Abstract:
Neonatal development of the testis sets the stage for reproductive health throughout life. During this critical life stage, the spermatogonia migrate to the basal lamina of the tubule and establish the spermatogonial niche, the Sertoli cells proliferate and form the blood-testis-barrier to prevent autoimmune response to eventual sperm, and the adult Leydig cell lineage emerges from the mesenchyme to jumpstart testosterone biosynthesis. The balance of these acts is so important for reproductive health, though methods to study them remain limited. Classically, in vivo animal models remain the go-to model for reproductive and developmental toxicology. However, as complex cell culture methods improve and can now support dynamic systems, there are exciting opportunities to move away from in vivo models to consider mechanistic toxicology and developmental biology with complex in vitro models. The research presented in this dissertation seeks to develop and characterize novel in vitro models of the developing testis to advance the adoption of these powerful reductionist approaches in reproductive and developmental toxicology.Chapter 1 presents a state-of-the-science on the use of various in vitro models in reproductive biology, ranging from classical immortalized cell lines, to intricate three-dimensional cell printing to recreate the testis architecture. Chapter 2 describes a microculture model of the testis that dramatically reduces rodent tissue needs while retaining the necessary biological relevance and multicellular complexity. Chapter 3 details a computational assessment of bulk RNA-sequencing deconvolution techniques to characterize the cell proportions within complex in vitro models, using a neonatal testis microculture and human duodenal enteroid model as case studies. Chapter 4 focuses on the spermatogonia present within developing testis at two life stages to identify if the dynamics of differentiation and proliferation can be maintained in vitro. The research presented in this dissertation supports that in vitro are a valuable way to approach reproductive and developmental toxicology. The developmental stage of tissue, culture method, and culture supplementation are found to be critical factors when considering the phenotype and response of a model, demonstrating the necessity of benchmarking for all in vitro models. The next exciting areas are discussed, together with pilot work on human induced pluripotent stem cells models and future human tissue use in mechanistic toxicology and biology of the testis.