Preparturient changes in plasma concentrations of estrone,estradiol-17β and estradiol-17α in the fetal calf: Effects of dexamethasone infusion

The concentrations of total estrogens in fetal calf plasma were determined during a 6–10 day period immediately before delivery. Comparison was made between levels found in untreated calves and calves infused with dexamethasone at the rate of 0.1, 1.0 and 10 mg/24 hours. In untreated calves the plasma estrone, estradiol-17β and estradiol-17α levels remained relatively constant at 38 ± 7 ng ml^?1 (mean ± SEM n = 3), 46 ± 6 ng ml^?1 and 29 ± 5 ng ml^?1 respectively. Infusion with dexamethasone at 0.1 mg/24 hr (3 calves) and 1.0 mg/24 hr (3 calves) was without dramatic effect on plasma estrogen levels. However, in one fetus infused with 10.0 mg/24 hr the dexamethasone treatment may have caused a transitory rise in the levels of all estrogens examined.     

The effect of fetal hypophysectomy and fetal death in gilts with small litters on concentrations of relaxin,progesterone and estradiol-17β

During late pregnancy concentrations of relaxin, progesterone (P) and estradiol-17β (E) in maternal plasma were measured in gilts with small litters of intact, hypophysectomized, partially hypophysectomized or dead fetuses and in gilts with litters of normal fetuses and numbers. To achieve a small litter size at parturition all but one or two fetuses were killed at surgery at Day 30 to 40 of gestation. Fetal hypophysectomy or sham procedures were attempted on Day 90 to 95. Gestation was prolonged in gilts carrying hypophysectomized or partially hypophysectomized fetuses (P<0.01). Lactation and farrowing did not occur if hypophysectomy was complete. Basal concentrations of E in plasma were lower (P<0.01), basal P appeared higher and basal relaxin was unchanged in gilts carrying hypophysectomized or dead fetuses as compared to gilts with intact fetuses. Near the end of pregnancy the concentration of E was 119.8 pg/ml in gilts with the normal number of fetuses, 32.6 pg/ml in the group with hypophysectomized fetuses, and 7.3 pg/ml in gilts with dead fetuses. The relaxin peak occurred near term in control pigs and was delayed in the groups with hypophysectomized and partially hypophysectomized fetuses. The concentration of relaxin at the peak in gilts with normal sized litters was 181.4±75.8 ng/ml as compared with 25.3±16.0 ng/ml in gilts with partially hypophysectomized fetuses and was 9.5±1.4 ng/ml in gilts with hypophysectomized fetuses and 10.6±3.3 ng/ml in gilts with one or two fetuses. In gilts with intact or partially hypophysectomized fetuses, or litters containing both types, which came into labor, the patterns of P and E were similar. In gilts with hypophysectomized fetuses, P and E at term showed little change from basal concentrations. The results confirm that the fetus influences basal concentrations of E and possibly P in late normal gestations. In addition, the presence of the fetal pituitary is associated with the peak in relaxin expected at term. These associations are likely to be related to pituitary function and/or the mass of the conceptus. Fetal hypophysectomy is clearly associated with maternal concentrations of P and E at Day 114 that are different from those in normal sows, suggesting that these two hormones may have an effect on the initiation of parturition in the pig.     

Autonomic innervation and plasma estradiol-17β and progesterone levels in rats with subcutaneous ovarian autografts

Summary Ovaries were removed from female rats and immediately autografted into a subcutaneous pouch in the flank in order to quantitate the relationship of graft re-innervation, steroid secretion and vaginal smear pattern. Animals were killed at three time periods: three days after grafting, on the first day a cornified vaginal smear appeared and at the first metestrus. In addition, control animals were killed at metestrus. Plasma samples were obtained from all rats and analyzed for estradiol-17 and progesterone concentration by radioimmunoassay.At the first day of vaginal cornification after grafting, plasma estradiol-17 (45.8±4.0 pg/ml) was elevated in comparison to controls at metestrus (24.0±2.6 pg/ml), but plasma progesterone (21.5±4.0 ng/ml) was not different (30.6±1.7 ng/ml). Subsequently, at the first metestrus following grafting, plasma estradiol-17 (23.0±3.5 pg/ml) was comparable to control values. In contrast, progesterone was decreased (17.5±1.9 ng/ml). A definite correlation was detected between the vaginal smear and plasma levels of steroid hormones in the castrated female rat with subcutaneous ovarian autografts.Histochemical techniques were used to study the adrenergic and cholinergic innervations of grafts three days after grafting, at the first day of vaginal cornification, and at the first metestrus. No correlation was shown between density of adrenergic or cholinergic innervation and plasma levels of estradiol-17 and progesterone or onset of a cycling vaginal smear.Supported in part by USPHS Grant T01-DE00241-04The authors wish to thank J. Canale, S. Hemelt, E. Schwartz and J. Skaggs for their technical and secretarial assistance. Anti-estradiol-17 antibody was obtained from Dr. I.H. Thorneycroft, University of Southern California School of Medicine, and anti-progesterone antibody from Dr. D. Tulchinsky, Harvard Medical Center     

Nitric oxide concentrations, estradiol-17β progesterone ratio in follicular fluid, and COC quality with respect to perifollicular blood flow in cows

The objectives were to investigate relationships among concentrations of nitric oxide (NO), estradiol 17 beta (E2), and progesterone (P4) in follicular fluids (FF), and quality of cumulus oocyte complexes (COCs) with respect to perifollicular blood flow (FBF). In Experiment I, follicles (138) were classified according to the presence or absence of FBF (assessed with transvaginal Doppler ultrasonography) and diameter of follicles (small, 2-4 mm; medium, 5-8 mm; and large, ≥9 mm). Concentrations of NO in FF did not differ significantly among these size categories. However, NO concentrations in FF with FBF (54.4 ± 7.4 μmol/l) were higher (P<0.05) than in those without FBF (36.6 ± 4.1 μmol/l). There was a positive correlation (r=0.30, P<0.05) between NO concentrations and the E2:P4 in FF. Rate of E2 active (E2:P4 ≥ 1) follicles were numerically 1.2 (0.8-1.8) times higher in follicles with FBF (38.1%) compared to those without FBF (25.0%). Moreover, rates of E2 active follicles were 6.1 (0.7-55.2) and 1.3 (0.1-17.3) times higher (P<0.06) in large (43.3%) and medium (14.3%) compared to small follicles (11.1%), respectively. In Experiment II, quality of COCs from 2 to 8 mm follicles, obtained by transvaginal ovum pick up (OPU), was investigated with respect to FBF. Odds ratio to obtain higher quality COCs from follicles with FBF (47.1%) was 3.3 (1.1-9.6) fold higher (P<0.05) compared to those from follicles without FBF (14.6%). In conclusion, E2:P4, and NO concentrations in FF, as well as FBF, could be used to determine the functionality of ovarian follicles in cows. Moreover, determination of FBF could be useful to predict quality of COCs in cattle.     

Interactions of estradiol-17β with amino acids

The solubility of estradiol-17β in phosphate buffer and in solutions containing phosphate buffer and amino acids was estimated. Arginine, aspartic acid, glutamic acid, lysine, tryptophan and tyrosine significantly increase the solubility of estradiol-17β. A slight increase in solubility of estradiol-17β was observed in solutions of proline and histidine, while other amino acids had no effect.     

Stimulation of pulses of 13,14-dihydro-15-keto-PGF2α (PGFM) with estradiol-17β and changes in circulating progesterone concentrations within a PGFM pulse in heifers

A single physiologic dose (0.1 mg) of estradiol-17β in sesame-oil vehicle or vehicle alone (n = 8) was given to heifers on day 14 after ovulation to study the effect on circulating 13-14-dihydro-15-keto-PGF2α (PGFM), PGFM pulses, and changes in progesterone concentrations within a PGFM pulse. Blood samples were collected hourly for 16 h after treatment. The estradiol group had a greater mean concentration of PGFM, greater number of heifers with PGFM pulses and number of pulses/heifer, and greater prominence of the PGFM pulses. Changes in progesterone concentrations were not detected during the 16 h sampling session in the vehicle group, indicating that the heifers were in preluteolysis. Progesterone decreased after 12 h in the estradiol group, indicating a luteolytic effect of the estradiol-induced PGF secretion as represented by PGFM concentrations. Intrapulse changes in progesterone were detected during a PGFM pulse in the estradiol group (P < 0.006), but not in the vehicle group. Progesterone increased (P < 0.01) between Hours −2 and −1 of an estradiol-induced PGFM pulse (Hour 0 = peak of pulse), decreased (P < 0.004) between Hours −1 and 0, and increased (P < 0.01) or rebounded between Hours 0 and 1. Results were compatible with previous reports of a role for estradiol in the induction of PGFM pulses in cattle and demonstrated intrapulse changes in progesterone concentrations during an induced PGFM pulse.     

Correlation of plasma estradiol-17β and progesterone levels with ultrastructure and histochemistry of ovarian follicles in the white-spotted char,Salvelinus leucomaenis

Summary Plasma estradiol-17 and progesterone profiles were correlated with morphological changes in ovarian follicles during the preovulatory and postovulatory periods in the white-spotted char, Salvelinus leucomaenis. Plasma estradiol levels were highest in September, and were followed by a sharp drop in October; they remained very low throughout the postovulatory period. There was a good correlation between plasma estradiol levels and the gonadosomatic index, thus suggesting that estradiol is responsible for the synthesis of vitellogenic proteins. Plasma progesterone levels were very low in August, began to rise in September and reached a peak soon after ovulation; progesterone remained high for several days after ovulation. A preovulatory rise in plasma progesterone levels was recorded, and this is discussed in relation to the induction of oocyte maturation.In the preovulatory follicles, neither granulosa cells nor special thecal cells (ST cells) showed ⁵, 3-hydroxysteroid dehydrogenase (3-HSD) activity. In the young postovulatory follicles, in contrast, the ST cells showed intense 3-HSD activity with extensive agranular endoplasmic reticulum and numerous large mitochondria, while granulosa cells did not show 3-HSD activity. These results strongly suggest that the ST cells are the major sites of progesterone synthesis during the postovulatory period.Nanae Fish Culture Experimental Station Contribution No 14     

Relationships between the incidence of pre-ovulatory behaviour and the concentrations of oestradiol-17β and progesterone in bovine plasma

The incidence of eleven components of behaviour exhibited by 24 British Friesian heifers, housed in three groups of eight, was recorded for 24 days. All observed behaviour involved the participation of two individuals, these being within groups. The identity of the heifer initiating each behavioural event and that of the one receiving it was recorded. Thus the incidence of 11 “initiating” components and 11 “receiving” components, in effect the incidence of 22 different (but related) components, was recorded throughout an oestrous cycle for all heifers.Components were classified into four groups: standing, mounting, initiating behaviour (other than mounting) and receiving behaviour (other than standing). There was a peak in the incidence of all four groups around the time of oestrus, standing being uniquely, and mounting very highly, associated with the state of oestrus.Plasma oestradiol-17β levels rose steadily during 4 days to a peak, at about the time of onset of the greatly increased incidence of behaviour at oestrus, when progesterone levels were low. Oestradiol-17β levels then declined rapidly to a low level within 12 h of the end of the period of increased incidence of behaviour. No correlation was found between the magnitude of individual, peak, pre-oestrus levels of plasma oestradiol-17β and the incidence of any of the four groups of behavioural components.It is suggested that the limits of the time period when oestrous behaviour is shown are controlled mainly by oestradiol-17β, probably by the timing of its retention by receptors in cells of target brain tissues. The incidence of behavioural events during this period is, however, in part controlled by the presence or absence of a second oestrous heifer.A second smaller peak of plasma oestradiol-17β occurred 5 days after oestrus, when plasma progesterone was about one-third of peak luteal phase levels. No increase in incidence of behaviour was associated with this oestradiol-17β peak.     

Effects of estradiol-17β and hCG supplementation on superovulatory responses and embryo quality in swamp buffalo (Bubalus bubalis) implanted with norgestomet

Twenty-four cycling swamp buffaloes with normal reproductive histories and 2–3 months postpartum were used to investigate the effect of addition of estradiol-17β and human chorionic gonadotrophin (hCG) to the superovulation regime on the level of ovarian stimulation and embryo production.The estrous cycles of buffaloes were synchronized by prostaglandin injection and then divided into two groups for superovulatory treatment. Those in Group 1 (n = 12) received a implant containing 3 mg norgestomet (Syncro-Mate-B) for 9 days (insertion day is Day 0), with 4000 IU of equine chorionic gonadotrophin (eCG) and 500 μg cloprostenol i.m. given at Day 7. Group 2 (n = 12) received the same regime as Group 1, together with 7.5 mg estradiol-17β given in three intramuscular injections on Days 3, 5 and 7 in decreasing doses (4.0, 2.5 and 1.0 mg, respectively) and 5000 I.U hCG i.v. coincidentally with the first insemination. Estrus was monitored visually and by placing treated animals with bulls. Each animal was inseminated twice with frozen sperm after standing estrus. The numbers of corpora lutea (CL) and follicles greater than 8 mm in diameter were recorded via palpation per rectum at 6 days after implant removal. Two days later 11 animals from Group 2 and two from Group 1 were slaughtered for direct observation of ovarian responses and for embryo collection.The mean number of CL were 0.91 ± 0.66 and 9.08 ± 5.0 for Groups 1 and 2, respectively. The average recovery rate based on CL counts at slaughter was 60% in Group 2. No embryos were recovered from the two animals in Group 1. Seventy-nine percent of the collected ova were fertilized and more than 60% of them had developed into hatched blastocysts. The percentages of buffalo with excellent and good estrus were 41.6 and 91.6% for Groups 1 and 2, respectively.These results showed that the supplementation of estradiol-17β and the hCG treatment significantly improved the level of ovarian stimulation in swamp buffalo.     

Effect of progesterone pretreatment on the uptake of estradiol-17β by the uterine epithelium of the rat

Summary Progesterone pretreatment of ovariectomised rats resulted in a moderate (44%) lowering of the level of nuclear estradiol receptors found in the uterine epithelium 2 h after a single injection of this estrogen.     

Plasma prolactin,progesterone and oestradiol-17β concentrations around parturition in the pig

Plasma concentrations of prolactin, progesterone and oestradiol-17β were measured by radioimmunoassay in samples taken from 2–15 days before until 1–4 days after spontaneous parturition in four sows and in one sow around prostaglandin F2α-induced parturition.Between Days ?15 and ?2 (Day 0 = parturition), prolactin concentrations in daily samples fluctuated somewhat, but exceeded 10 ng/ml only exceptionally. Plasma progesterone levels gradually declined or remained high until about 2 days before parturition. A final decrease of the progesterone concentrations coincided with distinct increases of the prolactin levels during the last 40 h of pregnancy.Maximal prolactin concentrations were measured before the onset of delivery of the piglets. Oestradiol-17β reached peak values around delivery. Prostaglandin F2α injection caused an immediate sharp increase of the prolactin concentration which lasted for about 6 h. During this period progesterone and oestradiol-17β concentrations did not change. A second elevation of prolactin levels was measured when progesterone finally decreased.Changes of prolactin concentrations found in this study were compared with those found in other domestic animals at the same reproductive stage.     

FSH-induced aromatase activity in hamster granulosa cells: effect of estradiol-17β in vitro

Summary We have shown previously that estradiol-17 (E₂) reduces number of ovulations in cyclic rats, induces atresia of the dominant preovulatory follicle in monkeys, and that the initial effects of this treatment include reduced viability and estrogen accumulation in vitro by aspirated granulosa cells (GC) from monkeys and hamsters. The present experiment was designed to determine whether the reduction in estrogen accumulation can be ascribed to a direct action of E₂ on the aromatization of androgen to estrogen in vitro. Female hamsters were injected with 30 I.U. pregnant-mare serum gonadotropin i.p. and sacrificed 3 days later. GC were aspirated from the largest follicles and incubated for 48 h (initial incubation period) in the presence of human pituitary follicle-stimulating hormone (hFSH, 100 ng/ml). Following initial incubation, GC were further incubated for up to 24 h (secondary incubation period). During this subsequent incubation, medium was supplemented with 100nM ³H-1-androstenedione (³H-A₄). Initial incubation with E₂ at doses of 10 ng/ml, 100 ng/ml and 1 m E₂/ml induced variability in GC response, and a maximal depression of 70%. The inhibition by E₂ of hamster GC function in vitro parallels that shown in vivo for both hamsters and monkeys, but contrasts with that shown for rats. Thus, hamsters may represent an appropriate model in which to study the atretogenic effects of E₂ directly on antral follicle development.     

Effects of oestradiol-17β and progesterone on total and nuclear-protein synthesis in epithelial and stromal tissues of the mouse uterus, and of progesterone on the ability of these tissues to bind oestradiol-17β

1. A method is described for separating uterine epithelium that is 80% pure and connective-tissue stroma that is 60% pure. This was used to study the effects of steroid hormones on total and nuclear-protein synthesis in these tissues. 2. Oestradiol-17beta given alone produces mitoses in the epithelium but not in the stroma. It stimulated incorporation in vitro of [(14)C]lysine into total protein, histones and acidic nuclear proteins to a greater extent in epithelium than stroma. Incorporation into acidic nuclear proteins was most markedly stimulated, reaching four to six times the normal value 4h after treatment, and then declining rapidly. This peak was only seen in epithelial preparations. 3. After pretreatment with progesterone, oestradiol-17beta has the reverse effect, producing mitoses only in stroma. Progesterone alone had no effect on the amounts or rates of incorporation of [(14)C]lysine into stromal nuclear proteins, but changes after oestradiol-17beta treatment were similar to those seen in epithelium with oestradiol-17beta alone. In the epithelium, progesterone alone depressed incorporation into histones and acidic nuclear proteins, but did not abolish the subsequent response to oestradiol-17beta. With this treatment there was a rapid, large and transient increase in incorporation into epithelial total protein not seen with oestradiol-17beta alone. 4. Progesterone had no qualitative effect on the distribution of specific oestrogen-binding proteins, as judged by sucrose-density-gradient centrifugation. However, progesterone treatment increased the uptake in vivo of [6,7-(3)H]oestradiol-17beta by stroma, and it is possible that this is important although the differences were not apparent after labelling in vitro.     

Effect of thyroid state on estradiol-17β metabolism in the rabbit

It is well known that the metabolic clearance rate (MCR) of a hormone is Influenced highly by the level of its specific binding protein. It was interesting, therefore, to study the metabolism of estradiol-17β (E₂) in an animal model such as the rabbit where there is a lack for a highly specific binding protein for the steroid. The kinetics of the hormone was studied in relation to the thyroid state, namely in rabbits receiving thyroxin or propylthiouracil.In the absence of any significant decrease of the level of the rabbit androgen binding protein (R-ABP), the accelerated MCR^E2 and the elevated conversion ratio of estradiol to estrone (CR ^E₂→E₁) observed in hyperthyroid rabbits were attributed to the important role of metabolizing enzymes in the liver and/or extrahepatic tissues. In hypothyroid rabbits, while the CR ^E₂→E₁ decreased significantly the MCR^E2 was not altered.     

Autoradiographic study on the localization of estradiol-17β in neonatal mouse uterus and cervix

Summary The uptake of ³H-estradiol-17 in the neonatal mouse uterus and cervix has been studied by an autoradiographic method. When the radio-active hormone is administered in vivo and in vitro, grains are found to be concentrated above the nuclei both in the uterine and cervical epithelium and stroma. Grain counts revealed that the nuclear concentration of grains is higher at 4 h than at 2 h after isotope injection. The cervical epithelium has a higher nuclear concentration than the uterine epithelium both in vivo and in vitro. In the stroma, this situation is reversed except after in vitro treatment of the tissues.In the cervix, more of the hormone seems to be located within the nucleus while in the uterus a higher proportion of the grains are found in the vicinity of the nuclear periphery.Although the nuclear concentration of grains is higher at 4 h than at 2 h, the number of grains above the sections is lower at 4 h. Both in vivo and in vitro, the number of grains is higher above the stromal than above the epithelial compartments of the uterus and cervix.Five days old animals showed the same labeling pattern. The differences in uptake and distribution of ³H-estradiol are discussed in relation to other known differences in the hormone responsiveness in these tissues.We are greatly indebted to Professor W.E. Stumpf and the Laboratories for Reproductive Biology, University of North Carolina Medical School for the opportunity to study the method of dry mount autoradiography. The work has been supported by the Norwegian Research Council for Science and the Humanities and by the Norwegian Cancer Society (Landsforeningen mot Kreft)     

Thyroid hormones are corequisites for estradiol-17β in vitro induction of Xenopus vitellogenin synthesis and secretion

Estradiol-17β administered to male frogs induces liver synthesis and secretion of vitellogenin, the precursor protein of the major egg yolk proteins. Estradiol-17β alone failed to induce this protein in cultures of liver tissue maintained for 1–2 weeks prior to addition of the hormone. If a “complex” defined culture medium, such as Coon's modified Ham's F12 medium, is used, efficient primary and secondary induction of vitellogenin synthesis and secretion occurs in the presence of estradiol-17β, triiodothyronine, and dexamethasone. Using Coon's medium we investigated the role of both triiodothyronine and dexamethasone as corequisites of estradiol-17β induction of secreted vitellogenin. Control cultures given no hormones showed a gradual decrease in the level of secreted albumin and fibrinogen. Addition of dexamethasone, alone, induced increased synthesis of secreted albumin and fibrinogen as well as other proteins. Cultures given thyroid hormones, alone, showed an increased level of secreted albumin and fibrinogen at early time points in the culture period. Thus, at early times thyroid hormones appear to enhance the activity of endogenous glucocorticoids. Independent of their interaction with glucocorticoids, thyroid hormones also enhance the activity of estrogens. Long-term cultures given estradiol-17β, alone, failed to synthesize and secrete vitellogenin. In contrast, cultures given the estrogen together with thyroid hormones showed vitellogenin synthesis. These results imply that similar interactions of several hormones occur in vivo in adult animals treated with estrogens. In the accompanying paper the interaction of dexamethasone with estradiol-17β and triiodothyronine is described (L. J. Wangh, 1982, Develop. Biol.89, 294–298).     

Radioimmunoassay of estradiol-17β in unextracted EWE plasma

A radioimmunoassay for estradiol-17β (E₂β) without solvent extraction is described. It can be used for plasma samples with concentrations higher than 10 pg/ml.Tritiated E₂β, and a specific antiserum in phosphate buffer were added to plasma samples, the total incubation volume being 0,5 ml. An identical volume of steroid free plasma to that assayed in unknowns (0.050 – 0.2 ml) was added to the standard curve. Immunoprecipitation was used to separate bound and free E₂β and the bound radioactivity counted in the polypropylene assay tube.The calculated regression of E₂β measured on plasma loaded with excess E₂β (y = 0.987x + 3.8; R = 0.99) and that of E₂β measured in the same sample by the direct assay on that of E₂β found by a reference extraction method (y = 0.998× + 14.9; R = 0.98) as well as the presence of parallelism between the standard curve and different volumes of plasma and acceptable inter and intra assay coefficients of variation show that this method is suitable for the measurement of E₂β in uteroovarian venous plasma. However, this method cannot be used for peripheral plasma of pregnant animals because it is not specific.The method was found useful in a study on the effect of gonadotrophin pulses on the ovary when many samples had to be analysed. Furthermore, there is a potential for automatization which would facilitate more detailed analyses of ovarian-hypophyseal relationships.     

Effects of dietary estradiol-17β on feminization,growth and body composition in the Japanese eel (Anguilla japonica)

• 1.1.Juvenile Japanese eels (Anguilla japonica) were fed on a diet supplemented with estradiol-17β (E₂) at doses of 25, 50 and 75 mg/kg. The effects on growth, sex distribution and body composition were investigated in two groups of gonadally undifferentiated stages (early and later juvenile stages).
• 2.2.Feminization (95–100%) was observed in all E₂-treated groups.
• 3.3.The growth rate of fish treated with 25 and 50 mg/kg E₂ diet at the early juvenile stage was significantly increased.
• 4.4.The amount of protein in muscle decreased and that of fat increased in the E₂-treated groups except in the early juvenile stage fed with 25 mg/kg E₂.