An enzymatic method for the isolation of mouse Leydig cells.
Mouse Leydig cells were obtained by dispersion of testes of adult animals (aged 6-15 months) with a neutral protease from B. polymxa (dispase; EC 3.4.24.4). The crude Leydig cell suspension was purified by centrifugation on a discontinuous Percoll gradient using a special centrifugation procedure similar to elutriation. The crude cell suspension obtained from 50 testes could be processed in one run. The combination of these two methods yielded 320000 +/- 53000 Leydig cells/testis (n = 554 testes). The purity of the Leydig cell fraction was greater than or equal to 95% (nucleated cells) based on morphological and histochemical (staining for naphthyl esterase) identification. The purified Leydig cells showed an excellent ultrastructural appearance. More than 98% excluded trypan blue. In the presence of NADPH, testosterone biosynthesis was increased only 1.15 +/- 0.1-fold yielding a "quality factor" of 34.8. Maximal hCG doses induced 10(6) purified Leydig cells to produce 5 nmol testosterone/hr. (40-fold stimulation in comparison to basal values). The Leydig cells showed 43100 +/- 2500 LH/hCG receptors and an association constant of Ka = 1.95 x 10(9) M-1. Due to the reproducibility of the method, to the yield as well as to the morphological and functional state of the purified Leydig cells at least 25% of laboratory animals could be saved.
Merkel
U
U
Department of Biochemistry II, University of Heidelberg, FRG.
Angermüller
S
S
Merz
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WE
D000818:Animals; D002469:Cell Separation; D002498:Centrifugation; D007985:Leydig Cells / Q000166:cytology* / Q000378:metabolism; D008297:Male; D051379:Mice; D011974:Receptors, LH / Q000378:metabolism; D013739:Testosterone / Q000096:biosynthesis
D011974:Receptors, LH; D013739:Testosterone