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.     

Comparison of embryo recovery, embryo quality, oestradiol-17 beta and progesterone profiles in domestic cats (Felis catus) at natural or induced oestrus

Domestic cats experiencing a natural or FSH-induced oestrus were studied. Mated cats produced fewer (P less than 0.01) unfertilized oocytes and more (P less than 0.01) morulato blastocyst-stage embryos of better quality after a natural oestrus than after FSH treatment. Serum oestradiol-17 beta concentrations were lower (P less than 0.05) and progesterone levels rose earlier (P less than 0.05) in the induced oestrus compared to the natural oestrus group. Morula/blastocyst-stage embryos from both groups transferred to 15FSH/hCG-treated recipients produced 3 pregnancies and 2 live-born litters (1 from a natural oestrus donor and 1 from an FSH-treated donor). These results indicate that fertilization rates and embryo quality in domestic cats appear to be compromised by the FSH treatment, probably because of altered oestradiol-17 beta and progesterone concentrations.     

Plasma levels of LH, FSH, prolactin, oestradiol-17beta and progesterone during natural and induced oestrus in the dairy goat

The patterns of LH, FSH, prolactin and oestradiol-17beta, before and during natural oestrus, and of progesterone during the following cycle were studied in four French Alpine dairy goats and compared with those obtained after synchronization of oestrus in the same animals. The highest concentration of oestradiol-17beta was measured at the beginning of oestrus and was followed 3 hours later by simultaneous rises of LH, FSH and prolactin. A second FSH peak was observed 48h after the first one. On D(3) (D(0) = day of oestrus) progesterone concentration was over 1 ng/ml. The luteal phase lasted 15 days. Peak concentrations of oestradiol-17beta and progesterone were higher in animals when oestrus was induced. This was attributed to their higher ovulation rate. The second FSH peak was lower, and the interval between oestradiol-17beta peak and gonadotrophin surge longer, than at natural oestrus.     

In-vitro zona-lytic activity in uterine fluid from ovariectomized mice treated with oestradiol-17 beta and progesterone

Mouse embryos collected before implantation were incubated in vitro for 24 h with fluid rinsed from the uteri of ovariectomized female mice injected with progesterone, oestradiol-17 beta + progesterone, oestradiol-17 beta + progesterone, or oestradiol-17 beta alone. Although none of the zonae was completely dissolved, those incubated in fluid from animals treated with oestradiol + progesterone were subsequently more soluble in sodium thiocyanate (NaSCN) than those incubated similarly in control buffer, indicating a sublytic change during the incubation with uterine washings. Zonae incubated in fluid from animals injected with either hormone alone did not undergo such a change.     

Changes in polyribosomes and poly(A)-containing polyribosomal RNA in the uterus of the ovariectomized rat in response to oestradiol-17 beta treatment.

Polyribosome formation and the characteristics of polyribosomal poly(A)-containing RNA from uteri of ovariectomized rats responding to a single dose of oestradiol-17 beta was investigated. The mean proportion of polyribosomes in the atrophic uterus was 65%. In response to 10 micrograms of oestradiol-17 beta/100 g body mass, the amount of polyribosomes increased to 88% 24 h after stimulation. Thereafter the proportion of polyribosomes decreased to a value of 48% at 72h. The pattern of amino acid incorporation in oocytes from Xenopus laevis injected with these polyribosomes was similar to the changes in polyribosome formation and degradation. The polyribosomal poly(A)-containing RNA from the controls consisted of a heterogeneous population of RNA with sedimentation values between 5S and 25S. The hormone stimulation resulted in an increase in both the amount and the size (13S to 35S) of the RNA.     

Production of oestrone and oestradiol-17 beta by different regions of the filamentous pig blastocyst

Pig blastocysts aged 14, 16 and 18 days were divided into 15 cm segments representing tissue adjacent to the embryonic disc, an intermediate section and the tip region. Whenever total blastocyst length allowed, the intermediate segment was divided into proximal and distal portions for separate culture. All were rinsed with buffer and incubated with dehydroepiandrosterone for 3 h. Rinsing buffer and incubation medium were subsequently assayed for concentrations of oestrone and oestradiol-17 beta. The highest production of oestrogen was found in the embryonic disc region. The intermediate regions had the lowest synthetic ability, while the tip region produced more oestrogens than the intermediate regions but less than the disc region. The production of oestrone was higher (P less than 0.05) in 18-day-old blastocysts than in younger ones while oestradiol-17 beta production was lower (P less than 0.05) on Day 16. The proportional role of the embryonic disc region as oestrogen-producing tissue increased over time. On Day 14, each intermediate region produced over 70% as much oestrogen as the disc region. These proportions declined on Days 16 and 18 to about 50 and 30% respectively. The regional variation in the ability of blastocysts to produce oestrogens may have some influence on the ability of the blastocyst to create an adequate microenvironment within the uterus which permits successful differentiation and placentation.     

Effects of oestradiol-17 beta and progesterone on the number of plasma cells in uteri of ovariectomized mice

Immunoglobulins A and G were localized by immunoperoxidase labelling in uteri of ovariectomized mice treated with oestradiol-17 beta and progesterone. The administration of oestradiol or progesterone alone to ovariectomized mice for 3 days increased the number of IgA plasma cells from about 1 to 14 per histological section. When the two hormones were administered simultaneously for 3 days the number of plasma cells per section was equal to or greater than with either hormone alone. Treatment with oestradiol followed by progesterone in a sequence that prepares the uterus for implantation resulted in about 31 IgA plasma cells per section. Counts of IgG plasma cells showed similar trends but the numbers were smaller. The results indicate that progesterone increases rather than decreases the number of plasma cells in the mouse uterus. This is consistent with observations on intact mice during oestrus and pregnancy and suggests that the marked increase in endometrial plasma cells at the time of implantation in mice is a response to progesterone acting on an oestrogen-primed uterus.     

Nature and characteristics of the binding of oestradiol-17beta to a uterine macromolecular fraction

The binding of oestradiol-17β to two proteins, namely serum albumin and a uterus fraction, was studied in vitro. The former protein has a physiological function in the transport of the hormone and the latter is involved in the selective uptake of the steroid by the target organ. The uterus fraction shows a high degree of stereospecificity for the binding of the steroid. Cortisone, oestradiol-17α and testosterone are bound negligibly and progesterone to a much smaller extent than is oestradiol-17β. This property is in contrast with the wide variety of ligands bound by the serum albumin. The temperature and the presence of the steroid influence markedly the binding properties. Oestradiol binding to the uterus fraction is optimum at 37° and at pH7–8·5. It is markedly decreased at pH values above or below this range, suggesting stringent conformational requirements. The tissue `receptor' protein is a macromolecule with a minimum molecular weight of 100000. The protein moiety is essential for the binding function. The probable concentration of the total binding sites for oestradiol in the ovariectomized-rat uterus cytoplasmic fraction as determined in vitro is about 1mμm at a steroid concentration of 50mμm.     

Effects of oestradiol-17 beta on peripheral plasma concentrations of LH and FSH in ovariectomized tammars (Macropus eugenii)

Two experiments, each using 8 animals, were conducted in the non-breeding and breeding seasons, respectively, and each animal was injected with 4 different doses of oestradiol benzoate over 4 trials. The resulting physiological concentrations of plasma oestradiol caused depression of both LH and FSH values. The highest dose elicited a biphasic response in LH with a pulse-like surge at 24 h after injection. There was no significant difference between the response of either hormone at the two times of the year and it is concluded that, in tammars, there is no seasonal difference in the responsiveness of the hypothalamus/pituitary to the negative feedback effect of oestradiol.     

Induction of male behaviour in ovariectomized ewes and ovariectomized-androgenized ewes chronically implanted with oestradiol-17β or testosterone

Normal mature ewes and ewes that had been androgenized with testosterone (T) between days 30–80 or 50–100 of fetal life were ovariectomized and given 100 mg implants of either oestradiol-17β (E) or T. The T implants caused a sustained elevation in plasma T levels but the E implants did not produce stable plasma levels of E. The implants were weighed on removal from the ewes and daily release rates for E and T were 14.4 ± 5.8 μg/kg/day and 24.2 ± 5.3 μg/kg/day respectively.The implants of E induced oestrous behaviour in both the non-androgenized and the androgenized ewes, some of these animals remaining in oestrus for up to 11 days. The ewes also began to mount each other after 1–9 days of treatments; the androgenized ewes also showed male-like aggressive behaviour whereas the non-androgenized ewes did not.The T implants induced oestrous behaviour in both androgenized and non-androgenized ewes. However, the non-androgenized ewes never mounted other ewes, nor did they show aggressive behaviour, whereas the androgenized ewes did.Prenatal androgenization clearly alters the ability of a ewe to respond to exogenous steroids by increasing its propensity to show masculine behaviour. Nevertheless, non-androgenized ewes may also show masculine behaviour during chronic steroid treatment.