Ad4BP (or SF-1) has been identified as a transcription factor which regulates all the steroidogenic P450 genes in the peripheral organs, and is encoded by the mammalian homologue of Drosophila FTZ-F1 gene. mRNA coding for Ad4BP was detected in the hypothalamus and pituitary of rats by RT-PCR. Immunohistochemical analyses using an antiserum to Ad4BP in the brain and pituitary revealed that the transcription factor is expressed in nuclei of the dorsomedial part of the ventromedial hypothalamus (dmVMH) and in some subpopulation of the adenohypophysial cells. Double immunostaining of the pituitary for Ad4BP and trophic peptide hormones, FSH, TSH, and ACTH, indicated a restricted localization of Ad4BP to the gonadotroph. Disruption of the mouse Ftz-FI gene was clarified to induce severe defects in the organization of the dmVMH and the function of the pituitary gonadotroph. However, some of the dm VMH neurons and pituitary gonadotrophs persisted, which provided a sharp contrast to complete agenesis of the peripheral steroidogenic tissues (adrenal and gonads) in the mutant mouse. Additional abnormalities were seen in the ventrolateral part of VMH and dorsomedial hypothalamic nucleus, both of which do not express Ad4BP but have strong reciprocal fiber-connections with the dmVMH. Aromatase P450-containing cells in the medial preoptico-amygdaloid region, which were devoid of Ad4BP, persisted even in the brain of the gene disrupted mice. The present results clearly showed that the hypothalamic and pituitary Ad4BPs are essential to normal development of the functional VMH and gonadotroph through some mechanism distinct from that in the peripheral steroidogenic tissues. 8 1995 Wiley-Liss, Inc.

To determine whether local estrogen production takes place in endometriotic or adenomyotic tissues, in eutopic endometrium from patients with endometriosis or adenomyosis, and in normal endometrium, tissue specimens were examined by immunohistochemistry, catalytic activity, and mRNA expression for aromatase cytochrome P450 (P450arom). P450arom was immunohistochemically localized in the cytoplasm of glandular cells of endometriotic and adenomyotic tissues, and of eutopic endometrium from patients with the respective diseases, whereas estrogen receptors and progesterone receptors were localized in the nuclei of the glandular cells and stroma. Aromatase activity in the microsomal fraction of adenomyotic tissues was inhibited by the addition of danazol, aromatase inhibitors, and anti-human placental P450arom monoclonal antibody (mAb3-2C2) in a manner similar to such inhibition in other human tissues. Reverse transcription polymerase chain reaction and Southern blot analysis also revealed P450arom mRNA in these tissues. However, neither P450arom protein activity nor mRNA was detected in endometrial specimens obtained from normal menstruating women with cervical carcinoma in situ but without any other gynecological disease. These results suggest that at a local level, endometriotic and adenomyotic tissues produce estrogens, which may be involved in the tissue growth through interacting with the estrogen receptor.

Estrone sulfatase (ES; 562 amino acids), one of the key enzymes responsible for maintaining high levels of estrogens in breast tumor cells, is associated with the membrane of the endoplasmic reticulum (ER). The structure of ES, purified from the microsomal fraction of human placentas, has been determined at 2.60-Å resolution by x-ray crystallography. This structure shows a domain consisting of two antiparallel ␣-helices that protrude from the roughly spherical molecule, thereby giving the molecule a "mushroom-like" shape. These highly hydrophobic helices, each about 40 Å long, are capable of traversing the membrane, thus presumably anchoring the functional domain on the membrane surface facing the ER lumen. The location of the transmembrane domain is such that the opening to the active site, buried deep in a cavity of the "gill" of the "mushroom," rests near the membrane surface, thereby suggesting a role of the lipid bilayer in catalysis. This simple architecture could be a prototype utilized by the ER membrane in dictating the form and the function of ER-resident enzymes.

No abstract

Estrogen production within the testis has been a subject of considerable controversy for many years. Several studies have shown that both Sertoli and Leydig cells produce estrogen during different stages of development. Therefore, we have conducted experiments to localize aromatase, a cytochrome P450 enzyme that converts androgen to estrogen, within the testis. First, P450 aromatase (P450arom) was localized in germ cells of the adult mouse testis by immunocytochemistry, using an antiserum generated against purified human placental cytochrome P450arom. In the germinal epithelium, P450arom was located primarily in the Golgi region of round spermatids, throughout the cytoplasm of elongating spermatids, and along the flagella of late spermatids. Second, localization of P450arom within the germinal epithelium was supported by Western blot analysis of isolated germ cells. Third, Northern blot analysis using a mouse P450arom cDNA probe indicated that the mRNA for the mouse P450arom was present in testicular germ cells. Fourth, P450arom activity was measured in germ cells by the 3H2O water assay. Based upon these observations, we conclude that germ cells are a site of estrogen synthesis in the adult mouse testis.