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Targeted disruption of galectin 3 in mice delays the first wave of spermatogenesis and increases germ cell apoptosis

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Abstract

Galectin 3 is a multifunctional lectin implicated in cellular proliferation, differentiation, adhesion, and apoptosis. This lectin is broadly expressed in testicular somatic cells and germ cells, and is upregulated during testicular development. Since the role of galectin 3 in testicular function remains elusive, we aimed to characterize the role of galectin 3 in testicular physiology. We found that galectin 3 transgenic mice (Lgals3−/−) exhibited significantly decreased testicular weight in adulthood compared to controls. The transgenic mice also exhibited a delay to the first wave of spermatogenesis, a decrease in the number of germ cells at postnatal day 5 (P5) and P15, and defective Sertoli cell maturation. Mechanistically, we found that Insulin-like-3 (a Leydig cell marker) and enzymes involved in steroid biosynthesis were significantly upregulated in adult Lgals3−/− testes. These observations were accompanied by increased serum testosterone levels. To determine the underlying causes of the testicular atrophy, we monitored cellular apoptosis. Indeed, adult Lgals3−/− testicular cells exhibited an elevated apoptosis rate that is likely driven by downregulated Bcl-2 and upregulated Bax and Bak expression, molecules responsible for live/death cell balance. Moreover, the percentage of testicular macrophages within CD45+ cells was decreased in Lgals3−/− mice. These data suggest that galectin 3 regulates spermatogenesis initiation and Sertoli cell maturation in part, by preventing germ cells from undergoing apoptosis and regulating testosterone biosynthesis. Going forward, understanding the role of galectin 3 in testicular physiology will add important insights into the factors governing the development of germ cells and steroidogenesis and delineate novel biomarkers of testicular function.

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Data availability

The datasets generated during and analysed during the current study are available from the corresponding author on reasonable request.

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Acknowledgements

The authors would like to thank Prof. Ralf Middendorff and Sabine Tasch for providing the PCNA antibody and acknowledge Prof. Thomas Linn and Dr. Qingkui Jiang for offering the MLTC-1 cell line. We are grateful to Yahia Almousa for assistance with the fixation protocol and tissue collection. We appreciate the support from our research assistants Petra Moschansky, Maria Daniltchenko and Gudrun Koch.

Funding

The financial support of the China Scholarship Council to Tao Lei and of the Medical Faculty of Justus-Liebig University is gratefully acknowledged. This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG) BL1115/2-1 and Heisenberg Program (BL1115/3-1-BL1115/7-1) to S.M.B.

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  1. Tao Lei

    Present address: Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China

Authors and Affiliations

  1. Department of Anatomy and Cell Biology, Justus-Liebig-University of Giessen, Aulweg 123, 35385, Giessen, Germany

    Tao Lei, Sudhanshu Bhushan, Eva Wahle, Andreas Meinhardt & Monika Fijak

  2. Department of Obstetrics and Fetal Medicine, AG Glycoimmunology, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20251, Hamburg, Germany

    Sandra M. Blois & Nancy Freitag

  3. Experimental and Clinical Research Center, A Cooperation between the Max Delbrück Center for Molecular Medicine in the Helmholtz Association, The Charité Universitätsmedizin Berlin, Berlin, Germany

    Sandra M. Blois & Nancy Freitag

  4. Division of General Internal and Psychosomatic Medicine, Berlin Institute of Health, Charité Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt Universität zu Berlin, Berlin, Germany

    Nancy Freitag

  5. Institute of Veterinary Anatomy, Histology, and Embryology, Justus-Liebig-University of Giessen, Giessen, Germany

    Martin Bergmann

  6. Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan

    Annie Chi-Chun Huang, Hung-Lin Chen & Fu-Tong Liu

  7. Steroid Research and Mass Spectrometry Unit, Pediatric Endocrinology and Diabetology, Center of Child and Adolescent Medicine, Justus-Liebig-University of Giessen, Giessen, Germany

    Michaela F. Hartmann & Stefan A. Wudy

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All procedures involving animals were carried out in strict accordance with guidelines for care and use of experimental animals of the German law of welfare. Organ collection from mice was approved by the local Animal Ethics Committee (Regierungspräsidium Giessen GI Nr. 646_M).

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18_2021_3757_MOESM1_ESM.pdf

Supplementary file1 (PDF 669 KB) Supplementary Figure 1. (A-F) Representative PCNA staining in testicular sections from Lgals3+/+ (A, C, E) and Lgals3-/- (B, D, F) mice at P5 (A, B), P15 (C, D) and P20 (E, F). The stars indicate the tubules without germ cells. Scale bars represent 80 µm.

18_2021_3757_MOESM2_ESM.pdf

Supplementary file2 (PDF 186 KB) Supplementary Figure 2. (A-B) Immunofluorescence staining for Sox9 (green; Sertoli cell marker) and Tra98 (red; germ cell marker) in testicular sections from Lgals3+/+ (A) and Lgals3-/- mice at P15 (B). (C) Relative expression of Wt1 mRNA was normalized to Actb and Gapdh at P15; (n = 3). The data represent the means ± SEM.

18_2021_3757_MOESM3_ESM.pdf

Supplementary file3 (PDF 956 KB) Supplementary Figure 3. Representative 11β - hydroxysteroid dehydrogenase (HSD11B1) DAB staining in testicular sections from Lgals3+/+ and Lgals3-/- mice at P50. The sections are counterstained with hematoxylin-eosin; (magnification x 100).

18_2021_3757_MOESM4_ESM.pdf

Supplementary file4 (PDF 368 KB) Supplementary Figure 4. Lgals3 siRNA knock down efficiency in MLTC-1 cells. Representative Western blot of galectin 3 protein levels in MLTC-1 cells 24, 48 and 72 h after transfection with Lgals3 siRNA or control siRNA or left untreated. β-actin was used as a loading control.

18_2021_3757_MOESM5_ESM.pdf

Supplementary file5 (PDF 145 KB) Supplementary Figure 5. The gating strategy used to analyse testicular macrophages by flow cytometry. (A-C) Detected cells were displayed by using forward scatter (FSC) and side scatter (SSC) (A). Doublets (B), cell debris, and non-viable cells (C) were gated out. (D-F) Testicular macrophages were defined as F4/80+CD11b+ cells in the Ly6C- fraction of CD45+ leukocytes.

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Lei, T., Blois, S.M., Freitag, N. et al. Targeted disruption of galectin 3 in mice delays the first wave of spermatogenesis and increases germ cell apoptosis. Cell. Mol. Life Sci. 78, 3621–3635 (2021). https://doi.org/10.1007/s00018-021-03757-2

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