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...

Blood testosterone levels were measured by RIA and gas chromatography-mass spectrometry in the pregnant mare. They were found to increase from the very beginning of pregnancy, reaching peak values 10 times higher than the basal values at the seventh month and then to return to basal values by the week after parturition. Testosterone binding by plasma proteins was investigated in nonpregnant and pregnant mares throughout gestation. Equilibrium dialysis and gel equilibration methods did not reveal any blood specific testosterone-binding activity at any gestational stage. Hence, blood testosterone increase cannot result as in the pregnant woman or guinea pig, from an increase in blood specific binding activity but more probably results from an increased steroid production. It is suggested that the maternal gonads would be responsible for the testosterone increase in early pregnancy, during the period of PMSG production, and the fetoplacental unit for the subsequent increase.

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Follicular fluid was obtained from equine follicles at different stages of development as determined by ultrastructural study. Gas chromatography-mass spectrometry associated with stable isotope dilution permitted the demonstration of high levels of 4-estrene-3,7-dione and 17 beta-hydroxy-4-estren-3-one, 17 beta-hydroxy-4-estren-3-one levels often being about 10 times higher than those of testosterone. These findings suggest that in the mare ovary, an aromatizing pathway may proceed using these 19-norsteroids as intermediates. As a consequence of this high level of 19-norsteroids, testosterone content may be most often overestimated by RIA. RIA of steroids in follicular fluid should be validated by a reference technique based on gas chromatography-mass spectrometry, associated with stable isotope dilution.