Donor splice-site mutations in WT1 are responsible for Frasier syndrome.

Frasier syndrome (FS) is a rare disease defined by male pseudo-hermaphroditism and progressive glomerulopathy. Patients present with normal female external genitalia, streak gonads and XY karyotype and frequently develop gonadoblastoma. Glomerular symptoms consist of childhood proteinuria and nephrotic syndrome, characterized by unspecific focal and segmental glomerular sclerosis, progressing to end-stage renal failure in adolescence or early adulthood. No case of Wilms' tumour has been reported, even in patients with extended follow-up. In contrast with FS patients, most individuals with Denys-Drash syndrome (DDS; refs 6,7) have ambiguous genitalia or a female phenotype, an XY karyotype and dysgenetic gonads. Renal symptoms are characterized by diffuse mesangial sclerosis, usually before the age of one year, and patients frequently develop Wilms' tumour. Mutations of the Wilms'-tumour gene, WT1, cause different pathologies of the urogenital system, including DDS. WT1 is composed of ten exons and encodes a protein with four zinc-finger motifs and transcriptional and tumour-suppressor activities. Alternative splicing generates four isoforms: the fifth exon may or may not be present, and an alternative splice site in intron 9 allows the addition of three amino acids (KTS) between the third and fourth zinc fingers of WT1 (ref. 17). Here we demonstrate that FS is caused by mutations in the donor splice site in intron 9 of WT1, with the predicted loss of the +KTS isoform. Examination of WT1 transcripts indeed showed a diminution of the +KTS/-KTS isoform ratio in patients with FS.

Evaluation of different markers of the ovarian reserve in patients presenting with premature ovarian failure.

Premature ovarian failure (POF) is a heterogeneous syndrome, possibly due to mutations of genes involved in the normal development of the ovary and/or the follicles. Based essentially on animal models, these mutations are associated with various ovarian histological phenotypes, from a complete absence of to a partial follicular maturation. The aims of our work were in one hand to determine if ovarian histology, compared to pelvic ultrasonography, would be helpful either in identifying which patients display an impaired follicular growth or in the orientation of the POF etiology; on the other hand, since developing follicles up to the antral stage are reported in POF and that Anti-Müllerian hormone (AMH) might be a good indicator of follicular presence, we decided to determine whether AMH should be a better marker to determine the presence of an ovarian reserve in POF patients. To try to answer to the first question, we studied first 166 patients suffering from POF with a normal karyotype. Vaginal ultrasonography (US) was performed in 134 patients and an ovarian biopsy was obtained in 67 women. The presence of follicles suggested at US was confirmed at histology in only 56% of the patients. Ovarian histology led to the distinction of two phenotypes (a) small-sized ovaries, deprived of follicles, and (b) normal-sized ovaries with partial follicular maturation. To confirm the value of ovarian biopsies, samples from 20 normal women have been studied, confirming that ovarian biopsy at random allow reliable assessment of follicular activity. To try to answer to the second question of our work, a cross sectional study analyzing serum AMH, ovarian histology and AMH immunoexpression in 48 POF patients, was performed. Serum AMH was significantly higher in women with more than 5 follicles at ovarian histology. Ovarian AMH immunostaining revealed a normal AMH expression in POF preantral follicles but a decrease expression at the early antral stages. In conclusion, ovarian histology appears to be a reliable tool to appreciate the follicular reserve, and helpful and complementary to clinical and hormonal phenotyping in order to orient the search for various genetic causes of POF syndrome. Finally, AMH levels in POF patients could identify women with persistent follicles.

Molecular pathology of the FSH receptor: new insights into FSH physiology.

Manipulations of mouse genome have helped to elucidate gonadotrophin function but important differences subsist between rodent and human reproduction. Studies of patients with mutations of gonadotrophins or gonadotrophin receptors genes allow understanding their physiological effects in humans. The correlation of the clinical phenotypes of patients with in vitro studies of the mutated receptor residual function and histological and immunohistological studies of the ovarian biopsies permits to understand which stages of follicular development are under FSH control. Total FSH receptor (FSHR) inactivation causes infertility with an early block of follicular maturation remarkably associated with abundant small follicles as in prepubertal ovaries and demonstrates the absolute requirement of FSH for follicular development starting from the primary stage. Partial FSHR inactivation, characterized by normal-sized ovaries, can sustain follicular development up to the early antral stages but incremental levels of FSH stimulation seem to be required for antral follicular growth before selection. These findings contrast with the traditional view of an initial gonadotrophin-independent follicular growth prior to the preantral-early antral stages. The presence of numerous reserve follicles in the ovaries of these patients may permit a future treatment of their infertility. The study of reduced FSHbeta or FSHR activity in genetically modified male mice models and in men suggests a minor impact of the FSHR on masculine fertility. Further studies on patients with a demonstrated total FSHbeta or FSHR inactivation are required to elucidate reported differences in spermatogenesis impairment. Finally, the studies of mutations of gonadotrophins and their receptors demonstrate differences in gonadotrophin function between genetically modified rodents and humans which suggest prudence in extrapolating observations in rodents to human reproduction. Ovarian hyperstimulation syndrome (OHSS) can infrequently arise spontaneously during pregnancy, but most often it is an iatrogenic complication of ovarian stimulation treatments with ovulation drugs for in vitro fertilization. The first genetic cause of familial recurrent spontaneous OHSS was identified as a broadening specificity of the FSHR for hCG due to naturally occurring heterozygous mutations located unexpectedly in the transmembrane domain of the FSHR. Broadening specificity of a G protein-coupled receptor is extremely rare. These observations led to the identification of the etiology of this previously unexplained syndrome and permitted to conceive novel models of FSHR activation. Susceptibility to iatrogenic OHSS or its clinical severity may be associated with FSHR polymorphisms with slightly different activities in vivo as suggested by several studies. The study of larger cohorts is needed to evaluate the clinical impact of these observations in the management of patients undergoing IVF protocols.

A novel phenotype related to partial loss of function mutations of the follicle stimulating hormone receptor.

A single natural loss of function mutation of the follicle stimulating hormone receptor (FSHR) has been described to date. Present in the Finnish population it markedly impairs receptor function, blocking follicle development at the primary stage and presenting as primary amenorrhea with atrophic ovaries. When Western European women with this phenotype were examined for FSHR mutations the result was negative, suggesting that other etiologies corresponding to this clinical pattern are markedly more frequent. We now describe a novel phenotype related to mutations provoking a partial loss of function of the FSHR. A woman with secondary amenorrhea had very high plasma gonadotropin concentrations (especially FSH), contrasting with normal sized ovaries and antral follicles up to 5 mm at ultrasonography. Histological and immunohistochemical examination of the ovaries showed normal follicular development up to the small antral stage and a disruption at further stages. The patient was found to carry compound heterozygotic mutations of the FSHR gene: Ile160Thr and Arg573Cys substitutions located, respectively, in the extracellular domain and in the third intracellular loop of the receptor. The mutated receptors, when expressed in COS-7 cells, showed partial functional impairment, consistent with the clinical and histological observations: the first mutation impaired cell surface expression and the second altered signal transduction of the receptor. This observation suggests that a limited FSH effect is sufficient to promote follicular growth up to the small antral stage. Further development necessitates strong FSH stimulation. The contrast between very high FSH levels and normal sized ovaries with antral follicles may thus be characteristic of such patients.

Luteinizing hormone pulsatility in patients with major ovarian hyperandrogenism.

Hyperandrogenism and ovulatory dysfunction are common in women with either polycystic ovary (PCOS) or ovarian virilizing tumor. However, contrasting with the numerous studies that have extensively described gonadotropin secretory abnormalities, principally increased LH pulse amplitude and frequency, few studies have concerned gonadotropin secretion in patients with ovarian virilizing tumors; low gonadotropin levels have occasionally been reported, but never extensively studied. The goal of the present study was to further evaluate the pulsatility of LH secretion in women with ovarian virilizing tumor compared with that of PCOS patients. Eighteen women with major hyperandrogenism (plasma testosterone level >1.2 ng/ml) were studied (5 women with ovarian virilizing tumor, 13 women with PCOS, and 10 control women). Mean plasma LH level, LH pulse number and amplitude were dramatically low in patients with ovarian tumors when compared to both PCOS (p<0.001) and controls (p<0.001). In case of major hyperandrogenism, LH pulse pattern differs markedly between women with ovarian virilizing tumor or PCOS, suggesting different mechanisms of hypothalamic or pituitary feedback.

trans-4-Hydroxytamoxifen concentration and metabolism after local percutaneous administration to human breast.

trans-4-Hydroxytamoxifen (4-OHTAM), a very active metabolite of the antiestrogen tamoxifen, was percutaneously administered to the affected breast of nine patients before surgery for breast cancer in order to evaluate 4-OHTAM absorption through the skin and its subcellular localization and metabolism. After percutaneous administration of 80 muCi, [3H]-4-OHTAM was detected in breast tissue. It was especially concentrated in tumor tissue and nuclear and cytosolic fractions, in which it remained unmetabolized except for limited isomerization from the trans to the cis form. In contrast to breast tissue, concentrations of radioactivity remained low in plasma but with a high proportion of metabolites. In another experiment [3H]tamoxifen was percutaneously administered over the breast of 3 patients, resulting in tissue retention weaker and shorter than after [3H]-4-OHTAM. In addition [3H]-4-OHTAM was administered to either breast or abdominal skin; the appearance of radioactivity in plasma and urine was delayed after administration to the breast in comparison with administration to the abdomen. It therefore appears that 4-OHTAM passes through the skin and is concentrated in receptor structures of breast tissue, thus avoiding the hepatic metabolism subsequent to p.o. administration. We suggest that local percutaneous administration of this active antiestrogen could be useful in the treatment of hormone-dependent benign breast diseases.

Estradiol 17 beta-hydroxysteroid dehydrogenase, a marker of breast cancer hormone dependency.

Intratumoral activity of the progesterone-dependent enzyme 17 beta-hydroxysteroid dehydrogenase (E2DH) was measured in 114 patients with breast cancer (33 pre- and 81 postmenopausal) before and/or after 8 days of a progestin treatment (lynestrenol, 10 mg/day). In 12 postmenopausal patients, the ability of E2DH to be stimulated by lynestrenol was compared to estradiol receptor (ER) and progesterone receptor (PR) levels. In premenopausal patients, E2DH was higher when tumors were excised in the luteal phase than when excised in the follicular phase. In postmenopausal patients, E2DH was higher after progestin treatment. However, E2DH stimulation by lynestrenol depended on receptor levels. It was most often markedly stimulated in ER-positive, PR-positive tumors. It remained low in ER-negative, PR-negative tumors. Intratumoral measurement of the progesterone-dependent enzyme E2DH in breast cancer after progestin treatment could therefore provide a fine and reliable index of the presence and functional character of PR and hormone dependency of the tumor.

Tamoxifen and hydroxytamoxifen isomers versus estradiol effects on normal human breast cells in culture.

Estradiol and triphenylethylene antiestrogen actions have been studied extensively in breast cancer cell lines. However, their effects are still poorly understood on normal human breast cells. We have developed a culture system of normal human breast epithelial (HBE) cells. It has been shown previously that cultured HBE cells were hormone dependent and well adapted for the study of hormone/antihormone actions. However, until now, no data were available on estradiol receptor (ER) in HBE cells. In this study, the presence of ER was demonstrated by (a) whole-cell biochemical assay on breast cells after enzymatic tissue dissociation and (b) an immunocytochemical method using an anti-ER monoclonal antibody both on enzymatically dissociated cells and on 8-day cultured cells. Immunostaining was nuclear and cell positivity was heterogeneous. However, the percentage of positive cells and staining intensity were far greater in the presence of estradiol in the culture, indicating estradiol stimulation of ER. Moreover, HBE cells were used to study the action on cell growth of estradiol versus trans-tamoxifen (TAM), trans-4-hydroxytamoxifen (trans-4OHTAM), and cis-4-hydroxytamoxifen (cis-4OHTAM) alone or added to estradiol. Cell growth was estimated daily by a histometric method and by DNA assay at the end of the 7-day study. When the medium was minimally supplemented with human serum (1%), estradiol stimulated cell growth in a dose-dependent manner at concentrations varying from 10(-9) to 10(-7) M. TAM and trans-4OHTAM clearly inhibited mammary cell division when estradiol was added to the medium and, to a lesser extent, in the absence of estradiol. This inhibitory effect was dose dependent. trans-4OHTAM was 100 times more active than trans-TAM. cis-4OHTAM also clearly inhibited breast cell division at 10(-7) and 10(-6) M concentrations but was 3-fold less efficient than trans-4OHTAM. In conclusion, (a) the presence and estradiol dependence of ER have been demonstrated in HBE cells, which constitute a fruitful model for the study of hormone/antihormone actions, and (b) in these normal cells, estradiol stimulates growth, whereas TAM and the 4OHTAM isomers are potent inhibitors of cell multiplication, as they are in breast cancer cell lines in culture.

Breast inflammatory gigantomastia in a context of immune-mediated diseases.

CONTEXT: Localized breast lesions have been described in lupic or diabetic patients. However, the description of breast gigantomastia in women presenting with autoimmune diseases has not been reported. SETTING: The study took place within the Department of Endocrinology and Reproductive Medicine, Necker Hospital, Paris, France. PATIENTS: We describe eight patients with inflammatory gigantomastia, occurring in a context of immune-mediated diseases: myasthenia, chronic arthritis, or thyroiditis. MAIN OUTCOME MEASURES: Together with hormonal, immunological, and breast magnetic resonance imaging (MRI) evaluation, breast histology enabled us to perform immunocytochemical and indirect immunofluorescence studies. Control sera were obtained from patients with (n = 10) and without (n = 7) antinuclear antibodies. RESULTS: Six of the eight patients developed gigantomastia either at puberty or during pregnancy. Neither a hormonal oversecretion nor a specific immunological pattern was observed. All patients except one presented antinuclear antibodies. Histological study revealed a diffuse, stromal hyperplasia and a severe atrophy of the lobules. A rarefaction of adipocytes was also noted, as previously suggested on MRI. There was a perilobular lymphocytic infiltrate made of CD3+ lymphocytes. Study of sera from five of six cases of gigantomastia showed a nuclear immunofluorescence pattern in normal mammary ductal and lobular glandular epithelium, as well as in kidney and intestine epithelial cells. In control sera, a nuclear signal was observed only when antinuclear antibodies were present. CONCLUSIONS: We suggest that breast tissue may be a target tissue in autoimmune diseases, this process being favored by the hormonal milieu. However, the precise mechanism of such association is not individualized. The fact that stromal hyperplasia is the main histological feature justifies the search for the involvement of growth factors in such a process.

New natural inactivating mutations of the follicle-stimulating hormone receptor: correlations between receptor function and phenotype.

Premature ovarian failure occurs in almost 1% of women under age 40. Molecular alterations of the FSH receptor (FSHR) have recently been described. A first homozygous mutation of the FSHR was identified in Finland. More recently, we described two new mutations of the FSHR in a woman presenting a partial FSH-resistance syndrome (patient 1). We now report new molecular alterations of the FSHR in another woman (patient 2) who presented at the age of 19 with primary amenorrhea contrasting with normal pubertal development. She had high plasma FSH, and numerous ovarian follicles up to 3 mm in size were evidenced by ultrasonography. Histological and immunohistochemical examination of ovarian biopsies revealed the presence of a normal follicular development up to the antral stage and disruption at further stages. DNA sequencing showed two heterozygous mutations: Asp224Val in the extracellular domain and Leu601Val in the third extracellular loop of FSHR. Cells transfected with expression vectors encoding the wild type or the mutated Leu601Val receptors bound hormone with similar affinity, whereas binding was barely detectable with the Asp224Val mutant. Confocal microscopy showed the latter to have an impaired targeting to the cell membrane. This was confirmed by its accumulation as a mannose-rich precursor. Adenylate cyclase stimulation by FSH of the Leu601Val mutant receptor showed a 12+/-3% residual activity, whereas in patient 1 a 24+/-4% residual activity was detected for the Arg573Cys mutant receptor. These results are in keeping with the fact that estradiol and inhibin B levels were higher in patient 1 and that stimulation with recombinant FSH did not increase follicular size, estradiol, or inhibin B levels in patient 2 in contrast to what was observed for patient 1. Thus, differences in the residual activity of mutated FSHR led to differences in the clinical, biological, and histological phenotypes of the patient.

Lupus nephropathy and pregnancy. Report of 104 cases in 36 patients.

The reciprocal influence of lupus nephropathy on the outcome of pregnancy and of pregnancy on the course of renal involvement was studied retrospectively in a series of 106 pregnancies observed during the past two decades in 36 patients with lupus nephropathy. The overall incidence of live births, corrected for induced abortions, was 54 (84%) in 64 pregnancies that began before clinical onset of systemic lupus erythematosus (SLE), 20 (87%) in 23 pregnancies that began after onset of SLE, and only four (57%) in seven cases where SLE was first manifested during or after gestation. Relapse or exacerbation of disease activity occurred in 12 (46%) of 26 pregnancies antedated by clinical onset of SLE, more frequently during gestation than post partum, with two cases (8%) of irreversible deterioration of renal function; clinical exacerbation of lupus disease was observed in 11 (66%) of 15 cases where SLE was clinically active at the time of conception, and in only one (9%) of 11 cases where SLE nephritis was in stable clinical remission for at least five months before conception. The data indicate that successful outcome of pregnancy may be expected even in the more severe forms of lupus nephritis if gestation begins after a sustained, complete clinical remission.

17 beta-Hydroxysteroid dehydrogenase activity in human breast epithelial cell and fibroblast cultures.

Primary cultures of human breast cells prepared from surgical specimens of reduction mammoplasty were used to study the activity of the enzyme 17 beta-hydroxysteroid dehydrogenase (E2DH) which converts estradiol (E2) into its less active metabolite estrone. This study was performed in both epithelial and stromal cells separated, after collagenase digestion of the tissue, on a Percoll gradient, and then cultured as monolayers in Ham's F 10 medium supplemented differently for epithelial cells and fibroblasts. E2DH activity was strikingly higher in epithelial cells than in fibroblasts, since after [3H]E2 incubation (2 nM), 600 fmol/micrograms DNA were metabolized to estrone in epithelial cells after 1 h, whereas an equivalent amount was hardly obtained in fibroblast cultures after 24 h. The affinity and capacity of E2DH were greater in epithelial cells with apparent Michaelis-Menten constant (Km) = 0.6 +/- 0.1 microM and maximum velocity (Vmax) = 250 to 360 pmol/micrograms DNA/h, whereas they were 10 +/- 1 microM and 50 to 70 pmol/micrograms DNA/h, respectively, in fibroblast cultures. Moreover, the E2DH activity was 2 to 5 times higher in epithelial cells cultured in the presence of the progestin medroxyprogesterone acetate, whereas it remained unchanged in fibroblasts cultured under the same conditions. This increase in E2DH activity was dose dependent from 10(-10) to 10(-7) M medroxyprogesterone acetate and inhibited by both actinomycin D and cycloheximide. This system of differential breast cell culture appears to be a fruitful tool for the study of the hormone dependence of normal breast growth and differentiation. Due to the presence of E2DH, epithelial cells are more apt to undergo and to moderate E2 action. Moreover, epithelial cells are a possible site of progesterone modulation of E2DH activity. Therefore, E2DH could be a good marker both for epithelial cells and their hormone dependence.

Treatment of hirsutism by oral cyproterone acetate and percutaneous estradiol.

Twenty hirsute women were treated with 50 mg cyproterone acetate orally, administered from the 5th to the 25th day of the menstrual cycle, along with 3 mg 17 beta-estradiol administered percutaneously from days 16-25. Percutaneously administered 17 beta-estradiol was used rather than ethinylestradiol in order to avoid the side effects of oral administration of synthetic estrogens. From a clinical point of view there was a dramatic improvement of hirsutism after 3-6 months of treatment. Biologically, plasma testosterone decreased markedly (P < 0.01) from 64.6 +/- 24.2 ng/dl (n = 20) to 25.2 +/- 11.8 (n = 20), 26.1 +/- 16.6 (n = 16), and 13.3 +/- 10.8 ng/dl (n = 14) after 3, 6, and 9 months of treatment. There was also a significant decrease in delta 4-androstenedione from 251.0 +/- 110.2 ng/dl to 129.9 +/- 66.5, 114.2 +/- 45.8, and 62.0 +/- 21.5 ng/dl after the same periods. From these results it may be assumed that this therapeutic combination has an antigonadotropic effect, as confirmed by the decrease in plasma estradiol, FSH, and LH and the absence of a significant progesterone level in all cases. Plasma and urinary cortisol, lipids, and hepatic tests remained normal. The good clinical and biological tolerance of this treatment makes it interesting to consider for use in the management of hirsutism.

Estradiol and progesterone receptors in human breast fibroadenomas.

Estradiol and progesterone receptors (ER and PR) were studied in 46 breast fibroadenomas obtained at different periods of the menstrual cycle (n = 38) or from patients under combined estrogen-progestagen contraceptive (n = 4) or substitutive progestagen treatment for progesterone insufficiency (n = 4). Cytosolic and nuclear ER (ERc and ERn) increased throughout the follicular phase and were at their maximal level in the preovulatory phase. They decreased during the luteal phase. PR levels were high in the follicular phase, especially in the cytosol (PRc). PRc then decreased while nuclear progesterone receptor (PRn) increased at the beginning of the luteal phase. Thereafter, PRc and PRn decreased and remained low during the luteal phase. PRc and PRn levels in fibroadenomas from patients under estrogen-progestagen therapy were similar to those found during the luteal phase of untreated patients. In patients receiving a substitutive progestagen treatment to correct progesterone insufficiency, PRn was markedly higher than PRc. The existence of ER and PR in breast fibroadenomas and the variations in their levels during the menstrual cycle or under hormonal treatment provide valuable information on the hormone dependency of breast fibroadenoma.

Contribution of plasma androstenedione to 5 alpha-androstanediol glucuronide in women with idiopathic hirsutism.

To confirm that plasma delta 4 androstenedione (delta 4) is the main precursor for 5 alpha-androstane-3 alpha, 17 beta-diol glucuronide (Adiol G) in patients with idiopathic hirsutism (IH), delta 4 was cutaneously applied to five normal women and five women with IH. Several parameters of androgen metabolism were assayed basally and throughout the studies. Those included plasma delta 4, testosterone, and dihydrotestosterone as well as urinary Adiol G and testosterone glucuronide excretion. Under basal conditions plasma testosterone, delta 4, and dihydrotestosterone did not differ significantly between the two groups of subjects. Urinary Adiol G excretion was significantly higher (P less than 0.01) in IH patients [123 +/- 36 (SE) micrograms/24 h] than in the normal women group (45 +/- 20 micrograms/24 h). After percutaneous administration of delta 4, plasma delta 4 increased in both groups by nearly 600% and there was a 300% increase in Adiol G excretion in IH patients (336 +/- 57 micrograms/24 h), whereas only a 50% increase occurred in normal women (65 +/- 17 micrograms/24 h). We postulate that plasma delta 4 may be the main precursor accounting for the increased production of urinary Adiol G in women with IH, in whom hirsutism may be due to a high 5 alpha-reductase activity. Indeed, 5 alpha-reductase as measured in vitro in pubic skin was significantly higher in hirsute patients (224 +/- 66 fmol/mg skin X h) than in normal women (45 +/- 15 fmol/mg skin X h).

Androgen receptor in human skin cytosol.

Human skin, an accessible tissue, is an androgen target organ. We have measured the androgen-binding capacity of human skin cytosol using either 5 alpha[3H]dihydrotestosterone ([3H]DHT) or [3H]methyltrienolone ([3H]R-1881) as ligand. Samples were incubated for 20 h at 0 C, and dextran-coated charcoal was used to separate bound from free steroids. The androgen receptor has a high affinity for both ligands (0.23 +/- 0.04 nM for [3H]DHT; 0.32 +/- 0.16 nM for [3H]R-1881). Testosterone, cyproterone acetate, and, to a lesser extent, estradiol also bind this protein. Progesterone displaces R-1881 from its binding sites, whereas its 5 alpha-reduced metabolite somewhat inhibits DHT binding. The highest binding capacity is measured in cytosol of skin from external genitalia (129.14 +/- 58.0 fmol/g skin; n = 34); it is lower in pubic skin (21.8 +/- 13 fmol/g skin; n = 6). There is no variation as a function of age or sex in genital skin; the higher concentrations observed in the cytosol of pubic skin of women compared to that of men are probably related to lower levels of endogenous steroids. Whereas most patients with the complete form of the testicular feminization syndrome do not have detectable concentrations of androgen receptor, one patient with apparent complete clinical androgen insensitivity had a normal androgen-binding capacity. The parity of values in genital skin from men and women, the absence of variation with age, and the presence of a cytosolic androgen receptor in some androgen-insensitive patients suggest that the androgen receptor in human skin cytosol is not regulated by androgens.

Progestin effect on cell proliferation and 17 beta-hydroxysteroid dehydrogenase activity in normal human breast cells in culture.

In contrast to cancer cell lines, normal human breast epithelial cells are infrequently studied. Such cells, now routinely cultured in our laboratory from tissue obtained at the time of reduction mammoplasty, were used to study the actions of estradiol (E2), the progestin promegestone (R5020), and the antiprogesterone RU486 on cell growth and progesterone-dependent 17 beta-hydroxysteroid dehydrogenase (E2DH) activity, which is considered good marker of epithelial differentiation as well as progesterone dependency. The studies were carried out using secondary cultures to assure equal initial cell distribution. Cell growth was estimated daily by a histometric method providing a growth index and DNA assay. E2 stimulation of cell growth was not found when the cells were grown in our usual culture medium, but E2 dose-dependent growth stimulation occurred in medium minimally supplemented with serum (1%), insulin; and epidermal growth factor. R5020 inhibited cell growth and stimulated E2DH activity in a dose-dependent manner. RU486 behaved as a pure but low potent progestin agonist concerning E2DH stimulation, but as an agonist with partial antagonist properties concerning cell growth inhibition. In conclusion, E2 stimulated proliferation of human breast epithelial cells in culture, whereas the progestin R5020 inhibited cell multiplication and favored differentiation. The antiprogesterone RU486 had a biphasic effect acting both as progestin agonist and partial antagonist.

Predominant expression of 5 alpha-reductase type 1 in pubic skin from normal subjects and hirsute patients.

Dihydrotestosterone (DHT), the 5 alpha-reduced metabolite of testosterone, is the active molecule triggering androgen action, and 5 alpha-reductase (5 alpha-R), the enzyme converting testosterone to DHT, is a key step in this mechanism. Skin, like prostate, is a DHT- dependent tissue. Our laboratory demonstrated, many years ago, that 5 alpha-R in external genitalia was not regulated by androgens, whereas it was androgen dependent in public skin. As two genes, 5 alpha-R types 1 and 2, encoding for 5 alpha-R enzymes have been recently cloned, we undertook the present study to determine whether the two enzymes we had postulated on the basis of regulation studies were coincident with the cloned isoforms. The expression of the two isoforms was studied in genital and pubic skin fibroblasts from normal men, normal women, and hirsute patients. Messenger ribonucleic acid analysis, using Northern blot and RT-PCR techniques, indicated that both 5 alpha-R1 and -2 messenger ribonucleic acids are expressed in genital skin as well as in public skin fibroblasts. In contrast, studies using specific inhibitors of 5 alpha-R1 (LY306089) and 5 alpha-R2 (finasteride) showed that 5 alpha-R2 is predominant in pubic skin of normal men, normal women, and hirsute patients. These data raise the question of the possible use of specific 5 alpha-R1 inhibitors in the treatment of idiopathic hirsutism.

Androgen binding capacity and 5 alpha-reductase activity in pubic skin fibroblasts from hirsute patients.

We have measured the total (cytosolic plus nuclear) androgen binding capacity of pubic skin fibroblasts from nine patients with hirsutism of various origin. Confluent intact cell monolayers were incubated with increasing concentrations (0.05-2 nM) of [3H]dihydrotestosterone ([3H]DHT) with or without a 200-fold excess of unlabeled DHT. The androgen binding capacities (mean +/- SD) were similar in normal men (411 +/- 171 fmol/mg DNA), women (310 +/- 103 fmol/mg DNA), and hirsute patients (313 +/- 141 fmol/mg DNA) regardless of the plasma androgen levels. In contrast, the 5 alpha-reductase level in pubic skin fibroblasts (mean +/- SD) was, as previously described, higher in hirsute women (3.3 +/- 2.6 fmol/micrograms DNA . h) than in normal women (1.1 +/- 0.6 fmol/microgram DNA . h; P less than 0.05). We conclude from these data that: 1) increased androgen binding capacity cannot be held responsible for hypersensitivity to androgens in hirsutism; 2) the androgen receptor is not regulated by androgens in human skin, as similar levels are observed in men, women, and hirsute patients; 3) this contrasts with 5 alpha-reductase activity and emphasizes the importance of this enzyme as an amplifier of androgen action in areas where it is stimulated by androgens, such as pubic skin.

Estradiol 17 beta-hydroxysteroid dehydrogenase activity in human breast fibroadenomas.

In the human endometrium, the presence of the progesterone-dependent enzyme 17 beta-hydroxysteroid dehydrogenase (E2DH) permits the conversion of an active estrogen, estradiol, into a less active one, estrone. This E2DH activity contributes to the antiestrogenic properties of progesterone. In the present study, E2DH activity was assayed in 54 surgically removed fibroadenomas. This benign breast disease was chosen since it offers rather homogeneous epithelial concentrations and still remains close to normal breast tissue from a pathological and hormonal point of view. E2DH activity was highest in fibroadenomas with high epithelial cell density. In addition, in these high epithelial cell density fibroadenomas (n = 18), E2DH activity increased markedly throughout the luteal phase of the menstrual cycle. Thus, it was 3- to 4-fold higher in fibroadenomas removed at the end of the luteal phase (1520 +/- 166 fmol/mg protein.h) than in those obtained during the follicular phase (375 +/- 95 fmol/mg protein.h). In addition, a striking increase in E2DH activity was observed in fibroadenomas from 5 patients treated with oral progestins (4080 +/- 650 fmol/mg protein.h) and 3 patients receiving progesterone topically applied upon the breast (3830 +/- 475 fmol/mg protein.h). E2DH activity, therefore, appears to be an important mechanism involved in the control by progesterone of estradiol action in breast tissue, as it is in the endometrium. It is also a good index of cellular differentiation and progesterone action at the molecular level. It is hypothesized that E2DH activity might be a specific marker of progesterone receptor itself and could be proposed in the evaluation of the hormone dependence of human breast tissue.