Gonadotrope-Specific Expression and Regulation of Ovine Follicle Stimulating Hormone Beta: Transgenic and Adenoviral Approaches Using Primary Murine Gonadotropes
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{"title"=>"Gonadotrope-Specific Expression and Regulation of Ovine Follicle Stimulating Hormone Beta: Transgenic and Adenoviral Approaches Using Primary Murine Gonadotropes", "type"=>"journal", "authors"=>[{"first_name"=>"Jingjing", "last_name"=>"Jia", "scopus_author_id"=>"55252903300"}, {"first_name"=>"Farideh", "last_name"=>"Shafiee-Kermani", "scopus_author_id"=>"6506242383"}, {"first_name"=>"William L.", "last_name"=>"Miller", "scopus_author_id"=>"55198267400"}], "year"=>2013, "source"=>"PLoS ONE", "identifiers"=>{"issn"=>"19326203", "pui"=>"369379100", "isbn"=>"1932-6203 (Electronic)\\r1932-6203 (Linking)", "pmid"=>"23874399", "sgr"=>"84880406798", "scopus"=>"2-s2.0-84880406798", "doi"=>"10.1371/journal.pone.0066852"}, "id"=>"59142833-3eea-3443-98b9-5addd7f98a4e", "abstract"=>"The beta subunit of follicle stimulating hormone (FSHB) is expressed specifically in pituitary gonadotropes in vertebrates. Transgenic mouse studies have shown that enhancers in the proximal promoter between -172/-1 bp of the ovine FSHB gene are required for gonadotrope expression of ovine FSHB. These enhancers are associated with regulation by activins and gonadotropin releasing hormone (GnRH). Additional distal promoter sequence between -4741/-750 bp is also required for expression. New transgenic studies presented here focus on this distal region and narrow it to 1116 bp between -1866/-750 bp. In addition, adenoviral constructs were produced to identify these critical distal sequences using purified primary mouse gonadotropes as an in vitro model system. The adenoviral constructs contained -2871 bp, -750 bp or -232 bp of the ovine FSHB promoter. They all showed gonadotrope-specific regulation since they were induced only in purified primary gonadotropes by activin A (50 ng/ml) and inhibited by GnRH (100 nM) in the presence of activin (except -232FSHBLuc). However, basal expression of all three viral constructs (in the presence of follistatin to block cellular induction by activin) was relatively high in pituitary non-gonadotropes as well as gonadotropes. Thus, gonadotrope-specific regulation associated with the proximal promoter was observed as expected, but the model was blind to distal promoter elements between -2871/-750 necessary for gonadotrope-specific expression of ovine FSHB in vivo. The new adenoviral-based in vitro technique did detect, however, a novel GnRH response element between -750 bp and -232 bp of the ovine FSHB promoter. We conclude that adenoviral-based studies in primary gonadotropes can adequately recognize regulatory elements on the ovine FSHB promoter associated with gonadotrope-specific regulation/expression, but that more physiologically based techniques, such as transgenic studies, will be needed to identify sequences between -1866/-750 bp of the ovine FSHB promoter that are also required for tissue/cell specific expression in vivo.", "link"=>"http://www.mendeley.com/research/gonadotropespecific-expression-regulation-ovine-follicle-stimulating-hormone-beta-transgenic-adenovi", "reader_count"=>6, "reader_count_by_academic_status"=>{"Student > Doctoral Student"=>2, "Researcher"=>2, "Student > Master"=>1, "Student > Bachelor"=>1}, "reader_count_by_user_role"=>{"Student > Doctoral Student"=>2, "Researcher"=>2, "Student > Master"=>1, "Student > Bachelor"=>1}, "reader_count_by_subject_area"=>{"Unspecified"=>1, "Biochemistry, Genetics and Molecular Biology"=>2, "Agricultural and Biological Sciences"=>3}, "reader_count_by_subdiscipline"=>{"Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>3}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>2}, "Unspecified"=>{"Unspecified"=>1}}, "reader_count_by_country"=>{"Canada"=>1}, "group_count"=>0}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/1121744"], "description"=>"<p>LβT2 cells (40,000 cells/well) were plated and infected with −232o<i>FSHBLuc</i>-V, −750o<i>FSHBLuc</i>-V or −2871o<i>FSHBLuc</i>-V. Different amounts of viral constructs (1, 3 and 10 ul/well of 10<sup>8</sup> ifu/ml) were tested. After a 12 hour infection, cells were treated with activin (A; 50 ng/ml) for 24 hours, and then assayed for luciferase activity. For each viral vector, data from different doses were normalized to the activity of the lowest dose. <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066852#pone-0066852-g004\" target=\"_blank\">Figure 4B:</a> Hormonal regulation of the LβT2 cells infected with viral constructs. LβT2 cells infected with 10 ul of 232o<i>FSHBLuc</i>-V, −750o<i>FSHBLuc</i>-V or −2871o<i>FSHBLuc</i>-V (see Fig. 4A) were treated with follistatin (F; 250 ng/ml; basal expression), activin (A; 50 ng/ml; maximal expression) or activin (50 ng/ml)+GnRH (A+G; 100 nM) (A+G; GnRH-regulated expression) respectively for 24 hours after a 12 hour infection, and then assayed for luciferase activity. The framed text indicates activin induction for each viral construct. For each viral construct, data from different doses were normalized to the activity of the lowest dose. Different letters indicate significant differences of means (P<0.05).</p>", "links"=>[], "tags"=>["Anatomy and physiology", "Reproductive system", "Reproductive physiology", "Sexual reproduction", "Biochemistry", "Biochemistry simulations", "hormones", "genetics", "gene expression", "DNA transcription", "chromatin", "Molecular genetics", "Gene regulation", "Animal genetics", "dose-response", "viral"], "article_id"=>747776, "categories"=>["Biological Sciences"], "users"=>["Jingjing Jia", "Farideh Shafiee-Kermani", "William L. Miller"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0066852.g004", "stats"=>{"downloads"=>0, "page_views"=>2, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_A_Dose_response_for_viral_infection_in_L_946_T2_cells_/747776", "title"=>"A: Dose-response for viral infection in LβT2 cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-07-18 01:51:51"}
  • {"files"=>["https://ndownloader.figshare.com/files/1121745"], "description"=>"<p>Primary gonadotropes (10,000 cells/well) were plated and infected with −232o<i>FSHBLuc</i>-V, −750o<i>FSHBLuc</i>-V or −2871o<i>FSHBLuc</i>-V. After a 24 hour infection, cells were treated with follistatin (F; 250 ng/ml; basal expression), activin (A; 50 ng/ml; maximal expression) or activin (50 ng/ml)+GnRH (100 nM) (A+G; GnRH-regulated expression) respectively for 48 hours. Experiments were performed in quadruplicate and repeated 3 times. <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066852#pone-0066852-g005\" target=\"_blank\"><b>Figure 5A</b></a> shows combined results for three individual experiments. Follistatin results (basal expression) were set a value of 1. The framed text indicates activin induction for each viral construct. Bars with different letters are significantly different (P<0.05). <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066852#pone-0066852-g005\" target=\"_blank\"><b>Figure 5B</b></a> shows non-normalized data from one of the three independent experimental replicates. The framed text indicates activin induction for each viral construct. Bars with different letters are significantly different (P<0.05).</p>", "links"=>[], "tags"=>["Anatomy and physiology", "Reproductive system", "Reproductive physiology", "Sexual reproduction", "Biochemistry", "Biochemistry simulations", "hormones", "genetics", "gene expression", "DNA transcription", "chromatin", "Molecular genetics", "Gene regulation", "Animal genetics", "purified"], "article_id"=>747777, "categories"=>["Biological Sciences"], "users"=>["Jingjing Jia", "Farideh Shafiee-Kermani", "William L. Miller"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0066852.g005", "stats"=>{"downloads"=>0, "page_views"=>6, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Adenoviral_infection_of_purified_gonadotropes_/747777", "title"=>"Adenoviral infection of purified gonadotropes.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-07-18 01:51:51"}
  • {"files"=>["https://ndownloader.figshare.com/files/1121746"], "description"=>"<p>Non-gonadotropes (10,000 cells/well) were plated and infected with −232o<i>FSHBLuc</i>-V, −750o<i>FSHBLuc</i>-V or −2871o<i>FSHBLuc</i>-V. After a 24 hour infection, cells were treated with follistatin (F; 250 ng/ml; basal expression), activin (A; 50 ng/ml; maximal expression) or activin (50 ng/ml)+GnRH (100 nM) (A+G; GnRH regulated expression) for 48 hours. Experiments were performed in quadruplicate and repeated 3 times. <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066852#pone-0066852-g006\" target=\"_blank\"><b>Figure 6A</b></a> shows combined results for three individual experiments. Follistatin results (basal expression) were adjusted to 1. Different letters indicate significantly different means (P<0.05). <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066852#pone-0066852-g006\" target=\"_blank\"><b>Figure 6B</b></a> represents data from one representative replicate. Different letters indicate significantly different means (P<0.05).</p>", "links"=>[], "tags"=>["Anatomy and physiology", "Reproductive system", "Reproductive physiology", "Sexual reproduction", "Biochemistry", "Biochemistry simulations", "hormones", "genetics", "gene expression", "DNA transcription", "chromatin", "Molecular genetics", "Gene regulation", "Animal genetics", "non-gonadotropes"], "article_id"=>747778, "categories"=>["Biological Sciences"], "users"=>["Jingjing Jia", "Farideh Shafiee-Kermani", "William L. Miller"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0066852.g006", "stats"=>{"downloads"=>0, "page_views"=>2, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Adenoviral_infection_of_non_gonadotropes_flow_through_cells_/747778", "title"=>"Adenoviral infection of non-gonadotropes (flow through cells).", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-07-18 01:51:51"}
  • {"files"=>["https://ndownloader.figshare.com/files/1121748"], "description"=>"<p>Tissues were harvested from mice at least 7 weeks old and lysates were assayed for luciferase activity and protein. Values are luciferase activity expressed as relative light units (RLU)×10<sup>−4</sup>/mg/protein. Values for the LS lines represent the mean ± SEM for three animals. LO data are from one mouse each.</p>", "links"=>[], "tags"=>["Anatomy and physiology", "Reproductive system", "Reproductive physiology", "Sexual reproduction", "Biochemistry", "Biochemistry simulations", "hormones", "genetics", "gene expression", "DNA transcription", "chromatin", "Molecular genetics", "Gene regulation", "Animal genetics", "ls", "lo", "transgenes"], "article_id"=>747780, "categories"=>["Biological Sciences"], "users"=>["Jingjing Jia", "Farideh Shafiee-Kermani", "William L. Miller"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0066852.t001", "stats"=>{"downloads"=>0, "page_views"=>6, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Expression_of_LS_and_LO_o_FSHBLuc_transgenes_in_mouse_tissues_/747780", "title"=>"Expression of LS and LO o<i>FSHBLuc</i> transgenes in mouse tissues.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2013-07-18 01:51:51"}
  • {"files"=>["https://ndownloader.figshare.com/files/1121739"], "description"=>"<p>4.7 kb of ovine <i>FSHB</i> promoter was aligned with 10 kb of human (Ensembl transcript ID: ENST0000025122) and 5.7 kb of porcine (NCBI Acc# D00621.1) <i>FSHB</i> promoters using BLAST 2. Regions between −3.6 and −2.5 kb of the ovine <i>FSHB</i> promoter are shown that correspond to similar sequences in the human (−5 to −6.1 kb) and porcine (−4.6 to −5.7 kb) <i>FSHB</i> promoters. A fourth region of high homology between porcine and ovine <i>FSHB</i> promoters was found between −2.48 kb and −2.07 kb.</p>", "links"=>[], "tags"=>["Anatomy and physiology", "Reproductive system", "Reproductive physiology", "Sexual reproduction", "Biochemistry", "Biochemistry simulations", "hormones", "genetics", "gene expression", "DNA transcription", "chromatin", "Molecular genetics", "Gene regulation", "Animal genetics", "ovine", "fshb", "promoter", "homology", "porcine"], "article_id"=>747771, "categories"=>["Biological Sciences"], "users"=>["Jingjing Jia", "Farideh Shafiee-Kermani", "William L. Miller"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0066852.g001", "stats"=>{"downloads"=>0, "page_views"=>1, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Regions_of_the_ovine_FSHB_promoter_with_8805_75_sequence_homology_to_porcine_and_or_human_5_8242_promoter_regions_/747771", "title"=>"Regions of the ovine FSHB promoter with≥75% sequence homology to porcine and/or human 5′ promoter regions.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-07-18 01:51:51"}
  • {"files"=>["https://ndownloader.figshare.com/files/1121740"], "description"=>"<p>Pituitary cells from transgenic mice expressing LS-o<i>FSHβLuc</i> were dispersed and plated at a density of 30,000/well. After 2 days, cells were treated with 250 ng/ml of follistatin (F; basal expression), 50 ng/ml of activin (A; maximal expression) or 50 ng/ml of activin +100 nM GnRH (A+G; GnRH-regulated expression) for 20 h. Cell lysates were assayed for luciferase activity. Values are luciferase activity expressed as relative light units (RLU) and represent the mean ± sem for three independent experiments each performed in triplicate.</p>", "links"=>[], "tags"=>["Anatomy and physiology", "Reproductive system", "Reproductive physiology", "Sexual reproduction", "Biochemistry", "Biochemistry simulations", "hormones", "genetics", "gene expression", "DNA transcription", "chromatin", "Molecular genetics", "Gene regulation", "Animal genetics", "dispersed", "pituitary"], "article_id"=>747772, "categories"=>["Biological Sciences"], "users"=>["Jingjing Jia", "Farideh Shafiee-Kermani", "William L. Miller"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0066852.g002", "stats"=>{"downloads"=>0, "page_views"=>2, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Hormonal_regulation_of_LS_o_FSHBLuc_in_dispersed_pituitary_cell_culture_/747772", "title"=>"Hormonal regulation of LS-o<b><i>FSHBLuc</i></b><b> in dispersed pituitary cell culture.</b>", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-07-18 01:51:51"}
  • {"files"=>["https://ndownloader.figshare.com/files/1121742"], "description"=>"<p>LβT2 cells (40,000 cells/well) were plated and transiently transfected with −195o<i>FSHBLuc</i>, −748o<i>FSHBLuc</i> or −2932o<i>FSHBLuc</i>. Different amounts of DNA (5, 15 or 50 ng/well) were tested. After a 12 hour transfection, cells were treated with activin (50 ng/ml) for 24 h, and then assayed for luciferase activity. For each plasmid, data from different doses were normalized to the activity of the lowest dose. <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066852#pone-0066852-g003\" target=\"_blank\">Figure 3B:</a> Hormonal regulation of LβT2 cells transfected with plasmids. LβT2 cells transfected with the 3 plasmid constructs at 50 ng/well (see Fig. 3A) were treated with follistatin (F; 250 ng/ml; basal expression), activin (A; 50 ng/ml; maximal expression) or activin (50 ng/ml)+GnRH (100 nM) (A+G; GnRH-regulated expression) for 24 hours after a 12 hour transfection, and then assayed for luciferase activity. The framed text indicates activin induction for each plasmid construct. For each plasmid, all the data were normalized to the basal activity (follistatin treatment). Different letters indicate significant differences in the means (P<0.05).</p>", "links"=>[], "tags"=>["Anatomy and physiology", "Reproductive system", "Reproductive physiology", "Sexual reproduction", "Biochemistry", "Biochemistry simulations", "hormones", "genetics", "gene expression", "DNA transcription", "chromatin", "Molecular genetics", "Gene regulation", "Animal genetics", "dose-response", "plasmid", "transfection"], "article_id"=>747774, "categories"=>["Biological Sciences"], "users"=>["Jingjing Jia", "Farideh Shafiee-Kermani", "William L. Miller"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0066852.g003", "stats"=>{"downloads"=>0, "page_views"=>6, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_A_Dose_response_of_plasmid_transfection_in_L_946_T2_cells_/747774", "title"=>"A: Dose-response of plasmid transfection in LβT2 cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-07-18 01:51:51"}

PMC Usage Stats | Further Information

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Relative Metric

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