Contrasting effects of estradiol-17 beta and 17 alpha-ethinyl estradiol-17 beta on cultured whole embryos

Estradiol-17 beta (E2) and 17 alpha-ethinyl estradiol-17 beta (EE) were compared in terms of their relative capacities to alter growth and developmental patterns of cultured whole embryos during the early stages of organogenesis. Embryos exhibited a notable differential susceptibility to the embryotoxic effects of parents E2 vs EE when these estrogens were added directly to the media at the onset of the culture period. At initial concentrations of 0.1 mM, E2 failed to produce statistically significant effects whereas EE elicited marked embryotoxicity. Inclusion of a P-450-dependent biotransformation system in the culture media resulted in a significant attenuation of the embryotoxic effects of parent E2 vs EE when these estrogens were added directly to the media at the onset of the culture period. At initial concentrations of 0.1 mM, E2 failed to produce statistically embryotoxicity by hepatic S9. The divergent results produced by the two steroids could not be attributed to differences in rates of catecholestrogen generation in the culture medium or by the conceptuses. The results demonstrate definitive dissimilarities between the effects of two steroidal estrogens on developmental parameters and document marked differences in the effects of biotransformation on their embryotoxic potential. The data strongly suggest that the embryotoxicity of these steroids is not mediated via interactions with estrogen receptors. Additionally, the data show that the differential capacity of these two steroids to produce embryotoxic effects is diametrically opposite to earlier reported patterns of their carcinogenic potential in the Syrian hamster kidney.     

Prostatic cytosol binding of 5 alpha-androstane-3 beta, 17 beta-diol and estradiol-17 beta

The goal of the present research was characterization of the interaction of 5 alpha-androstane-3 beta, 17 beta-diol (3 beta-diol) with prostatic estradiol-17 beta(E2) binding sites to address the role of this 5 alpha-dihydrotestosterone(DHT)a metabolite in prostatic regulation. Using dextran-charcoal assay we demonstrated specific 3 beta-diol and E2 binding sites in rat ventral prostate cytosol (RVPC) and dog prostate cytosol (DPC). In both cytosols, E2 binding is of high affinity (Ka congruent to 10(9) M-1; RVPC:68 fmol/mg protein), DPC:170 fmol/mg protein), and 3 beta-diol binding is of moderate affinity (Ka congruent to 10(8) M-1; RVPC:62 fmol/mg protein, DPC:165 fmol/mg protein). Unlabeled 3 beta-diol competes effectively for cytosolic 3H-E2 binding sites, whereas unlabeled DHT, 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-diol) and testosterone (T) are poor competitors for 3H-E2 binding sites. Using DNA-cellulose column chromatography, we separated prostatic androgen and estrogen binding activities. The E2 binding activity which adhered to DNA-cellulose was displaced by 100-fold excess 3 beta-diol but not by DHT. Thus data from two assay procedures show competition of 3 beta-diol for 3H-E2 binding sites in rat and dog prostate.     

Estradiol-17beta sulfotransferase activity in canine osteosarcoma D17 cells

Estrogen sulfatase and sulfotransferase (EST) activities are present in breast cancer tissues but there are no reports on EST in cancerous bone cells. We incubated [(3)H]estradiol-17beta with cells from a canine osteosarcoma D17 line for periods up to 24 h. Radioactive steroids were recovered from the media and separated into unconjugated and conjugated fractions using Sep-Pak C18 cartridges. The conjugate fraction was solvolyzed and the resulting free steroids were obtained from a second C18 cartridge. Little metabolism was apparent in 4 h of incubation, but by 24 h as much as one half of the radioactivity was seen in the conjugate fraction. Most of the conjugates were recovered as sulfates in all three experiments. HPLC profiles showed a limited metabolism of estradiol to other compounds except for estrone, which was clearly present in both free and sulfate fractions. These results suggest that EST may have a role in the local metabolism of estrogens in bone.     

Surface behaviour of oestradiol-17beta.

The absorption of [3H]oestradiol-17beta from its aqueous solutions has been measured in the range 0-10mug/ml. It is found that the first adsorbed molecules are parallel to the interface and occupy 100 A2. Those adsorbed in the range 6-10 mug/ml occupy 21 A2. They are presumably associated. When the adsorption occurs in the presence of a synthetic lecithin monolayer, the molecular area is equal to 16 A2. Surface tension measurements of the solutions of oestradiol-17beta and a parallel study of their fluorescence have been performed. No association of the hormone molecules has been observed in bulk. It is concluded that surfaces and liquid monolayers may favour molecular association of the oestradiol-17beta.     

Synthesis of 5 beta,17 alpha-19-norpregn-20-yne-3 beta,17-diol and of 5 beta,17 alpha-19-norpregn-20-yne-3 alpha,17-diol, human metabolites of norethindrone

A convenient synthesis of both 5 beta,17 alpha-19-norpregn-20-yne-3 beta,17-diol (1) and 5 beta,17 alpha-19-norpregn-20-yne-3 alpha,17-diol (2) in multigram quantities from estr-4-ene-3,17-dione is reported. Full characterization of these often-cited human metabolites of norethindrone is presented for the first time.     

Purification and interaction with estradiol-17beta.

The combination of polyacrylamide gel electrophoresis and Concanavalin-A-Sepharose affinity chromatography has permitted the isolation on a preparative scale, of four molecular forms of rat alpha1-fetoprotein: a "slow" and a "fast" fraction, each separable into Concanavalin-A-adorbed ("high carbohydrate", i.e. rich in accessible alphaD-Mannosyl and alphaD-Glucosyl residues) and a Concanavalin-A-non adsorbed ("low carbohydrate") fractions. These four iso-alpha-fetoproteins (iso-AFP) bind estradiol-17beta. However, they disclose differences in both their association constants and number of binding sites for this hormone. Very high affinity sites (10(9)) are mainly located on the "slow-low carbohydrate" form. Low affinity, high capacity sites are preferentially located on the "high carbohydrate" form. These results confirm the molecular and functional heterogeneity of rat AFP and suggest that the carbohydrate moiety of the protein may have a role in estrogen-AFP interactions.     

Oestradiol-17beta and progesterone in nymphomaniac cows

Oestradiol-17beta and progesterone were assayed in the plasma of 32 nymphomaniac cows. In 21 cases oestradiol-17beta concentrations were higher than those recorded during the preovulatory surge of normal cyclic cows. However, for a further 5 nymphomaniac cows oestradiol-17beta concentrations were within the range of those recorded in normal cows during the luteal phase. In 11 cases progesterone concentrations were higher than 1.5 ng/ml, but in only 5 of them could this have been due to a corpus luteum. The presence of progesterone, whether or not associated with a corpus luteum, did not determine the level of oestradiol-17beta. Therefore, nymphomania seems to be less a disease, per se, than a nonspecific symptom of ovarian perturbation.     

Structure determination of 3 beta, 17-dihydroxy-17 alpha-pregn-5-ene-20-one

The title compound was synthesized as part of an effort to determine the identity of an abnormal steroid metabolite present in the urine of a patient exhibiting pronounced gynecomastia. The X-ray investigation of the synthesized compound showed that the 20-carbonyl of the 17 alpha oriented side chain lies under the D ring, and does not participate in hydrogen bonding in the crystal lattice. This conformation appears to be stable and sufficiently shielded that it is unlikely to make a major contribution to possible protein interactions.     

Plasma estradiol-17beta level in the domestic fowl

Using healthy white Leghorn chickens the estradiol-17β level in plasma were determined by radioimmunoassay and the dependency on age and sex examined. In one and two year old laying hens average values between 48.0 and 54.4 pg/ml were found. The estradiol level in cocks of the same age were between 6.0 and 7.3 pg/ml. Significant difference with respect to the estradiol level were already noticeable in sexually immature 4 month old chickens ♀: 8.1 pg/ml, ♂: 1.0 pg/ml). The significance of the estradiol-17β level with respect to plasma lipids and electrophoretic mobility of serum lipoprotein is shown.     

Rat brain glycolysis regulation by estradiol-17 beta.

The effect of estradiol-17 beta on the activities of glycolytic enzymes from female rat brain was studied. The following enzymes were examined: hexokinase (HK, EC 2.7.1.1), phosphofructokinase (PFK, EC 2.7.1.11), aldolase (EC 4.1.2.13), glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), phosphoglycerate kinase (EC 2.7.2.3), phosphoglycerate mutase (EC 2.7.5.3), enolase (EC 4.2.1.11) and pyruvate kinase (PK, EC 2.7.1.40). The activities of HK (soluble and membrane-bound), PFK and PK were increased after 4 h of hormone treatment, while the others remained constant. The changes in activity were not seen in the presence of actinomycin D. The significant rise of the activities of the key glycolytic enzymes was also observed in the cell culture of mouse neuroblastoma C1300 treated with hormone. Only three of the studied isozymes, namely, HKII, B4 and K4 were found to be estradiol-sensitive for HK, PFK and PK, respectively. The results obtained suggest that rat brain glycolysis regulation by estradiol is carried out in neurons due to definite isozymes induction.     

Phosphorylation on tyrosine of oestradiol-17 beta receptor in uterus and interaction of oestradiol-17 beta and glucocorticoid receptors with antiphosphotyrosine antibodies

In whole rat uterus incubated in the presence of [32P]orthophosphate the oestradiol receptor is [32P]phosphorylated on tyrosine. This finding follows our previous observation that in vitro this receptor can be phosphorylated on tyrosine by a uterus kinase that endows the receptor with oestradiol-binding activity. The calf uterus oestradiol receptor interacts with high affinity with 2G8 and 1G2 antiphosphotyrosine antibodies coupled to Sepharose (Kd values of 0.28 and 1.1 nM, respectively). The interaction with 2G8 antibody has been exploited to purify the oestradiol receptor. This interaction disappears after inactivation of the oestradiol receptor by the nuclear phosphatase that hydrolyses phosphotyrosine of the receptor. This fact substantiates the evidence that the oestradiol receptor in uterus is phosphorylated on tyrosine and that this phosphorylation is required for hormone binding to the receptor. The rat liver glucocorticoid receptor also interacts with high affinity with 2G8 antiphosphotyrosine antibody coupled to Sepharose (Kd value of 0.21 nM). This receptor has been purified by using in sequence heparin-Sepharose and antiphosphotyrosine antibody-Sepharose.     

Active immunization of the cow against oestradiol-17beta

Six beef heifers were immunized over a 4-month period with an oestradiol-17beta-BSA conjugate in Freund's adjuvant. There was an interference with oestrus in the treated heifers; 2 ceased to exhibit oestrus, one exhibited one oestrus and three exhibited oestrus after Day 47 of treatment. The control heifers treated with Freund's adjuvant had normal oestrous cycles. The antiserum titre rose in all treated heifers and attained its highest level in the 2 animals in which oestrus did not recur. The temporal changes in plasma LH, progesterone and oestradiol were normal during the pretreatment period, but became abnormal during the 120 days after immunization. Although plasma oestradiol-17beta rose at the expected time of oestrus after treatment, it was apparently effectively neutralized by the antiserum induced by treatment as evidenced by the absence of an LH surge. Plasma progesterone levels fell to baseline and remained low, indicating lack of formation of corpora lutea.