Interrelationship between estrogen and thyroxine on the release of luteinizing hormone and gonadotropin-releasing hormone in vitro

J. sreroid Biochem. Vol. 28, No. 6, pp. 691496,

1987

Printed in Great Britain. All rights reserved

Copyright 0

OC224731/87 $3.00 + 0.00 1987 Pergamon Journals Ltd

INTERRELATIONSHIP BETWEEN ESTROGEN AND THYROXINE ON THE RELEASE OF LUTEINIZING HORMONE AND GONADOTROPIN-RELEASING HORMONE IN VITRO PAULUS

S. WANG,

HSIANG-TAI

CHAO* and SHYI-WU WANG~

Department of Physiology, National Yang-Ming Medical College and *Department of Gynecology and Obstetrics, Veterans General Hospital, Taipei, Taiwan, Republic of China (Received 3 January 1986)

Summary-The present experiments were designed to study the interaction between estradiol benzoate (EB) and thyroxine (T4) given in vivo on the responsiveness of pituitary luteinizing hormone (LH) to gonadotropin-releasing hormone (GnRH) and the release of GnRH in vitro. Ovariectomizedthyroidectomized (Ovx-TX) rats were injected S.C. with saline or T4 (2 fig/100 g b.wt), and oil or EB (0.1 pg) once daily for 40 days following a 2 x 2 factorial design. All animals were then decapitated and blood samples were collected. Anterior pituitaries (APs) were incubated in vitro with and without 0.1 ng GnRH at 37°C for 4 h. Mediobasal hypothalami (MBHs) were excised and then incubated with and without APs from Ovx donor rats. Concentrations of LH and GnRH in the medium and that of LH in the serum were measured by radioimmunoassay. The LH level in media containing MBHs and donor APs was used as the index of bioactive GnRH release. In Ovx-TX rats, T4 injections reduced the serum LH concentration, the pituitary LH response to GnRH, and the bioactive as well as the immunoreactive GnRH release. The serum LH levels and the spontaneous as well as the GnRH-stimulated release of LH in vitro were suppressed in Ovx-TX rats following administration of EB. By contrast, the serum LH concentration, as well as pituitary LH response to GnRH and GnRH release in vitro, were higher in the group treated with both T4 and EB than in that treated with saline and EB. These results suggest that the differential changes in the LH secretion after thyroidectomy of Ovx versus non-Ovx rats are due to

an antagonistic effect between T4 and estrogen on the response of pituitary LH to GnRH, and the release of GnRH.

INTRODUCTION

Thyroidectomy in rats with intact gonads result in a significant reduction of serum luteinizing hormone (LH) [l]. Administration of thyroxine (T4) in physiological doses to thyroidectomized (TX) rats restores serum LH to normal levels [l]. By contrast, the combination of thyroidectomy and ovariectomy causes an increase rather than a decrease in serum LH

when compared with the effect evoked by ovariectomy alone [l-5]. Neither the metabolic clearance rate of LH [3,4] nor the molecular form of LH [4] is altered by thyroidectomy in ovariectomized (Ovx) rats. Apparently, thyroid hormones stimulate LH release in the presence of the ovary but inhibit LH

release in the absence of the ovary. The reason for the decrease of serum LH in the TX rat with intact gonads and the increase of serum LH in the Ovx-TX rat is unclear. An interaction between T4 and estrogen on the regulation of LH secretion is strongly suggested. The present study was therefore designed to investigate the interaction of T4 and estrogen on the regulation of LH release.

tTo whom correspondence

should be addressed.

EXPERIMENTAL

Animals

Female Sprague-Dawley rats weighing 12C200 g were housed in constant temperature rooms with 14 h of artificial illumination daily (060@-2000) and were provided with water and food ad libitum. Experimental

design

After bilateral ovariectomy under ether anesthesia, rats were assigned as either experimental animals, or pituitary donor animals. The experimental animals were thyroparathyroidectomized (TX) immediately following ovariectomy and were injected S.C. with saline or thyroxine (T4), and oil or estradiol benzoate (EB) daily for 40 days according to a 2 x 2 factorial design. The simple effects (differences between treatments within the first factor at each level of the second factor) and main effects (averages of simple effects for each factor) of T4 and EB, and the interactions were examined [6]. An interaction between two factors (T4 and EB) reveals that the effect of one factor is not independent of the presence of a particular level of the other factor. The daily dose of EB was 0.1 pg in 0.1 ml peanut oil per rat, and the injected dose of T4 was 2 pg/lOO g b.wt/day. Twenty hours after the last injection, all rats were decapitated 691

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PAULUS S. WANG

and blood samples were collected. Serum was separated by centrifugation at 1,OOOg for 30 min and stored at -20°C until radioimmunoassay (RIA) for LH and thyroid-stimulating hormone (TSH). The anterior pituitary glands (APs) of experimental rats were removed, bisected and placed in flasks containing 2ml of Krebs-Ringer phosphate buffer containing 0.1% ascorbic acid, 0.1% bovine serum albumin, 10 mM glucose and 0.05% bacitracin (KRB) before aerating with 95% O2 and 5% CO, at room temperature. Two hemi-APs were placed in each of 4 flasks per group. Preincubation at 37°C for 1 h was performed after replacing the medium with 1 ml of fresh KRB. The tissues were incubated again with 1 ml of fresh KRB containing 0 or 0.1 ng GnRH at 37°C in a shaking water bath for 4 h. At the end of the incubation, the APs were blotted and weighed. The media was collected and stored at -20°C until assayed for LH by RIA. The mediobasal hypothalamus (MBH), excised from immediately behind the optic chiasma to the anterior margin of the mammillary bodies at a depth of less than 2mm, was bisected and incubated with APs of donor rats at 37°C for 4 h, the donor animals having been Ovx 40 days prior to sacrifice. Two hemi-APs and four hemi-MBHs were co-incubated in each flask containing 1 ml KRB. There were 6 flasks per group. The LH level in the media containing both MBH and donor APs was used as the index for bioactive GnRH release. In another experiment, the excised MBHs or cerebral cortices were incubated with KRB at 37°C for 4 h as previously described [7,8]. Eight hemi-MBHs or cerebral cortices with equivalent weights were used in each of 3 flasks per group. At the end of the incubation, the tissues were blotted and weighed. They were then homogenized with 0.1 N HCl and neutralized with 0.1 N NaOH. After centrifugation at 9500 g for 20 min, the supernatant was separated and stored at - 20°C until assayed for GnRH. The media was collected, extracted with 5 N-HCl and assayed for GnRH by RIA. RIA oJ_LH, TSH and GnRH

The immunoreactive LH and TSH in serum and medium samples were measured using the RIA kits provided by the Pituitary Agency of National Institute of Arthritis, Diabetes and Digestive and Kidney Diseases (NIADDK). Rat LH-I-5 and rat TSH-I-5 were used for iodination, and rat LH-RP-1 and rat TSH-RP-1 as standard preparations. Both LH and TSH were radioiodinated with “‘1 (1 mCi/lOpl, Amersham, U.K.) by a minor modification of the chloramine-T method [9]. A known amount of unlabeled standard preparation, or an aliquot of a sample was adjusted to a total volume of 0.3 ml with phosphate-buffered saline (PBS) containing 1% egg white, pH 7.5. Incubations were performed with 0.2ml of the antiserum appropriately diluted with 0.05 M ethylenediaminetetraacetic acid (EDTA) in

et al.

PBS containing 0.25% of normal rabbit serum, and 100 ~1 12SI-labeled hormone (approx 10,000 cpm) at 4°C for 48 h. Each sample was assayed in duplicate using two doses. Then 200 ~1 of sheep anti-rabbit gamma-globulin serum with 1: 20 dilution was added with a further incubation at 4°C for 48 h. At the end of incubation 2 ml of cold PBS were added, and the assay tubes were centrifuged at l,OOOg for 30 min. The supernatant was discarded and the radioactivity in the precipitates was counted in an automatic gamma counter (Beckman Gamma 8000, U.S.A.). Hormone concentrations in unknowns were calculated from the standard curve using a computer program (HP 9845, U.S.A.) for the log-logit transformation of the standard curve. Assay sensitivity was 5-10 ng/tube for LH RIA, and l&20 ng/tube for TSH RIA. The immunoreactive GnRH in the medium extract was measured by using a highly specific rabbit antiGnRH (Y 18-l 1) developed in our laboratory, against synthetic GnRH conjugated with bovine serum albumin using bisdiazotized-benzidine, as previously described [7, 81. The sensitivity was 5 pg/tube. The within- and between-assay CV was 2.1% (n = 6), and 10.5% (n = 13), respectively. The antiserum had less than 0.0005% cross reactivity with prostaglandin E, glutamic acid, serotonin, acetylcholine, epinephrine, norepinephrine, dopamine, rat LH, rat TSH, oxytocin, adrenocorticotropic hormone, somatostatin, thyrotropin-releasing hormone, and some GnRH analogues. Statistical

analysis

Data were analyzed by analysis of variance appropriate to factorial experiments [6].

RESULTS

Serum LH and TSH

Treatment with 0.1 ug EB failed to affect the serum TSH (P > 0.05, Fig. 1, center panel), but significantly increased the uterine weight (main effect, P < 0.01, Fig. 1, top panel) and decreased serum LH (main effect, P < 0.01, Fig. 1, bottom panel) in Ovx-TX rats. T4 replacement did not change uterine weight whereas serum TSH was reduced by T4 therapy (main effect, P < 0.01). In the absence of EB, the serum LH level was suppressed by T4 treatment (group I us group II, P < 0.01). Concentrations of serum LH were higher in Ovx-TX rats treated with both T4 and EB than those in Ovx-TX rats treated with EB alone (group III vs group IV, P < 0.05). An interaction between EB and T4 on serum LH concentration was observed (main effect, P < 0.01). In other words, the differential response of LH secretion to T4 effect is dependent on EB applied simultaneously. LH concentration in rat serum is inhibited by T4 in the absence of estrogen, but is stimulatory by T4 in the presence of estrogen.

693

Release of LH and GnRH in Ovx-TX rats

Group ho.w ceseofTLwl

0

Oose ol EBw

3-T

I

II 2

Ill

N

0

2

Fig. 1, Effects of thyroxine (T4) and estradiol benzoate (EB) on uterine weight (top panel), serum TSH (center panel) and serum LH (bottom panel) in ovariectomizedthyroidectomized (Ovx-TX) rats. Immediately after ovariectomy and thyroidectomy saline or T4 (2pg/lOOg b.wt/rat) and oil or EB (0.1 pg/rat) were injected S.C. once daily for 40 days. Twenty hours after the last injection, all rats were decapitated and blood samples were collected. Serum was separated by centrifugation and assayed for LH and TSH by radioimmunoassay (RIA). NIH rat LH-RP-I and rat TSH-RP-I were used as standards. The doses of T4 and EB are indicated on the abscissa. The summaries of the analysis of variance for main effects of T4 and EB are displayed at the right side of each panel. The significant simple effects are shown within panels. NS = not significant (P r 0.05). Each mean SEM is based on 12 replicates.

Pituitary LH response The spontaneous release of pituitary LH during the 4 h incubation was not reduced by T4 replacement (Fig. 2, top panel), but was significantly reduced by EB treatment (main effect, P < 0.01). The addition of GnRH (0.1 ng/ml) during incubation of pituitaries increased the LH concentration (4547%) in the medium, as shown in Fig. 2 (bottom panel, groups I and IV). Both T4 (main effect, P < 0.05) and EB (main effect, P < 0.01) reduced the release of LH in response to 0.1 ng GnRH. The LH released by APs from EB-treated Ovx-TX rats was enhanced by T4 replacement. Therefore, the effect of T4 on the release of LH in response to GnRH was estrogen-dependent. In the absence of estrogen, T4 was inhibitory for LH release in response to GnRH, whereas in the presence of estrogen, T4 was stimulatory for GnRH-induced LH release in vitro. An interaction (main effect, P < 0.01) between EB and T4 on the release of LH in response to GnRH was demonstrated. Release of GnRH

After 4 h incubation with donor APs, the LH in the media containing MBH from TCtreated animals was lower than LH released by pituitaries of salinetreated Ovx-TX rats (Fig. 3, group I vs group II, P c 0.01). This decrease reflected a reduction of GnRH release from the MBH of Ovx-TX rats after

Fig. 2. Spontaneous and GnRH-stimulated LH release in oitro from anterior pituitary glands (APs) of Ovx-TX rats treated in U&Jwith T4 and EB. After decapitation, APs were removed, bisected and incubated with 0 or 0.1 ng GnRH in 1 ml of fresh Krebs-Ringer phosphate buffer containing 0.1% ascorbic acid, 0.1% bovine serum albumin, 10 mM glucose and 0.05% bacitracin (KRB) at 37°C for 4 h. Two hemi-APs were placed in each flask. Concentrations of LH in the medium samples were measured by RIA. Each mean f SEM is based on 4 replicates. See legend to Fig. 1 for details.

T4 administration. Inasmuch as the release of bioactive GnRH from MBHs of the rats treated with both T4 and EB was greater (although nonsignificantly) than the level from those of MBHs from the rats treated with EB alone, there was an interaction between EB and T4 on the release of bioactive GnRH from rat MBH in vitro (main effect, P < 0.01). It was apparently that the effect of T4 on the release of bioactive GnRH was estrogen-dependent. The release of immunoreactive GnRH in vitro from MBH of Ovx-TX rats was reduced by the administration of T4 in vivo (Table 1, group I vs group II,

MBH:AP.2:1

Group N0.c.~ I kse d TL cm, 0

Dosed

E&p]

-8-

II 2

lU 0

N

2

xi-

Fig. 3. Concentrations of LH in the media after in vitro incubation of donor APs with mediobasal hypothalami (MBHs) of Ovx-TX rats administered with T4 and EB in uiuo. After decapitation, the excised MBH was bisected and incubated with APs of donor rats at 37°C for 4 h. The donor rats were Ovx 40 days prior to sacrifice. Two hemi-APs and four hemi-MBHs were co-incubated in each flask containing 1 ml KRB. Concentrations of LH in the medium samples were measured by RIA. Each mean k SEM is based on 6 replicates. See legend to Fig. 1 for details.

al.

PAULUS S. WANG et

694

Table 1. Effects of thyroxine (T4) and estradiol benzoate (EB) in oiuo on the release of immunoreactive GnRH from mediobasal hypothalamus (MBH) of ovariectomized-thyroidectomized (Ovx-TX) rats in vitro* GnRH Group no. I II III IV V

Treatment Saline and oil T4 and oil EB and saline EB and T4 Saline and oil

Tissue MBH MBH MBH MBH Cortex

concentration

Medium 4.1 2.6 3.6 4.3 0.2

f i + f *

0.25 0.29t 0.61 0.361 0.07

(pg/mg

Tissue 38.1 35.0 60.8 44.6 3.3

? + f k *

4.50 0.64 9.77 8.57 0.86

tissue)

Tissue plus medium 42.2 37.5 64.4 48.9 3.5

f 4.61 f 0.45 k 9.2lt & 8.31 f 0.92

*Ovx-TX rats were injected S.C. with saline or T4 (2 rg/lOO g b. w/rat) and oil or EB (0.1 pg/rat) once daily for 40 days. Twenty hours after the last injection, all rats were decapitated. The MBHs or cerebral cortices were excised, and then incubated with KRB at 37°C for 4 h. Eight hemi-MBHs or cerebral cortices with equivalent weights were used per flask. Concentration of GnRH in acid extracts of media and tissues was measured by radioimmunoassay. Each mean SEM is based on 3 replicates. tP < 0.05 vs Group no. I. IF’ < 0.05 vs Group no. 11.

P < 0.05). Compared with animals treated with T4 alone, combination of T4 and EB increased GnRH release from rat MBH (group II vs group IV, P < 0.05). An interaction (main effect, P < 0.05) between EB and T4 on the release of immunoreactive GnRH by rat MBH was observed. The quantity of GnRH remaining in MBH tissue after incubation was not significantly altered by hormone therapy. However, EB injection alone increased the content (tissue plus medium) of GnRH in rat MBH when compared with the group treated with solvent vehicle (group I vs group III, P < 0.05). The concentration of GnRH released from cerebral cortex after incubation with KRB was only 5% of that released from MBH. The concentration of GnRH in cerebral cortex tissue was 9% of that in MBH. These results reflect that the concentration of immunoreactive GnRH detected in the media was specifically released from MBH tissue.

DISCUSSION

These results suggest that (1) the inhibitory effect of estrogen on serum LH in Ovx-TX rats is mainly due to the suppression of spontaneous and GnRHstimulated LH release by the anterior pituitary, (2) T4 inhibits LH secretion in the absence of ovarian steroids at least in part by the reduction of GnRH release and pituitary response to GnRH, (3) T4 is an antagonist of estrogen on the regulation of LH secretion. The weight of the uterus was employed in the present study as an index of the effect of EB injection. A highly significant increase of uterine weight and a marked decrease of serum LH were observed in Ovx-TX rats after injection of EB (Fig. 1). Both spontaneous release and GnRH-stimulated discharge of LH were significantly reduced in vitro by the administration of EB in vivo. These results led to the postulation that at a dose of 0.1 pg, EB has an inhibitory effect on LH secretion by acting directly on gonadotrophs in the anterior pituitary gland. We

suggest that the number and/or affinity of pituitary GnRH receptors in Ovx-TX rats might be altered by a long-term administration of estrogen. This view is supported by the observation that the increased GnRH receptor in castrated rats is suppressed after EB administration [lo, 111. By contrast, EB alone did not affect the release of bioactive and immunoreactive GnRH. This result is consistent with the report of Sherwood and Fink [12], that GnRH levels in pituitary stalk plasma of ovariectomized and adrenalectomized (Ovx-Adx) rats were not altered by EB injection. The drop of immunoreactive GnRH levels in rat hypothalamus following ovariectomy has been verified repeatedly [13, 141. In our study, the subcutaneous injection of EB alone significantly enhanced GnRH concentration in the mediobasal hypothalamus. Since the release of GnRH was not influenced by EB injection, we speculated that estrogen stimulated the synthesis rather than the release of GnRH in the mediobasal hypothalamus of Ovx-TX rats. That T4 replacement reduces the serum LH in Ovx-TX rats has been well established [l, 3-51, and is reconfirmed in the present experiments. We have demonstrated repeatedly that the LH release in vitro in response to GnRH was significantly higher in the Ovx-TX group than those in the Ovx group [5, 151 and the T4 treated Ovx-TX group [15]. Although the APs of group II, the T4 treated Ovx-TX rats, did not respond to 0.1 ng GnRH (Fig. 2, Panel B), they did respond to 1,2.5 and 50 ng GnRH (data not shown). It is conceivable that the LH release responding to the lower GnRH dose (e.g. 0.1 ng) may be closer to the normal physiological response than the LH release responding to higher doses of GnRH. That T4 administration in vivo prevented the pituitary LH response to 0.1 ng GnRH in vitro illustrates a possibility of a direct effect of T4 on rat AP gonadotrophs. Boutrand et al. [16] showed that the plasma LH response after intravenous injection of GnRH was higher in TX rats than that in normal rats. Injection of GnRH caused a significantly greater increase of

Release of LH and GnRH in Ovx-TX rats

serum LH in hypothyroid than in euthyroid rats [ 171. These in uiuo observations agree with the data in our present in vitro studies. Since it has been shown that S-monodeiodination of T4 to triiodothyronine (T3) in rat AP occurs not only in thyrotrophs but also in somatotrophs and lactotrophs [18, 191,and that pituitary T4 S-deiodinase activity is increased in both hyperthyroid and hypothyroid rats [20]; it is suggestive that there must be an alteration of monodeiodination of T4 to T3 in gonadotrophs of Ovx-TX rats following the administration of T4. The suppression of bioactive and immunoreactive GnRH release in vitro by T4-treatment in vivo in Ovx-TX rats was demonstrated previously [S] and indicated that T4 might regulate LH secretion by acting on the mediobasal hypothalamus. Brown et al. [21] have shown that T4 treatments in male rats decrease the concentration of dopamine in anterior and middle hypothalamus and have suggested that a dopaminergic mechanism was involved in the action of T4 on rat hypothalamus. On the other hand, the release of immunoreactive GnRH in vitro from rat hypothalamus can be stimulated by potassium [22-241, ATP and magnesium [25], prostaglandin E, [23,26], dopamine [22] and norepinephrine [26,27]. Whether the regulation of T4 on the release of GnRH reflects a direct effect on hypothalamic GnRH neurons, or an indirect effect through catecholamine turnover, or an influx of ions is as yet unknown. In the present study a significant interaction between T4 and estrogen was demonstrated in serum LH concentrations, GnRH-stimulated release of LH, and the release of bioactive and immunoreactive GnRH. These results suggest that two mechanisms might be involved in the decrease of the concentration in the serum LH of intact rats following thyroidectomy, and in the increase in serum LH of Ovx rats following thyroidectomy. First, T4 blunts the reduction of pituitary LH response to GnRH caused by the presence of estrogen in Ovx-TX rats. Second, estrogen would seem to remove the inhibitory effects of T4 on the release of GnRH from rat hypothalamus. Chen and Walfish [24] reported that estrogen decreased circulating levels of T4 and increased circulating T3 in Ovx-TX rats injected with T4. They suggested that estrogen increased extrathyroid conversion of T4 and T3. Since T3 is thought to be more potent than T4 one might expect a greater effect when T4 is injected in the presence of estrogen than when T4 is injected in the absence of estrogen. This was not always the case in our studies. In summary, the results in this study clearly show that the differential changes in LH secretion after thyroidectomy of Ovx versus non-Ovx rats are apparently due to the antagonistic effects between T4 and estrogen, on pituitary LH response to GnRH and the release of GnRH. The precise mechanism by which these antagonizing effects between T4 and estrogen acting on the release of LH and GnRH is as yet still unknown.

695

Acknowledgements-The authors greatly appreciate the excellent technical assistance provided by Y. Y. Chen. The rat LH and THS RIA kits were kindly supplied by the NIADD& NIH, U.S.A. Thanks to Renon S. Mica1 for her assistance in the preparation of the manuscript. This work was supported by the National Science Council, Grant NSC71-0412-BOlO-18.

REFERENCES I. Bruni J. F., Marshall S., Dibbet J. A. and Meites J.:

Effects of hyper- and hypo-thyroidism on serum LH and FSH levels in intact and gonadectomized male and female rats. Endocrinology 91(1975) 558-563. 2. Greeley G. H. and Mahesh V. B.: Triiodothvronine (T31 modulation of gonadotropin secretion in the female rat. I

~---\--I

Endocr. Res. Commun. 5 (1978) 135-159.

3. LaRochelle Jr F. T. and Freeman M. E.: Superimposition of thyroid hormone regulation on gonadotropin secretion. Endocrinology 95 (1974) 379-387. 4. Freeman M. E., LaRochelle Jr F. T. and More R. B.: Thyroid hormone regulation of the pulsatile discharges of luteinizing hormone in ovariectomized rats. Endocrinology 97 (1975) 738-743. 5. Wang P. S., Liu T. C. and Jackson G. L.: Effects of thyroidectomy and thyroxine therapy on biosynthesis and release of luteinizing hormone by rat anterior pituitary glands in uifro. Biol. Reprod. 23 (1980) 752-759.

6. Steel R. G. D. and Torrie J. H.: Principles and Procedures of Sfaristics. McGraw-Hill, New York (1960) p. 194. 7. Wang P. S., Yu W. H. and Chen Y. Y.: Radioimmunoassay for gonadotropin-releasing hormone: Evaluation of method and applications. Chinese J. Physiol. 26 (1983) 93-106. 8. Wang S. W., Pu H. F., Chiang S. T., Ho L. T. and Wang P. S.: Effects of thyroidectomy and thyroxine replacement on the release of luteinizing hormone and gonadotropin-releasing hormone in vitro. Horm. Res. 25 (1987) 215-222. 9. Greenwood F. C., Hunter W. M. and Glover J. S.: The preparation of ‘r’I-labeled human growth hormone of high specific radioactivity. Biochem. J. 89 (1963) 114-123. 10. Heber D. and Ode11 W. D.: Estrogen modulation of pituitary LHRH receptor in the rat: in uiuo and in oitro studies. Am. J. Physiol. 237 (1972) E136El41. 11. Conne B. S., Scaglioni S., Lang U., Sizonenko P. C. and Aubert M. L.: Pituitary receptor sites for gonadotropinreleasing hormone: effect of castration and substitutive therapy with sex steroids in the male rat. Endocrinology 110 (1982) 7&79.

12. Sherwood N. M. and Fink G.: Effect of ovariectomy and adrenalectomy on luteinizing hormone-releasing hormone in pituitary stalk blood from female rats. Endocrinology 106 (1980) 363-367.

13. Araki S., Ferin M., Zimmerman E. A. and Vande Wiele R. L.: Ovarian modulation of immunoreactive gonadotropins-releasing hormone (Gn-RH) in the rat brain: evidence for a differential effect on the anterior and mid-hypothalamus. Endocrinology 96 (1975) 644650.

14. Wheaton J. E. and McCann S. M.: Luteinizing hormone-releasing hormone in peripheral plasma and hypothalamus of normal and- ovariectomized rats. Neuroendocrinology 20 (1976) 2963 IO. 15. Wang P. S., Chen Y. Y., Jea A. H., Chiang S. T., Ho L. T. and Wan W. C. M.: Effects of thyroidectomy on the responsiveness of the anterior pituitaries from ovariectomized rats to gonadotropin-releasing hormone. Narn. Sci. Count. Monthly, ROC 10 (1982) 981-987.

PAULUS

696

S. WANG et al.

16. Boutrand M., Pugeat M., Fevre M. and Tourniaire J.: Influence de I’hypothyroidie et de l’administration de thyroxine sur la reponse LH a LHRH chez le rat. Ann. endocr. (Paris) 38 (1977) 391-392.

17. Kalland G. A., Vera A., Peterson M. and Swerdloff R. S.: Reproductive hormonal axis of the male rat in experimental hypothyroidism. Endocrinology 102 (1978) 476484.

18. Obregon M. J., Pascual A., Escobar G. and Escobar Del a major role of L-thyroxine at in rats treated with iopanoic

Mallol J., Morreale De Rey F.: Evidence against the pituitary level: studies acid (Telepaque). Endo-

crinology 16 (1980) 1827-1836.

19. Koenig R. J., Leonard J. L., Senator D., Rappaport N., Watson A. Y. and Larsen P. R.: Regulation in thyroxine 5’-deiodinase activity by 3,5,3’-triiodo-thyronine in cultured rat anterior pituitary cells. Endocrinology 115 (1984) 324329.

20. Walker P.: Increased pituitary thyroxine 5’-deiodinase activity in adult rats rendered hyper- or hypo-thyroid during perinatal life. Can. J. Physiol. Pharmac. 63 (1985) 279-282. 21. Brown G. M., Krigstein

E., Dankova J. and Hornykiewicz 0.: Relationship between hypothalamic and median eminence catecholamines and thyroid function. Neuroendocrinology

10 (1972) 207-2 17.

22. Rotsztejn W. H., Charh J. L., Pattou E., Epelbaum J. and Kordon C.: In vitro release of luteinizing hormone-

releasing hormone (LHRH) from rat mediobasal hypothalamus: effects of potassium, calcium and dopamine. Endocrinology 99 (1976) 1663-1666. 23. Gallardo E. and Ramirez V. D.: A method for the

superfusion of rat hypothalami: Secretion of luteinizing hormone-releasing hormone (LH-RH). Proc. Sot. exp. Biol. Med. 155 (1977) 79-84. 24. Hartter D. E. and Ramirez V. D.: The effects of ions,

metabolic inhibitors, and colchicine on luteinizing hormone-releasing hormone release from superfused rat hypothalami. Endocrinology 107 (1980) 375-382. 25. Burrows G. H. and Barnea A.: Comparison of the effects of ATP, Mg’+, and MgATP on the release of luteinizing hormone-releasing hormone from isolated hypothalamic granules. J. Neurochem. 38 (1982) 569-573. 26. Heaulme M. and Dray F.: Noradrenaline

and prostaglandin Er stimulate LH-RH release from rat median eminence through distinct 1-alpha-adrenergic and PGE, receptors. Neuroendocrinology 39 (1984) 403407. 27. Miyake A., Tasaka K., Sakumoto T., Kawamura Y., Aono T. and Kurachi K.: Noreninenhrine induces releases of both LH-RH from the-hypothalamus and LH from the rat pituitary in uirro. Endocr. japon. 30 (1983) 509-512. 28. Chen H. J. and Walfish P. G.: Effects of estradiol

benzoate on thyroid-pituitary

function in female rats.

Endocrinology 103 (1978) 1023-1030.