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LncRNA Tug1 maintains blood–testis barrier integrity by modulating Ccl2 expression in high-fat diet mice

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Abstract

Sertoli cells are essential for spermatogenesis in the testicular seminiferous tubules by forming blood–testis barrier (BTB) and creating a unique microenvironment for spermatogenesis. Many lncRNAs have been reported to participate in spermatogenesis. However, the role of long noncoding RNAs (lncRNAs) in Sertoli cells has rarely been examined. Herein, we found that a high-fat diet (HFD) decreased sperm quality, impaired BTB integrity and resulted in accumulation of saturated fatty acids (SFAs), especially palmitic acid (PA), in mouse testes. PA decreased the expression of tight junction (TJ)-related proteins, increased permeability and decreased transepithelial electrical resistance (TER) in primary Sertoli cells and TM4 cells. Moreover, lncRNA Tug1 was found to be involved in PA-induced BTB disruption by RNA-seq. Tug1 depletion distinctly impaired the TJs of Sertoli cells and overexpression of Tug1 alleviated the disruption of BTB integrity induced by PA. Moreover, Ccl2 was found to be a downstream target of Tug1, and decreased TJ-related protein levels and TER and increased FITC–dextran permeability in vitro. Furthermore, the addition of Ccl2 damaged BTB integrity after overexpression of Tug1 in the presence of PA. Mechanistically, we found that Tug1 could directly bind to EZH2 and regulate H3K27me3 occupancy in the Ccl2 promoter region by RNA immunoprecipitation and chromatin immunoprecipitation assays. Our study revealed an important role of Tug1 in the BTB integrity of Sertoli cells and provided a new view of the role of lncRNAs in male infertility.

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All data generated or analysis during this study are available from the corresponding author upon reasonable request.

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Acknowledgements

We thank the other members of Dr. Yao’s laboratory for their discussion and help.

Funding

This work was supported by the National Key Research and Development Program of China (grant no. 2018YFC1004700), the National Natural Science Foundation of China (grant no. 81971373, 82001618, 81901547), the Natural Science Foundation of Jiangsu Province (BK20190252), and the 333 High-level Personnel Training Project of Jiangsu Province (grant no. BRA2019109).

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Author notes

  1. Shuxian Wang and Zhang Qian contributed equally to this work.

Authors and Affiliations

  1. Center of Reproductive Medicine, Nanjing Jinling Hospital, Clinical School of Medical College, Nanjing University, Nanjing, 210002, Jiangsu, China

    Shuxian Wang, Zhang Qian, Xie Ge, Chuwei Li, Mengqi Xue, Rujun Ma, Lei Ouyang, Lu Zheng, Jun Jing, Siyuan Cao, Yu Zhang, Jinzhao Ma & Bing Yao

  2. Center of Reproductive Medicine, Nanjing Jinling Hospital, The First School of Clinical Medicine, Southern Medical University, Nanjing, 210002, Jiangsu, China

    Kuan Liang & Bing Yao

  3. Basic Medical Laboratory, Nanjing Jinling Hospital, Clinical School of Medical College, Nanjing University, Nanjing, 210002, Jiangsu, China

    Yang Yang

  4. Immunology and Reproduction Biology Laboratory and State Key Laboratory of Analytical Chemistry for Life Science, Medical School, Nanjing University, Nanjing, 210093, China

    Yabing Chen

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  1. Shuxian Wang
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Contributions

S.W., Z.Q., C.L., L.Z. and K.L. carried out the experiments. R.M., X.G. and J.J. were involved in planning and supervising the work. M.X., L.O. and Y.Z. helped with the animal experiments. S.C., Y.C. and Y.Y. analyzed the data and designed the figures. S.W and Z.Q. wrote the manuscript with support from J.M. and B.Y.

Corresponding authors

Correspondence to Jinzhao Ma or Bing Yao.

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The authors declare that they have no competing interests.

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All animal experiments were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals, and all protocols were approved by the Ethics Committee of Nanjing Jinling Hospital.

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18_2022_4142_MOESM1_ESM.tif

Supplementary file1 Fig. S1 HFD impaired BTB integrity (a) The simple schematic diagram of animal experiment design in this study. (b-c) The immunofluorescence staining of BTB-related genes in the CD and HFD group. Scale bar = 50 μm. * p < 0.05 (TIF 2902 KB)

Supplementary file2 Fig. S2 The immunofluorescence staining of FSHR in primary Sertoli cells (TIF 1083 KB)

18_2022_4142_MOESM3_ESM.tif

Supplementary file3 Fig. S3 FSH supplementation did not rescue the sperm quality and BTB integrity in HFD group (n>5) (a) FSH level in control group, HFD group with or without FSH supplementation. (b-c) Sperm concentration and motility of control group, HFD group with or without FSH supplementation. (d) FITC tracing assay of control group, HFD group with or without FSH supplementation. FITC green fluorescence was seen in the lumen of HFD group and HFD group with FSH supplementation. * p < 0.05 (TIF 1011 KB)

18_2022_4142_MOESM4_ESM.tif

Supplementary file4 Fig. S4 GO and KEGG analysis of different expression genes of the testes of tug1-/- and wild type (WT) mice (a) GO terms of different expression genes of the testes of tug1-/- and WT mice. (b) The top 20 KEGG pathways of differentially expression genes. The right panel showed genes involved in tight junction pathway. (TIF 4240 KB)

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Wang, S., Qian, Z., Ge, X. et al. LncRNA Tug1 maintains blood–testis barrier integrity by modulating Ccl2 expression in high-fat diet mice. Cell. Mol. Life Sci. 79, 114 (2022). https://doi.org/10.1007/s00018-022-04142-3

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