0013-7227/79/1056-1303$02.00/0 Endocrinology Copyright © 1979 by The Endocrine Society
Vol. 105, No. 6 Printed in U.S.A.
A Role for the Ovaries in Maturational Processes of Hypothalamic Neurons Containing Luteinizing HormoneReleasing Hormone* AYALLA BARNEA, GLORIA CHO, AND JOHN C. PORTER Cecil H. and Ida Green Center for Reproductive Biology Sciences, Departments of Obstetrics and Gynecology and Physiology, The University of Texas Health Science Center at Dallas, Southwestern Medical School, Dallas, Texas 75235
ABSTRACT. The role of the ovaries in the maturation of the hypothalamic LHRH neuron has been investigated. Female rats were castrated at 4 weeks of age, and the subcellular distribution as weLl as the content of LHRH in the hypothalamic homogenates was analyzed 2.5, 12, and 20 weeks after ovariectomy. Hypothalami were homogenized in an isoosmotic sucrose solution, and a 900 X g supernatant fluid was prepared. Free granules and synaptosomes containing LHRH were separated by means of continuous sucrose density gradient centrifugation. In hypothalamic homogenates of intact immature female rats, 35% of the LHRH was associated with free granules and 65% was associated with synaptosomes; in homogenates of adult rats, 57% of the LHRH was associated with free granules and 43% was associated with synaptosomes. Ovariectomy did not alter the postnatal changes in the fractional distribution of LHRH between these two subcellular compartments. The LHRH content of the hypothalamus as well as the
W
ITHIN neurons of the adult rat hypothalamus, LHRH is sequestered in granules (1-3), part of which is entrapped in synaptosomes during homogenization in an isoosmotic sucrose solution and the remainder of which appears in the homogenization medium as free granules (3, 4). These findings can be taken as indicative of the presence of LHRH-containing granules in two subcellular sites in the LHRH neuron, viz. presynaptic terminals which give rise to synaptosomes and structures which do not form synaptosomes. In addition, the distribution of LHRH between these two subcellular sites is age dependent; in hypothalamic homogenates of neonatal and immature rats, most of the LHRH is entrapped in synaptosomes, whereas in homogenates of mature rats, LHRH is distributed equally between synaptosomes and free granules (4). Received April 11, 1979. Address all correspondence and requests for reprints to: Dr. Ayalla Barnea, Department of Obstetrics and Gynecology, The University of Texas Health Science Center at Dallas, Southwestern Medical School, 5323 Harry Hines Boulevard, Dallas, Texas 75235. * This work was supported by Research Grants AM-01237, AG00306, HD-11149, and HD-10358 from the NIH, Bethesda, MD.
amount of LHRH sequestered in free granules and in synaptosomes prepared from intact female rats increased as a function of age of the donor animals, but this increase was abolished by ovariectomy. In addition, ovariectomy resulted in a significant (P < 0.02) decrease in the LHRH content of the hypothalamus, which was evident 2.5 weeks postoperatively. This reduction appears to be a consequence of a preferential decrease in the amount of LHRH present in structures which give rise to synaptosomes, i.e. presynaptic terminals. It is concluded that 1) the increased accumulation of LHRH which occurs during maturation of the hypothalamic LHRH neuron is dependent on ovarian function, 2) the subcellular distribution of LHRH is independent of ovarian function, and 3) the changes in the subcellular distribution of LHRH which occur during postnatal development are independent of ovarian function but may be dependent on age. (Endocrinology 105: 1303, 1979)
It has been demonstrated that the amount of LHRH in hypothalami of adult rats is under the regulatory control of ovarian steroids. Ovariectomy of adult female rats results in a marked reduction in the hypothalamic content of LHRH (5-7), whereas administration of estradiol benzoate to ovariectomized rats leads to an increase in the LHRH content of the hypothalamus (5, 7, 8). The LHRH content of the hypothalamus increases progressively with postnatal age (4, 9, 10), an increase which occurs concomitantly with an increase in the content of other neuronal constituents in the hypothalamus [for example, TRH (4, 11), somatostatin (12), a-MSH,1 norepinephrine, and dopamine (13,14)]. It can be envisioned that the maturation process of the LHRH neuron, i.e. changes in the subcellular distribution and content of LHRH, is associated with the growth of hypothalamic neurons and, hence, is subjected to regulatory mechanisms governing growth. On the other hand, the maturation process of the LHRH neuron may be under regulatory control of ovarian steroids. In the present study, the following questions were 1
Barnea, A., G. Cho, and J. C. Porter, unpublished observation.
1303
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 November 2015. at 15:02 For personal use only. No other uses without permission. . All rights reserved.
Endo • 1979 Vol 105 • No 6
BARNEA, CHO, AND PORTER
1304
addressed. Is the age-related increase in hypothalamic content of LHRH dependent on ovarian hormones? If so, is the effect of withdrawal of ovarian hormones manifested at a specific subcellular site in the LHRH neuron? Is the age-related change in the distribution of LHRH within the hypothalamic LHRH neurons dependent on ovarian hormones? To address these questions, immature female rats were castrated at 4 weeks of age, and the hypothalamic content of LHRH, the amount of LHRH associated with free granules and synaptosomes, and the distribution of LHRH between these two subcellular compartments were determined 2.5, 12, and 20 weeks after ovariectomy. Materials and Methods
X gav (29,000 rpm) in a Beckman L5-75 ultracentrifuge using an SW-40 rotor (Beckman Instruments, Inc., Palo Alto, CA). At the end of each run, gradient fractions (0.6 ml/fraction) were collected using an ISCO model 640 density gradient fractionator (Instrumentation Specialties Co., Lincoln, NE). The gradient fractions were heated in a boiling water bath for 10 min and stored at -20 C to await assay (15). When this procedure is followed, free granules and synaptosomes containing LHRH band at 0.82 and 1.16 M sucrose, respectively (3, 4). An example of a typical separation is illustrated in Fig. 1. The quantity of LHRH associated with free granules and with synaptosomes was quantified by integrating the area under the corresponding peak of LHRH. The recoveries of LHRH from the sucrose density gradients did not vary with the age of the animals from which hypothalami were obtained and were 75 ± 2.5% (mean ± SE; n = 27) for the intact animals and 73 ± 2.8% (n = 17) for the ovariectomized animals.
Animals Virgin female rats (4-24 weeks of age) of the Long-Evans strain were used. All animals were housed in an air-conditioned room (21-24 C) with controlled lighting (14 h of light, 10 h of darkness) and were supplied with food and water ad libitum. Vaginal smears were taken daily for 2 weeks before the experiment, except in the case of the 6.5-week-old rats which were used within 1 week after vaginal opening. Adult female rats having 4- or 5-day estrous cycles were used when they exhibited leucocytic vaginal smears. Some animals were ovariectomized or sham ovariectomized at. 4 weeks of age. The animals were killed by decapitation 8-9 h after the beginning of the light period, and the brains were rapidly removed and placed in icecold 0.15 M NaCl. Blood was collected in cold tubes containing disodium-EDTA (1-2 mg/ml blood) and centrifuged. The plasma was aspirated and frozen at —20 C to await assay.
RIAs LHRH was quantified by RIA according to Nett et al. (17) using synthetic LHRH (Beckman Instruments) as the reference standard. Bacitracin was included with the standards in a concentration equal to that in the samples. Standard curves for LHRH were not affected by bacitracin. LH was determined according to the procedure of Niswender et al. (18), and FSH was assayed according to the procedure outlined by the Rat Pituitary Hormone Program, NIH (Bethesda, MD). The values for LH and FSH are expressed in terms of NIAMDD-Rat LH-
SYNAPTOSOMES
210 ID
\ i
p
30
o—o GRANULES A — * SYNAPTOSOMES
•—•GRANULES * — A SYNAPTOSOMES
15 AGE; weeks
FIG. 2. The effect of ovariectomy (OV'X) on the hypothalamic content of LHRH and plasma concentrations of LH and FSH. Female rats were ovariectomized at 4 weeks of age, and LHRH (A), LH (B), and FSH (C) were determined 2.5, 12, and 20 weeks thereafter. The numbers in parentheses denote the numbers of determinations. The horizontal arrow illustrates the age range of the intact animals. E3, Range of undetectable concentrations of FSH.
I 0
(6) (9)
i
Ov'x
I 8
1
I 16
(12) i
I 24
I
0
(6) (9) i I 8
i
(5) I 16
i
(3) I 24
Ov'x
AGE; week
FIG. 4. The effect of ovariectomy (OV'X) on the relative distribution of LHRH between free granules and synaptosomes. For details, see Fig. 3.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 November 2015. at 15:02 For personal use only. No other uses without permission. . All rights reserved.
BARNEA, CHO, AND PORTER
1306
LHRH between free granules and synaptosomes. In hypothalamic homogenates prepared from adult animals, 57% of the LHRH was associated with free granules and 43% was associated with synaptosomes. Ovariectomy, performed at 4 weeks of age, did not affect the temporal changes in the relative distribution of LHRH between free granules and synaptosomes (Fig. 4B).
Discussion During maturation of the female rat there is a progressive accumulation of LHRH (4, 9, 10) as well as other peptides (4, 11, 12) and biogenic amines (13, 14) within the hypothalamus. Since in the adult female rat castration leads to a marked reduction in the hypothalamic content of LHRH (5-7), the question arises whether the age-related accumulation of LHRH in the hypothalamus is dependent on ovarian hormones. In the present study, we found that ovariectomy in the immature female rat prevented the accumulation of LHRH in the hypothalamus (Fig. 2A). Moreover, this effect of ovariectomy was manifested in the subneuronal structures which give rise to free granules (Fig. 3A) and synaptosomes, i.e. presynaptic terminals (Fig. 3B). In addition, ovariectomy led to a reduction in the hypothalamic content of LHRH, which appeared to be a consequence of a preferential reduction in the amount of LHRH present in the presynaptic terminals (Fig. 3B). We speculate that a progressive increase in the rates of biosynthesis and storage of LHRH contributes to the increased accumulation of LHRH seen in the hypothalamic neurons of the maturing female rat. It is not known whether the ovariectomy-induced decrease in hypothalamic content of LHRH seen in this study is due primarily to a reduction in the storage of the peptide or whether it is a consequence of a concomitant decrease in the rates of biosynthesis and storage of LHRH. The correlated decrease in hypothalamic content of LHRH and the increase in plasma levels of LH and FSH seen in immature (the present study) as well as mature (6, 7) ovariectomized female rats is suggestive of an ovariectomy-induced increase in the secretion of LHRH from the hypothalamus. It is of interest that although ovarian hormones have profound effects on the process of accumulation of LHRH, they do not affect the distribution of LHRH within the LHRH neuron. Thus, the changes which occur during postnatal development in the subcellular distribution of LHRH appear to be independent of ovarian function. Since it is known that thyroid hormones are involved in the regulation of maturational processes in the brain, the question arises whether maturation of the hypothalamic LHRH neuron is under the regulation of thyroid hormones. Alternatively, it is possible that the subcellular distribution of LHRH is an inherent property
Endo 1979 Vol 105 , No 6
of the hypothalamic LHRH neuron and that the changes seen during development are a consequence of age.
Acknowledgments The authors thank Sherry Whitehurst, Sue Sherwin Martin, Linda Akers, Jodie Roberts, and Gaye Burnsed for excellent technical assistance and lone Crandell and Judy Wagers for valuable editorial assistance. The antiserum to rat FSH and the reference preparations used in the assay of FSH and LH were provided by the NIAMDD Rat Pituitary Hormone Program and Dr. A. Parlow. The antisera to LH and LHRH were gifts from Dr. G. D. Niswender.
References 1. Goldsmith, P. C, and W. F. Ganong, Ultrastructural localization of luteinizing hormone-releasing hormone in the median eminence of the rat, Brain Res 97: 181, 1975. 2. Styne, D. M., P. C. Goldsmith, S. R. Burstein, S. L. Kaplan, and M. M. Grumbach, Immunoreactive somatostatin and LRF in median eminence synaptosomes: detection by immunohistochemistry and quantification by radioimmunoassay, Endocrinology 101: 1099, 1977. 3. Barnea, A., W. B. Neaves, G. Cho, and J. C. Porter, A subcellular pool of hypo-osmotically resistant particles containing thyrotropin releasing hormone, a-melanocyte stimulating hormone, and luteinizing hormone releasing hormone in the rat hypothalamus, J Neurochem 30: 937, 1978. 4. Barnea, A., W. B. Neaves, and J. C. Porter, Ontogeny of the subcellular compartmentalization of thyrotropin releasing hormone and luteinizing hormone releasing hormone in the rat hypothalamus, Endocrinology 100: 1068, 1977. 5. Araki, S., M. Ferin, E. A. Zimmerman, and R. L. Vande Wiele, Ovarian modulation of immunoreactive gonadotropin-releasing hormone (Gn-RH) in the rat brain: evidence for a differential effect on the anterior and mid-hypothalamus, Endocrinology 96: 644, 1975. 6. Wheaton, J. E., and S. M. McCann, Luteinizing hormone-releasing hormone in peripheral plasma and hypothalamus of normal and ovariectomized rats, Neuroendocrinology 20: 296, 1976. 7. Kobayashi, R. M., K. H. Lu, R. Y. Moore, and S. S. C. Yen, Regional distribution of hypothalamic luteinizing hormone-releasing hormone in proestrous rats: effects of ovariectomy and estrogen replacement, Endocrinology 102: 98, 1978. 8. Kalra, S. P., Tissue levels of luteinizing hormone-releasing hormone in the preoptic area and hypothalamus, and serum concentrations of gonadotropins following anterior hypothalamic deafferentation and estrogen treatment of the female rat, Endocrinology 99: 101, 1976. 9. Araki, S., C. D. Toran-AUerand, M. Ferin, and R. L. Vande Wiele, Immunoreactive gonadotropin-releasing hormone (Gn-RH) during maturation in the rat: ontogeny of regional hypothalamic differences, Endocrinology 97: 693, 1975. 10. Dussault, J. H., P. Walker, J. D. Dubois, and F. Labrie, The development of the hypothalamo-pituitary axis in the neonatal rat: sexual maturation in male and female rats as assessed by hypothalamic LHRH and pituitary and serum LH and FSH concentrations, CanJPhysiol Pharmacol 55: 1091, 1977. 11. Dussault, J. H., and F. Labrie, Development of the hypothalamicpituitary-thyroid axis in the neonatal rat, Endocrinology 97: 1321, 1975. 12. Walker, P., J. H. Dussault, G. Alvarado-Urbina, and A. Dupont, The development of the hypothalamo-pituitary axis in the neonatal rat: hypothalamic somatostatin and pituitary and serum growth hormone concentrations, Endocrinology 101: 782, 1977. 13. Coyle, J. T., and J. Axelrod, Development of the uptake and storage of L-[3H]norepinephrine in the rat brain, J Neurochem 18: 2061, 1971. 14. Coyle, J. T., and D. Henry, Catecholamines in fetal and newborn rat brain, J Neurochem 21: 61, 1973.
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MATURATION OF HYPOTHALAMIC NEURONS 15. Barnea, A., C. Oliver, and J. C. Porter, Subcellular localization of a-melanocyte-stimulating hormone in the rat hypothalamus, J Neurochem 29: 619, 1977. X 16. Barnea, A., N. Ben-Jonathan, C. Colston, J. M. Johnston, and J. C. Porter, Differential sub-cellular compartmentalization of thyrotro
Vol. 105, No. 6 Printed in U.S.A.
A Role for the Ovaries in Maturational Processes of Hypothalamic Neurons Containing Luteinizing HormoneReleasing Hormone* AYALLA BARNEA, GLORIA CHO, AND JOHN C. PORTER Cecil H. and Ida Green Center for Reproductive Biology Sciences, Departments of Obstetrics and Gynecology and Physiology, The University of Texas Health Science Center at Dallas, Southwestern Medical School, Dallas, Texas 75235
ABSTRACT. The role of the ovaries in the maturation of the hypothalamic LHRH neuron has been investigated. Female rats were castrated at 4 weeks of age, and the subcellular distribution as weLl as the content of LHRH in the hypothalamic homogenates was analyzed 2.5, 12, and 20 weeks after ovariectomy. Hypothalami were homogenized in an isoosmotic sucrose solution, and a 900 X g supernatant fluid was prepared. Free granules and synaptosomes containing LHRH were separated by means of continuous sucrose density gradient centrifugation. In hypothalamic homogenates of intact immature female rats, 35% of the LHRH was associated with free granules and 65% was associated with synaptosomes; in homogenates of adult rats, 57% of the LHRH was associated with free granules and 43% was associated with synaptosomes. Ovariectomy did not alter the postnatal changes in the fractional distribution of LHRH between these two subcellular compartments. The LHRH content of the hypothalamus as well as the
W
ITHIN neurons of the adult rat hypothalamus, LHRH is sequestered in granules (1-3), part of which is entrapped in synaptosomes during homogenization in an isoosmotic sucrose solution and the remainder of which appears in the homogenization medium as free granules (3, 4). These findings can be taken as indicative of the presence of LHRH-containing granules in two subcellular sites in the LHRH neuron, viz. presynaptic terminals which give rise to synaptosomes and structures which do not form synaptosomes. In addition, the distribution of LHRH between these two subcellular sites is age dependent; in hypothalamic homogenates of neonatal and immature rats, most of the LHRH is entrapped in synaptosomes, whereas in homogenates of mature rats, LHRH is distributed equally between synaptosomes and free granules (4). Received April 11, 1979. Address all correspondence and requests for reprints to: Dr. Ayalla Barnea, Department of Obstetrics and Gynecology, The University of Texas Health Science Center at Dallas, Southwestern Medical School, 5323 Harry Hines Boulevard, Dallas, Texas 75235. * This work was supported by Research Grants AM-01237, AG00306, HD-11149, and HD-10358 from the NIH, Bethesda, MD.
amount of LHRH sequestered in free granules and in synaptosomes prepared from intact female rats increased as a function of age of the donor animals, but this increase was abolished by ovariectomy. In addition, ovariectomy resulted in a significant (P < 0.02) decrease in the LHRH content of the hypothalamus, which was evident 2.5 weeks postoperatively. This reduction appears to be a consequence of a preferential decrease in the amount of LHRH present in structures which give rise to synaptosomes, i.e. presynaptic terminals. It is concluded that 1) the increased accumulation of LHRH which occurs during maturation of the hypothalamic LHRH neuron is dependent on ovarian function, 2) the subcellular distribution of LHRH is independent of ovarian function, and 3) the changes in the subcellular distribution of LHRH which occur during postnatal development are independent of ovarian function but may be dependent on age. (Endocrinology 105: 1303, 1979)
It has been demonstrated that the amount of LHRH in hypothalami of adult rats is under the regulatory control of ovarian steroids. Ovariectomy of adult female rats results in a marked reduction in the hypothalamic content of LHRH (5-7), whereas administration of estradiol benzoate to ovariectomized rats leads to an increase in the LHRH content of the hypothalamus (5, 7, 8). The LHRH content of the hypothalamus increases progressively with postnatal age (4, 9, 10), an increase which occurs concomitantly with an increase in the content of other neuronal constituents in the hypothalamus [for example, TRH (4, 11), somatostatin (12), a-MSH,1 norepinephrine, and dopamine (13,14)]. It can be envisioned that the maturation process of the LHRH neuron, i.e. changes in the subcellular distribution and content of LHRH, is associated with the growth of hypothalamic neurons and, hence, is subjected to regulatory mechanisms governing growth. On the other hand, the maturation process of the LHRH neuron may be under regulatory control of ovarian steroids. In the present study, the following questions were 1
Barnea, A., G. Cho, and J. C. Porter, unpublished observation.
1303
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 November 2015. at 15:02 For personal use only. No other uses without permission. . All rights reserved.
Endo • 1979 Vol 105 • No 6
BARNEA, CHO, AND PORTER
1304
addressed. Is the age-related increase in hypothalamic content of LHRH dependent on ovarian hormones? If so, is the effect of withdrawal of ovarian hormones manifested at a specific subcellular site in the LHRH neuron? Is the age-related change in the distribution of LHRH within the hypothalamic LHRH neurons dependent on ovarian hormones? To address these questions, immature female rats were castrated at 4 weeks of age, and the hypothalamic content of LHRH, the amount of LHRH associated with free granules and synaptosomes, and the distribution of LHRH between these two subcellular compartments were determined 2.5, 12, and 20 weeks after ovariectomy. Materials and Methods
X gav (29,000 rpm) in a Beckman L5-75 ultracentrifuge using an SW-40 rotor (Beckman Instruments, Inc., Palo Alto, CA). At the end of each run, gradient fractions (0.6 ml/fraction) were collected using an ISCO model 640 density gradient fractionator (Instrumentation Specialties Co., Lincoln, NE). The gradient fractions were heated in a boiling water bath for 10 min and stored at -20 C to await assay (15). When this procedure is followed, free granules and synaptosomes containing LHRH band at 0.82 and 1.16 M sucrose, respectively (3, 4). An example of a typical separation is illustrated in Fig. 1. The quantity of LHRH associated with free granules and with synaptosomes was quantified by integrating the area under the corresponding peak of LHRH. The recoveries of LHRH from the sucrose density gradients did not vary with the age of the animals from which hypothalami were obtained and were 75 ± 2.5% (mean ± SE; n = 27) for the intact animals and 73 ± 2.8% (n = 17) for the ovariectomized animals.
Animals Virgin female rats (4-24 weeks of age) of the Long-Evans strain were used. All animals were housed in an air-conditioned room (21-24 C) with controlled lighting (14 h of light, 10 h of darkness) and were supplied with food and water ad libitum. Vaginal smears were taken daily for 2 weeks before the experiment, except in the case of the 6.5-week-old rats which were used within 1 week after vaginal opening. Adult female rats having 4- or 5-day estrous cycles were used when they exhibited leucocytic vaginal smears. Some animals were ovariectomized or sham ovariectomized at. 4 weeks of age. The animals were killed by decapitation 8-9 h after the beginning of the light period, and the brains were rapidly removed and placed in icecold 0.15 M NaCl. Blood was collected in cold tubes containing disodium-EDTA (1-2 mg/ml blood) and centrifuged. The plasma was aspirated and frozen at —20 C to await assay.
RIAs LHRH was quantified by RIA according to Nett et al. (17) using synthetic LHRH (Beckman Instruments) as the reference standard. Bacitracin was included with the standards in a concentration equal to that in the samples. Standard curves for LHRH were not affected by bacitracin. LH was determined according to the procedure of Niswender et al. (18), and FSH was assayed according to the procedure outlined by the Rat Pituitary Hormone Program, NIH (Bethesda, MD). The values for LH and FSH are expressed in terms of NIAMDD-Rat LH-
SYNAPTOSOMES
210 ID
\ i
p
30
o—o GRANULES A — * SYNAPTOSOMES
•—•GRANULES * — A SYNAPTOSOMES
15 AGE; weeks
FIG. 2. The effect of ovariectomy (OV'X) on the hypothalamic content of LHRH and plasma concentrations of LH and FSH. Female rats were ovariectomized at 4 weeks of age, and LHRH (A), LH (B), and FSH (C) were determined 2.5, 12, and 20 weeks thereafter. The numbers in parentheses denote the numbers of determinations. The horizontal arrow illustrates the age range of the intact animals. E3, Range of undetectable concentrations of FSH.
I 0
(6) (9)
i
Ov'x
I 8
1
I 16
(12) i
I 24
I
0
(6) (9) i I 8
i
(5) I 16
i
(3) I 24
Ov'x
AGE; week
FIG. 4. The effect of ovariectomy (OV'X) on the relative distribution of LHRH between free granules and synaptosomes. For details, see Fig. 3.
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BARNEA, CHO, AND PORTER
1306
LHRH between free granules and synaptosomes. In hypothalamic homogenates prepared from adult animals, 57% of the LHRH was associated with free granules and 43% was associated with synaptosomes. Ovariectomy, performed at 4 weeks of age, did not affect the temporal changes in the relative distribution of LHRH between free granules and synaptosomes (Fig. 4B).
Discussion During maturation of the female rat there is a progressive accumulation of LHRH (4, 9, 10) as well as other peptides (4, 11, 12) and biogenic amines (13, 14) within the hypothalamus. Since in the adult female rat castration leads to a marked reduction in the hypothalamic content of LHRH (5-7), the question arises whether the age-related accumulation of LHRH in the hypothalamus is dependent on ovarian hormones. In the present study, we found that ovariectomy in the immature female rat prevented the accumulation of LHRH in the hypothalamus (Fig. 2A). Moreover, this effect of ovariectomy was manifested in the subneuronal structures which give rise to free granules (Fig. 3A) and synaptosomes, i.e. presynaptic terminals (Fig. 3B). In addition, ovariectomy led to a reduction in the hypothalamic content of LHRH, which appeared to be a consequence of a preferential reduction in the amount of LHRH present in the presynaptic terminals (Fig. 3B). We speculate that a progressive increase in the rates of biosynthesis and storage of LHRH contributes to the increased accumulation of LHRH seen in the hypothalamic neurons of the maturing female rat. It is not known whether the ovariectomy-induced decrease in hypothalamic content of LHRH seen in this study is due primarily to a reduction in the storage of the peptide or whether it is a consequence of a concomitant decrease in the rates of biosynthesis and storage of LHRH. The correlated decrease in hypothalamic content of LHRH and the increase in plasma levels of LH and FSH seen in immature (the present study) as well as mature (6, 7) ovariectomized female rats is suggestive of an ovariectomy-induced increase in the secretion of LHRH from the hypothalamus. It is of interest that although ovarian hormones have profound effects on the process of accumulation of LHRH, they do not affect the distribution of LHRH within the LHRH neuron. Thus, the changes which occur during postnatal development in the subcellular distribution of LHRH appear to be independent of ovarian function. Since it is known that thyroid hormones are involved in the regulation of maturational processes in the brain, the question arises whether maturation of the hypothalamic LHRH neuron is under the regulation of thyroid hormones. Alternatively, it is possible that the subcellular distribution of LHRH is an inherent property
Endo 1979 Vol 105 , No 6
of the hypothalamic LHRH neuron and that the changes seen during development are a consequence of age.
Acknowledgments The authors thank Sherry Whitehurst, Sue Sherwin Martin, Linda Akers, Jodie Roberts, and Gaye Burnsed for excellent technical assistance and lone Crandell and Judy Wagers for valuable editorial assistance. The antiserum to rat FSH and the reference preparations used in the assay of FSH and LH were provided by the NIAMDD Rat Pituitary Hormone Program and Dr. A. Parlow. The antisera to LH and LHRH were gifts from Dr. G. D. Niswender.
References 1. Goldsmith, P. C, and W. F. Ganong, Ultrastructural localization of luteinizing hormone-releasing hormone in the median eminence of the rat, Brain Res 97: 181, 1975. 2. Styne, D. M., P. C. Goldsmith, S. R. Burstein, S. L. Kaplan, and M. M. Grumbach, Immunoreactive somatostatin and LRF in median eminence synaptosomes: detection by immunohistochemistry and quantification by radioimmunoassay, Endocrinology 101: 1099, 1977. 3. Barnea, A., W. B. Neaves, G. Cho, and J. C. Porter, A subcellular pool of hypo-osmotically resistant particles containing thyrotropin releasing hormone, a-melanocyte stimulating hormone, and luteinizing hormone releasing hormone in the rat hypothalamus, J Neurochem 30: 937, 1978. 4. Barnea, A., W. B. Neaves, and J. C. Porter, Ontogeny of the subcellular compartmentalization of thyrotropin releasing hormone and luteinizing hormone releasing hormone in the rat hypothalamus, Endocrinology 100: 1068, 1977. 5. Araki, S., M. Ferin, E. A. Zimmerman, and R. L. Vande Wiele, Ovarian modulation of immunoreactive gonadotropin-releasing hormone (Gn-RH) in the rat brain: evidence for a differential effect on the anterior and mid-hypothalamus, Endocrinology 96: 644, 1975. 6. Wheaton, J. E., and S. M. McCann, Luteinizing hormone-releasing hormone in peripheral plasma and hypothalamus of normal and ovariectomized rats, Neuroendocrinology 20: 296, 1976. 7. Kobayashi, R. M., K. H. Lu, R. Y. Moore, and S. S. C. Yen, Regional distribution of hypothalamic luteinizing hormone-releasing hormone in proestrous rats: effects of ovariectomy and estrogen replacement, Endocrinology 102: 98, 1978. 8. Kalra, S. P., Tissue levels of luteinizing hormone-releasing hormone in the preoptic area and hypothalamus, and serum concentrations of gonadotropins following anterior hypothalamic deafferentation and estrogen treatment of the female rat, Endocrinology 99: 101, 1976. 9. Araki, S., C. D. Toran-AUerand, M. Ferin, and R. L. Vande Wiele, Immunoreactive gonadotropin-releasing hormone (Gn-RH) during maturation in the rat: ontogeny of regional hypothalamic differences, Endocrinology 97: 693, 1975. 10. Dussault, J. H., P. Walker, J. D. Dubois, and F. Labrie, The development of the hypothalamo-pituitary axis in the neonatal rat: sexual maturation in male and female rats as assessed by hypothalamic LHRH and pituitary and serum LH and FSH concentrations, CanJPhysiol Pharmacol 55: 1091, 1977. 11. Dussault, J. H., and F. Labrie, Development of the hypothalamicpituitary-thyroid axis in the neonatal rat, Endocrinology 97: 1321, 1975. 12. Walker, P., J. H. Dussault, G. Alvarado-Urbina, and A. Dupont, The development of the hypothalamo-pituitary axis in the neonatal rat: hypothalamic somatostatin and pituitary and serum growth hormone concentrations, Endocrinology 101: 782, 1977. 13. Coyle, J. T., and J. Axelrod, Development of the uptake and storage of L-[3H]norepinephrine in the rat brain, J Neurochem 18: 2061, 1971. 14. Coyle, J. T., and D. Henry, Catecholamines in fetal and newborn rat brain, J Neurochem 21: 61, 1973.
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MATURATION OF HYPOTHALAMIC NEURONS 15. Barnea, A., C. Oliver, and J. C. Porter, Subcellular localization of a-melanocyte-stimulating hormone in the rat hypothalamus, J Neurochem 29: 619, 1977. X 16. Barnea, A., N. Ben-Jonathan, C. Colston, J. M. Johnston, and J. C. Porter, Differential sub-cellular compartmentalization of thyrotro
