Aromatase activity has been measured in Leydig cells and Sertoli cells from both immature and mature rats. Cytochrome P450 aromatase (P450arom) has been immunolocalized in germ cells of the rodent, bear, and rooster. Our purpose was to investigate expression of and to immunolocalize P450arom in adult rat testicular cells. After Western blotting with a specific anti-cytochrome P450arom antibody, we demonstrated the presence of a 55-kDa protein in mature rat seminiferous tubules and crude germ cell preparations. Immunoreactive aromatase was detected both in cultured rat Leydig cells and in testis sections (interstitial tissue and elongated spermatids showed positive immunoreactivity for P450arom). We next used reverse transcription-polymerase chain reaction to localize and quantify the P450arom mRNA in the various testicular cells. In rat Leydig cells, the amount of P450arom mRNA was 15 times higher than in Sertoli cells (34.1+/-3.2 to 2.3 +/-0.2 x 10(-3) amol/10(6) cells, respectively). In pachytene spermatocytes, round spermatids, and testicular spermatozoa the P450arom mRNA levels were 38.7+/-8.1, 20.4+/-3.8, and < 1.3 x 10(-3) amol/10(6) cells, respectively. The aromatase activity was 2.5-4 times higher in testicular spermatozoa (8.48+/-1.98 fmol/10(6) cells per hour) than in other germ cells. These results indicate that in mature rats, not only Leydig cells and Sertoli cells but also germ cells have the capacity to express functional P450arom. According to the germ cell maturation state, there was an inverse relationship between P450arom mRNA content and the biological activity of the protein. The expression of the functional P450arom in mature rat germ cells confirms the existence of an additional source of estrogens within the genital tract of the male.

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Estrogens are the major steroids produced by equine gonads.To identrfy the cells responsible for estrogen synthesis, an antiserum against purified equine testicular cytochrome P450 aromatase was produced in rabbits. The reactivity and specificity of the antiserum were assessed by ELISA, immuneblot analysis, and immunoneutralization studies. Immunofluorescence microscopy demonstrated that in the male gonad, cytochrome P450 aromtase (P45Oamm) was localized in the interstitial tissue, whereas, under the experimental conditions used, the Sertoli and germ cells did not show any specific staining. In the ovary, the granulosa cells of small follicles exhibited faint immunofluorescent staining for IntroductionThe conversion of androgens to estrogens, termed aromatization, is catalyzed by P45Oarom enzyme, the product of the gene called CYP19 in humans (1). Recently, Osawa et al. (2) showed that aromatase also acts as estrogen 2-hydroxylase and postulated that this enzyme could be responsible for the high levels of catechol estrogens and 19-hydroxyandrogens during pregnancy. The enzymatic complex, which is located in the endoplasmic reticulum, includes the heme glycoprotein P450arom and the flavoprotein NADPH-cytochrome P450 reductase.Estrogen biosynthesis occurs in several tissues and cells of mammals, including adipose tissue, skin fibroblasts, brain, pituitary, prostate, testis, ovary, placenta, and retina (3-10). P450arom has been immunohistochemically localized in the syncytiotrophoblast of the human placenta (11)(12)(13)(14)(15), in ovaries of human, rat, mice, golden hamster, guinea pig, and cow (11,14,(16)(17)(18)(19)(20)(21)(22), in testes of rat, mouse, Supported in part by a grant from the Association pour la Recherche sur le Cancer (grant no. 6933) and by a grant from the Ligue Nationale Fransaise contre le Cancer (Comitt de la Manche). and bear (23)(24)(25), in human prostate (6), in the brain of quail, rat, and mouse (26-28), and in the retina of goldfish (10).In mare, the concentration of estradiol-l7fl, which is the main estrogen in the follicular fluid, is greatly increased in the large follicles compared with the small follicles (8,29). The mare cyclic corpus luteum aromatizes androgens very efficiently (30), although only low levels of circulating estradioL17fl are present during the luteal phase (31,32). In comparison to other species (33,34), the stallion testis secretes very high amounts of estrogens, essentially as estrogen-3-sulfates. Plasma estrogens markedly increase with age (35), together with the number and volume of Leydig cells (36). However, the cellular localization of aromatase has not yet been established in equine ovary and testis.We have previously shown that equine aromatase exhibits some distinct properties compared with the human enzyme: the equine testicular, placental, and ovarian estrogen synthetase aromatizes androgens and 19-norandrogens with similar velocity, but not 16a-hydroxytestosterone or epitestosterone, in contrast to the human placental enzyme, which aromatizes androg...

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