Dynamic transcriptome profiles within spermatogonial and spermatocyte populations during postnatal testis maturation revealed by single-cell sequencing
Authors: Grive Kathryn J. , Hu Yang , Shu Eileen , Grimson Andrew , Elemento Olivier , Grenier Jennifer K. , Cohen Paula E. 
Published: November 2018

Spermatogenesis is the process by which male gametes are formed from a self-renewing population of spermatogonial stem cells (SSCs) residing in the testis. SSCs represent less than 1% of the total testicular cell population, but must achieve a stable balance between self-renewal and differentiation. Once differentiation has occurred, the newly formed and highly proliferative spermatogonia must then enter the meiotic program in which DNA content is doubled, then halved twice to create haploid gametes. While much is known about the critical cellular processes that take place during the specialized cell division that is meiosis, much less is known about how the spermatocytes in the “first-wave” compare to those that contribute to long-term, “steady-state” spermatogenesis. Given the strictly-defined developmental process of spermatogenesis, this study was aimed at exploring the transcriptional profiles of developmental cell stages over the age of the animal. Using a combination of comprehensive germ cell sampling with high-resolution, single-cell-mRNA-sequencing, we have generated a reference dataset of germ cell gene expression. We show that discrete developmental stages possess significant differences in the transcriptional profiles from neonates compared to juveniles and adults. Importantly, these gene expression dynamics are also reflected at the protein level in their respective cell types. We also show differential utilization of many biological pathways with age in both spermatogonia and spermatocytes, demonstrating significantly different underlying gene regulatory programs in these cell types over the course of testis development and spermatogenic waves. This dataset represents the first unbiased sampling of spermatogonia and spermatocytes in the developing testis over developmental age, at high-resolution, single-cell depth. Not only does this analysis reveal previously unknown transcriptional dynamics of a highly transitional cell population, it has also begun to reveal critical differences in biological pathway utilization in developing spermatogonia and spermatocytes, including response to DNA damage and double-strand breaks.

Author Summary

Spermatogenesis is the process by which male gametes – mature spermatozoa – are produced in the testis. This process requires exquisite control over many developmental transitions, including the self-renewal of the germline stem cell population, commitment to meiosis, and ultimately, spermiogenesis. While much is known about molecular mechanisms regulating single transitions at single time points in the mouse, much less is understood about how the spermatogenic progenitor cells, spermatogonia, or the meiotic cells, spermatocytes, of the testis change over developmental age.

Our single-cell-mRNA-sequencing analysis is the first to profile both spermatogonia and spermatocytes from neonatal mice through adulthood, revealing novel gene expression dynamics and differential utilization of biological pathways. These discoveries help us to understand how the spermatogenic progenitors of this population modulate their activity to adapt to a changing testicular environment. Furthermore, they also begin to explain previously-observed differences - and deficiencies - in spermatocytes that are derived from the first “wave” of spermatogenesis. Overall, this dataset is the first of its kind to comprehensively profile gene expression dynamics in male germ cell populations over time, enriching our understanding of the complex and highly-orchestrated process of spermatogenesis.