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PURIFICATION OF RAT LEYDIG CELLS: INCREASED YIELDS AFTER UNIT-GRAVITY SEDIMENTATION OF COLLAGENASE-DISPERSED INTERSTITIAL CELLS
Citation:
Salva, A., G R. Klinefelter, AND M. P. Hardy. PURIFICATION OF RAT LEYDIG CELLS: INCREASED YIELDS AFTER UNIT-GRAVITY SEDIMENTATION OF COLLAGENASE-DISPERSED INTERSTITIAL CELLS. JOURNAL OF ANDROLOGY 22(4):665-671, (2001).
Description:
Abstract
Procedures for purification of Leydig cells have facilitated studies of their regulatory biology. A multistep procedure, that includes a filtration with nylon mesh (100 micron pore size) to separate interstitial cells from the seminiferous tubules, combining centrifugal elutriation and Percoll density gradient sedimentation, has been used to obtain a 95% enrichment of rat Leydig cells. However, the number of recovered Leydig cells by this procedure represents only a small fraction of the 25 million that exist on average 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 unit gravity sedimentation (S method) for the filter step (F method). We also asked whether a greater number of Leydig cell clusters and/or macrophages would be recovered by this new method, and if the presence of Leydig cell clusters is associated with increased capacity for testosterone (T) 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 3b-HSD histochemical staining. Leydig cell clusters were also found in greater numbers with the S method both after collagenase dispersion and at the end of the purification. No differences were seen in T production or in the number of macrophages present in the Leydig cells that were prepared by the two methods. These results indicate that the new method recovers greater numbers of Leydig cells by collecting clustered Leydig cells that are systematically eliminated when a filtration step is used. Greater enrichment of clustered Leydig cells might improve our ability to assess structure and function of Leydig cells in hyperplasic nodules or Leydig cells in different states of reproductive development.