Abstract
BackgroundThe 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.ResultsGerm 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.ConclusionsThis 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2865-1) contains supplementary material, which is available to authorized users.
Highlights
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
For the C57BL/6J (B6) strain used in this study, this developmental window captures the initial differentiation of spermatogonia into early leptonema, through the stages of pachynema and diplonema
Meiotic substage-specific gene expression derived by covariance analysis To identify signatures for meiotic substage-specific gene expression, we developed a novel permutation-based maximum covariance analysis (PMCA), which maps groups of co-expressed genes to combinatorial cytological markerbased staging based antibodies to STRA8, SYCP3, phosphorylated H2AFX, and HISTH1T
Summary
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. Ball et al BMC Genomics (2016) 17:628 spermatogenesis includes regular and asynchronous initiation of subsequent waves of spermatogenic differentiation This asynchronous and continuous process of spermatogenesis has made it difficult to achieve molecular characterization of specific cell types in the lineage. This has been the case with respect to analysis of the defining process of gametogenesis, meiosis, which occurs in spermatocytes. The complex events of meiosis I prophase include recombination, homologous chromosome pairing, and synapsis, taking place as spermatocytes progress through the leptotene, zygotene, pachytene and diplotene substages. Because of the genetic importance of meiotic recombination for the production of chromosomally normal gametes and offspring, there has been great interest in elucidating the molecular hallmarks and their underlying transcriptional signatures that define the meiotic spermatocyte substages
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