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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.
Keywords
V-ATPase, JNK, c-Jun, DR5, hypoxia, apoptosis, spermatogenesis
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- 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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This preprint is under consideration at Cell & Bioscience. A preprint is a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research
yin jun
Third Military Medical University: Army Medical University
Corresponding Author
ORCiD: https://orcid.org/0000-0002-7541-1088
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Under Review
Version 1
Posted 17 Mar, 2021
No community comments so far
Editorial decision: Revise before peer review
On 15 Mar, 2021
Editor assigned
On 08 Mar, 2021
Editor invited
On 08 Mar, 2021
Submission checks complete
On 07 Mar, 2021
First submitted
On 04 Mar, 2021
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
General Cell Biology & Physiology
License:
This work is licensed under a CC BY 4.0 License. Read Full License
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.
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