BIOCHEMICAL AND P-31-NMR SPECTROSCOPIC EVALUATION OF IMMOBILIZED PERFUSED RAT SERTOLI CELLS

Cell immobilization and perfusion are used for physiologic studies of Sertoli cells with phosphorus 31 nuclear magnetic resonance (NMR) spec troscopy and biochemical methods. In this study the P-31 NMR spectra o f Sertoli cells isolated from 18-to 21-day-old rats and immobilized in agarose threads continuously perfused with oxygenated Dulbecco's modi fed Eagle medium were obtained at 81 MHz on an NMR system. Cytosolic C a2+, intracellular Mg2+, lactate and pyruvate, and oxygen consumption were measured with standard biochemical methods. Perfused Sertoli cell s maintain a stable intracellular adenosine triphosphate concentration for more than 10 hours. Sertoli cells placed in cold storage overnigh t and then subjected to perfusion partially regenerate cellular adenos ine triphosphate levels. Sertoli cells consume an average of 4.8 +/- 0 .4 nmol O-2/min/10(6) cells and maintain average ambient lactate and p yruvate levels of 7.1 +/- 0.8 mg/dl and 0.65 +/- 0.05 mg/dl, respectiv ely, with a lactate/pyruvate ratio in the range 8 to 12. The basal Ca- i(2+) of Sertoli cells is 98 +/- 0.7 nmol/L (n = 58), which declines t o a level less than 10 nmol/L when the Sertoli cells are perfused with a calcium-free medium. Perfusion of Sertoli cells with a sodium-free medium, with 10(-6) mol/L carbonyl cyanide P-trifluoromethoxy-thenylhy drozone, or with Ca2+ ionophore A23187 at a concentration of 10(-6) mo l/L increases the Ca-i(2+) to a level of 426 +/- 107 nmol/L, 274 +/- 2 9 nmol/L, or 282 +/- 57 nmol/L, respectively. A bioreactor for physiol ogic studies of Sertoli cells in real time with NMR spectroscopy has b een developed, These data demonstrate that isolated, immobilized, and perfused Sertoli cells are stable for prolonged periods. In addition, these data suggest that Sertoli cells possess a functional Na+-Ca2+ an tiporter and that they sequester extracellular Ca2+ in one or more int racellular compartments.