MAST4 controls cell cycle in spermatogonial stem cells

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.


| INTRODUCTION
Stem cells are characterized by their ability to self-renew and maintain their pool, remaining primarily quiescent in the body. 1 Self-renewal is the process by which stem cells divide to make more stem cells, maintaining the stem cell pool throughout life. This requires cell cycle control and cell cycle has been proposed as a gatekeeper for selfrenewal. 2 Dynamic changes in gene expression as a function of cell cycle progression are regulated by activities of specific cyclins and cyclin-dependent kinases (CDKs). 3 In the intestine, Wnt/β-catenin pathway upregulates nuclear translocation of β-catenin and promotes the expression of Wnt target genes, such as Ccnd1, Myc and Lgr5, which are associated with the self-renewal of intestinal stem cells. 4,5 In pancreatic β cells, cyclin D2 is required for postnatal β cell self-renewal in mice and the overexpression of cyclin D2 increases the self-renewal and cell mass of β cells. 6,7 Furthermore, BMI is required for the self-renewal of adult lung stem cells and multiple homeobox genes, such as cyclin-dependent kinase inhibitor 2b (p15), and 1c (p57), are increased in Bmi1 mutant lung cells. 8,9 Although several studies have reported stem cell self-renewal and maintenance, the molecular regulation about cell cycle progression of stem cells remains unclear.
Spermatogenesis originates in spermatogonial stem cells (SSCs), which are the only stem cells in the male germline that undergo selfrenewal and division. 10 SSCs give rise to undifferentiated spermatogonia, which undergo differentiation and further rounds of division to produce spermatocytes that enter the meiotic process, thereby supporting daily sperm production. 11,12 Spermatogenesis, supported by the self-renewal and differentiation of SSCs, is strictly controlled by a special microenvironment in the seminiferous tubules. 13 Sertoli cells, the only somatic cell type in the tubules, directly interact with SSCs to control their proliferation and differentiation through the secretion of specific factors such as glial cell line-derived neurotrophic factor and ETS variant 5 transcription factor (also known as ERM). [13][14][15][16] Although the foundation for SSC studies has been established based on morphological analyses, [17][18][19] it is vital to investigate relevant core concepts through molecular mechanisms involving cell-cell interactions or intracellular signalling pathways such as cell cycle regulation.
Promyelocytic leukaemia zinc finger (PLZF) protein belongs to the Krüppel-like zinc finger protein family, which is involved in the regulation of diverse cellular processes, including cell proliferation, apoptosis, differentiation and development. 20 PLZF is a negative regulator of cell division during embryogenesis, 21 and it is essential for the osteogenic differentiation of human mesenchymal stem cells during development. Furthermore, PLZF augments SSC self-renewal in the testes and the deletion of Plzf results in infertility. 22 PLZF, a zinc finger transcription factor, controls the expression of lineage-specific target genes, such as p21, p53, Ccna2 and c-Myc, 23-25 thereby instructing stem/progenitor cells to accept certain cell fate programs for self-renewal or differentiation.
Our previous study has shown that microtubule-associated serine/threonine kinase 4 (MAST4) regulates SSC self-renewal via the FGF2/ERM pathway. 26 The mechanism by which MAST4 regulates SSC self-renewal has been investigated in Sertoli cells intensively.

| Sperm count
To count sperm, both cauda epididymides from each mouse were collected, dissected and placed in 2 ml of Hanks' Balanced Salt Solution (HBSS; #14025-092, Life Technologies, USA) for 30 min to allow the release of motile cells (swim-out procedure). The total sperm number from suspension sperm was obtained using a haemocytometer.

| Immunohistochemistry
Samples were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) and then embedded in paraffin using standard procedures.
Sections (4-μm thickness) of the specimens were boiled in 10 mM citrate buffer (pH 6.0) and cooled at room temperature for 20 min. The specimens were incubated with primary antibodies at 4 C overnight.
Primary antibodies are listed in Table S1. The specimens were incubated with Alexa Flour secondary antibodies (Invitrogen, OR, USA; 1:200) for 2 h at room temperature and were counterstained with DAPI (D1306, Invitrogen, OR, USA; 1:1000). The sections were examined using a confocal laser microscope (TCS SP8, Leica, Germany).

| Western blot
Cell extracts from testis tissues were fractionated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane (PVDF; Millipore) using a transfer apparatus according to the manufacturer's protocols.

| Testis tissue culture
Testes from PN 1D WT and Mast4 KO male mice were decapsulated and gently fragmented into several pieces 1-3 mm in diameter.
Explants of testes were cultured as modified from the methods described by Sato et al. 29 Briefly, the tissues were placed in a Trowell- paraformaldehyde or harvested using TRIzol ® reagent.

| Statistical analysis
The graphic results were expressed as the mean ± SD. A GraphPad Prism 7 (GraphPad Software, San Diego, CA, USA) was used to analyse the data. Comparison of two groups was performed using an unpaired two-tailed t-test. Comparison of multiple groups was performed by one-way ANOVA followed by Tukey's multiple comparisons test. The p value < 0.05 was considered significant. arrowheads). PN 21M WT testes showed a phenotype similar to that of KO testes, with wide interstitial spaces and SCO tubules ( Figure 1H arrowheads). In addition, PLZF expression was examined to determine alterations in SSCs in the seminiferous tubules of WT and KO testes.
In PN 6W WT testes, PLZF was sparsely localized in the outermost layer of the seminiferous tubules ( Figure 1I), whereas the expression of PLZF decreased in KO testes ( Figure 1J). In PN 22W testes, PLZF expression increased overall compared to that in PN 6W testes, and KO testes showed decreased expression of PLZF compared to that in WT testes ( Figure 1K,L). Interestingly, in PN 21M WT testes, PLZF expression was lower than that in other KO testes ( Figure 1M). PN 6W mice showed no significant differences between WT and KO testes ( Figure 1N)

| Cell cycle alteration of SSCs in Mast4 KO testes
Since the self-renewal of SSCs was not properly regulated in Mast4  Table S3. To calculate the cell cycle, PLZF-positive cells were counted in one section slide, then IdUpositive/BrdU-negative cells were counted in adjacent serial section slide which co-localized with PLZF ( Figure 2K). IdU/BrdU injection and the formula for cell cycle calculation are described in detail in Figure S1. T c was not significantly different between PN 6W WT and KO SSCs ( Figure 2L); however, it significantly increased in PN 22W KO SSCs compared to that in the corresponding WT SSCs ( Figure 2M). T c of KO SSCs from both stages were not significantly different ( Figure 2N). In PN 21M WT SSCs, the cell cycle was similar to that in PN 22W WT SSCs, although these SSCs had the lowest PLZF expression ( Figure 2O). In addition, T c in PN 21M WT SSCs was shorter than that in the KO SSCs ( Figure 2P,Q). Taken together, T c of SSCs increased not in pubertal stages but in adult stages of Mast4 KO mice, indicating that cell cycle decreased in adult Mast4 KO SSCs.

| Effect of MAST4 on cell cycle in SSCs
To determine how MAST4 regulates cell cycle in SSCs, the molecular mechanism of this protein was investigated. CDK2 is a key regulator of G1-S transition in stem cells by phosphorylating PLZF. 23 An immunoprecipitation assay with Flag-PLZF and HA-CDK2 co-transfected into HEK293T cells confirmed the interaction between PLZF and CDK2 ( Figure 3A). In addition, the expression of CDK2 and PLZF tar-  Figure 3B,C arrowhead). Western blotting of PN 22W WT and KO testes also showed that the expression of CDK2 decreased in KO testes ( Figure 3D). It has been reported that PLZF binds to the promoter region of p21, p53 and Ccna2, and represses their expression. 23,25 In KO testes, the expression of p21 significantly increased, and p53 expression was similar to that in WT testes ( Figure 3D). Furthermore, RT-qPCR data indicated that the expression of p21, p53 and Ccna2 significantly increased in KO testes ( Figure 3E-G). The expression of Ccnd1, which represents cells in the G1 phase and G1-S transition, decreased in KO testes ( Figure 3H). Immunohistochemical analysis of p21 and p53 showed similar results ( Figure 3I-L). Taken together, CDK2 directly interacts with PLZF, and MAST4 regulates the expression of CDK2 and PLZF, and subsequently, the transcriptional level of PLZF target genes.
F I G U R E 4 Legend on next page.
3.4 | FGF2-MAST4-CXCL12 pathway straightens disturbed cell cycle of SSCs in Mast4 KO testes To determine whether the reduced cell cycle progression in SSCs could be rescued by the FGF2-MAST4-CXCL12 pathway, 26 an in vitro tissue culture system was used. Postnatal day 1 (PN 1D) testes of WT and Mast4 KO mice were fragmented and cultured. To induce the MAST4 pathway, exogenous FGF2 and CXCL12, which are upstream and downstream of MAST4, respectively, were added to the culture medium ( Figure 4A). One week after culture, WT testes had wellorganized seminiferous tubular structures, as observed by H&E staining ( Figure 4B). However, KO testes had an irregular spermatogonia structure and a wider interstitial space than WT testes did ( Figure 4C).
Exogenous FGF2 and CXCL12 rescued the enlarged interstitial space in KO testes, similar to that in WT testes ( Figure 4D). Quantification of the interstitial space area indicated that the interstitial space of KO testes significantly increased compared to that of WT testes, and KO testes cultured with FGF2 and CXCL12 (KO + F + C) showed a decreased interstitial space area compared to that of KO testes ( Figure 4E). The expression of SSC-and cell cycle-related proteins was examined using immunohistochemistry. The expression of PLZF and c-Kit, which is a marker of differentiating spermatogonia, decreased to the extent that it was invisible in KO testes and recovered in KO + F + C testes, similar to that in WT testes ( Figure 4F-K).
Subsequently, the expression of PLZF target genes, p21 and p53, was examined. Their expression is rarely observed in spermatogonia ( Figure 4L,O); however, the expression of p21 and p53 significantly increased in KO testes and recovered in KO + F + C testes ( Figure 4M,N,P,Q). Cyclin D1 was expressed in differentiating spermatogonia of WT testes ( Figure 4R), whereas its expression decreased in KO testes. KO + F + C testes expressed cyclin D1, similar to WT testes ( Figure 4T). These results were also supported by the RT-qPCR analyses.
The expression of p21 and Ccna2 increased in KO mice and recovered in KO + F + C testes ( Figure 4U,V). Although p53 expression did not significantly change in KO testes, it decreased in KO + F + C testes compared to that in other groups ( Figure 4W). However, the expression of Ccnd1 was not significant ( Figure 4X). The interaction between PLZF and CDK2 increased after CXCL12 treatment ( Figure 4Y). Taken together, the FGF2-MAST4-CXCL12 pathway is crucial for maintaining cell cycle in SSCs by regulating PLZF and its target gene expression.

| DISCUSSION
Mammalian spermatogenesis is a classic adult stem cell-based process that is regulated by mitosis and meiosis, and is supported by the self- In conclusion, MAST4 is closely related to the maintenance of SSCs by regulating their cell cycle ( Figure 5). In Sertoli cells, MAST4 phosphorylates ERM and subsequently regulates the transcription of Cxcl12, which is the target gene of ERM. CXCL12 migrates to SSCs and transmits signals involved in SSC selfrenewal. PLZF is phosphorylated by CDK2 and suppresses the transcription of p21, p53 and Ccna2. Inhibition of the transcription of PLZF target genes enables SSCs to maintain their cell cycle.
Taken together, this study provides new insights into a novel mechanism by which MAST4 regulates CDK2 and PLZF involved in SSC self-renewal. Moreover, our findings not only suggest the significance of apprehending spermatogenesis but also reflect its potential therapeutic usage in spermatogenic dysregulation.

AUTHOR CONTRIBUTIONS
Seung-Jun Lee contributed to design, data acquisition, analysis, inter-