Skp1-Cullin-F-box (SCF)-type ubiquitin ligase FBXW7 negatively regulates spermatogonial stem cell self-renewal

Abstract

Spermatogonial stem cells (SSCs) undergo self-renewal divisions to support spermatogenesis throughout life. Although several positive regulators of SSC self-renewal have been discovered, little is known about the negative regulators. Here, we report that F-box and WD-40 domain protein 7 (FBXW7), a component of the Skp1-Cullin-F-box-type ubiquitin ligase, is a negative regulator of SSC self-renewal. FBXW7 is expressed in undifferentiated spermatogonia in a cell cycle-dependent manner. Although peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 (PIN1), essential for spermatogenesis, is thought to destroy FBXW7, Pin1 depletion decreased FBXW7 expression. Spermatogonial transplantation showed that Fbxw7 overexpression compromised SSC activity whereas Fbxw7 deficiency enhanced SSC colonization and caused accumulation of undifferentiated spermatogonia, suggesting that the level of FBXW7 is critical for self-renewal and differentiation. Screening of putative FBXW7 targets revealed that Fbxw7 deficiency up-regulated myelocytomatosis oncogene (MYC) and cyclin E1 (CCNE1). Although depletion of Myc/Mycn or Ccne1/Ccne2 compromised SSC activity, overexpression of Myc, but not Ccne1, increased colonization of SSCs. These results suggest that FBXW7 regulates SSC self-renewal in a negative manner by degradation of MYC.

Fig. 1.

Expression of FBXW7 in spermatogonia. (A–C) Expression of FBXW7 protein in CDH1⁺ (A), EPCAM⁺ (B), or KIT⁺ (C) cells during postnatal testis development. At least 12, 15, and 14 tubules were counted in 1-, 10-, and 42-d-old testes, respectively. (D) Triple immunohistochemistry of FBXW7, MKI67, and CDH1 in 42-d-old testes. At least 17 tubules were counted. Arrows indicate CDH1⁺MKI67⁻ cells with FBXW7 expression. Arrowheads indicate CDH1⁺MKI67⁺ cells without FBXW7 expression. (E) Proportion of CDH1⁺ cells with FBXW7 expression. (F) Real-time PCR analysis of Fbxw7 expression following depletion of indicated genes by shRNA (n = 6). (G) Western blot analysis of FBXW7 expression following Pin1 depletion. (Scale bar: D, 20 μm.)

Fig. 2.

Decreased SSC activity with Fbxw7 overexpression. (A) RT-PCR analysis of Fbxw7 isoform expression in GS cells and mouse embryonic fibroblasts (MEFs). (B) Colony counts after Fbxw7α overexpression and transplantation of green mouse testis cells. Results of two experiments (n = 12). (C) Western blot analysis of Fbxw7α expression in GS cells transduced with Fbxw7α. Cells were recovered 4 d after transfection. (D) Flow cytometric analysis of GS cells transduced with Fbxw7α. Cells were analyzed at the indicated time points. Green lines indicate GS cells without transfection. (E) Proliferation of GS cells following Fbxw7α overexpression.

Fig. 3.

Impaired spermatogenesis in Fbxw7^f/f Stra8-Cre mice. (A) Testis weight of 2-mo-old Fbxw7^f/f Stra8-Cre mice (n = 8, control; n = 6, Fbxw7^f/f Stra8-Cre). (B) Histological appearance of Fbxw7^f/f Stra8-Cre testis. (C) Tubules with spermatogenesis, defined as the presence of multiple layers of germ cells in the entire circumference of the tubules. At least 207 tubules were counted. (D) Expression of FBXW7 in CDH1⁺ cells. At least 20 tubules were counted. (E) Expression of SYCP3 in KIT⁺ cells. At least 22 tubules were counted. (F) Ratio of CDH1⁺ spermatogonia and GATA4⁺ Sertoli cells. At least 34 tubules were counted. (G) Ratio of KIT⁺ spermatogonia and GATA4⁺ Sertoli cells. At least 8 tubules were counted. (H) The number of TUNEL⁺ cells in seminiferous tubules. At least 11 tubules were counted. (I) The number of KIT⁺ cells undergoing apoptosis. At least 8 tubules were counted. (J) The number of EPCAM⁺ cells undergoing apoptosis. At least 10 tubules were counted. (K) Expression of MKI67 in CDH1⁺ cells. Eighteen tubules were counted. (L) Expression of MKI67 protein in KIT⁺ cells. Twenty tubules were counted. (Scale bar: B, 50 μm.) Stain, hematoxylin/eosin (B).

Fig. 4.

Enhanced SSC activity in Fbxw7 KO testis cells. (A) Experimental procedure. Testis cells from Fbxw7^f/f mice were dissociated and incubated with AxCANCre overnight and then injected into the seminiferous tubules of W testes. (B) Macroscopic appearance of recipient testis. Histological appearance (Inset). (C) Colony counts after Cre transfection and transplantation of Fbxw7^f/f mouse testes. Results of three experiments (n = 18). (D) RT-PCR analysis of spermatogenic gene expression in recipient testes. (E) Colony counts after Pin1 depletion and transplantation of green mouse testes. Results of two experiments (n = 16). (Scale bars: B, 1 mm; B, Inset, 50 μm.)

Fig. 5.

Characterization of Fbxw7 KO GS cells for target identification. (A) Enhanced proliferation of GS cells after Cre transfection. After overnight incubation with AxCANCre, virus supernatant was removed, and cells were replated in a new dish. Cell number was determined 3 d after replating. AxCANLacZ was used as a control (n = 4). Results of two experiments. (B) Quantification of GS cells expressing MKI67. At least 732 cells in four different fields were counted. (C) Western blot analysis of molecules involved in proliferation of GS cells. (D) Western blot analysis of FBXW7 substrates. (E) Expression of MYC in CDH1⁺ cells in Fbxw7^f/f Stra8-Cre testes. Fifteen tubules were counted. (F) Expression of CCNE1 in CDH1⁺ cells in Fbxw7^f/f Stra8-Cre testes. Fifteen tubules were counted. (G) Expression of CDK4 in CDH1 cells in Fbxw7^f/f Stra8-Cre testes. At least 8 tubules were counted. (H) Experimental procedure. Immature testis cells from Fbxw7^f/f mice were infected with lentiviruses expressing shRNA against Myc/Mycn or Ccne1/Ccne2. Culture medium was changed on the next day after infection. Two days after shRNA infection, cells were infected with AxCANCre and transplanted 24 h after AxCANCre infection. (I) Colony counts after depleting the indicated genes (n = 8). Results of two experiments. (J) Colony counts after overexpression of indicated genes and transplantation of green mouse testis cells. Results of two experiments (n = 12). (K) Summary of experiments. Loss of FBXW7 increases self-renewal by up-regulation of MYC and suppresses differentiation.