lv jagged1  (Santa Cruz Biotechnology)

Journal: Endocrinology

Article Title: Neurotrophins Acting Via TRKB Receptors Activate the JAGGED1-NOTCH2 Cell-Cell Communication Pathway to Facilitate Early Ovarian Development

doi: 10.1210/en.2011-1465

Figure Lengend Snippet: RT-PCR primer list

Article Snippet: Assessment of cell proliferation The ovaries from 3-d-old TrkB −/− mice, incubated with LV- Jagged1 or LV-no Jagged1 for 4 d, were fixed in Zamboni's fixative, embedded in paraffin, sectioned at 14 μm, and subjected to immunohistochemistry for proliferating cell nuclear antigen (PCNA), as reported ( 21 , 43 ), using a monoclonal antibody to PCNA (Mab PC-10, 1:100; Santa Cruz Biotechnology, Inc.) and developing the immunoreaction with a diaminobenzidine, H 2 O 2 , and nickel chloride solution, followed by counterstaining with Nuclear Fast Red (undiluted, 10 min at room temperature; Vector Laboratories, Burlingame, CA).

Techniques:

Journal: Endocrinology

Article Title: Neurotrophins Acting Via TRKB Receptors Activate the JAGGED1-NOTCH2 Cell-Cell Communication Pathway to Facilitate Early Ovarian Development

doi: 10.1210/en.2011-1465

Figure Lengend Snippet: Absence of TRKB receptors result in reduced expression of Jagged1 and NOTCH target genes in the mouse ovary. Panel A, Decrease in Jagged1 mRNA content in the ovary of 7-d-old TrkB−/− mice detected using cDNA microarrays. Changes in mRNA content are expressed as fold-decrease with respect to mRNA values in TrkB+/+ mice of the same age. Filled squares represent the values detected in independent microarray determinations. Panel B, Jagged1 mRNA content was reduced in the ovary of 7-d-old TrkB−/− mice, as assessed by real-time PCR. Inset, Jagged1 mRNA content increased in 7-d-old WT ovaries treated in vitro with BDNF (100 ng/ml, 8 h) compared to control (C) ovaries incubated with vehicle. Relative mRNA values are expressed as arbitrary units (AU), normalized using 18s RNA or Ppia mRNA values as the normalizing unit. Panel C, Hes1 and Hey2, but not Notch, mRNA abundance, was also reduced in TrkB-null ovaries. Panel D, JAGGED1 immunoreactive material (green color) mostly localizes to oocytes in the ovary from 7-d-old TrkB+/+ mice. Panel E, JAGGED1 immunoreactivity was noticeably decreased in oocytes of TrkB−/− mice. Panel F, Section incubated without JAGGED1 antibodies. Cell nuclei stained with the DNA-binding dye Hoechst are shown in blue. White arrows point to examples of oocytes showing JAGGED1 staining. Columns in B and C represent means from four to five animals per group, and vertical lines are sem. *, P < 0.05; **, P < 0.01 vs. WT controls. Scale bars, 50 μm.

Article Snippet: Assessment of cell proliferation The ovaries from 3-d-old TrkB −/− mice, incubated with LV- Jagged1 or LV-no Jagged1 for 4 d, were fixed in Zamboni's fixative, embedded in paraffin, sectioned at 14 μm, and subjected to immunohistochemistry for proliferating cell nuclear antigen (PCNA), as reported ( 21 , 43 ), using a monoclonal antibody to PCNA (Mab PC-10, 1:100; Santa Cruz Biotechnology, Inc.) and developing the immunoreaction with a diaminobenzidine, H 2 O 2 , and nickel chloride solution, followed by counterstaining with Nuclear Fast Red (undiluted, 10 min at room temperature; Vector Laboratories, Burlingame, CA).

Techniques: Expressing, Microarray, Real-time Polymerase Chain Reaction, In Vitro, Incubation, Staining, Binding Assay

Journal: Endocrinology

Article Title: Neurotrophins Acting Via TRKB Receptors Activate the JAGGED1-NOTCH2 Cell-Cell Communication Pathway to Facilitate Early Ovarian Development

doi: 10.1210/en.2011-1465

Figure Lengend Snippet: Changes in ovarian content of Jagged1, Notch2, Hes1, and Hey2 mRNA during the first postnatal week of life of the mouse, as assessed by real-time PCR. A, Jagged1 mRNA. B, Notch2 mRNA. C, Hes1 mRNA. D, Hey2 mRNA. Relative mRNA values are expressed as arbitrary units (AU), normalized using 18s RNA values as the normalizing unit. E–J, In situ hybridization, using a mouse-specific 35S-uridine triphosphate-labeled Jagged1 cRNA probe, shows that Jagged1 mRNA is exclusively expressed in oocytes and that the abundance of Jagged1 mRNA increases during the first 12 d of postnatal life. Bright field images are shown in E–G and dark field images in H–J. Black and white arrows point to examples of Jagged1 mRNA-containing oocytes. Bars represent the mean of four to five mice per group, and vertical lines are sem. *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. 0-d-old group. Scale bar, 50 μm.

Article Snippet: Assessment of cell proliferation The ovaries from 3-d-old TrkB −/− mice, incubated with LV- Jagged1 or LV-no Jagged1 for 4 d, were fixed in Zamboni's fixative, embedded in paraffin, sectioned at 14 μm, and subjected to immunohistochemistry for proliferating cell nuclear antigen (PCNA), as reported ( 21 , 43 ), using a monoclonal antibody to PCNA (Mab PC-10, 1:100; Santa Cruz Biotechnology, Inc.) and developing the immunoreaction with a diaminobenzidine, H 2 O 2 , and nickel chloride solution, followed by counterstaining with Nuclear Fast Red (undiluted, 10 min at room temperature; Vector Laboratories, Burlingame, CA).

Techniques: Real-time Polymerase Chain Reaction, In Situ Hybridization, Labeling

Journal: Endocrinology

Article Title: Neurotrophins Acting Via TRKB Receptors Activate the JAGGED1-NOTCH2 Cell-Cell Communication Pathway to Facilitate Early Ovarian Development

doi: 10.1210/en.2011-1465

Figure Lengend Snippet: Lentiviral-mediated delivery of Jagged1, using the Gdf9 promoter to target expression of a JAGGED1-HA fusion protein to oocytes, correctly targets JAGGED1 to the cell membrane of oocytes. A, Map of the lentiviral delivery construct (LV-Jagged1) used in this study. The lentiviral vector employed has been previously described (79). The 3′LTR of this vector contains a 400-bp deletion that results in the self-inactivation (SIN) of the vector. The other components include the packaging signal (ψ), the Rev response element binding site (RRE), the central polypurine tract (cPPT), and the woodchuck-hepatitis-virus posttranslational regulatory element (wPRE). The LV-Jagged1 construct contains a bicistronic transgene cassette in which expression of a Jagged1-HA cDNA is driven by the rat Gdf9 promoter (Gdf9p). The Jagged1-HA cDNA is linked to an enhanced green fluorescent protein (eGFP) cDNA via an internal ribosome entry site (IRES). A construct lacking Jagged1-HA (LV-no Jagged1) was used as a negative control. B–D, Immunohistofluorescent images of sections from 3-d-old mouse ovaries cultured for 4 d in the presence of LV-Jagged1 and stained with monoclonal antibodies against the HA epitope. E, Section from an ovary not infected with LV. F, Section from an ovary infected with LV-Jagged1 and incubated without HA antibodies. G, Section from an ovary infected with LV-no Jagged1. JAGGED1 immunoreactive cells are seen in red, and cell nuclei stained with the DNA-binding dye Hoechst are shown in blue. Scale bar, 50 μm.

Article Snippet: Assessment of cell proliferation The ovaries from 3-d-old TrkB −/− mice, incubated with LV- Jagged1 or LV-no Jagged1 for 4 d, were fixed in Zamboni's fixative, embedded in paraffin, sectioned at 14 μm, and subjected to immunohistochemistry for proliferating cell nuclear antigen (PCNA), as reported ( 21 , 43 ), using a monoclonal antibody to PCNA (Mab PC-10, 1:100; Santa Cruz Biotechnology, Inc.) and developing the immunoreaction with a diaminobenzidine, H 2 O 2 , and nickel chloride solution, followed by counterstaining with Nuclear Fast Red (undiluted, 10 min at room temperature; Vector Laboratories, Burlingame, CA).

Techniques: Expressing, Construct, Plasmid Preparation, Binding Assay, Negative Control, Cell Culture, Staining, Infection, Incubation

Journal: Endocrinology

Article Title: Neurotrophins Acting Via TRKB Receptors Activate the JAGGED1-NOTCH2 Cell-Cell Communication Pathway to Facilitate Early Ovarian Development

doi: 10.1210/en.2011-1465

Figure Lengend Snippet: Oocyte-specific restoration of JAGGED1 expression, via lentiviral-mediated gene transfer, rescues the deficit in follicle growth of TrkB−/− ovaries. A, Increased number of secondary follicles in TrkB−/− ovaries incubated for 4 d with LV-Jagged1 in comparison with TrkB−/− ovaries infected with LV-no Jagged1. B, Section from a TrkB−/− ovary infected with LV-no Jagged1. C, Section from a TrkB−/− ovary infected with LV-Jagged1. Arrows point to secondary follicles, which contain an oocyte surrounded by two layers of GC. Scale bar, 50 μm. Columns represent the mean of four mice per group, and vertical lines are sem. One ovary from each animal was infected with LV-Jagged1 and the contralateral ovary from the same animal with LV-no Jagged1. *, P < 0.05.

Article Snippet: Assessment of cell proliferation The ovaries from 3-d-old TrkB −/− mice, incubated with LV- Jagged1 or LV-no Jagged1 for 4 d, were fixed in Zamboni's fixative, embedded in paraffin, sectioned at 14 μm, and subjected to immunohistochemistry for proliferating cell nuclear antigen (PCNA), as reported ( 21 , 43 ), using a monoclonal antibody to PCNA (Mab PC-10, 1:100; Santa Cruz Biotechnology, Inc.) and developing the immunoreaction with a diaminobenzidine, H 2 O 2 , and nickel chloride solution, followed by counterstaining with Nuclear Fast Red (undiluted, 10 min at room temperature; Vector Laboratories, Burlingame, CA).

Techniques: Expressing, Incubation, Infection

Journal: Endocrinology

Article Title: Neurotrophins Acting Via TRKB Receptors Activate the JAGGED1-NOTCH2 Cell-Cell Communication Pathway to Facilitate Early Ovarian Development

doi: 10.1210/en.2011-1465

Figure Lengend Snippet: Oocyte-specific restoration of JAGGED1 expression, via lentiviral-mediated gene transfer, rescues the deficit in GC proliferation of TrkB−/− ovaries. A, Percent of follicles showing at least one PCNA-positive GC. B, Number of PCNA-positive GC per follicle (primary and secondary). C, Image of a section from a TrkB−/− ovary incubated for 4 d with LV-no Jagged1. D, A section from a TrkB−/− ovary incubated for 4 d with a LV-Jagged1. E, Ovarian section immunostained in absence of primary antibodies. Columns represent the mean of four mice per group, and vertical lines are sem. In each group, four sections per ovary were used for quantification. One ovary from each animal was infected with LV-Jagged1 and the contralateral ovary from the same animal with LV-no Jagged1. Scale bar, 50 μm. ***, P < 0.001 vs. LV-no Jagged1.

Article Snippet: Assessment of cell proliferation The ovaries from 3-d-old TrkB −/− mice, incubated with LV- Jagged1 or LV-no Jagged1 for 4 d, were fixed in Zamboni's fixative, embedded in paraffin, sectioned at 14 μm, and subjected to immunohistochemistry for proliferating cell nuclear antigen (PCNA), as reported ( 21 , 43 ), using a monoclonal antibody to PCNA (Mab PC-10, 1:100; Santa Cruz Biotechnology, Inc.) and developing the immunoreaction with a diaminobenzidine, H 2 O 2 , and nickel chloride solution, followed by counterstaining with Nuclear Fast Red (undiluted, 10 min at room temperature; Vector Laboratories, Burlingame, CA).

Techniques: Expressing, Incubation, Infection

Journal: Endocrinology

Article Title: Neurotrophins Acting Via TRKB Receptors Activate the JAGGED1-NOTCH2 Cell-Cell Communication Pathway to Facilitate Early Ovarian Development

doi: 10.1210/en.2011-1465

Figure Lengend Snippet: TrkB signaling sustains c-Myc and Odc1 expression but not the expression of core regulatory components of the cell cycle. Panel A, c-Myc mRNA content was reduced in 7-d-old TrkB−/− ovaries as compared with WT animals. Inset, In vitro exposure of WT ovaries to NT4/5 (100 ng/ml, 8 h) increased c-Myc mRNA abundance as compared to control (C) ovaries incubated with vehicle. Panel B, Odc1 mRNA abundance was also decreased in TrkB−/− ovaries. Inset, NT4/5 increased Odc1 mRNA abundance in WT ovaries. Panels C and D, The content of mRNA encoding cyclins (CycD2 and CycE1), CDK (Cdk2 and Cdk4), and the CKI of the INK4 family (p15INK4b, p16INK4a, p18INK4c, and p19INK4d) and CIP/KIP family (p21Cip1, p27Kip1, and p57Kip2) remain unaltered in TrkB−/− ovaries as compared with TrkB+/+ mice. Panel E, c-Myc mRNA levels were increased in TrkB−/− ovaries after oocyte-specific restoration of JAGGED1 synthesis. The ovaries from 3-d-old mice were incubated for 4 d with a lentiviral construct carrying the Jagged1-coding region under the control of the Gdf9 promoter. Panel F, Neither p19INK4D nor p27Kip1 mRNA levels changed after lentiviral-mediated restoration of JAGGED1 synthesis. Control ovaries were infected with a LV lacking Jagged1 cDNA (LV-no Jagged1). Each column represents the mean of four to five mice per group, and vertical lines are sem. One ovary from each animal was infected with LV-Jagged1 and the contralateral ovary with LV-no Jagged1. *, P < 0.05; **, P < 0.01 vs. their respective controls. AU, Arbitrary units.

Article Snippet: Assessment of cell proliferation The ovaries from 3-d-old TrkB −/− mice, incubated with LV- Jagged1 or LV-no Jagged1 for 4 d, were fixed in Zamboni's fixative, embedded in paraffin, sectioned at 14 μm, and subjected to immunohistochemistry for proliferating cell nuclear antigen (PCNA), as reported ( 21 , 43 ), using a monoclonal antibody to PCNA (Mab PC-10, 1:100; Santa Cruz Biotechnology, Inc.) and developing the immunoreaction with a diaminobenzidine, H 2 O 2 , and nickel chloride solution, followed by counterstaining with Nuclear Fast Red (undiluted, 10 min at room temperature; Vector Laboratories, Burlingame, CA).

Techniques: Expressing, In Vitro, Incubation, Construct, Infection