Estrogenic Molecules Research Articles

Influence of Human Infant Formulas Containing Phytoestrogens on Brain Aromatase Activity in Adult Rats

The biosynthesis of estrogens from androgen precursors is regulated by the aromatase enzyme, and estrogenic molecules are known to be significant factors in the formation of sexually dimorphic brain structures in the anterior hypothalamus. Phytoestrogens have been shown to inhibit aromatase enzyme activity in peripheral tissues. In this study, phytoestrogen-containing human infant formulas were examined to determine whether these estrogen mimics influence brain aromatase levels in adult rats.

Identification of estrogen-modified nucleosides from calf thymus DNA reacted with 6-hydroxyestrogen 6-sulfates.

Two estrogen sulfates, pyridinium 3-methoxyestra-1,3, 5(10)-trien-6alpha-yl sulfate (3MeE-6alpha-S) and its 6beta-isomer (3MeE-6beta-S), synthesized as model compounds to demonstrate the carcinogenesis of estrogen, were found to react with calf thymus DNA to produce steroid-modified DNA adducts. Digestion of the DNA by nuclease P1 and phosphodiesterase I followed by alkaline phosphatase gave a deoxyribonucleoside fraction, of which N2-[3-methoxyestra-1,3, 5(10)-trien-6alpha-yl]deoxyguanosine, N2-[3-methoxyestra-1,3, 5(10)-trien-6beta-yl]deoxyguanosine, N6-[3-methoxyestra-1,3, 5(10)-trien-6beta-yl]deoxyadenosine, and N6-[3-methoxyestra-1,3, 5(10)-trien-6alpha-yl]deoxyadenosine (identified as a base adduct) were identified using HPLC by comparing them with authentic specimens prepared by reacting dG and dA with both sulfates. No steroid-dC adduct was detected in the digestion products of the DNA adduct, although dC reacted with the sulfates to form N4-[3-methoxyestra-1,3,5(10)-trien-6beta-yl]deoxycytidine. These results mean that estrogen 6-sulfate has an ability to modify DNA via the amino group of a guanine or adenine residue in DNA. The present studies imply that a sequential metabolism (hydroxylation and sulfation) at the C6-position of the estrogen molecule causes damage to DNA.

Heterogeneity of binding sites of estrogens and proliferative effect of estrogenic molecules in human colon cancer cell lines

Heterogeneity of binding sites of estrogens and proliferative effect of estrogenic molecules in human colon cancer cell lines

Contraception as prevention and therapy: Sex steroids and the brain

The brain is one of the specific target tissues for sex steroid hormones. Estrogens, progestins and androgens are able to induce several effects in brain areas of the central nervous system (CNS), through the binding with specific receptors. Specific receptors for gonadal steroids have been identified in the amygdala, hippocampus, basal forebrain cortex, cerebellum, locus ceruleus, midbrain rafe nuclei, glial cells, pituitary gland, hypothalamus and central gray matter. At the hypothalamic level, the principal target for sex steroids is those neurons producing the pulsatile release of the gonadotropin releasing hormone (GnRH), localized in the mediobasal hypothalamus and the arcuate nucleus. The GnRH release depends on the complex and co-ordinated interrelationships among gonadal steroids, pituitary gonadotropins and neuroactive transmitters, such as the noradrenaline, dopamine, opioid peptides (β-endorphin), acetylcholine, serotonin, γ-aminobutyrric acid, corticotropin releasing hormone and neuropeptides Y. The interplay of these control mechanisms is governed by peripheral feedback signals; as well as the input from higher brain centers they may modify the GnRH secretion. The anterior pituitary lobe is the best known target tissue for endogenous or exogenous sex steroid hormones, because it is possible to detect luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels in blood, as the expression of the pituitary cells' activity. The synthesis and release of FSH and LH by the gonadotropic cells depend upon the peripheral control of gonadal hormones and the GnRH hypothalamic release.In summary, during a woman's reproductive life, the interaction between neurotransmitters, neuropeptides and gonadal hormones modulates the hypothalamo—pituitary—gonadal axis by acting selectively on the synthesis and release of GnRH and of pituitary gonadotropic hormones. The increased use of oral contraceptives in the last 30 years and, in general, of sex steroid hormone derivative therapies, has led to the study of the biochemical and metabolic properties of the different progestin molecules available in hormonal therapies by focusing attention on the interactions between estrogens and progestins in the modulation of the hypothalamo—pituitary-gonadal axis. The different kinds of estrogen and progestin molecules used in oral contraceptives inhibit the ovulatory process and may interfere with other sex steroid hormone receptors, thus exerting multiple effects in each target tissue.

Promoter and species specific differential estrogen-mediated gene transcription in the uterus and cultured cells using structurally altered agonists.

Certain types of estrogenic compounds have been shown to have tissue-specific actions. In addition, some tissues may exhibit differential gene regulation by agonists and antagonists. Our previous studies using structurally modified estrogenic molecules had indicated differential effects on specific estrogen responses, indicating that the activity of the estrogen receptor protein can be altered depending not only upon the structure of the bound ligand but also the regulated gene itself. The mechanism of differential induction, however, was not determined, and might involve altered binding to the estrogen response element (ERE), altered transcription, or post-transcriptional modification of gene products. Our previous studies indicated that differential induction by modified diethylstilbestrol (DES) agonists could not be accounted for by differences in ligand affinity for the estrogen receptor (ER) or differential binding of the ER to a consensus vitellogenin A2 (vit A2) ERE. To determine if this differential hormonal responsiveness was reflected at the level of transcription, we analyzed mouse uterine mRNA of several estrogen-responsive genes, including glucose-6-phosphate dehydrogenase (G6PD), ornithine decarboxylase (ODC) and lactoferrin, by Northern blot following injection with the modified agonists DES, indenestrol A (IA), indenestrol B (IB) and Z-pseudo DES (ZPD). All compounds induced the G6PD message, although IB and ZPD induced expression only transiently, while DES and IA maintained the message for 24 h. No difference in induction was seen for ODC message, which was induced equally by all the compounds. In contrast, lactoferrin, a highly estrogen-responsive gene, was induced only by DES and IA and not by the other agonists IB or ZPD, showing that the lactoferrin gene was differentially regulated by these compounds. To determine whether this difference was due to altered transcriptional activity, the mouse lactoferrin estrogen-responsive module (mERM) linked to a chloramphenicol acetyl transferase (CAT) reporter gene was tested in transfected cells. Using the mouse estrogen receptor in RL95 cells, DES and IA induced expression of CAT, but IB did not, confirming the differential response seen in vivo. To show whether this difference in transcription occurred because of altered binding to the lactoferrin ERE, which is not a perfect consensus ERE a gel shift assay was used to examine DNA binding of ER bound to the agonists. All ligands produced equivalent binding to the lactoferrin ERE suggesting that differential regulation was not a result of altered DNA binding. Taken together, these observations indicate that the differential induction of lactoferrin by these compounds occurs via altered activation of the transcriptional components unique to lactoferrin and is likely to involve altered interaction with co-activators. Surprisingly, unlike the mouse ER, the human estrogen receptor activated and induced expression of lactoferrin estrogen-responsive module-CAT with all the compounds. Mouse ER is also known to vary from the human ER in its activity with the triphenylethylene estrogen tamoxifen, which has agonist activity with the mouse ER but mixed antagonist/agonist activity with the human ER. The data show that human and mouse estrogen receptors are activated differently by this group of stilbestrol estrogen ligands when assayed on the lactoferrin response element, which is the first description of this type of gene and species specific difference. Lactoferrin gene regulation by estrogen receptor can be used as a model to study the mechanism of differential gene activation by different estrogen agonists and antagonists using a more physiological situation than commonly used with in vitro gene reporter systems.

A developmental switch in expression from blastocyst to endometrial/placental-type cytochrome P450 aromatase genes in the pig and horse.

Pig blastocysts exhibit a transient period of estrogen production at periimplantation, with a second, more sustained period of estrogen synthesis occurring in endometrium and placenta at later pregnancy. Previously we reported the isolation of cDNA clones encoding a novel isoform of cytochrome P450 aromatase (the terminal enzyme in the estrogen biosynthetic pathway) from porcine periimplantation blastocysts. The present study investigated pregnancy-associated expression, in blastocysts and maternal reproductive tract tissues of this and an additional mRNA transcript encoding a distinct P450 aromatase isoform. Restriction endonuclease and nucleotide sequence analyses of 44 cDNA clones demonstrated that the major aromatase mRNA in periimplantation blastocysts and early-pregnancy endometrium and placenta (blastocyst-type) differed in sequence from the major aromatase mRNA expressed in endometrium and placenta at midpregnancy (endometrial-type). The deduced blastocyst and endometrial aromatase isoform protein sequences had 93% similarity. A third type of aromatase mRNA, deleted in exons 4-6 sequences, also was cloned from blastocysts. This cDNA was identical in nucleotide sequence to the blastocyst full-length aromatase cDNA and specified an open reading frame of 354 amino acids for a putative aromatase-related protein containing the heme-binding domain. Expression of this shorter mRNA in blastocysts was confirmed by reverse transcription polymerase chain reaction. The 5'-untranslated exon sequences in the transcripts encoding the blastocyst-type aromatase isoform were distinct from that of the endometrial type, consistent with differential expression of multiple chromosomal genes. In periimplantation equine embryos, however, embryonic and placental 5'-untranslated exon-containing transcripts were coexpressed. Results identify an aromatase isoform expressed in the endometrium and placenta at midpregnancy, demonstrate a transition in synthesis of aromatase isoform-specific mRNAs during placental development, and suggest the preferential involvement of the blastocyst aromatase isoform in synthesis of estrogenic molecules that may function in embryo-maternal signaling at periimplantation.

A review of brain aromatase cytochrome P450

Aromatase cytochrome P450 (P450 AROM ) enzyme activity catalyzes the conversion of androgens to estrogens in specific brains areas. During central nervous system development local estrogen formation influences the sexual differentiation of neural structures (i.e., by increasing neurite growth and establishing neural circuitry) and modulates neuroendocrine/reproductive functions and sexual behavior. More than 20 years ago, in 1970, Naftolin et al. provided preliminary direct evidence for the aromatization of androgens by central neuroendocrine tissues. This work created the foundation for the brain aromatase hypothesis. A review of past and recent data reveals the importance of brain aromatase in the development and function of the central nervous system. This review re-examines the aromatase hypothesis in light of recent data and a theoretical proposal is presented in reference to the aromatase mechanism. The metabolic pathway of androgen metabolism by the aromatase cytochrome P450 pathway, cell type, distribution, developmental profile, and regulation of brain aromatase is also presented. The complex nature of brain aromatase is exemplified by recent molecular biology studies examining the expression of aromatase cytochrome P450 during prenatal/postnatal development. Data derived from these studies provide insight into the regulation of the brain aromatase cytochrome P450 gene and suggest an additional level of control for the expression of brain aromatase. These findings present evidence for the utilization of alternative promoter(s) in man and rodents in driving aromatase gene expression in brain. It is clear that molecular mechanism(s) account for the diverse expression of aromatase in different neural tissue sites and during various physiological states or developmental periods. Therefore, further study is necessary in order to understand the significance of the regulation of local estrogen biosynthesis by the aromatase cytochrome P450 gene during prenatal and postnatal development due to the dramatic impact these estrogen molecules have on neural development and their influence on reproductive function and behavior.

Estrogens in unexpected places: possible implications for researchers and consumers.

Estrogenic activity originating in unexpected places was encountered on three occasions during an investigation of whether Saccharomyces cerevisiae synthesized estrogens. In each instance, estradiol found in the conditioned yeast culture medium originated from an exogenous source and was not synthesized by the yeast. In the first instance, yeast grown in the laboratory showed a time-dependent increase in estradiol in the conditioned medium. However, the culture medium supplement Bacto-peptone was found to contain large amounts of estrone. When added to yeast cultures in the form of YPD medium (yeast extract, Bacto-peptone, and dextrose), S. cerevisiae converted the estrone to estradiol leading to the accumulation of estradiol over time. In the second instance, commercially purchased S. cerevisiae grown in a molasses medium exhibited substantial amounts of estradiol. However, corn and beet molasses contained sufficient estrone and estradiol to account for the findings. As in the first instance, the yeast converted the estrone into estradiol. In the third instance, autoclaving culture medium in polycarbonate plastic flasks was found to cause an estrogenic substance to be added to the medium, whether yeast were present or not. It was determined that the autoclaving process leached bisphenol-A (BPA) out of the polycarbonate plastic. BPA was shown to bind to estrogen receptors and to induce estrogenic activity, including stimulation of MCF-7 breast cancer-cell proliferation and induction of the expression of progesterone receptors. The three instances highlight potential problems for investigators who might inadvertently add estrogens to experimental systems confounding their results. The BPA findings raise concerns about the possible addition of this estrogenic molecule to the food supply since polycarbonate plastic is used in myriad applications in the packaging of food and beverages. Although we are unaware of the substantial contamination of food products with BPA, we believe this possibility should be carefully investigated.

Estrogen-specific 17 beta-hydroxysteroid oxidoreductase type 1 (E.C. 1.1.1.62) as a possible target for the action of phytoestrogens.

Several plant estrogens, especially coumestrol and genistein, were found to reduce the conversion of [3H]estrone to [3H] 17 beta-estradiol catalyzed by estrogen-specific 17 beta-hydroxysteroid oxidoreductase Type 1 (E.C. 1.1.1.62) in vitro. Coumestrol, the most potent inhibitor in our experiments, is the best inhibitor of the enzyme known to date. All compounds with inhibitory effects were also estrogenic. However, structural demands for 17 beta-HSOR Type 1 inhibition and estrogenicity of tested compounds in breast cancer cells (judged by increased cell proliferation) were not identical. Zearalenone and diethylstilbestrol, both potent estrogens, did not inhibit 17 beta-HSOR Type 1. Thus, changes in the estrogen molecule may discriminate between active sites of 17 beta-HSOR Type 1 and estrogen binding sites of the ER. The effects of these compounds in vivo cannot be predicted on the basis of these results. Inhibition of 17 beta-HSOR Type 1 enzyme could lead to a decrease in the availability of the highly active endogenous estrogen. However, these compounds are estrogenic per se, and they may thus replace endogenous estrogens. Additional studies are needed to further understand the role of these plant estrogens in the etiology of hormone-dependent cancers. It is not easily conceivable how the chemopreventive action of Asian diets, possibly mediated by phytoestrogens in soya products, can be based on the inhibition of estrone reduction at the target cells by phytoestrogens or related compounds, unless they are "incomplete estrogens" (i.e., unable to induce all effects typical of endogenous estrogens).

13th Immunology of Diabetes Workshop

Although compelling data have demonstrated the effectiveness of estrogen replacement therapy for the treatment of accelerated bone loss in postmenopausal osteoporosis and ovariectomized animals, the mechanisms by which estrogens reduce bone resorption remain to be elucidated. To address this issue, in the present study we investigated whether estrogens were able to induce programmed cell death or apoptosis in osteoclast precursors. To this purpose, a preosteoclastic cell line (FLG 29.1) was cultured in the absence or presence of nanomolar concentrations of 17β-estradiol (17βE2). Using time-lapse videomicroscopy, it was shown that 17βE2induced FLG 29.1 cell apoptosis in a dose- and time-dependent manner. Furthermore, a significant increase in the activity of caspase 3 enzyme and in the number of nuclei undergoing DNA fragmentation was observed in FLG 29.1 cells treated with 17βE2compared to untreated cells. Finally, transmission electron microscopy of the treated cells showed typical apoptotic morphology. These data indicate that 17βE2is able to promotein vitroapoptosis in preosteoclastic cells and suggest that estrogenic molecules may exertin vivoa direct role in negatively modulating the pool of undifferentiated bone marrow cells capable ultimately of maturing into osteoclasts.

Distribution of estradiol‐17β hydroxysteroid oxidoreductase in the urogenital tract of control and neonatally estrogenized male mice: Immunohistochemical, enzymehistochemical, and biochemical study

The present study was conducted to investigate the regional distribution of the enzymes catalyzing the interconversion of the hydroxyl and carbonyl groups at C-17 of the estrogen molecule within the male urogenital tract of adult mouse and to test the hypothesis, whether regional differences in the distribution are critical for estrogen responses. The highest ratios of NADPH-dependent 3H-estrogen reduction to oxidation at C-17 of cell-free homogenates were obtained from coagulating gland and seminal vesicle as well as from the prostatic and lower intrapelvic urethra, which are considered the most estrogen-sensitive parts of the male urogenital tract. Both NADP- and NAD-dependent oxidation of 3H-17 beta-estradiol were low or nondetectable at these sites. The epithelium of the lower and prostatic urethra as well as the periurethral collecting ducts were stained with the antibody prepared against human placental 17 beta-hydroxysteroid oxidoreductase. The NAD-dependent 3H-estradiol-17 beta oxidase activity was highest in the bladder epithelium, and the activity declined sharply in the urinary tract from the bladder downward. The lowest detectable activities were found in vas deferens and prostate (combined ventral and dorsolateral lobes). The uneven distribution of estradiol-17 beta oxidase activity may provide additional explanation for the regional differences of estrogen responses. The NADPH-dependent 17 beta-reduction of estrone and the immunohistochemical staining of the human placental estradiol-17 beta oxidoreductase antigenicity were not significantly altered after neonatal estrogenization. These findings do not lend any support to the idea that the increased estrogen sensitivity observed after neonatal estrogenization is associated with changes in 17 beta-oxidoreduction. However, the possibility remains that there are specific sites (e.g., epithelium of prostatic urethra and collecting ducts) in which the changes in 17 beta-oxidoreduction of estrogen does play a role in the regulation of estrogen action.

Blocking Breast Cancer

H idden within every breast tumor lies a clue to its character: the presence or absence of receptors that bind with the hormone estrogen. Receptor-rich tumors depend on estrogen for their growth, and some scientists believe they represent a milder form of breast cancer than tumors without estrogen receptors. Furthermore, women with estrogen-dependent tumors are considered candidates for a relatively nontoxic drug called tamoxifen, which can halt tumor by blocking estrogen binding. Tamoxifen's discovery in the 1970s was hailed as both a lifesaver and a deliverance from the terrible side effects of cell-killing chemotherapy Yet the presence of estrogen receptors in a breast tumor may, in fact, offer a false and perhaps fatal promise. New research suggests that the estrogen receptors in some breast tumors are defective, allowing the cancer to proliferate and spread aggressively Estrogen receptors work by snaring estrogen molecules entering a tumor cell from the bloodstream. The hormone spurs the tumor cell to proliferate at a steady but relatively slow pace. If an active receptor instead encounters tamoxifen estrogen's chemical lookalike it binds the drug rather than the hormone, and thus has no impetus for growth. But the new findings suggest that tumors with flawed receptors have lost their ability to respond to estrogen or to tamoxifen, and that their growth switch stays on at all times. These tumors no longer need estrogen to grow, the researchers speculate. The result: a rapidly proliferating malignancy requiring harsh chemotherapy In separate work, other scientists report finding tamoxifen-like substances in soybeans that may block tiny seeds of estrogen-dependent breast cancer early in the disease process, before estrogen receptors have a chance to go bad. The two studies fit into a hypothetical scenario in which functioning estrogen receptors, while allowing breast tumors to grow, also restrain the pace of proliferation. But these receptors may gradually lose their binding ability -a process that leads to rapid, out-of-control cell growth. If this theory is confirmed, physicians will need to distinguish between functional and flawed receptors in order to select the best treatment for women with receptor-containing breast tumors. In addition, potential preventive measures such as the soybean compound could become especially important for women at high risk of developing breast cancer, which ranks as the second leading cause of cancer deaths in U.S. women. Samuel Broder, director of the National Cancer Institute, calls this concept of receptor malfunction exciting because it may explain why some receptor-containing breast cancers do not respond to tamoxifen and instead spread to distant body parts. Still, Broder cautions that evidence supporting the theory remains preliminary A lot of interesting studies don't pan out. We should get some answers on this in a couple of years, he says.

Estradiol membrane binding sites on human breast cancer cell lines. Use of a fluorescent estradiol conjugate to demonstrate plasma membrane binding systems

A fluorescent estradiol macromolecular complex was used to study and to characterize steroid binding to membranes of living target cells. Ligand binding to plasma membranes was quantitated with a sensitivity of 0.1 nM. In this way, we found two types of estradiol-binding sites on hormone sensitive MCF-7 cells. Type A sites (8000–16000 sites per cell) were rapidly saturated at low concentrations of the estradiol-bovine serum albumin-fluorescein isothiocyanate macromolecular complex (E 2-BSA-FITC). They had a greater affinity for the complex than did the type B sites for which a phenomenon of cooperative fixation was shown. The complex binding was displaced by estrogenic molecules, but not by non-estrogenic compounds, such as cortisol or progesterone. We also studied complex binding on another breast cancer cell line, MDA-MB-231 (MDA), without intracellular estrogen receptors. These cells showed a specific plasma membrane binding system for estrogen, but lacked the high affinity type A binding site. Then, we report the effects of enzyme treatments (trypsin, phospholipase A2 and neuraminidase) on E 2-BSA-FITC binding to MCF-7 cell membranes. The quantity of complex bound to membranes decreased after phospholipase and neuraminidase treatments and increased after trypsin. But, in the three cases, the binding was no longer specific because it could not be displaced by E 2-BSA or by estradiol. The enzymatic effects were reversible and specific binding was totally restored within 24 h. However, in the presence of the protein synthesis inhibitor, cycloheximide, no restoration of specific binding occurred on trypsin-treated cells. Estrogen binding to MCF-7 and MDA cell plasma membranes thus possesses the three characteristics of all mediated transport processes across biological membranes: saturability, substrate specificity, and specific inhibition. However, the high affinity type A binding site was found only on the estrogen-sensitive cell line, MCF-7.

Effects of 2-Fluoroestradiol on lipid metabolism in the ovariectomized rat

Biological activities of estrogen molecules are altered by fluorination of ring A, and the resulting impairment to form catechols. 2-fluoroestradiol (2-F-E 2) has been found to be devoid of carcinogenic action despite its high estrogenic potency; its metabolic effects are so far unknown. This study was designed to investigate the effects of 2-F-E 2 on lipid metabolism, as compared to those of estradiol-17β(E 2). Ovariectomized rats received E 2 or 2-F-E 2 by s.c. injection at a dose of 60 μg for three consecutive days. Parameters measured were weights of parametrial fat depots, fat cell volumes, levels of triacylglycerol and acylcholesterol in plasma, and enzymatic responses to the estrogens in isolated parametrial fat cells as evaluated in terms of lipoprotein lipase (LPL) and hormone-sensitive lipase (HSL) activities. 2-F-E 2 and E 2 were found to produce comparable decreases in fat depots, cell volumes and plasma levels of acylcholesterol whereas plasma triacylglycerol was unchanged. Both estrogens decreased LPL, and increased HSL activities to the same extent. Thus, 2-F-E 2 and E 2 exhibited comparable effects on lipid metabolism. These effects appeared to depend mainly on the estrogenic potency of these molecules, and to be distinct from their carcinogenic action. Despite its high estrogenic potency, 2-F-E 2 was found to be slightly less estrogenic than E 2.

Use of fluorescent sex-steroid conjugates for the histochemical detection of breast cancer cell receptors: a critique.

All current techniques (autoradiography excepted) for the detection of nuclear or cytosol steroid hormone receptors require homogenization of tissue, and thereby sophisticated equipment not available at all centers. Several methods have been developed to trace estrogen (ER) and progesterone receptors (PgR) under the microscope (see [2] for review). In this regard the ligands employed so far consist of: 1. Steroid hormones linked to a carrier molecule (bovine serum albumin) which is labeled with fluorescent dyes (∼2 × 10-4 M effective bound steroids) [10, 12] 2. Fluorescein-labeled estrogen molecule, without a protein carrier (∼ 10-6 M bound steroid) [5, 14]

Estrogen induced appearance of N-terminal glycine from chromatographically purified fibrinogen.

Abstract Purified human fibrinogen or agarose gel fractionated fibrinogen preparations were subjected to N-terminal amino acid analysis before and after incubation with 17-ethynylestradiol. Incubation of fibrinogen with thrombin produced a fibrin molecule containing 4 moles of N-terminal glycine and 2 moles of N-terminal tyrosine. Incubation of fibrinogen with 17-ethynylestradiol converted fibrinogen to a fibrin like molecule containing 2 moles of N-terminal tyrosine and almost twice as much N-terminal glycine as observed with thrombin. Diethylstilbestrol induced the same changes as thrombin; however, other unidentified N-terminal amino groups were also released. These results, which suggest a thrombin like proteolytic function for the estrogen molecule, do not appear to be due to contamination of the steroid and fibrinogen preparations.

Hormone dependence in breast cancer

Both prolactin and estrogen are involved in regulating the growth and functions of the mammary gland, and many mammary tumors retain their dependence on these hormones. Specific binding of prolactin to mammary gland can be shown in vivo. Binding of estrogen in vivo and in vitro depends on the presence of a cytoplasmic receptor protein that acts in the nucleus after binding estrogen molecules. This estrogen receptor is present in both rat and human hormone-dependent mammary tumors, but is usually missing in autonomous mammary tumors. The presence or absence of estrogen receptor may thus be an indicator of the retention or partial loss of the complete endocrine regulatory unit, which mediates the interacting effects of many hormones including prolactin and estrogen on the normal mammary gland. In agreement with preliminary case series, presence of the estrogen receptor in breast tumors would therefore be expected to correlate with hormone dependence and a favorable response to hormone manipulation.

The dose-time relationship of estradiol-17β and lung cholinesterase

those treated with clomiphene alone, while ovulation occurred in 29 (70.7 per cent) and pregnancy in 8 (21.9 per cent) in those treated with clomiphene and Quinestrol (see Table I). Twenty-eight of the 41 women treated (68.2 per cent) showed quantitative and qualitative improvement of cervical mucus. Results of the Huhner test changed and became positive in 16 cases (39 per cent). Many changes in the estrogen molecule in an attempt to improve cervical mucus only led to decrease in ovulation. But the addition of an aliphatic ether to ethinyl estradio12 causes: (a) twoto threefold increase in estrogen effect over ethinyl estradiol; (b) increased duration of action, i.e., all excreted within 8 days as opposed to 24 hours for ethinyl estradiol; and (c) lack of suppression of ovulation. We found Quinestrol to be successful in the treatment of hostile cervical mucus.s In 75 per cent of our cases there was improved threadibility and in 52 per cent increase in sperm penetration. These findings agree with those of other investigators. We therefore gave combined Quinestrol and clomiphene therapy to those cases of failed clomiphene therapy with a later development of impaired cervical mucus. Small doses of Quinestrol (25 ag) were insufficient and large doses (more than 150 pg) caused suppression of ovulation and cystic hyperplasia. The dose of 50 fig from the sixth to sixteenth day of the cycle was found to be suitable. Of the women in our group 21.9 per cent became pregnant despite their long-term sterility and various treatments including clomiphene over the years, Nausea in a small number of cases was the only side effect of the combined treatment with Quinestrol and clomiphene. We conclude, therefore, that where clomiphene is unsuccessful in promoting pregnancy, and there is deterioration of the cervical mucus, combined clomiphene and Quinestrol therapy will give a much higher rate of success.

Effect of sulfate and phosphate esters of estrogens on the level of aminotransferases in the perfused rat heart.

Abstract Phosphate and sulfate esters of estradiol and diethylstilbestrol, which in vitro inhibit the recombination of apo -aminotransferases with PALP without affecting the enzymatic activity of the reconstituted holoenzyme, negatively influence the level of aspartate aminotransferase, alanine aminotransferase and tyrosine aminotransferase in the perfused rat heart. It seems that in this system as in vitro , the estrogen esters compete with PALP for the apoenzyme. The effect is fast, specific for estrogenic anionic molecules, and reveals that about 50% of the enzymes is present as apoenzyme in the rat heart muscle.

Stereospecific binding of estrogens in the rat uterus

After administration of low levels of 6,7-H 3-estradiol-17β to immature rats, the isotope was found to be incorporated in all subcellular fractions of the rat uterus, but predominantly in the nuclear-myofibrillar and soluble fractions. The incorporation of H 3-estradiol could be inhibited by steroid and nonsteroid estrogens but not by nonestrogenic steroids. The inhibition appears to be competitive and suggests that the uterus contains a binding site which is stereospecific for estrogenic molecules. Bound H 3-estradiol could be released from the nuclear-myofibrillar fraction by digestion with proteases or by incubation at extreme pH. The authors conclude that estradiol-17β is probably bound to a protein in the rat uterus which is stereospecific for estrogenic molecules.