title: Fractionation of human spermatogenic cells using STA-PUT gravity sedimentation and their miRNA profiling.
abstract: Human spermatogenic cells have not yet been isolated, and notably, their global miRNA profiles remain unknown. Here we have effectively isolated human spermatogonia, pachytene spermatocytes and round spermatids using STA-PUT velocity sedimentation. RT-PCR, immunocytochemistry and meiosis spread assays revealed that the purities of isolated human spermatogonia, pachytene spermatocytes, and round spermatids were 90%, and the viability of these isolated cells was over 98%. MiRNA microarrays showed distinct global miRNA profiles among human spermatogonia, pachytene spermatocytes, and round spermatids. Thirty-two miRNAs were significantly up-regulated whereas 78 miRNAs were down-regulated between human spermatogonia and pachytene spermatocytes, suggesting that these miRNAs are involved in the meiosis and mitosis, respectively. In total, 144 miRNAs were significantly up-regulated while 29 miRNAs were down-regulated between pachytene spermatocytes and round spermatids, reflecting potential roles of these miRNAs in mediating spermiogenesis. A number of novel binding targets of miRNAs were further identified using various softwares and verified by real-time PCR. Our ability of isolating human spermatogonia, pachytene spermatocytes and round spermatids and unveiling their distinct global miRNA signatures and novel targets could provide novel small RNA regulatory mechanisms mediating three phases of human spermatogenesis and offer new targets for the treatment of male infertility.
authors: State Key Laboratory of Oncogenes and Related Genes, Renji- Med X Clinical Stem Cell Research Center, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.
authors: Yun
authors: Y
authors: Liu
authors: State Key Laboratory of Oncogenes and Related Genes, Renji- Med X Clinical Stem Cell Research Center, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.
authors: Minghui
authors: M
authors: Niu
authors: State Key Laboratory of Oncogenes and Related Genes, Renji- Med X Clinical Stem Cell Research Center, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.
authors: Chencheng
authors: C
authors: Yao
authors: State Key Laboratory of Oncogenes and Related Genes, Renji- Med X Clinical Stem Cell Research Center, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.
authors: Yanan
authors: Y
authors: Hai
authors: State Key Laboratory of Oncogenes and Related Genes, Renji- Med X Clinical Stem Cell Research Center, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.
authors: Qingqing
authors: Q
authors: Yuan
authors: State Key Laboratory of Oncogenes and Related Genes, Renji- Med X Clinical Stem Cell Research Center, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.
authors: Yang
authors: Y
authors: Liu
authors: State Key Laboratory of Oncogenes and Related Genes, Renji- Med X Clinical Stem Cell Research Center, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.
authors: Ying
authors: Y
authors: Guo
authors: Department of Urology, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai Human Sperm Bank, Shanghai Institute of Andrology, 145 Shangdong Road, Shanghai 200001, China.
authors: Zheng
authors: Z
authors: Li
authors: 1] State Key Laboratory of Oncogenes and Related Genes, Renji- Med X Clinical Stem Cell Research Center, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China [2] Department of Urology, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai Human Sperm Bank, Shanghai Institute of Andrology, 145 Shangdong Road, Shanghai 200001, China [3] Shanghai Key Laboratory of Assisted Reproduction and Reproductive Genetics, Shanghai 200127, China [4] Shanghai Key Laboratory of Reproductive Medicine, Shanghai 200025, China.
authors: Zuping
authors: Z
authors: He
mesh_terms: D000328:Adult; D001483:Base Sequence; D002458:Cell Fractionation / Q000379:methods*; D002469:Cell Separation; D048430:Cell Shape; D002498:Centrifugation / Q000379:methods*; D016000:Cluster Analysis; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D006112:Gravitation; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D049471:Pachytene Stage; D010641:Phenotype; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D013087:Spermatids / Q000166:cytology / Q000378:metabolism; D013090:Spermatocytes / Q000166:cytology / Q000378:metabolism; D013093:Spermatogonia / Q000166:cytology* / Q000378:metabolism*; D055815:Young Adult
chemical_list: D035683:MicroRNAs; D012333:RNA, Messenger
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