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Specific deficiency of Plzf paralog, Zbtb20, in Sertoli cells does not affect spermatogenesis and fertility in mice.

Jiang X, Zhang H, Yin S, Zhang Y, Yang W, Zheng W, Wang L, Wang Z, Bukhari I, Cooke HJ, Iqbal F, Shi Q - Sci Rep (2014)

Bottom Line: Ztbt20 is a POK family transcription factor and primarily functions through its conserved C2H2 Krüppel type zinc finger and BTB/POZ domains.Immunohistochemical studies revealed that ZBTB20 protein was localized specifically in the nuclei of Sertoli cells in seminiferous tubules.Taken together, though specific deletion of transcription factor Zbtb20 in Sertoli cells has no apparent influence on spermatogenesis, its specific localization in Sertoli cells makes Zbtb20 a useful marker for the identification of Sertoli cells in seminiferous tubules.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular and Cell Genetics, CAS Key Laboratory of Innate Immunity and Chronic Disease, CAS Hefei Institutes of Physical Science, Hefei National Laboratory for Physical Sciences at Microscale, School of Life Sciences, University of Science &Technology of China, Hefei, China.

ABSTRACT
Ztbt20 is a POK family transcription factor and primarily functions through its conserved C2H2 Krüppel type zinc finger and BTB/POZ domains. The present study was designed to define the function of the Zbtb20, in vivo, during mouse spermatogenesis. Immunohistochemical studies revealed that ZBTB20 protein was localized specifically in the nuclei of Sertoli cells in seminiferous tubules. To investigate its role during spermatogenesis, we crossed Amh-Cre transgenic mice with Zbtb20 floxp mice to generate conditionally knockout mice (cKO) in which Zbtb20 was specifically deleted in Sertoli cells. The cKO mice were fertile and did not show any detectable abnormalities in spermatogenesis. Taken together, though specific deletion of transcription factor Zbtb20 in Sertoli cells has no apparent influence on spermatogenesis, its specific localization in Sertoli cells makes Zbtb20 a useful marker for the identification of Sertoli cells in seminiferous tubules.

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Conditional deletion of Zbtb20 in Sertoli cells.(A). Hybrid scheme used to develop Zbtb20 cKO mice. (B). Western blot analysis of ZBTB20 in isolated Sertoli cells from 21-dpp control and cKO mice. GAPDH is served as a protein loading control. The image shown is a representative result from three independent experiments. (C). Quantitative results of Western blot experiments shown in (B). The ZBTB20 protein level was normalized and plotted against GAPDH, and was arbitrarily set as 1 in controls. Data are presented as mean ± SEM from three independent experiments. **P < 0.01, Student's t-test. (D). Cellular localization of ZBTB20 and SOX9 in sections of control and cKO testes. Localization of ZBTB20 (green) and SOX9 (red) in sections of testes of 21-dpp mice were shown by immunofluorescence. ZBTB20 localization was lost in cKO Sertoli cells. The shown are representative images from experiments that were repeated thrice using samples from different sets of testes and yielded similar results. Scale bars, 50 μm.
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f2: Conditional deletion of Zbtb20 in Sertoli cells.(A). Hybrid scheme used to develop Zbtb20 cKO mice. (B). Western blot analysis of ZBTB20 in isolated Sertoli cells from 21-dpp control and cKO mice. GAPDH is served as a protein loading control. The image shown is a representative result from three independent experiments. (C). Quantitative results of Western blot experiments shown in (B). The ZBTB20 protein level was normalized and plotted against GAPDH, and was arbitrarily set as 1 in controls. Data are presented as mean ± SEM from three independent experiments. **P < 0.01, Student's t-test. (D). Cellular localization of ZBTB20 and SOX9 in sections of control and cKO testes. Localization of ZBTB20 (green) and SOX9 (red) in sections of testes of 21-dpp mice were shown by immunofluorescence. ZBTB20 localization was lost in cKO Sertoli cells. The shown are representative images from experiments that were repeated thrice using samples from different sets of testes and yielded similar results. Scale bars, 50 μm.

Mentions: In order to assess the Zbtb20 functions in Sertoli cells during spermatogenesis, we generated mice in which the Zbtb20 gene was specifically disrupted in testicular Sertoli cells. We combined a conditional floxp Zbtb20 allele with the Cre-expressing line Amh-Cre (Fig. 2A). To study the CRE recombinase activity, Amh-cre mice were crossed with fluorescence reporter mice (mT/mG), in which Amh-Cre mediated recombination resulted in excision of the RFP cassette and expression of the GFP reporter21. Based on the analysis of Amh-Cre recombined mT/mG reporter mice, we confirmed that the Amh-Cre mice had strong recombinase activities in Sertoli cells (Supplementary Fig. S2). Zbtb20 deletion efficiency in Sertoli cells was analyzed by detecting the Zbtb20 mRNA and protein levels in the isolated Sertoli cells (Supplementary Fig. S3). We found that the Zbtb20 disruption resulted in a drastic reduction in Zbtb20 mRNA and protein levels in the cKO Sertoli cells (Supplementary Fig. S4 and Fig. 2B–C). As shown in Fig. 2D, immunofluorescent analysis of the cKO testes revealed that the localization of ZBTB20 in Sertoli cell was abolished (Fig. 2D) without affecting its localization in Leydig cells (Supplementary Fig. S5), which further confirms the efficient and specific disruption of Zbtb20 in Sertoli cells.


Specific deficiency of Plzf paralog, Zbtb20, in Sertoli cells does not affect spermatogenesis and fertility in mice.

Jiang X, Zhang H, Yin S, Zhang Y, Yang W, Zheng W, Wang L, Wang Z, Bukhari I, Cooke HJ, Iqbal F, Shi Q - Sci Rep (2014)

Conditional deletion of Zbtb20 in Sertoli cells.(A). Hybrid scheme used to develop Zbtb20 cKO mice. (B). Western blot analysis of ZBTB20 in isolated Sertoli cells from 21-dpp control and cKO mice. GAPDH is served as a protein loading control. The image shown is a representative result from three independent experiments. (C). Quantitative results of Western blot experiments shown in (B). The ZBTB20 protein level was normalized and plotted against GAPDH, and was arbitrarily set as 1 in controls. Data are presented as mean ± SEM from three independent experiments. **P < 0.01, Student's t-test. (D). Cellular localization of ZBTB20 and SOX9 in sections of control and cKO testes. Localization of ZBTB20 (green) and SOX9 (red) in sections of testes of 21-dpp mice were shown by immunofluorescence. ZBTB20 localization was lost in cKO Sertoli cells. The shown are representative images from experiments that were repeated thrice using samples from different sets of testes and yielded similar results. Scale bars, 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f2: Conditional deletion of Zbtb20 in Sertoli cells.(A). Hybrid scheme used to develop Zbtb20 cKO mice. (B). Western blot analysis of ZBTB20 in isolated Sertoli cells from 21-dpp control and cKO mice. GAPDH is served as a protein loading control. The image shown is a representative result from three independent experiments. (C). Quantitative results of Western blot experiments shown in (B). The ZBTB20 protein level was normalized and plotted against GAPDH, and was arbitrarily set as 1 in controls. Data are presented as mean ± SEM from three independent experiments. **P < 0.01, Student's t-test. (D). Cellular localization of ZBTB20 and SOX9 in sections of control and cKO testes. Localization of ZBTB20 (green) and SOX9 (red) in sections of testes of 21-dpp mice were shown by immunofluorescence. ZBTB20 localization was lost in cKO Sertoli cells. The shown are representative images from experiments that were repeated thrice using samples from different sets of testes and yielded similar results. Scale bars, 50 μm.
Mentions: In order to assess the Zbtb20 functions in Sertoli cells during spermatogenesis, we generated mice in which the Zbtb20 gene was specifically disrupted in testicular Sertoli cells. We combined a conditional floxp Zbtb20 allele with the Cre-expressing line Amh-Cre (Fig. 2A). To study the CRE recombinase activity, Amh-cre mice were crossed with fluorescence reporter mice (mT/mG), in which Amh-Cre mediated recombination resulted in excision of the RFP cassette and expression of the GFP reporter21. Based on the analysis of Amh-Cre recombined mT/mG reporter mice, we confirmed that the Amh-Cre mice had strong recombinase activities in Sertoli cells (Supplementary Fig. S2). Zbtb20 deletion efficiency in Sertoli cells was analyzed by detecting the Zbtb20 mRNA and protein levels in the isolated Sertoli cells (Supplementary Fig. S3). We found that the Zbtb20 disruption resulted in a drastic reduction in Zbtb20 mRNA and protein levels in the cKO Sertoli cells (Supplementary Fig. S4 and Fig. 2B–C). As shown in Fig. 2D, immunofluorescent analysis of the cKO testes revealed that the localization of ZBTB20 in Sertoli cell was abolished (Fig. 2D) without affecting its localization in Leydig cells (Supplementary Fig. S5), which further confirms the efficient and specific disruption of Zbtb20 in Sertoli cells.

Bottom Line: Ztbt20 is a POK family transcription factor and primarily functions through its conserved C2H2 Krüppel type zinc finger and BTB/POZ domains.Immunohistochemical studies revealed that ZBTB20 protein was localized specifically in the nuclei of Sertoli cells in seminiferous tubules.Taken together, though specific deletion of transcription factor Zbtb20 in Sertoli cells has no apparent influence on spermatogenesis, its specific localization in Sertoli cells makes Zbtb20 a useful marker for the identification of Sertoli cells in seminiferous tubules.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular and Cell Genetics, CAS Key Laboratory of Innate Immunity and Chronic Disease, CAS Hefei Institutes of Physical Science, Hefei National Laboratory for Physical Sciences at Microscale, School of Life Sciences, University of Science &Technology of China, Hefei, China.

ABSTRACT
Ztbt20 is a POK family transcription factor and primarily functions through its conserved C2H2 Krüppel type zinc finger and BTB/POZ domains. The present study was designed to define the function of the Zbtb20, in vivo, during mouse spermatogenesis. Immunohistochemical studies revealed that ZBTB20 protein was localized specifically in the nuclei of Sertoli cells in seminiferous tubules. To investigate its role during spermatogenesis, we crossed Amh-Cre transgenic mice with Zbtb20 floxp mice to generate conditionally knockout mice (cKO) in which Zbtb20 was specifically deleted in Sertoli cells. The cKO mice were fertile and did not show any detectable abnormalities in spermatogenesis. Taken together, though specific deletion of transcription factor Zbtb20 in Sertoli cells has no apparent influence on spermatogenesis, its specific localization in Sertoli cells makes Zbtb20 a useful marker for the identification of Sertoli cells in seminiferous tubules.

Show MeSH
Related in: MedlinePlus