Reproduction Abstracts

Introduction: Early developmental events in the testis set the scene for future spermatogenesis. After birth, gonocytes, the precursors to type-A spermatogonia reproliferate and become transiently motile in a poorly understood process. Aberrations are associated with altered fertility or pre-malignant states. Gonocytes exist as overlapping subpopulations at varying developmental stages, which makes them difficult to study.

Materials and methods: Transgenic mice carrying enhanced green fluorescent protein under the control of the Oct4 promoter (Oct4:GFP) and a ubiquitous mTd Tomato gene were used. Isolated seminiferous tubules were explanted from fetal and neonatal testes with gonocyte development visualised using 4D time-lapse videomicroscopy. Using immunohistochemistry, qRT-PCR, and FACS analysis, markers associated with differentiation, apoptosis, and proliferation confirmed imaging events.

Results and discussion: An ex vivo testicular model in mice was used to follow gonocyte development in real time. After a period of quiescence, gonocytes proliferated and migrated to the basement membrane making extensive cell:cell contacts via lamellopodia which extended and retracted from their surface (days 1–4 postpartum). A novel process of cytoplasmic shedding was captured dynamically with a concomitant decrease in gonocyte size in a subpopulation found at the basement membrane. Additionally, a peak of apoptosis was seen at day 4 postpartum and confirmed with TUNEL staining. An understanding of gonocyte subpopulations behaviour is limited. This novel approach circumvents difficulties in studying testicular development by facilitating a dynamic study of gonocytes topographically organised within their microenvironment. For first time, cytoplasmic shedding is described in gonocytes making the transition to type A spermatogonia.