Figure 4.

Differentiation in vitro of post-natal day (PND) 10 rat Leydig cells in the absence (open bars) or presence (filled bars) of hCG. (A) Cells were purified from the abdominal testes of PND10 male Sprague Dawley rats by mechanical dispersion followed by unit sedimentation, then cultured in serum-free medium at 400,000 cells per well of 12-well plates at 37°C. Medium was changed every 2–3 days, with aliquots collected exactly 48 h after the last medium change, for measurement of INSL3 using rat INSL3-specific TRFIA (10). (B) Cells prepared as above were seeded in parallel at 30,000 cells per well into 96-well plates and subjected to the WST-1 (4-[3-(4-Iodophenyl)-2-(4-nitro-phenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) assay to measure cell numbers, as described by the manufacturers (Roche Applied Science (Castle Hill, NSW, Australia). The inset in the upper panel indicates the fold-increase in INSL3 secretion calculated on a per cell basis for key times relative to basal expression on day 1, thus representing the differentiation of the individual Leydig cells, discrete from any effects on cell proliferation or cell death. This shows that while hCG has a marked effect on Leydig cell proliferation and/or survival, it is not essential for cell differentiation, though it does augment it. Animal experimentation was conducted under the terms of permit S-2010-102 of the Animal Ethics Committee, University of Adelaide.