Receptors In Endometrium Research Articles

A double immunofluorescent method for simultaneous analysis of progesterone-dependent changes in proliferation and the oestrogen receptor in endometrium of rhesus monkeys

A double immunofluorescent technique that permitted the nuclear localization of both the oestrogen receptor and the Ki-67 antigen (marker of cell proliferation) within the same tissue section was developed and used to determine the relationship between the oestrogen receptor and proliferation within the major zones of the primate endometrium. Endometrial tissue was obtained from ovariectomized rhesus monkeys in which the hormonal pattern of oestradiol and progesterone was simulated with Silastic implants containing each hormone. The oestrogen receptor was detected using the H222 rat monoclonal antibody and Texas Red conjugated streptavidin. Immunofluorescent staining of a mouse monoclonal antibody to the Ki-67 antigen was detected by an antibody to mouse IgG conjugated with fluorescein isothiocyanate. Primary antibodies were co-incubated with tissue sections followed by sequential detection of the oestrogen receptor and Ki-67. A dual wavelength filter was used to monitor simultaneously both immunofluorescent patterns. Application of this technique to study regulation of endometrial proliferation and oestrogen receptor content by progesterone showed that the endometrium exhibited two different responses to progesterone: luminal and glandular epithelia and stromal cells in the functionalis and zone III of the basalis showed coincident decreases of oestrogen receptor and proliferation, whereas the oestrogen receptor in glandular epithelia of zone IV (basalis) was not downregulated and there was an increase in proliferation.

Changes in progesterone and oestrogen receptor mRNA and protein and oxytocin receptors in endometrium of ewes after intrauterine injection of ovine trophoblast interferon.

This study determined whether twice-daily intrauterine injections of ovine conceptus secretory proteins (oCSP) containing type-I trophoblast interferon (25 micrograms/uterine horn) from day 11 to day 15 post-oestrus (oestrus = day 0) could alter the binding capacities of endometrial receptors for oxytocin, progesterone and oestrogen in cyclic ewes when compared with control ewes receiving serum protein (SP) injections. Injections of oCSP on days 11-15 post-oestrus decreased concentrations of oestrogen receptors (P < 0.06), oestrogen receptor mRNA (P < 0.05) and progesterone receptors (P < 0.08) in endometrium on day 16 when compared with SP-infused control ewes, which were undergoing corpus luteum regression on days 14-16. Injection of oCSP also decreased the number (P < 0.10) and affinity (P < 0.06) of oxytocin receptors. Inositol phosphate formation induced in the endometrium on day 16 by 100 nM oxytocin in vitro was highly correlated with the concentration (r > or = 0.93, P < 0.001) and Kd (r = -0.91, P < 0.01) of oxytocin receptors in SP-infused ewes, but was not as highly correlated with concentration (r < or = 0.83, P < 0.06) and Kd (r < or = 0.40, P > 0.40) of oxytocin receptors in oCSP-infused ewes. This indicates that oCSP disrupted the relationship between oxytocin receptor binding and oxytocin-induced activation of its second messenger system. These results indicate that antiluteolytic type-I trophoblast interferon may prevent oxytocin-induced luteolytic pulsatile secretion of prostaglandin F2 alpha during maternal recognition of pregnancy in sheep, by reducing the synthesis and affinity of endometrial oxytocin receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Concentrations of progesterone and oxytocin receptors in endometrium of postpartum cows expected to have a short or normal oestrous cycle.

Luteal lifespan is short after first postpartum ovulation in early-weaned beef cows unless cows are pretreated with a progestogen. Regression of the short-lived corpus luteum in the postpartum beef cow is due to a premature release of prostaglandin F2 alpha (PGF2 alpha) from the uterus. The premature release of PGF2 alpha may be mediated through lower concentrations of receptors for progesterone, higher concentrations of oxytocin receptors, or both, in the endometrium. Thirty-one beef cows were randomly assigned to four groups at parturition. Calves from cows assigned to the short cycle group (n = 6; control) and the short cycle/endometrium group (n = 10) were weaned at 30-32 days post partum. Cows in the normal cycle group (n = 5; control) and the normal cycle/endometrium group (n = 10) received norgestomet implants for 9 days beginning 21-23 days post partum and calves were weaned at implant insertion. Duration of oestrous cycle (x +/- SEM; P < 0.01) following first postpartum ovulation for the short cycle group was 11.5 +/- 1.9 days compared with 18.8 +/- 0.6 days for the normal cycle group. On day 5 following first postpartum ovulation, cows in the short cycle/endometrium and the normal cycle/endometrium groups were hysterectomized and endometrial tissue collected for measurement of progesterone and oxytocin receptors. Mean number of total progesterone receptors per cell was lower (P < 0.05) in the short cycle/endometrium group than in the normal cycle/endometrium group. Mean concentration of oxytocin receptors (fmol mg-1 protein) in the short cycle/endometrium group was higher (P < 0.05) than that in the normal cycle/endometrium group.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of tamoxifen alone and in combination with RU 486 on the endometrium in the mid-luteal phase.

The effects of RU 486 combined with tamoxifen and tamoxifen alone on hormonal parameters and endometrial development at the time of implantation were studied. Measurements of cytosolic oestrogen and progesterone receptors in endometrium and placental protein 14 (PP14) in plasma were also included. Three dosage schedules were used: single oral dose of 40 mg tamoxifen alone and in combination with 200 mg RU 486, and 40 mg tamoxifen for three consecutive days starting on the first day after the luteinizing hormone (LH) surge. The combined treatment prolonged the luteal phase (P < 0.05) and increased the plasma levels of progesterone. A single dose of tamoxifen did not affect the bleeding pattern and plasma hormone levels, but raised plasma oestradiol and LH with the 3-day treatment. The endometrium was retarded after the combined and the 3-day treatment with tamoxifen. Concentrations of cytosolic progesterone receptors were higher after the combined therapy, but were unaffected after tamoxifen only. PP14 levels were higher (P < 0.05) after repeated tamoxifen doses than in controls, but were lower with combined treatment. Progesterone and oestrogen are evidently necessary for endometrial maturation during the secretory phase of the menstrual cycle. PP14 levels in plasma cannot be used for clinical assessments of endometrial function because high levels coincide with disturbed endometrial development.

Epidermal growth factor receptor expression in normal endometrium and endometriosis: An immunohistochemical study

Objective To study epidermal growth factor (EGF) receptor expression in endometrium throughout the menstrual cycle and to compare EGF-receptor expression in endometrium from patients with endometriosis with receptor expression in synchronously sampled endometriosis and in endometrium from healthy women. Design An immunohistochemical study of receptor expression using murine monoclonal antibodies and timed endometrial and endometriotic biopsies. Subjects 25 healthy women and 27 patients with a diagnosis of endometriosis. Results Positive staining for EGF receptors was observed in 24 of 25 samples from normal women and in 26 of 27 endometrial samples from patients with endometriosis. In neither group was there any variation in the intensity of staining throughout the menstrual cycle and both glands and stroma were stained. EGF-receptor expression was observed in the glands of 15 out of 17 endometriotic lesions and in 12 of these biopsies positive staining was also present within endometriotic stroma. Conclusion This study shows no difference in the intensity of staining of EGF receptors in endometrium throughout the menstrual cycle or between the glands of normal endometrium and those of endometriosis.

Increase in concentration of uterine oxytocin receptors and decrease in response to 13,14-dihydro-15-keto prostaglandin F2 alpha in ewes after withdrawal of exogenous progesterone.

Ovariectomized ewes were treated with progesterone and oestradiol to induce oestrus (day of expected oestrus = day 0) and with progesterone on days 1 to 12. The concentrations of endometrial oxytocin receptors and the 13,14-dihydro-15-keto prostaglandin F2 alpha (PGFM) response induced by oxytocin were measured on days 12, 14, 16 and 18 after the cessation of progesterone treatment on day 12, by a receptor binding assay and direct radioimmunoassay, respectively. During the period of treatment, the concentrations of plasma progesterone were high and remained above 2 ng ml-1 until day 13 when they dropped rapidly to less than 0.5 ng ml-1 by day 14. The concentrations of oxytocin receptors in endometrium of control ewes were high (820.7 +/- 91.7 (SEM) fmol mg-1 protein). Treatment with progesterone significantly (P < 0.01) reduced the concentrations of the receptors on days 12 and 14 (144.1 +/- 65.0 and 200.4 +/- 45.4 fmol mg-1 protein, respectively). The receptor concentrations then increased to relatively high values on day 16 (1021.4 +/- 216.6 fmol mg-1 protein) and remained high until day 18 (677.7 +/- 103.4 fmol mg-1 protein).(ABSTRACT TRUNCATED AT 250 WORDS)

Epidermal growth factor receptor expression in normal endometrium and endometriosis: an immunohistochemical study.

To study epidermal growth factor (EGF) receptor expression in endometrium throughout the menstrual cycle and to compare EGF-receptor expression in endometrium from patients with endometriosis with receptor expression in synchronously sampled endometriosis and in endometrium from healthy women. An immunohistochemical study of receptor expression using murine monoclonal antibodies and timed endometrial and endometriotic biopsies. 25 healthy women and 27 patients with a diagnosis of endometriosis. Positive staining for EGF receptors was observed in 24 of 25 samples from normal women and in 26 of 27 endometrial samples from patients with endometriosis. In neither group was there any variation in the intensity of staining throughout the menstrual cycle and both glands and stroma were stained. EGF-receptor expression was observed in the glands of 15 out of 17 endometriotic lesions and in 12 of these biopsies positive staining was also present within endometriotic stroma. This study shows no difference in the intensity of staining of EGF receptors in endometrium throughout the menstrual cycle or between the glands of normal endometrium and those of endometriosis.

Concentrations of Nuclear Progesterone Receptors in Endometrium of Virgin and Repeat Breeder Heifers after Embryo Transfer

The present study aimed to correlate the repeat breeder syndrome in the bovine with an impaired or suboptimal uterine progestational response. Concentrations of nuclear (i.e. transformed) receptors for progesterone (PRn) were determined with a binding and exchange method in endometrial samples from virgin (VH) and repeat breeder (RBH) heifers 15 days post oestrus. The heifers were recipients of Day 7 demi-embryos collected from donors with normal fertility and transferred 8 days prior to tissue sampling. Results were compared with both the type of heifer, the condition of the embryo present within the uterus and the temporal relationship to the hormone plasma levels. The binding data for PRn indicated that a single class of high-affinity, low-capacity sites existed. The amount of PRn in VH endometria holding embryonic structures was significantly greater than in RBH, but no statistical differences were found in their plasma progesterone levels. PRn concentrations were also higher in the uterine horn in which an elongated (greater than 15 mm), morphologically normal embryo was present, when compared to cornua with small (less than 5 mm) embryos, regardless of recipient group. Furthermore, in endometria with an elongated embryo in the lumen, the relative amount of PRn was significantly greater in VH than in RBH. The present results indicate that the RBH recipients of Day 7 demi-embryos by Day 15 have fewer numbers of specific receptors for maternal progesterone, which could explain in part the poorer development of the transferred embryos compared with that in virgin heifers.

Oestrogen and progestogen receptors in endometrium and myometrium at the time of blastocyst implantation in pregnant diabetic rats.

A suitable hormonal environment is a prerequisite for blastocyst implantation. Experimental diabetes was previously shown to modify the hormonal milieu and produce alterations in oestrogen receptor kinetics in the uterine tissue. In the present work, oestrogen and progestogen receptor levels were measured on the morning of day 6 of pregnancy in normal and in streptozotocin-induced diabetic rats, both in implantation sites and in interembryonic segments of endometrium and myometrium. Receptor levels were different in the implantation sites compared to the interembryonic segments of endometrium, both in the control and in the diabetic animals. Indeed, implantation sites were characterized by lower oestrogen receptor levels in cytosol and higher progestogen receptor levels in cytosol and nuclei. However, compared to the control rats, the diabetic rats had lower oestrogen receptor levels in implantation sites, both in cytosol and nuclei. In the myometrium, the differences between sites or between types of rats were minimal. Plasma levels of oestradiol were lower in diabetic rats than in control animals, whereas progesterone levels were similar. A 20% lower implantation rate was found in diabetic rats, compared to normal rats. These results show that the specific distribution of oestrogen and progestogen receptors between implantation sites and interembryonic segments was preserved in the diabetic rats; however the absolute level of oestrogen receptor was lower. This abnormal endocrine milieu might arise from a lower oestradiol level and a decreased oestradiol/progesterone ratio in the circulating blood. Whether the lower implantation rate in diabetic rats might be a consequence of the overall disturbed hormonal status remains to be elucidated.

Effect of continuous infusion of oxytocin on length of the oestrous cycle and luteolysis in cattle

In Exp. I oxytocin (60 micrograms/100 kg/day) was infused into the jugular vein of 3 heifers on Days 14-22, 15-18 and 16-19 of the oestrous cycle respectively. In Exp. II 5 heifers were infused with 12 micrograms oxytocin/100 kg/day from Day 15 of the oestrous cycle until clear signs of oestrus. Blood samples were taken from the contralateral jugular vein at 2-h intervals from the start of the infusion. The oestrous cycle before and after treatment served as the controls for each animal. Blood samples were taken less frequently during the control cycles. In Exp. III 3 heifers were infused with 12 micrograms oxytocin/100 kg/day for 50 h before expected oestrus and slaughtered 30-40 min after the end of infusion for determination of oxytocin receptor amounts in the endometrium. Three other heifers slaughtered at the same days of the cycle served as controls. Peripheral concentrations of oxytocin during infusion ranged between 155 and 641 pg/ml in Exp. I and 18 and 25 pg/ml in Exp. II. In 4 our of 8 heifers of Exps I and II, one high pulse of 15-keto-13,14-dihydro-prostaglandin F-2 alpha (PGFM) appeared soon after the start of oxytocin infusion followed by some irregular pulses. The first PGFM pulse was accompanied by a transient (10-14 h) decrease of blood progesterone concentration. High regular pulses of PGFM in all heifers examined were measured between Days 17 and 19 during spontaneous luteolysis. No change in length of the oestrous cycle or secretion patterns of progesterone, PGFM and LH was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Subcellular distribution, properties and interrelationship of oestrogen receptors in endometrium and other target tissues.

More than half of the extranuclear receptor content of resting cells is associated with cytoplasmic structures. Subfractionation of microsomes reveals a preponderance of "basic" (low electrophoretic mobility) receptor in rough endoplasmic reticulum. Surfynol-dithiothreitol extracts of smooth membranes are rich in "acidic" (high electrophoretic mobility) receptor. Trypsin increases the yields up to seven-fold, and this increase is correlated (r = 0.993) with the acidic receptor content and 5'-nucleotidase activity of these microsomal preparations. Acidic microsomal "cytosolic" and nuclear receptor can be degraded to the "basic" variety of streptomyces hyaluronidase. All forms give rise to a tryptic fragment with unchanged affinity for oestradiol and dimerization ability. Basic receptor isolated after enzymatic conversion of acidic receptor is microheterogenous on isoelectric focusing, but gives rise to only one precipitation arc versus the IgG fraction of a polyclonal antiserum. The precipitation arc can be recharged with labelled oestradiol and autoradiographed. Non-immune IgG's from (unspecific) soluble complexes with oestrogen receptors, but not with their tryptic fragments. The polyclonal antioestrogen receptor IgG fraction precipitates the oestradiol-tagged tryptic receptor fragment from all subcellular sources of all homologous (porcine) and heterologous (human, ovine, bovine, goat, rabbit, guinea pig, rat) target tissues tested. Organ specificity can therefore be excluded and a high degree of phylogenetic conservation of the oestrogen receptor's protein moiety is implied. The presence, in the immune IgG fraction, of steroid releasing antibodies, which apparently distort the binding site, should spur the search for monoclonal antibodies with similar properties.

Postmenopausal hormone replacement therapy with estrogen periodically supplemented with antiestrogen

To inhibit endometrial stimulation during postmenopausal estrogen therapy, 25 women with climacteric symptoms were treated with a daily dose of 1.25 mg of conjugated estrogens for 7 weeks followed by a period of 10 days with clomiphene citrate administration (50 mg per day). This combination was repeated three times during the 6-month trial. The marked rellef of climacteric symptoms with estrogen was slightly less during clomiphene treatment. Uterine bleeding occurred five times during estrogen treatment periods but never during or after clomiphene supplementation. Histologic examination revealed endometrial atrophy in 41% of the samples after the first estrogen treatment, whereas after the first and third elements periods this was increased to 77% and 73%, respectively. The first clomiphene treatment significantly decreased the concentrations of cytosol estrogen and progestin receptors in endometrium, as compared with the levels recorded at the end of the processing estrogen therapy. The depression in the cytosol estrogen receptor concentration was permitted whereas cytosol progestin receptor concentration tended to increase during the subsequently strogen-plus-clomiphene treatment. Estrogen declined serum concentration of follicle-stimulating hormone (FSH), whereas the concentration of luteinizing hormone (LH) and prolactin remained unchanged. Clomiphene did not change the levels of these hormones from those observed during the estrogen treatment. The concentration of free fatty acids in serum was increased during the estrogen and clomiphene treatments, whereas the levels of cholesterol and high-density lipoprotein-cholesterol did not change. Our results suggest that this treatment regimen relieves climacteric symptoms without endometrial stimulation or other adverse effects. Thus, clomiphane appears to be a practical alternative to progestin for interruption of the postmenopausal endometrial effect of estrogen.

Uteroglobin mRNA and levels of nuclear progesterone receptor in endometrium

To investigate the mechanism of induction of uteroglobin by progesterone, the relationship between uteroglobin mRNA (mRNA ug) activity and nuclear progesterone receptor levels has been examined in rabbit endometrium during early pregnancy. mRNA ug activity was assessed by translation in vitro of poly A-rich endometrial RNA and immunoprecipitation of the synthesized peptides using anti-uteroglobin antibodies. Progesterone receptor was determined in purified endometrial nuclei by Scatchard analysis of the specific binding of 3H-R5020 under exchange conditions. mRNA ug activity reached a peak on Day 4 of pregnancy and declined thereafter up to Day 8. Nuclear progesterone receptor levels showed a slight rise on Day 2, then returned to baseline levels. When mRNA ug activity was high, progesterone receptor levels had declined. A cause-and-effect relationship between nuclear progesterone receptors and specific mRNA or protein synthesis remains to be established.

Uteroglobin mRNA activity and levels of nuclear progesterone receptor in endometrium

To investigate the mechanism of induction of uteroglobin by progesterone, the relationship between uteroglobin mRNA (mRNA UG) activity and nuclear progesterone receptor levels has been examined in rabbit endometrium during early pregnancy. mRNA UG activity was assessed by translation in vitro of poly A-rich endometrial RNA and immunoprecipitation of the synthesized peptides using anti-uteroglobin antibodies. Progesterone receptor was determined in purified endometrial nuclei by Scatchard analysis of the specific binding of 3H-R5020 under exchange conditions. mRNA UG activity reached a peak on Day 4 of pregnancy and declined thereafter up to Day 8. Nuclear progesterone receptor levels were uniformly low through this period, except for a slight rise on Day 2. When mRNA UG activity was high, progesterone receptor levels were low. A cause-and-effect relationship between nuclear progesterone receptors and specific mRNA or protein synthesis remains to be established.

Progesterone inhibition of estrogen receptor replenishment in ovine endometrium.

The influence of exogenous progesterone and estradiol on the concentration of total cytoplasmic and nuclear estrogen receptor in endometrium of ovariectomized ewes was studied and the effect of the respective steroid treatments on the receptor-mediated translocation of estrogen to the nucleus determined. 16 ovariectomized ewes were assigned to treatment groups as follows: Group 1 controls; Group 2 estradiol-17beta (EZ) implants (silastic capsules packed with 54 + or -3 mg); Group 3 progesterone (P) (15 mg twice daily); and Group 4 EZ + P. Control and P-treated animals were implanted with empty silastic capsules. Blood samples were collected on Days 0 and 2-7 for analysis for EZ and P. Ewes were sacrificed on Day 7 and the uteri were removed. Total specific nuclear and cytoplasmic estrogen binding sites were determined by use of tritiated estradiol exchange assay. Tritiated E2 bound to total cytoplasmic and nuclear receptors was greater (p less than .05) in endometirum of E2 treated ewes (10.4 + or -7.27 and .23 + or -.06 fmoles/mcg DNA respectively) than in endometrium of control P- and E2 + P-treated animals (1.62 + or -.26 and .13 + or -.02 fmoles/mcg DNA respectively). An average of 81 + or -1% of the tritiated E2 bound to available cytoplasmic receptors was translocated to the nucleus during incubation of endometrium of ewes from all treatment groups. The concentration of tritiated E2 specifically bound in the cytoplasm and translocated to the nucleus during incubation was greater in the endometrium of controls P-treated and E2 + P-treated groups. Concentrations of labeled E2 in the cytoplasm after in vitro translocation was greater in the endometrium of E2-treated ewes than in the endometrium of the other treatment groups. It is suggested that P antagonizes estrogen binding in the uterus by decreasing the concentration of cytoplasmic estrogen receptor.

Estradiol and 20alpha-dihydroprogesterone dehydrogenase activities in human endometrium during the menstrual cycle.

Estradiol dehydrogenase E2-DH)and 20α-dihydroprogesterone dehydrogenase (20αDHP-DH) activities in human endometrium at various phases of the menstrual cycle were measured by incubating 3H-estradiol or 3H-20α-dihydroprogesterone (20αDHP) with an 800 × g supernatant of tissue and excess NAD*. The enzymatic activities, expressed as nmoles of product formed per hr and per mg of protein, showed a significant increase during the secretory phase. The average values of E2-DH activities were 1.2 in proliferative endometrium and 14 in secretory tissue. The corresponding 20αDHP-DH activities were lower (0.3 and 3.0). The total amount of E2 receptors in endometrium, determined by the method of superfusion, was found o t decline from 3.2 pmole/mg DNA in the proliferative phase to 0.6 pmole/mg DNA at the end of the secretory phase. The increase in E2-DH levels leads to a faster removal of E2 from endometrium since the conversion of E2 to E2 is only partially reversible and E1 rather than E2 is released by the tissue. T...