Increased incorporation of 3H-lysine in specific hypothalamic nuclei following castration in the male rat

EXPERIMENTAL

NEUROLOGY

40,

309-315 (1973)

Increased Incorporation of 3H-Lysine in Specific Hypothalamic Nuclei Following Castration in the Male Rat M. LITTERIA Neurology Service, Veterans Administration

l Hospital, Downey, Illinois 60064

Received January 29,1973 The purpose of the present quantitative radioautographic investigation was to determine if castration in the adult male rat is followed by alterations in the incorporation of 8H-lysine into neurons of the preoptic and specific hypothalamic nuclei. Five adult, intact male rats and five male rats castrated for a period of 5 wk were each injected ip with 2 &i/g body wt of L-4,5-3H (n) lysine monohydrochloride (sp act = 7.5 Ci/mmol). Thirty minutes later the rats were perfused with a formaldehyde fixing solution and 6-&m sections cut from paraplast-embedded brains were processed for radioautography. Silver grains per neuron were determined for the medial and lateral preoptic nuclei and for eight hypothalamic nuclei, i.e., anterior, arcuate, dorsomedial, paraventricular, periventricular, suprachiasmatic, supraoptic, and ventromedial. Incorporation of ‘H-lysine was significantly increased in neurons of the arcuate, periventicular, paraventricular, and supraoptic nuclei following castration. Enhancement of protein synthesis in specific hypothalamic nuclei 5 wk following removal of the major source of androgen suggests that these nuclei represent androgen sensitive, negative feedback sites.

INTRODUCTION Electron microscopic (1, 24, 25) as well as biochemical studies (20) indicate alterations in protein synthesis of specific regions of the brain following castration in the rat. Electron micrographs of the supraoptic and paraventricular nuclei (24) and arcuate nucleus (25) from male and female rats castrated for either 1 or 6 mo demonstrated hypertrophy of the endoplasmic reticulum and an increase in the number of ribosomes. Additional structural changes which may be associated with enhanced protein synthesis have been described for the arcuate nucleus at various intervals 1 Supported in part by a Grant from the Rockefeller Foundation to the Laboratories for Reproductive Biology, University of North Carolina at Chapel Hill and by VA Hospital, Downey, Illinois. 309 Copyright 0 1973 hy AcademicPress,Inc. All rights of reproduction in any form reserved.

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following castration in the male rat (1). The in tivo incorporation of radioactive phenylalanine into proteins of the hypothalamus of male rats was significantly increased 6 wk following castration (20). Treatment with testosterone prevented this increase in the castrated rats (20). The purpose of the present investigation was to localize by quantitative radioautography hypothalamic nuclei responding to androgen removal by alterations in protein synthesis. The utilization of radioautography as a technique for the localization of newly synthesized proteins following the injection of a labeled amino acid has been extensively reviewed (4, 18). For example, following the injection of a labeled amino acid, approximately 96% of the radioactivity remaining in tissue sections after histologic processing is bound to protein by peptide bonds, any remaining free amino acid being removed during tissue preparation (4). While many factors influence protein synthesis, e.g., RNA translation and transcription as well as the size of the intracellular free amino acid pool, the observed reduced silver grain represents a terminal event, namely, the incorporation of amino acid into protein. The initial events responsible for any alterations are not defined by a radioautographic study. METHODS Holtzman male rats were maintained on a 12/12 hour light/dark schedule and received Purina rat chow and water ad lib. At 2 mo of age some of the rats were castrated and the remaining intact rats served as the control group. *Five weeks after castration, each rat per group received an injection of 2 &i/g body wt, ip, of L-[4,5-3H(n)]-lysine monohydrochloride (sp act = 7.5 Ci/mmol, Amersham/Searle) . The advantages in utilizing labeled lysine in this study have been discussed (17). Thirty minutes later sodium pentobarbital (25 mg/lOO g body wt, ip) was injected and within 3 min the degree of anesthesia was sufficient to allow perfusion of the brain via the left ventricle of the heart at a constant pressure of 90 mm Hg (15). After perfusion, the brains were stored in 10% formalin for 2-3 wk. Coronal sections were cut at 6 pm from paraplast-embedded brains. These were deparaffinized and coated with Kodak liquid emulsion NTB 2 according to the radioautographic technique of Kopriwa and Leblond (16). After an exposure period of 7 wk at 4 C, the sections were developed and stained with methylgreen pyronin. Silver grains per neuron (nucleus and cytoplasm) for the preoptic and hypothalamic nuclei listed in Table 1 were determined by the same person in all focus planes at a magnification of 1600. Grains were recorded for each group of five rats from 150 neurons (30 neurons/rat) for each nucleus. Background radiation assessed from comparable brain sections

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~~YP~THALAMIC

311

from noninjected rats was negligible (ca. O-2 grains/neuron). The slides were labeled in such a manner that the experimental and control groups were not known until the final compilation of the data. Significance of differences was determined between the castrated and intact groups by Student’s t test; P 5 0.05 was considered significant. RESULTS The data in Table 1 indicate that incorporation proteins was very significantly increased (P < 0.001) TABLE SILVER

GRAINS

PER NEURON NUCLEI FROM

of 3H-lysine into for neurons of the

1

FOR THE PREOPTIC AND SELECTED HYPOTHALAMIC INTACT AND CASTRATED MALE RATS Silver

grains

per neurona

P

Nucleus Intact Arcuate Paraventricular Periventricular Supraoptic Anterior Dorsomedial Lateral preoptic Medial preoptic Suprachiasmatic Ventromedial a Mean zk standard error; per group. b P value is not significant.

17.65 f 55.20 f 16.35 f 51.80 f 19.00 f 19.40 f 18.43 f 18.25 f 12.47 f 20.13 f the mean

Castrated .54 .71 .31 1.01 .53 .47 .40 56 .40 .49

represents

31.68 71.37 23.63 70.22 18.23 18.57 18.73 18.62 12.50 20.67 the value

f f f f f f f f f f

1.30 2.01 1.15 1.30 .16 .68 .40 .49 .41 .44 obtained


five rat brains

arcuate, paraventricular, periventricular, and supraoptic nuclei following castration of adult male rats for 5 wk. Incorporation of 3H-lysine was greatest for neurons of the paraventricular and supraoptic nuclei. The ability of these nuclei to synthesize large amounts of protein from amino acids has been previously reported (22)) and may be related to the high neurosecretory activity or the rich vascularity (or both) of these two nuclei. As expected, silver grains were distributed over the neuronal nucleus and cytoplasm (4), however, no attempt was made to obtain a distribution ratio per neuron.

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DISCUSSION Changes in neuronal nucleolar volume have been associated with changes in cellular protein synthetic activity, i.e., an increase in nucleolar size is indicative of enhanced activity whereas a decrease in nucleolar size represents depressed activity (5). This technique has been utilized to localize hypothalamic feedback sites responding to castration (11) and gonadotropic hormone replacement ( 12)) however, interpretation of these data has presented difficulties (11, 12). More direct experimental approaches have provided both structural and biochemical evidence of an enhancement of protein synthesis in the hypothalamus following remova of the gonads in the rat (1, 20, 24, 25). The present investigation corroborates and extends these studies. Of the eight hypothalamic nuclei examined (Table 1 ), only neurons of the arcuate, periventricular, paraventricular, and supraoptic demonstrated an increase in the incorporation of 3H-lysine into proteins 5 wk following castration. This data, with the exception of the periventricular nucleus, is in agreement with the studies with the electron microscope providing structural evidence for the enhancement of protein synthesis following castration, i.e., hypertrophy of the endoplasmic reticulum and an increase in the number of ribosomes (24, 25). The periventricular nucleus, which represents an extension of the arcuate nucleus along the walls of the third ventricle, was not examined in those studies. The structural changes indicative of enhanced protein synthesis in the supraoptic and paraventicular nuclei following castration have been associated with an increase in the synthesis of the polypeptide neurohypophysial hormones, i.e., oxytocin and vasopressin (24, 26). Since lysine is absent from rat neurohypophysial hormones (7), the increase in the incorporation of 3H-lysine into nonspecific protein of the supraoptic and paraventricular nuclei suggests that androgens exert a general effect on protein synthesis in specific hypothalamic nuclei. This is supported by the observation that treatment of the castrated male rats with testosterone reversed the above described structural changes (24). The arcuate and periventricular nuclei are part of the so-called “hypophysiotropic area” of the hypothalamus implicated in the synthesis of the polypeptide gonadotropin releasing hormone(s) (GRH) which regulate the secretion of the pituitary gonadotropins (10). Both nuclei responded to castration with an increase in the incorporation of 3H-lysine into protein (Table 1) . Although the structure of rat GRH is not known, it is tempting to speculate that if lysine is present in rat GRH, then removal of androgens stimulates the synthesis or turnover of GRH. In this regard it has been noted that castration of male rats is followed by an increase in the hypo-

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thalamic content of follicle stimulating-releasing hormone and that treatment with testosterone depressed the levels of follicle stimulating and luteinizing hormone-releasing hormones (‘2). These observations are consistent with the well-known increase in the synthesis and secretion of pituitary gonadotropins following castration (6). In addition to the structural changes indicative of enhanced protein synthesis, there was a marked increase in the number of granulated vesicles in the neurons of the arcuate nucleus following castration (25). Although speculative, the dense core vesicles may contain catecholamines (25) or GRH, or both, ( 13). However, the interrelationships between hypothalamic catecholamines and GRH regulating the secretion of the pituitary gonadotropins have not been clarified (19). In addition to the eight hypothalamic nuclei, the preoptic nuclei (Table 1) were also evaluated because of the involvement of the medial preoptic nucleus in the regulation of sexual behavior in the male rat (3). The medial and lateral preoptic nuclei did not demonstrate alterations in the incorporation of SH-lysine into proteins. The specific activity of L- [ SH] -phenylalanine incorporated into hypothalamic proteins was significantly increased 6 wk following castration in male rats (20). However, 3 wk following castration the specific activity of L- [ *HI -phenylalanine was decreased below that of the intact controls. The significance of this biphasic effect is not clear; however, the decrease in specific activity observed 3 wk following castration was at the lower limit of significance, i.e., P < 0.05 (20). Nevertheless, replacement therapy with testosterone returned the values to those of the intact controls for both castration intervals (20). In a recent study (21), fetal male rats were either castrated or sham operated in ufero on the twenty-first day of pregnancy. Twenty-four hours later they were injected in utero with SH-leucine, and 1 hr later the brains were processed for radioautography. Silver grains were determined for the arcuate, paraventricular, supraoptic, and ventromedial nuclei. A significant increase in the incorporation of SH-leucine into proteins of the arcuate nucleus was found following castration. Since the arcuate nucleus has been implicated in the synthesis of the GRH, it was proposed that the enhanced incorporation of SH-leucine into neurons of the arcuate nucleus following castration was associated with an increase in the synthesis of the GRH (21). In contrast to the enhancement of protein synthesis in specific hypothalamic nuclei following castration of the adult and fetal male rat, castration at 2 days of age was followed by an inhibition in the accumulation of DL-[3H]-lysine in the protein containing fraction of the brain 5 wk later (14). The presence of androgen during the first days after birth is essential

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for the development of the male “acyclic” hypothalamus and normal sexual behavior in the rat (9). Therefore, it is not surprising that alterations in protein metabolism occur following castration during this critical period of neural development. Considerable difficulties are encountered in attempting to correlate the uptake of a labeled steroid with the site of hormone action (8)) nevertheless, 3H-testosterone has been found to accumulate in neurons of the male anterior hypothalamus, the arcuate and ventromedial nuclei, and hippocampus (23). In summary, the increase in the incorporation of 3H-lysine into proteins of specific hypothalamic nuclei, i.e., arcuate, paraventricular, periventricular, and supraoptic 5 wk following castration of adult male rats suggests that these nuclei represent androgen sensitive feedback sites. Studies are in progress to determine if other areas of the brain respond to castration by alterations in protein synthesis. REFERENCES 1. BRAWER, J. R. 1971. The role of the arcuate nucleusin the brain-pituitarygonadaxis. 3. Camp.Near. 143: 411-446. 2. DONOVAN, B. T. 1970.“‘MammalianNeuroendocrinology,” pp. 121-122.McGrawHill, New York. 3. DARNER, G. F. D~CKE, and S. MOUSTAFA. 1968. Differential localization of a male and female hypothalamic mating centre. J. Reprod. Fe&. 17: 583-586. 4. DROZ, B., and H. L. KOENIG. 1970, Localization of protein metabolism in neurons, pp. 93-108. In “Protein Metabolism of the Nervous System.” A. Lajtha [Ed.]. Plenum Press, New York. 5. EDSTRGM, J. E., and D. EICHNER. 1958. Relation between nucleolar volume and cell body content of ribonucleic acid in supraoptic neurons. Nature (London) 181: 619. 6. GAY, V. L., and A. R. MIDCLEY. 1969. Response of the adult rat to orchidectomy and ovariectomy as determined by LH radioimmunoassay. Endocrinology 84: 1359-1364. 7. GOREMAN, A., and H. A. BERN. 1962. “Textbook of Comparative Endocrinology,” pp. 72-73. Wiley, New York. 8. GORSKI, R. A., and L. G. CLEMENS. 1970. Action of gonadal hormones, pp. 429Vol. 4. A. Lajtha [Ed.]. Plenum 449. In “Handbook of Neurochemistry,” Press, New York. 9. GORSKI, R. A. 1971. Gonadal hormones and the perinatal development of neuroendocrine function, pp. 237-290. 1% “Frontiers of Neuroendocrinology.” L. Martini and W. F. Ganong [Eds.]. Oxford University Press, New York. 10. HALASZ, B., L. PUPP, S. UHLARIK, and L. TIMA. 1965. Further studies on the hormone secretion of the anterior pituitary transplanted into the hypophysiotrophic area of the rat hypothalamus. Endocrinology 77: 343-355. 11. IFFT, J. D., 1964. The effect of endocrine gland extirpations on the size of nucleoli in rat hypothalamic neurons. Anat. Rec. 148: 599-603. 12. IFFT, J. D., 1%6. Further evidence on an “internal” feedback from the adenohypophysis to the hypothalamus. Neuroendocrinology 1: 350-357.

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