title: Regulatory complexity revealed by integrated cytological and RNA-seq analyses of meiotic substages in mouse spermatocytes.
abstract: The continuous and non-synchronous nature of postnatal male germ-cell development has impeded stage-specific resolution of molecular events of mammalian meiotic prophase in the testis. Here the juvenile onset of spermatogenesis in mice is analyzed by combining cytological and transcriptomic data in a novel computational analysis that allows decomposition of the transcriptional programs of spermatogonia and meiotic prophase substages.
  
  
  
  Germ cells from testes of individual mice were obtained at two-day intervals from 8 to 18 days post-partum (dpp), prepared as surface-spread chromatin and immunolabeled for meiotic stage-specific protein markers (STRA8, SYCP3, phosphorylated H2AFX, and HISTH1T). Eight stages were discriminated cytologically by combinatorial antibody labeling, and RNA-seq was performed on the same samples. Independent principal component analyses of cytological and transcriptomic data yielded similar patterns for both data types, providing strong evidence for substage-specific gene expression signatures. A novel permutation-based maximum covariance analysis (PMCA) was developed to map co-expressed transcripts to one or more of the eight meiotic prophase substages, thereby linking distinct molecular programs to cytologically defined cell states. Expression of meiosis-specific genes is not substage-limited, suggesting regulation of substage transitions at other levels.
  
  
  
  This integrated analysis provides a general method for resolving complex cell populations. Here it revealed not only features of meiotic substage-specific gene expression, but also a network of substage-specific transcription factors and relationships to potential target genes.
authors: The Jackson Laboratory, Bar Harbor, ME, USA.
authors: Robyn L
authors: RL
authors: Ball
authors: The Jackson Laboratory, Bar Harbor, ME, USA.
authors: Yasuhiro
authors: Y
authors: Fujiwara
authors: The Jackson Laboratory, Bar Harbor, ME, USA.
authors: Fengyun
authors: F
authors: Sun
authors: The Jackson Laboratory, Bar Harbor, ME, USA.
authors: Jianjun
authors: J
authors: Hu
authors: The Jackson Laboratory, Bar Harbor, ME, USA.|Department of Computer Science, Trinity University, San Antonio, TX, USA.
authors: Matthew A
authors: MA
authors: Hibbs
authors: The Jackson Laboratory, Bar Harbor, ME, USA. maryann.handel@jax.org.
authors: Mary Ann
authors: MA
authors: Handel
authors: The Jackson Laboratory, Bar Harbor, ME, USA. greg.carter@jax.org.
authors: Gregory W
authors: GW
authors: Carter
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D002843:Chromatin / Q000378:metabolism; D053263:Gene Regulatory Networks; D005854:Germ Cells / Q000166:cytology; D008297:Male; D008540:Meiosis; D051379:Mice; D008810:Mice, Inbred C57BL; D025341:Principal Component Analysis; D012313:RNA / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D017423:Sequence Analysis, RNA; D013090:Spermatocytes / Q000166:cytology / Q000378:metabolism*; D013091:Spermatogenesis; D013737:Testis / Q000166:cytology; D014157:Transcription Factors / Q000378:metabolism; D059467:Transcriptome
chemical_list: D002843:Chromatin; D014157:Transcription Factors; D012313:RNA
keywords: Maximum covariance analysis; Meiosis; Mouse; RNA-seq; Spermatogenesis; Transcriptome