Electrophysiological study of single Leydig cells freshly isolated from rat testis. I. Technical approach and recordings of the membrane potential in standard solution

Abstract

Single Leydig cells were isolated from rat testis by a collagenase digestion procedure and purified through a 21,000 g self generated densities gradient of 35% Percoll. A method including collagen and fibronectin was proposed to attach freshly prepared Leydig cells to the bottom of plastic Petri dishes. Four hours after the isolation of the cells, it was simultaneously possible to determine their membrane potential by a standard electrophysiological technique using intracellular microelectrodes and to judge cellular integrity by direct microscopic observations. In standard Earle's solution, changes of membrane potentials appeared to be biphasic. On 198 impaled cells, 18 +/- 1 S after the impalement was effective, the membrane potential reached a most negative value (MP1) (-37.6 +/- 0.7 mV), followed by a gradual depolarization to a steady state (MP2) (-25.1 +/- 0.6 mV) which remained constant for a few minutes. In standard Earle's solution, the membrane resistance was low or decreasing towards the most negative potential, then it increased towards the steady potential. At this state, the average value of the cell input resistance was 65.9 +/- 6.0 M omega (n = 16). No action potential was observed either in standard Earle's solution or under a depolarizing current state. It was concluded that the electrophysiological characteristics of the Leydig cell are similar to those of fibroblasts and macrophages, three types of cells with the same mesenchymal origin, present in the interstitial tissue of the rat testis. But the resting potential of the Leydig cell is higher and this secreting cell does not elicit hyperpolarizing oscillations at the steady state, under mechanical or electrical stimuli.