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MAST4 controls cell cycle in spermatogonial stem cells - PubMed

MAST4 controls cell cycle in spermatogonial stem cells

Seung-Jun Lee et al. Cell Prolif. 2023 Apr.

Abstract

Spermatogonial stem cell (SSC) self-renewal is regulated by reciprocal interactions between Sertoli cells and SSCs in the testis. In a previous study, microtubule-associated serine/threonine kinase 4 (MAST4) has been studied in Sertoli cells as a regulator of SSC self-renewal. The present study focused on the mechanism by which MAST4 in Sertoli cells transmits the signal and regulates SSCs, especially cell cycle regulation. The expression of PLZF, CDK2 and PLZF target genes was examined in WT and Mast4 KO testes by Immunohistochemistry, RT-qPCR and western blot. In addition, IdU and BrdU were injected into WT and Mast4 KO mice and cell cycle of SSCs was analysed. Finally, the testis tissues were cultured in vitro to examine the regulation of cell cycle by MAST4 pathway. Mast4 KO mice showed infertility with Sertoli cell-only syndrome and reduced sperm count. Furthermore, Mast4 deletion led to decreased PLZF expression and cell cycle progression in the testes. MAST4 also induced cyclin-dependent kinase 2 (CDK2) to phosphorylate PLZF and activated PLZF suppressed the transcriptional levels of genes related to cell cycle arrest, leading SSCs to remain stem cell state. MAST4 is essential for maintaining cell cycle in SSCs via the CDK2-PLZF interaction. These results demonstrate the pivotal role of MAST4 regulating cell cycle of SSCs and the significance of spermatogenesis.

© 2023 The Authors. Cell Proliferation published by Beijing Institute for Stem Cell and Regenerative Medicine and John Wiley & Sons Ltd.

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Conflict of interest statement

The authors declare no competing interest.

Figures

FIGURE 1
FIGURE 1

The dysfunction of spermatogenesis and decreased PLZF expression in Mast4 KO testes. (A) Life history stages in C57BL/6 mice in comparison to those in human. (B) Mast4 KO testes reduced in size compared to WT testes in both stages. The testes of PN 21M WT mice are larger than other Mast4 KO testes. (C) The sperm count in Mast4 KO testes decreased by a quarter compared to that in the WT. In PN 21M WT testes, sperm count is more than double of that in the KO testes. (D–H) HE staining of testes in WT and Mast4 KO mice. WT testes from (D) PN 6W and (F) PN 22W mice have well‐organized seminiferous tubules. All types of germ cells are observed in the seminiferous tubules. KO testes from (E) PN 6W and (G) PN 22W mice have SCO tubules. Germ cells are depleted and only Sertoli cells are observed (arrowheads). (H) PN 21M mice testes have SCO tubules similar to those in Mast4 KO testes (arrowheads). (I–M) Immunohistochemistry of PLZF in WT and Mast4 KO testes. PLZF is localized in spermatogonial stem cells in the outermost layer of the seminiferous tubules. Compared to (I) PN 6W and (K) PN 22W WT testes, PLZF expression in (J) PN 6W and (L) PN 22W KO testes is decreased, respectively. (M) In PN 21M WT testes, PLZF expression is significantly decreased. (N) Quantification of PLZF expression in WT and Mast4 KO testes (n = 15, five images per group of three mice). (O) Western blot of PLZF in WT and Mast4 KO testes. (P, Q) RT‐qPCR of Plzf in WT and Mast4 KO testes. (N–Q) Expression of PLZF is significantly decreased in PN 22W KO mice but not in PN 6W KO mice. Scale bars; B, 2 mm, D–M, 100 μm, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns; no significance

FIGURE 2
FIGURE 2

Analyses of T c in SSCs between WT and Mast4 KO testes. (A–J) Immunohistochemistry of IdU/BrdU and PLZF for cell cycle calculation in SSCs in WT and Mast4 KO testes. T c was calculated from the co‐localization of IdU‐only labelled and PLZF‐labelled cells by serial sectioning. (K) Schematic diagram of cell cycle calculation in IdU/BrdU staining with PLZF‐expressing SSCs. For immunohistochemistry of PLZF and IdU/BrdU by serial sectioning after IdU/BrdU injection, (K‐i) PLZF‐positive cells were counted per unit area (Pcells, arrowhead). (K‐ii) In adjacent serial sectioning, IdU‐only labelled cells were counted under co‐localization with PLZF (Lcells, arrowhead). T c was calculated using the formula in Figure S1 for cell cycle. (L–Q) Statistical analysis T c at each stage (n = 15). (L) T c in PN 6W KO SSCs is similar to that in WT SSCs. (M) T c dramatically increases in PN 22W KO SSCs compared to that in WT SSCs. (N) T c differences are not shown between PN 6W and PN 22W KO SSCs. (O) Among WT groups, T c in PN 6W SSCs is longer than that in other SSCs. PN 22W and PN 21M SSCs have similar T c values. (P, Q) T c in PN 21M WT SSCs is shorter than that in (P) PN 6W and (Q) PN 22W KO SSCs. Scale bars; 100 μm, **p < 0.01, ****p < 0.0001, ns; no significance

FIGURE 3
FIGURE 3

Effect of MAST4 on CDK2‐PLZF mechanisms. (A) Interaction between PLZF and CDK2 was examined. Flag‐PLZF and HA‐CDK2 were transiently co‐transfected into HEK293T cells, followed by HA pull‐down. Note the interaction between PLZF and CDK2. (B) In PN 22W WT testes, CDK2 is expressed in the cytoplasm of spermatogonia located in the outermost layer of seminiferous tubules. (C) In PN 22W Mast4 KO testes, CDK2 expression significantly decreases compared to that in WT testes (arrowhead). (D) Western blot analysis of PN 22W WT and Mast4 KO testes indicates that CDK2 expression decreases in PN 22W Mast4 KO testes. The expression of p21 increases in PN 22W Mast4 KO testes, while p53 levels does not significantly change. (E–H) RT‐qPCR analyses of PN 22W WT and Mast4 KO testes indicate that the expression of (E) p21, (F) p53 and (G) Ccna2 significantly increases in PN 22W Mast4 KO testes. (H) Ccnd1 expression decreases in PN 22W Mast4 KO testes. (I) In PN 22W WT testes, p21 is rarely expressed in seminiferous tubules. (J) In PN 22W Mast4 KO testes, p21 expression significantly increases compared to that in WT testes. (K) In PN 22W WT testes, p53 is rarely expressed in seminiferous tubules. (L) In PN 22W Mast4 KO testes, p53 expression slightly increases compared to that in WT testes. Scale bars; 100 μm, *p < 0.05, **p < 0.01

FIGURE 4
FIGURE 4

FGF2‐MAST4‐CXCL12 pathway modulates cell cycle in SSCs. (A) Schematic diagram for testes tissue culture procedure. (B–D) HE staining of PN 1D testes cultured for 1 week in vitro. (B) WT testes show well‐organized seminiferous tubules. (C) Mast4 KO testes show irregular tubular structure and wider interstitial space compared to those in WT testes. (D) KO + F + C testes have similar seminiferous tubular structure compared to that in WT testes. (E) Quantitative analysis of interstitial space in testes tissue culture. KO testes show significantly widened area compared to that in WT testes. KO + F + C testes have decreased interstitial space. (F–T) Immunohistochemistry analyses of PN 1D testes cultured for 1 week. (F) In WT testes, PLZF is sparsely localized in the outermost layer of seminiferous tubules. (G) PLZF expression is not observed in KO testes. (H) In KO + F + C testes, PLZF expression is similar to that in WT testes. (I) In WT testes, c‐Kit localizes partially in the outermost layer of seminiferous tubules. (J) KO testes do not express c‐Kit. (K) Expression of c‐Kit is observed in KO + F + C testes. (L) p21 is expressed sparsely in the spermatogonia of seminiferous tubules in WT testes. (M) p21 expression dramatically increases in KO testes. (N) The expression of p21 in KO + F + C testes is similar to that in WT testes. (O) p53 is rarely expressed in seminiferous tubules in WT testes. (P) p53 expression significantly increases in KO testes. (Q) In KO + F + C testes, p53 expression is similar to that in WT testes. (R) Cyclin D1 is partially expressed in spermatogonia of WT testes. (S) Although the expression of cyclin D1 decreases in KO testes, (T) its expression in KO + F + C testes is similar to that in WT testes. (U) p21 expression increases in KO testes and decreases in KO + F + C testes, similar to that in WT testes (n = 3). (V) Ccna2 expression increases in KO testes and decreases in KO + F + C testes, similar to that in WT testes (n = 3). (W) While there is no significant difference in p53 expression between WT and KO testes, p53 expression significantly decreases in KO + F + C testes compared to that in the other groups (n = 3). (X) Ccnd1 expression is not significant among all groups (n = 3). (Y) The effect of CXCL12 on PLZF and CDK2 interaction was examined. Flag‐PLZF and HA‐CDK2 were transiently co‐transfected into HEK293T cells, followed by CXCL12 treatment (100 ng/ml) for 24 h. CXCL12 increases the interaction between PLZF and CDK2. F; FGF2, C; CXCL12, WCL; Whole cell lysates. Scale bars; 100 μm. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns; no significance

FIGURE 5
FIGURE 5

Maintenance of cell cycle in SSCs through MAST4‐PLZF mechanism. MAST4 phosphorylates ERM and subsequently regulates the transcription of Cxcl12, which is the target gene of ERM in Sertoli cells. CXCL12 migrates to SSCs and transmits signals involved in SSC self‐renewal. PLZF is phosphorylated by CDK2 and suppresses the transcription of p21, p53 and Ccna2. Inhibition of Ccna2 transcription enables SSCs to maintain their cell cycle. Mast4 KO decreases the transcription of Cxcl12 and interaction between PLZF and CDK2. Inhibition of PLZF could not suppress the transcription of its target genes, leading to cell cycle arrest in SSCs.

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