P450 AROMATASE MESSENGER-RIBONUCLEIC-ACID EXPRESSION IN MALE-RAT GERM-CELLS - DETECTION BY REVERSE TRANSCRIPTION-POLYMERASE CHAIN-REACTION AMPLIFICATION

We have previously demonstrated that cytochrome P450 aromatase (P450ar om) protein, an estrogen-synthesizing enzyme, is present and active in germ cells of the adult mouse testis. To establish that P450arom mRNA is expressed in germ cells of other species, we examined expression o f P450arom in adult rat germ cells by employing reverse transcription- polymerase chain reaction (RT-PCR), Total RNA was extracted from Sta-p ut separated germ cells and reverse transcribed. The resulting cDNA wa s amplified by nested PCR reactions using oligonucleotide primers sele cted from a highly conserved region of the P450arom gene, RT-PCR analy sis yielded cDNA products of 334 bp in length that corresponded to the predicted size expected from the final nested amplification. The iden tity of the germ cell P450arom PCR products was confirmed by restricti on enzyme analysis and direct nucleotide sequencing. Rat genomic DNA w as subjected to PCR to verify that P450arom DNA products were not obta ined from genomic DNA contamination. Rat genomic DNA yielded a nested PCR product for P450arom of approximately 2000 bp, suggesting that, as is the case with the human P450arom gene, the rat P450arom gene conta ins an intron in the amplified region. In addition, a semiquantitative technique was utilized to eliminate the possibility that the P450arom RT-PCR products were derived from Leydig cell contamination of Sta-pu t-separated germ cell preparations. RT-PCR for P450arom and 3-beta-hyd roxysteroid dehydrogenase (3 beta-HSD), a Leydig cell-specific steroid ogenic enzyme, was carried out on Sta-put-separated germ cells and int erstitial cell preparations containing Leydig cells. P450arom and SP-H SD RT-PCR reactions were stopped at three cycle intervals to detect an d compare the earliest appearance of RT-PCR reaction products in vario us cell types. Results indicated that P450arom mRNA is detected in rou nd spermatids before it is detected in interstitial cells, whereas 3 b eta-HSD was detected only in interstitial cells, suggesting that the P 450arom mRNA detected in germ cells is not due to interstitial cell co ntamination of germ cell preparations. Therefore, our results indicate that P450arom mRNA is expressed in adult rat germ cells and that test icular germ cells are a potential source of estrogen in the male repro ductive tract.