41 research outputs found

    Indispensable Roles of COUP-TFII in Progenitor Leydig Cell Differentiation.

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    <p>A) Evaluation of Leydig cell differentiation in the mutant testes, indicated by Leydig cell marker 3β-HSD immunostaining. The samples were collected at P14, P21, P28, P60 and P90, which correspond to 0, 7, 14, 46 and 76 days after tamoxifen injection, respectively. B) Testosterone treatment could not rescue the hypoplasia of Leydig cells. There were no increases in the intensity of P450Scc (C) and EST (D) signals or localization changes of 3β-HSD (B) signals in the rescued animal. E) qRT-PCR analysis of the expression of Leydig cell marker. RNA was isolated from 3-months-old littermates implanted with control or testosterone pellet (n = 6). Expression levels of each gene were normalized to the levels of the 18sRNA. Data in (E) indicate mean±SD. * P<0.05; ** P<0.01</p

    <i>COUP-TFII</i> Null Mice Display Leydig Cell Hypoplasia.

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    <p>(A) Immunohistological detection of COUP-TFII expression in the testes from adult wild type mice. Green, COUP-TFII; Blue: DAP1 (B) <i>COUP-TFII</i> deletion efficiency was examined by qRT-PCR. Testes were collected from the littermates. N = 6; ** P<0.01 (C) Serum testosterone, LH, and FSH levels in 3-month-old males: F/F; Tam, Cre/+ F/F; Oil and Cre/+ F/F; Tam. (n = 8, 10, and 7, respectively). (D-I) H&E staining of paraffin-embedded testes (D). Immunohistochemistry results of Leydig cell marker P450Scc (E), EST (F), 3β-HSD (G) and CYP19 (H) indicated that mutant mice display Leydig cell hypoplasia. However, Sertoli cells in the null mice are normal (I).</p

    Inducible Ablation of <i>COUP-TFII</i> at Pre-puberty Stage Leads to Infertility and Hypogonadism.

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    <p>A) Scheme of inducible ablation of <i>COUP-TFII</i> at Pre-puberty stage. Tamoxifen or oil was intraperitonealy injected into P14 animals to induce the deletion of <i>COUP-TFII</i> gene, and mice were sacrificed at P90. B) Immunoblotting of COUP-TFII was performed to examine the deletion efficiency in mutant mice, and tissues were collected from the comparison littermates. F/F, Tam: <i>COUP-TF<sup>flox/flox</sup></i> treated with tamoxifen: Cre/+ F/F, Oil: <i>Cre-ER<sup>TM (+/−)</sup> COUP-TFII<sup>flox/flox</sup></i> treated with oil and Cre/+ F/F, Tam: <i>Cre-ER<sup>TM (+/−)</sup> COUP-TFII<sup>flox/flox</sup></i> treated with tamoxifen. C) The photograph depicts the appearance of male reproduction organs from 3-month-old littermate. D) Relative weight of reproduction organs normalized with body weight. Results are expressed as the mean (±SD) of the ratios for each genotype. Statistical comparison was done with a student test. * P<0.05, ** P<0.01</p

    COUP-TFII is not Essential for Maintenance of Leydig Cell Function.

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    <p>A) Histological examination of testis and epididymis. Spermatazoa (arrow) were observed in testes and epididymis of 4-month-old control and Cre/+ F/F mice, which were treated with tamoxifen at the age of two months. B) Immunohistochemistry for Leydig cell markers, 3β-HSD, P450Scc and EST. C) Immunoblotting for COUP-TFII, 3β-HSD, P450Scc and EST. Tam OD indicates the day before tamoxifen injection when mice are two month old. Tam 60D means 60 days after tamoxifen injection.</p

    COUP-TFII Plays Roles in Testis Organogenesis and Progenitor Leydig cell Formation.

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    <p>A) Immunohistochemistry for COUP-TFII at embryonic 18.5 (E18.5) and P7. Tamoxifen was injected to pregnant mothers at E18.5. B) Immunohistochemistry for Leydig cell markers, 3β-HSD at E18.5, P14 and P21. Arrow indicated progenitor Leydig cells, and arrowhead was fetal Leydig cells. C) Quantitative results of progenitor Leydig cell number peri-seminiferous tubule. Data in (C) indicate mean±SD. * P<0.05; ** P<0.01. D) H& E staining of the testes and epididymis from P60 littermate of control and mutant mice. Spermatazoa was indicated by arrow. Immunohistochemistry result for Leydig cell markers, 3β-HSD and EST. E) The photograph depicts the appearance of testes from control and mutant mice at E18.5 and P14.</p

    Arrest of Spermatogenesis at the Round Spermatid Stage in the Null Mice.

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    <p>H&E staining of paraffin-embedded testes (A, A') and epididymis (B). A' is the large magnification of the box area in the A. (C) Quantitative realtime RT-PCR analysis of the germ cell differentiation markers. RNA was isolated from 3-months-old littermates. Expression levels of each gene were normalized to the levels of the 18sRNA (n = 6). Data in (C) indicate the mean±SD. * P<0.05; ** P<0.01</p

    Hypogonadism and Spermatogenesis Defects were Rescued by Testosterone Replacement.

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    <p>Gross appearance of testes and accessory glands after testosterone treatment. The size of testes, epididymis, seminal vesicles (A) and prostate (B) grew markedly in Cre/+ F/F mice implanted with testosterone pellet compared with the null mice implanted with control pellet. H& E staining demonstrates the resumption of spermatogenesis in the null mine treated with testosterone. The elongated spermatid or spermatozoa could be observed in the mutant testes (C, D) and epididymis (E). In addition, loss of secretary protein in seminal vesicles could be observed in the rescued mice (F; arrow). A, anterior prostate; V ventral prostate; DL, dorsal-lateral prostate.</p

    Expression and function profiling of orphan nuclear receptors using bacterial artificial chromosome (BAC) transgenesis.-1

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    <p><b>Copyright information:</b></p><p>Taken from "Expression and function profiling of orphan nuclear receptors using bacterial artificial chromosome (BAC) transgenesis."</p><p>Nuclear Receptor Signaling 2003;1():-.</p><p>Published online 16 Jun 2003</p><p>PMCID:PMC1402220.</p><p>Copyright © 2003, Nemoz-Gailliard et al. This is an open-access article distributed under the terms of the Creative Commons Non-Commercial Attribution License, which permits unrestricted non-commercial use, distribution and reproduction in any medium, provided the original work is properly cited. </p>construct will place the expression of the reporter gene under the control of the promoter and regulatory regions present in the BAC and are likely to recapitulate the expression pattern of the NR considered. Note: it is also possible to generate knock-out constructs with this approach, providing that a eukaryotic promoter (Pr.) is introduced in the construct in order to allow selection of recombinant ES cells with an appropriate selection marker (Sm* – dual selection marker, for prokaryotic and eukaryotic selection). Homologous recombination allows for more complex designs than the one presented here. B. Activity trap setting. This particular design results in the replacement of the endogenous DNA-binding domain (DBD) by the Gal4 DBD. Activation of a reporter gene upon binding of the chimeric protein to Gal4 UAS sequences (inset) should parallel the activity of the endogenous NR. C. Ligand trap setting. A version slightly different from B. The expression of this fusion gene will activate reporter gene expression upon binding of LBD by (putative) ligand(s). A, B: regions of homology designed for recombination; Sm: Selection marker; Pr.: Eukaryotic promoter; DBD: DNA-Binding Domain; LBD: Ligand Binding Domain ; NR: Nuclear Receptor; frt: recombination sites for the Flt recombinase (for removal of the selection marker in a subsequent step)

    Expression and function profiling of orphan nuclear receptors using bacterial artificial chromosome (BAC) transgenesis.-0

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    <p><b>Copyright information:</b></p><p>Taken from "Expression and function profiling of orphan nuclear receptors using bacterial artificial chromosome (BAC) transgenesis."</p><p>Nuclear Receptor Signaling 2003;1():-.</p><p>Published online 16 Jun 2003</p><p>PMCID:PMC1402220.</p><p>Copyright © 2003, Nemoz-Gailliard et al. This is an open-access article distributed under the terms of the Creative Commons Non-Commercial Attribution License, which permits unrestricted non-commercial use, distribution and reproduction in any medium, provided the original work is properly cited. </p>on construct is flanked by two homologous regions (A and B) for the targeted BAC clone and contains a prokaryotic selection marker (Sm) for the selection of recombinant-containing bacteria. Electrocompetent cells cells containing the BAC (BAC or PAC) are prepared and induced to express the recombination function (rec+). The recombinant construct is then introduced in these cells by electroporation and the recombinants are selected against the selection marker (Sm). B. Modification of a BAC plasmid by homologous recombination in the bacteria: circular recombination. The targeting plasmid consists of a selectable and counter-selectable marker (Sm–Csm), a temperature-sensitive origin of replication (ts-ori), the recA gene and two homology regions (A and B). In the first round of homologous recombination, recombination can take place through either homology arm (A or B; for simplicity, only recombination through A is shown here), which results in the formation of a co-integrant. The co-integrant is identified by selection for the selectable gene at a temperature that is not permissive for the ts-ori. In the second round of homologous recombination, resolution of the cointegrant is driven by applying counter-selection against the counter-selectable gene at a temperature that is permissive for the ts-ori. Resolution can either revert the co-integrant to the original target molecule or generate the recombinant
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