Progesterone stimulation of in vitro GnRH release from the hypothalamus of the estrogen-primed, ovariectomized rat: serotoninergic role

Neurochem. Int. Vol. 13, No. 4, pp. 469-474, 1988 Printed in Great Britain. All rights reserved

0197-0186/88 $3.00 + 0.00 Copyright © 1988 Pergamon Press pie

PROGESTERONE STIMULATION OF IN VITRO GnRH RELEASE FROM THE HYPOTHALAMUS OF THE ESTROGEN-PRIMED, OVARIECTOMIZED RAT: SEROTONINERGIC ROLE THOMAS S. KING* and INN SO0 KANG Departments of CSB and OB-GYN, University of Texas Health Science Center, San Antonio, TX 78284, U.S.A. (Received I0 March 1988; accepted 2 June 1988)

Abstract--The ability of ovarian steroids to affect luteinizing hormone secretion is closely related to the influence of these steroids on the activities of several neurotransmitter systems within specific areas of the hypothalamus and associated brain areas. The purpose of this study was to characterize /n vitro progestagenic effects on serotonin (5-hydroxytryptamine, 5-HT) and gonadotropin-releasing hormone (GnRH) release from hypothalamic slices from estrogen-primed, ovariectomized rats. Results of this study show that (1) progesterone can stimulate/n vitro GnRH and 5-HT release from hypothalamic tissue slices of ovariectomized rats primed with estrogen and (2) the 5-HT receptor antagonist mianserin blocks the ability of progesterone to augment in vitro GnRH release from these tissue slices. This suggests that the influence of progesterone on the estrogen-induced LH surge is, at least in part, via progestagenic release of 5-HT and the subsequent effect of this neurotransmitter on the release of GnRH within the hypothalamus.

Under basal conditions in the rat, luteinizing hormone (LH) is secreted in low amplitude, episodic pulses. On the afternoon of proestrus, LH is secreted as rapid, high amplitude pulses constituting the "preovulatory LH surge". The latter lasts for 4 - 6 h , resulting in ovulation (Gay et al., 1970; Kalra et al., 1971; Gallo, 1981). This pattern of LH secretion is produced by an increased frequency in the pulsatile release of the decapeptide gonadotropin-releasing hormone (GnRH) from the hypothalamus. Patterns in G n R H release are, in turn, governed primarily by ovarian steroid feedback. Although the mechanisms for such steroidal feedback on G n R H release are not currently well understood, various hypothalamic neurotransmitter systems are believed to play integrating roles in the interactions between ovarian steroids and G n R H release. One of these hypothalamic neurotransmitter systems is 5-hydroxytryptamine (5-HT; serotonin). Estrogen-induced LH surges in the female rat can be suppressed by electrolytic lesion of medial and dorsal raphe nuclei (Hery et al., 1978), by basal mediopontine transection (Hery

et al., 1978) or by microinjection of the 5-HT-

selective neurotoxin 5,7-dihydroxytryptamine into the medial preoptic area of the hypothalamus (Johnson and Crowley, 1986). Thus, hypothalamic 5-HT would seem to represent a necessary component of the central mechanism regulating LH secretion. Circulating levels of progesterone increase concurrently with the prcovulatory LH surge in cycling animals (Butcher et al., 1974). Progesterone advances and amplifies estradiol (E2)-induced LH surges in the ovariectomized rat (Kalra and Kalra, 1979; Simpkins, et ai., 1980; Wise et al., 1981). Progesteronc (P) has also been shown to increase 5-HT synthesis in selected hypothalamic areas of these rats (King et al., 1986b). The purpose of the present study was to determine (1) the effect of progesterone on in vitro G n R H and concurrent 5-HT release and (2) the effect of a 5-HT2 receptor antagonist, mianserin, on progestagenic stimulation o f / n vitro G n R H release from the hypothalamus of E2-primed, ovariectomized rats. EXPERIMENTAL PROCEDURES

*Send correspondence to: Dr Thomas S. King, Department of CSB, University of Texas Health Science Center, San Adult female rats (160-1808) obtained from Charles Antonio, TX 78284, U.S.A. River Laboratories, Inc. (Wilmington, Mass.) were housed 469

470

I'ttOMAS S. KENt; and INN Sty) KANt;

3 ~ in clear-plastic cages. The animals were kept in a light/dark cycle consisting of 14h:10h L : D (lights on at 06.00 daily). Soon after their arrival at this institution, all of the rats were bilaterally ovariectomized under ether anesthesia. The rats were provided food (Wayne Lab-Blox) and fresh tap water ad libitum. Materials Progesterone, 17/~-estradiol benzoate, 5-HT, N-methyltryptamine and other compounds used in the in vitro perifusion were purchased from Sigma Chemical Company (St Louis, Mo.). Mianserin HCI was purchased from Research Biochemicals, Inc. (Wayland, Mass.). The liquid chromatography column (Biophase ODS C18, 25 cm x 4.6 mm; 5 tam pore size) was purchased from Bioanalytical Systems (West Lafayette, Ind.). All reagents used in our liquid chromatography system were o f " H P L C Grade Quality". Experiment 1. Three weeks after ovariectomy, one group of rats was injected subcutaneously on 2 consecutive days (09.00 h) with sesame oil diluent only. The second group of rats was injected with 50 pg/kg of estradiol benzote (E=) in sesame oil subcutaneously on each these 2 days (09.00 h). The third group of rats was injected with E 2 as was the second group and the day following the second E 2 injection the rats were injected subcutaneously with 2mg/kg of progesterone in sesame oil at 09.00 h and decapitated 4 h later. The mediobasal hypothalamus (1 2 m m in depth; 2 3 mm in length; weighing 5 -10 mg each) including median eminence, anterior hypothalamus and medical preoptic area were rapidly removed and sliced longitudinally (300-500/~m slices). The preincubation period of the subsequent in vitro perifusion was followed by three 5 min pulses o f 60 mM K* over a 100 min incubation period (5 min on; 20 min off). Release of 5-HT was measured in sample fractions collected every 5 min. Experiment H. These rats were also injected with E 2 (or oil diluent only) as described in the previous section but were not injected with progesterone. Instead, following the preincubation period, one group of hypothalamic tissue slices from E2-primed rats was exposed to three 10min pulses of 10-TM progesterone over a 100 min incubation period (10min on; 20min off). The other two groups (E 2 and oil only) were not exposed to pulses of any substances during the incubation period. Release of 5-HT was measured in 5 min sample intervals. Experiment III. These rats were also injected either with oil alone or E 2 as described previously. Tissue slices from E2-primed rats were divided into three subgroups: (I) those receiving no pulsed substances in vitro; (2) those receiving three 10min pulses of 10-TM progesterone in vitro as described previously; and (3) those receiving the three progesterone pulses during a 90min incubation period in which 10-TM mianserin (5-HT2 receptor antagonist) was delivered continuously. Immunoassayable GnRH was measured in 5 min sample fractions. In vitro perifusion The slices from each hypothalamus were quickly placed in 0.1 ml microchambers through which modified Krebs-Ringers buffer (pH 7.4 at 25°C) with 0.1 mg ascorbic acid, 0.15mM pargyline and 0.11 mM bacitracin was perifused at a flow rate of 6.12ml/h via a programmable

perifusion system (Endotronics, Inc., Coon Rapids, Minn.i The microchambers were maintained at a constant 37~'(? b~ means of an automatically controlled, circulating water bath. The medium was bubbled with a gas mixture of 95% air/5% CO 2 which provided the following gas partial pressures: Po_,= 180-210mmHg and Pco.~= 28-46 mmHg. Unlike this air~CO, mixture, a mixture of 95°/5 O3~5% CO~ produced Po,, values of 480-550 mmHg which, considering the physiologic Po2 range of 100-1 I0 mmHg, was considered undesirably high. A preincubation period consisting of 40 min of media-only perifusion was maintained to allow the tissue slices to equilibrate to the in vitro conditions. At the end of the 100 min incubation period of Experiments 11 and Ill, tissue slice "viability" was tested by nonspecitic release response to a 5 min 60 mM K + pulse. Results from tissue slices having a negative response to the K ' pulse were eliminated from the study. Sample tubes contained 100 ~1 of 0.1 N perchloric acid with 10 3 M sodium metabisulfite and 50ng/~l N-methylserotonin (internal standard for 5-HF measurements). The samples were "'snap-frozen '~ immediately after collection in an acetone dry ice bath and stored at - 7 0 C until assayed for 5-HT or GnRH levels 5 - H T measurements Twenty i~1 aliquots from each sample were injected into a liquid chromatographic system with electrochemical detection (LCED). 5-HT was isolated on a C18 reverse phase column using a mobile phase consisting of 0.1 M sodium acetate, 0.1 M citric acid and 10% methanol at a flow-rate of 1.5 ml/min. The indole was then detected using a glassy carbon electrode set at a potential of 0.71 V vs an Ag/AgC1 reference electrode. Concentrations were determined by comparing peak heights of the unknown samples with those of standards using a programmed integrator interfaced with the detector unit. Values are corrected for recovery of the internal standard from samples, which averaged 96% or better. Intra-assay variations ranged from 3 to 6% while interassay variation (Experiment I vs II) averaged 4.7%. Data are expressed as ng of 5-HT per mg of hypothalamic tissue slices (wet wt) released into each 5 min media fraction. G n R H measurements Sample aliquots were adjusted to pH 7.4 with IN NaOH and subsequently analyzed by a single antibody (EL-14; 1:750,000 dilution) (Ellinwood et al., 1985)-ethanol precipitation RIA method (Negro-Vilar et al., 1979). Sensitivity of the RIA was 0.8 pg o f G n R H per tube. Intra- and interassay coefficients of variation were < 10% for a 2 pg concentration of synthetic GnRH. Data are expressed in terms of pg of G n R H per mg of hypothalamic tissue slices (wet wt) released into each 5 min media fraction. Analysis o f data Each tissue chamber contained the MBH from a single rat. Each point on each graph represents the mean + SE of that mean for six MBH. The data were analyzed initially by one-way analysis of variance among multiple means. This was followed by analysis of the statistical significance of differences between specific means using the Neuman-Keuls multiple range test (Zar, 1974). Significant pulses are defined as a minimum increase of four times the relevant individual intra-assay coefficient of variation.

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RESULTS 2.5

The results from Experiment I are shown in Fig. 1. Five minute pulses of 60 mM K + stimulated/n vitro 5-HT release from hypothalamic tissue slices of both OVX and OVX + E 2 treated rats equally. Ten minute pulses of 10 -7 M progesterone stimulated/11 vitro 5-HT release from hypothalamic tissue slices of OVX + E2 treated rats (Fig. 2). Similarly, these progesterone pulses stimulated/n vitro G n R H release from hypothalamic tissue slices of OVX + F_q rats (Fig. 3). Progesterone stimulation of /n vitro G n R H release was blocked by exposure of the tissue slices to the 5-HT reoeptor antagonist mianserin, although this effect was not seen with the third progesterone pulse (Fig. 3).

DISCUSSION

Our studies demonstrate that (l) progesterone can stimulate both 5-HT and G n R H release from the hypothalamus and that (2) progesterone's ability to stimulate G n R H release within the hypothalamus can be blocked with the 5-HT2 receptor antagonist (mianserin). The latter finding suggests that a 5-HT mechanism mediates progesterone stimulation of CmRH release. Progesterone has been shown previously to enhance E2-induced G n R H release from the hypothalamus (Drouva et aL, 1985; Kim and Ramirez, 1985).

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Fig. 3. Effect of 10 7M progesterone (P) (10min on; 20min off) on in vitro GnRH release from hypothalami of OVX rats and of OVX rats treated with E2. The lower light panel illustrates the effect of the continuous presence of the 5-HT receptor antagonist mianserin on in vitro progesterone stimulated GnRH release. Pre-incubation period (40 min) is not shown. Each value represents the mean + SE for 6 MBH (n = 6). *Significantlydifferent vs OVX rats and OVX rats treated with E2.

Our results also indicate that K+-induced G n R H release is potentiated in hypothalamic slices from OVX rats treated both with E2 and P in contrast to E 2 alone or to a lack of steroid treatment. This suggests that progesterone enhances K+-stimulated G n R H release from the E2-primed hypothalamus. Drouva et aL (1985) similarly reported that K+-induced G n R H release was enhanced in hypothalamic tissue (from OVX + E2 rats) superfused with medium containing 10-TM progesterone in contrast to that superfused with medium less the progesterone. Whether these effects of progesterone on K+-stimulated G n R H release are facilitory or additive in nature can not be determined on the basic of results from our study or that of Drouva and colleagues. Progesterone is known to amplify (and temporally advance) E2-induced in vivo G n R H activity and LH secretion (Wise et al., 1981) as well as the activities of several hypothalamic neurotransmitters related to this system (King et al., 1986a,b). However, the mechanism(s) by which progesterone amplifies (and advances) estrogenic stimulation of the G n R H - L H system is presently undetermined. Our results suggest that 5-HT plays an important role in that mechanism.

Mianserin, a tetracyclic antidepressant, has been shown to decrease 5-HT-sensitive [3H]spiperone binding in cortical membranes with a preferential reduction in the number of high affinity binding sites (Kendall and Nahorski, 1983). Our observation that mianserin failed to block increased G n R H release in responses to a third progesterone pulse suggests that a component or components of the 5-HT system regulating G n R H release can be overridden or bypassed. How this occurs and exactly what components of the GnRH regulatory system are involved is not clear at this time. Like most other 5-HT receptor antagonists, mianserin can affect other neurotransmitter systems. In higher concentrations mianserin has been shown to have anti-histaminergic activity (Brogden et al., 1978). Mianserin is also considered an st adrenergic receptor antagonist, acting at these presynaptic receptors to inhibit norepinephrine (NE) re-uptake (Brogden et al., 1978). Norepinephrine is known to play a major role in stimulation of hypothatamic GnRH release (Barraclough and Wise, 1982)• If mianserin as an NE re-uptake inhibitor had increased noradrenergic activity in our in vitro system, then we would have expected to see increased G n R H release

Progesterone stimulation of GnRH rather than the observed decreases. Mianserin is not thought to have any significant effect on dopaminergic (Goodlet et al., 1977) or cholinergic (Brogden et ai., 1978) activities in the brain. Accordingly, we believe that our results using mianserin reflect a 5-HT role in mediating progesterone's enhancement of E2-induced GnRH release from the hypothalamus. Johnson and Crowley (1986) have also recently suggested that 5-HT in the medial preoptic area of the hypothalamus mediates progesterone's effects on the E2 promoted LH surge. Those investigators also showed that the opioid receptor antagonist naloxone did not affect hypothalamic 5-HT activity in E2 primed, ovariectomized rats even though LH secretion was increased in those animals. However, those animals were not treated with progesterone which we have both suggested acts to stimulate 5-HT activity in the medial preoptic area. Thus, it would not be surprising that naloxone failed to alter "ambient" 5-HT activity within the medial preoptic area. In fact, Johnson and Crowley (1984) have previously suggested that opioids do influence 5-HT activity in the medial preoptic area although they believed this to be more important in the regulation of prolactin, rather than LH, secretion. Some of the earlier, seemingly conflicting views of 5-HT's role in the regulation of GnRH secretion could perhaps be resolved with the hypothesis that hypothalamic 5-HT has a modulatory influence on the regulation of GnRH (and thus LH) secretion via a bimodal mechanism (McEwen and Parsons, 1982; Hery et al., 1975; Kordon and Glowinski, 1972). Whether hypothalamic 5-HT acted in the stimulation (or in the inhibition) of GnRH release would depend on the existing steroidal milieu and its interactions with GnRH neurons and/or other neurotransmitter systems within specific hypothalamic areas. Thus, a stimulatory 5-HT mechanism within the medial preoptic area would appear to be time-dependent, demonstrating a "critical period" during which 5-HT has a positive modulating influence on the late proestrous LH surge (Coen and MacKinnon, 1979; Walker, 1980, 1983; Walker and Wilson, 1983). Inhibition of 5-HT synthesis or 5-HT receptor antagonism blocks the late proestrous LH surge and subsequent ovulation only if such pharmacological intervention is done during proestrus. Treatment with quipazine, a 5-HT2 receptor agonist, induces a premature LH surge of greater than normal amplitude only during early proestrus. In addition to its known ability to block the late proestrous LH surge, administration of the antiprogestagenic drug WIN 32729 also suppresses hypo-

473

thalamic 5-HT activity (Walker and Wilson, 1983). Our previous observations that an E2 + P induced increase in 5-HT synthesis in the medial prenptic area of OVX rats at the time of the LH surge (King et al., 1986a) as well as during the proestrous LH surge in intact female rats (King et al., 1986b) complement and extend Walker's (1980) observation of increased hypothalamic 5-HT synthesis at the time of the proestrous LH surge. All of those studies suggest that changes in 5-HT activity within the medial preoptic area mediate or, in some key fashion, influence the progestagenic effects on E2-induced LH surges. Our in vitro results further support that contention, demonstrating that in vitro progestagenic effects on GnRH secretion can be attenuated by 5-HT receptor blockade. Acknowledgements--The technical assistance of Diana

Frasier and assistance of Terry Harder in preparing this manuscript are gratefully acknowledged. This work was supported by NIH Grant HD 10202.

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