Brain Research. 548 (1991) 50--54 © 1991 Elsevier Science Publishers B.V. 0006-8993/91/$1)3.50 ADONIS 11006899391165587
50
BRES 16558
The effect of estrogen on the estrogen receptor-immunoreactive cells in the rat medial preoptic nucleus Kazunari Yuri and Mitsuhiro Kawata Department of Anatomy, Kyoto Prefectural University of Medicine, Kyoto (Japan) (Accepted 20 November 1990)
Key words: Estrogen receptor; Immunohistochemistry; Medial preoptic nucleus; Estradiol; Ovariectomy
The localization of estrogen receptor-immunoreactive (ER-IR) cells in the medial preoptic nucleus (MPN) and the effect of estrogen upon these cells were quantitatively analyzed by immunohistochemistry using a monoclonal antibody to estrogen receptor (ER) protein in rat. ER was distributed in the nucleus of MPN neurons and was not detected in either cytoplasm or glial cells. There were more ER-IR cells with higher immunoreactivities in ovariectomized (OVX) rats than in estradiol (E2)-treated rats. The number of ER-IR cells in E2-treated compared with OVX rats was reduced by 43%. In particular, the number of ER-IR cells in the central part of the MPN was largely decreased. Our data indicate that the central part of the MPN is most sensitive to estrogen.
INTRODUCTION It has b e e n elucidated by in vivo autoradiography that the brains of vertebrates including mammals have estrogen concentrating neurons. These n e u r o n s are mainly located in the preoptic area, hypothalamus and central grey of the midbrain, and form a n e u r o n a l network related to the sexual behavior of the animal 16'19"2°. Recently, a m o n o c l o n a l antibody to estrogen receptor (ER) protein has b e e n raised ~° and immunohistochemical studies using this antibody have shown that the distribution of estrogen receptor-immunoreactive ( E R - I R ) cells was almost identical to that of autoradiography using radiolabeled estrogen 3"13'21. The medial preoptic nucleus (MPN) exhibits sexual dimorphism in the brain and detailed research has been performed with regard to sex differences in the size of the nucleus t'6, the n u m b e r , size and shape of n e u r o n s 7 and in the distribution of active substances such as amines 4"17"18 and peptides 22'23 in afferent fibers in the MPN. However, the distribution of E R in the MPN in different hormonal circumstances has hitherto not been investigated. In the present study, the influence of estrogen o n the MPN and its subnuclei in the ovariectomized (OVX) rat was quantitatively analyzed using an a n t i - E R monoclonal antibody.
MATERIALS AND METHODS
Animals
Wistar rats (virgin female, n = 5, not including 28 female rats for preliminary test) weighing 150-200 g were ovariectomized (OVX) while anesthetized with sodium pentobarbital, housed at 20 ~C and maintained on a 12.00 h/12.00 h light/dark cycle for 4 weeks before use. Food and water were available ad libitum.
Hormone administration To study the effect of estrogen on brain ER,IR cells, 5 0 V X animals were divided into two groups. The estradiol (E2)-treated group (n = 3) was injected subcutaneously with 30 /~g of 17restradiol (Nakarai, Kyoto, Japan) in 0.3 ml sesame oil and the remaining (OVX rats, n = 2) received 0.3 ml vehicle only.
Brain preparation Twenty-four hours after the administration of E 2 or vehicle, the animals were anesthetized with sodium pentobarbital and peffused with 50 ml phosphate-buffered saline (PBS, pH 7.2) followed by 600 ml ice-cold fixative containing 4% paraformaldehyde and 0.2% picric acid in 0.1 M phosphate buffer (pH 7.2). Brains were removed, postfixed with the same fixative for 24 h and transferred to 20% sucrose for 48 h. The brain blocks were sectioned into 20 ~m thick slices by cryostat at -20 °C and stored in PBS containing 0.3% Triton X-100 at 4 °C for 24 h for immunohistochemistry.
Immunohistochemicalprocedures The sections from all the brains were incubated simultaneously in the following antisera. Free-floating sections were immersed in 1% normal goat serum to decrease non-specific staining. The sections were incubated in anti-ER monoclonal antibody (Abbott, Chicago, IL, working dilution 1:20) for 168 h at 4 °C, immersed in anti-rat IgG (KPL, Gaithersburg, MD, working dilution 1:100) for 2 h, then PAP complex (Polysciences, Warrington, PA, working dilution
Correspondence: K. Yuri, Department of Anatomy, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602, Japan. Fax: (81) (75) 211-7093.
51
'OC
Fig. 1. Schematic drawing of the MPN for counting cells in a specified area. Grids were set on the MPN and the ER-IR cells were counted. Each grid has a 50-/~m side. AC: anterior commissure, OC: optic chiasm, MPN: medial preoptic nucleus, V: third ventricle.
Fig. 2. Immunoreactivity was recognized within the nucleus of MPN neurons. The ER-IR cell is indicated by arrowheads. C: cytoplasm, N: nucleus, bar = 10 ~m.
1:300) for 3 h at room temperature. The sections were reacted with 3,3'-diaminobenzidine (DAB) (Dojin, Kumamoto, Japan) intensifled by nickel: 20 mg DAB and 80 mg nickel (II)-ammonium sulphate, hexahydrate (Nakalai tesque, Kyoto, Japan) in 100 ral of 0.05 M Tris-HCl buffer containing 7 /~l of 30% H202. Primary antibody was omitted in some sections to check the non-specific reaction.
Fig. 3. ER-IR cells were elucidated in the MPN of the OVX group (A) and E:-treated group (B). Immunoreactivity was stronger in the OVX group than in the hormone-treated group. Boundary of the MPN is indicated by a broken line. Bar = 100/~m.
52
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Fig. 5. The number of ER-IR cells in the MPN was 1286 + 79/mm~ in the OVX group and 751 +_ 58/mm~ in the E2-treated group. Values (mean _+ S.E.M.) represent significant differences between the OVX group and E2-treated group (*P < 0.01, Student's t-test).
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Fig. 4. The central part of MPN was intensely immunostained in the OVX group (A) but not in the E2-treated group (B). Bar = 30/~m.
Both in the O V X and E2-treated rats, E R - I R cells were observed in the p r e o p t i c area which includes not only the M P N but also the periventricular preoptic nucleus and areas b e y o n d the MPN. T h e r e was no
Quantitative analysis
difference in the total area of the M P N b e t w e e n the O V X
A comparison was made between OVX and hormone treatment groups in 6 sets of equivalent sections. These sections were selected according to the criteria which were based on the shape of the anterior commissure, the optic chiasm and the third ventricle. Adjacent sections were Nissl-stained with Cresyl violet (Chroma, Stuttgart, F.R.G.); the sections were immersed into this solution for 10 rain at 36 °C. Immunohistochemical sections were photographed and the MPN boundary was defined by the adjacent Nissl-stained section. The number of ER-IR cells within the MPN was counted and the area of the MPN was measured by an image analyser MOP-1 (Kontron, Munich, F.R.G. ). Four sets of equivalent sections including the central part of the MPN were used for further analysis of the MPN; grids were prepared and set on the side of the third ventricle 500 gm above the bottom of the ventricle. The grids consisted of 6 x 14 = 84 square grids and each grid corresponded to a 50 x 50 gm section of the brain (Fig. 1). The number of ER-IR cells was counted in each grid, and a histogram constructed. All the sections were coded to prevent experimental bias during cell count analysis. The MPN was subdivided according to the classification and nomenclature of Simerly et al. 17. Immunohistochemical data were analysed using the Student t-test and differences were considered significant,
and E2-treated rats: the average area was 0.21 m m 2. The size of M P N neurons (the m a j o r axis) was 15.3 + 0.6 p m (mean + S . E . M . ) in the O V X and 17.2 _+ 0.7/~m (mean __- S . E . M . ) in the E2-treated rats. N e i t h e r d e g e n e r a t i o n nor abnormalities were o b s e r v e d in the neurons. In the M P N , E R i m m u n o r e a c t i v i t y was o b s e r v e d within the nucleus of neurons (Fig. 2), but not in the cytoplasm nor in any glial cells. E R - I R cells were m o r e i m m u n o r e a c t i v e in the O V X than in the E2-treated rats (Fig. 3 A , B ) . This tendency was a feature of the central part of the M P N (Fig. 4 A , B ) . The n u m b e r of E R - I R cells in the M P N was 1286 +_ 79/mm 2 (mean +_ S . E . M . ) in the O V X and 751 + 58/ram 2 (mean + S . E . M . ) in the E2-treated rats, indicating a 43% reduction in E R - I R cells by E 2 (Fig. 5). Cell counts revealed that the largest n u m b e r of E R - I R cells was in a grid 150-200 g m from the third ventricle in the O V X ,
53
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Fig. 6. The number of ER-IR cells counted in the grid presented in Fig. 1, was 7.2/grid 150-200/am from the ventricle in the OVX group (A), however, this peak significantlydecreased to 3.3/grid in the E2-treated group (B). *P < 0.05, **P < 0.01, values that are significantly different from those in the OVX group (Student's t-test),
which corresponds to the central part of the MPN. There was a gradual decrease in number of ER-IR cells toward the lateral end of the MPN. The number of ER-IR cells in the E2-treated rats was lower than that of the OVX and there was no conspicuous increase in number, particularly in the central part of the MPN. It was found that the number of ER-IR cells in the central part of the MPN of the OVX was 7.2 + 1.0/grid (mean + S.E.M.), compared with 3.3 _+ 0.5/grid (mean _+ S.E.M.) in the E2-treated rats. This was a statistically (P < 0.05) significant decrease (Fig. 6A,B). DISCUSSION The monoclonal antibody to E R was prepared from the human breast cancer cell line MCF-7 ~° and it has been widely used to elucidate the localization of ER in many organs including the central nervous system in mammals, The results have been consistent 3'13'21. We examined the specificity of this antibody and ascertained that it reacted
with E R specifically. In the present study, the effects of E 2 on OVX rat brains were quantitatively analyzed with the monoclonal antibody. Intact female rats should not be used as a control because the plasma E 2 level changes during the estrous cycle2. Immunoreactivity was observed in the nucleus of the neurons in the preoptic area including the MPN. There have been many debates as to whether the E R was localized in the cytoplasm or in the nucleus 9'24. Recent immunohistochemical studies have indicated that ER exists in the nucleus of target cells 3'12'13'21. In this study, we observed immunoreactivity only in the nucleus of the MPN neuron (Fig. 2). The loss of E R or other constituents in the section might be possible during 168 h incubation in the primary antibody, however, it is not related to antigenicity of E R because we observed stronger immunoreactivity in the 168-h incubated section than that of 48 h or 120 h in the preliminary studies. ER-IR cells were detected in 10year-old sections of the peripheral tissue 11. Both the number of ER-IR cells and the intensity of immunoreactivity in the MPN decreased in the E2-treated rats (Figs. 3 and 4). These observations were consistent in our preliminary studies directed at optimizing the immunohistochemical method using 28 female rats. At present, there are two explanations for these phenomena 3. One is that the reduction in the E R number was caused by the E2 treatment and the other is that a decrease in antigenicity of the E R occurred because it was transformed into a different three-dimensional structure after coupling with E 2. Immunoassay has demonstrated that the volume of E R in the preoptic area decreases after E 2 treatment 15 and in situ hybridization histochemistry has revealed that E R mRNA levels are also reduced TM. Therefore, a decrease in E R mRNA production after E2 treatment may cause the reduction in E R number. Further molecular biological studies will be required to solve the problem completely. The injection of 30/~g of E 2 induces a high s e r u m E 2 level and it is sufficient to saturate the E R in the neurons. A twenty-four-hour interval after injection of E 2 was appropriate because it was reported that the effect of E 2 on the neurons was strongest at 24 h 5'14. The MPN is known to be sexually dimorphic. Sex steroids affect the size and number of neurons that constitute this n u c l e u s 6'7. T h e MPN consists of 3 parts: a medial, central and lateral part 17. Of these 3, the central part corresponds to the sexually dimorphic nucleus of the preoptic area which was proposed by Gorski et al. and is thought to be the most sensitive to sex steroids 7A7. In the present study, ER-IR cells decreased significantly in the central part (Fig. 6A,B), indicating that it is definitely the part that is most sensitive to estrogen treatment.
54
REFERENCES 1 Bleier, R., Byne, W. and Siggelkow, I., Cytoarchitectonic sexual dimorphisms of the medial preoptic and anterior hypothalamic areas in guinea pig, rat, hamster, and mouse, J. Comp. Neurol., 212 (1982) 118-130. 2 Brown-Grant, K., Exley, D. and Naftolin, E , Peripheral plasma oestradiot and luteinizing hormone concentrations during the oestrous cycle of the rat, J. Endocrinol., 48 (1970) 295-296. 3 Cintra, A., Fuxe, K., H~irfstrand, A., Agnati, L.E, Miller, L.S., Greene, J.L. and Gustafsson, J.-A., On the cellular localization and distribution of estrogen receptors in the rat tel- and diencephalon using monoclonal antibodies to human estrogen receptor, Neurochem. Int., 8 (1986) 587-595. 4 Commins, D. and Yahr, P., Acetylcholinesterase activity in the sexually dimorphic area of the gerbil brain: sex differences and influences of adult gonadal steroids, J. Comp. Neurol., 224 (1984) 123-131. 5 Giordano, A.L., Siegel, H.I. and Rosenblatt, J.S., Nuclear estrogen binding in the preoptic area and hypothalamus of pregnancy-terminated rats: correlation with the onset of maternal behavior, Neuroendocrinology, 50 (1989) 248-258. 6 Gorski, R.A., Gordon, J.H., Shryne, J.E. and Southam, A.M., Evidence for a morphological sex difference within the medial preoptic area of the rat brain, Brain Research, 148 (1978) 333-346. 7 Gorski, R.A., Harlan, R.E., Jacobson, C.D., Shryne, J.E. and Southam, A.M., Evidence for the existence of a sexually dimorphic nucleus in the preoptic area of the rat, J. Comp. Neurol., 193 (1980) 529-539. 8 Omitted. 9 Gorski, J., Welshons, W. and Sakai, D., Remolding the estrogen receptor model, Mol. Cell. Endocrinol., 36 (1984) 11-15. 10 Greene, G.L., Nolan, C., Engler, J.P. and Jensen, E.V., Monoclonal antibodies to human estrogen receptor, Proc. Natl. Acad. Sci. U.S.A., 77 (1980) 5115-5119. 11 Jackson, P., Teasdale, J. and Cowen, P.N., Development and validation of a sensitive immunohistochemical oestrogen receptor assay for use on archival breast cancer tissue, Histochemistry, 92 (1989)149-152. 12 King, W.J. and Greene, G.L., Monoclonal antibodies localize oestrogen receptor in the nuclei of target cells, Nature, 307 (19841 745-747.
13 Koch, M. and Ehret, G., Immunocytochemical localization and quantitation of estrogen-binding cells in the male and female (virgin, pregnant, lactating) mouse brain, Brain Research, 489 (19891 101-112. 14 Lauber, A.H., Romano, G.J., Mobbs, C.V., Chambon, P. and Pfaff, D.W., Estradiol regulation of estrogen receptor mRNA in rat hypothalamus, Soc. Neurosci. Abstr., 15 (1989) 984. 15 Lustig, R.H., Mobbs, C.V., Bradlow, H.L., McEwen, B.S. and Pfaff, D.W., Differential effect of estradiol and 16a-hydroxyestrone on pituitary and preoptic estrogen receptor regulation, Endocrinology, 125 (19891 2701-2709. 16 Pfaff, D. and Keiner, M., Atlas of estradiol-concentrating cells in the central nervous system of the female rat, J. Comp. Neurol., 151 (1973) 121-158. 17 Simerly, R.B., Swanson, L.W. and Gorski, R.A., Demonstration of a sexual dimorphism in the distribution of serotoninimmunoreactive fibers in the medial preoptic nucleus of the rat, J. Comp. NeuroL, 225 (1984) 151-166. 18 Sladek, Jr., J.R., Fields, J.A. and Jacobson, C.D., The sexually dimorphic nucleus of the preoptic area: catecholamine innervation, Anat. Rec., 205 (1983) 187A-188A. 19 Stumpf, W.E., Estradiol-concentrating neurons: topography in the hypothalamus by dry-mount autoradiography, Science, 162 (1968) 1001-1003. 20 Stumpf, W.E., Sar, M. and Keefer, D.A., Atlas of estrogen target cells in rat brain. In: W.E. Stumpf and L.D. Grant (Eds.), Anatomical Neuroendocrinology, Based on the International Conference on Neurobiology of CNS-Hormone Interactions, Karger, Basel, 1975, pp. 1(14-119. 21 Warembourg, M., Jolivet, A. and Milgrom, E., Immunohistochemical evidence of the presence of estrogen and progesterone receptors in the same neurons of the guinea pig hypothalamus and preoptic area, Brain Research, 480 (1989) 1-15. 22 Watson, Jr., R.E. and Hoffmann, G.E.,Thesexuallydimorphic nucleus of the preoptic area: peptidergic components, Anat. Rec., 205 (19831 210A-211A. 23 Watson, Jr., R.E., Hoffmann, G.E. and Wiegand, S.J., Sexually dimorphic opioid distribution in the preoptic area: manipulation by gonadal steroids, Brain Research, 398 (1986) 157-163. 24 Welshons, W.V., Lieberman, M.E. and Gorski, J., Nuclear localization of unoccupied oestrogen receptors, Nature, 307 (1984) 747-749.
50
BRES 16558
The effect of estrogen on the estrogen receptor-immunoreactive cells in the rat medial preoptic nucleus Kazunari Yuri and Mitsuhiro Kawata Department of Anatomy, Kyoto Prefectural University of Medicine, Kyoto (Japan) (Accepted 20 November 1990)
Key words: Estrogen receptor; Immunohistochemistry; Medial preoptic nucleus; Estradiol; Ovariectomy
The localization of estrogen receptor-immunoreactive (ER-IR) cells in the medial preoptic nucleus (MPN) and the effect of estrogen upon these cells were quantitatively analyzed by immunohistochemistry using a monoclonal antibody to estrogen receptor (ER) protein in rat. ER was distributed in the nucleus of MPN neurons and was not detected in either cytoplasm or glial cells. There were more ER-IR cells with higher immunoreactivities in ovariectomized (OVX) rats than in estradiol (E2)-treated rats. The number of ER-IR cells in E2-treated compared with OVX rats was reduced by 43%. In particular, the number of ER-IR cells in the central part of the MPN was largely decreased. Our data indicate that the central part of the MPN is most sensitive to estrogen.
INTRODUCTION It has b e e n elucidated by in vivo autoradiography that the brains of vertebrates including mammals have estrogen concentrating neurons. These n e u r o n s are mainly located in the preoptic area, hypothalamus and central grey of the midbrain, and form a n e u r o n a l network related to the sexual behavior of the animal 16'19"2°. Recently, a m o n o c l o n a l antibody to estrogen receptor (ER) protein has b e e n raised ~° and immunohistochemical studies using this antibody have shown that the distribution of estrogen receptor-immunoreactive ( E R - I R ) cells was almost identical to that of autoradiography using radiolabeled estrogen 3"13'21. The medial preoptic nucleus (MPN) exhibits sexual dimorphism in the brain and detailed research has been performed with regard to sex differences in the size of the nucleus t'6, the n u m b e r , size and shape of n e u r o n s 7 and in the distribution of active substances such as amines 4"17"18 and peptides 22'23 in afferent fibers in the MPN. However, the distribution of E R in the MPN in different hormonal circumstances has hitherto not been investigated. In the present study, the influence of estrogen o n the MPN and its subnuclei in the ovariectomized (OVX) rat was quantitatively analyzed using an a n t i - E R monoclonal antibody.
MATERIALS AND METHODS
Animals
Wistar rats (virgin female, n = 5, not including 28 female rats for preliminary test) weighing 150-200 g were ovariectomized (OVX) while anesthetized with sodium pentobarbital, housed at 20 ~C and maintained on a 12.00 h/12.00 h light/dark cycle for 4 weeks before use. Food and water were available ad libitum.
Hormone administration To study the effect of estrogen on brain ER,IR cells, 5 0 V X animals were divided into two groups. The estradiol (E2)-treated group (n = 3) was injected subcutaneously with 30 /~g of 17restradiol (Nakarai, Kyoto, Japan) in 0.3 ml sesame oil and the remaining (OVX rats, n = 2) received 0.3 ml vehicle only.
Brain preparation Twenty-four hours after the administration of E 2 or vehicle, the animals were anesthetized with sodium pentobarbital and peffused with 50 ml phosphate-buffered saline (PBS, pH 7.2) followed by 600 ml ice-cold fixative containing 4% paraformaldehyde and 0.2% picric acid in 0.1 M phosphate buffer (pH 7.2). Brains were removed, postfixed with the same fixative for 24 h and transferred to 20% sucrose for 48 h. The brain blocks were sectioned into 20 ~m thick slices by cryostat at -20 °C and stored in PBS containing 0.3% Triton X-100 at 4 °C for 24 h for immunohistochemistry.
Immunohistochemicalprocedures The sections from all the brains were incubated simultaneously in the following antisera. Free-floating sections were immersed in 1% normal goat serum to decrease non-specific staining. The sections were incubated in anti-ER monoclonal antibody (Abbott, Chicago, IL, working dilution 1:20) for 168 h at 4 °C, immersed in anti-rat IgG (KPL, Gaithersburg, MD, working dilution 1:100) for 2 h, then PAP complex (Polysciences, Warrington, PA, working dilution
Correspondence: K. Yuri, Department of Anatomy, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602, Japan. Fax: (81) (75) 211-7093.
51
'OC
Fig. 1. Schematic drawing of the MPN for counting cells in a specified area. Grids were set on the MPN and the ER-IR cells were counted. Each grid has a 50-/~m side. AC: anterior commissure, OC: optic chiasm, MPN: medial preoptic nucleus, V: third ventricle.
Fig. 2. Immunoreactivity was recognized within the nucleus of MPN neurons. The ER-IR cell is indicated by arrowheads. C: cytoplasm, N: nucleus, bar = 10 ~m.
1:300) for 3 h at room temperature. The sections were reacted with 3,3'-diaminobenzidine (DAB) (Dojin, Kumamoto, Japan) intensifled by nickel: 20 mg DAB and 80 mg nickel (II)-ammonium sulphate, hexahydrate (Nakalai tesque, Kyoto, Japan) in 100 ral of 0.05 M Tris-HCl buffer containing 7 /~l of 30% H202. Primary antibody was omitted in some sections to check the non-specific reaction.
Fig. 3. ER-IR cells were elucidated in the MPN of the OVX group (A) and E:-treated group (B). Immunoreactivity was stronger in the OVX group than in the hormone-treated group. Boundary of the MPN is indicated by a broken line. Bar = 100/~m.
52
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Fig. 5. The number of ER-IR cells in the MPN was 1286 + 79/mm~ in the OVX group and 751 +_ 58/mm~ in the E2-treated group. Values (mean _+ S.E.M.) represent significant differences between the OVX group and E2-treated group (*P < 0.01, Student's t-test).
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Fig. 4. The central part of MPN was intensely immunostained in the OVX group (A) but not in the E2-treated group (B). Bar = 30/~m.
Both in the O V X and E2-treated rats, E R - I R cells were observed in the p r e o p t i c area which includes not only the M P N but also the periventricular preoptic nucleus and areas b e y o n d the MPN. T h e r e was no
Quantitative analysis
difference in the total area of the M P N b e t w e e n the O V X
A comparison was made between OVX and hormone treatment groups in 6 sets of equivalent sections. These sections were selected according to the criteria which were based on the shape of the anterior commissure, the optic chiasm and the third ventricle. Adjacent sections were Nissl-stained with Cresyl violet (Chroma, Stuttgart, F.R.G.); the sections were immersed into this solution for 10 rain at 36 °C. Immunohistochemical sections were photographed and the MPN boundary was defined by the adjacent Nissl-stained section. The number of ER-IR cells within the MPN was counted and the area of the MPN was measured by an image analyser MOP-1 (Kontron, Munich, F.R.G. ). Four sets of equivalent sections including the central part of the MPN were used for further analysis of the MPN; grids were prepared and set on the side of the third ventricle 500 gm above the bottom of the ventricle. The grids consisted of 6 x 14 = 84 square grids and each grid corresponded to a 50 x 50 gm section of the brain (Fig. 1). The number of ER-IR cells was counted in each grid, and a histogram constructed. All the sections were coded to prevent experimental bias during cell count analysis. The MPN was subdivided according to the classification and nomenclature of Simerly et al. 17. Immunohistochemical data were analysed using the Student t-test and differences were considered significant,
and E2-treated rats: the average area was 0.21 m m 2. The size of M P N neurons (the m a j o r axis) was 15.3 + 0.6 p m (mean + S . E . M . ) in the O V X and 17.2 _+ 0.7/~m (mean __- S . E . M . ) in the E2-treated rats. N e i t h e r d e g e n e r a t i o n nor abnormalities were o b s e r v e d in the neurons. In the M P N , E R i m m u n o r e a c t i v i t y was o b s e r v e d within the nucleus of neurons (Fig. 2), but not in the cytoplasm nor in any glial cells. E R - I R cells were m o r e i m m u n o r e a c t i v e in the O V X than in the E2-treated rats (Fig. 3 A , B ) . This tendency was a feature of the central part of the M P N (Fig. 4 A , B ) . The n u m b e r of E R - I R cells in the M P N was 1286 +_ 79/mm 2 (mean +_ S . E . M . ) in the O V X and 751 + 58/ram 2 (mean + S . E . M . ) in the E2-treated rats, indicating a 43% reduction in E R - I R cells by E 2 (Fig. 5). Cell counts revealed that the largest n u m b e r of E R - I R cells was in a grid 150-200 g m from the third ventricle in the O V X ,
53
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Fig. 6. The number of ER-IR cells counted in the grid presented in Fig. 1, was 7.2/grid 150-200/am from the ventricle in the OVX group (A), however, this peak significantlydecreased to 3.3/grid in the E2-treated group (B). *P < 0.05, **P < 0.01, values that are significantly different from those in the OVX group (Student's t-test),
which corresponds to the central part of the MPN. There was a gradual decrease in number of ER-IR cells toward the lateral end of the MPN. The number of ER-IR cells in the E2-treated rats was lower than that of the OVX and there was no conspicuous increase in number, particularly in the central part of the MPN. It was found that the number of ER-IR cells in the central part of the MPN of the OVX was 7.2 + 1.0/grid (mean + S.E.M.), compared with 3.3 _+ 0.5/grid (mean _+ S.E.M.) in the E2-treated rats. This was a statistically (P < 0.05) significant decrease (Fig. 6A,B). DISCUSSION The monoclonal antibody to E R was prepared from the human breast cancer cell line MCF-7 ~° and it has been widely used to elucidate the localization of ER in many organs including the central nervous system in mammals, The results have been consistent 3'13'21. We examined the specificity of this antibody and ascertained that it reacted
with E R specifically. In the present study, the effects of E 2 on OVX rat brains were quantitatively analyzed with the monoclonal antibody. Intact female rats should not be used as a control because the plasma E 2 level changes during the estrous cycle2. Immunoreactivity was observed in the nucleus of the neurons in the preoptic area including the MPN. There have been many debates as to whether the E R was localized in the cytoplasm or in the nucleus 9'24. Recent immunohistochemical studies have indicated that ER exists in the nucleus of target cells 3'12'13'21. In this study, we observed immunoreactivity only in the nucleus of the MPN neuron (Fig. 2). The loss of E R or other constituents in the section might be possible during 168 h incubation in the primary antibody, however, it is not related to antigenicity of E R because we observed stronger immunoreactivity in the 168-h incubated section than that of 48 h or 120 h in the preliminary studies. ER-IR cells were detected in 10year-old sections of the peripheral tissue 11. Both the number of ER-IR cells and the intensity of immunoreactivity in the MPN decreased in the E2-treated rats (Figs. 3 and 4). These observations were consistent in our preliminary studies directed at optimizing the immunohistochemical method using 28 female rats. At present, there are two explanations for these phenomena 3. One is that the reduction in the E R number was caused by the E2 treatment and the other is that a decrease in antigenicity of the E R occurred because it was transformed into a different three-dimensional structure after coupling with E 2. Immunoassay has demonstrated that the volume of E R in the preoptic area decreases after E 2 treatment 15 and in situ hybridization histochemistry has revealed that E R mRNA levels are also reduced TM. Therefore, a decrease in E R mRNA production after E2 treatment may cause the reduction in E R number. Further molecular biological studies will be required to solve the problem completely. The injection of 30/~g of E 2 induces a high s e r u m E 2 level and it is sufficient to saturate the E R in the neurons. A twenty-four-hour interval after injection of E 2 was appropriate because it was reported that the effect of E 2 on the neurons was strongest at 24 h 5'14. The MPN is known to be sexually dimorphic. Sex steroids affect the size and number of neurons that constitute this n u c l e u s 6'7. T h e MPN consists of 3 parts: a medial, central and lateral part 17. Of these 3, the central part corresponds to the sexually dimorphic nucleus of the preoptic area which was proposed by Gorski et al. and is thought to be the most sensitive to sex steroids 7A7. In the present study, ER-IR cells decreased significantly in the central part (Fig. 6A,B), indicating that it is definitely the part that is most sensitive to estrogen treatment.
54
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