Influence of the blood concentration of prolactin on the length of the sensitive period for establishing maternal behavior in sheep at parturition

The fading of postpartum maternal interest for the neonate (sensitive period) in ewes separated from their young at lambing is delayed when parturition is induced with 20 mg of estradiol benzoate (EB). An experiment was carried out to investigate the role of prolactin in this phenomenon. The sensitive period was studied in three groups of parturient ewes. In all groups lambs were removed at birth and reintroduced to their mothers 24 hr later. Maternal acceptance was tested at this time. In group 1 (dexamethasone D), ewes were induced to lamb with dexamethasone (15 mg im). In group 2 (EB), ewes were treated with 20 mg of estradiol benzoate (im). In group 3 (EB + CB 154) ewes received 20 mg of EB as in group 2 and 1 mg of CB 154 (sc) every 12 hr to prevent the enhanced secretion of prolactin which normally occurs after EB injection. The concentration of prolactin was highest in group 2 (EB), lowest in group 3 (EB + CB), and intermediate in group 1 (D) (p ? 0.001 between groups). By contrast, the proportion of ewes showing maternal behavior was similar in groups 2 and 3 ($\text{15}\text{23}$ and $\text{17}\text{22}$), both of which differed from group 1 ($\text{3}\text{22}\text{; p ? 0.005}$). It is concluded that the lengthening of the sensitive period for establishing maternal behavior in sheep following EB induced parturition is not related with high levels of prolactin in the peripheral circulation.     

Effects of estrogens on aryl hydrocarbon hydroxylase mediated binding of benzo(a) pyrene metabolites to DNA in vitro

$\text{In vivo}$ and $\text{in vitro}$ studies were carried out to determine the effects of estradiol and other steroid hormones on aryl hydrocarbon hydroxylase-mediated binding of benzo(a)pyrene metabolites to DNA. Injection of female $\text{C57}\text{B1}\text{6J}$ mice with 0.2 mg or 2 mg of estradiol 24 hours prior to, during and 24 hours after injection of 3-methylcholanthrene resulted in a significant decrease in the capacity of hepatic microsomes from these animals to mediate the binding of benzo(a)pyrene metabolites to DNA when compared to microsomes from animals receiving 3 methylcholanthrene treatment only. Binding of benzo(a) pyrene metabolites was inhibited between 22 and 50%, depending on the dose of estradiol used. The enzyme and cytochrome components of the aryl hydrocarbon hydroxylase multienzymic complex were not affected by either estradiol treatment. The data suggests that estradiol inhibits aryl hydrocarbon hydroxylase mediated binding of benzo(a)pyrene metabolites to DNA by activity as a non-competitive inhibitor of aryl hydrocarbon hydroxylase activity.     

Rapid modulation by progesterone and tamoxifen of estradiol effects on nuclear histone acetylation in the uterus of the fetal guinea pig

The effect of estradiol, progesterone, tamoxifen, estradiol + progesterone or estradiol + tamoxifen on the [³H]acetylation of histones in the fetal uterus of guinea pig was studied. The fetuses were injected subcutaneously ‘in situ’ with the hormones or $\text{tamoxifen}\text{+ [}{}^3\text{H}\text{]}\text{acetate}$ alone. In 10 min, estradiol stimulated the acetylation of histone 10–12-times with respect to the control animals. Progesterone and tamoxifen blocked this effect. It is suggested that histone acetylation is an early step induced by estrogen action during intrauterine life and that progesterone and tamoxifen suppress this mechanism very effectively.     

Onset of the receptive and proceptive components of feminine sexual behavior in rats following the intravenous administration of progesterone

The present study was carried out in order to assess the time course of action of progesterone (P) in the facilitation of complete feminine sexual behavior. Female rats (estrogen primed via 5% E₂ Silastic capsules) were given 200 μg of P either intravenously (iv) or subcutaneously (sc), and tested for estrous behavior at $\text{1}\text{4}$, $\text{1}\text{2}$, 1, 2, and 4 hr after treatment. Among iv-treated animals, significant amounts of lordosis behavior were seen as early as $\text{1}\text{2}$ hr, and a dramatic rise in solicitation behavior was observed at 2 hr. Although sc-treated animals displayed significant amounts of lordosis and solicitation behavior at 2 hr, the behavior was not maximal until 4 hr. Intravenous administration of 400 μg P was equipotent to 200 μg P, whereas 50 μg of iv P was relatively ineffective. A dual mechanism hypothesis pertaining to progesterone's actions in the facilitation of both the receptive and preceptive components of feminine sexual behavior in rats is discussed.     

Conditions for the measurement of nuclear estrogen receptor at low temperature

This study was undertaken to determine optium conditions for the extraction and measurement of uterine nuclear estrogen receptor at low temperature. We measured the influence of glycero, 0.5 M KCl, 10 mM pyridoxal 5′-phosphate, and 0.5 M NaSCN on the dissocation of estradiol from the receptor at 0°C. The half-time (${}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) of estradiol dissociation from the receptor in 0.5 M KCl nuclear extracts containing 30% glycerol was very slow (greater than 250 h). Exclusion of glycerol from the extract (Tris buffer) increased the dissociation rate ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 35}\text{h}$). The inhibitory effect of glycerol on estradiol dissociation kinetics predominated over the mild stimulatory effect of KCl; and both effects were independent of the electrical conductivity of the buffer. When pyridoxal phosphate was added to a nuclear KCl extract (barbital fubber) lacking glycerol, dissociation of the estrogen-receptor complex increased such that the $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) decreased from 20 to 7.6 h; the receptor extracted from nuclei with 10 mM pyridoxal phosphate exhibited these same rapid dissociation kinetics. The $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of estradiol dissociation from the receptor at 0°C in the presence of 0.5 M NaSCN was 5.6 h. Following extraction of uterine receptro by KCl, pyridoxal phosphate, or NaSCN, we measured the number of estradiol binding sites at each of two incubation temperatures: 30°C for 1 hr and 0°C for 24 h. We verified that unoccupied receptors was measured reliability in KCl extract during incubation at 0°C in the presence of glycerol. Total receptor can be determined using either pyridoxal phosphate extract or NaSCN extract at low temperature. However, the number of sites recovered in either pyridoxal phosphate or NaSCN extract was twice the number obtained with the KCl procedure at elevated temperature. It is noteworthy that pyridoxal phosphate and NaSCN increased the number of sites when added directly to nuclear KCl extract, and the effect of pyridoxal phosphate and NaSCN was reversed by treatment with L-lysine and dialysis against KCl, respectively. Thus, the lower receptor recovery with the KCl procedure is not due to the inability of KCl to extract these sites from the nucleus but rather is ascribable to the assay procedure itself. Although total receptor can be measured at low temperature with either NaSCN or pyridoxal phosphate, the pyridoxal phosphate method can be used to assay nuclear progesterone receptor in tha same extract.     

Androgen on the estrogen receptor I — Binding and in vivo nuclear translocation

In the immature rat uterus, high concentrations of androgens competed specifically with estradiol on the estrogen receptor (RE). This competition was stereospecific for C₁₉ steroids bearing a 17β and/or 3 hydroxyl group. Very low affinity ligands, such as testosterone, could not compete with estradiol at equilibrium but decreased the association rate of estradiol on its receptor. High doses (> 0.4mg) of 5 α aihydrotestosterone provoked $\text{in vivo}$ as $\text{in vitro}$ the nuclear translocation of RE. The nuclear receptor thus formed displayed the same 5.2 S sedimentation constant as that induced by estradiol. We conclude that the weak affinity binding of androgens to the estrogen receptor is sufficient to induce its nuclear translocation $\text{in vivo}$ provided androgen concentration is high enough in uterus to occupy the estradiol binding site. Conversely, progesterone which does not bind RE could not provoke its nuclear translocation.     

Control of estrogen and androgen receptors in the rat pineal gland by catecholamine transmitter

Sucrose gradient studies of rat pineal cytosol incubated with ³H-estradiol (female pineals) or ³H-5 α -dihydrotestosterone (male pineals) revealed a radioactivity peak in the 8 S region which disappeared after superior cervical ganglionectomy or incubation with excess unlabeled hormone. Ganglionectomy decreased significantly estradiol and testosterone uptake by the pineal gland $\text{in vitro}$ as well as high affinity binding to pineal cytoplasmic and nuclear components. Norepinephrine treatment counteracted all the effects of ganglionectomy but was unable to modify hormone uptake and binding by the pineal gland of sham-operated controls. Pre-treatment with actinomycin D or propranolol but not with phentolamine impaired norepinephrine effects; propranolol blockage however was only partial. Administration of isoproterenol, L-dopa or phentolamine increased hormone uptake by denervated pineals. The effects of isoproterenol were also observed $\text{in vitro}$ and were blocked by propranolol. These results indicate that sex steroid receptors in the pinealocytes are controlled by norepinephrine via beta-adrenergic receptors and that depletion of neural norepinephrine enhanced responsiveness of pineal hormone receptors to exogenous catecholamines.     

Cytochrome P-450 and NADPH cytochrome c reductase in rat brain: formation of catechols and reactive catechol metabolites.

Microsomes isolated from whole rat brain were found to contain cytochreme P-450 (0.025 to 0.051 nmoles/mg) and NADPH cytochrome $\text{c}$ reductase activity (26.0 to 55.0 nmoles/mg/min). The oxidation of estradiol to a reactive metabolite that became covalently bound to rat brain microsomal protein was inhibited 63% by an atmosphere of CO:O₂ (9:1), indicating the involvement of a cytochrome P-450 oxygenase. In contrast, this atmosphere had no effect on the binding of either the catechol estrogen, 2-hydroxyestradiol, or several catecholamines to rat brain microsomes. An antibody prepared against NADPH cytochrome $\text{c}$ reductase was found to decrease significantly both the formation of 2-hydroxyestradiol from estradiol by rat brain microsomes and the covalent binding of the catechol estrogen and catecholamines to rat brain microsomal protein.     

Estrogen causes a rapid, large and prolonged rise in the level of nuclear estrogen receptor in Xenopus laevis liver.

In male $\text{Xenopus laevis}$, a single large injection of estradiol causes a large rise in the level of estradiol receptor in liver nuclei. The rise is almost certainly due to synthesis, and the newly-synthesized receptor is indistinguishable from pre-existing receptor. The high level of receptor induced by estradiol persists for over 30 days, well after the vitellogenin synthesis that is also induced has disappeared.     

Steroid induction of nerve growth factor synthesis in cell culture

Nerve growth factor (NGF) is a peptide hormone which is necessary for the development of sympathetic neurons. Exposing a rat central nervous system glioma cell line (C-6) to the steroid hormone 17β-estradiol increases the amount of NGF secreted by these cells into the surrounding medium. This induction is highly specific to 17β-estradiol in that similar steroids do not increase NGF levels. Both NGF activity and protein levels increase upon estradiol stimulation and there is a parallel increase in NGF $\text{de}$$\text{novo}$ synthesis. The estradiol effect can be blocked with actinomycin D but not with puromycin or cycloheximide. This is the first report demonstrating regulation of NGF synthesis by a steroid hormone in a clonal cell line of glial origin. We propose this system as a model system for the study of the regulation of NGF synthesis and the isolation and analysis of putative precursors to the NGF molecule.     

Prostaglandin levels in preovulatory follicles from rabbit ovaries perfused invitro

Prostaglandin (PG) levels in follicular fluid from preovulatory follicles of rabbit ovaries perfused $\text{in}$$\text{vitro}$ were measured in order to compare PG changes in this model system with those that occur $\text{in}$$\text{vivo}$ and in isolated, LH-treated follicles $\text{in}$$\text{barvitro}$. One ovary from each rabbit was perfused without further treatment (control). The other ovary was exposed to LH (0.1 or 1 ug/ml) beginning 1 hour (h) after initiation of perfusion. Samples of perfusion medium were taken at frequent intervals for measurement of PGE, PGF, progesterone and estradiol 17β. The perfusions were terminated when the first ovulation occurred or appeared imminent as judged by changes in the size and shape of the follicles. Follicular fluid was then rapidly aspirated from all large follicles on both ovaries for PGE and PGF measurement.Ovulations occurred only in the LH-treated ovaries. Progesterone and estradiol levels were significantly elevated in the perfusion medium within 1 h of LH treatment in comparison to controls. PG levels in perfusion medium from the control and LH-treated ovaries were not different throughout perfusion and increased in both groups. In contrast, PG levels measured in follicular fluid from LH-treated ovaries were 4- to 5-fold greater than in fluid from control ovaries. It is concluded that ovulation induced by LH in this experimental model is accompanied by an increase in follicular PG levels similar to that seen in other $\text{in}$$\text{vivo}$ and $\text{in}$$\text{vitro}$ models. This difference in follicular PG levels between the LH-treated and control ovaries is, however, not reflected in the perfusion medium.     

Effect of synthetic estrogen on platelet aggregation and vascular release of PGI2-like material in the rabbit

Treatment of adult female New Zealand white rabbits with ethinyl estradiol, the synthetic estrogen used in many oral contraceptives, results in a significant increase in $\text{in vivo}$ aggregation. This alteration in platelet behavior is accompanied by diminished vascular release of antiaggregatory PGI₂ (prostacyclin)-like material. Addition of a progestin prevents the change in platelet aggregation seen with the estrogen alone. Diminished vascular PGI₂ release may be an important factor in the pathogenesis of thrombotic occurrences experienced by some oral contraceptive users. $\text{In vivo}$ platelet aggregation may be of value in identifying individuals at risk of developing thrombotic disturbances while taking oral contraceptives.     

Independence of the differentiation of masculine and feminine sexual behavior in rats.

Male rats received Silastic implants of the aromatase inhibitor, 1,4,6-androstatriene-3, 17-dione (ATD), on days 2–10 of life. Controls received blank implants. There were no differences in the masculine sexual behavior of ATD and control males when they were tested as gonadally intact adults. In contrast, even without exogenous hormone treatment, nine of 14 ATD males exhibited lordosis behavior, whereas only one of 12 controls did so. In addition, during a sexual preference test in which access was provided to both a sexually receptive female and to a stud male, there was no difference in the proportions of ATD ($\text{11}\text{14}$) and control ($\text{7}\text{12}$) males that copulated with the stimulus female; however, seven of the ATD males also exhibited feminine sexual behavior including some instances of solicitation. Only one of the control males showed any lordosis behavior. In general, all animals spent more time with the stimulus female than with the stud male. At the termination of preference testing, all animals were castrated and then tested twice for feminine sexual behavior under exogenous estradiol benzoate and progesterone. All of the ATD males showed lordosis behavior with a mean lordosis quotient (LQ) of 85; and 11 of the 14 also showed solicitation behavior. Only five of 12 control males exhibited lordosis ($\text{X}\text{?}\text{LQ}\text{= 59}$) and only one showed solicitation behavior. These results indicate that the propensity of males to show feminine sexual behavior can be manipulated independently of the capacity for masculine sexual behavior. Moreover, our results suggest that the process of defeminization may occur primarily postnatally in rats since treatment during that period results in substantial increments in later feminine sexual behavior including solicitation behaviors.     

Two high affinity estrogen binding proteins of different specificity in the immature rat uterus cytosol

The presence of two high affinity estrogen binding proteins in the uterine cytosol of the immature rat has been observed.Besides the 8 S cytosol estrogen $\text{receptor}$, there is a 4–5 S fraction binding estradiol and estrone with a large capacity. In fact, the two binding systems have a different affinity for estradiol and estrone, the receptor binding more the former and the 4–5 S fraction more the latter. Exposure of the cytosol to specific anti-α₁-Fetoprotein antibodies suppresses a large part of the 4–5 S binder, if not the totality. Moreover the estrogen binding 4–5 S fraction decreases with increasing age until puberty, while the $\text{receptor}$ increases. These results suggest therefore that the estradiol-estrone binding 4–5 S peak of the uterine cytosol is mainly made up of Estradiol Binding Plasma Protein-α₁-Fetoprotein (EBP-AFP). Also they confirm that “cytosol” should be taken as an operational fraction which may include extracellular components.During the course of these experiments, it has been observed that the increase of the estradiol $\text{receptor}$ is more rapid than the other uterine cytosol proteins until the 8th day, and that there is a second period of growth when it follows the development of the uterus and of the animal, as if it had reached a constant number of binding sites per cell.     

Mechanism of the enzyme-catalyzed bioluminescent oxidation of coelenterate-type luciferin.

The use of a fully active, synthetic analogue of coelenterate-type luciferin labeled in the carbonyl position with ¹⁴C and ¹⁸O was used to probe the mechanism of the $\text{Renilla}$ luciferase catalyzed oxidative decarboxylation of this compound. In the presence of ¹⁷O₂, the CO₂ produced in this oxidation can be shown to contain approximately one ¹⁷O atom per CO₂ molecule. This result is consistent with a cyclic peroxide or dioxetanone-type mechanism. In the presence of luciferase, the oxygen in the luciferin carbonyl group is rapidly exchanged with solvent water prior to the production of CO₂. Thus, the $\text{reaction}$ CO₂ contains considerable oxygen derived from water, via exchange with the carbonyl group, and about one oxygen from O₂ via a cyclic peroxide.     

The aging leydig cell: VII. Cytoplasmic estradiol receptors

Plasma estradiol and cytosolic estradiol receptor levels of testes were determined in a group of young (2–3 months) and old (24 months) Sprague-Dawley rats. Estradiol binding sites for the young rats averaged 5.6 ± 0.3 fmol/mg protein ($\text{x}\text{±}\text{SE, n}\text{=12}$), which was comparable to that of the old rats, 5.7 ± 0.3 fmol/mg protein (n=12). Using Scatchard analyses, the association constants at equilibrium of estradiol receptor binding of the old and young rats were the same, 6.1 × 10¹⁰M^?1. Plasma estradiol levels were also similar in both groups-19.6 ± 2.8 pg/ ml (n=14) for the young and 19.2 ± 2.6 pg/ml (n=10) for the old rats. Our results suggest that impaired testosterone biosynthesis in old rats was not due to elevated plasma estradiol levels or to differences in testicular estradiol receptor content.     

Synthesis of α,ω-bis(diphenylphosphino)alkane and α,ω-bis(diphenylphosphino)(poly)ether ligands and complexes of rhodium(I)

α,ω-Bis(diphenylphosphino)alkane and α,ω-bis(diphenylphosphino)(poly)ether ligands can be prepared in very high yields via reaction of the appropriate dihalide with two equivalents of LiPPh₂. For the [Rh(COD)($\text{P P}$)][ClO₄] complexes of these ligands, the $\text{P P}$ ligands with five or less atoms in the alkane or ether bridge form monomeric complexes via η²-coordination. In general the ligands with eight or more atoms in the bridge give di- or polynuclear species. In addition the long chain diphosphino-polyethers form – to a small extent – monomeric species by η²-coordination.     

Stimulation of the estrogen synthesizing system of the immature rat ovary by exogenous and endogenous gonadotropins

Immature rat ovaries increase their secretion of estradiol (E₂) when stimulated by gonadotropins but only after a lag period of several hours. Once established, estrogen secretion can be maintained, or increased, by the continued presence of gonadotropin. A combination of ovine FSH+LH given at 2 hr intervals stimulated the estrogen synthesizing system ($\text{ESS}$) of the ovary and serum E₂ showed a pronounced rise between 16 and 20 hrs after the initial injection. When given every 2 hrs for 5 doses (0–8 hrs) serum E₂ was undetectable. However, it was increased if 20 IU PMS was injected at the time of the last dose of FSH+ LH. Endogenous FSH&LH, increased by hourly injections of LH-releasing hormone for a period of 8 hrs, stimulated the $\text{ESS}$; serum E₂ increased at the expected time when this treatment was followed by an injection of PMS.Anti-PMS antiserum given 12 hrs after PMS, prevented the expected rise in serum E₂ at 24 hrs. However, FSH, LH or a combination of the two given every 2 hrs beginning at the time of the anti-PMS produced an increase in E₂ secretion; the combination was more effective than either hormone alone.These results are consistent with the interpretation that a combined FSH-LH action is responsible for induction of the $\text{ESS}$ in the immature rat ovary. The combination of hormones is also very effective in maintaining estrogen secretion but some function appears possible with FSH or LH alone.     

Topography of nucleic acid helices in solutions. 28. Evidence for a dynamic structure of DNA in solution

Proton magnetic resonance (pmr), ultraviolet absorption, induced circular dichroism (CD), and viscometric evidence is presented which show that reporter molecules $\text{1}$ and $\text{2}$ bind to DNA via an intercalation process. Preliminary kinetic studies show that the DNA·$\text{1}$ complex forms rapidly (i.e., <1 msec), whereas the DNA·$\text{2}$ complex forms at a considerably slower rate ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{> 100 msec}$). The kinetic results, and the steric requirements for intercalation of $\text{2}$ can be explained on the basis of a dynamic structure of DNA.     

Variant T47D human breast cancer cells with high progesterone-receptor levels despite estrogen and antiestrogen resistance

In target tissues for estrogen, including breast cancer cells, the synthesis of progesterone receptors (PRs) is controlled by estradiol acting through estrogen receptors (ERs). We describe studies with T47D human breast cancer cells, whose PRs are not regulated by estradiol, though present in extraordinary amounts (300,000 sites per cell). These cells have no ERs sedimenting at 8S on sucrose density gradients, and no unfilled cytoplasmic or nuclear ERs; some apparently hormone-filled nuclear sites, with $\text{K}{}_{\mathrm{<mtext>D</mtext>}}\text{? 0.7}$ nM, can be demonstrated by exchange. The nuclear ER sites are not processed after estradiol treatment. Nafoxidine, however, doubles nuclear estrogen binding in 6 hr, in a cycloheximide-insensitive step that may represent a reversal of processing. T47D cells are profoundly resistant to estrogens and antiestrogens; estradiol does not stimulate PRs, and nafoxidine concentrations that are cytotoxic to ER-positive cells have no effect on cell growth or on PR levels. Yet the PRs are normal by several criteria, and they can be stoichiometrically translocated to, and extracted from, nuclei in the first 3 min after progesterone addition. If progesterone treatment exceeds 10 min, rapid nuclear turnover prevents quantitative PR recovery. Cytoplasmic PRs are replenished in 10 to 24 hr, and this cycloheximide-sensitive step is also estrogen- and nafoxidine-resistant. However, despite their insensitivity to estradiol or antiestrogen, PRs are not constitutively synthesized; 5-bromodeoxyuridine and sodium butyrate can selectively inhibit PR production. Thus, since PRs retain some characteristics of inducible proteins, the persistent nuclear estrogen-binding sites may be stimulating PRs continuously, even in the absence of exogenous estradiol.