Cytochrome P450 aromatase in male germ cells

The ability of the male gonad to convert androgens into estrogens is well known; the microsomal enzymatic complex involved in this transformation is named aromatase and is composed of a specific cytochrome P450 aromatase (P450arom) and an ubiquitous reductase. According to age, aromatase activity has been already measured in immature and mature rat Leydig cells as well as in Sertoli cells. Recently, in different studies, a cytochrome P450arom has even been immunolocalized not only in Leydig cells but also in germ cells of mouse, brown bear and rooster whereas in pig, ram and human the aromatase is mainly present in Leydig cells. Our purpose was to investigate the testicular cell distribution of cytochrome P450arom mRNA in adult rat using RT-PCR. With two highly specific primers located on exons 8 and 9, we have been able to amplify a 289 bp aromatase fragment not only in Leydig cells and Sertoli cells but also in highly-enriched preparations of pachytene spermatocytes, round spermatids and testicular spermatozoa. These amplified products showed 100% homology with the corresponding fragment of the rat ovary cDNA. In parallel, using an anti-human cytochrome P450arom antibody we have demonstrated the presence of a 55 kDa protein in seminiferous tubules and crude germ cells (pachytene spermatocytes and round spermatids) of the mature rat. After incubation with tritiated androstenedione, the aromatase activity in the microsomal fractions of purified testicular spermatozoa was 2.96 pmoles/mg/h and was found to be 5-fold higher when compared to that of either purified pachytene spermatocytes or round spermatids. Using a quantitative RT-PCR method with a standard cDNA 29 bp shorter, we have compared the amount of cytochrome P450arom mRNA in mature rat Leydig cells and Sertoli cells. In purified Leydig cells from mature rats the P450arom mRNA level was: 36 x 10(-3) amoles/microgram RNA whereas in Sertoli cells the mRNA level was 10 fold lower. In pachytene spermatocytes, round spermatids and testicular spermatozoa the P450arom mRNA levels were respectively 367, 117 and < 1 x 10(-3) amoles/microgram RNA. Therefore, we evidenced that not only the Leydig cells but also the Sertoli cells of the rat during the testicular maturation have the capacity to express the gene of the cytochrome P450 aromatase. More importantly a biologically active cytochrome P450 aromatase is also present in germ cells (pachytene spermatocytes, round spermatids and spermatozoa). The existence of an additional source of estrogens within the genital tract of the male is now well documented and that suggests a putative role for these hormones during the male germ cell development and maturation not only in the testis but also in the epididymis.