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Autoradiogsaphic study of (6.7-3H)oestradiol-17β incorporation into rat uterus
661
AUTORADIOGRAPHICSTUDY OF (6,7-3H)OESTPADIOL-17~ INCORPORATIONINTO RAT UTERUS.
And&s
TchernitchinV.
Institute of Physiology, University of Chile, Santiago Chile Obstetrical Clinic, J. J. Aguirre Hospital, University of Chile Received April 4, 1967 ABSTRACT
(6,7-3H) oestradiol-17)3incorporationby the rat uterus has been studied using autoradiography. The cytoplasm and/or the cellular membrane of uterine eosinophilic cells seems to incorporate oestradiol in a much greater quantity than the rest of the uterus. The characteristicsand localization of these cells are described. Oestradiol incorporation is not modified by changes in incubation temperature (4O or 37OC), by the presence or absence of glucose in the incubation medium, nor by the presence or absence of oxygen. Fixation in formalin, prior to incubation, does not alter labeling.
After systemic injection of (6,7-3H) oestradiol-17p , rat uterus retains oestrogens for a considerable period. The greater part (if not all) of the retained material is 17P
- oes-
tradiol 1*2. It was suggested' that the oestrogens receptor is in the non epithelial cells. King and Gordon3 demonstratedby scintillation counting that there was no difference between the concentration of oestradiol in the epithelium as compared to the rest of the uterus. They also found that there was oestradiol exchange between the nucleus and cytoplasm and that this exchange favors cytoplasm. A major part of the epithelial oestradiol was in the soluble fraction, although there was also radioactivity associated to the nucleus. host of the oestradiol of the soluble fraction was associated with a compound of high molecular weight, possibly a protein.
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Nothing is known about non-epithelial uterine oestradiol. Inman, Banfield and King'l,using autoradiographsfollowing injection of tritiated oestradiol to rats "in viva", found autoradiographic reaction in the nuclear membrane of the eosinophils of the anterior pituitary and in the cytoplasm of hepatic cells, but did not obtain any satisfactory results in the uterus. The aim of this paper is to study the incorporation and localization of tritiated oestradiol in the uterus, using autoradiographic methods in an "in vitro" system. Artefacts were kept to a minimum by incubation of cryostat sections that avoids oestradiol displacement or extraction brought about by organic solvent in standard tissue preparation.
MATERIAL AND METHODS _Animals:- Female albino rats weighing around 200 grs. bred in the Institute of Physiology, were used. The stage of oestral cycle was recorded through vaginal smears. &7-3H)Oestradiol-17fi :- Stock solution:- 1.0 millicurics: 0.00714 mg. in 1.0 ml 10% methanol-benzene(New England Nuclear Corp.) . Specific activity 38.1 Curies/millimole.Incubation media: were prepared starting from the stock solution diluted in ethanol (1:lO). This was again diluted in phosphate buffer at pH 7.4 with or without 0.1% glucose in three different concentrations of oestradiol: sol. A - 2.5 microcurie/ml. so1.B - 0.25 microcurie/ml, sol.C-O.025 microcurie/ml. IIcubation:- Preliminary experiments, in which l-2 mm sections of uterus were incubated with tritated oestradiol, gave insatisfactory results as only certain cells on the surface of the section showed incorporation. In view of this finding we adopted a method using thin cryostat 14 micra sections, as employed for assay of cell enzyme activity5. Rats were sacrificed by decapitation, and the uterus were immediately frozen in dry ice and cut in a cqtostat (-2O'C) at 14 micra. The fresh frozen sections were placed on glass slides and then kept at 37°C. 0.1 ml of the incubation solution was dropped
Dec. 1967
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663
over the tissue section. Time of incubation was 10 min at 37OC and a relative humidity of 100% (in order to avoid evaporation). Then, thoroughly washed in distilled water and dried at 37OC. Autoradiography:- Tissue sections were dipped in Ilford G-5 photographic emulsion diluted 2:l and placed at 40°C, dried and kept in light tight boxes at 20*C. Times of exposure were 5, 15 or 45 days and then were developed in D-19 (Ilford) for 5 minutes at 20°C, fixed and maintained in distilled water 30 minutes until posterior staining with either eosine-hematoxiline,Toluidine blue or Maximow. Autoradiographywas performed for the following groups of experiments: 1 .- Three different concentrationsof radioactive oestradiol for incubation. 2.- Rats in proestrus, estrus, diestrus; prepuber, castrated or pregnant rats. 3.- Fixation in formalin of the sections prior to incubation, or without fixation. 4.- Incubation with or without glucose. 5.- Incubation in air or in nitrogen atmosphere. 6.- Different incubation temperatures (4O or 37OC). me Histochemistry:-In some experiments, histochemical tests were also carried out for the demonstrationof the following dehydrogenases: Lactic and succinic dehydrogenase,glucose-6-phosphate dehydrogenase and 3P-hydroxysteroid dehydrogenase5.In these experiments, the (6,7-3H) oestradiol-17/jsolutions A, B or C were added 1:l to the enzyme substrate incubation media to determine its incorporationby the areas showing enzymatic activity.
RESULTS _In all the uterine sections of all the studied rats there are cells whose cytoplasm and/or cytoplasmaticmembrane incorporate (6,7-3H)oestradiol-17fi in a considerably greater proportion than the rest of the uterus. (Figs. 2,3,4). Resolution of the method does not allow the discriminationof the precise sites of incorporation in the cytoplasm. However the distribution of the autoradiographic granules suggests a preferential localization on the cell surface (Figs. 3,4). No nuclear labeling was detected. The labeled cells can be identified as eosinophils, and have the following characteristics:a) Intensely eosinophilic and
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slightly granulated, rounded or irregular cytoplasm. b) A big, round and sometimes multiilobulatednucleus (Fig. 4). They contain orthochromatic granules that stain with Toluidine blue and were negative to enzymatic reactions for lactic and succinic dehydrogenase,glucose6-phosphate dehydrogenase and 3/j -hydroxysteroiddehydrogenase. These cells are found predominantly in the deep chorion of the mucose and in the connective tissue between the muscular layers; a few cells are also seen between muscular fibres, outside the muscular layer or in some other localization (Fig. 1). They gradually decrease in number towards the uterine cervix at which level they totally disappear. Changes in incubation temperature (37O or 4" C), the presence or absence of glucose in the incubation medium or changes in the composition of the incubation atmosphere (20% oxygen or pure nitrogen), seem to have no significant influence in the tritiated oestradiol incorporation.It is particularly interesting that fixation of the section in 10% neutral formalin during lo-15 minutes, prior to incubation, does not seem to modify incorporation. Incorporationof oestradiol also varies, for each of these cells, according to the concentration of radioactive oestradiol in the in the incubation solution (Fiq. 2 and 3). XSCUSSIC,r: This paper shows that in the deep chorion and in the connective tissue of the uterus, exist cells with eosinophilic properties whose cytoplasm and/or cytoplasmaticmembrane incorporates (6,7-3H)oestradiol-17,fi . Stone and Baggett6, using scintillationcounting, found that incorporationof tritiated oestradiol by the uterus does not
STEROIDS
Dec. 1967
change significantlyif preincubation is made with or without cose
or
665
glu-
in an oxygen or nitrogen atmosphere - a fact confirmed by
us in this work - and they conclude that oestradiol incorporation by its receptors would be a process not requiring energy. Further support for this conclusion comes from the absence of temperature effect observed by us (4O or 37°C). Finding that previous fixation with formalin does not modify incorporation,would suggest that incorporation does not depend on an enzymatic process. Stone and Baggett6 found, through sciytll:.ationcounting, that treatment of the animal with oestradiol, 12-36 hrs before incubation with radioactive estrogen, increases incorporationof the latter. This might be related to a variation of the number of cells that incorporate oestrogen in accordance with the hormonal state of the animal. This hypothesis is under study. It has been found that the antioestrogendimethylstilbestrol inhibits oestradiol incorporation in vivo6, and that in rat previously treated with clomiphene, this agent seems to compete with the natural oestrogen for this receptor located in the uterus and pituitary, preventing oestrogen fixation and probably displacing it from the receptive zones7. Preliminary results obtained by us strongly suggest that incorporationby the eosinophilic cells depends on the animal oestrogen contents. This would suggest that (6,7-3H)oestradiol-17/3 competes for receptors with oestrogens produced by the animal or with estrogenic or antiestrogenicsubstances administered to it. This will be explained in future work.
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Fig. 1. Cross section of rat uterus in diestrus incubated with 2.5 microcuries/mlof (6,7-3H) oestradiol-17 . Each black dot correspond& to a number of silver grains over one eosinophilic cell. MagnificationX 72.
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Fig. 2. Autoradiographyof cells showing incorporationof oestrogen in the deep chorion of rat uterus. Experimental conditions as above. The heavy labeling masks cell structural details. Hagnification x 420.
Fig. 3. Incorporationof oestrogen in the deep chorion of rat uterus. The incubation was in solution C (0.025 microcuries/ml of . Some silver grains are located in the (6,7-3H) oestradiol-17 cell surface (arrow). Magnification x 720.
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Fig. 4. Three micrographs of representativeeosinophilic cells showing cell morphology and silver grains localization. Magnification x 1800.
REFERENCES
1 .- JEXSEZT,E. V. and JACOBSfIN,H.X. - Recent Prog. Harm. Res. 1962, la& 387-414. 2.- KING, R.J.B., GORDON, J. and INMAN, D. R. - J. Ehdocrin. 1965, 32, 9-15. 3.- KING, R.J.B. and GORDON, J. - J. Endocrin. 1966, 34, 431-437. 4 .- INMAN, D. R., BWIELD, 1965, 32, 17-22.
R.E.W. and KING, R.J.B. - J. Endocrin.
5.- PUPKIN, M., BUTT, M., WEISZ, J., LLOYD, C.W., BALOGH, K. Endocrinology,1966, 79, 316-327. 6 .- STONE, G.M., and BAGGETT, B. - Steroids, 1965, 2, 809-826. 7 .- ROY, S., MAHESH, V.B., GREENBLATT, R.B. - Acta endocrin, 1964, 2, 669-675.
AUTORADIOGRAPHICSTUDY OF (6,7-3H)OESTPADIOL-17~ INCORPORATIONINTO RAT UTERUS.
And&s
TchernitchinV.
Institute of Physiology, University of Chile, Santiago Chile Obstetrical Clinic, J. J. Aguirre Hospital, University of Chile Received April 4, 1967 ABSTRACT
(6,7-3H) oestradiol-17)3incorporationby the rat uterus has been studied using autoradiography. The cytoplasm and/or the cellular membrane of uterine eosinophilic cells seems to incorporate oestradiol in a much greater quantity than the rest of the uterus. The characteristicsand localization of these cells are described. Oestradiol incorporation is not modified by changes in incubation temperature (4O or 37OC), by the presence or absence of glucose in the incubation medium, nor by the presence or absence of oxygen. Fixation in formalin, prior to incubation, does not alter labeling.
After systemic injection of (6,7-3H) oestradiol-17p , rat uterus retains oestrogens for a considerable period. The greater part (if not all) of the retained material is 17P
- oes-
tradiol 1*2. It was suggested' that the oestrogens receptor is in the non epithelial cells. King and Gordon3 demonstratedby scintillation counting that there was no difference between the concentration of oestradiol in the epithelium as compared to the rest of the uterus. They also found that there was oestradiol exchange between the nucleus and cytoplasm and that this exchange favors cytoplasm. A major part of the epithelial oestradiol was in the soluble fraction, although there was also radioactivity associated to the nucleus. host of the oestradiol of the soluble fraction was associated with a compound of high molecular weight, possibly a protein.
STEROIDS
662
lo:6
Nothing is known about non-epithelial uterine oestradiol. Inman, Banfield and King'l,using autoradiographsfollowing injection of tritiated oestradiol to rats "in viva", found autoradiographic reaction in the nuclear membrane of the eosinophils of the anterior pituitary and in the cytoplasm of hepatic cells, but did not obtain any satisfactory results in the uterus. The aim of this paper is to study the incorporation and localization of tritiated oestradiol in the uterus, using autoradiographic methods in an "in vitro" system. Artefacts were kept to a minimum by incubation of cryostat sections that avoids oestradiol displacement or extraction brought about by organic solvent in standard tissue preparation.
MATERIAL AND METHODS _Animals:- Female albino rats weighing around 200 grs. bred in the Institute of Physiology, were used. The stage of oestral cycle was recorded through vaginal smears. &7-3H)Oestradiol-17fi :- Stock solution:- 1.0 millicurics: 0.00714 mg. in 1.0 ml 10% methanol-benzene(New England Nuclear Corp.) . Specific activity 38.1 Curies/millimole.Incubation media: were prepared starting from the stock solution diluted in ethanol (1:lO). This was again diluted in phosphate buffer at pH 7.4 with or without 0.1% glucose in three different concentrations of oestradiol: sol. A - 2.5 microcurie/ml. so1.B - 0.25 microcurie/ml, sol.C-O.025 microcurie/ml. IIcubation:- Preliminary experiments, in which l-2 mm sections of uterus were incubated with tritated oestradiol, gave insatisfactory results as only certain cells on the surface of the section showed incorporation. In view of this finding we adopted a method using thin cryostat 14 micra sections, as employed for assay of cell enzyme activity5. Rats were sacrificed by decapitation, and the uterus were immediately frozen in dry ice and cut in a cqtostat (-2O'C) at 14 micra. The fresh frozen sections were placed on glass slides and then kept at 37°C. 0.1 ml of the incubation solution was dropped
Dec. 1967
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663
over the tissue section. Time of incubation was 10 min at 37OC and a relative humidity of 100% (in order to avoid evaporation). Then, thoroughly washed in distilled water and dried at 37OC. Autoradiography:- Tissue sections were dipped in Ilford G-5 photographic emulsion diluted 2:l and placed at 40°C, dried and kept in light tight boxes at 20*C. Times of exposure were 5, 15 or 45 days and then were developed in D-19 (Ilford) for 5 minutes at 20°C, fixed and maintained in distilled water 30 minutes until posterior staining with either eosine-hematoxiline,Toluidine blue or Maximow. Autoradiographywas performed for the following groups of experiments: 1 .- Three different concentrationsof radioactive oestradiol for incubation. 2.- Rats in proestrus, estrus, diestrus; prepuber, castrated or pregnant rats. 3.- Fixation in formalin of the sections prior to incubation, or without fixation. 4.- Incubation with or without glucose. 5.- Incubation in air or in nitrogen atmosphere. 6.- Different incubation temperatures (4O or 37OC). me Histochemistry:-In some experiments, histochemical tests were also carried out for the demonstrationof the following dehydrogenases: Lactic and succinic dehydrogenase,glucose-6-phosphate dehydrogenase and 3P-hydroxysteroid dehydrogenase5.In these experiments, the (6,7-3H) oestradiol-17/jsolutions A, B or C were added 1:l to the enzyme substrate incubation media to determine its incorporationby the areas showing enzymatic activity.
RESULTS _In all the uterine sections of all the studied rats there are cells whose cytoplasm and/or cytoplasmaticmembrane incorporate (6,7-3H)oestradiol-17fi in a considerably greater proportion than the rest of the uterus. (Figs. 2,3,4). Resolution of the method does not allow the discriminationof the precise sites of incorporation in the cytoplasm. However the distribution of the autoradiographic granules suggests a preferential localization on the cell surface (Figs. 3,4). No nuclear labeling was detected. The labeled cells can be identified as eosinophils, and have the following characteristics:a) Intensely eosinophilic and
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slightly granulated, rounded or irregular cytoplasm. b) A big, round and sometimes multiilobulatednucleus (Fig. 4). They contain orthochromatic granules that stain with Toluidine blue and were negative to enzymatic reactions for lactic and succinic dehydrogenase,glucose6-phosphate dehydrogenase and 3/j -hydroxysteroiddehydrogenase. These cells are found predominantly in the deep chorion of the mucose and in the connective tissue between the muscular layers; a few cells are also seen between muscular fibres, outside the muscular layer or in some other localization (Fig. 1). They gradually decrease in number towards the uterine cervix at which level they totally disappear. Changes in incubation temperature (37O or 4" C), the presence or absence of glucose in the incubation medium or changes in the composition of the incubation atmosphere (20% oxygen or pure nitrogen), seem to have no significant influence in the tritiated oestradiol incorporation.It is particularly interesting that fixation of the section in 10% neutral formalin during lo-15 minutes, prior to incubation, does not seem to modify incorporation. Incorporationof oestradiol also varies, for each of these cells, according to the concentration of radioactive oestradiol in the in the incubation solution (Fiq. 2 and 3). XSCUSSIC,r: This paper shows that in the deep chorion and in the connective tissue of the uterus, exist cells with eosinophilic properties whose cytoplasm and/or cytoplasmaticmembrane incorporates (6,7-3H)oestradiol-17,fi . Stone and Baggett6, using scintillationcounting, found that incorporationof tritiated oestradiol by the uterus does not
STEROIDS
Dec. 1967
change significantlyif preincubation is made with or without cose
or
665
glu-
in an oxygen or nitrogen atmosphere - a fact confirmed by
us in this work - and they conclude that oestradiol incorporation by its receptors would be a process not requiring energy. Further support for this conclusion comes from the absence of temperature effect observed by us (4O or 37°C). Finding that previous fixation with formalin does not modify incorporation,would suggest that incorporation does not depend on an enzymatic process. Stone and Baggett6 found, through sciytll:.ationcounting, that treatment of the animal with oestradiol, 12-36 hrs before incubation with radioactive estrogen, increases incorporationof the latter. This might be related to a variation of the number of cells that incorporate oestrogen in accordance with the hormonal state of the animal. This hypothesis is under study. It has been found that the antioestrogendimethylstilbestrol inhibits oestradiol incorporation in vivo6, and that in rat previously treated with clomiphene, this agent seems to compete with the natural oestrogen for this receptor located in the uterus and pituitary, preventing oestrogen fixation and probably displacing it from the receptive zones7. Preliminary results obtained by us strongly suggest that incorporationby the eosinophilic cells depends on the animal oestrogen contents. This would suggest that (6,7-3H)oestradiol-17/3 competes for receptors with oestrogens produced by the animal or with estrogenic or antiestrogenicsubstances administered to it. This will be explained in future work.
STEROIDS
666
lo:6
Fig. 1. Cross section of rat uterus in diestrus incubated with 2.5 microcuries/mlof (6,7-3H) oestradiol-17 . Each black dot correspond& to a number of silver grains over one eosinophilic cell. MagnificationX 72.
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Fig. 2. Autoradiographyof cells showing incorporationof oestrogen in the deep chorion of rat uterus. Experimental conditions as above. The heavy labeling masks cell structural details. Hagnification x 420.
Fig. 3. Incorporationof oestrogen in the deep chorion of rat uterus. The incubation was in solution C (0.025 microcuries/ml of . Some silver grains are located in the (6,7-3H) oestradiol-17 cell surface (arrow). Magnification x 720.
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Fig. 4. Three micrographs of representativeeosinophilic cells showing cell morphology and silver grains localization. Magnification x 1800.
REFERENCES
1 .- JEXSEZT,E. V. and JACOBSfIN,H.X. - Recent Prog. Harm. Res. 1962, la& 387-414. 2.- KING, R.J.B., GORDON, J. and INMAN, D. R. - J. Ehdocrin. 1965, 32, 9-15. 3.- KING, R.J.B. and GORDON, J. - J. Endocrin. 1966, 34, 431-437. 4 .- INMAN, D. R., BWIELD, 1965, 32, 17-22.
R.E.W. and KING, R.J.B. - J. Endocrin.
5.- PUPKIN, M., BUTT, M., WEISZ, J., LLOYD, C.W., BALOGH, K. Endocrinology,1966, 79, 316-327. 6 .- STONE, G.M., and BAGGETT, B. - Steroids, 1965, 2, 809-826. 7 .- ROY, S., MAHESH, V.B., GREENBLATT, R.B. - Acta endocrin, 1964, 2, 669-675.