Endocrine Abstracts

JOINT3366

Introduction: Previous research has demonstrated that inhibition of RANKL signalling with the monoclonal antibody denosumab results in increased sperm count and testis weight in a humanized mouse model. Additionally, when culturing adult testis from the humanized RANKL mouse, inhibition of RANKL resulted in an increase in germ cell proliferation. Based on these effects we hypothesized that RANKL signalling may also play a role in prepubertal and foetal testis. Therefore, this project aims to establish an ex vivo culture model for prepubertal and foetal mouse testis tissue to examine the involvement of RANKL signalling in testicular development and in the onset of spermatogenesis.

Materials and Methods: Three different ex vivo culture approaches were tested using prepubertal and foetal mouse testis tissue, namely hanging drop, porous membranes and agarose gel fragments. The prepubertal testis tissue was cultured for 48 hours, 4 days and 1 week. Foetal testis tissue was cultured for 48 hours or 1 week. After the culture period, the tissue was fixed, and paraffin embedded. Hematoxylin and eosin (H&E) staining was performed to evaluate tissue morphology, and immunohistochemical stainings and analyses was subsequently conducted to assess cell proliferation (BrdU incorporation) and apoptosis (cleaved PARP).

Results: Analysis of the three culture methods and culture lengths are currently ongoing. The optimal culture method will be determined based on preservation of tissue morphology, sustained cell proliferation, and minimal apoptosis.

Discussion: Preliminary results show that ex vivo culture of both prepubertal and foetal mouse testis tissue is feasible with the tested approaches. Ongoing analysis will identify the most effective culture method. The establishment of ex vivo models for both developmental stages will provide a crucial framework to investigate the role of RANKL signalling in testicular development.