Date Published: January 15, 2019
Publisher: Impact Journals
Author(s): Zhiping Zhu, Qiuling Yue, Jie Xie, Shuya Zhang, Wenxiu He, Shun Bai, Suwen Tian, Yingwen Zhang, Mengneng Xiong, Zheng Sun, Chaoyang Huang, Yuebei Li, Ke Zheng, Lan Ye.
Mechanistic target of rapamycin (mTOR) controls cell growth and metabolism in response to environmental and metabolic signals. Rapamycin robustly extends the lifespan in mammals and has clinical relevance in organ transplantation and cancer therapy but side effects include male infertility. Here, we report that chronic rapamycin treatment causes spermatogenic arrest in adult male mice due to defects in sex body formation and meiotic sex chromosome inactivation (MSCI). Many sex chromosome-linked genes were up-regulated in isolated pachytene spermatocytes from rapamycin-treated mice. RNA-Seq analysis also identified mRNAs encoding the core piRNA pathway components were decreased. Furthermore, rapamycin treatment was associated with a drastic reduction in pachytene piRNA populations. The inhibitory effects of rapamycin on spermatogenesis were partially reversible, with restoration of testis mass and sperm motility within 2 months of treatment cessation. Collectively, we have defined an essential role of mTOR in MSCI and identified a novel function as a regulator of small RNA homeostasis in male germ cells.
Mechanistic target of rapamycin (mTOR) is a conserved serine/threonine protein kinase that regulates cell survival and metabolism in response to extra- and intracellular signals that include nutrients, growth factors, cellular energy, and stress. mTOR resides in two structurally and functionally distinct multiprotein complexes, mTORC1 and mTORC2 [1–4]. The mTORC1 complex requires the mTOR-associated adaptor protein Raptor and activates ribosomal biogenesis and protein translation by phosphorylation of the ribosomal protein S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) [5,6]. The mTORC2 complex incorporates the adaptor protein Rictor and exerts metabolic control through AKT and serum/glucocorticoid induced kinase (SGK) [7,8]. mTORC2 also regulates the cytoskeleton through protein kinase C alpha (PKC-α) signaling .
Here, to study the potential function of mTOR signaling in male germ cell development, we used chronic rapamycin treatment at approximately the same dose that was used to extend lifespan, and found that rapamycin-mediated prolonged inhibition of mTOR signaling causes male infertility, specifically, meiotic defects resulting from disruption in the meiotic silencing of sex chromosomes. Further analysis revealed that the recruitment of the essential silencing factor ATR to the sex chromatin was attenuated at the pachytene stage. ATR is a key mediator of meiotic silencing, and it is a member of the phosphatidylinositol-3 kinase-like kinase (PIKK) family which also includes mTOR . Importantly, ATR catalyzes H2AX phosphorylation, regulates localization of a set meiotic silencing components at unsynapsed axes, and is required to induce repressive epigenetic modifications . We recently showed that the accumulation of ATR on sex chromatin was also diminished in pachytene spermatocytes from testis-specific Raptor (mTORC1 subunit) knockout mice, which arrested at pachytene stage with defects in meiotic sex chromosome inactivation . However, meiotic progression and recruitment of silencing factors to sex chromosomes was normal in testes with conditional knockout of mTORC2 component Rictor  (Supplementary Fig. S4
). These results suggest that rapamycin-mediated defects in meiosis and meiotic silencing of sex chromosomes is mTORC1-dependent. Previous studies have reported that rapamycin inhibits the proliferation of cultured mouse spermatogonial stem cells (SSCs) , but it strengthens the self-renewal potential of undifferentiated spermatogonia in mice with conditional ablation of Plzf, a transcription factor essential for the maintenance of undifferentiated spermatogonia [73,74]. Rapamycin has also been found to reduce testis size by blocking the differentiation of spermatogonia in neonatal mice at P4 and P8 . This mechanism is unlikely to be important in our adult mouse model because long-term rapamycin treatment did not affect the level of leptotene spermatocytes but specifically late pachytene and diplotene spermatocytes. Furthermore, the initial recruitment of DNA damage pathway proteins at the leptotene/zygotene stage and the formation of early recombination intermediates occurred normally in rapamycin-treated testes. However, this mechanism may play a role in the shorter-term effects of rapamycin on germ cell development in neonatal testes, when spermatogonial cells are enriched.