Brain Research, 171 (1979) 503-510 © Elsevier/North-Holland Biomedical Press
503
S E X - R E L A T E D A N D C Y C L I C V A R I A T I O N OF T R A C E E L E M E N T S I N R A T HYPOTHALAMUS AND PITUITARY
GEO RG E R. MERRIA M*, LEWIS L. N U N N E L L E Y * *, JAMES W. V. TRISH and FREDERICK
NAFTOLIN*** Laboratory of Human Reproduction and Reproductive Biology, Harvard Medical School, Boston, Mass. 02115, and (L.L.N. and J. W. V. 7".) Department of Physics, University of Colorado, Boulder, Colo. 80302 (U.S.A.).
(Accepted November 30th, 1978)
SUMMARY The concentrations of 7 trace elements - - iron, copper, zinc, arsenic, selenium, bromine, and rubidium - - in rat hypothalami and anterior pituitaries were measured by X-ray fluorescence spectrometry. Male, cycling female, and oophorectomized animals were studied under different conditions of reproductive function. Hypothalamic zinc and copper concentrations both rose between proestrus and estrus days and fell again at diestrus. After castration, concentrations of iron, copper, and arsenic were decreased in both pituitary and hypothalamus, while zinc concentrations rose. Male rats had lower pituitary concentrations of iron, copper, zinc, arsenic, and rubidium than cycling females. Under these hormonal manipulations, hypothalamic zinc concentration and gonadotropin secretion appear to be correlated. While injections of copper salts into the hypothalamus can also stimulate gonadotropin release, we did not observe any consistent relation between endogenous hypothalamic copper concentrations and gonadotropins.
INTRODUCTION The participation of several abundant cations, particularly calcium and potassium, in coupling the stimulation of an endocrine cell to the secretion of its product * Present address (for all correspondence): National Institute of Child Health and Human Development, NIH, Bethesda, Md. 20205, U.S.A. ** Present address: Department of Chemistry, Chemeketa College, Salem, Oreg., U.S.A. *** Present address: Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, Conn., U.S.A.
504 has been well documented in a variety of organs, including the anterior and posterior pituitary gland and the hypothalamus 2. In addition, various elements present in only trace concentrations can alter the secretion of certain neuroendocrine and pituitary hormones. The most extensively studied of these trace element effects is the stimulation of luteinizing hormone secretion by copper ions. Forty years ago, Fevold et al. noted that intravenous injections of copper salts produced ovulation in rabbits 4. Whether this was a hypothalamic, a pituitary, or a direct ovarian effect remained uncertain for many years; but the more recent work of Suzuki and his colleagues has pointed primarily to the hypothalamus as the site of action. As little as 100/zg CuSO4 implanted into the posterior median eminence is sufficient to induce ovulation in rabbits 6 and rats 11. Several other trace metal ions have similar but less potent effects. In their original study, Fevold and colleagues found that zinc and iron were capable of inducing ovulation when given in high doses, and it has been claimed that zinc is synergistic with copper in this effect9. Microiontophoresis of 200 nmol of ferrous or ferric chloride into the rat hypothalamus also stimulates ovulation 3. Other ions affect different hormones; for example, prolactin secretion is inhibited by nickel s All of these are pharmacologic effects. Copper, zinc, iron, nickel, and other elements are present in trace quantities in the pituitary and hypothalamus, but it is not known whether these ions participate in the physiological control of hypothalamic or pituitary function. If they do, we thought it possible that their concentrations in these tissues might change under different hormonal conditions. In this study, we measured the levels of several trace elements in rat pituitaries and hypothalami in different states of reproductive function: in normal adult males, in cycling females at each stage of the estrus cycle, and in castrated females. We employed the technique of X-ray fluorescence spectroscopy, in which tissue samples are bombarded with an X-ray beam, and induced X-ray emission at wavelengths characteristic for each element is observed and counted. This method is highly sensitive and permits the simultaneous measurement of many elements in a single sample. Nickel levels were too low to be studied accurately; but iron, copper, zinc, arsenic, selenium, bromine, and rubidium could be measured with consistency. Significant variations in all these elements except selenium were found. METHODS Sprague-Dawley rats - - adult females weighing approximately 175 g, adult males, and oophorectomized females - - were maintained in an environment lighted for 14 h of the day. Vaginal smears from cycling female animals were obtained daily at 08.00 h for cytological confirmation of the stage of the estrus cycle; only animals which demonstrated 3 regular 4-day cycles were used. Sequential smears from oophorectomized rats were also obtained for at least 4 successive days to confirm the absence of estrogenic effect. Animals were stunned by cervical dislocation before 10.00 h; blood samples were withdrawn by cardiac puncture, and anterior pituitaries and
505 hypothalami removed within 2 rain. Hypothalami were excised with scissors as diamond-shaped blocks bounded by the anterior edge of the optic chiasm and the mamillary bodies, and approximately 3 mm deep. Wet weights of samples were recorded, and the samples were dried and homogenized in small agate mortars. The powdered homogenate (250 /zg) was placed between two layers of Formvar foil, mounted in an aluminum ring, and studied in an X-ray fluorescence apparatus, as previously described 1. Typically, samples were exposed to a collimated and filtered 55 keV X-ray beam 7 mm in diameter ( ~ 20 mA, Emax -- 29 keV) for 10 rain. Characteristic X-ray emission was counted with a lithium-doped silicon detector and then converted to concentrations, using an iterative computer program to correct for internal X-ray absorption and fluorescence with standard correction formulae lz. Serum was separated and stored frozen. At analysis, 10/~1 aliquots of serum samples were placed directly on the Formvar sheets, allowed to dry, and analyzed in the same X-ray system. All tools and glassware were cleaned prior to use in a solution of spectroscopic grade 70 ~ HNO3 and washed in glass-distilled water. Blank samples handled and stored in the same manner showed no measurable contamination with any of the elements studied. The data were examined statistically with a standard one-way analysis of variance program and Duncan's multiple range test. P values for significant differences were calculated using a t-test modified for multiple comparisons. RESULTS AND DISCUSSION Concentrations of the 7 elements studied are shown in Tables 1 and II. There are few published values with which to compare these results, but hypothalamic levels of copper are close to the concentrations reported by Gekht using atomic absorption techniques 5. Variations of most elements during the estrus cycle were slight. Hypothalamic zinc showed a significant rise at estrus and metestrus; copper appeared to fall at proestrus and then rise again by the morning of estrus; but the statistical significance of the difference is marginal (P = 0.06). Hypothalamic rubidium was higher at estrus and metestrus. Levels in the pituitary gland showed no significant cyclic variations except for small elevations in bromine and rubidium at estrus. By contrast, pituitary concentrations showed a general sex-related difference, while hypothalamic levels were largely identical in males and females. Iron, copper, zinc, arsenic, and rubidium were lower in male pituitaries, while bromine in males was higher. Castration lowered the levels of all these elements except selenium - - which showed no significant variations under any circumstances - - and zinc, which alone rose after oophorectomy. These differences were generally greater than the sex-related variations, excepting pituitary copper and rubidium. Values of serum copper and zinc are shown in Table III. The concentrations are all much lower than tissue levels. In contrast to results obtained for plasma by atomic absorption TM, there was no significant variation in copper during the estrus cycle, a difference possibly due to proteins removed during coagulation.
18.3 ~ 3.0
70.2 ± 2
4.8 i
2.1 ± 0.2 15.0 ± 1.7
31.3 ± 1.5
Copper
Zinc
Arsenic
Selenium Bromine
Rubidium
32.8 ± 1.8
2.1 ± 0.3 16.1 ± 1.8
3.8 ± 1.1
70.7 ± 1.9
16.8 ± 1.8
265 :~ 32 (11)
Estrus
31.3 j : 1.5
1.9 i 0.2 •3.8 ~: 0.7
3.5 ± 1.0
68.3 ± 3.0
17.6 ± 2.6
257 ± 31 (8)
Metestrus
30.0 ± 2.2
2.1 i 0.2 15.3 ± 1.8
4.4 = 1.7
68.3 ± 1.9
16.8 ± 2.4
285 ± 56 (10)
Diestrus 32 (6)
23.4 ± 4.6
1.8 ± 0.6 16.5 ~: 3.1
2.9 ~ 0.9
59.1 ± 7.8
t l . 9 ± 1.4
23l i
Males
1.4
29.8 ± 2.1
1.9 ± 0.1 12.8 ± 1.1
1.0 ± 0.5
74.7 i 2.2
13.6 i
147 ~ 35 (8)
Castrated females
** P < 0.01, *** P
503
S E X - R E L A T E D A N D C Y C L I C V A R I A T I O N OF T R A C E E L E M E N T S I N R A T HYPOTHALAMUS AND PITUITARY
GEO RG E R. MERRIA M*, LEWIS L. N U N N E L L E Y * *, JAMES W. V. TRISH and FREDERICK
NAFTOLIN*** Laboratory of Human Reproduction and Reproductive Biology, Harvard Medical School, Boston, Mass. 02115, and (L.L.N. and J. W. V. 7".) Department of Physics, University of Colorado, Boulder, Colo. 80302 (U.S.A.).
(Accepted November 30th, 1978)
SUMMARY The concentrations of 7 trace elements - - iron, copper, zinc, arsenic, selenium, bromine, and rubidium - - in rat hypothalami and anterior pituitaries were measured by X-ray fluorescence spectrometry. Male, cycling female, and oophorectomized animals were studied under different conditions of reproductive function. Hypothalamic zinc and copper concentrations both rose between proestrus and estrus days and fell again at diestrus. After castration, concentrations of iron, copper, and arsenic were decreased in both pituitary and hypothalamus, while zinc concentrations rose. Male rats had lower pituitary concentrations of iron, copper, zinc, arsenic, and rubidium than cycling females. Under these hormonal manipulations, hypothalamic zinc concentration and gonadotropin secretion appear to be correlated. While injections of copper salts into the hypothalamus can also stimulate gonadotropin release, we did not observe any consistent relation between endogenous hypothalamic copper concentrations and gonadotropins.
INTRODUCTION The participation of several abundant cations, particularly calcium and potassium, in coupling the stimulation of an endocrine cell to the secretion of its product * Present address (for all correspondence): National Institute of Child Health and Human Development, NIH, Bethesda, Md. 20205, U.S.A. ** Present address: Department of Chemistry, Chemeketa College, Salem, Oreg., U.S.A. *** Present address: Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, Conn., U.S.A.
504 has been well documented in a variety of organs, including the anterior and posterior pituitary gland and the hypothalamus 2. In addition, various elements present in only trace concentrations can alter the secretion of certain neuroendocrine and pituitary hormones. The most extensively studied of these trace element effects is the stimulation of luteinizing hormone secretion by copper ions. Forty years ago, Fevold et al. noted that intravenous injections of copper salts produced ovulation in rabbits 4. Whether this was a hypothalamic, a pituitary, or a direct ovarian effect remained uncertain for many years; but the more recent work of Suzuki and his colleagues has pointed primarily to the hypothalamus as the site of action. As little as 100/zg CuSO4 implanted into the posterior median eminence is sufficient to induce ovulation in rabbits 6 and rats 11. Several other trace metal ions have similar but less potent effects. In their original study, Fevold and colleagues found that zinc and iron were capable of inducing ovulation when given in high doses, and it has been claimed that zinc is synergistic with copper in this effect9. Microiontophoresis of 200 nmol of ferrous or ferric chloride into the rat hypothalamus also stimulates ovulation 3. Other ions affect different hormones; for example, prolactin secretion is inhibited by nickel s All of these are pharmacologic effects. Copper, zinc, iron, nickel, and other elements are present in trace quantities in the pituitary and hypothalamus, but it is not known whether these ions participate in the physiological control of hypothalamic or pituitary function. If they do, we thought it possible that their concentrations in these tissues might change under different hormonal conditions. In this study, we measured the levels of several trace elements in rat pituitaries and hypothalami in different states of reproductive function: in normal adult males, in cycling females at each stage of the estrus cycle, and in castrated females. We employed the technique of X-ray fluorescence spectroscopy, in which tissue samples are bombarded with an X-ray beam, and induced X-ray emission at wavelengths characteristic for each element is observed and counted. This method is highly sensitive and permits the simultaneous measurement of many elements in a single sample. Nickel levels were too low to be studied accurately; but iron, copper, zinc, arsenic, selenium, bromine, and rubidium could be measured with consistency. Significant variations in all these elements except selenium were found. METHODS Sprague-Dawley rats - - adult females weighing approximately 175 g, adult males, and oophorectomized females - - were maintained in an environment lighted for 14 h of the day. Vaginal smears from cycling female animals were obtained daily at 08.00 h for cytological confirmation of the stage of the estrus cycle; only animals which demonstrated 3 regular 4-day cycles were used. Sequential smears from oophorectomized rats were also obtained for at least 4 successive days to confirm the absence of estrogenic effect. Animals were stunned by cervical dislocation before 10.00 h; blood samples were withdrawn by cardiac puncture, and anterior pituitaries and
505 hypothalami removed within 2 rain. Hypothalami were excised with scissors as diamond-shaped blocks bounded by the anterior edge of the optic chiasm and the mamillary bodies, and approximately 3 mm deep. Wet weights of samples were recorded, and the samples were dried and homogenized in small agate mortars. The powdered homogenate (250 /zg) was placed between two layers of Formvar foil, mounted in an aluminum ring, and studied in an X-ray fluorescence apparatus, as previously described 1. Typically, samples were exposed to a collimated and filtered 55 keV X-ray beam 7 mm in diameter ( ~ 20 mA, Emax -- 29 keV) for 10 rain. Characteristic X-ray emission was counted with a lithium-doped silicon detector and then converted to concentrations, using an iterative computer program to correct for internal X-ray absorption and fluorescence with standard correction formulae lz. Serum was separated and stored frozen. At analysis, 10/~1 aliquots of serum samples were placed directly on the Formvar sheets, allowed to dry, and analyzed in the same X-ray system. All tools and glassware were cleaned prior to use in a solution of spectroscopic grade 70 ~ HNO3 and washed in glass-distilled water. Blank samples handled and stored in the same manner showed no measurable contamination with any of the elements studied. The data were examined statistically with a standard one-way analysis of variance program and Duncan's multiple range test. P values for significant differences were calculated using a t-test modified for multiple comparisons. RESULTS AND DISCUSSION Concentrations of the 7 elements studied are shown in Tables 1 and II. There are few published values with which to compare these results, but hypothalamic levels of copper are close to the concentrations reported by Gekht using atomic absorption techniques 5. Variations of most elements during the estrus cycle were slight. Hypothalamic zinc showed a significant rise at estrus and metestrus; copper appeared to fall at proestrus and then rise again by the morning of estrus; but the statistical significance of the difference is marginal (P = 0.06). Hypothalamic rubidium was higher at estrus and metestrus. Levels in the pituitary gland showed no significant cyclic variations except for small elevations in bromine and rubidium at estrus. By contrast, pituitary concentrations showed a general sex-related difference, while hypothalamic levels were largely identical in males and females. Iron, copper, zinc, arsenic, and rubidium were lower in male pituitaries, while bromine in males was higher. Castration lowered the levels of all these elements except selenium - - which showed no significant variations under any circumstances - - and zinc, which alone rose after oophorectomy. These differences were generally greater than the sex-related variations, excepting pituitary copper and rubidium. Values of serum copper and zinc are shown in Table III. The concentrations are all much lower than tissue levels. In contrast to results obtained for plasma by atomic absorption TM, there was no significant variation in copper during the estrus cycle, a difference possibly due to proteins removed during coagulation.
18.3 ~ 3.0
70.2 ± 2
4.8 i
2.1 ± 0.2 15.0 ± 1.7
31.3 ± 1.5
Copper
Zinc
Arsenic
Selenium Bromine
Rubidium
32.8 ± 1.8
2.1 ± 0.3 16.1 ± 1.8
3.8 ± 1.1
70.7 ± 1.9
16.8 ± 1.8
265 :~ 32 (11)
Estrus
31.3 j : 1.5
1.9 i 0.2 •3.8 ~: 0.7
3.5 ± 1.0
68.3 ± 3.0
17.6 ± 2.6
257 ± 31 (8)
Metestrus
30.0 ± 2.2
2.1 i 0.2 15.3 ± 1.8
4.4 = 1.7
68.3 ± 1.9
16.8 ± 2.4
285 ± 56 (10)
Diestrus 32 (6)
23.4 ± 4.6
1.8 ± 0.6 16.5 ~: 3.1
2.9 ~ 0.9
59.1 ± 7.8
t l . 9 ± 1.4
23l i
Males
1.4
29.8 ± 2.1
1.9 ± 0.1 12.8 ± 1.1
1.0 ± 0.5
74.7 i 2.2
13.6 i
147 ~ 35 (8)
Castrated females
** P < 0.01, *** P
