Purification of rat Leydig cells: Increased yields after unit-gravity sedimentation of collagenase-dispersed interstitial cells

Procedures for purification of Leydig cells have facilitated studies of the ir regulatory biology. A multistep procedure that includes a filtration wit h nylon mesh (100-micron pore size) to separate interstitial cells from the seminiferous tubules, combining centrifugal elutriation and Percoll densit y gradient sedimentation, has been used to obtain a 95% enrichment of rat L eydig cells, However, the number of recovered Leydig cells by this procedur e represents only a small fraction of the 25 million, on average, that exis t in the adult rat testis. The objective of this study was to test whether the yield of purified Leydig cells might be enhanced by substitution of uni t-gravity sedimentation (S method) for the filter step (F method). We also asked whether a greater number of Leydig cell clusters, macrophages, or bot h would be recovered by this new method, and if the presence of Leydig cell clusters is associated with increased capacity for testosterone production in vitro. The number of purified Leydig cells was 1.9-fold higher for the S method than for the F method. with no differences in purity assessed by S P-hydroxysteroid dehydrogenase histochemical staining. Leydig cell clusters were also found in greater numbers with the S method both after collagenas e dispersion and at the end of the purification. No differences were seen i n testosterone production or in the number of macrophages present in the Le ydig cells that were prepared by the 2 methods. These results indicate that the new method recovers greater numbers of Leydig cells by collecting clus tered Leydig cells that are systematically eliminated when a filtration ste p is used.