Cell-cell interactions between germ cells and the essential somatic supporting cells, Sertoli cells, are essential for the maintenance of male fertility. Intracellular calcium (Ca2+) signaling controls many processes in mammalian physiology, including important aspects of fertility and reproduction. Importantly, disruption of Ca2+ channels in mice can result in infertility. Despite the clear importance of this pathway in male fertility, it remains unknown exactly which cells in the male gonads express the Ca2+ channels that are indispensable for normal gonadal function, how these channels are activated by components of the extracellular environment and whether Ca2+ signals are propagated between neighboring cells and cell communities. Therefore, this project has two aims: 1) to visualize intercellular Ca2+ signaling between both germ cells and Sertoli cells and, 2) to define the 3D architecture of testicular cells with a focus on cellular connections. To do this, we use genetically engineered mouse models that express fluorescent sensors in either germ cells or Sertoli cells and visualize stable and dynamic signals by confocal microscopy. Preliminary results show that upon agonist stimulation Sertoli cells exhibit robust intracellular Ca2+ signals, which can propagate across defined regions of the tissue through mechanisms yet to be elucidated. Through volumetric imaging we have also started to map cell-cell connectivity patterns at various regions of the seminiferous tubules. Overall, our study is working toward defining cellular connectivity in the testis, shedding important insights into mechanisms of cellular information flow. Ultimately, this work will provide critical new knowledge on normal mammalian reproductive development and cell synchronization and may provide new avenues for modulating male fertility.