Interstitial cell heterogeneity in rat testes. II. Purification of cells by Percoll and metrizamide gradient centrifugation with preferential localization of gonadotropin binding sites in light cell fraction and hormone-induced steroidogenesis in heavier cell fraction.
The ability of 125I-labeled human chorionic gonadotropin (125I-labeled hCG) to bind and stimulate steroidogenesis was studied in light cells (density, 1.053-1.065 g/cm3) and heavier cells (density, 1.090-1.110 g/cm3) purified from collagenase-dispersed rat testicular interstitial cells by unit gravity sedimentation (Bhalla, V.K., Rajan, V.P., Burgett, A.C., and Sohal, G.S. (1987) J. Biol. Chem. 262, 5313-5321). Preferential localization of gonadotropin binding sites was demonstrated on light cells, and the heavier cells produced testosterone in response to hCG without occupancy of high affinity (Kd = 2.02 X 10(-10) M) binding sites. In this study, established methods for interstitial cell purification involving gradient centrifugation were utilized to demonstrate the cell heterogeneity. Light cells bound hCG with high affinity (Kd = 3 X 10(-10) M) without manifestation of steroidogenic response. The heavier cells responded to hCG with elicitation of steroidogenesis, but the occupancy was negligible. Stimulation of steroidogenesis by hCG in heavier cells was dose and time dependent. Dibutyryl and bromo cyclic AMP (1 mM) also promoted steroidogenesis comparable to a level stimulated by the tropic hormone (700% stimulation). The concept of spare receptors was tested in purified cell fractions. Upon cell purification, no saturable high affinity binding sites were observed in the heavier cell fraction. Autoradiographic analyses at the electron microscopical level supported this conclusion. Our data suggest that target cell activation is not preceded by hormone occupancy of high affinity binding sites. A model for defining the functional domains of the physiological receptor for hCG is presented.
D000818:Animals; D002469:Cell Separation; D002499:Centrifugation, Density Gradient / Q000379:methods; D000242:Cyclic AMP / Q000096:biosynthesis; D007700:Kinetics; D007985:Leydig Cells / Q000166:cytology* / Q000187:drug effects; D008297:Male; D008793:Metrizamide; D008854:Microscopy, Electron; D011205:Povidone; D051381:Rats; D011974:Receptors, LH / Q000032:analysis*; D012822:Silicon Dioxide; D013737:Testis / Q000166:cytology*; D013739:Testosterone / Q000096:biosynthesis*
D011974:Receptors, LH; D013739:Testosterone; C016039:Percoll; D012822:Silicon Dioxide; D000242:Cyclic AMP; D011205:Povidone; D008793:Metrizamide