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Abstract

Purification of rat Leydig cells: Increased yields after unit-gravity sedimentation of collagenase-dispersed interstitial cells 
Salva,Antonio; Klinefelter,Gary R.; Hardy,Matthew P.
Journal of Andrology 22(4): 665-671
Publication date: 2001


Procedures for purification of Leydig cells have facilitated studies oftheir regulatory biology. A multistep procedure that includes a filtrationwith nylon mesh (100-micron pore size) to separate interstitial cells fromthe seminiferous tubules, combining centrifugal elutriation and Percolldensity gradient sedimentation, has been used to obtain a 95% enrichment ofrat Leydig cells. However, the number of recovered Leydig cells by thisprocedure represents only a small fraction of the 25 million, on average,that exist in the adult rat testis. The objective of this study was to testwhether the yield of purified Leydig cells might be enhanced bysubstitution of unit-gravity sedimentation (S method) for the filter step(F method). We also asked whether a greater number of Leydig cell clusters,macrophages, or both would be recovered by this new method, and if thepresence of Leydig cell clusters is associated with increased capacity fortestosterone production in vitro. The number of purified Leydig cells was1.9-fold higher for the S method than for the F method, with no differencesin purity assessed by 3beta-hydroxysteroid dehydrogenase histochemicalstaining. Leydig cell clusters were also found in greater numbers with theS method both after collagenase dispersion and at the end of thepurification. No differences were seen in testosterone production or in thenumber of macrophages present in the Leydig cells that were prepared by the2 methods. These results indicate that the new method recovers greaternumbers of Leydig cells by collecting clustered Leydig cells that aresystematically eliminated when a filtration step is used.