Transcription Factor GLIS3: A New and Critical Regulator of Postnatal Stages of Mouse Spermatogenesis

Abstract

In this study, we identify a novel and essential role for the Krüppel-like zinc finger transcription factor GLI-similar 3 (GLIS3) in the regulation of postnatal spermatogenesis. We show that GLIS3 is expressed in gonocytes, spermatogonial stem cells (SSCs) and spermatogonial progenitors (SPCs), but not in differentiated spermatogonia and later stages of spermatogenesis or in somatic cells. Spermatogenesis is greatly impaired in GLIS3 knockout mice. Loss of GLIS3 function causes a moderate reduction in the number of gonocytes, but greatly affects the generation of SSCs/SPCs, and as a consequence the development of spermatocytes. Gene expression profiling demonstrated that the expression of genes associated with undifferentiated spermatogonia was dramatically decreased in GLIS3-deficient mice and that the cytoplasmic-to-nuclear translocation of FOXO1, which marks the gonocyte-to-SSC transition and is necessary for SSC self-renewal, is inhibited. These observations suggest that GLIS3 promotes the gonocyte-to-SSC transition and is a critical regulator of the dynamics of early postnatal spermatogenesis. Stem Cells 2016;34:2772-2783.

Keywords: GLIS3; Gene expression; Spermatogenesis; Spermatogonial stem cell.

Figure 1

GLIS3 is expressed in gonocytes and undifferentiated spermatogonial cells. Glis3-EGFP knock-in mouse, in which GLIS3 is expressed as a fusion protein with EGFP, was generated to analyze the pattern of GLIS3-EGFP expression. Sections of PND1 (A), PND5 (B), and PND7 (C) testes were examined by immunohistochemistry with antibodies against GLIS3-EGFP (green) and the pan germ cell marker TRA98 (Red). Nuclei were stained with Dapi. Merged images are indicated. (D): E-cadherin positive (ECAD⁺; red) undifferentiated spermatogonia stained Glis3-EGFP⁺. (E, F): Images of sections of PND7 (E) and PND14 (F) testes immunostained with antibodies against GLIS3-EGFP and ZBTB16 (PLZF) (red). Arrows indicate ZBTB16 and GLIS3-EGFP double positive cells; arrowheads indicate PLZF16 single positive cells. (G): Percentage of ZBTB16⁺ cells in seminiferous tubules of PND4, 7, 14 testes that were GLIS3-EGFP⁺. (H, I): Representative images of sections of PND5 or PND7 testes immunostained with antibodies against GLIS3-EGFP and GFRA1 (red). Arrows in (I) indicate GFRA1 and GLIS3-EGFP double positive cells; arrowheads indicate GLIS3-EGFP single positive cells. (J): Percentage of GLIS3-EGFP⁺ cells in seminiferous tubules of PND4, 7, 14 testes that were GFRA1⁺. Bars indicate 50 lm. Abbreviation: GLIS3, GLI-similar 3.

Figure 2

GLIS3 is not expressed in differentiated spermatogonia. (A–D): Representative images of a section of PND14 testes stained with antibodies against GLIS3-EGFP and KIT (red; B). Nuclei were stained with Dapi. Merged images are indicated. Bar indicates 50 μm. (E): Cells isolated from PND7 Glis3-EGFP testis, were sorted by Flow cytometry to isolate EGFP- and KIT enriched cell populations as described in M&M. These subpopulations were subsequently examined for Kit, Zbtb16, Nanos3, and Glis3 mRNA expression by QRT-PCR. Data present mean ± SEM. (F): Proposed scheme of GLIS3 expression during early postnatal spermatogenesis. GLIS3 expression was compared to that of ZBTB16, FOXO1, GFRA1, and KIT. GLIS3 is expressed in gonocytes and most undifferentiated spermatogonia. It is repressed during the A_al stage after the downregulation of GFRA1, but before the downregulation of ZBTB16. G, gonocytes; A_s, spermatogonia cell A-single; A_pr, A-paired; A_al, A-aligned; A1, type A1 spermatogonia. Abbreviations: GLIS3, GLI-similar 3; QRT-PCR, Quantitative real time PCR.

Figure 3

GLIS3 is required for normal spermatogenesis. (A, B): Relative weights (A) and size (B) of testes from WT and Glis3KO2 mice (n ≥ 3). Images of whole testes from 4 weeks-old WT and Glis3KO2 mice are shown in (B). Weight and size of Glis3KO2 testes are greatly reduced. Data present mean ± SEM. * p <.05. (C): Representative images of H&E stained sections of testes from 7-weeks old WT, heterozygous, and Glis3KO2 mice showing that spermatogenesis is greatly impaired in Glis3KO2 mice.

Figure 4

The number of undifferentiated spermatogonial cells is dramatically reduced in Glis3KO2 testis. (A): Representative images of sections of PND1 WT and Glis3KO2 testes stained with antibodies against TRA98 (red). Nuclei were stained with Dapi. Merged images are indicated. (B): Representative images of sections of PND7 WT and Glis3KO2 testes immunostained with antibodies against ZBTB16 (red) and Dapi. (C): The number of TRA98⁺ germ cells in seminiferous tubules of Glis3KO2 testes (n ≥ 4) was greatly decreased during early postnatal spermatogenesis. (D, E) Representative images of sections of testes from 2-weeks old WT and Glis3KO2 mice stained with antibodies against KIT (red; A) and HSPA2 (red; B). Nuclei were stained with Dapi. Bars indicate 50 μm. Data present mean ± SEM. * p <.05.

Figure 5

GLIS3 deficiency causes dramatic changes in gene expression in testes from PND7 and 4 weeks old mice. (A, B): Heatmaps generated from gene expression profiles obtained by microarray analyses of testes from PND7 (A) and 4 week-old (B) WT and Glis3KO2 mice. Several groups of genes associated with different cell types are indicated. (C): The expression of several genes associated with the regulation of early spermatogenesis, in testis from PND7 WT and Glis3KO2 mice (n = 3) were compared by Quantitative real time PCR analysis. Data present mean ± SEM. * p <.05.

Figure 6

GLIS3 deficiency reduces the generation of GFRA1⁺ cells. (A, C): Representative images of sections of PND4 and PND7 WT and Glis3KO2 testes stained with antibodies against GFRA1 (red) and ZBTB16 (Plzf, green) and Dapi. (B, D): The percentage of ZBTB16⁺ cells staining positively for GFRA1 in PND4 and PND7 WT and Glis3KO2 testes (n = 4) was calculated and plotted. Bars indicate 50 μm. Data present mean 6SEM. * p <.05.

Figure 7

GLIS3 deficiency impairs the subcellular localization of Foxo1. (A): Representative images of sections of PND7 WT and Glis3KO2 testes stained with antibodies against FOXO1 (green) and TRA98 (red), and Dapi. (B): The subcellular localization of FOXO1 in TRA98⁺ cells was analyzed and the number of cells expressing FOXO1 largely in the cytoplasm (C) or largely in the nucleus or about equally in both (C + N) was counted and plotted. Bars indicate 50 μm.