The Generation of a Testicular Peritubular Cell Line from Giant Pandas (Ailuropoda melanoleuca)

Giant pandas (Ailuropoda melanoleuca), a flagship endangered species under priority preservation in China, remain poorly understood in terms of their testicular physiology and the mechanisms underlying spermatogenesis. Testicular peritubular cells (TPTCs), a crucial somatic cell type surrounding seminiferous tubules, secrete growth factors such as GDNF and CSF1 and release inflammatory factors such as IL-6 and IL-1β, contributing to the testicular niche and immune homeostasis. The contraction of TPTCs also facilitates the transport of sperm towards the epididymis. Nonetheless, TPTCs tend to undergo replicative senescence in vitro, which is a hinderance to their in-depth study. Here, we generated an immortalized monoclonal cell line with TPTC identities from giant pandas via lentiviral transduction of SV40 large T antigen into the cells and the subsequent clonal isolation through limiting dilution. The generated cell line, designated PD-TPTCs, demonstrated unlimited proliferative capacity and has been cultured for over five months and passaged more than 50 times to date. Characterization of PD-TPTCs revealed stable expression of key TPTC markers including ACTA2, MYH11, CNN1, and AR. Moreover, PD-TPTCs could respond to ATP and forskolin (FSK) stimulation with a pro-inflammatory gene expression profile and increased steroidogenic activity, respectively, and they were also amenable to lipofection. As such, the generated PD-TPTC line represents a promising cellular model for future mechanistic studies on the testicular niche, spermatogenesis, and reproductive disorders in giant pandas, laying the foundation for the development of novel assisted reproductive technology (ART) in this endangered species.