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Endometrium estradiol receptors type I and type II during early pregnancy of rat
Life Sciences 78 (2006) 2919 – 2922 www.elsevier.com/locate/lifescie
Endometrium estradiol receptors type I and type II during early pregnancy of rat Edith L. Salazar a,*, Leobardo Calzada b b
a Medical Research Unit in Endocrine Disease, Medical Research Coordination, Social Security Mexican Institute (IMSS), Mexico Health Center (T-III) Dr. Manuel Escontria, Sanitary Jurisdiction Alvaro Obregon of the Public Health Service of Distrito Federal, Mexico
Received 23 May 2005; accepted 15 November 2005
Abstract By centrifugation in a sucrose density gradient we studied the citosol 17h-estradiol binding sites of blastocyst receptive and non-receptive endometrial zones, as well as uterine horn endometrium whose ovary was extirpated three weeks before pregnancy. The cytosol was prelabelled with {3H}-17h-estradiol 2 and 25 nM. In this work two incubation temperatures were studied. On the other hand, at 4 -C unoccupied receptors were identified as different from the classic receptor 8S type I. At the same time, we found that 25 -C is the optimal temperature for the assay of total receptors to achieve complete exchange of {3H}-17h-estradiol by 17h-estradiol in the binding sites. In these conditions, the major component was the 4S type II receptor, mainly in the endometrium from ovariectomized uteri. Furthermore, 17h-estradiol content was determined in the total homogenized by radioimmunoassay and the results were: 1.42 T 0.16, 1.22 T 0.15 and 1.75 T 0.27 pmol/g wet tissue for receptive, non-receptive and ovariectomized uteri, respectively. D 2005 Elsevier Inc. All rights reserved. Keywords: Estradiol receptors; Endometrium; Sucrose gradient; Rats
Introduction Binding properties of two proteins to intracellular specific receptors to 17h-estradiol in different target tissue of rat and humans have been reported (Calzada et al., 1993; Clark et al., 1978; Panko et al., 1981). They are described as union site or receptors type I and type II, respectively. The type I corresponds to the classic receptor and is located in the region of 8S in a sucrose density gradient, characterized by having high affinity and low binding capacity; 4S type II has opposite properties. Furthermore it is not translocated (Lopes et al., 1987) and is identified more easily by exchange analysis, which consists on incubating at 25 -C in presence of 25 nM {3H}-17h-estradiol. At this temperature, the dissociation and reassociation of the receptor esteroid complex is more active and the {3H}-17hestradiol substitutes the endogenous 17h-estradiol (Sanborn et al., 1971). It has been suggested that this receptor would * Corresponding author. PO Box 86-056 Me´xico D.F. CP 14391. Tel./fax: +52 5588 7521. E-mail address: [email protected] (E.L. Salazar). 0024-3205/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2005.11.018
constitute a reserve mechanism of 17h-estradiol for the receptor type I (Lopes et al., 1987). In the oestrus cycle and pregnancy, the rat endometrium tissue shows functional changes in short periods of time in relation to ovarian steroid fluctuations. In day five of rat pregnancy, the endometrium presents regularly spaced zones that have undergone a series of biochemical changes (Dey et al., 1991; Heald, 1976), which permit the blastocyst implantation that will be identified easily in the fifth day, showing greater permeability to molecules of great size (trypan blue – albumin complex). These changes are part of the functional differentiation of those zones of the endometrium. The mechanism that begins and maintains this process is not known, although it is known that increasing levels of progesterone from the beginning of pregnancy and an estrogenic environment are required for implantation, at least from day three (Dickmann et al., 1976; Yoshinaga, 1994). On the other hand, it has been proposed that the endometrium requires additional quantities of estradiol which would be produced by the blastocyst (Dickmann et al., 1977) to complete the functional differentiation of the endometrium.
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Fig. 1. Analysis by sucrose gradient of receptors type I and type II to 17hestradiol in endometrium receptive to the blastocyst. The cytosol prelabelled using 2 and 25 nM of {3H}-17h-estradiol (•-----•) or the same concentration of {3H}-17h-estradiol more 200 times the molar concentration of 17h-estradiol not labelled (°-----° ), was incubated at 4 and 25 -C, respectively, during 2 h. They are applied on sucrose gradient and the distribution of radioactivity is shown in the figures.
The close vicinity between two functionally different zones [blastocyst receptive (BR) and non-receptive (BNR) endometrial zones] within a same estrogenic environment, possibly requires a receptor complex system to express selectively the target cell function (Psychoyos, 1973). In this article we propose to study the characteristics of the receptors to 17h-estradiol 8S type I and 4S type II (this last in exchange conditions) in the BR, BNR and endometrium of uterine horn submitted to unilateral oophorectomy (UO) previous to pregnancy, and the content of 17h-estradiol tissue in the same samples were determined. Material and methods Female rats of the Long Evans Strain of an average weight of 220 T 15 g and regular oestrus cycles were used under controlled light–dark conditions. Water and pellet food were supplied ad libitum. The rats were anesthetized with phenobarbital, unilateral oophorectomized and maintained for 21 days prior to study. The pregnant rats were obtained after housing the mature females in groups of two with one male of the same strain. Vaginal smears were taken daily. Day zero of pregnancy was designated when spermatozoa were found in the smear and then males were removed. On day five between 9 and 11 AM, a group of pregnant rats was injected (i.v.) 1 ml of 1% trypan blue in saline. Thirty minutes later, these rats were sacrificed by cervical dislocation, then the rats were perfused with saline solution and their uterus were obtained. In this experiment the blue-stained regions represented the implanted sites, whereas the non-stained tissue was considered as non-implanted regions (Heald, 1976; Sanborn et al., 1971). The samples were frozen in liquid nitrogen and stored. The uteruses were thawed at 4 -C, cut lengthwise and gently agitated with glass pearls to detach the epithelial cells. The cells were homogenized in a Tris buffer at 4 -C (Tris –HCl 10 mM, EDTA 1.5 mM, glycerol 10%, monothioglycerol 10 mM) in a proportion of 150 mg of wet
weight/ml using a glass – glass homogenizer (2 pulses, each for 30 s at 150 revolutions/min with intervals of 60 s). The homogenate was centrifuged for 10 min at 800 g. The supernatant was reserved and the pellet was washed two times with the same buffer and recentrifuged. Supernatants were collected and centrifuged for 30 min at 105,000 g to obtain the cytosol, as well as adjusted the protein content to 2– 4 mg protein /ml. Type I estrogen receptors were determined incubating 500 Al of cytosol for 2 h at 4 -C, with 2 nM or 25 nM of 3H-17h-estradiol. For the determination of type II estrogen receptors, identical batches were prepared and incubated for the same period of time at 25 -C. Non-specific binding was assayed using the same radioactive ligand plus a 200-fold excess of no radioactive ligand. Bound and free estradiol was separated using a dextran-coated charcoal separation method (Calzada et al., 1993). Five hundred microliters of prelabelled [3H-estradiol] cytosol were layered on a linear 5% to 20% sucrose gradient prepared in the same Tris buffer and centrifuged at 45,000 rpm for 15 h. After centrifugation 44 fractions were collected by puncturing the bottom of the tube. The distribution of radioactivity was measured by liquid scintillation counted on a Packard Tri-Carb Model 3380. [14C] bovine serum albumin was used to estimate the location of the 4S region of the gradient in control experiments. Tissue 17h-estradiol determinations were made in 200 Al of homogenized (approximately 30 mg of wet tissue) extracted with ether and measurements by radioimmunoassay by duplicate with commercial kits (cis Sorin, France). Results were expressed as femtomoles per milligram protein. Specific saturation data was plotted by the Scatchard method (Scatchard, 1949). Receptor levels were calculated by the graphical method of Rosenthal (Rosenthal, 1967) . Protein values were determined using Lowry method (Lowry et al., 1951). Results To establish the optimum conditions for the complex receptor formation analysis, the system was studied in different
Fig. 2. Analysis by sucrose gradient of receptors, type I and type II to 17hestradiol in endometrium non-receptive to blastocyst. The cytosol was prelabelled as is described in Fig. 1. Total incorporation of {3H}-17h-estradiol (•-----•), incorporation of {3H}-17h-estradiol in presence of 200 times the concentration molar of 17h-estradiol not labelled (°-----°).
E.L. Salazar, L. Calzada / Life Sciences 78 (2006) 2919 – 2922
Fig. 3. Analysis by sucrose gradient of receptors, type I and type II to 17hestradiol in uterine horn endometrium that it has been submitted to oophorectomy. The cytosol was prelabelled as is described in Fig. 1. Total incorporation of {3H}-17B-estradiol (•-----•), incorporation of {3H}-17hestradiol in presence of 200 times the molar concentration of 17h-estradiol not labelled (°-----° ).
temperatures (12 – 37 -C), we found that the maximum incorporation was at 25 -C. Representative data is presented in Figs. 1– 3 of three regions of the endometrium studied corresponding to the radioactivity profile in absence and presence of not labelled 17h-estradiol to 200 times the concentration of the labelled. Section A of each figure shows the incubation with 2 nM of {3H}-17h-estradiol at 4 -C, section B shows the incubation with 25 nM of {3H}-17h-estradiol at 25 -C. In the two analysis systems two radioactivity peaks clearly defined were observed, located in the areas 4S and 8S. All the cases studied in exchange conditions show a greater peak in the 4S region. This sucrose gradient analysis suggests the distribution and proportion between the two binding sites in the endometrium. The results of these experiments present a higher quantity of union sites in the BR 4S region, than in the BNR 4S region and UO endometrium, (section B of Figs. 2 and 3). On the other hand, a high content of binding sites was observed in the UO 8S region. At the same time, the binding of {3H}-17h-estradiol measured by dextran-coated charcoal method in the samples used for the sucrose gradient was 124, 79 and 182 fmol/mg of protein for BR, BNR and UO, respectively (section A of Figs. 1 –3). On the other hand, in exchange conditions (section B of the same figures) the results were 277, 129 and 426 fmol/mg protein, respectively too. Finally, the content of 17h-estradiol in the tissues studied was as follows: 1.42 T 0.16, 1.22 T 0.15 and 1.75 T 0.27 pmol/g of wet weight for BR, BNR and UO, respectively. These differences were significant only (Student’s t-test P < 0.05) when UO was compared to BNR. Discussion The functional difference between regularly spaced zones in the rat endometrium, at day five of pregnancy (Dey et al., 1991), that would permit the implantation of the blastocyst, could correspond, at least partially, to a concerted action between 17h-estradiol– progesterone at a receptor complex
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system (Heald, 1976). To that respect, the existence of two types of specific union sites for 17h-estradiol in the cytosol of the endometrium (Sanborn et al., 1971) has been suggested. Through sucrose gradient analysis, we observed two types of molecules in all samples of the endometrium. These two types of molecules (4S and 8S) are bound to the 17hestradiol, which is specifically analyzed for the study of the classic receptor (8S). Therefore our data agreed with the results cited by other authors for target tissue to 17hestradiol (Clark et al., 1978; Lopes et al., 1987). This study suggests different binding properties in the two types of receptors to 17h-estradiol between different zones of the endometrium. The significant differences in affinity and in union capacity to 17h-estradiol, analyzed by the method of Scatchard (Scatchard, 1949) and Rosenthal are also shown (Rosenthal, 1967). At the same time, the significance that the results present in UO endometrium (in ‘‘rest’’ condition, by the absence of blastocysts) shows receptors with greater specific activity and greater radioactivity peak in the areas 4S and 8S that the BR and BNR (by the presence of blastocysts) furthermore show an amount greater than that of 17h-estradiol (1.75 T 0.27 pmol/g of wet weight). The gradient concentration of 17h-estradiol in the same rat endometrium (same circulating level and free diffusion between the cells) is difficult to explain, but it could be thought that the UO in ‘‘rest’’ condition is depleting the steroid. Possibly due to not being translocated to the nucleus in the same proportion as in the endometrium when it is in contact with blastocysts. On the other hand, our results would seem opposite to the proposed (Dickmann et al., 1977) about the ER requiring additional quantities of 17h-estradiol, which would be supplied by the blastocyst to culminate the differentiation process. In our data we found that in absence of the blastocyst, the content of 17h-estradiol tissue is greater, possibly due to methodological differences. Our results suggest that the participation of estradiol in the determination of functional zones (BR and BNR) of the endometrium during early pregnancy, could be guided by a complex interaction of at least two union site types or receptor types in the cell, by a regulation through levels of steroids and by a distribution and interaction between these. There is still a lot to search about it. References Calzada, L., Martı´nez, M., Salazar, E.L., 1993. Evaluation of glaucolide receptor assays in human breast cancer tissues. Medical Science Research 21 (17), 645 – 646. Clark, J.H., Hardin, J.W., Upchurch, S., Eriksson, H., 1978. Heterogeneity of estrogen binding sites in the cytosol of the rat uterus. The Journal of Biological Chemistry 253 (21), 7630 – 7634. Dey, S.K., Paria, B.C., Andrews, G.K., 1991. In: Strauss III, J.F., Lyttle, C.R. (Eds.), Uterine and Embryonic Factors in Early Pregnancy. Press New York, pp. 51 – 69. Dickmann, Z., Dey, S.K., Gupta, J.S., 1976. A new concept: control of early pregnancy by steroid hormones originating in the preimplantation embryo. Vitamins and Hormones 34, 215 – 242.
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Dickmann, Z., Gupta, J.S., Dey, S.K., 1977. Does ‘‘blastocyst estrogen’’ initiate implantation. Science 195 (4279), 687 – 688. Heald, P.J., 1976. Biochemical aspects of implantation. Journal of Reproduction and Fertility 25, 29 – 52R (Suppl). Lopes, M.T., Liberato, M.H., Widman, A., Brentani, M.M., 1987. Occupied and unoccupied type II estrogen binding sites in human breast cancer. Journal of Steroid Biochemistry 26 (2), 219 – 226. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the folin phenol reagent. The Journal of Biological Chemistry 193, 265 – 275. Panko, W.B., Watson, C.S., Clark, J.H., 1981. The presence of a second, specific estrogen binding site in human breast cancer. Journal of Steroid Biochemistry 14 (12), 1311 – 1316.
Psychoyos, A., 1973. Hormonal control oovoimplantation. Vitamins and Hormones 31, 201 – 256. Rosenthal, H.H., 1967. A graphic method for the determination and presentation of binding parameters in a complex system. Biochemistry 6 (1), 525 – 532. Sanborn, B.M., Rao, B.R., Korenman, S.G., 1971. Interaction of 17-h-estradiol and its specific uterine receptor. Evidence for complex kinetic and equilibrium behaviour. Biochemistry 10 (26), 4955 – 4962. Scatchard, G., 1949. The attraction of proteins for small molecules and ions. Annals of the New York Academy of Science 51, 660 – 672. Yoshinaga, K., 1994. Endocrinology of implantation. In: Tulchinsky, D., Little, A.B. (Eds.), Maternal-Fetal Endocrinology. W.B. Saunders Company Philadelphia, Pennsylvania, pp. 335 – 349.
Endometrium estradiol receptors type I and type II during early pregnancy of rat Edith L. Salazar a,*, Leobardo Calzada b b
a Medical Research Unit in Endocrine Disease, Medical Research Coordination, Social Security Mexican Institute (IMSS), Mexico Health Center (T-III) Dr. Manuel Escontria, Sanitary Jurisdiction Alvaro Obregon of the Public Health Service of Distrito Federal, Mexico
Received 23 May 2005; accepted 15 November 2005
Abstract By centrifugation in a sucrose density gradient we studied the citosol 17h-estradiol binding sites of blastocyst receptive and non-receptive endometrial zones, as well as uterine horn endometrium whose ovary was extirpated three weeks before pregnancy. The cytosol was prelabelled with {3H}-17h-estradiol 2 and 25 nM. In this work two incubation temperatures were studied. On the other hand, at 4 -C unoccupied receptors were identified as different from the classic receptor 8S type I. At the same time, we found that 25 -C is the optimal temperature for the assay of total receptors to achieve complete exchange of {3H}-17h-estradiol by 17h-estradiol in the binding sites. In these conditions, the major component was the 4S type II receptor, mainly in the endometrium from ovariectomized uteri. Furthermore, 17h-estradiol content was determined in the total homogenized by radioimmunoassay and the results were: 1.42 T 0.16, 1.22 T 0.15 and 1.75 T 0.27 pmol/g wet tissue for receptive, non-receptive and ovariectomized uteri, respectively. D 2005 Elsevier Inc. All rights reserved. Keywords: Estradiol receptors; Endometrium; Sucrose gradient; Rats
Introduction Binding properties of two proteins to intracellular specific receptors to 17h-estradiol in different target tissue of rat and humans have been reported (Calzada et al., 1993; Clark et al., 1978; Panko et al., 1981). They are described as union site or receptors type I and type II, respectively. The type I corresponds to the classic receptor and is located in the region of 8S in a sucrose density gradient, characterized by having high affinity and low binding capacity; 4S type II has opposite properties. Furthermore it is not translocated (Lopes et al., 1987) and is identified more easily by exchange analysis, which consists on incubating at 25 -C in presence of 25 nM {3H}-17h-estradiol. At this temperature, the dissociation and reassociation of the receptor esteroid complex is more active and the {3H}-17hestradiol substitutes the endogenous 17h-estradiol (Sanborn et al., 1971). It has been suggested that this receptor would * Corresponding author. PO Box 86-056 Me´xico D.F. CP 14391. Tel./fax: +52 5588 7521. E-mail address: [email protected] (E.L. Salazar). 0024-3205/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2005.11.018
constitute a reserve mechanism of 17h-estradiol for the receptor type I (Lopes et al., 1987). In the oestrus cycle and pregnancy, the rat endometrium tissue shows functional changes in short periods of time in relation to ovarian steroid fluctuations. In day five of rat pregnancy, the endometrium presents regularly spaced zones that have undergone a series of biochemical changes (Dey et al., 1991; Heald, 1976), which permit the blastocyst implantation that will be identified easily in the fifth day, showing greater permeability to molecules of great size (trypan blue – albumin complex). These changes are part of the functional differentiation of those zones of the endometrium. The mechanism that begins and maintains this process is not known, although it is known that increasing levels of progesterone from the beginning of pregnancy and an estrogenic environment are required for implantation, at least from day three (Dickmann et al., 1976; Yoshinaga, 1994). On the other hand, it has been proposed that the endometrium requires additional quantities of estradiol which would be produced by the blastocyst (Dickmann et al., 1977) to complete the functional differentiation of the endometrium.
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Fig. 1. Analysis by sucrose gradient of receptors type I and type II to 17hestradiol in endometrium receptive to the blastocyst. The cytosol prelabelled using 2 and 25 nM of {3H}-17h-estradiol (•-----•) or the same concentration of {3H}-17h-estradiol more 200 times the molar concentration of 17h-estradiol not labelled (°-----° ), was incubated at 4 and 25 -C, respectively, during 2 h. They are applied on sucrose gradient and the distribution of radioactivity is shown in the figures.
The close vicinity between two functionally different zones [blastocyst receptive (BR) and non-receptive (BNR) endometrial zones] within a same estrogenic environment, possibly requires a receptor complex system to express selectively the target cell function (Psychoyos, 1973). In this article we propose to study the characteristics of the receptors to 17h-estradiol 8S type I and 4S type II (this last in exchange conditions) in the BR, BNR and endometrium of uterine horn submitted to unilateral oophorectomy (UO) previous to pregnancy, and the content of 17h-estradiol tissue in the same samples were determined. Material and methods Female rats of the Long Evans Strain of an average weight of 220 T 15 g and regular oestrus cycles were used under controlled light–dark conditions. Water and pellet food were supplied ad libitum. The rats were anesthetized with phenobarbital, unilateral oophorectomized and maintained for 21 days prior to study. The pregnant rats were obtained after housing the mature females in groups of two with one male of the same strain. Vaginal smears were taken daily. Day zero of pregnancy was designated when spermatozoa were found in the smear and then males were removed. On day five between 9 and 11 AM, a group of pregnant rats was injected (i.v.) 1 ml of 1% trypan blue in saline. Thirty minutes later, these rats were sacrificed by cervical dislocation, then the rats were perfused with saline solution and their uterus were obtained. In this experiment the blue-stained regions represented the implanted sites, whereas the non-stained tissue was considered as non-implanted regions (Heald, 1976; Sanborn et al., 1971). The samples were frozen in liquid nitrogen and stored. The uteruses were thawed at 4 -C, cut lengthwise and gently agitated with glass pearls to detach the epithelial cells. The cells were homogenized in a Tris buffer at 4 -C (Tris –HCl 10 mM, EDTA 1.5 mM, glycerol 10%, monothioglycerol 10 mM) in a proportion of 150 mg of wet
weight/ml using a glass – glass homogenizer (2 pulses, each for 30 s at 150 revolutions/min with intervals of 60 s). The homogenate was centrifuged for 10 min at 800 g. The supernatant was reserved and the pellet was washed two times with the same buffer and recentrifuged. Supernatants were collected and centrifuged for 30 min at 105,000 g to obtain the cytosol, as well as adjusted the protein content to 2– 4 mg protein /ml. Type I estrogen receptors were determined incubating 500 Al of cytosol for 2 h at 4 -C, with 2 nM or 25 nM of 3H-17h-estradiol. For the determination of type II estrogen receptors, identical batches were prepared and incubated for the same period of time at 25 -C. Non-specific binding was assayed using the same radioactive ligand plus a 200-fold excess of no radioactive ligand. Bound and free estradiol was separated using a dextran-coated charcoal separation method (Calzada et al., 1993). Five hundred microliters of prelabelled [3H-estradiol] cytosol were layered on a linear 5% to 20% sucrose gradient prepared in the same Tris buffer and centrifuged at 45,000 rpm for 15 h. After centrifugation 44 fractions were collected by puncturing the bottom of the tube. The distribution of radioactivity was measured by liquid scintillation counted on a Packard Tri-Carb Model 3380. [14C] bovine serum albumin was used to estimate the location of the 4S region of the gradient in control experiments. Tissue 17h-estradiol determinations were made in 200 Al of homogenized (approximately 30 mg of wet tissue) extracted with ether and measurements by radioimmunoassay by duplicate with commercial kits (cis Sorin, France). Results were expressed as femtomoles per milligram protein. Specific saturation data was plotted by the Scatchard method (Scatchard, 1949). Receptor levels were calculated by the graphical method of Rosenthal (Rosenthal, 1967) . Protein values were determined using Lowry method (Lowry et al., 1951). Results To establish the optimum conditions for the complex receptor formation analysis, the system was studied in different
Fig. 2. Analysis by sucrose gradient of receptors, type I and type II to 17hestradiol in endometrium non-receptive to blastocyst. The cytosol was prelabelled as is described in Fig. 1. Total incorporation of {3H}-17h-estradiol (•-----•), incorporation of {3H}-17h-estradiol in presence of 200 times the concentration molar of 17h-estradiol not labelled (°-----°).
E.L. Salazar, L. Calzada / Life Sciences 78 (2006) 2919 – 2922
Fig. 3. Analysis by sucrose gradient of receptors, type I and type II to 17hestradiol in uterine horn endometrium that it has been submitted to oophorectomy. The cytosol was prelabelled as is described in Fig. 1. Total incorporation of {3H}-17B-estradiol (•-----•), incorporation of {3H}-17hestradiol in presence of 200 times the molar concentration of 17h-estradiol not labelled (°-----° ).
temperatures (12 – 37 -C), we found that the maximum incorporation was at 25 -C. Representative data is presented in Figs. 1– 3 of three regions of the endometrium studied corresponding to the radioactivity profile in absence and presence of not labelled 17h-estradiol to 200 times the concentration of the labelled. Section A of each figure shows the incubation with 2 nM of {3H}-17h-estradiol at 4 -C, section B shows the incubation with 25 nM of {3H}-17h-estradiol at 25 -C. In the two analysis systems two radioactivity peaks clearly defined were observed, located in the areas 4S and 8S. All the cases studied in exchange conditions show a greater peak in the 4S region. This sucrose gradient analysis suggests the distribution and proportion between the two binding sites in the endometrium. The results of these experiments present a higher quantity of union sites in the BR 4S region, than in the BNR 4S region and UO endometrium, (section B of Figs. 2 and 3). On the other hand, a high content of binding sites was observed in the UO 8S region. At the same time, the binding of {3H}-17h-estradiol measured by dextran-coated charcoal method in the samples used for the sucrose gradient was 124, 79 and 182 fmol/mg of protein for BR, BNR and UO, respectively (section A of Figs. 1 –3). On the other hand, in exchange conditions (section B of the same figures) the results were 277, 129 and 426 fmol/mg protein, respectively too. Finally, the content of 17h-estradiol in the tissues studied was as follows: 1.42 T 0.16, 1.22 T 0.15 and 1.75 T 0.27 pmol/g of wet weight for BR, BNR and UO, respectively. These differences were significant only (Student’s t-test P < 0.05) when UO was compared to BNR. Discussion The functional difference between regularly spaced zones in the rat endometrium, at day five of pregnancy (Dey et al., 1991), that would permit the implantation of the blastocyst, could correspond, at least partially, to a concerted action between 17h-estradiol– progesterone at a receptor complex
2921
system (Heald, 1976). To that respect, the existence of two types of specific union sites for 17h-estradiol in the cytosol of the endometrium (Sanborn et al., 1971) has been suggested. Through sucrose gradient analysis, we observed two types of molecules in all samples of the endometrium. These two types of molecules (4S and 8S) are bound to the 17hestradiol, which is specifically analyzed for the study of the classic receptor (8S). Therefore our data agreed with the results cited by other authors for target tissue to 17hestradiol (Clark et al., 1978; Lopes et al., 1987). This study suggests different binding properties in the two types of receptors to 17h-estradiol between different zones of the endometrium. The significant differences in affinity and in union capacity to 17h-estradiol, analyzed by the method of Scatchard (Scatchard, 1949) and Rosenthal are also shown (Rosenthal, 1967). At the same time, the significance that the results present in UO endometrium (in ‘‘rest’’ condition, by the absence of blastocysts) shows receptors with greater specific activity and greater radioactivity peak in the areas 4S and 8S that the BR and BNR (by the presence of blastocysts) furthermore show an amount greater than that of 17h-estradiol (1.75 T 0.27 pmol/g of wet weight). The gradient concentration of 17h-estradiol in the same rat endometrium (same circulating level and free diffusion between the cells) is difficult to explain, but it could be thought that the UO in ‘‘rest’’ condition is depleting the steroid. Possibly due to not being translocated to the nucleus in the same proportion as in the endometrium when it is in contact with blastocysts. On the other hand, our results would seem opposite to the proposed (Dickmann et al., 1977) about the ER requiring additional quantities of 17h-estradiol, which would be supplied by the blastocyst to culminate the differentiation process. In our data we found that in absence of the blastocyst, the content of 17h-estradiol tissue is greater, possibly due to methodological differences. Our results suggest that the participation of estradiol in the determination of functional zones (BR and BNR) of the endometrium during early pregnancy, could be guided by a complex interaction of at least two union site types or receptor types in the cell, by a regulation through levels of steroids and by a distribution and interaction between these. There is still a lot to search about it. References Calzada, L., Martı´nez, M., Salazar, E.L., 1993. Evaluation of glaucolide receptor assays in human breast cancer tissues. Medical Science Research 21 (17), 645 – 646. Clark, J.H., Hardin, J.W., Upchurch, S., Eriksson, H., 1978. Heterogeneity of estrogen binding sites in the cytosol of the rat uterus. The Journal of Biological Chemistry 253 (21), 7630 – 7634. Dey, S.K., Paria, B.C., Andrews, G.K., 1991. In: Strauss III, J.F., Lyttle, C.R. (Eds.), Uterine and Embryonic Factors in Early Pregnancy. Press New York, pp. 51 – 69. Dickmann, Z., Dey, S.K., Gupta, J.S., 1976. A new concept: control of early pregnancy by steroid hormones originating in the preimplantation embryo. Vitamins and Hormones 34, 215 – 242.
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E.L. Salazar, L. Calzada / Life Sciences 78 (2006) 2919 – 2922
Dickmann, Z., Gupta, J.S., Dey, S.K., 1977. Does ‘‘blastocyst estrogen’’ initiate implantation. Science 195 (4279), 687 – 688. Heald, P.J., 1976. Biochemical aspects of implantation. Journal of Reproduction and Fertility 25, 29 – 52R (Suppl). Lopes, M.T., Liberato, M.H., Widman, A., Brentani, M.M., 1987. Occupied and unoccupied type II estrogen binding sites in human breast cancer. Journal of Steroid Biochemistry 26 (2), 219 – 226. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the folin phenol reagent. The Journal of Biological Chemistry 193, 265 – 275. Panko, W.B., Watson, C.S., Clark, J.H., 1981. The presence of a second, specific estrogen binding site in human breast cancer. Journal of Steroid Biochemistry 14 (12), 1311 – 1316.
Psychoyos, A., 1973. Hormonal control oovoimplantation. Vitamins and Hormones 31, 201 – 256. Rosenthal, H.H., 1967. A graphic method for the determination and presentation of binding parameters in a complex system. Biochemistry 6 (1), 525 – 532. Sanborn, B.M., Rao, B.R., Korenman, S.G., 1971. Interaction of 17-h-estradiol and its specific uterine receptor. Evidence for complex kinetic and equilibrium behaviour. Biochemistry 10 (26), 4955 – 4962. Scatchard, G., 1949. The attraction of proteins for small molecules and ions. Annals of the New York Academy of Science 51, 660 – 672. Yoshinaga, K., 1994. Endocrinology of implantation. In: Tulchinsky, D., Little, A.B. (Eds.), Maternal-Fetal Endocrinology. W.B. Saunders Company Philadelphia, Pennsylvania, pp. 335 – 349.