Spermatogenesis is a well-organized and tightly regulated biological process composed of four distinct biological activities: 1) Continuous stem cell self-renewal and the generation of transit amplifying progenitor spermatogonia 2) the formation of fully differentiated, mitotic spermatogonia from the progenitors, 3) meiosis, and 4) the orderly and coordinated differentiation of haploid cells into spermatozoa (spermiogenesis). The transition between these developmental states is precisely timed, and is dependent upon both germ cell intrinsic and extrinsic factors secreted by supporting cells (e.g. Sertoli, Leydig, Myoid and Immune cells). In the past, the spatial organization of these cells across the seminiferous tubule has been studied histologically, and their contribution to spermatogenesis has been explored through the purification of predefined cell types, followed by gene expression profiling of the cell populations. However, these approaches rely on known markers to define cell population. Furthermore, past genomic profiling methods reveal average properties of cell populations ? not individual cells. Hence, the existing view of spermatogenesis fails to: 1) describe the full extent of inter-cellular heterogeneity 2) identify rare cell populations e.g., transitional states, or 3) integrate and reconcile variation in gene expression of a cell depending on its specific stage of the cycle of the seminiferous epithelium. To overcome these limitations, we will use the newly available single-cell RNA-sequencing technology to create an unbiased cellular catalog of the adult testis. However, single cell RNA-seq, as currently performed, requires the dissociation of complex tissues and the loss of spatial information. Therefore, to begin addressing and resolving the complexity of the seminiferous tubule, we will integrate gene expression profiles from individual cells with their spatial location using single molecule RNA fluorescent in-situ hybridization (smFISH). To demonstrate the power of combining single-cell sequencing and smFISH we will begin with the development of a high-resolution map of Sertoli cells (SC), across the seminiferous tubule? a cell type known to exhibit stage-specific transcriptional dynamics. The smFISH on the SC populations will resolve the distribution of individual subtypes of SC and potentially highlight their changes during different stages of the seminiferous epithelium cycle. In short, this proposal will provide a systematic analysis of germ cells and the somatic environment in the mouse testis. We will provide a complete inventory of major cell types, mRNA markers, and will specifically map subtypes of Sertoli cells to known stages of the seminiferous epithelium at an unprecedented resolution. All genomic data and spatial maps will be shared with the community as a resource essential for understanding the structural and functional diversity of germ cell development, helping to answer new mechanistic questions regarding the regulation and signaling processes that guide this important developmental process. The information gained from these studies will provide a foundation for future efforts to drive in vitro gametogenesis or restore germ cell in vivo.

Public Health Relevance

Infertility affects 1 in 6 couples worldwide. Known genetic factors account for ~10-15% of male factor infertility cases, whereas, the remaining ~35-40% of the male factor cases have an unknown etiology. Therefore, a comprehensive understanding of the molecular mechanism / pathways involved in proper germ cell development is required for better a diagnoses or developing effective therapies.

National Institute of Health (NIH)
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Exploratory/Developmental Grants (R21)
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Cellular, Molecular and Integrative Reproduction Study Section (CMIR)
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Moss, Stuart B
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University of Michigan Ann Arbor
Schools of Medicine
Ann Arbor
United States
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Green, Christopher Daniel; Ma, Qianyi; Manske, Gabriel L et al. (2018) A Comprehensive Roadmap of Murine Spermatogenesis Defined by Single-Cell RNA-Seq. Dev Cell 46:651-667.e10