0013-7227/90/1274-1928$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society

Vol. 127, No. 4 Printed in U.S.A.

Hypothalamic Prolactin Stimulates the Release of Luteinizing Hormone-Releasing Hormone from Male Rat Hypothalamus* N. AZAD, L. DUFFNER, E. B. PALOYAN, D. REDA, L. KIRSTEINS, N. V. EMANUELE, AND A. M. LAWRENCE Research and Medical Services, Veterans Administration Hines Hospital, Hines, Illinois 60141; and the Departments of Medicine and Biochemistry, Loyola University of Chicago, Stritch School of Medicine, Maywood, Illinois 60153

nation from pituitary PRL. Therefore, we repeated the experiment using hypothalami from animals that had been hypophysectomized 2 weeks before death. Again, PRL antibody significantly inhibited the release of LHRH compared with that by hypothalami incubated in normal rabbit serum. Since testosterone is important to LHRH synthesis, a third experiment was carried out using hypothalami from hypophysectomized male rats that had been implanted sc with testosterone-containing capsules 72 h before death. By 72 h serum testosterone levels had normalized. PRL antibody added to medium containing hypothalamic explants from these animals substantially inhibited in vitro LHRH release, a pattern essentially similar to that seen in intact and hypophysectomized animals without testosterone replacement. From these studies we have concluded that hypothalamic PRL is an important neuromodulator that promotes the release of LHRH from the hypothalamus. Testosterone, at least under the experimental conditions employed, appears not to be essential in this hypothalamic PRL-LHRH interaction. (Endocrinology 127: 1928-1933, 1990)

ABSTRACT. Previous works from our laboratory and others have shown that there is a PRL-like immunoreactive protein with immunological, chromatographic, and biological characteristics identical to those of pituitary PRL, and this is widely distributed in the rat central nervous system. Since pituitary PRL is important in controlling hypothalamic LHRH release, we have hypothesized that hypothalamic PRLlike immunoreactive protein might serve a similar role, that of an endogenous neuromodulator influencing hypothalamic LHRH release. To this end, we have examined the effect of PRL antiserum and normal rabbit serum on the release of immunoreactive LHRH from rat hypothalamic fragments cultured in vitro. In the first experiment, LHRH release from hypothalami of intact rats, bathed in PRL antiserum (1:200 in Krebs-Ringer bicarbonate buffer), was significantly lower than that from hypothalami bathed in normal rabbit serum (1:200 in Krebs-Ringer bicarbonate buffer) for 90 min of incubation. It was, however, possible that the PRL, immunoneutralized in the first experiment, was material that represented contami-

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HE PRESENCE of a PRL-like immunoreactive protein (PLIP) in rat brain and spinal cord (1) has been previously reported by our laboratory as well as by several other investigators, based on immunocytochemistry (2-5), RIA (6-8), and bioassay (9) data. The identification of PRL mRNA in the hypothalamus (10) indicates the likelihood of local synthesis in the brain, and PLIP has been shown to be widely distributed in the rat central nervous system, with the highest concentration found in the mediobasal hypothalamus. Immunocytochemical data reveals that PLIP-containing neuronal cell bodies are mainly localized in the mediobasal hypothalReceived March 19, 1990. Address all correspondence and requests for reprints to: A. M. Lawrence, M.D., Ph.D., Box 455, Hines, Illinois 60141. * This work was supported by the V.A. Medical Research Service, V.A. Hospital, Hines, IL; the Department of Veterans Affairs (to N.A. and N.V.E.), NIH Grant ROl-DK-39361 (to N.V.E.), and the Claire and Leonard Tow Foundation.

amus, with fiber distribution leading to many areas of the brain, including the olfactory tubercle, caudate-putamen, stria terminalis, frontal cortex, and spinal cord (2-4). Spinal cord and brain PLIP molecules behave chromatographically in a manner similar to pituitary PRL (1, 7, 8), and the results of sodium dodecyl sulfatepolyacrylamide gel electrophoresis and Western blot analysis indicate that hypothalamic PLIP has a molecular size of 24 kD, similar to that of pituitary PRL (11), a finding confirmed with immunoblots by two-dimensional gel electrophoresis (4). The concentration of hypothalamic PLIP is higher in female rats than in males and does not change substantially with hypophysectomy in male rats (9,12). Restraint stress leads to an insignificant decrease in hypothalamic PLIP, while serum PRL, which presumably is coming from the pituitary gland, increases 4-fold (9). PLIP is mainly concentrated in membrane-bound secretory granules of nerve endings

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HYPOTHALAMIC PRL AND LHRH and is released by potassium-induced depolarization in a calcium-dependent fashion (6, 7). Furthermore, hypothalamic PLIP concentration in hypophysectomized rats is increased by estrogen in a dose-dependent manner (13). This is interesting, since 30-40% of PLIP-containing neurons also contain receptors for estradiol, which may indicate an important central role for hypothalamic PLIP in modulation of reproduction (5). Taken together, these findings suggest that hypothalamic PLIP is a locally synthesized central nervous system neuromodulator that is responsive at least in part to peripheral endocrine signals. What precise neuromodulatory function(s) PLIP might serve is not clear. Because of the known modulatory role of pituitary PRL on hypothalamic LHRH (1418), we have hypothesized that hypothalamic PLIP might function in a similar manner, as an endogenous regulator of LHRH. Therefore, the following experiments were designed to study the in vitro effect of immunoneutralization of hypothalamic PLIP on the release of LHRH from the hypothalamus.

Materials and Methods Male Sprague-Dawley rats, 60-70 days old, either intact or 2 weeks after hypophysectomy, were used. The intact animals were purchased from Harlan Laboratory (Indianapolis, IN), and the hypophysectomized rats from Hormonal Assay Laboratories (Chicago, IL). The animals were kept in a 12-h light, 12-h dark cycle and given rat chow and tap water ad libitum. In addition, hypophysectomized animals had free access to 5% dextrose H2O. Testosterone (Ciba, Summit, NJ) capsules were made using Silastic tubing (id, 0.062 mm; od, 0.125 mm) purchased from Dow-Corning (Midland, MI) (19). In the third experiment, hypophysectomized rats were implanted sc with a 3-cm long testosterone-filled Silastic capsule under light anesthesia with halothane. The animals were killed by instantaneous decapitation 72 h after capsule implantation. The completeness of hypophysectomy was confirmed by the animals' weight, visual inspection of the sella turcica, and serum analysis, which showed undetectable levels of PRL in randomly selected animals. The hypothalami were quickly dissected out and sliced into six pieces by one sagittal and two coronal sections. The hypothalamic dissection included an area 2 mm anterior to the optic chiasm anteriorly to the mammillary bodies posteriorly. Dissection laterally followed the groove that separates the hypothalamus from the olfactory tubercle rostrally, the hypothalamus from the amygdala at midlevels, and the hypothalamus from the optic tract caudally. Each hypothalamus was 3 mm thick, and two sliced hypothalami per vial were incubated in Krebs-Ringer bicarbonate (KRB) buffer, pH 7.4, at 37 C in a metabolic shaker. After the medium from a 30-min stabilization period was discarded, fresh medium was added, subsequently changed every 30 min for 2 h, and stored at -20 C for LHRH RIA. In the first 30 min, the medium (KRB) consisted of 118.5 mM NaCl, 25 mM NaHCO3, 4.75 mM KC1,1.18 mM MgSO4-7H20,0.96 mM KH2PO4, 2.52 mM CaCl2, plus 10 mM dextrose, 0.1% bovine albumin, and 0.05% bacitra-

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cin. Thereafter, during the subsequent two incubation periods, the medium was supplemented with either rabbit antirat PRL antiserum (1:200) or normal rabbit serum (NRS; 1:200). The PRL antiserum was obtained from the NIDDK, the National Hormone and Pituitary Program, and the University of Maryland School of Medicine through Drs. A. F. Parlow and S. Raiti. In the final 300-min incubation the medium was similar to that used in the two prior 30-min incubations (i.e. KRB containing either PRL antiserum or NRS), but the K+ concentration was raised to 60 mM, with a proportional decrease in sodium concentration. This was done to stimulate LHRH release by inducing cellular membrane depolarization and provide, in some measure, an assessment of the viability of the hypothalamic fragments. In all experiments it was possible to show that these hypothalamic explants, in short term culture, were viable and responded to the increased K+ with significantly augmented LHRH release, thus validating the viability of the explants throughout the incubation period. LHRH RIA The LHRH RIA was conducted using antiserum graciously supplied by Drs. Victor Ramirez and Dean Dluzen of the University of Illinois (Champaign-Urbana, IL). This particular antiserum was rabbit anti-LHRH CRR11B73. Briefly, each assay tube contained 100 X of either rabbit anti-LHRH (initial dilution, 1:20,000) or buffer (0.01 M PBS, 0.05 M EDTA, and 1% NRS, pH 7.4), 200 X standard (synthetic LHRH, obtained from Sigma Chemical Co., St. Louis, MO) or unknown, and 100 X [125I]LHRH (10,000 cpm/tube) iodinated by the chloramine-T method. [125I]LHRH was prepared in 0.1% gelatin0.01% PBS buffer. The iodinated hormone was purified on a 8 x 200-mm column containing Sephadex G-25 (fine) with 0.01 M acetic acid. The mixture was incubated at 4 C for 24 h, and then precipitated with 1.5 ml 95% ethanol after 20-min incubation at 4 C. The tubes were centrifuged at 2,000 rpm (1,720 x g) at 4 C for 10 min, the supernatants were aspirated, and the pellets were counted for 1 min each and counts calculated on a log/logit curve. Assay sensitivity was 15.6 pg/ml, with an interassay coefficient of variation of 12% and an intraassay coefficient of variation of 5%. For the statistical analysis, comparisons between incubation periods within each group were performed using Student's t test for paired data. Comparisons between groups at each time point were performed using Student's t test for independent groups. Analysis of covariance was used to adjust for differences at the first incubation period. Data are expressed as mean picograms of LHRH released per 30 min ± SE.

Results Exp 1 (Fig. 1) LHRH release during the first incubation period (baseline) from hypothalami of pituitary-intact rats bathed in medium with PRL antisera was 225 ± 53 pg/30 min. During the next two 30-min intervals with PRL antiserum diluted 1:200 in KRB, secretion fell to 161 ± 41 pg/30 min and further still to 100 ± 16 pg/30 min, with

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HYPOTHALAMIC PRL AND LHRH

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lypophysectomized Mole Rats

IntGct Mole Rets F HD NRS, N = 8 400 L H i Brolcctin Antiserum, N=10

Endo • 1990 Voll27«No4

V77\

P=0.04

Baseline

f C H NRS, N=16 • ™ 1 Proloctin Antiserum, N=15 150 f

P77

r

P=0.03

300 100 LHRH pg/30 min.

^HRH pg/30 min. 200

50 100

0 -

30

30

FIG. 1. The effect of PRL antiserum on the release of immunoreactive

LHRH from the hypothalami of pituitary-intact adult male rats. H, Baseline for NRS-treated group,E3, baseline for PRL antiserum-treated group.

the latter being significantly lower than the release during the first incubation period (P = 0.04). In contrast to the progressive and significant fall in LHRH secretion from hypothalami bathed in PRL antisera, LHRH secretion from hypothalami incubated with medium containing NRS during the latter two 30-min periods did not decline (185 ± 38 pg/30 min during the first 30 min and 233 ± 60 and 220 ± 55 pg/30 min during subsequent periods; P = NS). When secretion at each of the three time intervals was compared between PRL antibody- and NRS-treated groups, the release during the third 30-min interval was significantly lower in PRL antibody-treated compared to NRS-treated hypothalami (100 ± 16 vs. 220 ± 55 pg/30 min, respectively; P = 0.03).

60

90

Incubation Time (Min.)

Incubation Time (Min.

FlG. 2. The effect of PRL antiserum on the release of immunoreactive

LHRH from the hypothalami of hypophysectomized adult male rats. M, Baseline for NRS-treated group; E2, baseline for PRL antiserumtreated group.

When secretion at each of these three time intervals was compared between the PRL antiserum- and NRStreated groups by analysis of covariance, LHRH release during the third 30-min interval was significantly lower in PRL antibody-treated than in NRS-treated hypothalami (31 ± 5 vs. 68 ± 14 pg/30 min, respectively; P = 0.04). Parenthetically, we have confirmed the work of others which has shown that the LHRH content of the hypothalamus is lower in hypophysectomized than in pituitary-intact animals (Fig. 4) (20, 21). In addition, we have also found that hypothalamic LHRH release from hypophysectomized rats is significantly lower than that from intact rats (P < 0.001; Figs. 1 and 2). Exp 3 (Fig. 3)

Exp 2 (Fig. 2) To eliminate the possibility that the immunoneutralized PRL in Exp 1 might be from the pituitary, the experiment was repeated using hypothalami from hypophysectomized animals. LHRH release during the first incubation period from hypothalami bathed in medium containing no PRL antibody was 47 ± 5 pg/30 min. During the next two 30-min intervals, during which antiserum was added, secretion fell to 37 ± 7 and 31 ± 5 pg/30 min, respectively. LHRH release was significantly lower during the third (last incubation) compared to the first (baseline) incubation period (P = 0.05). In contrast to this progressive and significant fall in LHRH secretion, LHRH from hypothalami of hypophysectomized rats incubated in medium containing normal rabbit serum during the latter two 30-min periods remained unchanged (64 ± 14, 80 ± 29, and 68 ± 14 pg/30 min; periods 1, 2, and 3, respectively; P = NS).

Because of the importance of testosterone to LHRH synthesis and release, we decided to test whether testosterone was involved in hypothalamic PLIP regulation of LHRH release, as demonstrated in Exp 1 and 2. To this end, hypophysectomized animals were sc implanted with testosterone-containing Silastic capsules and killed 72 h later. The reason for the choice of a 72-h testosterone treatment interval was 2-fold. First, we found that this length of treatment restored hypothalamic LHRH content to control values (Fig. 4). Second, this achieved physiological serum concentrations of testosterone. In intact animals, testosterone levels ranged from 20-26 ng/ ml, and in hypophysectomized, 72-h testosterone pellettreated animals, testosterone concentrations were virtually identical to control values, ranging from 20-29 ng/ ml. These hypothalami were incubated in medium containing either PRL antiserum or NRS, as in Exp 1 and 2.

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HYPOTHALAMIC PRL AND LHRH Hypophysectomized Male Rats, Treated with Testosterone CZD NRS, N = 9 • i Prolactin Antiserum, N = 8

200

V7A

Baseline

P=0.09 150 LHRH pg/30 min.

P=0.06

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was compared between PRL antibody- and NRS-treated groups, the LHRH release during the third interval was lower in the PRL antibody-exposed group than in the NRS-exposed group, approaching statistical significance (42 ± 10 vs. 78 ± 14 pg/30 min; P = 0.06). Parenthetically, we found that although treatment with testosterone normalized hypothalamic LHRH content in hypothalami of hypophysectomized rats (Fig. 4), LHRH release in the presence of PRL antiserum was still significantly lower than that in the intact animals (Fig. 3).

50

Discussion 30

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Incubation Time (Min.)

FIG. 3. The effect of PRL antiserum on the release of immunoreactive LHRH from the hypothalami of hypophysectomized male rats treated with testosterone. H, Baseline for NRS-treated group;S3, baseline for PRL antiserum-treated group. Male Rats 3000

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