Fig 1.
Transmission electron microscopy of wild-type and Stra8-deficient testes.
(A, B) Testicular cross sections from wild-type (A) and Stra8-deficient (B) mice at 21 d.p.p.. (Aa, Ba) Hematoxylin-eosin-staining of testicular cross section from wild-type (Aa) and Stra8-deficient (Ba) mice. (Ab, Bb) Transmission electron microscopy of testicular cross section from wild-type (Ab) and Stra8-deficient (Bb) mice. Left of the red dashed line is the spermatogonia compartment (Spg). Right of the dashed line is the spermatocyte compartment (Spc). (Ac-e, Bc-e) Detailed views of spermatogonia (Ac, Bc), spermatocyte (Ad, Bd), and Sertoli cells (Ae, Be) in wild-type (Ac-e) and Stra8-deficient (Bc-e) testes. (Bd) A detailed view of the luminal compartment in Stra8-deficient testes. (Bf-j) Autophagosomes identified in Stra8-deficient testes (arrows).
Fig 2.
In vivo RFP-GFP-LC3 reporter in wild-type and Stra8-deficient testes.
(A) Whole-mount seminiferous tubules from RFP-GFP-LC3 transgenic mouse testes in juvenile wild-type and Stra8-deficient backgrounds by confocal imaging (upper panels). Quantification of vesicle numbers per imaging area relative to wild-type samples is shown. Total, RFP-positive vesicles. AL (Autolysosome), RFP-positive GFP-negative vesicles. All values are means ± SD. n = 3 mice per genotype. *P < 0.05 (Student’s t test). (B) Testicular cross sections of RFP-GFP-LC3 transgenic mouse testes in juvenile wild-type and Stra8-deficient backgrounds. Dashed lines indicate seminiferous tubules. Arrows indicate autophagosome. Arrowheads indicates autolysosome (autophagosome maturation). Quantification of vesicle numbers per imaging area relative to wild-type samples is shown. Total, RFP-positive vesicles. AL (Autolysosomes), RFP-positive GFP-negative vesicles. All values are means ± SD. n = 3 mice per genotype. *P < 0.05 (Student’s t test).
Fig 3.
Rapid autophagic degradation of p62 in Stra8-deficient testicular germ cells.
(A) Immunohistochemistry of p62 in wild-type and Stra8-deficient testes at 21 d.p.p.. Numbers indicate the percentages of seminiferous tubules containing germ cells with nuclear p62 accumulation. (B) qRT-PCR analysis of Sqstm1 in wild-type and Stra8-deficient testes normalized to β-actin. Data represent mean ± SD; n = 3 per group. n.s.: not significant. (C) Dual immunofluorescence staining of p62 and MVH in Stra8-deficient testes treated with vehicle (PBS; left panel) or chloroquine (CQ; right panel) treatment for 7 days. (D) Quantification of the number of MVH+ germ cells exhibiting cytoplasmic p62 expression per seminiferous tubule with or without chloroquine treatment in Stra8-deficient testes. Data represent mean ± SD; n = 3 animals per group; 326 seminiferous tubules were examined from Stra8-deficient testes without chloroquine treatment. 296 seminiferous tubules were examined from Stra8-deficient testes with chloroquine treatment. *P < 0.05 (Student’s t test).
Fig 4.
Transcriptional upregulation of selective autophagy-lysosome genes in Stra8-deficient testes.
qRT-PCR analysis of autophagy and lysosome genes in wild-type and Stra8-deficient testes at 10 d.p.p. normalized to β-actin. Data represent mean ± SD; n = 5 per group. *P < 0.05 (Student’s t test).
Fig 5.
STRA8 inhibits de novo autophagosome formation upon autophagy induction.
(A) Cell lysates from F9 cells stably expressing GFP (Ctrl) or STRA8 (tagged with GFP) treated with EBSS for 2 hours were subjected to Western blot analyses using antibodies as indicated. Graph shows quantification of LC3-II to actin ratio. Data represent mean ± s.e.m; n = 3 independent experiments; *P < 0.05 (Student’s t test). (B) Cell lysates from F9 cells stably expressing GFP (Ctrl) or STRA8 (tagged with GFP) treated with vehicle or rapamycin (Rapa; 0.1 μM) for 2 hours were subjected to Western blot analyses using antibodies as indicated. Graph shows quantification of LC3-II to actin ratio. Data represent mean ± s.e.m; n = 3 independent experiments; *P < 0.05 (Student’s t test). (C) Cell lysates from F9 cells stably expressing GFP (Ctrl) or STRA8 (tagged with GFP) treated with vehicle or metformin (Met; 2 mM) for 2 hours were subjected to Western blot analyses using antibodies as indicated. Graph shows quantification of LC3-II to actin ratio. Data represent mean ± s.e.m; n = 3 independent experiments; *P < 0.05 (Student’s t test).
Fig 6.
STRA8 inhibits autophagosome maturation under basal condition (no autophagy induction).
(A) Cell lysates from F9 cells that stably express GFP (Ctrl) or STRA8 (tagged with GFP) at normal conditions were subjected to Western blot analyses by using antibodies as indicated. Graphs show quantification of LC3-II to actin ratio. Data represent mean ± s.e.m.; n = 3 independent experiments; *P < 0.05 (Student’s t test). (B) Fluorescence microscope images of F9 cells expressing GFP (Ctrl) and STRA8 stained with antibodies against LC3 treated with or without chloroquine for 2 hours. Graph shows quantification of LC3-positive puncta per cell. Black dots represent LC3 puncta. The number of LC3-positive puncta in each cell was counted for 100 cells in each group. Data represent mean ± s.e.m.; n = 3 independent experiments; *P < 0.05 (Student’s t test). (C) Representative fluorescence images of HeLa cells stably expressing GFP-mRFP-LC3 transfected with empty (Ctrl) or STRA8-expressing plasmid. Transfection efficiency of the plasmid into HeLa cells was approximately 80–85%. Graph shows the percentage of autolysosome vesicles (GFP-negative RFP-positive) in total vesicles (mRFP-positive). 100 cells were analyzed in each group. Data represent mean ± s.e.m.; n = 3 independent experiments; *P < 0.05 (Student’s t test). (D, E) Cell lysates from F9 cells that stably express GFP (Ctrl) and STRA8 (tagged with GFP) treated with vehicle or chloroquine (CQ; 20 μM) for 2 hours were subjected to Western blot analyses using LC3 (D) or p62 (E) antibodies. Graph shows quantification of LC3-II (D) or p62 (E) to actin ratio. Data represent mean ± s.e.m.; n = 3 independent experiments; *P < 0.05 (Student’s t test). (F) qRT-PCR analysis of autophagy and lysosome genes in control F9 cells and F9 cells expressing stably STRA8. Data represent mean ± SD; n = 3 cultures per group. *P < 0.05 (Student’s t test).
Fig 7.
STRA8 represses Nr1d1 expression.
(A) Genes upregulated and downregulated by transient ectopic STRA8 expression detected by RNA-seq analysis. Total RNA was collected from cells transfected with empty vector (pCMV6) or STRA8 after 24 hours. (B) In situ hybridization of Nr1d1 in testicular cross sections from age-matched wild-type and Stra8-deficient testes at 10 d.p.p.. (C) qRT-PCR analysis of Nr1d1 expression relative to β-actin level in undifferentiated (c-Kit-negative integrin α6-high) and differentiating spermatogonia (c-Kit-positive integrin α6-low) isolated from juvenile wild-type and Stra8-deficient testes by FACS. Data represent mean ± SD; n = 3 mice per group; *P < 0.05 (Student’s t test). (D) Dual immunofluorescence staining of NR1D1 and MVH in wild-type and Stra8-deficient testes. Arrows in left panel indicate germ cells that do not express appreciable level of NR1D1 in wild-type testes. Arrows in right panel indicate germ cells in Stra8-deficient testes that express NR1D1. Note the germ cells in Stra8-deficient testes that express NR1D1 exhibit doublet nucleus, typical of what is frequently observed in preleptotene spermatocytes. A minimum of 60 cells in testicular cross sections of 2 mice was analyzed in each genotype. Data represent mean ± SD; *P < 0.05 (Student’s t test). (E) Schematic of the NR1D1 promoter in human, mouse, rat, and zebrafish showing conserved E-box. (F) Upper, schematic of the primer set targeting the E-box in the human NR1D1 promoter for ChIP analysis. Lower left, ChIP analysis of STRA8 WT, mNLS and mHelix binding to the NR1D1 promoter at the E-Box in 293T cells after transient transfection. Graph represent mean ± SD from duplicate PCR reactions. Lower right, ChIP analysis shows absence of STRA8 WT binding to the exon 8 of NR1D1 gene.
Fig 8.
NR1D1 is required for the upregulated Ulk1 expression in Stra8-deficient testes.
(A) In situ hybridization of Ulk1 in testicular cross sections from age-matched wild-type and Stra8-deficient testes at 10 d.p.p.. (B) qRT-PCR analysis of Ulk1 expression relative to β-actin level in undifferentiated (c-Kit-negative integrin α6-high) and differentiating (c-Kit-positive integrin α6-low) spermatogonia isolated from juvenile wild-type and Stra8-deficient testes by FACS. Data represent mean ± SD; n = 3 mice per group; *P < 0.05 (Student’s t test). (C) Left panel, schematic of the primer sets targeting RORE sites in the mouse Ulk1 promoter for ChIP analysis. Lower, ChIP analysis of NR1D1 binding to the Ulk1 promoter at the RORE sites in mouse testicular lysates at 21 d.p.p.. Graph represent mean ± SD from triplicate PCR reactions. (D) qRT-PCR analysis of testicular Ulk1 expression relative to β-actin levels in testes with indicated genotypes. Data represent mean ± SD; n = 3 mice per group; *P < 0.05 (Student’s t test).
Fig 9.
Genetic and pharmacological NR1D1 inhibition rescues meiosis initiation arrest in Stra8-deficient testes.
(A) Photomicrographs of hematoxylin/eosin-stained testicular cross sections from testes with indicated genotypes at 10 d.p.p.. Insets show germ cells exhibiting chromosome condensation at early meiotic prophase. Percentages of tubules containing germ cells exhibiting chromosome condensation at early meiotic prophase are shown underneath. Data represent mean ± SD. n = 2–3 mice per genotype. (B) Dual immunofluorescence staining of γ-H2AX and SYCP3 in Stra8-/-;Nr1d1+/+ and Stra8-/-;Nr1d1-/- testes at 10 d.p.p.. Note the brighter foci of SYCP3 staining in Stra8-/-;Nr1d1+/+ testes indicative of premeiotic status of their germ cells in contrast to the germ cells with nuclear distribution of SYCP3 and foci of γ-H2AX found in Stra8-/-;Nr1d1-/- testes. (C) qRT-PCR analysis of testicular Spo11, Dmc1, and Sycp3 expression normalized to β-actin from mice with indicated genotypes at 10 d.p.p.. Data are mean ± SD; n = 2–3 mice per group. *P < 0.05 (Student’s t test). (D) Photomicrographs of hematoxylin/eosin-stained testicular cross sections from wild-type and Stra8-deficient testes treated with vehicle or SR8278 (100 mg/kg) for 3 days. Testes were collected at 10 d.p.p.. Insets show germ cells exhibiting chromosome condensation at early meiotic prophase. Percentages of tubules containing germ cells exhibiting chromosome condensation at early meiotic prophase are shown underneath. Data represent mean ± SD. n = 3 mice per genotype. (E) Dual immunofluorescence staining of γ-H2AX and SYCP3 in Stra8-deficient testes treated with vehicle or SR8278. (F) qRT-PCR analysis of testicular Spo11, Dmc1, and Sycp3 expression normalized to β-actin from mice treated with vehicle or SR8278. Data are mean ± SD; n = 4–5 mice per group. *P < 0.05 (Student’s t test).
Fig 10.
Loss of STRA8 results in aberrant autophagosome formation and upregulation of autophagy-lysosome gene expression in fetal ovarian germ cells during the developmental window of meiotic initiation.
(A, B) Transmission electron microscopy images of ovarian cross sections from wild-type (A) and Stra8-deficient (B) mice at embryonic day 14.5 (E14.5) that show primordial germ cells (PGC). Arrows indicate autophagosomes. (C, D) qRT-PCR analysis of autophagy and lysosome gene expression in wild-type and Stra8-deficient fetal ovaries (C) and testes (D) at E14.5 normalized to germ cell content by Mvh levels. Data represent mean ± SD; n = 3 embryos per group. *P < 0.05 (Student’s t test).
Fig 11.
Schematic model of STRA8-mediated meiosis initiation by suppressing autophagy through NR1D1-ULK1 axis.