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Lactation but not prolactin increases the levels of pre-proNPY mRNA in the rat arcuate nucleus
MOLECULAR
AND CELLULAR
Lactation
3, 286-290
NEUROSCIENCES
(1992)
but Not Proiactin Increases the Levels of pre-proNPY mRNA in the Rat Arcuate Nucleus GEORGES PELLETIER
MRC
Group
in Molecular
Endocrinology,
CHUL Received
Research for publication
Evidence mostly obtained from pharmacological studies shows that neuropeptide Y (NPY) is involved in neuroendocrine regulation of reproduction functions. In the present study, we have studied the influence of lactation, a physiological condition associated with high plasma levels of prolactin (PRL) as well as the effects of hyperprolactinemia on pre-proNPY gene expression in the rat arcuate nucleus. The amounts of pre-proNPY mRNA were measured by in situ hybridization using a 36S-labeled cRNA probe encoding for pre-proNPY. In lactating animals killed 4 days after parturition, pre-proNPY mRNA levels were 50% higher than those detected in virgin adult females. Twenty-four hours after pup removal the mRNA levels returned to those observed in virgin animals. Hypophysectomy performed 2 weeks previously decreased by 50% pre-proNPY mRNA levels. Chronic haloperidol treatment which induced high levels of circulating prolactin increased the amounts of preproNPY mRNA by 70 and 66% in intact and hypophysectomized animals, respectively. Intracerebroventricular injections of PRL produced no significant changes in the hybridization signal. Chronic hyperprolactinemia obtained by pituitary implants under the kidney capsule did not induce any modifications in the pre-proNPY mRNA levels. These data demonstrate that pre-proNPY gene expression in the rat arcuate nucleus is increased during lactation and that this effect is probably not a consequence of hyperprolactinemia. Moreover, the stimulatory effect of haloperidol treatment on pre-proNPY mRNA levels does not appear to be mediated by the pituitary gland. 0 1992 Academic PEWS, 1~.
INTRODUCTION Neuropeptide Y (NPY) is a tyrosine-rich 36-amino acid neuropeptide that is abundant in several brain areas, including the hypothalamus where the major NPY-immunoreactive neuronal population is located in the arcuate nucleus (l-3). This neuropeptide has been shown to be involved in neuroendocrine regulation of reproduction functions in the rat (for reviews, see (4, 5)). Depending upon the status of gonadal steroidal milieu, intracerebroventricular injection of NPY either inhibits or increases 1044-7431/92
Copyright All rights
$5.00
0 1992 by Academic Press, Inc. of reproduction in any form reserved.
AND
Center
YIAI
TONG
and Laual
March
University,
Quebec,
Canada
Gl V 4G2
13. 1992
the release of luteinizing hormone (LH) (6-8). Intravenous injection of NPY has also been shown to induce an increase in prolactin (PRL) release (6). Moreover, it has been very recently reported (9) that in the rat the number of NPY-immunoreactive nerve fibers in the median eminence was increased during lactation, a physiological condition associated with high levels of PRL and low levels of estrogen and progesterone (10). We have also demonstrated that injection of haloperidol, a dopaminergic antagonist which stimulates PRL secretion, produced a marked increase in NPY immunoreactivity and pre-proNPY mRNA levels in the rat arcuate nucleus (11, 12). These studies suggest that PRL might be involved in the regulation of NPY neurons in the arcuate nucleus where PRL binding sites have been localized (13). In order to study the role of lactation and PRL in the regulation of NPY gene expression in the arcuate nucleus, we evaluated the influence of lactation as well as the effects of intracerebroventricular injection of PRL and chronic hyperprolactinemia induced by haloperidol treatment and anterior pituitary implants under kidney capsule on pre-proNPY mRNA levels. MATERIALS
AND METHODS
Lactation Twenty primiparous pregnant Sprague-Dawley rats (purchased from Charles River Canada, Inc.) gave birth in our animal facility. Ten lactating animals (with 8-10 actively suckling pups) were killed 4 days postpartum. Ten other lactating rats that had been separated from their pups at Day 4 of postpartum were sacrificed 24 h after pup removal. As a control, 10 virgin rats were sacrificed at the diestrus II stage of the estrous cycle. Intracerebroventricular
Injection
of PRL
Groups (10 animals per group) of intact adult female rats (weighing 140-160 g) at the diestrus II stage of the estrous cycle and adult female rats hypophysectomized 2 weeks previously were used. Two groups were injected into the third ventricle with 3 ~1 of 0.9% NaCl solution containing either 3 wg of ovine PRL (NIDDK-oPRL-19; 286
LACTATION
AND
PRE-PRONPY
biopotency, 31 IU/mg) (14, 15) or bovine serum albumin (BSA) (control). They were sacrificed 4 h after the injection. Two other groups received two intracerebroventricular injections of either PRL or BSA at 12-h intervals and were killed 4 h after the second injection.
GENE
rats were treated twice daily with haloperidol (3 mg/kg/ day) for 10 days. In hypophysectomized animals, the treatment was initiated 4 days after surgery. Control animals received only vehicle. Prolactin
Pituitary
Implants
To achieve chronic hypoprolactinemia, a group of 10 adult female rats (weighing 140-160 g) at the diestrus II stage of the estrous cycle received three anterior pituitary implants under the left kidney capsule. As a control, another group of 10 adult female rats was sham-operated. All the animals were sacrificed 2 weeks after surgery. Haloperidol
Measurement
To measure serum levels of PRL, 4 ml of blood was taken by intracardiac puncture from each animal before vascular perfusion for histological fixation. Serum PRL was measured by double-antibody radioimmunoassay using rat prolactin-I-l for iodination, PRL-RP-1 as standard, and rabbit antiserum (anti-PRL-Sl), kindly supplied by the National Pituitary Program (Baltimore, MD, U.S.A.).
Treatment
Groups (10 animals per group) of intact and hypophysectomized adult female rats at the diestrus II stage of the estrous cycle and hypophysectomized adult female
FIG. nucleus. Control
287
EXPRESSION
In Situ Hybridization All the animals were fixed by intracardiac perfusion with 300 ml of 4% paraformaldehyde in 0.1 M phosphate
1. Typical X-ray autoradiographs illustrating hybridization signal in hypothalamic sections through (A) Virgin diestrus II female. (B) Lactating animal, 4 days postpartum. (C) Five-day postpartum section that had been hybridized with the labeled sense probe. No reaction can be detected.
the central region animal, 24 h after
of the rat arcuate pup removal. (D)
288
PELLETIER
buffer (pH 7.4). The brains were removed, postfixed in the same fixative for an additional period of 2 h at 4”C, and then rinsed overnight in 0.05 M saline phosphate buffer (PBS) containing 15% sucrose. Thereafter, the brains were quickly frozen in isopentane chilled in liquid nitrogen. Ten-micrometer coronal sections were cut throughout the complete length of the arcuate nucleus. One section out of two was collected. The sections were mounted on gelatin- and polylysine-coated glass slides and kept at -70°C until use. In situ hybridization using a 35S-labeled cRNA probe (supplied by Dr. J. Allen) (16) was performed as previously described (11). After hybridization, the sections were dehydrated and exposed to Kodak X-Omat films for 5 days. To test the specificity of hybridization, identically treated serial sections were hybridized with the 35S-labeled antisense probe and with the 35S-labeled sense probe (control). Some sections were also treated with RNase (30 pg/ ml) for 60 min at 37°C prior to the hybridization procedure. Densitometric measurement of autoradiographs of arcuate nuclei from all the experimental groups (10 animals per group) was obtained using a digitized Amersham RAS image analysis system. At least 30 sections throughout the complete length of the arcuate nucleus per animal were analyzed. Statistical significance was determined according to the multiple-range test of DuncanKramer (17). RESULTS
As previously described in the hypothalamus (ll), hybridization signal was exclusively observed in the arcuate nucleus (Figs. 1 and 3). In sections that had been hybridized with the sense strand 35S-labeled probe, no signal could be detected (Fig. 1D). Also, no reaction could be found in sections that had been treated with RNase before hybridization (data not shown). In lactating rats, killed 4 days after parturition, mRNA levels were 50% higher than those observed in virgin adult females (Figs. lA, lB, and 2). In lactating animals that had had their pups removed for 24 h, the hybridization signal returned to values observed in virgin animals (Figs. lA-1C and 2). Serum PRL concentration which was high (55 & 7.2 rig/ml) in lactating animals markedly dropped to 4.5 + 1.4 rig/ml 24 h after pup removal, becoming then very close to the serum level measured in virgin animals (3.2 + 0.9 rig/ml). As illustrated in Figs. 3A, 3C, and 4, hypophysectomy performed 2 weeks previously induced a 50% decrease in hybridization signal. Administration of haloperidol for 10 days increased the amounts of pre-proNPY mRNA by 70 and 66% in intact and hypophysectomized rats, respectively (Figs. 3B, 3D, and 4). In haloperidol-treated intact animals, the levels of serum PRL were much higher (130.5 t 16.2 rig/ml) than those observed in vehicle-injected animals (2.6 + 0.3 rig/ml). In hypophysectomized animals
AND
TONG
FIG. 2. Effect of lactation in the relative pre-proNPY mRNA levels in the arcuate nucleus. (A) Virgin diestrus II female. (B) Lactating animal, 4 days postpartum. (C) Five-day postpartum rat, 24 h after pup removal. Data are expressed as the mean i- SEM in optical density (OD) units. ***P < 0.001, virgin female (A) vs other groups.
treated or not with haloperidol, serum PRL levels were not detectable. Intracerebroventricular injection of 3 1.18of PRL performed 4 h before sacrifice did not significantly modify the levels of pre-proNPY mRNA in both intact and hypophysectomized animals, the levels being 12.1 ? 2.2 and 7.1 + 1.8 OD units for intact and hypophysectomized control rats, respectively, and 13.0 * 2.1 and 6.6 -+ 1.8 OD units for PRL-injected intact and hypophysectomized animals, respectively. Similarly, in intact and hypophysectomized animals that received two injections of PRL at 12-h intervals and were killed 4 h after the second injection, the levels of pre-proNPY mRNA were not significantly modified by the treatment. In animals that had been bearing anterior pituitary implants for 2 weeks, serum PRL were markedly increased (118.6 +- 1.5 rig/ml vs 2.1 f 0.2 rig/ml for sham-operated rats). Pituitary implants did not significantly modify preproNPY mRNA in the arcuate nucleus, the levels being 14.1 + 2.8 OD units for sham-operated animals and 13.2 ? 1.9 OD units for pituitary implant-bearing animals. DISCUSSION
The present data demonstrate for the first time that pre-proNPY mRNA levels are increased in neurons in the arcuate nucleus during lactation in the rat. It has already been shown that the number of endings containing immunoreactive NPY material was increased in the external zone of the median eminence during lactation (9). This increase in stained endings could be explained by either enhanced synthesis of the peptide or inhibition of its release without any increase in its production. The present results together with previous observations (9) strongly suggest that the biosynthesis of NPY is increased during lactation. Pre-proNPY mRNA levels rapidly decreased after suckling had been stopped since the hybridization signal was similar to that observed in virgin animals 24 h after
LACTATION
FIG. 3. The reaction
Typical X-ray autoradiographs is weaker in hypophysectomized
AND
PRE-PRONPY
GENE
demonstrating the effects of HAL treatment rats and is increased by HAL treatment
EXPRESSION
in intact (A, B) and hypophysectomized in both intact and hypophysectomized
289
(C, D) animals. animals.
injection of NPY induced an increase in PRL release (6) and that this peptide stimulated PRL release from female rat anterior pituitary cells in culture (18) might suggest that in some circumstances NPY might act as a PRLstimulating factor. This possible role of NPY remains to be investigated. Considering the fact that during lactation there is a marked increase in PRL release (10, 19) and that haloperidol, which markedly stimulates PRL release (19), has been shown to induce an increase in pre-proNPY mRNA levels in the arcuate nucleus (ll), we have investigated the effect of intracerebroventricular injections of PRL as well as the effect of a chronic elevation of PRL plasma levels on NPY gene expression. Intracerebroventricular injections of PRL performed 4 and 28 h before sacrifice produced no effect on the amounts of pre-proNPY mRNA in intact or hypophysectomized rats. This absence of effect might be explained by the incapacity of PRL to reach the HAL arcuate nucleus or by time intervals that were too short to significantly influence pre-proNPY mRNA levels. The INTACT ) HYPOPHYSECTOMUED I dose of PRL as well as the time intervals used in the FIG. 4. Effect of chronic treatment with haloperidol (HAL) in intact present experiment were probably adequate since we have ***P < 0.001, intact control animals and hypophysectomized animals. recently observed that the intracerebroventricular injec(C) vs all the other experimental groups. tt?P < 0.001, hypophysectotion of 3 pg of PRL 4 h before sacrifice was able to decrease mized animals vs HAL-treated hypophysectomized animals.
pup removal. These observations suggest that there is a relationship between suckling and high levels of preproNPY mRNA levels during lactation. The role of NPY or pre-proNPY-related peptides in lactation is still unknown. Our previous results indicating that intravenous
.. T
290
PELLETIER
POMC mRNA levels in the arcuate nucleus (20). That PRL is not a factor involved in the regulation of arcuate NPY neurons is also supported by the present findings showing that chronic hyperprolactinemia induced by anterior pituitary implants under the kidney capsule could not modify pre-proNPY mRNA levels. It remains to identify the factor(s) responsible for the increase in preproNPY gene expression during lactation. The present results also demonstrate that the stimulating effect of the dopamine receptor antagonist haloperidol in pre-proNPY gene expression is not prevented by hypophysectomy. This clearly indicates that the effect of haloperidol in NPY mRNA levels is not solely mediated by variations in the secretion of pituitary hormones and that the chronic hyperprolactinemia induced by haloperidol in intact animals cannot be responsible for the increase in pre-proNPY mRNA levels. Concerning the depressing effect of hypophysectomy, the present experiments do not allow the identification of the hormonal deficiencies responsible for this effect. PRL is probably not involved since, as shown by the present results, hyperprolactinemia does not modify pre-proNPY mRNA. A likely candidate would be ACTH since it has been shown that hypocorticoidism induced by bilateral adrenalectomy produced a reduction in pre-proNPY mRNA levels in the arcuate nucleus (21). Gonadal steroid deficiency should also be considered responsible for decreasing the activity of NPY neurons since it has shown that ovariectomy decreased and 17/3-estradiol replacement restored immunoreactive NPY levels in the hypothalamus (22). In summary, we have demonstrated that pre-proNPY gene expression in the rat arcuate nucleus is increased during lactation and that this effect is probably not a consequence of hyperprolactinemia. Moreover, the stimulatory effect of haloperidol on pre-proNPY mRNA levels does not appear to be mediated by the pituitary gland. REFERENCES 1.
2.
3.
4.
Chronwall, B. M., D. A. Di Maggio, V. J. Massari, V. M. Pickel, D. A. Ruggiero, and T. L. Donohue (1985). The anatomy of neuropeptide Y-containing neurons in the rat brain. Neuroscience 15: 1159-1181. Pelletier, G., J. Guy, Y. S. Allen, and J. M. Polack (1984). Electron microscope immunocytochemical localization of neuropeptide Y (NPY) in the rat brain. Neuropeptides 4: 319-324. Guy, J., and G. Pelletier (1988). Neuronal interactions between neuropeptide Y (NPY) and catecholaminergic systems in the rat arcuate nucleus as shown by dual immunocytochemistry. Peptides 9: 567-570. Kalra, S. P., A. Sahu, P. S. Kalra, and W. R. Crowley (1990). Hypothalamic neuropeptide Y: A circuit in the regulation of gonadotropin secretion and feeding behavior. Ann. N. Y. Acad Sci.
611:273-283.
AND
TONG
5.
McDonald, Neurobiol.
6.
Kerkerian, L., J. Guy, G. Lefkvre, and G. Pelletier of neuropeptide Y (NPY) on the release of anterior mones in the rat. Peptides 6: 1201-1204.
I.
Guy, L., S. Li, and G. Pelletier (1988). Studies in the physiological role and mechanism of action of neuropeptide Y in the regulation of luteinizing hormone secretion in the rat. Regul. Pept. 23: 209216.
8.
Kalra, S. P., and W. R. Crowley (1984). Norepinephrine-like of neuropeptide Y on LH release in the rat. Z,ife Sci. 35: 1176.
9.
Ciofi, P., J. H. Fallon, D. Croix, J. M. Polak, and G. Tram” (1991). Expression of neuropeptide Y precursor-immunoreactivity in the hypothalamic dopaminergic tubero-infundibular system during lactation in rodents. Endocrinology 128: 823-834. McNeilly, A. S. (1988). Suckling and the control of gonadotropin secretion. In The Physiology of Reproduction (E. Knobil and J. Neill, Eds.), pp. 2323-2349. Raven Press, New York.
10.
11.
12.
13.
14.
15.
16.
17.
18.
J. K. (1988). 4: 97-135.
NPY
and
related
substances.
(‘rit.
Kerr.
(1985). Effects pituitary hor-
effects 1173-
Pelletier, G., and J. Simard (1991). Dopaminergic regulation of pre-proNPY mRNA levels in the rat arcuate nucleus. Neurosci. Lett. 127: 96-98. Li, S., and G. Pelletier (1986). The role of dopamine in the control of neuropeptide Y neurons in the rat arcuate nucleus. Neurosci. Lett. 69: 74-77. Muccioli, G., C. Ghe, and R. Di Carol (1991). Distribution and characterization of prolactin binding sites in the male and female rat brain: Effects of hypophysectomy and ovariectomy. Neuroendocrinology 53: 47-53. Nicholson, G., G. H. Greely, Jr, J. Humm, W. W. Young-Blood, and d. S. Kizer (1980). Prolactin in cerebrospinal fluid: a probable site of prolactin autoregulation. Brain Res. 190: 447-457. Gudelsky, G. A., and J. C. Porter (1980). Release of dopamine from tuberoinfundibular neurons into pituitary stalk blood after prolactin or haloperidol administration. Endocrinology 106: 526-529. Allen, J., J. Novotny, J. Martin, and G. Heinrich (1987). Molecular structure of mammalian neuropeptide Y: Analysis by molecular cloning and computer-aided comparison with crystal structure of avian homologues. Proc. N&l. Acad. Sci. USA 84: 2532-2536. Kramer C. Y. (1956) Extension of multiple-range tests to group means with unequal numbers of replications. Biometrics 12: 307310. Chabot, J. G., A. Enjalbert, P. Pelletier, P. M. Duvois, and G. Morel (1988). Evidence for a direct action of neuropeptide Y in the rat pituitary gland. Neuroendocrinology 47: 511-517.
19.
Ben-Jonathan, roendocrine 339-447.
N., L. A. Arbogast, regulation of prolactin
and J. F. Hyde (1989). release. Bog. Neurobiol.
Neu33:
20.
Tong, Y., and G. Pelletier (1992). Prolactin regulation of POMC gene expression in the arcuate nucleus of the rat hypothalamus, Neuroendocrinology, in press.
21.
White, B. D., R. G. Dean, and R. J. Martin decreases neuropeptide Y mRNA levels Brain Res. Bull. 25: 711-715.
22.
Crowley, W. R., R. E. Tessel, T. L. O’Donohue, B. A. Allen, and S. P. Kalra (1985). Effects of ovarian hormones on the concentration of immunoreactive neuropeptide Y in discrete brain regions of the female rat: Correlation with serum LH and median eminence LHRH. Endocrinology 117: 1151-1155.
(1990). Adrenalectomy in the arcuate nucleus.
AND CELLULAR
Lactation
3, 286-290
NEUROSCIENCES
(1992)
but Not Proiactin Increases the Levels of pre-proNPY mRNA in the Rat Arcuate Nucleus GEORGES PELLETIER
MRC
Group
in Molecular
Endocrinology,
CHUL Received
Research for publication
Evidence mostly obtained from pharmacological studies shows that neuropeptide Y (NPY) is involved in neuroendocrine regulation of reproduction functions. In the present study, we have studied the influence of lactation, a physiological condition associated with high plasma levels of prolactin (PRL) as well as the effects of hyperprolactinemia on pre-proNPY gene expression in the rat arcuate nucleus. The amounts of pre-proNPY mRNA were measured by in situ hybridization using a 36S-labeled cRNA probe encoding for pre-proNPY. In lactating animals killed 4 days after parturition, pre-proNPY mRNA levels were 50% higher than those detected in virgin adult females. Twenty-four hours after pup removal the mRNA levels returned to those observed in virgin animals. Hypophysectomy performed 2 weeks previously decreased by 50% pre-proNPY mRNA levels. Chronic haloperidol treatment which induced high levels of circulating prolactin increased the amounts of preproNPY mRNA by 70 and 66% in intact and hypophysectomized animals, respectively. Intracerebroventricular injections of PRL produced no significant changes in the hybridization signal. Chronic hyperprolactinemia obtained by pituitary implants under the kidney capsule did not induce any modifications in the pre-proNPY mRNA levels. These data demonstrate that pre-proNPY gene expression in the rat arcuate nucleus is increased during lactation and that this effect is probably not a consequence of hyperprolactinemia. Moreover, the stimulatory effect of haloperidol treatment on pre-proNPY mRNA levels does not appear to be mediated by the pituitary gland. 0 1992 Academic PEWS, 1~.
INTRODUCTION Neuropeptide Y (NPY) is a tyrosine-rich 36-amino acid neuropeptide that is abundant in several brain areas, including the hypothalamus where the major NPY-immunoreactive neuronal population is located in the arcuate nucleus (l-3). This neuropeptide has been shown to be involved in neuroendocrine regulation of reproduction functions in the rat (for reviews, see (4, 5)). Depending upon the status of gonadal steroidal milieu, intracerebroventricular injection of NPY either inhibits or increases 1044-7431/92
Copyright All rights
$5.00
0 1992 by Academic Press, Inc. of reproduction in any form reserved.
AND
Center
YIAI
TONG
and Laual
March
University,
Quebec,
Canada
Gl V 4G2
13. 1992
the release of luteinizing hormone (LH) (6-8). Intravenous injection of NPY has also been shown to induce an increase in prolactin (PRL) release (6). Moreover, it has been very recently reported (9) that in the rat the number of NPY-immunoreactive nerve fibers in the median eminence was increased during lactation, a physiological condition associated with high levels of PRL and low levels of estrogen and progesterone (10). We have also demonstrated that injection of haloperidol, a dopaminergic antagonist which stimulates PRL secretion, produced a marked increase in NPY immunoreactivity and pre-proNPY mRNA levels in the rat arcuate nucleus (11, 12). These studies suggest that PRL might be involved in the regulation of NPY neurons in the arcuate nucleus where PRL binding sites have been localized (13). In order to study the role of lactation and PRL in the regulation of NPY gene expression in the arcuate nucleus, we evaluated the influence of lactation as well as the effects of intracerebroventricular injection of PRL and chronic hyperprolactinemia induced by haloperidol treatment and anterior pituitary implants under kidney capsule on pre-proNPY mRNA levels. MATERIALS
AND METHODS
Lactation Twenty primiparous pregnant Sprague-Dawley rats (purchased from Charles River Canada, Inc.) gave birth in our animal facility. Ten lactating animals (with 8-10 actively suckling pups) were killed 4 days postpartum. Ten other lactating rats that had been separated from their pups at Day 4 of postpartum were sacrificed 24 h after pup removal. As a control, 10 virgin rats were sacrificed at the diestrus II stage of the estrous cycle. Intracerebroventricular
Injection
of PRL
Groups (10 animals per group) of intact adult female rats (weighing 140-160 g) at the diestrus II stage of the estrous cycle and adult female rats hypophysectomized 2 weeks previously were used. Two groups were injected into the third ventricle with 3 ~1 of 0.9% NaCl solution containing either 3 wg of ovine PRL (NIDDK-oPRL-19; 286
LACTATION
AND
PRE-PRONPY
biopotency, 31 IU/mg) (14, 15) or bovine serum albumin (BSA) (control). They were sacrificed 4 h after the injection. Two other groups received two intracerebroventricular injections of either PRL or BSA at 12-h intervals and were killed 4 h after the second injection.
GENE
rats were treated twice daily with haloperidol (3 mg/kg/ day) for 10 days. In hypophysectomized animals, the treatment was initiated 4 days after surgery. Control animals received only vehicle. Prolactin
Pituitary
Implants
To achieve chronic hypoprolactinemia, a group of 10 adult female rats (weighing 140-160 g) at the diestrus II stage of the estrous cycle received three anterior pituitary implants under the left kidney capsule. As a control, another group of 10 adult female rats was sham-operated. All the animals were sacrificed 2 weeks after surgery. Haloperidol
Measurement
To measure serum levels of PRL, 4 ml of blood was taken by intracardiac puncture from each animal before vascular perfusion for histological fixation. Serum PRL was measured by double-antibody radioimmunoassay using rat prolactin-I-l for iodination, PRL-RP-1 as standard, and rabbit antiserum (anti-PRL-Sl), kindly supplied by the National Pituitary Program (Baltimore, MD, U.S.A.).
Treatment
Groups (10 animals per group) of intact and hypophysectomized adult female rats at the diestrus II stage of the estrous cycle and hypophysectomized adult female
FIG. nucleus. Control
287
EXPRESSION
In Situ Hybridization All the animals were fixed by intracardiac perfusion with 300 ml of 4% paraformaldehyde in 0.1 M phosphate
1. Typical X-ray autoradiographs illustrating hybridization signal in hypothalamic sections through (A) Virgin diestrus II female. (B) Lactating animal, 4 days postpartum. (C) Five-day postpartum section that had been hybridized with the labeled sense probe. No reaction can be detected.
the central region animal, 24 h after
of the rat arcuate pup removal. (D)
288
PELLETIER
buffer (pH 7.4). The brains were removed, postfixed in the same fixative for an additional period of 2 h at 4”C, and then rinsed overnight in 0.05 M saline phosphate buffer (PBS) containing 15% sucrose. Thereafter, the brains were quickly frozen in isopentane chilled in liquid nitrogen. Ten-micrometer coronal sections were cut throughout the complete length of the arcuate nucleus. One section out of two was collected. The sections were mounted on gelatin- and polylysine-coated glass slides and kept at -70°C until use. In situ hybridization using a 35S-labeled cRNA probe (supplied by Dr. J. Allen) (16) was performed as previously described (11). After hybridization, the sections were dehydrated and exposed to Kodak X-Omat films for 5 days. To test the specificity of hybridization, identically treated serial sections were hybridized with the 35S-labeled antisense probe and with the 35S-labeled sense probe (control). Some sections were also treated with RNase (30 pg/ ml) for 60 min at 37°C prior to the hybridization procedure. Densitometric measurement of autoradiographs of arcuate nuclei from all the experimental groups (10 animals per group) was obtained using a digitized Amersham RAS image analysis system. At least 30 sections throughout the complete length of the arcuate nucleus per animal were analyzed. Statistical significance was determined according to the multiple-range test of DuncanKramer (17). RESULTS
As previously described in the hypothalamus (ll), hybridization signal was exclusively observed in the arcuate nucleus (Figs. 1 and 3). In sections that had been hybridized with the sense strand 35S-labeled probe, no signal could be detected (Fig. 1D). Also, no reaction could be found in sections that had been treated with RNase before hybridization (data not shown). In lactating rats, killed 4 days after parturition, mRNA levels were 50% higher than those observed in virgin adult females (Figs. lA, lB, and 2). In lactating animals that had had their pups removed for 24 h, the hybridization signal returned to values observed in virgin animals (Figs. lA-1C and 2). Serum PRL concentration which was high (55 & 7.2 rig/ml) in lactating animals markedly dropped to 4.5 + 1.4 rig/ml 24 h after pup removal, becoming then very close to the serum level measured in virgin animals (3.2 + 0.9 rig/ml). As illustrated in Figs. 3A, 3C, and 4, hypophysectomy performed 2 weeks previously induced a 50% decrease in hybridization signal. Administration of haloperidol for 10 days increased the amounts of pre-proNPY mRNA by 70 and 66% in intact and hypophysectomized rats, respectively (Figs. 3B, 3D, and 4). In haloperidol-treated intact animals, the levels of serum PRL were much higher (130.5 t 16.2 rig/ml) than those observed in vehicle-injected animals (2.6 + 0.3 rig/ml). In hypophysectomized animals
AND
TONG
FIG. 2. Effect of lactation in the relative pre-proNPY mRNA levels in the arcuate nucleus. (A) Virgin diestrus II female. (B) Lactating animal, 4 days postpartum. (C) Five-day postpartum rat, 24 h after pup removal. Data are expressed as the mean i- SEM in optical density (OD) units. ***P < 0.001, virgin female (A) vs other groups.
treated or not with haloperidol, serum PRL levels were not detectable. Intracerebroventricular injection of 3 1.18of PRL performed 4 h before sacrifice did not significantly modify the levels of pre-proNPY mRNA in both intact and hypophysectomized animals, the levels being 12.1 ? 2.2 and 7.1 + 1.8 OD units for intact and hypophysectomized control rats, respectively, and 13.0 * 2.1 and 6.6 -+ 1.8 OD units for PRL-injected intact and hypophysectomized animals, respectively. Similarly, in intact and hypophysectomized animals that received two injections of PRL at 12-h intervals and were killed 4 h after the second injection, the levels of pre-proNPY mRNA were not significantly modified by the treatment. In animals that had been bearing anterior pituitary implants for 2 weeks, serum PRL were markedly increased (118.6 +- 1.5 rig/ml vs 2.1 f 0.2 rig/ml for sham-operated rats). Pituitary implants did not significantly modify preproNPY mRNA in the arcuate nucleus, the levels being 14.1 + 2.8 OD units for sham-operated animals and 13.2 ? 1.9 OD units for pituitary implant-bearing animals. DISCUSSION
The present data demonstrate for the first time that pre-proNPY mRNA levels are increased in neurons in the arcuate nucleus during lactation in the rat. It has already been shown that the number of endings containing immunoreactive NPY material was increased in the external zone of the median eminence during lactation (9). This increase in stained endings could be explained by either enhanced synthesis of the peptide or inhibition of its release without any increase in its production. The present results together with previous observations (9) strongly suggest that the biosynthesis of NPY is increased during lactation. Pre-proNPY mRNA levels rapidly decreased after suckling had been stopped since the hybridization signal was similar to that observed in virgin animals 24 h after
LACTATION
FIG. 3. The reaction
Typical X-ray autoradiographs is weaker in hypophysectomized
AND
PRE-PRONPY
GENE
demonstrating the effects of HAL treatment rats and is increased by HAL treatment
EXPRESSION
in intact (A, B) and hypophysectomized in both intact and hypophysectomized
289
(C, D) animals. animals.
injection of NPY induced an increase in PRL release (6) and that this peptide stimulated PRL release from female rat anterior pituitary cells in culture (18) might suggest that in some circumstances NPY might act as a PRLstimulating factor. This possible role of NPY remains to be investigated. Considering the fact that during lactation there is a marked increase in PRL release (10, 19) and that haloperidol, which markedly stimulates PRL release (19), has been shown to induce an increase in pre-proNPY mRNA levels in the arcuate nucleus (ll), we have investigated the effect of intracerebroventricular injections of PRL as well as the effect of a chronic elevation of PRL plasma levels on NPY gene expression. Intracerebroventricular injections of PRL performed 4 and 28 h before sacrifice produced no effect on the amounts of pre-proNPY mRNA in intact or hypophysectomized rats. This absence of effect might be explained by the incapacity of PRL to reach the HAL arcuate nucleus or by time intervals that were too short to significantly influence pre-proNPY mRNA levels. The INTACT ) HYPOPHYSECTOMUED I dose of PRL as well as the time intervals used in the FIG. 4. Effect of chronic treatment with haloperidol (HAL) in intact present experiment were probably adequate since we have ***P < 0.001, intact control animals and hypophysectomized animals. recently observed that the intracerebroventricular injec(C) vs all the other experimental groups. tt?P < 0.001, hypophysectotion of 3 pg of PRL 4 h before sacrifice was able to decrease mized animals vs HAL-treated hypophysectomized animals.
pup removal. These observations suggest that there is a relationship between suckling and high levels of preproNPY mRNA levels during lactation. The role of NPY or pre-proNPY-related peptides in lactation is still unknown. Our previous results indicating that intravenous
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POMC mRNA levels in the arcuate nucleus (20). That PRL is not a factor involved in the regulation of arcuate NPY neurons is also supported by the present findings showing that chronic hyperprolactinemia induced by anterior pituitary implants under the kidney capsule could not modify pre-proNPY mRNA levels. It remains to identify the factor(s) responsible for the increase in preproNPY gene expression during lactation. The present results also demonstrate that the stimulating effect of the dopamine receptor antagonist haloperidol in pre-proNPY gene expression is not prevented by hypophysectomy. This clearly indicates that the effect of haloperidol in NPY mRNA levels is not solely mediated by variations in the secretion of pituitary hormones and that the chronic hyperprolactinemia induced by haloperidol in intact animals cannot be responsible for the increase in pre-proNPY mRNA levels. Concerning the depressing effect of hypophysectomy, the present experiments do not allow the identification of the hormonal deficiencies responsible for this effect. PRL is probably not involved since, as shown by the present results, hyperprolactinemia does not modify pre-proNPY mRNA. A likely candidate would be ACTH since it has been shown that hypocorticoidism induced by bilateral adrenalectomy produced a reduction in pre-proNPY mRNA levels in the arcuate nucleus (21). Gonadal steroid deficiency should also be considered responsible for decreasing the activity of NPY neurons since it has shown that ovariectomy decreased and 17/3-estradiol replacement restored immunoreactive NPY levels in the hypothalamus (22). In summary, we have demonstrated that pre-proNPY gene expression in the rat arcuate nucleus is increased during lactation and that this effect is probably not a consequence of hyperprolactinemia. Moreover, the stimulatory effect of haloperidol on pre-proNPY mRNA levels does not appear to be mediated by the pituitary gland. REFERENCES 1.
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