| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ARTICLES |
Department of Biomedical Sciences (R.S.B., B.A.R., E.M.B.), Tufts University School of Veterinary Medicine, North Grafton, Massachusetts 01536; and Division of Biomedical Sciences (L.Y., A.M.W.), University of California, Riverside, California 92521
Address all correspondence and requests for reprints to: Robert S. Bridges, Department of Biomedical Sciences, Tufts University School of Veterinary Medicine, 200 Westboro Road, North Grafton, Massachusetts 01536. E-mail: robert.bridges{at}tufts.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Earlier studies in our laboratory demonstrated that the rapid onset of maternal behavior induced by steroid treatment in virgin rats could be delayed by the systemic administration of bromocriptine, a dopamine agonist, which suppresses endogenous PRL secretion (3). Moreover, central infusions of PRL into the lateral ventricle (LV) or medial preoptic area (MPOA) of ovariectomized, nulliparous rats, concurrently treated with steroids plus bromocriptine, facilitated a rapid onset of maternal care (4). Recent work in mice has indicated that the PRL receptor mediates the expression of maternal care (5). Specifically, female mice with null mutations for the PRL receptor exhibited deficits in maternal behavior, both as homozygous virgins and heterozygous primiparous dams.
The possible involvement of the PRL receptor in the hormonal induction of maternal behavior, however, has not been elucidated. The development of the PRL receptor antagonist, S179D-PRL, a mutant of wild-type human PRL (wt hPRL) in which the serine residue at position 179 is replaced with aspartate, provides a molecular tool to examine the role of PRL in neural processing (6). Use of S179D-PRL has the potential to bypass the possible neurological side effects of bromocriptine on brain dopaminergic systems and their possible effects on maternal care (3). Moreover, evaluation of the effectiveness of S179D-PRL within the brain will tell us whether this compound can act as a behavioral and neurobiological receptor antagonist, a finding which may prove to be clinically important.
In the present study, three experiments were conducted that examined the possible central involvement of PRL receptors in the induction of maternal behavior. In the first study, it was asked whether direct infusions of the PRL receptor antagonist, S179D-PRL, into the ventricular system of steroid-primed, nulliparous rats would delay the onset of maternal behavior displayed toward foster young. The second study investigated whether one site of action of the PRL receptor antagonist was the MPOA. In this experiment, S179D-PRL was infused bilaterally into the MPOA of steroid-primed, nulliparous rats, and maternal behavior was measured. The third study asked whether the possible inhibitory effects of the PRL receptor antagonist were specific to the properties of the antagonist. To test this possibility, wt hPRL was infused into the MPOA, and its effects were compared with those of vehicle-treated controls. The results of these studies demonstrate that central infusions of S179D-PRL into either the ventricles or MPOA delay the onset of maternal care. The effects within the MPOA can be attributed to the biological properties of the PRL antagonist, because infusions of the wt hPRL did not interfere with the expression of maternal behavior.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Steroid treatment
One to 2 weeks after arriving in our laboratory, all
experimental animals were ovariectomized under ketamine/xylazine (100
µl/100 g) anesthesia. After a 1-week recovery period (on day 1 of
steroid treatment), animals were anesthetized with Isoflurane inhalant
and implanted sc with three 30-mm P4-filled
SILASTIC capsules. On day 11 (ketamine/xylazine anesthesia),
P4 implants were removed, and one 2-mm
E2 SILASTIC capsule was implanted. This steroid
regimen has previously been shown to stimulate a rapid onset of
maternal behavior in behaviorally inexperienced, nulliparous rats,
reducing latencies from about 5 days to 1–2 days (7).
Preparation of the PRL receptor antagonist
Both PRL preparations were produced and characterized as
previously described (6). The proteins used in this study
were expressed and purified in parallel and were expressed at similar
levels (6). The recombinant PRLs were then tested for
proliferative activity (wt hormone) in the Nb2 assay, or antagonism of
this (S179D PRL). Recombinant PRL preparations were concentrated in
saline using Amicon Centripreps (Amicon, Danvers, MA).
Experimental treatments
Exp 1: The effect of infusions of
the PRL receptor antagonist, S179D-PRL, into the LV on the rate of
induction of maternal behavior in steroid-primed, nulliparous rats.
The goal of the first study was to determine whether central
infusion of the PRL receptor antagonist, S179D-PRL, into the brain via
the ventricular system would delay the rate of onset of maternal
behavior. Animals (n = 9–10/group) were implanted with a sc
miniosmotic pump (Model no.1007D; Alza Corp., Palo Alto,
CA) connected via a 10-cm polyethylene (PE60) catheter tube to a
28-gauge cranial connector cannula (Plastics One Inc., Roanoke, VA)
directed at the right LV at the time the steroid capsules were changed
on treatment day 11. The stereotaxic coordinates based upon bregma for
placement of unilateral cannulae in the LV was AP = -0.8; ML
= -1.5; DV = +3.5 (8). The miniosmotic pumps,
which were preincubated for 24 h before implantation, contained
one of two doses (0.115 mg/ml or 1.15 mg/ml) of the PRL receptor
antagonist [S179D-PRL (6)] or vehicle (0.85% NaCl). The infusion
rate of the pumps was 0.5 µl/h. On day 12 of treatment, approximately
24 h after the miniosmotic pumps were implanted, behavioral
testing began. Testing was conducted once daily for a maximum of 6
days. At the completion of testing, brains were perfused and stored at
-80 C until histological verification of cannula placement sites.
Exp 2: The effects of MPOA infusions of the PRL
receptor antagonist, S179D-PRL, on the induction of maternal behavior.
The objective of the second study was to assess
whether direct infusions of S179D-PRL into the MPOA of steroid-primed,
nulliparous rats would delay the onset of maternal behavior. On day 11
of treatment, at the time that E2 capsules were
inserted, animals were sc implanted with miniosmotic pumps (Model no.
1002), each of which delivered its contents at a rate of 0.25
µl/h for 14 days. Each pump was fitted with an 8-cm length PE60
tubing that was attached to a Y connector, which in turn was attached
to the bilateral cannula. The connector cannulae were directed at the
MPOA (coordinates based upon bregma: AP = -0.3; ML = ±1.0;
DV = +9.0; 8). Pumps were primed before implantation, and tubing
was filled with the infusate before surgery. The MPOA infusates
consisted of S179D-PRL (0.115 mg/ml or 1.15 mg/ml) or vehicle (0.85%
NaCl). Behavioral testing was conducted once daily for 6 days from
treatment days 12–17 or until an animal reached the criterion for
being fully maternal. Brains were collected at the end of testing to
verify cannula placements.
Exp 3: Effects of infusions of wt hPRL into the
MPOA on the induction of maternal behavior.
The goal of the third experiment was to determine
whether infusions of a control substance, wt PRL, into the MPOA would
interfere with the rate of onset of maternal behavior in
steroid-primed, nulliparous rats.
Adult nulliparous female rats were implanted with P4-filled SILASTIC capsules on treatment day 1, 1 week after ovariectomy. On day 11, the P4 capsules were removed, a single E2-filled capsule was implanted sc, and a 14-day Alzet miniosmotic pump (Model no. 1002) was connected to a bilateral connector cannula directed at the MPOA, as described in Exp 2. Pumps were filled with either wt hPRL (1.15 mg/ml) or vehicle (0.85% NaCl). Behavioral testing began on treatment day 12 and continued for a maximum of 6 days as described below. At the end of testing, brains were collected for subsequent histological analysis of cannula placement sites.
Testing and histology
In each experiment, maternal behavior testing began on
treatment day 12, between 0900 and 1200 h. To initiate a test
session, three foster pups, 3–9 days of age, were placed in separate
quadrants of the cage, avoiding the test animal’s nest site. Latencies
for the test female to contact, retrieve, group, and crouch over the
foster test young were recorded continuously for 15 min, then checked
every 15 min for pup and subject positions, throughout the daily 1-h
test session (7). Subjects were tested for 6 consecutive
days or until an animal displayed full maternal behavior (FMB) for 2
consecutive days. On test days 2–6, the previous test day’s pups were
removed from the test cage, an hour before testing.
At the end of each experiment, animals were rapidly decapitated, and brains were removed and fixed in 10% formalin for histological exam-ination. Brains were sectioned at 40 microns and then stained with cresyl violet before viewing cannula placements.
Statistical analyses
One-way ANOVAs and Newman-Keuls tested the latencies to
retrieve, group, and crouch over the test pups and display FMB. The
Fisher’s exact probability test was used to compare the numbers of
animals displaying specific behaviors on designated test days.
Statistical significance was noted by a P value 
0.05.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, fewer low- and high-dose females
retrieved a single or two pups during the first 15 min of testing than
did vehicle controls (P < 0.05). This effect persisted
in the high-dose group at the end of the 1-h test session on test day
1, given that 44% of vehicle-infused animals retrieved 1 or 2 pups,
compared with none of the high-dose antagonist group (P
< 0.05; see Fig. 1B). No statistical differences were found in the
other measures of maternal behavior at 15 min or 1 h on the first
test day. The cumulative percentages of animals that displayed FMB over
the 6 test days are shown in Fig. 2. On
the second test day, significantly more vehicle-infused females (7 of
9) were fully maternal than were high-dose-treated animals (2 of 9;
P < 0.05). Hence, infusion of the high dose of the PRL
receptor antagonist directly into the ventricular system delayed the
rapid onset of full maternal care on this test day.
|
|
Exp 2: The effects of MPOA infusions of the PRL receptor
antagonist, S179D-PRL, on the induction of maternal behavior
Direct bilateral infusions of the PRL antagonist into the
MPOA of steroid-primed, nulliparous rats resulted in a more profound
delay in the onset of maternal behavior than that found when S179D-PRL
was infused into the LV in Exp 1. As shown in Fig. 3A, fewer animals treated with the low
and high doses of S179D-PRL retrieved, grouped, and crouched over
the three test pups than did vehicle controls during the initial 15-min
test period on treatment day 12 (P values <
0.05–0.02). A similar response pattern was present at the end of the
first 1-h test session (see Fig. 3B), except that only the low-dose and
vehicle groups differed statistically from each other at the end of the
1-h test (8 of 11 vehicle rats retrieved 3 pups, compared with 3 of 13
low-dose animals; P < 0.02).
|
. Treatment with either dose of
S179D-PRL blocked the expression of FMB on test day 1. On day 1 of
testing, 7 of 11 vehicle-infused, steroid-primed rats were fully
maternal within the 1-h test session, whereas 2 of 13 low-dose and 1 of
10 high-dose animals were fully maternal. On test day 2, only animals
in the high-dose group continued to display lower incidences of FMB
than did controls (P < 0.05). Therefore, infusions of
the antagonist into the MPOA delayed the onset of maternal care, with
the higher dose being more effective than the lower dose.
|
Cannula placements for animals in each experimental group are depicted
in Fig. 5. Only data from animals with
bilateral placements within or contiguous with the MPOA were
included in the statistical analyses. Four females (3 high-dose and 1
low-dose) were excluded from the study because of inaccurate cannula
placements. Similarly, 11 of 12 vehicle-infused animals had bilateral
cannula placements within the MPOA.
|
, A and B, respectively. No behavioral
differences were found in the number of animals displaying various
aspects of maternal behavior at either 15 or 60 min on the first test
day. The cumulative percentages of animals that were fully maternal
over the 6 test days are shown in Fig. 7.
Again, there were no statistical differences in the proportion of
animals displaying FMB between these 2 groups on any individual test
day. Whereas only 1 of 12 wt hPRL MPOA-infused females was fully
maternal on test day 1, by the third test day, 10 of 12 subjects were
fully maternal. In comparison, 4 of 11 vehicle animals were fully
maternal on day 1, whereas on test day 3, 6 of 11 were responsive. By
the sixth test day, all wt hPRL and 8 of 11 vehicle MPOA-infused
animals were fully responsive.
|
|
). Thus, on the first day of FMB,
females given MPOA infusions of wt hPRL responded faster than did
controls. All bilateral cannula placements for the wt hPRL and vehicle
groups are shown in Fig. 5
.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In the present study, we administered S179D-PRL, wt hormone, or vehicle using miniosmotic pumps that continuously delivered these solutions for 1 week. Chronic infusions of S179D-PRL into the LV in P4-plus-E2-treated, ovariectomized rats resulted in a delay in the development of maternal behavior. Infusions of the PRL receptor antagonist into the MPOA resulted in a similar, yet slightly longer, delay in maternal care. In contrast, MPOA infusions of wt hPRL, as expected, did not affect the rate of onset of maternal behavior. These findings demonstrate that blocking access of neural PRL receptors to endogenous ligands interferes with the rapid onset of maternal care brought about by exposure to a pregnancy-like steroid regimen (7).
It was somewhat surprising that, in both Exp 1 and Exp 2, a majority of the animals treated with S179D-PRL began to display maternal behavior by the third test day. One possible explanation that might account for this escape from the inhibitory actions of S179D-PRL by test day 3 may be that test animals were exposed to progressively elevated levels of endogenous PRL from test day 1 to 6, which could override the actions of the PRL receptor antagonist. Plasma PRL levels have been shown to increase over the course of testing in the presence of E2 in rats treated with this identical steroid regimen (3). Therefore, it is possible that, by test day 3, elevated titers of endogenous PRL were able to occupy a sufficient number of MPOA PRL receptors to stimulate the induction of maternal behavior. It is also possible that the onset of maternal behavior can be induced through some other neural site or is not dependent on PRL after the second day of testing. This latter possibility seems less likely to account for the behavioral responses after day 2, because continued suppression of endogenous PRL with bromocriptine prevents most steroid-treated females from responding maternally for 4–5 test days (3).
The possible actions of the wt hPRL on maternal behavior were examined to determine whether the inhibitory actions of S179D-PRL were specific for the purported antagonist or whether the infusion of the native protein would have similar effects. The results indicate that the inhibitory actions of S179D-PRL in the prior experiments were attributable to its biological properties as an antagonist. Interestingly, infusion of the native hormone seemed to stimulate certain aspects of maternal behavior beyond the level induced by the steroid-treatment. Specifically, comparisons of retrieval latencies between wt and vehicle controls revealed that animals given MPOA infusions of wt hPRL retrieved the first and second test pup significantly faster than did vehicle-infused controls on the first day that animals displayed FMB. The stimulation of retrieval behavior by wt hPRL is consistent with earlier studies that found the repeated injections of ovine PRL in hypophysectomized female rats stimulated the initial induction of pup retrieval (11).
Similar comparisons of the actions of the PRL antagonist on maternal responsiveness on the first day of full maternal care indicated that animals treated with the low doses of S179D-PRL, infused into either the ventricles or MPOA, responded somewhat slower to the test pups than did vehicle-infused controls. The lack of a similar effect with the high dose of S179D-PRL may be attributable to the large degree of variability in the response latencies of high-dose animals. Further studies are needed to understand how varying doses of this antagonist act upon neurons to regulate neural processing.
The finding that central infusions of wt hPRL facilitate pup retrieval, whereas the PRL receptor antagonist delays retrieval responses, lends support to the concept that central exposure to PRL may potentiate the expression of maternal behavior once it is established, a finding that runs contrary to existing dogma in the field, i.e. that ongoing maternal care is regulated independent of hormonal control. It is established that cells and fibers that contain anti-PRL immunoreactive protein are present within the rat central nervous system, with high densities of immunoreactivity present in the MPOA (12), an area that is sensitive to PRL regulation of maternal behavior (4). What the relative contributions of brain and endocrine PRL to the onset and maintenance of maternal behavior are, however, remain to be established. One possibility is that the regulation of maternal behavior may shift from an endocrine to a neural PRL dependence after parturition, a change that might help account for the diminished role for circulating hormones in regulating maternal behavior during lactation and in previously parous females.
The actions of S179D-PRL seemed to persist for a longer period when infused directly into the MPOA, a neural site known to regulate PRL and placental lactogen-stimulated maternal behavior (4, 13), than when infused into the ventricular system. Although the present results have established that the MPOA is one neural site involved in PRL receptor-mediated regulation of maternal behavior, an examination of other possible neural sites of PRL action is warranted. Given the recently identified role of the ventromedial hypothalamus (VMH) in the neural regulation of maternal behavior (14), the abundance of PRL receptors present in the VMH (15), and the role of the VMH in feeding (16, 17), it would be of interest to determine whether this region might be a site of PRL regulation of feeding behavior during lactation.
One significant advantage in using this PRL receptor antagonist to examine the role of PRL is that S179D-PRL presumably acts directly on the PRL receptor to interfere with maternal care, rather than acting indirectly via the dopaminergic system to suppress PRL secretion (3). Given that maternal behavior and many other behaviors, including locomotion and appetitive behaviors (18), can be affected by the activity of the endogenous dopaminergic system, use of this new PRL receptor antagonist may bypass the possible confound that could result from a more general stimulation of neural dopaminergic activity after systemic bromocriptine administration.
Finally, S179D-PRL is a molecular mimic of phosphorylated PRL, which is a natural product of the rat pituitary gland (19, 20, 21). Phosphorylated PRL itself acts as a PRL antagonist under certain conditions (22). The proportion of unmodified-to-phosphorylated PRL is physiologically regulated (23, 24), with higher proportions of unmodified PRL being characteristic of the latter two-thirds of pregnancy (23). This proportional change during pregnancy is dependent on ovarian rather than placental hormones (23) and can be reproduced by the administration of estrogen to ovariectomized animals (A. M. Walker, unpublished data). At least part of the estrogen priming effect on maternal behavior, therefore, could be due to changes in the form of PRL produced. On the basis of these studies, one would also suggest that appropriate changes in the form of PRL during pregnancy and lactation may be necessary for normal maternal behavior and that abnormal ratios of the two forms of PRL could be responsible for diminished maternal behavior in some animals.
In summary, the results of the present study demonstrate that S179D-PRL can act within the brain to alter the expression of a PRL-regulated behavior. Chronic infusion of S179D-PRL into the LV or MPOA of steroid-primed virgin rats delayed the onset of maternal care. Central administration of this PRL receptor antagonist provides researchers with an endocrine preparation with which to assess the central actions of PRL and other lactogenic molecules that bind to PRL receptors.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Received June 30, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  D. A. Slattery and I. D. Neumann No stress please! Mechanisms of stress hyporesponsiveness of the maternal brain J. Physiol., January 15, 2008; 586(2): 377 - 385. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Bachelot and N. Binart Reproductive role of prolactin Reproduction, February 1, 2007; 133(2): 361 - 369. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. M. Anderson, D. R. Grattan, W. van den Ancker, and R. S. Bridges Reproductive Experience Increases Prolactin Responsiveness in the Medial Preoptic Area and Arcuate Nucleus of Female Rats Endocrinology, October 1, 2006; 147(10): 4688 - 4694. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. J. Naylor, S. R. Oakes, M. Gardiner-Garden, J. Harris, K. Blazek, T. W. C. Ho, F. C. Li, D. Wynick, A. M. Walker, and C. J. Ormandy Transcriptional Changes Underlying the Secretory Activation Phase of Mammary Gland Development Mol. Endocrinol., July 1, 2005; 19(7): 1868 - 1883. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Goffin, S. Bernichtein, P. Touraine, and P. A. Kelly Development and Potential Clinical Uses of Human Prolactin Receptor Antagonists Endocr. Rev., May 1, 2005; 26(3): 400 - 422. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Shingo, C. Gregg, E. Enwere, H. Fujikawa, R. Hassam, C. Geary, J. C. Cross, and S. Weiss Pregnancy-Stimulated Neurogenesis in the Adult Female Forebrain Mediated by Prolactin Science, January 3, 2003; 299(5603): 117 - 120. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Bernichtein, S. Kinet, S. Jeay, M. Llovera, D. Madern, J. A. Martial, P. A. Kelly, and V. Goffin S179D-Human PRL, a Pseudophosphorylated Human PRL Analog, Is an Agonist and Not an Antagonist Endocrinology, September 1, 2001; 142(9): 3950 - 3963. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
