Dose response relationship between Denosumab treatment and germ cell proliferation of humanized RANKL mice in vivo and in testicular tissue cultures

Today there exist no treatment that can improve semen quality in more than 85% of infertile men. Interaction between germ and Sertoli cells is critical for sperm production, and Denosumab, an inhibitor of RANKL, has previously been shown to stimulate sperm production in a fraction of infertile men. Here, we show in a humanized RANKL mouse that Denosumab dose dependently increase germ cell proliferation and testicular weight. RANKL was modified so it can be blocked by Denosumab. Fragments of mouse testis were cultured with the RANKL inhibitor Denosumab and compared with vehicle treatment. Testis tissue were cultured for 48 hours. Germ cell proliferation was assessed by BrdU incorporation and Denosumab treatment 1-100 ng/ml increased germ cell proliferation compared with vehicle although no effect on germ cell apoptosis was observed. This effect is physiologically relevant as the seminal fluid concentration reached 120 ng/ml following Denosumab injection of 60 mg dosing sc. once. Mice with humanized RANKL were treated with Denosumab 0,1-3 mg/kg once and sacrificed after 6 weeks. All mice treated with Denosumab had increased testicular weight and an increased serum AMH and Inhibin B although only statistically significant for 0.1 mg/kg. This shows that transient RANKL inhibition leads to increased germ cell output after 1 full spermatogenesis that takes 35 days in mice. This study smggests that even low doses of Denosumab can stimulate mouse spermatogenesis; however, further investigation is critical for understanding the interplay between Sertoli cell function, spermatogenesis, and RANKL inhibition in humans.