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An LH-responsive Leydig cell preparation (containing 6 ± 2% Leydig cells) was obtained by collagenase treatment of rat testis. Centrifugation of this cell preparation through a 13% Ficoll solution for 10 min at 1500 g resulted in a four times purification of the Leydig cells, with a concomitant increase in steroidogenic activity. Addition of 0·2% albumin to the 13% Ficoll solution, adjusted to 280 mosmol/l, resulted in a further twofold purification of the Leydig cells paralleled by a twofold increase in steroidogenic activity. Centrifugation of these Ficoll-albumin-purified Leydig cells through a 6% dextran solution for 2 min at 100 g resulted in a further 1·7 times purification of the Leydig cells. A combination of the two centrifugation steps resulted in a 12·5 times purification of Leydig cells compared with the original crude cell suspension, while an increase in steroidogenic activity of 22·5 times was obtained. This final cell preparation contained 59 ± 17% Leydig cells (mean ± s.d., n = 6). The recovery of Leydig cells was 29%.

Collagenase treatment of testes deficient in spermatogenesis resulted in a cell preparation with the same steroidogenic activity as Ficoll-purified cells from normal testes. Centrifugation of these cells through a 13% Ficoll solution gave only a limited increase in the steroidogenic activity. Isopycnic centrifugation of the crude cell preparation on a discontinuous Ficoll metrizoate gradient resulted in two discrete peaks of Leydig cells, one peak at a density of 1·039–1·055 g/ml and one at a density of 1·068–1·088 g/ml. Both types of cells produced testosterone. In the presence of LH, cyclic AMP production in both types of Leydig cells increased, but testosterone production was only increased by LH in the 'denser' Leydig cells and not in the 'light' Leydig cells.

No difference in sensitivity to LH could be observed between the Leydig cell preparations of different purity. Using a 60 min pre-incubation period the highest testosterone response was obtained with 100–1000 ng LH/ml. The same maximum testosterone response was obtained with 10–100 ng LH/ml when the pre-incubation period was omitted.