The molecular building blocks of human cells have been increasingly mapped in large-scale projects by emerging high-throughput antibody profiling and sequencing methods. These transformational efforts have shown remarkable progress in resolving the expression levels and spatial locations of proteins in many human cells. This thesis aimed to characterize the spatial protein expression at the single-cell level in human reproductive tissues, testis and fallopian tube (FT), and additionally study how aberrantly expressed testis-proteins repurposed in non-small cell lung cancer (NSCLC) affect the immune microenvironment.

In Paper I, more than 500 proteins with elevated RNA expression levels in the testis were profiled in eight different cell types with immunohistochemistry (IHC). Several poorly characterized proteins, so-called “missing proteins,” were localized at the cell-type level at various stages of spermatogenesis, providing novel insights into their possible function.

In Paper II, a spatiotemporal map of human spermatogenesis was created by combining single-cell transcriptomics and multiplex IHC. High-throughput image analysis determined the cell state-specific protein expression for almost 500 proteins. By examining RNA and protein correlation dynamics, we highlighted the complex spatiotemporal landscape of the human testis. These proteins serve as targets for functional studies.

In Paper III, protein-coding genes elevated in FT based on RNA levels were profiled by IHC, and most proteins were functionally related to cilia motility, a mechanism necessary for creating the tubal flow essential for fertilization. Of 133 proteins annotated at the cell-type level, most were exclusive to ciliated cells, including several proteins previously not described in motile cilia.

In Paper IV, cancer-testis antigens (CTA) were characterized by IHC on more than 300 immunophenotyped NSCLC patients. CTAs are typically expressed in the testis and harbor immunogenic properties that may be used as treatment targets due to aberrant expression in NSCLC. CTAs were associated with immune profiles, such as macrophage and plasma cell infiltration, possibly demonstrating an in situ immunogenic effect. These associations can be studied and exploited as potential immunotherapy targets.

This thesis defines the spatial proteome of reproductive tissues at the single-cell resolution and identifies many proteins with previously unknown functions in reproduction. The integrative approach to mapping tissue-specific cellular diversity at the molecular level shows the importance of combining RNA and protein detection methods. The thesis developed emerging methods like multiplexed staining and bioimage analysis, which hold promise for large-scale efforts. This work significantly contributes to the cellular atlas of reproductive tissues, which historically have not been well-studied.