[Frontiers In Bioscience, Landmark, 24, 1316-1329, June 1, 2019]

Kruppel-like factor 6 regulates Sertoli cell blood-testis barrier

Xiu-Xia Wang1, Yan Zhang1,2, Xiao-Yu Li1, Jian Li1, Ji-Xin Tang1, Yuan-Yuan Li1,3, Shou-Long Deng1, Chuen-Yan Cheng4, Yi-Xun Liu1,2

1State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beichen west Road, Chaoyang District, Beijing 100101, China, 2Changsha Reproductive Medicine Hospital, Changsha, China, 3University of Chinese Academy of Sciences, Beijing 100049, China, 4The Mary M. Wohlford Laboratory for Male Contraceptive Research, Center for Biomedical Research, Population Council, 1230 York Ave, New York 10065, USA


1. Abstract
2. Introduction
3. Materials and methods
3.1. Animals
3.2. Primary mouse SC isolation
3.3. Knockdown of Klf6 by siRNA and shRNA
3.4. TUNEL assay
3.5. RNA extraction and RT-PCR
3.6. Western blot analysis
3.7. Immunofluorescent staining
3.8. Assessment of SC tight junction permeability barrier in vitro
3.9. Electron microscopy
3.10. Luciferase assay
3.11. Statistics
4. Results
4.1. Klf6 is expressed in Sertoli cells of mouse testis
4.2. Klf6 is required for the proliferative activity of cultured SCs
4.3. Klf6 regulates the integrity and permeability of BTB
4.4. Klf6 regulates the expression of BTB proteins in SCs
4.5. Zo-1 and Claudin-3 are direct targets of transcription factor Klf6
5. Discussion
6. Acknowledgement
7. References


Blood-testis barrier (BTB) that is constructed by testicular Sertoli cells (SCs) is essential for spermatogenesis. Krüppel-like factor 6 (Klf6), a nuclear transcription regulator, is reported to be associated with tight junction molecules of BTB between SCs during spermatogenesis; however, the specific regulatory role and mechanism of Klf6 in BTB regulation are still unknown. Here, we primarily confirmed the temporal and spatial expression patterns of Klf6 in mouse testes. Then, Klf6 was silenced in mouse cultured SCs using either Klf6-siRNA or Klf6-shRNA lentivirus. We mainly found that: (i) Klf6 was indispensable for the proliferative activity of mouse SCs; (ii) Klf6 regulated the integrity and permeability of BTB; (iii) Klf6 knockdown led to the significant upregulation of Zo-1, Claudin-11 and Vimentin, and downregulation of Claudin-3. Furthermore, Zo-1 and Claudin-3, participated in the tight junction remolding, were determined as targets of transcription factor Klf6 by luciferase assay. In summary, our findings suggest that Klf6 regulates the BTB assembly and disassembly via mainly targeting Zo-1 and Claudin-3 in mouse SCs.


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Key Words: Kruppel-like factor 6, Klf6, Blood-testis barrier, Sertoli cells, Zo-1; Claudin-3

Send correspondence to: Xiu-Xia Wang, 1State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China, Tel: 86-010-64807038; Fax: 86-010-64807038, E-mail: wangxiuxia@ioz.ac.cn