NOTE. Department of Anatomy, Tokai University Medical School, Isehara City Jaian

Transcription

1 Endocrinol. Japon. 1978, 25 (3), NOTE Accumulation and Binding of 3H-Estradio1-17ƒÀ by Lymphoid Tissues of Castrated Mice KANJI SEIKI, YosHlo IMANISHI AND YASUO HARUKI Department of Anatomy, Tokai University Medical School, Isehara City Jaian Synopsis The in vivo uptake of 3H-estradiol-17ƒÀ by the various lymphoid tissues, fat tissue, skeletal muscle and circulating blood of castrated male and female mice of C57BL strain was studied after an intravenous injection of the hormone. In both sexes the highest uptake was by the bone marrow and fat tissue, followed by the spleen, thymus and mesenteric lymph node. The lowest was by the muscle and the blood serum. The lymphoid tissues in the female took up a slightly more amount of radioactivity than those in the male until 1 hr after the injection, but thereafter the uptake was at almost the same level between both sexes. The lymphoid tissues showed a relative and long-term retention of the radioactivity, as compared with that in the muscle and blood serum. The binding of 3H-estradiol-17ƒÀ by the lymphoid tissue cytosol and their binding specificity for the hormone were examined in vitro by Sephadex G-100 column analysis. All the tissue cytosols tested contained estradiol-binding component(-s). Among those the thymic cytosol showed to contain the component(-s) which is fairly specific to this hormone. These results suggest that there is a relatively high uptake and retention of estrogen in the lymphoid tissues in which it is bound to cytoplasmic binding component(-s). Numerous data have accumulated concerning estrogen target tissues such as uterus, vagina, oviduct, mammary gland and pituitary of various species of animals (see review by Liao, 1975). Those tissues preferentially take up and retain the hormone from blood, by which it palys a role in the alteration of cellular function in the tissues. That is, the first step of estrogen action is the entry of the hormone into target cells where it binds to cytoplasmic receptor proteins (Jensen et al., 1968; Gorski et al., 1968; Jensen and DeSombre, 1973), Following this hormone-receptor interaction, the hormone-receptor complex is transported into the cell nucleus where it interacts with Received November 29, some specific part of the genome and initiates the transcription of specific gene, resulting in the alteration of cellular function. There have been many observations on the physiological or immunological effects of estrogen on lymphoid tissues such as the thymus, lymph node, spleen and bone marrow. For example, Gardner et al., (1944) reported that a large dosis of estrogen caused lymphoid tissue proliferation in mice. Sherman et al. (1963) reported that estrogen administration to neonatally thymectomized hamsters prevented wasting, whereas oophorectomy in neonatally thymectomized females permitted wasting. On the contrary, Dougherty (1952), Kappas and Palmer (1963) and Thompson et al.(1966) have shown

2 SEIKI et al. that estrogen administration caused thymic involution in the intact animals, but its effect on lymphoid tissues other than the thymus was minimal and quite variable, depending on the species used. When estrogen was administered to newborn mice, it induced wasting syndrome with central and peripheral lymphoid hypoplasia (Thompson and Russe, 1965). In addition, Ablin el al.(1974) have recently reported that the incorporation of 3H-thymidine by phytohemagglutinin-stimulated blood lymphocytes was significantly suppressed by estrogen. However, the mode of action of estrogen in the lymphoid tissues through which the physiological and immunological effects are brought about is unclear. As an initial step to investigate the mode of action in the tissues we examined a property of estrogen-binding proteins as well as an aspect of uptake and retention of the hormone in the tissues. Animals Materials and Methods Male and female mice of C57BL strain (purchased from CLEA-Japan, Inc., Tokyo), 4 weeks old and weighing 17-18g, were used throughout the experiment. They were castrated under ether anesthesia and maintained on purina chow pellet and water ad libitum. Endocrinol. June 1978 Japon. measured in a liquid scintillation counter (Aloka LSC-653 type, Aloka Co., Tokyo) with 50% efficiency for 3H. Another part of each sample was homogenized in Tris-DETA buffer (10mM Tris-HCl, ph 7.4, with 1mM EDTA) in an all-glass homogenizer. The homogenate was extracted 3 times with ethyl ether, and the extract was chromatographed on a thin-layer plate coated with Wakogel B-5 (Wako Pure Chemical Industries, Ltd., Osaka) using a solvent system of benzene-methanol (9:1, v/v). The estradiol-17ƒà spot in each extract was located by the simultaneous development of standard estradiol-17ƒà. The spot was scraped off and then offered for radioactivity measurement. Sephadex G-100 column chromatography Three days after the castration the bone marrow, spleen, thymus and mesenteric lymph node were obtained in the same manner as in the uptake study. The tissues were rinsed and homogenized in cold Tris-EDTA buffer. The supernatant fraction,"cytosol", was obtained by centrifuging the homogenate at 800 ~g for 10min, followed by 107,000 ~g for 1hr. The cytosol was diluted with Tris-EDTA buffer to obtain the protein concentration of 10mg/ml (measured by the method of Lowry et al., 1951). The cytosol was then incubated for 12hr at 4 Ž in 10-7M 3H-estradiol-17ƒÀ in the presence or in the absence of 10-5M unlabeled steroids such as estradiol- 17ƒÀ, progesterone, testosterone and corticosterone (all purchased from Sigma Chemical Co., USA). A 0.2-ml aliquot of each incubate was layered at 4 Ž on Sephadex G-100 column (25 cm ~0.8cm), and 0.5 ml fractions were collected at a flow rate of 1ml/8 min. The optical density was measured for each fraction, and then the radioactivity was measured in Bray's scintillator (Bray, 1960) in the liquid scintillation counter with 32% efficiency for 3H. Uptake study Three days after the castration each animal was injected through tail vein with 5ƒÊCi] 2, 4, 6, 7-3H]- estradiol-17ƒà (sp. act. 50 Ci/m mole, New England Nuclear, Boston) dissolved in 50ƒÊl physiological saline containing 10% ethanol. At 0.5, 1, 3, 6 and 12hr after the injection, blood samples were taken via the femoral artery, and then the thymus, mesenteric lymph node, spleen, gastrocnemius muscle, fat tissue, and the tibial and femoral bones were excised. The bone marrow was collected by washing out the marrow cavity with physiological saline through an injection needle. A part of each sample was weighed and burned in the Packard Model 306 Tri-Carb Sample Oxidizer (Packard Instrument Co., Inc., USA) to collect tritiated water vapor in a scintillation fluid (Monophase-40, Packard Instrument Co., Inc., U.S.A.). The radioactivity was then Results The distribution of radioactivity in various tissues after 3H-estradiol-17ƒÀ administration is shown in Fig. 1. The radioactivity in each tissue of both sexes reached a maximum within 0.5hr, then it gradually dropped in nearly the same pattern as each other until 12 hr, at which time substantial radioactivity was still detected. In both sexes the tissues with the highest concentrations with time after the injection were the bone marrow and fat tissue. The lymphoid tissues such as the spleen, thymus and lymph node also accumulated fairly

3 Vol.25, No.3 ESTRADIOL UPTAKE BY LYMPHOID TISSUES larger amounts of radioactivity than did the muscle and blood serum. The radioactivity in the lymphoid tissues were slightly at the higher levels in females than in males until 1hr after the injection, but thereafter it was at almost the same level between both sexes. It was revealed from the thin-layer chromatographic analysis that 25, 25, 25, 17, 30, 38 and 40% of the radioactivity in the bone marrow, fat tissue, spleen, thymus, mesenteric lymph node, muscle and blood serum, respectively, were from the radioactive estradiol-17ƒà. The ratio of the radioactivity concentration between the tissues and blood serum in male and female mice after 3H-estradiol- 17ƒÀ injection (calculated from the data in Fig. 1) is listed in Table 1. Among the tissues studied in both sexes the ratio was the highest in the bone marrow and fat tissue with time after the injection. The Table 1. Ratio of the radioactivity concentration between the tissues and blood serum of C57BL mice after an intravenous injection of 3Hestradio1-17ƒÀ*. * Calculated from the data in Fig. 1. Values are average of 10 determinations hr. after injection Fig. 1. Radioactivity pattern in various tissues and blood serum of castrated C57BL mice after an intravenous injection of 3H-estradiol-17ƒÀ. =male, =female. and œ= standard error based on 10 determinations in male and female, respectively. B =bone marrow, F = fat tissue, Sp = spleen, T=thymus, L=lymph node, M=muscle, S=blood serum.

4 SEIKI et al. Endocrinol. June 1978 Japon. remaining lymphoid tissues showed almost the same ratio between each other with time, and the muscle showed the lowest. In addition, the four lymphoid tissues in both sexes showed the same pattern of changes in the ratio with time after the injection ; first decreasing until 1hr, then increasing until 6 hr and finally decreasing thereafter, giving a relative and long-term retention of the radioactivity in those tissues, as compared with that in the muscle and blood serum. The Sephadex column assays of the cytosols from the thymus, spleen, mesenteric lymph node and bone marrow of both male and female mice all yielded one radioactive peak which was eluted within void volume and congruent with a protein peak determined by optical density at 280nm. In the thymic cytosols (Fig. 2 and 3), the radioactive peak was greatly diminished by a 100-fold amount of unlabeled estradiol- 17ƒÀ (50% reduction in males and 40% reduction in females), but not by the other steroids used. The radioactive peak in the female bone marrow cytosol was not diminished by any of the unlabeled steroids used (Fig. 4), but the peak in the male cytosol was reduced by estradiol- 17ƒÀ and progesterone (both 50% reduction). In the female lymph node cytosol the peak was reduced by all the steroids used (ranging 30-50%), whereas in the male cytosol it was reduced only by progesterone (50% reduction) and testosterone (70% reduction). The radioactive peak in the splenic cytosols of both sexes was not diminished by any of the steroids used. Fig. 3. Sephadex G-100 column chromatography of the thymic cytosol from ovariectomized C57BL mice. The cytosol (0.2ml containing 2mg protein) was incubated with 10-7M 3H-estradiol-17ƒÀ in the presence of 10-5M unlabeled steroids. Symbols are same as in Fig. 1. Fig. 2. Sephadex G-100 column chromatography of the thymic cytosol from orchiectomized C57BL mice. The cytosol (0.2ml containing 2mg protein) was incubated with 10-7M 3H- estradiol- 17ƒÀ in the Fig. 4. Sephadex G-100 column chromatography of presence of 10-5M unlabeled steroids. = 3Hestradiol-17ƒÀ the bone marrow cytosol from ovariectomized alone, œ3= H-estradiol-17ƒÀ+ estradiol- C57BL mice. The cytosol (0.2ml containing 2mg 17ƒÀ, =3H-estradiol-17ƒÀ+ corticosterone, = 3Hestradiol-17ƒÀ+ protein) was incubated with 10-7M 3H-estradiol- progesterone, = 3H-estradiol- 17ƒÀ 17ƒÀ in the presence of 10-5M unlabeled steroids. Symbols are same as in Fig. 1.

5 Vol.25, No.3 ESTRADIOL UPTAKE BY LYMPHOID TISSUES Discussion The present in vivo experiment showed the high uptake and long-term retention of the injected radioactive eatradiol-17ƒà by the mouse lymphoid tissues such as the thymus, spleen and mesenteric lymph node, as compared with those by the muscle and circulating blood. In addition, the present in vitro study demonstrated the presence of estradiol-binding component(-s) in those tissues. Among those tissues the thymic cytosol showed to contain the component(-s) which is fairly specific to estradiol-17ƒà, whereas those in the spleen and mesenteric lymph node were quite variable to bind the hormone. Dougherty (1952) and Kappas and Palmer (1963) reviewed that estrogenic hormones regularly caused acute thymic involution in the intact animals. Their effects on the lymphoid tissues other than the thymus were minimal, quite variable and related to the species used. Thompson et al. (1965) reported almost the same effects of estradiol on the lymphoid tissues of the adult normal female mice. Thompson et al. (1966) reported that estradiol, like X- irradiation, caused thymic involution which was characterized by diminished nucleic acid content and DNA synthesis, although the hormone not only altered nucleic acid content or synthesis in the mesenteric lymph node but also enhanced DNA synthesis in the spleen. The present results, although preliminary, are in obvious parallel with the above reports and suggest that physiological or immunological effects of the estrogen on the lymphoid tissues are initiated by its binding to the cytosols of these tissues, especially to the thymic cytosol. The present experiment demonstrated the high uptake of 3H-estradiol-17ƒÀ by the fat tissue in both sexes, which has been reported also by Eisenfeld and Axelrod (1965) and Maurer et al. (1970). These results strongly indicate that the fat tissue is very sensitive to estrogen and its functions are greatly influenced by this hormone. A more detailed explanation for these results may be found in the review by Grodsky (1973) that in the fat tissue estrogen-dependent transhydrogenase which catalizes the transfer of hydrogen from NADPH brings about an increased rate of biologically useful energy, and this energy stimulates the incorporation of acetate into lipid molecules in this tissue. Finally, the present study demonstrated that the radioactivity taken up by the lymphoid tissues was slightly higher in the female mice than in the male mice at the early period of time after the injection of 3H-estradiol -17ƒÀ, but thereafter it was at almost the same level between each sex. This might mean that there is essentially no difference in estrogen uptake by the lymphoid tissues between both sexes, which, in turn, indicates that the hormonal influences on the lymphoid tissues are almost the same degree between both sexes. References Ablin, R. J., G. R. Bruns, P. Guinan and I. M. Bush (1974). J. Immunol. 113, 705. Bray, G. A.(1960). Analyt. Biochem. 1, 279. Dougherty, T. F.(1952). Physiol. Review 32, 379. Eisenfeld, A. J. and J. A. Axelrod (1965). J. Pharmacol. Exp. Therap. 150, 469. Gardner, W. U., T. F. Dougherty and W. T. Williams (1944). Cancer Res. 4, 73. Gorski, J., D. O. Toft, G. Shyamala and A. Notides (1968). Rec. Prog. Hormone Res. 24, 45. Grodsky, G. M. Review of Physiological Chemistry (edited by H. A. Harper), Lange Medical Publishers, San Francisco, p ,(1973). Jensen, E. V., T. Suzuki, T. Kawashima, W. E. Stumpf, P. W. Jungblut and E. R. DeSombre (1968). Proc. Nat. Acad. Sci. 59, 632. Jensen, E. V. and E. R. DeSombre (1973). Science 182, 126. Kappas, A. and R. H. Palmer (1963). Pharmacol. Review 15, 123. Liao, S.(1975). Intern. Review Cytol. 41, 87. Lowry, O. H., N. J. Rosebrough, A. L. Farr and R. J. Randall (1951). J. Biol. Chem. 193, 265.

6 SEIKI et al. Maurer, R. A., D. E. Woolley and M. M. Saari (1970). Proc. West. Pharmacol. Soc. 13, 47. Sherman, J. D., M. M. Adner and W. Dameshek (1963). Blood 22, 469. Thompson, J. S., R. W. Reilly, M. Crawford and H. P. Russe (1965). Radiation Res. 26, 567. Thompson, J. S., C. D. Severson and R. W. Reilly (1966). Radiation Res. 29, 537. Thompson, J. S. and H. P. Russe (1965). Fed. Proc. 24, 161. Endocrinol. June Japon.