Estetrol (E4) is a natural estrogen produced exclusively by the human fetal liver during pregnancy. Its physiological activity remains unknown. In contrast to ethinyl estradiol and estradiol (E2), E4 has a minimal impact on liver cell activity and could provide a better safety profile in contraception or hormone therapy. The aim of this study was to delineate if E4 exhibits an activity profile distinct from that of E2 on mammary gland. Compared with E2, E4 acted as a low-affinity estrogen in both human in vitro and murine in vivo models. E4 was 100 times less potent than E2 to stimulate the proliferation of human breast epithelial (HBE) cells and murine mammary gland in vitro and in vivo respectively. This effect was prevented by fulvestrant and tamoxifen, supporting the notion that ERα (ESR1) is the main mediator of the estrogenic effect of E4 on the breast. Interestingly, when E4 was administered along with E2, it significantly antagonized the strong stimulatory effect of E2 on HBE cell proliferation and on the growth of mammary ducts. This study characterizes for the first time the impact of E4 on mammary gland. Our results highlight that E4 is less potent than E2 and exhibits antagonistic properties toward the proliferative effect of E2 on breast epithelial cells. These data support E4 as a potential new estrogen for clinical use with a reduced impact on breast proliferation.
C Gérard, S Blacher, L Communal, A Courtin, E Tskitishvili, M Mestdagt, C Munaut, A Noel, A Gompel, C Péqueux and J M Foidart
Josephine F Trott, Katherine C Horigan, Julia M Gloviczki, Kristen M Costa, Bradley A Freking, Chantal Farmer, Kanako Hayashi, Thomas Spencer, Joseph E Morabito and Russell C Hovey
Prolactin (PRL) acts through its receptor (PRLR) via both endocrine and local paracrine/autocrine pathways to regulate biological processes including reproduction and lactation. We analyzed the tissue- and stage of gestation-specific regulation of PRL and PRLR expression in various tissues of pigs. Abundance of pPRLR-long form (LF) mRNA increased in the mammary gland and endometrium during gestation while in other tissues it remained constant. There was a parallel increase in the abundance of the pPRLR-LF protein in the mammary gland and endometrium during gestation. We determined the hormonal regulation of pPRLR-LF mRNA expression in various tissues from ovariectomized, hypoprolactinemic gilts given combinations of the replacement hormones estrogen (E2), progestin (P), and/or haloperidol-induced PRL. Abundance of pPRLR-LF mRNA in kidney and liver was unaffected by hormone treatments. Expression of uterine pPRLR-LF mRNA was induced by E2 whereas the effect of E2 was abolished by co-administering P. The expression of pPRLR-LF mRNA in the mammary gland stroma was induced by PRL, whereas E2 induced its expression in the epithelium. In contrast to these changes in pPRLR expression, pPRL expression was relatively constant and low during gestation in all tissues except the pituitary. Taken together, these data reveal that specific combinations of E2, P, and PRL differentially regulate pPRLR-LF expression in the endometrium and mammary glands, and that the action of PRL on its target tissues is dependent upon pPRLR-LF abundance more so than the local PRL expression.
M Baratta, S Grolli, A Poletti, R Ramoni, M Motta and C Tamanini
Androgens have been found in mammary epithelium and in milk throughout the cycle of the mammary gland in vivo. The aim of this study was to investigate the possible role of these substances in mammary epithelial growth and differentiation in the mouse HC11 cell line. Cells were stimulated with testosterone, dihydrotestosterone, androstenedione and 5alpha-androstane-3alpha,17beta-diol at concentrations ranging between 0.3 nM and 30 nM. Cyproterone acetate or flutamide, androgen receptor antagonists, (3 microM) were used to block specific androgen effects. Proliferative effects were measured by an MTT (tetrazolium blue) conversion test and [(3)H]thymidine uptake. HC11 cells were transfected with pbetacCAT, a chimeric rat beta-casein gene promoter-chloramphenicol acetyl transferase (CAT) gene construct and CAT ELISA was used to determine gene expression. RT-PCR was performed to detect androgen receptor expression. After 24, 48 and 72 h androgens significantly (P<0.05) increased proliferation. Androgen antagonists significantly (P<0.05) reduced the proliferative effects. Furthermore androgens potentiated the lactogenic effect of prolactin, insulin and dexamethasone (P<0.05). Finally, the androgen receptor gene was expressed in both proliferating and differentiated HC11 cells. These observations lead us to hypothesize an activity of this class of steroids in mammary physiology. In particular, androgens stimulate cell proliferation and beta-casein gene expression; this influence appears to be mediated by androgen receptors.
ME Dunbar, P Dann, CW Brown, J Van Houton, B Dreyer, WP Philbrick and JJ Wysolmerski
We have previously demonstrated that overexpression of parathyroid hormone-related protein (PTHrP) in the mammary glands of transgenic mice results in defects in ductal elongation and branching during puberty and in lobuloalveolar development during pregnancy. In addition, we have shown that PTHrP is necessary for the formation of the initial ductal tree during embryonic mammary development. In order to examine the effect of varying the timing of PTHrP overexpression on mammary development, we created tetracycline-regulated, K14-tTA/Tet(O)-PTHrP double transgenic mice. In this report, we document that this 'tet-off' system directs transgene expression to the mammary gland and that it is fully repressed in the presence of tetracycline. Using these mice, we demonstrate that transient overexpression of PTHrP before birth causes defects in ductal branching during puberty and that overexpression of PTHrP during puberty decreases the rate of ductal elongation. Furthermore, we demonstrate that if PTHrP overexpression is initiated after ductal morphogenesis is completed, lobuloalveolar development is unaffected. Finally, we demonstrate that the impairment in ductal elongation caused by PTHrP is associated with an increase in the basal rate of epithelial cell apoptosis in terminal end buds and a failure to increase end bud cell proliferation and decrease apoptosis in response to estrogen and progesterone.
H. L. Buttle
Ovariectomy, but not removal of the corpora lutea, of gilts at mid-pregnancy delayed the onset of lobuloalveolar development in the mammary glands. Lactogenesis at the time of parturition was delayed by both ovariectomy and removal of the corpora lutea.
J. Endocr. (1988) 118,41–45
B. FREEDMAN and R. A. HAWKINS
In rat mammary tumours and uteri it was found that reaction time, ionic strength, reproductive state and storage in liquid nitrogen affected the form of the oestrogen receptor detected by sucrose density-gradient analysis using a vertical-tube rotor. A standard method of gradient analysis was defined. The sedimentation profile of the oestrogen receptor was compared in three types of experimental rat mammary tumour of known hormonal sensitivity. These were two lines of ovary-independent transplantable tumours and those tumours induced by dimethylbenz(a)anthracene (DMBA) which are mainly ovary-dependent. In all three types of tumour the 8S receptor was the predominant molecular form (32 out of 36 tumours induced by DMBA and 11 out of 13 transplantable tumours). Sedimentation profiles of the oestrogen receptor were also examined in 46 human breast carcinomata found to be receptor-positive by the dextran-coated charcoal technique. Of these, 17 were found to be receptor-negative by gradient analysis and 28 out of 29 receptor-positive tumours contained 8S receptor. It was concluded that (1) 8S receptor is the predominant molecular form in both human and rat mammary tumours, (2) molecular form of receptor is not related to ovarian dependence in these rat mammary tumours and (3) gradient analysis seems unlikely to provide additional discrimination in the prediction of response to endocrine therapy over that provided by simpler methods.
A. BORKOWSKI, M. L'HERMITE, P. DOR, E. LONGEVAL, M. ROZENCWEIG, C. MUQUARDT and E. VAN CAUTER
The endocrine response to prolonged dexamethasones treatment was investigated in six postmenopausal women with generalized mammary carcinoma. Plasma cortisol levels decreased rapidly and became undetectable whereas significant concentrations of plasma dehydroepiandrosterone and androstenedione persist throughout the study, even in two ovariectomized patients, indicating a certain degree of autonomy or a greater resistance of adrenal 'androgens' to the inhibition of ACTH secretion.
Except in the ovariectomized patients, plasma testosterone did not fall significantly whereas the plasma oestrogens tended progressively towards undetectable concentrations. A similar response was found in six normal postmenopausal women although the disappearance of their oestrogens was relatively rapid. This indicates that much of the testosterone patient after the menopause could still be produced by the ovaries whereas the ovarian production of oestrogens becomes negligible. The delayed disappearance of oestrogens in the patients with mammary carcinoma indicates that the persisting adrenal 'androgens' remained efficient precursors of oestrogen synthesis within the peripheral tissues and presumably within the mammary tumour itself. Plasma dihydrotestosterone behaved like the plasma oestrogens. Despite the fall in plasma oestrogens, plasma gonadotrophins did not increase further but plasma prolactin rose progressively.
The persistence of steroid sex hormones and the rise of plasma prolactin might explain the poor response to dexamethasone treatment in mammary carcinoma.
H. M. Buckler, D. L. Healy and H. G. Burger
Ovarian inhibin production is stimulated by the administration of human menopausal gonadotrophins or following a rise in endogenous LH and FSH. In order to determine whether FSH specifically stimulates inhibin secretion in vivo, immunoassayable serum inhibin levels were measured following the administration of a highly purified preparation of urinary FSH free of significant contamination with LH. Ten anovulatory women underwent a protocol of induction of ovulation with purified FSH and human chorionic gonadotrophin (hCG). During the induction of ovulation, blood samples were taken for radioimmunoassay of FSH, LH, oestradiol, progesterone and inhibin. During the administration of FSH there were increases in plasma concentrations of FSH, oestradiol and inhibin (P < 0·01) but no significant change in the concentration of LH. Oestradiol and inhibin concentrations rose in parallel and were closely correlated (τ = 0·920, n = 110, P < 0·001). There was also a direct correlation between the measured level of FSH and inhibin (τ = 0·512, n = 110, P < 0·05), but there was no correlation between LH and oestradiol, inhibin or FSH. Inhibin (τ− = 0·702, n = 10, P < 0·01) and oestradiol (τ− = 0·691, n = 10, P < 0·01) were correlated with the number of follicles seen on ovarian ultrasound. Levels of oestradiol and inhibin reached a peak on the day of hCG administration or on the following day. Inhibin levels then fell over the next 2 days in all cycles. In an ovulatory cycle resulting in conception, inhibin and oestradiol then rose in parallel with progesterone. We conclude that inhibin appears to be a follicular product which, in the follicular phase of the cycle, is stimulated by FSH alone, with granulosa cells being the probable site of production.
Journal of Endocrinology (1989) 122, 279–285
R. C. HALLOWES, D. Y. WANG, D. J. LEWIS, C. R. STRONG and R. DILS
Explants of mammary glands and of subcutaneous body fat from sexually mature virgin and from 19-day pregnant Sprague-Dawley rats and of mammary gland from 5-day lactating Sprague-Dawley rats, were maintained in organ culture for up to 96 h. The effects of insulin (I), corticosterone (B), prolactin (P) and growth hormone (G) on the rate of fatty acid synthesis were measured by the incorporation of [14C]acetate. The effect of these hormones on the synthesis of various carbon-chain length fatty acids was measured by radio gas-liquid chromatography.
Explants from both tissues had a reduced rate of fatty acid synthesis after 24 h in medium 199, but this rate was increased by the addition of insulin. In explants of subcutaneous fat from virgin rats, the rate was further increased by culture in IBP or IBG, but this increase was not blocked by actinomycin D. In explants from subcutaneous fat of 10-day pregnant rats the rate was not increased by the addition of B, P or G to the insulin-containing medium. In mammary gland explants from virgin rats, IBP stimulated a greater rate of fatty acid synthesis than did insulin alone. In mammary gland explants from 10-day pregnant rats, the rate of fatty acid synthesis was increased by both IBP and IBG. In mammary gland explants from rats on the 5th day of lactation, both IBP and IBG increased the rate of fatty acid synthesis compared with insulin or IB. Actinomycin D blocked the increased fatty acid synthesis produced by prolactin or growth hormone but not that produced by insulin alone.
Mammary gland explants from rats on the 5th day of lactation were cultured for the first 4 h after excision in medium 199 that contained sodium [14C]acetate. Sixty-eight per cent of the 14C was incorporated into C8-C12 fatty acids. In explants from subcutaneous fat none of the hormones tested increased 14C incorporation in these fatty acids. In mammary gland explants from virgin or 10-day pregnant rats, insulin, corticosterone and prolactin increased the incorporation in these fatty acids. Growth hormone was less efficient than prolactin in stimulating C8-C12 fatty acid synthesis.
C M Allan, Y Wang, M Jimenez, B Marshan, J Spaliviero, P Illingworth and D J Handelsman
Ovarian primordial follicle reserve is considered hormonally independent or subject to depletion by FSH-driven follicle recruitment. To explore specific in vivo effects of FSH on early follicle populations in the absence of luteinizing hormone (LH) activity, we examined mature hypogonadal (hpg), gonadotrophin-deficient mice expressing transgenic (tg) human FSH. Sustained expression of tg-FSH (5.3 ± 0.3 IU/l) increased ovary weights fourfold and significantly elevated total primordial follicle numbers twofold in tg-FSH hpg (4209 ± 457) relative to non-tg hpg (2079 ± 391) and wild-type (2043 ± 195) age-matched ovaries. Absolute primary follicle numbers in tg-FSH hpg ovaries were similar to non-tg hpg and wild-type ovaries. Furthermore, tg-FSH quantitatively increased secondary and antral follicles in hpg ovaries to numbers equivalent to wild-type, but did not induce ovulation, indicating a selective FSH response without LH. Circulating inhibin B and inhibin A levels were significantly increased in tg-FSH hpg females compared with hpg controls, and inhibin B correlated with antral number, consistent with FSH-driven antral follicle formation. These findings revealed that sustained pituitary-independent FSH activity, in the absence of endogenous gonadotrophins, promotes an increase in primordial follicle reserve despite also stimulating follicular growth in mature females. Therefore, the tg-FSH hpg ovary presents a novel paradigm to evaluate specific gonadotrophin effects on follicle reserve and recruitment.