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yin jun
Third Military Medical University: Army Medical University
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ORCiD: https://orcid.org/0000-0002-7541-1088
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Spermatocyte apoptosis is the primary cause of poor outcome after hypoxia-triggered spermatogenesis reduction (HSR). The vacuolar H+-ATPase (V-ATPase) has been found to be involved in the regulation of hypoxia-induced GC-2 cells apoptosis. However, the mechanism of V-ATPase regulating spermatocyte apoptosis after HSR hasnot been well elucidated. In this study, HSRmodel was established by hypoxia exposure in vivo in V-ATPase-knockout (V-ATPase-/-) and wild-type (WT) mice to investigate theeffectof V-ATPase deficiency on spermatocyte apoptosis. GC-2, amouse pachytene spermatocyte-derived cell line, was introduced in vitro experiments. The sperm count and spermatogenic apoptosis were recorded after 60 d of hypoxia exposure in HSR model. The apoptosis of GC-2 cells was detected by flow cytometry and TUNEL staining. The expression of JNK/c-Jun was evaluated by RNA-seq or western blot. The expression of DR5 and caspase-8 was evaluated by RT-qPCR and western blot. The expression of V-ATPase was determined by western blot in the presence and absence ofLenti-transcription factor EB (TFEB).C-Jun interference was used for evaluating the role of JNK in regulating the apoptosis of GC-2 cells byTUNEL and flow cytometry. The in vivo results suggested that hypoxia induced spermatogenesis reduction and downregulation of V-ATPase. Moreover, V-ATPase deficiency resulted in moresevere spermatogenesis reduction after hypoxia exposure. The spermatogenesis reduction was associated with exacerbation of spermatocyte apoptosis. Hypoxia down-regulated the transcription of V-ATPase through inhibiting TFEB and its nuclear translocation. The mRNA and protein expressions of V-ATPaseincreased after TFEB overexpression in GC-2 cells. Moreover, V-ATPase deficiency enhanced JNK/c-Jun activation and related DR-apoptotic pathwayin GC-2 cells.However,inhibition of c-Jun attenuated V-ATPase deficiency-induced GC-2 cells apoptosis in vitro and HSR in vivo. In conclusion, JNK/c-Jun was involved in the enhancement of V-ATPase-mediated HSR in V-ATPase -/- mice. V-ATPase deficiency aggravates spermatocyte apoptosis, which may account forthe poor spermatogenesis outcomes of V-ATPase-/- mice. The discoveredfunction of V-ATPase modulating spermatocyte apoptosis indicates its potential therapeutic effect against HSR.
Keywords
V-ATPase, JNK, c-Jun, DR5, hypoxia, apoptosis, spermatogenesis
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This is a list of supplementary files associated with this preprint. Click to download.
- FigureS1.jpg
Fig.S1 Hypoxia induces apoptosis of spermatogeic cells in mice at different times. Hypoxic mice were raised in a hypobaric chamber for 0, 3, 5 or 15 d under 10% O2 condition. (A) Pathological structures of the seminiferous tubules by haematoxylin and eosin staining show spermatogenic cells. Scale bar = 100 µm. (B) TUNEL staining of seminiferous tubule: apoptotic nuclei featured condensed or fragmented DNA that was brightly stained with TUNEL. Original magnification: 400×. The apoptosis rate of spermatogenic cells was counted by TUNEL staining under a high-magnification field.
- FigureS2.jpg
Fig.S2 V-ATPase overexpression attenuates apoptosis of spermatogeic cells under hypoxia exposure. Hypoxic mice were raised in a hypobaric chamber for 15 dunder 10% O2 condition after injection of LV-V-ATPase. (A) Pathological structures of the seminiferous tubules by haematoxylin and eosin staining show spermatogenic cells. Scale bar = 100 µm. (B) TUNEL staining of seminiferous tubule: apoptotic nuclei featured condensed or fragmented DNA that was brightly stained with TUNEL. Original magnification: 400×. The apoptosis rate of spermatogeniccells was counted by TUNEL staining under a high-magnification field.
- FigureS3.jpg
Fig.S3 Hypoxia induces downregulation of V-ATPase and the cellular apoptosis of GC-2 cells. GC-2 cells were seeded at 5×105/ml for 0, 24 or 48 h in hypoxic cultures.(A) The TUNEL staining resultsof GC-2 cells: apoptotic nuclei were featured by condensed or fragmented DNA that wasbrightly stained with TUNEL. Original magnification:200×.(B) Representative graphs obtained from the flow cytometry analysis of cellular apoptosis afterdouble staining with annexin V-FITC and propidium iodide. Apoptotic incidences of GC-2 cells were measured by double staining with annexin V-FITC and propidium iodide. (C) The representative western blot assays for V-ATPase, DR5 and caspase-8 protein expression in mouse GC-2 cellsthat were subjected to 1% oxygen for 0h, 24h and48h.
- FigureS4.jpg
Fig.S4 Sustain hypoxia induces inactivation of TFEB in GC-2 cells. GC-2 cells were seeded at 5×105/ml for 0, 3, 6, 12, 24 or 48 h in hypoxic cultures. (A) Immunofluorescence images showing the distribution of TFEB in cells, as detected by laser confocal microscopy using antibodies against TFEB (red). Nuclei are labeled with DAPI (blue). Scalebar: 20μm. (B) The mRNA expression of TFEB by RT-PCR in mouse GC-2 cells. The values from treated cells have been normalized to β-actin measurement and then expressed as a ratio of normalized values to mRNA in control cells (n=4). *p<0.05 vs.control. (C) The time-dependent changes of TFEB expression from 3 h to 48 h after hypoxia treatment. (D) Nuclear and cytoplasmic subfractions of GC-2 cells were examined in the same gel to test the migrating bands and the shift in size of TFEB.
- FigureS5.jpg
Fig.S5 Hypoxia upregulates mRNA of JNK/c-Jun related positive markers in GC-2 cells. GC-2 cells were seeded at5×105/ml for48 h in normoxic or hypoxic culture. (A) A representative diagram of the common up/downregulated genes detected by RNA-seq in GC-2 cellsthat were subjected to 1% oxygen for 48 h. (B) Heatmap of altered genes and the KEGG enrichment analysis. JNK and other MAPK positive regulatory genes were robustly upregulated in response to 1% oxygen.
- FigureS6.jpg
Fig.S6 c-Jun deficiency alleviates apoptosis of spermatogenic cells under hypoxia exposure. Hypoxic mice transfected with c-Jun siRNA were raised in a hypobaric chamber for 15 days under 10% oxygen condition. (A) Pathological structures of the testicular seminiferous tubules transfected with control siRNA (CTL siRNA) or V-ATPase siRNA (siV-ATPase) by haematoxylin and eosin staining show spermatogenic cells. Scale bar, 100μm.(B) TUNEL staining of seminiferous tubule: apoptotic nuclei featured condensed or fragmented DNA that was brightly stained with TUNEL. Original magnification: 400×. The apoptosis rate of spermatogenic cells wascounted by TUNEL staining under a high-magnification field.
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yin jun
Third Military Medical University: Army Medical University
Corresponding Author
ORCiD: https://orcid.org/0000-0002-7541-1088
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Spermatocyte apoptosis is the primary cause of poor outcome after hypoxia-triggered spermatogenesis reduction (HSR). The vacuolar H+-ATPase (V-ATPase) has been found to be involved in the regulation of hypoxia-induced GC-2 cells apoptosis. However, the mechanism of V-ATPase regulating spermatocyte apoptosis after HSR hasnot been well elucidated. In this study, HSRmodel was established by hypoxia exposure in vivo in V-ATPase-knockout (V-ATPase-/-) and wild-type (WT) mice to investigate theeffectof V-ATPase deficiency on spermatocyte apoptosis. GC-2, amouse pachytene spermatocyte-derived cell line, was introduced in vitro experiments. The sperm count and spermatogenic apoptosis were recorded after 60 d of hypoxia exposure in HSR model. The apoptosis of GC-2 cells was detected by flow cytometry and TUNEL staining. The expression of JNK/c-Jun was evaluated by RNA-seq or western blot. The expression of DR5 and caspase-8 was evaluated by RT-qPCR and western blot. The expression of V-ATPase was determined by western blot in the presence and absence ofLenti-transcription factor EB (TFEB).C-Jun interference was used for evaluating the role of JNK in regulating the apoptosis of GC-2 cells byTUNEL and flow cytometry. The in vivo results suggested that hypoxia induced spermatogenesis reduction and downregulation of V-ATPase. Moreover, V-ATPase deficiency resulted in moresevere spermatogenesis reduction after hypoxia exposure. The spermatogenesis reduction was associated with exacerbation of spermatocyte apoptosis. Hypoxia down-regulated the transcription of V-ATPase through inhibiting TFEB and its nuclear translocation. The mRNA and protein expressions of V-ATPaseincreased after TFEB overexpression in GC-2 cells. Moreover, V-ATPase deficiency enhanced JNK/c-Jun activation and related DR-apoptotic pathwayin GC-2 cells.However,inhibition of c-Jun attenuated V-ATPase deficiency-induced GC-2 cells apoptosis in vitro and HSR in vivo. In conclusion, JNK/c-Jun was involved in the enhancement of V-ATPase-mediated HSR in V-ATPase -/- mice. V-ATPase deficiency aggravates spermatocyte apoptosis, which may account forthe poor spermatogenesis outcomes of V-ATPase-/- mice. The discoveredfunction of V-ATPase modulating spermatocyte apoptosis indicates its potential therapeutic effect against HSR.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
This is a list of supplementary files associated with this preprint. Click to download.
- FigureS1.jpg
Fig.S1 Hypoxia induces apoptosis of spermatogeic cells in mice at different times. Hypoxic mice were raised in a hypobaric chamber for 0, 3, 5 or 15 d under 10% O2 condition. (A) Pathological structures of the seminiferous tubules by haematoxylin and eosin staining show spermatogenic cells. Scale bar = 100 µm. (B) TUNEL staining of seminiferous tubule: apoptotic nuclei featured condensed or fragmented DNA that was brightly stained with TUNEL. Original magnification: 400×. The apoptosis rate of spermatogenic cells was counted by TUNEL staining under a high-magnification field.
- FigureS2.jpg
Fig.S2 V-ATPase overexpression attenuates apoptosis of spermatogeic cells under hypoxia exposure. Hypoxic mice were raised in a hypobaric chamber for 15 dunder 10% O2 condition after injection of LV-V-ATPase. (A) Pathological structures of the seminiferous tubules by haematoxylin and eosin staining show spermatogenic cells. Scale bar = 100 µm. (B) TUNEL staining of seminiferous tubule: apoptotic nuclei featured condensed or fragmented DNA that was brightly stained with TUNEL. Original magnification: 400×. The apoptosis rate of spermatogeniccells was counted by TUNEL staining under a high-magnification field.
- FigureS3.jpg
Fig.S3 Hypoxia induces downregulation of V-ATPase and the cellular apoptosis of GC-2 cells. GC-2 cells were seeded at 5×105/ml for 0, 24 or 48 h in hypoxic cultures.(A) The TUNEL staining resultsof GC-2 cells: apoptotic nuclei were featured by condensed or fragmented DNA that wasbrightly stained with TUNEL. Original magnification:200×.(B) Representative graphs obtained from the flow cytometry analysis of cellular apoptosis afterdouble staining with annexin V-FITC and propidium iodide. Apoptotic incidences of GC-2 cells were measured by double staining with annexin V-FITC and propidium iodide. (C) The representative western blot assays for V-ATPase, DR5 and caspase-8 protein expression in mouse GC-2 cellsthat were subjected to 1% oxygen for 0h, 24h and48h.
- FigureS4.jpg
Fig.S4 Sustain hypoxia induces inactivation of TFEB in GC-2 cells. GC-2 cells were seeded at 5×105/ml for 0, 3, 6, 12, 24 or 48 h in hypoxic cultures. (A) Immunofluorescence images showing the distribution of TFEB in cells, as detected by laser confocal microscopy using antibodies against TFEB (red). Nuclei are labeled with DAPI (blue). Scalebar: 20μm. (B) The mRNA expression of TFEB by RT-PCR in mouse GC-2 cells. The values from treated cells have been normalized to β-actin measurement and then expressed as a ratio of normalized values to mRNA in control cells (n=4). *p<0.05 vs.control. (C) The time-dependent changes of TFEB expression from 3 h to 48 h after hypoxia treatment. (D) Nuclear and cytoplasmic subfractions of GC-2 cells were examined in the same gel to test the migrating bands and the shift in size of TFEB.
- FigureS5.jpg
Fig.S5 Hypoxia upregulates mRNA of JNK/c-Jun related positive markers in GC-2 cells. GC-2 cells were seeded at5×105/ml for48 h in normoxic or hypoxic culture. (A) A representative diagram of the common up/downregulated genes detected by RNA-seq in GC-2 cellsthat were subjected to 1% oxygen for 48 h. (B) Heatmap of altered genes and the KEGG enrichment analysis. JNK and other MAPK positive regulatory genes were robustly upregulated in response to 1% oxygen.
- FigureS6.jpg
Fig.S6 c-Jun deficiency alleviates apoptosis of spermatogenic cells under hypoxia exposure. Hypoxic mice transfected with c-Jun siRNA were raised in a hypobaric chamber for 15 days under 10% oxygen condition. (A) Pathological structures of the testicular seminiferous tubules transfected with control siRNA (CTL siRNA) or V-ATPase siRNA (siV-ATPase) by haematoxylin and eosin staining show spermatogenic cells. Scale bar, 100μm.(B) TUNEL staining of seminiferous tubule: apoptotic nuclei featured condensed or fragmented DNA that was brightly stained with TUNEL. Original magnification: 400×. The apoptosis rate of spermatogenic cells wascounted by TUNEL staining under a high-magnification field.
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