Figure 1.

The fetal testis assay (FeTA). Human (6–12 GW), rat (14.5 dpc) or mouse (12.5 dpc) fetal testes are cultured on Millicell-CM Biopore membranes (pore size 0.4 μm, Millipore, Billerica, MA, USA) floating on 320 μl culture medium in tissue culture dishes at 37 °C in a humidified atmosphere containing 95% air/5% CO₂. The culture medium is phenol red-free DMEM/Ham F12 (1:1) without biological factors and hormones. The culture medium is completely changed every 24 h. Whole mouse and rat testes are cultured on the membrane, while human testes are cut into small pieces (around 0.2 mm³) due to their larger size and three to four pieces are randomly placed on the membrane (two to eight wells per testis). The secretion of various molecules in the medium (testosterone, INSL3, AMH, transferrin, lactate, etc.) can be quantified every day. At the end of the culture, explants can be fixed and tissue sections can be used to assess, for instance, the total number of cells per testis, apoptotic activity (cleaved caspase 3, TUNEL assay) or mitotic activity (Ki67, BrdU incorporation), expression of Leydig cell (steroidogenic actors, INSL3, LH Receptors, etc.), Sertoli cell (AMH, SOX9, etc.) and germ cell markers (pluripotency genes, such as OCT3-4, NANOG and differentiation genes such as c-KIT, NANOS2 and DNMT3L).