular barrier of activation rather than to an increase of binding affinity. Here we report the first germline mutation responsible for sOHSS (c.383C>A, p.Ser128Tyr), located in the ECD of the FSHR. Contrary to the mutations described previously, the p.Ser128Tyr FSHR mutant displayed increase in affinity and sensitivity toward hCG and did not show any constitutive activity, nor promiscuous activation by TSH. Thus, sOHSS can be achieved from different molecular mechanisms involving each functional domains of the FSHR. Based on the structure of the FSHR/FSH complex and site-directed mutagenesis studies, we provide robust molecular models for the GPH/GPHR complexes and we propose a molecular explanation to the binding characteristics of the p.Ser128Tyr mutant.

  • GRIS: Glycoprotein-Hormone Receptor Information System
    Molecular endocrinology (Baltimore Md.), 2006
    Co-Authors: Joost Van Durme, Sabine Costagliola, Florence Horn, Gert Vriend, Gilbert Vassart
    Abstract:

    The glycoprotein-hormone Receptor information system (GRIS) presents a comprehensive view on all available molecular data for the lutropin/choriogonadotropin Receptor, Follitropin Receptor, and thyrotropin Receptor G protein-coupled Receptors. It features a mutation database presently containing 696 point mutations, combined with all sequences and the associated homology models. The mutation information was automatically extracted from the literature and manually augmented with respect to constitutivity, surface expression, sensitivity to hormones, and binding affinity. All information in this integrated system is presented in a G protein-coupled Receptor specialist-friendly way. A series of interactive tools such as rotamer analysis, mutation prediction, or cavity visualization aids with the design and interpretation of experiments. A universal residue numbering system has been introduced to ease database searches as well as the use of the information in conjunction with literature data from diverse origins. Users can upload new mutations. GRIS is freely accessible at http://gris.ulb.ac.be/.

  • a mutation in the follicle stimulating hormone Receptor as a cause of familial spontaneous ovarian hyperstimulation syndrome
    The Journal of Clinical Endocrinology and Metabolism, 2004
    Co-Authors: Lucia Montanelli, Anne Delbaere, Costantino Di Carlo, Carmine Nappi, Guillaume Smits, Gilbert Vassart, Sabine Costagliola
    Abstract:

    Ovarian hyperstimulation syndrome (OHSS) occurs mainly after excessive stimulation of the ovaries by exogenous gonadotropins administrated in the context of in vitro fertilization procedures (iatrogenic OHSS). Recently, spontaneous and recurrent occurrence of the disease (spontaneous OHSS) was shown in two families to be caused by mutations affecting the Follitropin Receptor (FSHr). The two mutant FSHr (T449I, D567N) harbor aminoacid substitutions in the serpentine portion of the Receptor and display abnormally high sensitivity to the pregnancy hormone hCG, thus providing a satisfactory explanation to the phenotype. In addition, mutant D567N showed also increased sensitivity to thyrotopin (TSH) and displayed increase in basal (ligand-independent) activity. In this report, we describe a new familial case of recurrent OHSS. The affected women were heterozygous for a different mutation involving codon 449, where an alanine was substituted for threonine. Similar to D567N, the T449A FSHr mutant shows an increa...