Purification of rat leydig cells: increased yields after unit-gravity sedimentation of collagenase-dispersed interstitial cells

• Published in: Journal of andrology Vol. 22; no. 4; pp. 665 - 671
• Main Authors: Salva, A, Klinefelter, G. R, Hardy, M. P
• Format: Journal Article
• Language: English
• Published: Oxford, UK Am Soc Andrology 01.07.2001; Blackwell Publishing Ltd; American Society of Andrology
• Subjects: Animals; Antibodies, Monoclonal - pharmacology; Biological and medical sciences; Cell Communication; Cell Count; Cell Separation - methods; Clusters; Collagenases; Fundamental and applied biological sciences. Psychology; Gap Junctions; Gravitation; Immunohistochemistry; Leydig Cells - cytology; Leydig Cells - metabolism; macrophages; Macrophages - cytology; Macrophages - immunology; Male; Mammalian male genital system; Morphology. Physiology; Rats; Rats, Sprague-Dawley; Testosterone - biosynthesis; Vertebrates: reproduction; Gravity separation; Purification; Rat; Rodentia; Cluster; Testicle; Male genital system; Gap junction; Leydig cell; Vertebrata; Mammalia; Investigation method; Animal
• ISSN: 0196-3635; 1939-4640
• DOI: 10.1002/j.1939-4640.2001.tb02227.x

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, on average, that exist 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, macrophages, or both 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 3β‐hydroxysteroid dehydrogenase 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 testosterone production or in the number of macrophages present in the Leydig cells that were prepared by the 2 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.