Abstract

Cellular receptors for sex steroids (SSRs) were studied in an unselected series of 55 human pituitary tumors. Cytosolic receptors for estrogen (ERcs) and progesterone (PgRcs) were determined in all cases and cytosolic androgen receptors (ARcs) in 47 cases. Nuclear receptors (ERns, PgRns, ARns) were also studied in 33 cases. ERs and PgRs were determined by an ELISA and ARs by [³H]methyltrienolone binding. Where both cytosolic and nuclear receptors were studied (n=33), ERs, PgRs and ARs were found in at least one subcellular fraction in 66·7, 60·6 and 81·8% of cases respectively, ERs and ARs being mainly recovered from the cytosol and PgRs from the nucleus. No linear correlation was found between preoperative plasma steroid hormones and their specific cellular receptors. Nonetheless, the differential expression of SSRs according to sex and gonadal status at the time of surgery strongly supports their regulation by the steroid environment in vivo: PgRcs were more frequent in tumors found in women (41·4 vs 15·4%, P<0·05), whereas a high expression of ERcs and ARcs (>15 fmol/mg protein) was more common in tumors found in men (34·5 vs 10·3%, P<0·05 and 54·5 vs 24·0% respectively). PgRs were positively correlated with ERns, indicating the possibility of estrogen priming of their expression, and negatively correlated with ARs in nuclear fractions. SSRs appeared to be widely distributed among pituitary tumors, although, compared with other hormone-secreting groups, prolactinomas displayed a higher ERc expression (34·8 ± 11·3 vs 4·8 ± 5·1 fmol/mg protein, P=0·007) and gonadotroph cell adenomas lower ARc values (1·3 ± 0·8 vs 38·2 ± 10·6 fmol/mg protein, P=0·048). Microadenomas were characterized by a higher PgR expression than macroadenomas, whereas hemorrhagic (macro)adenomas were characterized by a high ER expression (>90%). The present results indicate that most pituitary tumors are targets for sex steroids, SSR expression being partially triggered by the steroid environment itself. Possible physiopathological and therapeutic implications of these findings are discussed.

Journal of Endocrinology (1996) 151, 175–184

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.

Abstract

The protein hormone relaxin is secreted by the ovaries throughout the second half of the 23 day pregnancy in the rat. We recently reported that neutralization of endogenous relaxin with monoclonal antibodies for rat relaxin decreases water consumption during the daily light period during the second half of pregnancy in rats. The apparent effects of relaxin on water consumption, however, were extremely modest. One explanation for the failure to observe a greater relaxin-dependent effect on water consumption is failure of the monoclonal antibody for rat relaxin to neutralize all circulating relaxin. A second explanation is that circulating relaxin has only slight effects on water consumption. This investigation was conducted with an experimental model in which circulating relaxin was removed in order to re-examine the effects of relaxin on water consumption during the daily light period in late pregnancy in rats.

On day 9 (D9) of pregnancy, before the presence of relaxin (R) in the circulation, primiparous pregnant rats were ovariectomized (O) or sham ovariectomized (C). Throughout the remainder of pregnancy, rats were treated with combinations of either progesterone (P) and estrogen (E, group OPE) or progesterone, estrogen and porcine relaxin (group OPER) in doses that restore physiological parameters to values similar to those that occur during the second half of pregnancy in intact rats. Progesterone and estrogen were administered by Silastic tubing implants and porcine relaxin was administered via miniature osmotic pump. Sham-ovariectomized animals received either the hormone vehicles (group SC) or no implants (group IC). Water consumption was measured daily from D4 to D20 at both 0700 and 2100 h which was when the lights went on and off respectively.

Water consumption increased as pregnancy continued from D10 to D20 during the daily 10 h dark periods (P<0·01), but not during the 14 h light periods for all four groups. Daily water consumed by rats in group OPE was significantly lower (P<0·05) than that consumed by shamovariectomized rats from D17 to D20 and lower than that consumed by rats in group OPER on D20. During the dark period there was no difference in water consumption among groups. During the light period, however, group OPE consumed significantly less water (P<0·05) than group C from D18 to D22. Moreover, there was a consistent tendency (P<0·13) for the water consumption to be greater in rats in group OPER than in those in the relaxin-deficient group OPE during the daily light period from D11 to D20 of pregnancy.

We conclude that the increase in water consumption that occurs during the daily dark periods during the second half of pregnancy is not attributable to circulating relaxin. Circulating relaxin promotes only modest increases in water consumption during the daily light periods during late pregnancy in the rat.

Journal of Endocrinology (1997) 153, 33–40

We examined 24p3 expression in the mouse uterus at various stages of the natural estrous cycle and during the preimplantation period. The level of 24p3 mRNA appeared intensively in proestrus and estrus, then declined sharply from metestrus to diestrus. Consistent with this observation, 24p3 protein was abundant in proestrus, decreased from estrus to metestrus and declined to a very low level in diestrus. The uterine 24p3 expression closely overlapped with the estradiol (E2) surge in proestrus and estrus but it was suppressed when progesterone (P4) rose to a high level during the reproductive cycle. Neither the protein nor its message was detected in the uteri of immature mice or ovariectomized adult animals. While an injection of P4 to these animals was unable to initiate uterine 24p3 expression, administration of estrogenic steroids to these animals markedly stimulated the gene expression. Treatment of these animals with E2 together with P4, on the other hand, did not stimulate the gene expression. In pregnant animals (day 1 (D1)=day of vaginal plug), 24p3 mRNA remained at a high level on D1 and D2 but dropped to an almost undetectable level on D3 and D4. This was accompanied by a decrease in 24p3 protein from D1 to D2 and a decline in the protein to undetectable levels from D3 to D4. The staining patterns of both the immunohistochemical localization of 24p3 protein and in situ hybridization for the detection of 24p3 mRNA in the uterine sections showed that 24p3 expression took place mainly in the luminal and glandular epithelial cells of the endometrium. This together with our previous observation that 24p3 protein is found in uterine luminal fluid indicates that the protein is secreted primarily from these cells to their respective luminal surfaces during proestrus and estrus.

The role of epidermal growth factor (EGF) in the regulation of estrogen receptor-alpha (ER-alpha) gene expression in the human breast cancer cell line MCF-7 was investigated. Treatment of cells with 0.4 ng/ml EGF resulted in an approximately 60% decrease in ER-alpha protein concentration by 6 h and the amount of receptor remained suppressed for 24 h. Ligand binding assays demonstrated that the decrease in ER-alpha protein corresponded to a similar decrease (approximately 50%) in estradiol binding sites. Although EGF treatment resulted in a decrease in the number of binding sites, it had no effect on the binding affinity of ER-alpha. The dissociation constant of the estradiol-ER-alpha complex in the presence or absence of EGF was the same (K(d)=2.3x10(-)(10) M in control cells versus K(d)=1.98x10(-)(10) M in EGF-treated cells). The decrease in ER-alpha protein concentration paralleled a decrease in the steady-state amount of ER-alpha mRNA. By 9 h there was an approximately 60% decrease in ER-alpha mRNA. The amount of ER-alpha mRNA remained suppressed for 48 h. Transcription run-on experiments demonstrated that there was a decrease of approximately 70% in ER-alpha gene transcription upon EGF treatment, suggesting that the mechanism by which EGF regulates ER-alpha gene expression is transcriptional. In addition to regulating the amount of ER-alpha, EGF affected the activity of the receptor. At high concentrations, EGF induced progesterone receptor. Estradiol and high concentrations of EGF had an additive effect on progesterone receptor. In contrast to high concentrations, low concentrations of EGF had no effect on progesterone receptor and blocked estradiol induction. The effects of EGF on ER-alpha expression were inhibited by tyrophostins and wortmannin, suggesting that the effects of the growth factor are mediated by the EGF receptor and protein kinase B. When the cells were placed in serum-free medium and then treated with EGF, there was no effect on ER-alpha protein concentration or activity. However, increasing concentrations of serum restored the effects of EGF on ER-alpha, suggesting that an additional serum factor was required for the EGF-mediated effect on the decrease in ER-alpha protein concentration.

Development of the functional secretory epithelium in the mammary gland of the female mouse requires the elongation of the anlage through the mammary fat pad to form the primary/secondary ductal network from which tertiary ductal side-branches and lobuloalveoli develop. In this study we examined the hormonal requirements for the spatial development of the primary/secondary epithelial network and tertiary side-branches by quantifying ductal growth and epithelial cell proliferation in normal and hormone-treated BALB/c mice between 21 and 39 days of age. In normal mice, an allometric increase in ductal length commenced at 31 days of age and resulted in completion of the primary/secondary ductal network by 39 days of age. Concurrent with this allometric growth was a significant increase in cellular proliferation in the terminal end-buds (TEBs) of the ductal epithelium from 29 days of age, as determined by 5-bromo-2'-deoxyuridine (BrdU) incorporation. A level of cellular proliferation similar to that in the TEBs of 33-day-old control mice could be induced in the TEBs of 25-day-old mice following treatment for 1 day with estrogen (E), or progesterone (P) or both (E/P), indicating that both E and P were mitogenic for epithelial cells of the peripubertal TEBs. However, the period of allometric ductal growth in untreated mice did not correspond to an increase in serum E or P (which might have been expected during the estrous cycle). In addition, epithelial growth was not observed in mammary glands from 24-day-old mice that were cultured in vitro with E, P or E/P. In contrast to treatment with E, treatment with P promoted a dramatic increase, relative to control mice, in the number of tertiary branch points upon the primary/secondary ductal network. BrdU labeling of mammary glands from 24- 33-day-old mice pelleted with cholesterol (C), E, P or E/P confirmed the greater mitogenicity of P on the epithelial cells of the secondary/tertiary ducts as compared with C or E. Concurrent with these changes, localized progesterone receptor (PR) expression in clusters of cells in the ductal epithelium was associated with structures that histologically resembled early branch points from ductules. In conclusion, our results suggest that additional endocrine growth factor(s) other than E and P contribute to the development of the primary/secondary ductal network, and that P is responsible for the formation of tertiary side-branches in the mammary glands of mice during puberty.

The number of epidermal growth factor (EGF) binding sites was determined by competitive binding assays in a series of 46 pituitary macroadenomas. A single concentration of 125I-EGF (1 nM) was used for all experiments. In four cases, a displacement curve was obtained by adding increasing concentrations of cold EGF, and Scatchard analysis showed the presence of two classes of EGF binding sites, with Kd1 = 0.62 +/- 0.23 nM and Kd2 = 53.8 +/- 8.2 nM for the high- and low-affinity binding sites respectively. The distribution of EGF binding sites was studied in 42 cases by a single-point assay, in the presence and in the absence of a 100-fold cold EGF excess. A non-parametric distribution of EGF binding sites was observed (median 10.2 fmol/mg membrane protein, range 0.0-332.0). EGF-receptor positivity, defined as EGF binding > or = 10.0 fmol/mg protein, was observed in 23 samples (54.8%), especially in prolactinomas (76.5%, P < 0.05 vs other tumors taken together) and in gonadotrope adenomas (62.5%). EGF binding was higher in invasive than in non-invasive adenomas (median: 12.8 vs 0.0 fmol/mg membrane protein, P = 0.047), and especially in adenomas invading the sphenoid sinus (median 26.7 fmol/mg membrane protein, P = 0.008 vs other adenomas). EGF binding also tended to increase with the grade of supra/extrasellar extension according to Wilson (P = 0.15). Sex steroid receptors (SSRs) were simultaneously determined in both cytosolic and nuclear fractions of 31 pituitary adenomas. Estrogen and progesterone receptors were determined by an enzyme-linked immunoassay and androgen receptors by a competitive binding assay with [3H]methyltrienolone. No correlation could be found between EGF binding and either the gender and gonadal status of the patients, or the expression of SSRs by the adenomas. We conclude that the EGF family of growth factors may play a role in the evolution of a significant subset of human pituitary adenomas, especially in their invasiveness, and that a high EGF binding capacity may represent an additional marker of aggressiveness for these tumors. Sex steroids do not appear to have a significant role in the regulation of EGF binding in vivo in these tumors.

High doses of lipopolysaccharide (LPS) induce transient hyperglycemia, then chronic hypoglycemia and increased insulin resistance. In addition, appetite is reduced, while body temperature and concentrations of cortisol and tumor necrosis factor alpha (TNFalpha) are elevated. Furthermore, concentrations of GH and IGF-I are reduced in cattle. The objectives of this study were to determine whether a gonadal steroid implant (20 mg estrogen and 200 mg progesterone) given to endotoxemic steers would: (1) reduce hyperglycemia, reduce hypoglycemia, reduce insulin resistance, (2) reduce changes in concentrations of GH and IGF-I, (3) reduce inappetence and reduce concentrations of blood urea nitrogen (BUN) and non-esterified fatty acids (NEFA), and (4) reduce fever and concentrations of TNFalpha and cortisol. Holstein steers were assigned within a 2x2 factorial arrangement of treatments as follows (n=5 per group): C/C, no steroid and vehicle; S/C, steroid and vehicle; C/E, no steroid and LPS (1 microg/kg body weight (BW), i.v.); S/E, steroid and endotoxin. Steroid implants were given at 20 weeks of age (day 0) and serial blood samples (15 min) were collected on day 14 for 8 h, with vehicle or LPS injected after 2 h. Intravenous glucose tolerance tests (100 mg/kg BW) were carried out at 6 h and 24 h. Hyperglycemia was 67% lower (P<0.05) in S/E- compared with C/E-treated steers between 30 and 150 min after i.v. injection of LPS. Hypoglycemia developed after 4 h and insulin resistance was greater in S/E- compared with C/E-treated steers (P<0. 05) at 6 and 24 h. Concentrations of IGF-I were restored earlier in steroid-treated steers than in controls. Concentrations of GH were not affected by steroids, but increased 1 h after injection of LPS, then were reduced for 2 h. Appetite was greater (P<0.05) in S/E- (2.1% BW) compared with C/E-treated steers (1.1% BW) (pooled s.e.m.=0.3). Concentrations of NEFA increased after injecting LPS, but concentrations were lower (P<0.05) in S/E- compared with C/E-treated steers. LPS did not affect concentrations of BUN, but concentrations were lower in steroid-treated steers. Steroids did not affect body temperature or concentrations of TNFalpha and cortisol. In summary, gonadal steroids reduce hyperglycemia, reduce inappetence and tissue wasting, but increase insulin resistance. Furthermore, concentrations of IGF-I are restored earlier in steroid-treated than in non-steroid-treated steers injected with LPS. It is concluded that gonadal steroids reduce severity of some endocrine and metabolic parameters associated with endotoxemia. However, it is unlikely that gonadal steroids acted via anti-inflammatory and immunosuppressive actions of glucocorticoids or through reducing concentrations of cytokines.