1971). The injection of 6-OH-dopamine results in selective degeneration. puberty. The local microinjection of 6-OH-dopamine into the locus coeruleus

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1 Central Research Division, Postgraduate Medical School, Budapest, Hungary EFFECT OF THE DEPLETION OF BRAIN NORADRENALINE ON THE PLASMA FSH AND GROWTH HORMONE LEVELS IN OVARIECTOMIZED RATS By E. Endr\l=o"\czi,I. Marton, Z. Radnai and J. Bir\l=o'\ ABSTRACT The electrolytic lesion of locus coeruleus reduced the hypothalamic noradrenaline (NE) content to 28 % of the control values and resulted in a marked decrease of the plasma FSH level in ovariectomized rats before puberty. The local microinjection of 6-OH-dopamine into the locus coeruleus led to a reduction in the hypothalamic NE content to 21 % of the control level and induced a decrease of the compensatory ovarian hypertrophy and that of the plasma FSH level. The electrolytic lesion of the locus coeruleus or the 6-OH-dopamine treatment failed to influence the plasma growth hormone (GH) level. The observations support the hypothesis that ventral noradrenergic projection is involved in controlling the pituitary FSH secretion. There is histofluorescent evidence that the hypothalamus contains NE projec tion of the ventral ascending adrenergic pathway, which innervates the neurosecretory elements of the basal-medial hypothalamus (Fuxe et al. 1968; Ungerstedt 1971). The injection of 6-OH-dopamine results in selective degeneration of central catecholaminergic pathways (Ungerstedt 1971). Thus, the anterolateral injection of 6-OH-dopamine in the hypothalamus produces a significant reduction in the NE content (Sechzer et al. 1973). Other observations support the hypothesis that NE fibers are involved in the hypothalamic mechanism activating the pituitary gonadotrophin release

2 - (Kalra 8c McCann 1973; Sawyer et al. 1974; Marion 8c Endröczi 1976). Preg nant mare serum-induced ovulation in the immature rat can be blocked by the depletion of brain catecholamines with reserpine or a-methyl-p-tyrosine (Coppola et al. 1966). In the present investigations we have studied the effect of the depletion of brain catecholamines after electrolytic lesion of the locus coeruleus or local injection of 6-OH-dopamine, on the plasma FSH and GH levels and on the compensatory hypertrophy of the ovaries in immature rats. A total of 84 MATERIALS AND METHODS female R-Amsterdam rats were used in the study. The animals were housed in groups of 6-8 and kept under constant light and dark periods; food and water was provided ad libitum. Two types of brain surgery were performed under pentobarbital anaesthesia (4 mg/100 g, ip). Electrolytic lesion. At days of age, the animals were ovariectomized and the locus coeruleus was destroyed by electrolytic lesions with the aid of a stereotaxic instrument. The bilateral lesions were produced by a current intensity of 2.5 nia for 7 seconds through a stainless-steel electrode with an uninsulated tip of 0.5 mm. The site of the lesions is shown in Fig. 1. LC PCS Fig. 1. Schematic illustration of electrolytic lesion destroying the locus coeruleous region. FR: formatio reticularis. LC: nucleus linearis caudalis. NtVD: nucleus trigemini ventralis-dorsalis. PCS: pedunculus cerebri superior.

3 - - - Using 6-OH-dopamine injection. a 30 gauge cannula and a 50 ja syringe, the 6-OH-dopamine was injected bilaterally into the locus coeruleus of 22-day old rats with the aid of a stereotaxic apparatus. The 6-OH-dopamine was dissolved in cold saline with 0.1 "la ascorbic acid, and 20 jig in 2.5 til was injected into each side. All the rats were hemiovariectomized at the same stage of the operation; the ovary was cleaned and weighed on a torsion balance with an accuracy of 0.2 mg. Analytical procedures. The plasma FSH and GH levels were determined by a specific radioimmunoassay for rat FSH and GH, using the reagents supplied by the NIAMDD and using the systems as described previously (Endröczi et al., in press). The values of the plasma hormone levels are given in terms of NIAMDD-RP-1. For determination of NE content after decapitation of rats, the brain was quickly removed and the hypothalamus was dissected on ice and weighed on a torsion balance with an accuracy of 0.2 mg. The caudal portion of the brain was fixed in 10 %> formalin and the sites of the electrolytic lesion and the injection of 6-OH-dopamine were controlled in 50 A thick frozen sections. The hypothalamic NE content was deter mined by the method of Udenfriend 8c Zaltzman-Nirenberg (1963). In both the the Experiment I and II, the animals were sacrificed 2 weeks after ovariectomy and brain surgery both operations being made at the time of the opera tion. At the end of the experiments, the animals were decapitated and the trunk blood was collected in a tube containing 500 U heparin. After centrifugation with 3000 r. p. m, the plasma was frozen at -20 C until the determination of hormones. For sham-opera tion (with hemi- or bilateral ovariectomy), the skull was opened and the electrodes were lowered down to the appropriate brain area in the absence of an electric current flow. The experimental data were evaluated by Student's i-test. RESULTS Experiment I. Bilateral electrolytic lesions of locus coeruleus reduced the hypothalamic NE content to 28 % of the control values and led to a marked decrease of the plasma FSH level (Table 1). The ovariectomy induced an in crease in the plasma FSH level that was reduced to the range of the intact animals by the lesion of locus coeruleus. There was no change in the plasma GH level after destruction of locus coeruleus. Experiment II. Bilateral injections of 6-OH-dopamine into the locus coeruleus led to a 79 % reduction of the hypothalamic NE content and pro duced a marked decrease of the plasma FSH level (Table 2). In comparison to the electrolytic lesioned values, 6-OH-dopamine induced a decrease of the plasma FSH level which was greater, and the values were below the intact controls. The 6-OH-dopamine injection resulted in a marked decrease of com pensatory ovarian hypertrophy (P < 0.01). Like the electrolytic lesioned group, the 6-OH-dopamine injection into the locus coeruleus failed to influence the plasma GH level (Table 2). The histological sections showed a bilateral destruction of the periventri-

4 Table 1. Changes in the plasma FSH and GH levels and hypothalamic NE content after bilateral electrolytic lesion of the locus coeruleus in the rat. No. of rats Plasma FSH Plasma GH NE ng/g Intact (12) Ovariectomized control (12) Lesion + ovariectomy (12) 322 ± 32 P< ± 27 P < ± ± ± ± ± ± P < ± cular grey and the lesions partly destroyed the fibers of the medial longitudinal fasciculus. There was no marked necrosis after local injection of 6-OH-dopa mine: the track of the cannula could be traced to the rostral portion of the locus coeruleus. Table 2. Changes in the plasma FSH and GH levels and compensatory ovarian hypertrophy after intracerebral administration of 6-OH-dopamine in rats. No. of rats Plasma FSH Plasma GH Ovarian hypertrophy in per cent NE ng/g Intact (12) Control, hemiovariectomy (12) 6-OH-dopamine + hemiovariectomy (12 6-OH-dopamine + bilateral ovariectomy (12) 322 ± ± ± ± P < 0.05 P < ± ± ± ± P < P < 0.01 P < ± ± ± ± P < P < 0.001* 268 ± ± ± Control, hemiovariectomy vs. 6-OH-dopamine + bilateral ovariectomy.

5 DISCUSSION The present observations indicate that a marked reduction in the hypothalamic NE content does not induce changes in the basal plasma GH level in immature rats. Halász et al. (1970) after total, Mitchell et al. (1972) after incomplete deafferentation of the hypothalamus found no change in the plasma GH levels in rats. Depletion of brain catecholamines by a-methyl-p-trypsine results in a rise of the basal plasma GH level but it can be restored by L-DOPA, sug gesting that dopamine, but not NE in the basal medial hypothalamus plays an inhibitory role in GH secretion (Chibara et al. 1975). A large number of studies support the hypothesis that catecholamines are involved in the regulation of the secretion of pituitary gonadotrophins. Ad ministration of the catecholamine-depleting agents, reserpine or a-methyl-ptyrosine, results in the occurrence of pseudopregnancy (Coppola et al. 1965), the blockade of spontaneous ovulation (Barraclough 8c Sawyer 1957), and de layed the vaginal opening (Khazan et al. 1960). Moreover, it has been found that catecholamine depletion blocks the pregnant mare serum-induced ovula tion in the immature animal (Coppola et al. 1966). Bilateral injections of 6-OH-dopamine into the anterolateral hypothalamic area induces constant vaginal cornification, polyfollicular ovaries and uterine hypertrophy in cyclic adult female rats (Benedetti et al. 1976). This, and earlier findings support the hypothesis that noradrenaline is involved in the ovulatory discharge of LH (Kalra 8c McCann 1973; Sawyer et al. 1974). In recent studies we have found that the injection of NE into the basal-medial hypothalamus is followed by a marked increase in the plasma FSH and a decrease in the pituitary FSH content in ovariectomized rats. The NE-induced rise of the plasma FSH level could be blocked by phenoxybenzamine, suggesting the involvement of a-adrenergic receptors in the FSH-RH release (Marton 8c Endröczi 1976). Depletion of hypothalamic NE led to a marked decrease of the plasma FSH level in ovariectomized rats and prevented the compensatory ovarian hypertrophy. These observations are in accordance with the view that ascending NE neurones are involved in controlling the FSH-RH producing neurosecretory elements in the anterior and basal-medial hypothalamus. These observations a role in led to the assumption that the ventral noradrenergic pathway plays the control of both tonic and phasic regulation of the pituitary gonadotrophins. REFERENCES Barraclough C. A. Sc Sawyer C. H.: Endocrinology 61 (1957) 341. Benedetti W. L., Sala M. A. 8- Otegui J. T.: Neuroendocrinology 21 (1976) 297. Chibara K., Kato Y., Ohgo S. Se Imura H.: Neuroendocrinology 18 (1975) 192.

6 Coppola J. A., Leonardi R. G. Se Lippman W.: Endocrinology 65 (1966) 563. Coppola J. A., Leonardi R. G., Lippman W., Perrine J. W. Se Ringler L: Endocrinology 77 (1965) 485. Fuxe K., Hornberger B. Sc Hiikfelt T.: Brain Res. 8 (1968) 125. Halász B., Schalch D. S. Se Gorski R. A.: Endocrinology 89 (1970) 198. Kalra S. Se McCann S. M.: Endocrinology 93 (1973) 356. Khazan N., Salman F. Se Winnik H.: Proc. Soc. exp. Biol. (N. Y.) 105 (1960) 201. Marton I. Se Endröczi E. In: Endrüczi E., Ed. Cellular and Molecular Bases of Neuroendocrinology, Akadémiai Kiadó, Budapest (1976) 111. Mitchell J. A., Smyrl R., Hutchins M., Schindler W. J. Se Critchlow V.: Neuroendocrinology 10 (1972) 31. Sawyer C. H., Hilliard J., Kanematsu S., Scaramuzzi R. Sc Blake C. A.: Neuroendocrinology 15 (1974) 328. Sechzer J. A., Ervin G. N. 8c Smith G. P.: Exp. Neurol. 41 (1973) 723. Udenfriend S. Sc Zaltzman-Nirenberg P.: Science 142 (1963) 394. Ungerstedt U'.: Acta physiol. scand. Suppl. 367 (1971) 1. Received on April 15th, 1977.

Endocrinology Division, National Cancer Center Research Institute, Tsukiji 5-chome, Chuo-ku, Tokyo 104

Sterility and Delayed Vaginal Opening Following Electrolytic Lesions Placed Neonatally in the Medial Preoptic Area in Female Rats SHINJI HAYASHI AND TOMOKO ORIMO Endocrinology Division, National Cancer