DISTRIBUTION OF AROMATASE CYTOCHROME-P450 MESSENGER-RIBONUCLEIC-ACID IN ADULT RHESUS-MONKEY BRAINS

The conversion of androgens to estrogens by aromatase cytochrome P450 (P450(arom)) is an important step in the mechanism of androgen action in the brain. However, the distribution of P450(arom) mRNA in adult rh esus monkey brains has not been studied because specific probes have n ot been available. To address this deficit, we cloned and sequenced a 455-basepair segment of the 5' coding region of the rhesus P450(arom) cDNA. Total RNA was extracted from a rhesus monkey placenta (Day 47 of gestation) and subjected to reverse transcriptase (RT) polymerase cha in reaction (PCR) using consensus oligonucleotide primers selected fro m published human and rat P450(arom) DNA sequences. The RT-PCR product was subcloned into a vector and sequenced. The monkey P450(arom) cDNA was 97% identical to the human sequence but shared only 86% homology with the rat sequence. We then developed a ribonuclease protection ass ay using a monkey P450(arom) cDNA and studied the distribution of P450 (arom) mRNA in adult monkey brains. This assay protected two RNA fragm ents, one 455 nucleotides (nt) in length and the second approximately 300 nt. The relative distribution of P450(arom) mRNA (the 455-nt fragm ent) between brain areas of the adult (n = 3) was high in the bed nucl eus of the stria terminalis > medial preoptic/anterior hypothalamus > amygdala; intermediate in the medial basal hypothalamus (infundibular nucleus, median eminence, ventromedial nucleus) > lateral preoptic/ant erior hypothalamus; and low in the septum > lateral-dorsal-medial hypo thalamus. P450(arom) mRNA was undetectable in cingulate and parietal c ortex, hippocampus, and cerebellum. P450(arom) activity, as measured b y the (H2O)-H-3 assay, correlated well with the distribution of P450(a rom) mRNA (the 455-nt protected fragment; r = 0.9) in the same tissues . A shorter protected RNA fragment was found in the medial basal hypot halamus, the bed nucleus of the stria terminalis, the amygdala, and th e cingulate and parietal cortex but not in the other brain areas inves tigated. Its presence did not correlate with aromatase activity in bra in tissue. This study describes the development of a ribonuclease prot ection assay using a monkey cDNA produced by RT-PCR and its usefulness for studying the distribution of P450(arom) mRNA in brains of nonhuma n primates.