A spatio-temporal atlas of human spermatogenesis based on single-cell transcriptomics and multiplex antibody imaging

• Testis is one of the most complex human organs, with spermatogenesis involving thousands of genes and proteins that are activated or repressed through multiple cell states, from spermatogonial stem cells to mature sperms. Understanding the intricate functions and mechanisms at each step of this process requires a multi-dimensional approach that integrates both quantitative and qualitative methods. Here, we used integration of multiple datasets based on scRNA-seq and multiplex, antibody-based spatial proteomics with automated image analysis to generate a high-resolution spatio-temporal map of human testis. Based on transcriptomics analysis we identified 12 distinct states of germ cells corresponding to subsets of spermatogonia, spermatocytes, and spermatids. A large-scale fluorescent-based multiplex immunohistochemistry pipeline was utilized to execute an in-depth characterization of approximately 500 proteins expressed in different germ cells and map them to their spatial localization. The generated data allowed us to cluster the proteins into expression groups based on common patterns of cell state-specific expression, forming the basis for downstream functional analysis. In total, we identified 18 different cell type clusters and we showed that the proteins in these groups were linked to germ cell-specific functions related to differentiation and cell cycle regulation at different phases of spermatogenesis. The analysis has the potential to characterize numerous poorly defined proteins not previously described in the context of male sperm development. By also studying the mRNA and protein correlation dynamics along spermatogenesis, we identified a large number of proteins with low expression correlation coefficients to the known germ cell specific proteins, thus demonstrating the complex spatio-temporal nature of the transcriptional dynamics in this tissue. In summary, we present a spatio-temporal single-cell type reference map of adult human testis that decodes the complexity of human germ cells, tissue heterogeneity, and homeostasis. The current automated staining-to-image analysis pipeline makes it possible to extract quantitative data from germ cells in human testis tissue. The data is presented as an open-access resource as part of the Human Protein Atlas (www.proteinatlas.org), thus contributing to the understanding of the male reproductive tissue and providing a knowledge-based for processes linked to infertility or various disorders of the male gonadal system.

Nyckelord

testis

multiplex immunohistochemistry

spermatogonial stem cells

male germ cell differen-tiation

Publikations- och innehållstyp

vet (ämneskategori)

ovr (ämneskategori)