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A method involving centrifugal elutriation followed by density gradient centrifugation and incubation with a macrophage monoclonal antibody has been investigated to separate and characterize Leydig cells and macrophages from adult rat testes. After dispersion of the testes with collagenase, the isolated interstitial cells were found to contain 18% Leydig cells and 12% macrophages. These cells were then separated by centrifugal elutriation into eight fractions (F1–F8) (9 to 74 ml/min at 386 g). Each of these fractions was then further purified by density gradient centrifugation on 0–90% Percoll gradients. After centrifugal elutriation, the macrophages were mainly eluted in the first three fractions (F1–F3), whereas the Leydig cell percentage increased in each fraction with increasing flow rate. After further purification of each fraction on Percoll gradients, high percentages of macrophages (11–20%) were found in fractions F1–F3 (average density 1·045 g/ml), containing 11–37% Leydig cells. Less than 3% of the cells in fraction F4–F8 (average density 1 ·075 g/ml) were macrophages and more than 95% were Leydig cells. Heterogeneity of Leydig cells with respect to sedimentation velocities and function was found. Leydig cells from elutriated-and Percoll-purified fractions F4–F8 were heterogeneous with respect to testosterone and cyclic AMP (cAMP) production but showed a similar binding capacity for ¹²⁵I-labelled human chorionic gonadotrophin. Leydig cells with the highest sedimentation velocity (35·7 mm/h-g) from fractions F7 and F8 were approximately twofold more responsive to LH (3·3 nmol/l) with respect to testosterone and cAMP production compared with Leydig cells with the lowest sedimentation velocity (20·7 mm/h-g).

The elutriated and Percoll-purified cells (corresponding to fractions F4–F8) were further purified by incubation with magnetic beads coated with a macrophage monoclonal antibody; this yielded very pure Leydig cells containing <0·3% macrophages. The incubation temperature (room temperature or 4 °C) during the purification with magnetic beads did not affect the degree of purity or the responsiveness of the Leydig cells to LH. The removal of the remaining macrophages with magnetic beads did not have any significant effect on the Leydig cell responsiveness to LH.

It was concluded that Leydig cells purified by elutriation and density gradient centrifugation are heterogeneous with respect to their sedimentation velocities and responses to LH; the higher the sedimentation velocity, the higher is their capacity to respond to LH. Leydig cells free from macrophages can be prepared by further purification using magnetic beads coated with a macrophage monoclonal antibody.

Journal of Endocrinology (1991) 130, 357–365

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