DEVELOPMENTAL-CHANGES IN LEVELS OF LUTEINIZING-HORMONE RECEPTOR AND ANDROGEN RECEPTOR IN RAT LEYDIG-CELLS

To further assess the hormonal response capabilities of Leydig cell progenitors (PLC) from 21-day-old rats, their levels of LH and androgen receptors (LH-R and AR) were measured and compared to those of isolated immature (ILC) and adult Leydig cells (ALC) from 35- and 90-day-old rats, respectively. Levels of LH receptor were estimated by Scatchard analysis of binding to [I-125]hCG, and levels of LH receptor mRNA were determined by Northern blot analysis using a rat LH receptor antisense RNA probe. The numbers of LH receptors per cell measured by the binding study were 2,623 +/- 1,110 in PLC, 9,024 +/- 1,992 in ILC, and 39,896 +/- 15,234 in ALC (mean +/- SEM Of four replicate experiments; ALC significantly greater than either PLC or ILC at P < 0.05). The Northern blotting revealed three major bands [6.7, 2.6, and 2.3 kilobases (kb)] that were present in Leydig cells at all three ages and were not detected in HepG2 cells. When the steady state levels of the predominant 6.7-kb species were normalized to actin mRNA, PLC were 6.3-fold lower than ILC and 1.7-fold lower than ALC (n = 3 replicate isolations of poly(A) RNA). The 2.6- and 2.3-kb species exhibited similar trends. Levels of AR were estimated by immunoblotting using a polyclonal antibody against a synthetic peptide of the receptor (residues 14-32) that detected a 110-kilodalton AR protein. Levels of AR mRNA were estimated by Northern blot analysis, using a rat AR antisense RNA probe that detected a single 10-kb AR mRNA. The relative levels of AR protein were 1.0, 1.5, and 0.5 in PLC, ILC, and ALC, respectively (n = 3). Similar trends were observed for AR mRNA (n = 3). The observation that both LH and AR levels were lower in PLC compared to ILC is consistent with the hypothesis that the former are progenitors of Leydig cells.