title: Rapamycin (Sirolimus) alters mechanistic target of rapamycin pathway regulation and microRNA expression in mouse meiotic spermatocytes.
abstract: Mechanistic target of rapamycin (mTOR) is a signal transduction pathway that modulates translation initiation in several animals including mammals. Rapamaycin, an allosteric inhibitor of mTOR pathway, is often used as an immunosuppressive drug following kidney transplantation and causes gonadal dysfunction and defects in spermatogenesis. The molecular mechanism behind rapamycin-mediated testicular dysfunction is not known. We have therefore explored the contribution of rapamycin in mTOR regulation and microRNA (miRNA) expression in mouse spermatocytes, the intermediate stage of spermatogenesis, where meiosis takes place. In the present study, we optimized the isolation of highly pure and viable spermatocytes by flow sorting, treated them with rapamycin, and investigated the expression of mTOR and downstream effector molecules. Western blot and immunocytochemical analysis confirm that rapamycin treatment suppresses mTOR and phopsphorylated P70S6 kinase activities in spermatocytes, but not that of phosphorylated 4E-binding protein 1. Also, rapamycin treatment modulates the expression of several spermatocyte-specific miRNAs. To complement these finding an in vivo study was also performed. In silico prediction of target genes of these miRNAs and their functional pathway analysis revealed that, several of them are involved in crucial biological process, cellular process and catalytic activities. miRNA-transcription factor (TF) network analysis enlisted different TFs propelling the transcription machineries of these miRNAs. In silico prediction followed by quatitative real-time PCR revealed two of these TFs namely, PU.1 and CCCTC binding factor (CTCF) are down and upregulated, respectively, which may be the reason of the altered expression of miRNAs following rapamycin treatment. In conclusion, for the first time, the present study provides insight into how rapamycin regulates mTOR pathway and spermatocyte-specific miRNA expression which in turn, regulate expression of target genes post-transcriptionally.
authors: Division of Molecular Immunology and Microbiology, National Institute for Research in Reproductive Health, Indian Council of Medical Research, Mumbai, India.
authors: A
authors: A
authors: Mukherjee
authors: Division of Molecular Immunology and Microbiology, National Institute for Research in Reproductive Health, Indian Council of Medical Research, Mumbai, India.
authors: S
authors: S
authors: Koli
authors: Division of Molecular Immunology and Microbiology, National Institute for Research in Reproductive Health, Indian Council of Medical Research, Mumbai, India.
authors: K V R
authors: KV
authors: Reddy
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D000076246:CCCTC-Binding Factor; D002352:Carrier Proteins / Q000378:metabolism; D018797:Cell Cycle Proteins; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D039642:Eukaryotic Initiation Factors; D008297:Male; D051379:Mice; D035683:MicroRNAs / Q000096:biosynthesis* / Q000235:genetics; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis; D051381:Rats; D060888:Real-Time Polymerase Chain Reaction; D012097:Repressor Proteins / Q000096:biosynthesis; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000037:antagonists & inhibitors; D015398:Signal Transduction / Q000187:drug effects; D020123:Sirolimus / Q000494:pharmacology*; D013090:Spermatocytes / Q000166:cytology / Q000378:metabolism*; D013091:Spermatogenesis / Q000187:drug effects*; D058570:TOR Serine-Threonine Kinases / Q000037:antagonists & inhibitors*; D013737:Testis / Q000166:cytology; D015534:Trans-Activators / Q000096:biosynthesis; D000099232:Proto-Oncogene Protein Spi-1
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000076246:CCCTC-Binding Factor; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D039642:Eukaryotic Initiation Factors; D035683:MicroRNAs; D010750:Phosphoproteins; D011518:Proto-Oncogene Proteins; D012097:Repressor Proteins; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D020123:Sirolimus; D058570:TOR Serine-Threonine Kinases; D015534:Trans-Activators; D000099232:Proto-Oncogene Protein Spi-1; C000619698:Ctcf protein, mouse; C499527:Eif4ebp1 protein, mouse; C546843:mTOR protein, mouse
keywords: mechanistic target of rapamycin; microRNA; rapamycin; spermatocyte; transcription factor