1. mTOR/P70S6K promotes spermatogonia proliferation and spermatogenesis in Sprague Dawley rats 
Hao Xu, Lianju Shen, Xuemei Chen, Yubin Ding, Junlin He, Jing Zhu, Yingxiong Wang, Xueqing Liu 
Reproductive BioMedicine Online 
Volume 32, Issue 2, Pages (February 2016)
DOI: /j.rbmo
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2. Figure 1
Haematoxylin and eosin staining and the expression of the mammalian target of rapamycin (mTOR) signalling pathway proteins in various developmental stage testes. (A) (a) The histological morphology was normal in 8-week-old rat testes; (b) a portion of seminiferous tubules showed atrophy and vacuolation in 38-week-old rat testes; (c) the phenomenon of atrophy and vacuolation were more serious in 80-week-old rat testes; (d–f) magnification (200×) of boxed area shown in panels a–c, respectively. The arrows in A and B show vacuolation in testis. (B) mTOR immunohistochemistry in the testis at several developmental stages; (a) 8-week-old rat testes; (b) 38-week-old rat testes; (c) 80-week-old rat testes; (d–f) magnification (200×) of boxed area shown in panels a–c, respectively; (C) a quantitative analysis of mTOR immunohistochemistry integrated option density (IOD). Values are mean ± SD; (D) the protein expression of mTOR and its target in rat testes as determined by Western blot. The ratio of Phospho-p70s6k/p70s6k, Phospho-RPS6/RPS6 and Phospho-4ebp1/4ebp1 was significantly lower in the 80-week-old group than the 8-week-old group; (b–d) a quantitative presentation of the data is shown in (a). *P < 0.05 compared with 8-week-old. Each group has 10 rats, and these experiments were repeated three times.
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3. Figure 2
Testicular histological change and mammalian target of rapamycin (mTOR) signalling pathway activity after treatment with rapamycin or after rapamycin withdrawal. (A) Testicular histological changes after treatment with rapamycin. The seminiferous tubules developed normally, and all stages of spermatogenic cells were visible in blank group (a) and dimethyl sulphoxide (DMSO) group (b). The seminiferous tubules displayed atrophy and vacuolation after treatment with rapamycin (c); (d–f) magnification (400×) of boxed area shown in panels a–c, respectively. The arrows indicate vacuolation in testis; (B) testicular histological change after rapamycin withdrawal; (a–c) blank, DMSO and rapamycin groups, respectively. Vacuolated seminiferous tubules were rescued to some extent in the withdrawal group (d); (C) mTOR signalling pathway activation in rat testis after rapamycin withdrawal. The ratio of Phospho-p70s6k/p70s6k and Phospho-RPS6/RPS6 was rescued after rapamycin withdrawal, which indicates that the activity of the mTOR signalling pathway was rescued; (D) the protein expression of mTOR and its target as determined by Western blot. The ratio of Phospho-p70s6k/p70s6k and Phospho-RPS6/RPS6 was significantly lower after treatment with rapamycin, and the ratio of Phospho-4ebp1/4ebp1 was not different. (b–d) A quantitative presentation of the data is shown in (a). *P < 0.05 compared with blank and DMSO group. Each group had 10 rats, and these experiments were repeated three times.
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4. Figure 3
Spermatogonia proliferation. (A) Ki67 immunohistochemistry in the testes of the 8-week-old (a), 38-week-old (b) and 80-week-old groups (c); (d–f) Ki67 immunohistochemistry in the blank, dimethyl sulphoxide group and rapamycin groups, respectively. The arrows indicate the spermatogonia without Ki67 expression; (B) a quantitative analysis of Ki67. Ki67 expression gradually decreased with ageing (a); *P < 0.05 compared with 8-week-old group, and decreased significantly after rapamycin treatment; (b) *P < 0.05 compared with blank and dimethyl sulphoxide group. Each group had 10 rats, and these experiments were repeated three times. DMSO, dimethyl sulphoxide. IOD, integrated option density.
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5. Figure 4
Cell cycle analysis after treatment with rapamycin and the activity of the mammalian target of rapamycin (mTOR) signalling pathway in spermatogonia after treatment with rapamycin, okadaic acid, or both; (A) spermatogonia morphology by optical microscopy; (B) flow cytometry analysis using c-kit surface expression; (C) flow cytometry analysis of the cell cycle, which showed that spermatogonia were blocked in G1 after rapamycin treatment; (D) a quantitative presentation of the data shown in C. *P < 0.05 compared with blank and dimethyl sulphoxide group; (E) the protein expression of mTOR and its target after various treatments by Western blot. After treatment with rapamycin or rapamycin and okadaic acid, the ratio of Phospho-p70s6k and Phospho-RPS6/RPS6 were significantly lower than that in the control or okadaic acid group, but the ratio of Phospho-4ebp1/4ebp1 did not change; (b–d) a quantitative presentation of the data is shown in (a). *P < 0.05 compared with blank and dimethyl sulphoxide group. Each group had 10 rats, and these experiments were repeated three times. DMSO, dimethyl sulphoxide.
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6. Figure 5
PI3K activation in testes of different age and treatment groups and activation status of the mTOR signalling pathway after treatment of spermatogonia, isolated from 9-day-old male rats, with the PI3K inhibitor LY (A) PI3K phosphorylation was decreased in the 80-week-old group, but there was no difference in the rapamycin group; (b–c) a quantitative presentation of the data shown in (a). *P < 0.05 compared with 8-week-old group; (B) after LY treatment, the ratio of Phospho-Akt/Akt was significantly lower compared with the blank and dimethyl sulphoxide groups, which confirms that PI3K was inhibited by LY The ratio of Phospho-4ebp1/4ebp1, Phospho-p70s6k/p70s6k and Phospho-RPS6/RPS6 was significantly lower in LY group compared with blank groups; (b–e) a quantitative presentation of the data is shown in (a). *P < 0.05 compared with blank and dimethyl sulphoxide group. Each group had 10 rats, and these experiments were repeated three times. DMSO, dimethyl sulphoxide.
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7. Figure 6
Mammalian target of rapamycin (mTOR) signalling pathways in cancer cell and spermatogonia proliferation; (A) in most types of cancer cells, mTOR regulates protein translation and cell proliferation by activating p70s6k and 4ebp1; (B) in spermatogonia, mTOR regulates p70s6k activity, whereas PI3K indirectly or directly regulates 4ebp1 activation status, according to the present results.
Reproductive BioMedicine Online  , DOI: ( /j.rbmo )
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