Distribution of aromatase cytochrome P450 messenger ribonucleic acid in adult rhesus monkey brains

Abstract

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 rhesus monkey brains has not been studied because specific probes have not 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 chain reaction (PCR) using consensus oligonucleotide primers selected from 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 assay using a monkey P450(arom) cDNA and studied the distribution of P450(arom) mRNA in adult monkey brains. This assay protected two RNA fragments, one 455 nucleotides (nt) in length and the second approximately 300 nt. The relative distribution of P450(arom) mRNA (the 455-nt fragment) between brain areas of the adult (n = 3) was high in the bed nucleus of the stria terminalis > medial preoptic/anterior hypothalamus > amygdala; intermediate in the medial basal hypothalamus (infundibular nucleus, median eminence, ventromedial nucleus) > lateral preoptic/anterior hypothalamus; and low in the septum > lateral-dorsal-medial hypothalamus. P450(arom) mRNA was undetectable in cingulate and parietal cortex, hippocampus, and cerebellum. P450(arom) activity, as measured by the 3H2O assay, correlated well with the distribution of P450(arom) mRNA (the 455-nt protected fragment; r = 0.9) in the same tissues. A shorter protected RNA fragment was found in the medial basal hypothalamus, the bed nucleus of the stria terminalis, the amygdala, and the cingulate and parietal cortex but not in the other brain areas investigated. Its presence did not correlate with aromatase activity in brain tissue. This study describes the development of a ribonuclease protection assay using a monkey cDNA produced by RT-PCR and its usefulness for studying the distribution of P450(arom) mRNA in brains of nonhuman primates.