Functional and morphological studies on isolated Leydig cells: purification by centrifugal elutriation and metrizamide fractionation.
Rat interstitial cells were fractionated by centrifugal elutriation to facilitate the purification of Leydig cells for analysis of mechanisms of gonadotropin action in vitro. By this procedure, 10(9) collagenase-dispersed interstitial cells from adult rat testes were separated into 12 fractions in about 1 h. Fractions 1-7 (sedimentation velocities, 1.9-12 mm/h.g) comprised 80-85% of the total cells applied, including erythrocytes, lymphocytes, germinal cells, macrophages, endothelial cells, damaged Leydig cells, and contained less than 4% intact Leydig cells. Fractions 8-12 (sedimentation velocities, greater than 12 mm/h.g) comprised 15-20% of the original cells and contained 90-95% intact Leydig cells. Despite their different sedimentation velocities, the Leydig cell-rich fractions were similar in their LH receptor content (mean +/- SD, 36,115 +/- 4,815 sites/cell) and showed similar 5-fold increases above the original cell preparation in testosterone and cAMP responses to hCG. The pooled Leydig cell-rich fractions (8-12) were further resolved on 16-24% Metrizamide gradients into 5 bands. Bands II-V (density range, 1.075-1.110 g/ml) contained pure Leydig cells, and band I (1.048 g/ml) contained pachytene spermatocytes, the contaminating cell type present in the Leydig cell-rich fractions obtained by elutriation. Each of the 4 Leydig cell-rich bands showed similar morphology and functional activity. Essentially similar results were observed using 14-32% Metrizamide gradients. Leydig cells desensitized in vivo by hCG treatment and isolated by elutriation were also resolved by Metrizamide gradients into 4 bands, but showed a redistribution in the gradient, due to the shift of about 50% of the cells originally present in the heaviest bands to lower density fractions. However, in spite of their changes in density, the Leydig cell bands still showed similar degrees of receptor down-regulation and impairment of the steroid responses to hCG in vitro. This study has demonstrated that centrifugal elutriation is a rapid and effective method for obtaining large quantities of purified (greater than 90%) and active Leydig cells. Further resolution of the Leydig cell-rich fractions in Metrizamide gradients has allowed complete Leydig cell purification, which is not achieved by density gradient centrifugation alone. Since less responsive or inactive Leydig cells displayed various degrees of structural damage, such cells should not be considered as a population of physiological significance.(ABSTRACT TRUNCATED AT 400 WORDS)
D000818:Animals; D002469:Cell Separation / Q000379:methods; D002499:Centrifugation, Density Gradient / Q000379:methods; D006063:Chorionic Gonadotropin / Q000378:metabolism; D000242:Cyclic AMP / Q000096:biosynthesis; D005640:Follicle Stimulating Hormone / Q000378:metabolism; D007985:Leydig Cells / Q000166:cytology / Q000502:physiology*; D007986:Luteinizing Hormone / Q000378:metabolism; D008297:Male; D008793:Metrizamide; D011284:Pregnenolone / Q000096:biosynthesis; D051381:Rats; D011919:Rats, Inbred Strains; D011956:Receptors, Cell Surface / Q000378:metabolism; D011962:Receptors, FSH; D011974:Receptors, LH; D013739:Testosterone / Q000096:biosynthesis
D006063:Chorionic Gonadotropin; D011956:Receptors, Cell Surface; D011962:Receptors, FSH; D011974:Receptors, LH; D013739:Testosterone; D011284:Pregnenolone; D007986:Luteinizing Hormone; D005640:Follicle Stimulating Hormone; D000242:Cyclic AMP; D008793:Metrizamide