Specific binding sites for prolactin (PRL) and gonadotrophins on ventral and dorsolateral prostate as well as on Leydig cells and tubules of testes of rats at different ages were examined. The binding sites for PRL were found in greatest number in ventral prostate and in Leydig cells. LH binding sites were also more numerous than FSH binding sites in the latter. FSH sites were greater than LH sites in tubular preparations obtained from the testis. Specific binding (SB) of PRL in the Leydig cells reached a maximum at 45 days (4%) and in the case of LH a maximum of 12% was obtained at 70 days. In both preparations SB of FSH exhibited a plateau between 20 and 40 days (11 %) followed by a gradual decline to 6% at 100 days. Following 20 days of treatment with Bromocriptin beginning at 20 days serum PRL was suppressed and SB of LH to the Leydig cells was significantly decreased, whereas SB of PRL and FSH was unaffected.These studies suggest that despite decreases in serum PRL, the number of PRL and FSH receptors remain unaltered. On the contrary, LH receptors in the rat testis are modulated by changes in serum PRL.

A variety of cell lines were examined by Northern blot hybridization for the expression of PRL or PRL-related mRNAs. We found that a human B-lymphoblast cell line transcribed a mRNA which hybridized to human PRL cDNA under high stringency conditions. The human lymphoblast cell line of interest is a variant subline of the IM-9 line that we have designated IM-9-P. The lymphoblast-derived PRL mRNA is approximately 150 bases longer than that produced by the human pituitary as determined by Northern blot analysis. IM-9-P PRL was immunoaffinity purified from conditioned medium and found to be identical in mol wt by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to human pituitary PRL. Moreover, IM-9-P PRL is biologically active in the rat Nb2 lymphoma mitogenic assay. Ribonuclease-H digestion of mRNA poly(A) tracts indicated that the size difference between pituitary and IM-9-P PRL transcripts was not due to an elongated poly(A) tail on the lymphoid PRL mRNA. Genomic Southern blot analysis showed no major rearrangements of the PRL gene in IM-9-P cells compared to the parent IM-9 line and human placenta DNA. Thus, it is highly likely that an elongation of the 5' and/or 3' untranslated regions of IM-9-P PRL mRNA account for the size difference with pituitary PRL mRNA. The PRL-producing IM-9-P line was cloned by limiting dilution, and a high PRL-producing clone IM-9-P3 and a non-PRL producer IM-9-P6 were isolated for further analysis. IM-9-P3 cells were found to secrete 40-50 ng PRL/10(6) cells.24 h regardless of cell density. The level of PRL mRNA also remained constant during exponential growth of IM-9-P3 cells. The existence of the PRL-producing IM-9-P3 clone and the IM-9-P6 clone which does not produce PRL as well as the IM-9 progenitor line provides a unique system with which to analyze the molecular mechanism of ectopic human PRL expression.

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