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Daniel J. Bernard - One of the best experts on this subject based on the ideXlab platform.
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Human Follicle-Stimulating Hormone ß Subunit Expression Depends on FOXL2 and SMAD4.
Endocrinology, 2020Co-Authors: Luisina Ongaro, Gauthier Schang, Mathias Treier, Ulrich Boehm, Chu-xia Deng, T. Rajendra Kumar, Ziyue Zhou, Daniel J. BernardAbstract:Follicle-stimulating hormone (FSH), an essential regulator of mammalian fertility, is synthesized by pituitary gonadotrope cells in response to activins. In mice, activins signal via SMAD3, SMAD4, and FOXL2 to regulate transcription of the FSHβ subunit (FSHB) gene. Gonadotrope-specific deletion of Foxl2, alone or in combination with Smad4, renders mice FSH-deficient. Whether human FSHB expression is similarly regulated is not known. Here, we used a combination of transgenic and conditional knockout mouse strains to assess the roles of activins, FOXL2, and SMAD4 in regulation of the human FSHB gene. First, we cultured pituitaries from mice harboring a human FSHB transgene (hFSHB mice) and measured both murine FSHB and human FSHB messenger ribonucleic acid (mRNA) expression in response to exogenous activins or two antagonists of endogenous activin-like signaling (follistatin-288 and SB431542). Both murine FSHB and human FSHB expression were stimulated by activins and reduced by the inhibitors. Next, we analyzed human FSHB expression in hFSHB mice carrying floxed Foxl2 and Smad4 alleles. Cre-mediated ablation of FOXL2 and SMAD4 strongly reduced basal and activin-stimulated murine FSHB and human FSHB expression in cultured pituitaries. Finally, the hFSHB transgene was previously shown to rescue FSH production and fertility in FSHB knockout mice. However, gonadotrope-specific Foxl2/Smad4 knockout females carrying the hFSHB transgene have significantly reduced murine FSHB and human FSHB pituitary mRNA levels and are hypogonadal. Collectively, these data suggest that similar to FSHB regulation in mice, FOXL2 and SMAD4 play essential roles in human FSHB expression.
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HDAC inhibitors impair FSHB subunit expression in murine gonadotrope cells.
Journal of molecular endocrinology, 2019Co-Authors: Gauthier Schang, Chirine Toufaily, Daniel J. BernardAbstract:Fertility is dependent on follicle-stimulating hormone (FSH), a product of gonadotrope cells of the anterior pituitary gland. Hypothalamic gonadotropin-releasing hormone (GnRH) and intra-pituitary activins are regarded as the primary drivers of FSH synthesis and secretion. Both stimulate expression of the FSH beta subunit gene (FSHB), although the underlying mechanisms of GnRH action are poorly described relative to those of the activins. There is currently no consensus on how GnRH regulates FSHB transcription, as results vary across species and between in vivo and in vitro approaches. One of the more fully developed models suggests that the murine FSHB promoter is tonically repressed by histone deacetylases (HDACs) and that GnRH relieves this repression, at least in immortalized murine gonadotrope-like cells (LβT2 and αT3-1). In contrast, we observed that the class I/II HDAC inhibitor trichostatin A (TSA) robustly inhibited basal, activin A-, and GnRH-induced FSHB mRNA expression in LβT2 cells and in primary murine pituitary cultures. Similar results were obtained with the class I specific HDAC inhibitor, entinostat, whereas two class II-specific inhibitors, MC1568 and TMP269, had no effects on FSHB expression. Collectively, these data suggest that class I HDACs are positive, not negative, regulators of FSHB expression in vitro and that, contrary to earlier reports, GnRH may not stimulate FSHB by inhibiting HDAC-mediated repression of the gene.
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Follicle-stimulating hormone synthesis and fertility depend on SMAD4 and FOXL2
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 2014Co-Authors: Jérôme Fortin, Mathias Treier, Ulrich Boehm, Chu-xia Deng, Daniel J. BernardAbstract:Follicle-stimulating hormone (FSH) is an essential regulator of gonadal function and fertility. Loss-of-function mutations in the FSHB/FSHB gene cause hypogonadotropic hypogonadism in humans and mice. Both gonadotropin-releasing hormone (GnRH) and activins, members of the transforming growth factor β (TGFβ) superfamily, stimulate FSH synthesis; yet, their relative roles and mechanisms of action in vivo are unknown. Here, using conditional gene-targeting, we show that the canonical mediator of TGFβ superfamily signaling, SMAD4, is absolutely required for normal FSH synthesis in both male and female mice. Moreover, when the Smad4 gene is ablated in combination with its DNA binding cofactor Foxl2 in gonadotrope cells, mice make essentially no FSH and females are sterile. Indeed, the phenotype of these animals is remarkably similar to that of FSHB-knockout mice. Not only do these results establish SMAD4 and FOXL2 as essential master regulators of FSHB transcription in vivo, they also suggest that activins, or related ligands, could play more important roles in FSH synthesis than GnRH.—Fortin, J., Boehm, U., Deng, C.-X., Treier, M., Bernard, D. J. Follicle-stimulating hormone synthesis and fertility depend on SMAD4 and FOXL2.
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cycloheximide inhibits follicle stimulating hormone β subunit transcription by blocking de novo synthesis of the labile activin type ii receptor in gonadotrope cells
Cellular Signalling, 2013Co-Authors: Carlis Rejon, Xiang Zhou, Ying Wang, Daniel J. Bernard, Catherine C Ho, Terence E. HébertAbstract:Abstract The pituitary gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), play essential roles in the regulation of vertebrate reproduction. Activins and inhibins have opposing actions on FSH (but not LH) synthesis, either inducing or inhibiting transcription of the FSHβ subunit gene ( FSHB ). The translational inhibitor cycloheximide (CHX) produces inhibin-like effects in cultured pituitary cells, selectively suppressing FSH production. Using the murine gonadotrope-like cell line, LβT2, as a model, we tested the hypothesis that a component of the activin pathway is highly labile in gonadotrope cells and that its rapid loss following CHX treatment impairs activin-stimulated FSHB transcription. Treatment of cells with CHX for 6 h, but not 1 h, blocked activin A-stimulated FSHB transcription. Pre-treatment of LβT2 cells with CHX for as few as 2–3 h inhibited activin A-stimulated SMAD2/3 phosphorylation without altering total SMAD2/3 protein levels. These data indicated that CHX affects activin signalling upstream of SMAD proteins, most likely at the receptor level. Indeed, CHX rapidly reduced activin A binding to LβT2 cells. We went on to show that activin A signals via the type II receptor ACVR2, rather than ACVR2B, to regulate FSHB transcription and that the receptor has a half life of ~ 2 h in LβT2 cells. The mechanism of ACVR2 turnover remains undefined, but appears to be ligand-, proteasome-, and lysosome-independent. Collectively, these data indicate that CHX produces inhibin-like effects in gonadotropes by preventing de novo synthesis of the highly labile ACVR2, thereby blocking activin signaling to the FSHB promoter.
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NR5A2 regulates Lhb and FSHB transcription in gonadotrope-like cells in vitro, but is dispensable for gonadotropin synthesis and fertility in vivo.
PloS one, 2013Co-Authors: Jérôme Fortin, Derek Boerboom, Ulrich Boehm, Xiang Zhou, Ying Wang, Vikas Kumar, Johan Auwerx, Kristina Schoonjans, Daniel J. BernardAbstract:Successful mammalian reproduction depends on proper synthesis of the pituitary-derived glycoprotein hormones, luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Several transcription factors cooperate to activate cell-specific and hormone-regulated expression of the gonadotropin beta subunits (Lhb and FSHB). Among these, NR5A1 (steroidogenic factor 1; SF1) has been shown to directly bind to the Lhb promoter, mediate basal and gonadotropin-releasing hormone (GnRH)-stimulated Lhb transcription, and possibly directly regulate FSHB expression. Recently, the closely-related NR5A2 was shown to activate the rat Lhb promoter in vitro. Here, we further characterized the role of NR5A2 in regulating gonadotropin synthesis. Ectopically expressed NR5A2 directly activated the murine Lhb promoter in a manner identical to that of NR5A1, whereas neither factor activated the murine FSHB promoter. In LβT2 gonadotrope-like cells, depletion of endogenous NR5A1 or NR5A2 impaired basal and GnRH-stimulated Lhb and FSHB transcription. To analyze the physiological role of NR5A2 in gonadotropes in vivo, we generated mice with a gonadotrope-specific deletion of Nr5a2. In contrast with our in vitro data, these mice had normal pituitary Lhb and FSHB expression and intact fertility. Together, our data establish that NR5A2 can act in a non-redundant manner to regulate Lhb and FSHB transcription in vitro, but is dispensable in vivo.
Pankaj Lamba - One of the best experts on this subject based on the ideXlab platform.
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SMADs and FOXL2 Synergistically Regulate Murine FSHβ Transcription Via a Conserved Proximal Promoter Element
Molecular endocrinology (Baltimore Md.), 2011Co-Authors: Stella Tran, Ying Wang, Pankaj Lamba, Daniel J. BernardAbstract:Pituitary FSH regulates ovarian and testicular function. Activins stimulate FSHβ subunit (FSHB) gene transcription in gonadotrope cells, the rate-limiting step in mature FSH synthesis. Activin A-induced murine FSHB gene transcription in immortalized gonadotropes is dependent on homolog of Drosophila mothers against decapentaplegic (SMAD) proteins as well as the forkhead transcription factor FOXL2 (FOXL2). Here, we demonstrate that FOXL2 synergizes with SMAD2, SMAD3, and SMAD4 to stimulate murine FSHB promoter-reporter activity in heterologous cells. Moreover, SMAD3-induction of FSHB promoter activity or endogenous mRNA expression is dependent upon endogenous FOXL2 in homologous cells. FOXL2/SMAD synergy requires binding of both FOXL2 and SMAD3 or SMAD4 to DNA. Of three putative forkhead-binding elements identified in the murine FSHB promoter, only the most proximal is absolutely required for activin A induction of reporter activity in homologous cells. Additionally, mutations to the minimal SMAD-binding element adjacent to the proximal forkhead-binding element abrogate activin A or FOXL2/SMAD3 induction of reporter activity. In contrast, a mutation that impairs an adjacent PBX1/PREP1 (pre-B cell leukemia transcription factor 1-PBX/knotted-1 homeobox-1) binding site does not alter activin A-stimulated promoter activity in homologous cells. Collectively, these and previous data suggest a model in which activins stimulate formation of FOXL2-SMAD2/3/4 complexes, which bind to the proximal murine FSHB promoter to stimulate its transcription. Within these complexes, FOXL2 and SMAD3 or SMAD4 bind to adjacent cis-elements, with SMAD3 brokering the physical interaction with FOXL2. Because this composite response element is highly conserved, this suggests a general mechanism whereby activins may regulate and/or modulate FSHB transcription in mammals.
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Activin A Regulates Porcine Follicle-Stimulating Hormone β-Subunit Transcription via Cooperative Actions of SMADs and FOXL2
Endocrinology, 2010Co-Authors: Pankaj Lamba, Ying Wang, Stella Tran, Tamara Ouspenskaia, Vanessa Libasci, Terence E. Hébert, Gregory J. Miller, Daniel J. BernardAbstract:Activins stimulate FSH synthesis and secretion by pituitary gonadotrope cells. Activin A induction of porcine and murine FSHβ (FSHB) gene transcription in immortalized gonadotropes is dependent on homolog of Drosophila mothers against decapentaplegic (SMAD) proteins as well as the forkhead transcription factor L2 (FOXL2). Using both heterologous and homologous cell models, we demonstrate that FOXL2 functionally synergizes with SMAD3/4 to stimulate porcine FSHB promoter-reporter activity. We further show that endogenous FOXL2 and SMAD2/3 physically interact in homologous cells. We identify two composite cis-elements of adjacent FOXL2 and SMAD binding sites in the proximal porcine FSHB promoter that mediate activin A, FOXL2, and SMAD3 actions. FOXL2 can bind these elements independently of SMADs, whereas SMAD3/4 binding requires high-affinity FOXL2 binding. Conversely, FOXL2 alone is insufficient to regulate FSHB transcription and requires SMADs to induce promoter activity. Collectively, our data suggest a model in which activins stimulate formation and nuclear accumulation of SMAD3/4 complexes, which interact with FOXL2 bound to at least two proximal promoter elements. This association stabilizes SMAD3/4 binding to adjacent SMAD binding elements. SMAD-FOXL2 complexes then mediate activation of transcription through a currently unknown mechanism. Conservation of one of the two composite cis-elements suggests that this may form part of a general mechanism whereby activins regulate FSHB subunit transcription and FSH synthesis.
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A Novel Role for the Forkhead Transcription Factor FOXL2 in Activin A-Regulated Follicle-Stimulating Hormone β Subunit Transcription
Molecular endocrinology (Baltimore Md.), 2009Co-Authors: Pankaj Lamba, Ying Wang, Jérôme Fortin, Stella Tran, Daniel J. BernardAbstract:Selective synthesis and release of FSH from pituitary gonadotropes is regulated by activins. Activins directly stimulate murine FSHβ (FSHB) subunit gene transcription through a consensus 8-bp Sma- and Mad-related protein-binding element (SBE) in the proximal promoter. In contrast, the human FSHB promoter is relatively insensitive to the direct effects of activins and lacks this SBE. The proximal porcine FSHB promoter, which is highly conserved with human, similarly lacks the 8-bp SBE, but is nonetheless highly sensitive to activins. We used a comparative approach to determine mechanisms mediating differential activin induction of human, porcine, and murine FSHB/FSHB promoters. We mapped an activin response element in the proximal porcine promoter and identified interspecies variation in a single base pair in close proximity that conferred strong binding of the forkhead transcription factor FOXL2 to the porcine, but not human or murine, promoters. Introduction of the human base pair into the porcine promoter abolished FOXL2 binding and activin A induction. FOXL2 conferred activin A induction to the porcine promoter in heterologous cells, whereas knockdown of the endogenous protein in gonadotropes inhibited the activin A response. The murine FSHB promoter lacks the high-affinity FOXL2-binding site, but its activin induction is FOXL2 sensitive. We identified a more proximal FOXL2-binding element in the murine promoter, which is conserved across species. Mutation of this site attenuated activin A induction of both the porcine and murine promoters. Collectively, the data indicate a novel role for FOXL2 in activin A-regulated FSHB transcription.
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Activator Protein-1 and Smad Proteins Synergistically Regulate Human Follicle-Stimulating Hormone β-Promoter Activity
Endocrinology, 2008Co-Authors: Ying Wang, Pankaj Lamba, Jérôme Fortin, Marco Bonomi, Luca Persani, Mark S. Roberson, Daniel J. BernardAbstract:GnRH1 stimulates the synthesis and secretion of FSH and LH from the anterior pituitary gland. The molecular mechanisms through which GnRH1 produces these effects in humans have not been determined. Here, we examined transcriptional regulation of the human FSHBeta (FSHB) subunit using reporter assays in immortalized murine gonadotrope cells. GnRH1 dose and time dependently stimulated FSHB promoter activity, with peak stimulation occurring at 8 h. GnRH1 rapidly stimulated various MAPK cascades, though the ERK1/2 and p38 pathways appeared to be most critical for FSHB induction. Indeed, constitutively active forms of both Raf1 kinase and MAP2K6 (MKK6) were sufficient to stimulate reporter activity. GnRH1 stimulated activator protein-1 (AP-1) (FosB, c-fos, JunB, and cJun) synthesis and complex formation, the latter of which bound to a conserved cis-element within -120 bp of the transcription start site. A second, lower affinity, site was mapped more proximally. Mutations of both cis-elements diminished GnRH1-stimulated promoter activity, though disruption of the higher affinity site had a more dramatic effect. A dominant-negative Fos protein dose dependently inhibited GnRH1-stimulated FSHB transcription, confirming a role for endogenous AP-1 proteins. MAPK kinase 1 (MEK1) and p38 inhibitors significantly attenuated GnRH1-stimulated c-fos, FosB, and JunB synthesis, suggesting a mechanism whereby the ERK1/2 and p38 signaling pathways regulate FSHB transcription. Activins and inhibins potently regulate FSH synthesis in rodents, but their roles in FSH regulation in humans are less clear. Activin A, though weak on its own, synergized with GnRH1 to stimulate human FSHB promoter activity. In contrast, activin A partially inhibited GnRH1-stimulated LHbeta subunit (LHB) transcription. The GnRH1 and activin A signaling pathways appear to converge at the level of the high-affinity AP-1 site. Fos and Jun proteins synergistically regulate reporter activity through this element, and their effects are potentiated by coexpression of either Smad2 or Smad3, effectors in the activin signaling cascade. In summary, GnRH1 and activin A synergistically regulate human FSHB subunit transcription. The combined actions of AP-1 and Smad proteins acting through a conserved AP-1 element provide a candidate mechanism for this effect. The ability of activins to potentiate selectively the effects of GnRH1 on FSHB expression suggests a model for preferential increases in FSH secretion at the luteal-follicular transition of the menstrual cycle.
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Paired-Like Homeodomain Transcription Factors 1 and 2 Regulate Follicle-Stimulating Hormone β-Subunit Transcription through a Conserved cis-Element
Endocrinology, 2008Co-Authors: Pankaj Lamba, Vishal Khivansara, Ana C. D'alessio, Michelle M. Santos, Daniel J. BernardAbstract:Paired-like homeodomain transcription factors (PITX) regulate the activity of pituitary hormone-encoding genes. Here, we examined mechanisms through which the family of PITX proteins control murine FSH beta-subunit (FSHB) transcription. We observed that endogenous PITX1 and PITX2 isoforms from murine LbetaT2 gonadotrope cells could bind a highly conserved proximal cis-element. Transfection of PITX1 or PITX2C in heterologous cells stimulated both murine and human FSHB/FSHB promoter-reporter activities, and in both cases, mutation of the critical cis-element abrogated these effects. In homologous LbetaT2 cells, the same mutation decreased basal reporter activity and greatly reduced activin A-stimulated transcription from murine and human promoter-reporters. Transfecting dominant-negative forms of PITX1 or PITX2C or knocking down PITX1 or -2 expression by RNA interference in LbetaT2 cells inhibited murine FSHB transcription, confirming roles for endogenous PITX proteins. Both PITX1 and PITX2C interacted with Smad3 (an effector of the activin signaling cascade in these cells) in coprecipitation experiments, and the PITX binding site mutation greatly inhibited Smad2/3/4-stimulated FSHB transcription. In summary, both PITX1 and PITX2C regulate murine and human FSHB/FSHB transcription through a conserved cis-element in the proximal promoter. Furthermore, the data indicate both common and distinct mechanisms of PITX1 and PITX2C action.
Ying Wang - One of the best experts on this subject based on the ideXlab platform.
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Conditional Deletion of FOXL2 and SMAD4 in Gonadotropes of Adult Mice Causes Isolated FSH Deficiency.
Endocrinology, 2018Co-Authors: Yining Li, Gauthier Schang, Mathias Treier, Ulrich Boehm, Chu-xia Deng, Adrien Levasseur, Xiang Zhou, Alexandre Boyer, Ying Wang, Derek BoerboomAbstract:The glycoprotein FSH, a product of pituitary gonadotrope cells, regulates ovarian follicle development in females and spermatogenesis in males. FSH is a heterodimer of the common α gonadotropin subunit and the hormone-specific FSHβ subunit (a product of the FSHB gene). Using a conditional knockout approach (Cre-lox), we previously demonstrated that FSHB expression in mice depends on the transcription factors forkhead box L2 (FOXL2) and SMAD4. Deletion of Foxl2 or Smad4 alone led to FSH deficiency, female subfertility, and oligozoospermia in males. Simultaneous deletion of the two genes yielded a greater suppression of FSH and female sterility. The Cre-driver used previously was first active during embryonic development. Therefore, it is unclear whether FOXL2 and SMAD4 play important roles in the development or adult function of gonadotropes, or both. To address this question, we developed a tamoxifen-inducible Cre-driver line, which enabled Foxl2 and Smad4 gene deletions in gonadotropes of adult mice. After tamoxifen treatment, females with previously demonstrated fertility exhibited profound reductions in FSH levels, arrested ovarian follicle development, and sterility. FSH levels were comparably reduced in males 1 or 2 months after treatment; however, spermatogenesis was unaffected. These data indicate that (1) FOXL2 and SMAD4 are necessary to maintain FSH synthesis in gonadotrope cells of adult mice, (2) FSH is essential for female reproduction but appears to be unnecessary for the maintenance of spermatogenesis in adult male mice, and (3) the inducible Cre-driver line developed here provides a powerful tool to interrogate gene function in gonadotrope cells of adult mice.
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cycloheximide inhibits follicle stimulating hormone β subunit transcription by blocking de novo synthesis of the labile activin type ii receptor in gonadotrope cells
Cellular Signalling, 2013Co-Authors: Carlis Rejon, Xiang Zhou, Ying Wang, Daniel J. Bernard, Catherine C Ho, Terence E. HébertAbstract:Abstract The pituitary gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), play essential roles in the regulation of vertebrate reproduction. Activins and inhibins have opposing actions on FSH (but not LH) synthesis, either inducing or inhibiting transcription of the FSHβ subunit gene ( FSHB ). The translational inhibitor cycloheximide (CHX) produces inhibin-like effects in cultured pituitary cells, selectively suppressing FSH production. Using the murine gonadotrope-like cell line, LβT2, as a model, we tested the hypothesis that a component of the activin pathway is highly labile in gonadotrope cells and that its rapid loss following CHX treatment impairs activin-stimulated FSHB transcription. Treatment of cells with CHX for 6 h, but not 1 h, blocked activin A-stimulated FSHB transcription. Pre-treatment of LβT2 cells with CHX for as few as 2–3 h inhibited activin A-stimulated SMAD2/3 phosphorylation without altering total SMAD2/3 protein levels. These data indicated that CHX affects activin signalling upstream of SMAD proteins, most likely at the receptor level. Indeed, CHX rapidly reduced activin A binding to LβT2 cells. We went on to show that activin A signals via the type II receptor ACVR2, rather than ACVR2B, to regulate FSHB transcription and that the receptor has a half life of ~ 2 h in LβT2 cells. The mechanism of ACVR2 turnover remains undefined, but appears to be ligand-, proteasome-, and lysosome-independent. Collectively, these data indicate that CHX produces inhibin-like effects in gonadotropes by preventing de novo synthesis of the highly labile ACVR2, thereby blocking activin signaling to the FSHB promoter.
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NR5A2 regulates Lhb and FSHB transcription in gonadotrope-like cells in vitro, but is dispensable for gonadotropin synthesis and fertility in vivo.
PloS one, 2013Co-Authors: Jérôme Fortin, Derek Boerboom, Ulrich Boehm, Xiang Zhou, Ying Wang, Vikas Kumar, Johan Auwerx, Kristina Schoonjans, Daniel J. BernardAbstract:Successful mammalian reproduction depends on proper synthesis of the pituitary-derived glycoprotein hormones, luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Several transcription factors cooperate to activate cell-specific and hormone-regulated expression of the gonadotropin beta subunits (Lhb and FSHB). Among these, NR5A1 (steroidogenic factor 1; SF1) has been shown to directly bind to the Lhb promoter, mediate basal and gonadotropin-releasing hormone (GnRH)-stimulated Lhb transcription, and possibly directly regulate FSHB expression. Recently, the closely-related NR5A2 was shown to activate the rat Lhb promoter in vitro. Here, we further characterized the role of NR5A2 in regulating gonadotropin synthesis. Ectopically expressed NR5A2 directly activated the murine Lhb promoter in a manner identical to that of NR5A1, whereas neither factor activated the murine FSHB promoter. In LβT2 gonadotrope-like cells, depletion of endogenous NR5A1 or NR5A2 impaired basal and GnRH-stimulated Lhb and FSHB transcription. To analyze the physiological role of NR5A2 in gonadotropes in vivo, we generated mice with a gonadotrope-specific deletion of Nr5a2. In contrast with our in vitro data, these mice had normal pituitary Lhb and FSHB expression and intact fertility. Together, our data establish that NR5A2 can act in a non-redundant manner to regulate Lhb and FSHB transcription in vitro, but is dispensable in vivo.
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Activin A induction of murine and ovine follicle-stimulating hormone β transcription is SMAD-dependent and TAK1 (MAP3K7)/p38 MAPK-independent in gonadotrope-like cells.
Cellular signalling, 2012Co-Authors: Ying Wang, Daniel J. BernardAbstract:Abstract Activins stimulate follicle-stimulating hormone (FSH) β subunit (FSHB) gene transcription in pituitary gonadotrope cells. Previous studies suggest that activins signal via homolog of Drosophila mothers against decapentaplegic (SMAD) proteins to stimulate murine or porcine FSHB promoter activity in the gonadotrope-like cell line, LβT2. In contrast, activins were suggested to regulate the ovine FSHB promoter via a SMAD-independent pathway involving TGFβ associated kinase 1 (TAK1, MAP3K7) and p38 mitogen activated protein kinase (MAPK). Here, we examined roles for TAK1 and p38 in activin A-stimulated murine and ovine FSHB transcription. The TAK1 inhibitor 5Z-7-Oxozeanol (Oxo) significantly impaired fold activin A induction of murine and ovine FSHB promoter-reporters (FSHB-luc) in LβT2 cells, but only at concentrations 50–100 fold greater than its IC50 for TAK1. Moreover, Oxo failed to inhibit activin A induction of endogenous FSHB mRNA levels or fold induction of FSHB-luc activity by a constitutively active form of the activin type I receptor (ALK4). Oxo, at a concentration 5–10 fold greater than its IC50 for TAK1, attenuated TAK1/TAB2 stimulation of a p38-dependent reporter in the same cells. A Map3k7 siRNA impaired TAK1/TAB2-stimulated p38-dependent reporter activity, but failed to antagonize activin A-stimulated FSHB-luc. Though TAK1 was previously suggested to act via p38 to stimulate the ovine FSHB promoter, activin A failed to stimulate p38 phosphorylation in LβT2 cells. In apparent contrast, however, the p38 inhibitors SB203580 and SB202190 concentration-dependently attenuated activin A-induced FSHB-luc activity. Given the lack of p38 activation, we postulated that the inhibitors might non-selectively antagonize ALK4 activity. Indeed, both attenuated activin A-stimulated SMAD2 phosphorylation, consistent with direct antagonism of ALK4 kinase activity. Finally, we observed that RNA-mediated suppression of Smad4, and to a lesser extent Smad3, attenuated activin A induction of both murine and ovine FSHB promoter-reporters. Collectively, these data suggest that activin A signals via SMAD proteins, but not TAK1 or p38, to regulate murine and ovine FSHB transcription in gonadotrope-like cells.
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SMADs and FOXL2 Synergistically Regulate Murine FSHβ Transcription Via a Conserved Proximal Promoter Element
Molecular endocrinology (Baltimore Md.), 2011Co-Authors: Stella Tran, Ying Wang, Pankaj Lamba, Daniel J. BernardAbstract:Pituitary FSH regulates ovarian and testicular function. Activins stimulate FSHβ subunit (FSHB) gene transcription in gonadotrope cells, the rate-limiting step in mature FSH synthesis. Activin A-induced murine FSHB gene transcription in immortalized gonadotropes is dependent on homolog of Drosophila mothers against decapentaplegic (SMAD) proteins as well as the forkhead transcription factor FOXL2 (FOXL2). Here, we demonstrate that FOXL2 synergizes with SMAD2, SMAD3, and SMAD4 to stimulate murine FSHB promoter-reporter activity in heterologous cells. Moreover, SMAD3-induction of FSHB promoter activity or endogenous mRNA expression is dependent upon endogenous FOXL2 in homologous cells. FOXL2/SMAD synergy requires binding of both FOXL2 and SMAD3 or SMAD4 to DNA. Of three putative forkhead-binding elements identified in the murine FSHB promoter, only the most proximal is absolutely required for activin A induction of reporter activity in homologous cells. Additionally, mutations to the minimal SMAD-binding element adjacent to the proximal forkhead-binding element abrogate activin A or FOXL2/SMAD3 induction of reporter activity. In contrast, a mutation that impairs an adjacent PBX1/PREP1 (pre-B cell leukemia transcription factor 1-PBX/knotted-1 homeobox-1) binding site does not alter activin A-stimulated promoter activity in homologous cells. Collectively, these and previous data suggest a model in which activins stimulate formation of FOXL2-SMAD2/3/4 complexes, which bind to the proximal murine FSHB promoter to stimulate its transcription. Within these complexes, FOXL2 and SMAD3 or SMAD4 bind to adjacent cis-elements, with SMAD3 brokering the physical interaction with FOXL2. Because this composite response element is highly conserved, this suggests a general mechanism whereby activins may regulate and/or modulate FSHB transcription in mammals.
Michelle M. Santos - One of the best experts on this subject based on the ideXlab platform.
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Paired-Like Homeodomain Transcription Factors 1 and 2 Regulate Follicle-Stimulating Hormone β-Subunit Transcription through a Conserved cis-Element
Endocrinology, 2008Co-Authors: Pankaj Lamba, Vishal Khivansara, Ana C. D'alessio, Michelle M. Santos, Daniel J. BernardAbstract:Paired-like homeodomain transcription factors (PITX) regulate the activity of pituitary hormone-encoding genes. Here, we examined mechanisms through which the family of PITX proteins control murine FSH beta-subunit (FSHB) transcription. We observed that endogenous PITX1 and PITX2 isoforms from murine LbetaT2 gonadotrope cells could bind a highly conserved proximal cis-element. Transfection of PITX1 or PITX2C in heterologous cells stimulated both murine and human FSHB/FSHB promoter-reporter activities, and in both cases, mutation of the critical cis-element abrogated these effects. In homologous LbetaT2 cells, the same mutation decreased basal reporter activity and greatly reduced activin A-stimulated transcription from murine and human promoter-reporters. Transfecting dominant-negative forms of PITX1 or PITX2C or knocking down PITX1 or -2 expression by RNA interference in LbetaT2 cells inhibited murine FSHB transcription, confirming roles for endogenous PITX proteins. Both PITX1 and PITX2C interacted with Smad3 (an effector of the activin signaling cascade in these cells) in coprecipitation experiments, and the PITX binding site mutation greatly inhibited Smad2/3/4-stimulated FSHB transcription. In summary, both PITX1 and PITX2C regulate murine and human FSHB/FSHB transcription through a conserved cis-element in the proximal promoter. Furthermore, the data indicate both common and distinct mechanisms of PITX1 and PITX2C action.
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Bone morphogenetic protein 2 and activin A synergistically stimulate follicle-stimulating hormone β subunit transcription
Journal of Molecular Endocrinology, 2007Co-Authors: Katharine B Lee, Stuart C. Sealfon, Pankaj Lamba, Vishal Khivansara, Michelle M. Santos, Tony Yuen, Daniel J. BernardAbstract:Transforming growth factor beta superfamily ligands regulate pituitary FSH production and secretion. The best-described examples are the activins and inhibins, which respectively stimulate and hinder FSHB subunit transcription in gonadotrope cells. More recently, members of the bone morphogenetic protein (BMP) sub-family were shown to regulate FSH production in a manner analogous to the activins. Here, we used the murine gonadotrope cell line, LbetaT2, to investigate mechanisms through which BMP2 regulates the FSHB gene. Although expressed at low levels in LbetaT2 cells, Bmp2 mRNA was readily detected in adult murine pituitary gland. Recombinant BMP2 stimulated FSHB promoter-reporter activity, although its effects were weaker than those of equimolar activin A or B. BMP4 stimulated transcription comparably with BMP2, but BMPs 6 and 7 were about tenfold less potent. Remarkably, BMP2 and activin A synergistically upregulated FSHB transcription and endogenous FSHB mRNA levels in LbetaT2 cells. Although functionally cooperative, the two ligands appeared to use distinct intracellular mechanisms to mediate their responses because neither ligand altered the timing or magnitude of the other's effects. Receptor overexpression analyses suggested that BMP2 may preferentially signal through complexes of the type II receptor, BMPR2, and the type I receptor, activin receptor like kinase (ALK2; Acvr1), to stimulate FSHB transcription. BMP2 rapidly activated the Smad1/5/8 intracellular signaling cascade and Smad8 overexpression potentiated BMP2's effects. In summary, BMPs regulate FSHB transcription in LbetaT2 cells and can amplify the already robust effects of the activins through a distinct signaling mechanism. Because BMP2 is expressed in the adult mouse pituitary, it may act as critical paracrine co-regulator of FSH synthesis by gonadotropes.
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Bone morphogenetic protein 2 and activin A synergistically stimulate follicle-stimulating hormone beta subunit transcription.
Journal of molecular endocrinology, 2007Co-Authors: Katharine B Lee, Stuart C. Sealfon, Pankaj Lamba, Vishal Khivansara, Michelle M. Santos, Tony Yuen, Daniel J. BernardAbstract:Transforming growth factor beta superfamily ligands regulate pituitary FSH production and secretion. The best-described examples are the activins and inhibins, which respectively stimulate and hinder FSHB subunit transcription in gonadotrope cells. More recently, members of the bone morphogenetic protein (BMP) sub-family were shown to regulate FSH production in a manner analogous to the activins. Here, we used the murine gonadotrope cell line, LbetaT2, to investigate mechanisms through which BMP2 regulates the FSHB gene. Although expressed at low levels in LbetaT2 cells, Bmp2 mRNA was readily detected in adult murine pituitary gland. Recombinant BMP2 stimulated FSHB promoter-reporter activity, although its effects were weaker than those of equimolar activin A or B. BMP4 stimulated transcription comparably with BMP2, but BMPs 6 and 7 were about tenfold less potent. Remarkably, BMP2 and activin A synergistically upregulated FSHB transcription and endogenous FSHB mRNA levels in LbetaT2 cells. Although functionally cooperative, the two ligands appeared to use distinct intracellular mechanisms to mediate their responses because neither ligand altered the timing or magnitude of the other's effects. Receptor overexpression analyses suggested that BMP2 may preferentially signal through complexes of the type II receptor, BMPR2, and the type I receptor, activin receptor like kinase (ALK2; Acvr1), to stimulate FSHB transcription. BMP2 rapidly activated the Smad1/5/8 intracellular signaling cascade and Smad8 overexpression potentiated BMP2's effects. In summary, BMPs regulate FSHB transcription in LbetaT2 cells and can amplify the already robust effects of the activins through a distinct signaling mechanism. Because BMP2 is expressed in the adult mouse pituitary, it may act as critical paracrine co-regulator of FSH synthesis by gonadotropes.
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Activin B can signal through both ALK4 and ALK7 in gonadotrope cells
Reproductive biology and endocrinology : RB&E, 2006Co-Authors: Daniel J. Bernard, Katharine B Lee, Michelle M. SantosAbstract:Background: Activins stimulate pituitary FSH synthesis via transcriptional regulation of the FSHBeta subunit gene (FSHB). Like other members of the TGFbeta superfamily, these ligands signal through complexes of type I and type II receptor serine/threonine kinases. The type I receptors, or activin receptor-like kinases (ALKs), propagate intracellular signals upon ligand binding and phosphorylation by associated type II receptors. ALK4 is generally regarded as the type I receptor for activins; however, recent data suggested that activin B and AB might also signal through ALK7. Here, we examined a role for ALK7 in activin B-regulated FSHB transcription. Methods: We analyzed ALK7 mRNA expression in immortalized gonadotrope cells, LbetaT2, and adult murine pituitary by RT-PCR. We next transfected LbetaT2 cells with wild-type and kinasedeficient (Lys to Arg, KR) forms of ALK4 and ALK7 and examined the effects of these receptors on activin A and B stimulated FSHB promoter-reporter activity. Cells were also transfected with constitutively active (Thr to Asp, TD) forms of the receptors and their effects on endogenous FSHB mRNA levels and phosphorylation of transfected Smad2/3 were measured by RT-PCR and Western blot, respectively. Finally, we measured ALK4(TD) and ALK7(TD) stimulation of FSHB transcription when endogenous Smad3 levels were depleted using short hairpin RNAs. Results: ALK7 mRNA was expressed in LbetaT2 cells and pituitary gland. Transfection of ALK4 cDNA potentiated the effects of both activin A and activin B on FSHB promoter-reporter activity in LbetaT2 cells. In contrast, ALK7 transfection selectively potentiated activin B's effects. Transfection of ALK4(KR) and ALK7(KR) partly inhibited basal and activin B-stimulated reporter activity, whereas ALK4(TD) and ALK7(TD) potently stimulated the FSHB promoter and endogenous mRNA levels. Transfection of both ALK4(TD) and ALK7(TD) stimulated Smad2/3 phosphorylation, and the effects of both receptors on FSHB promoter activity were inhibited by depletion of endogenous Smad3 protein levels. Conclusion: These data suggest that immortalized gonadotropes express ALK7 and that activin B can signal through this receptor to stimulate FSHB transcription. The relative roles of endogenous ALK4 and ALK7 receptors in mediating activin B's effects in these cells have yet to be determined.
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Acute regulation of murine follicle-stimulating hormone β subunit transcription by activin A
Journal of molecular endocrinology, 2006Co-Authors: Pankaj Lamba, Michelle M. Santos, Daniel P Philips, Daniel J. BernardAbstract:In rodents, activins stimulate immediate-early increases in pituitary follicle-stimulating hormone (FSHB) subunit transcription. Here, we investigated the underlying signaling mechanisms using the mouse gonadotrope cell line, LT2. Activin A increased mouse FSHB-luciferase reporter activity within 4 h through a Smad-dependent signaling pathway. The ligand rapidly stimulated formation of SMAD2/3/4 complexes that could interact with a consensus palindromic Smad binding element (SBE) in the proximal FSHB promoter. SMAD over-expression potently stimulated transcription, with the combination of SMADs 2, 3 and 4 producing the greatest synergistic activation. A mutation in the SBE that abolished Smad binding greatly impaired the effects of acute (4 h) activin A treatment and SMAD over-expression on promoter activity, but did not abolish the effects of chronic (24 h) activin A exposure. Within activated SMAD complexes, SMADs 3 and 4 appeared to bind the SBE simultaneously and the binding of both was required for maximal transcriptional activation. Interestingly, the human FSHB promoter, which lacks the consensus SBE, was neither rapidly stimulated by activin A nor by over-expressed SMADs, but was activated by 24 h activin A. Addition of the SBE to the human promoter increased both SMAD2/3/4-sensitivity and acute regulation by activin A, though not to levels observed in mouse. We postulate that short reproductive cycles in female rodents, particularly the brief interval between the primary and secondary FSH surges of the estrous cycle, require the FSHB promoter in these animals to be particularly sensitive to the rapid, Smad-dependent actions of activins on transcription. The human FSHB promoter, in contrast, is chronically regulated by activins seemingly through a SMAD-independent pathway.
T. Rajendra Kumar - One of the best experts on this subject based on the ideXlab platform.
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Human Follicle-Stimulating Hormone ß Subunit Expression Depends on FOXL2 and SMAD4.
Endocrinology, 2020Co-Authors: Luisina Ongaro, Gauthier Schang, Mathias Treier, Ulrich Boehm, Chu-xia Deng, T. Rajendra Kumar, Ziyue Zhou, Daniel J. BernardAbstract:Follicle-stimulating hormone (FSH), an essential regulator of mammalian fertility, is synthesized by pituitary gonadotrope cells in response to activins. In mice, activins signal via SMAD3, SMAD4, and FOXL2 to regulate transcription of the FSHβ subunit (FSHB) gene. Gonadotrope-specific deletion of Foxl2, alone or in combination with Smad4, renders mice FSH-deficient. Whether human FSHB expression is similarly regulated is not known. Here, we used a combination of transgenic and conditional knockout mouse strains to assess the roles of activins, FOXL2, and SMAD4 in regulation of the human FSHB gene. First, we cultured pituitaries from mice harboring a human FSHB transgene (hFSHB mice) and measured both murine FSHB and human FSHB messenger ribonucleic acid (mRNA) expression in response to exogenous activins or two antagonists of endogenous activin-like signaling (follistatin-288 and SB431542). Both murine FSHB and human FSHB expression were stimulated by activins and reduced by the inhibitors. Next, we analyzed human FSHB expression in hFSHB mice carrying floxed Foxl2 and Smad4 alleles. Cre-mediated ablation of FOXL2 and SMAD4 strongly reduced basal and activin-stimulated murine FSHB and human FSHB expression in cultured pituitaries. Finally, the hFSHB transgene was previously shown to rescue FSH production and fertility in FSHB knockout mice. However, gonadotrope-specific Foxl2/Smad4 knockout females carrying the hFSHB transgene have significantly reduced murine FSHB and human FSHB pituitary mRNA levels and are hypogonadal. Collectively, these data suggest that similar to FSHB regulation in mice, FOXL2 and SMAD4 play essential roles in human FSHB expression.
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FSHB Knockout Mouse Model, Two Decades Later and Into the Future.
Endocrinology, 2018Co-Authors: T. Rajendra KumarAbstract:In 1997, nearly 20 years ago, we reported the phenotypes of follicle-stimulating hormone (FSH) β (FSHB) null mice. Since then, these mice have been useful for various physiological and genetic studies in reproductive and skeletal biology. In a 2009 review titled "FSHβ Knockout Mouse Model: A Decade Ago and Into the Future," I summarized the need for and what led to the development of an FSH-deficient mouse model and its applications, including delineation of the emerging extragonadal roles of FSH in bone cells by using this genetic model. These studies opened up exciting avenues of research on osteoporosis and now extend into those on adiposity in postmenopausal women. Here, I summarize the progress made with this mouse model since 2009 with regard to FSH rerouting in vivo, deciphering the role of N-glycosylation on FSHβ, roles of FSH in somatic-germ cell interactions in gonads, and provide a road map that is anticipated to emerge in the near future. Undoubtedly, the next 10 years should be an even more exciting time to explore the fertile area of FSH biology and its implications for basic and clinical reproductive physiology research.
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Evaluation of in vivo bioactivities of recombinant hypo- (FSH21/18) and fully- (FSH24) glycosylated human FSH glycoforms in FSHB null mice.
Molecular and cellular endocrinology, 2016Co-Authors: Huizhen Wang, George R. Bousfield, Jacob May, Viktor Y. Butnev, Bin Shuai, Jeffrey V. May, T. Rajendra KumarAbstract:Abstract The hormone - specific FSHβ subunit of the human FSH heterodimer consists of N-linked glycans at Asn 7 and Asn 24 residues that are co-translationally attached early during subunit biosynthesis. Differences in the number of N-glycans (none, one or two) on the human FSHβ subunit contribute to macroheterogeneity in the FSH heterodimer. The resulting FSH glycoforms are termed hypo-glycosylated (FSH 21/18 , missing either an Asn 24 or Asn 7 N-glycan chain on the β - subunit, respectively) or fully glycosylated (FSH 24 , possessing of both Asn 7 and Asn 24 N-linked glycans on the β - subunit) FSH. The recombinant versions of human FSH glycoforms (FSH 21/18 and FSH 24 ) have been purified and biochemically characterized. In vitro functional studies have indicated that FSH 21/18 exhibits faster FSH- receptor binding kinetics and is much more active than FSH 24 in every assay tested to date. However, the in vivo bioactivity of the hypo-glycosylated FSH glycoform has never been tested. Here, we evaluated the in vivo bioactivities of FSH glycoforms in FSHB null mice using a pharmacological rescue approach. In FSHB null female mice, both hypo- and fully-glycosylated FSH elicited an ovarian weight gain response by 48 h and induced ovarian genes in a dose- and time-dependent manner. Quantification by real time qPCR assays indicated that hypo-glycosylated FSH 21/18 was bioactive in vivo and induced FSH-responsive ovarian genes similar to fully-glycosylated FSH 24 . Western blot analyses followed by densitometry of key signaling components downstream of the FSH-receptor confirmed that the hypo-glycosylated FSH 21/18 elicited a response similar to that by fully-glycosylated FSH 24 in ovaries of FSHB null mice. When injected into FSHB null males, hypo-glycosylated FSH 21/18 was more active than the fully-glycosylated FSH 24 in inducing FSH-responsive genes and Sertoli cell proliferation. Thus, our data establish that recombinant hypo-glycosylated human FSH 21/18 glycoform elicits bioactivity in vivo similar to the fully-glycosylated FSH. Our studies may have clinical implications particularly in formulating FSH-based ovarian follicle induction protocols using a combination of different human FSH glycoforms.
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A human FSHB transgene encoding the double N-glycosylation mutant (Asn(7Δ) Asn(24Δ)) FSHβ subunit fails to rescue FSHB null mice.
Molecular and cellular endocrinology, 2016Co-Authors: Huizhen Wang, Vladimir Y. Butnev, George R. Bousfield, T. Rajendra KumarAbstract:Follicle-stimulating hormone (FSH) is a gonadotrope-derived heterodimeric glycoprotein. Both the common α- and hormone-specific β subunits contain Asn-linked N-glycan chains. Recently, macroheterogeneous FSH glycoforms consisting of β-subunits that differ in N-glycan number were identified in pituitaries of several species and subsequently the recombinant human FSH glycoforms biochemically characterized. Although chemical modification and in vitro site-directed mutagenesis studies defined the roles of N-glycans on gonadotropin subunits, in vivo functional analyses in a whole-animal setting are lacking. Here, we have generated transgenic mice with gonadotrope-specific expression of either an HFSHB(WT) transgene that encodes human FSHβ WT subunit or an HFSHB(dgc) transgene that encodes a human FSHβ(Asn7Δ 24Δ) double N-glycosylation site mutant subunit, and separately introduced these transgenes onto FSHB null background using a genetic rescue strategy. We demonstrate that the human FSHβ(Asn7Δ 24Δ) double N-glycosylation site mutant subunit, unlike human FSHβ WT subunit, inefficiently combines with the mouse α-subunit in pituitaries of FSHB null mice. FSH dimer containing this mutant FSHβ subunit is inefficiently secreted with very low levels detectable in serum. FSHB null male mice expressing HFSHB(dgc) transgene are fertile and exhibit testis tubule size and sperm number similar to those of FSHB null mice. FSHB null female mice expressing the mutant, but not WT human FSHβ subunit-containing FSH dimer are infertile, demonstrate no evidence of estrus cycles, and many of the FSH-responsive genes remain suppressed in their ovaries. Thus, HFSHB(dgc) unlike HFSHB(WT) transgene does not rescue FSHB null mice. Our genetic approach provides direct in vivo evidence that N-linked glycans on FSHβ subunit are essential for its efficient assembly with the α-subunit to form FSH heterodimer in pituitary. Our studies also reveal that N-glycans on FSHβ subunit are essential for FSH secretion and FSH in vivo bioactivity to regulate gonadal growth and physiology.
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FSHB-iCre mice are efficient and specific Cre deleters for the gonadotrope lineage
Molecular and cellular endocrinology, 2015Co-Authors: Huizhen Wang, William L. Miller, Richard C. Hastings, T. Rajendra KumarAbstract:Genetic analysis of development and function of the gonadotrope cell lineage within mouse anterior pituitary has been greatly facilitated by at least three currently available Cre strains in which Cre was either knocked into the Gnrhr locus or expressed as a transgene from Cga and Lhb promoters. However, in each case there are some limitations including CRE expression in thyrotropes within pituitary or ectopic expression outside of pituitary, for example in some populations of neurons or gonads. Hence, these Cre strains often pose problems with regard to undesirable deletion of alleles in non-gonadotrope cells, fertility and germline transmission of mutant alleles. Here, we describe generation and characterization of a new FSHB-iCre deleter strain using 4.7 kb of ovine FSHB promoter regulatory sequences driving iCre expression exclusively in the gonadotrope lineage within anterior pituitary. FSHB-iCre mice develop normally, display no ectopic CRE expression in gonads and are fertile. When crossed onto a loxP recombination-mediated red to green color switch reporter mouse genetic background, in vivo CRE recombinase activity is detectable in gonadotropes at more than 95% efficiency and the GFP-tagged gonadotropes readily purified by fluorescence activated cell sorting. We demonstrate the applicability of this FSHB-iCre deleter strain in a mouse model in which Dicer is efficiently and selectively deleted in gonadotropes. We further show that loss of DICER-dependent miRNAs in gonadotropes leads to profound suppression of gonadotropins resulting in male and female infertility. Thus, FSHB-iCre mice serve as a new genetic tool to efficiently manipulate gonadotrope-specific gene expression in vivo.
