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Department of Anatomy and Structural Biology and Neuroscience Research Center, University of Otago School of Medical Sciences, Dunedin, New Zealand
Address all correspondence and requests for reprints to: Dr. David Grattan, Department of Anatomy and Structural Biology, University of Otago School of Medical Sciences, P.O. Box 913, Dunedin, New Zealand. E-mail: david.grattan{at}stonebow.otago.ac.nz
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Abstract |
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Introduction |
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During early pregnancy in rats, PRL secretion occurs in a semicircadian rhythm, which is characterized by a large nocturnal surge and a slightly smaller diurnal surge (6, 7, 8). Both of these surges are necessary for the initiation and maintenance of luteal function (9). During this time TIDA neurons exhibit a daily pattern of activity that is inversely related to PRL secretion. Dopamine levels in the portal blood (10), tyrosine hydroxylase activity in the median eminence (8, 11, 12), and expression of Fos-related antigens in neuroendocrine dopaminergic neurons (13) are all reduced during the PRL surges and elevated in the intersurge interval when PRL is low. These PRL surges begin to reduce in magnitude on day 8 of pregnancy and are completely inhibited by day 10 of pregnancy (7, 14). The reduction in PRL surges during midpregnancy coincides with the onset of placental lactogen secretion, which inhibits PRL secretion from the maternal pituitary gland (15, 16, 17, 18) and replaces PRL as the major luteotropic factor in mid- and late pregnancy. As placental lactogen has a high degree of structural homology with PRL (19), the increased secretion of placental lactogen at midpregnancy mimics the negative feedback action of PRL to produce chronic stimulation of the TIDA neurons (2, 18, 20). This increase in TIDA activity is responsible for terminating maternal PRL secretion (16) and maintaining low levels of PRL throughout the second half of pregnancy (2, 21).
During late pregnancy, plasma PRL levels remain low until a large PRL surge occurs during the dark period immediately preceding parturition (22). The magnitude and timing of this antepartum surge are similar to the nocturnal surges of early pregnancy, except that the antepartum PRL surge is much broader (22, 23). Although plasma placental lactogen begins to decline slightly during late pregnancy (24, 25), placental lactogen levels remain relatively high at the end of pregnancy. Thus, sustained feedback inhibition of pituitary PRL secretion by placental lactogen would be expected (17, 20). Elevated PL, however, does not appear to inhibit the PRL secretion during the nocturnal antepartum surge (26, 27). Moreover, in rats bearing intrahypothalamic anterior pituitary grafts to induce central hyperprolactinemia, the antepartum PRL surge was unaffected (28). These observations suggest that the mechanisms involved in negative feedback regulation of PRL secretion become unresponsive during the antepartum period in the rat. A similar lack of responsiveness to PRL has been described in lactating rats (29, 30). This effect is not due to the decreased sensitivity of the lactotrophs to dopamine in the pituitary, as bromocriptine, a dopamine agonist, completely abolishes the antepartum PRL surge (28). Thus, the lack of feedback regulation is likely to be mediated by a change in the responsiveness of TIDA neurons to PRL or placental lactogen during the last days of gestation.
The few studies examining TIDA neuronal activity during late pregnancy provide evidence suggesting a gradual reduction of dopamine synthesis and secretion at this time. Dopamine levels in the portal blood showed a slight, but not significant, decrease on the day before parturition (31). Similarly, tyrosine hydroxylase messenger RNA expression in the arcuate nucleus was reduced on day 20 of pregnancy, although tyrosine hydroxylase activity at this time was not significantly different from that at other time points throughout pregnancy (11). Both of these studies, however, collected only a single sample and did not account for the nocturnal PRL surge. Hence, the present study was designed to characterize TIDA neuronal activity specifically during the antepartum PRL surge and to compare this with known changes in TIDA activity that occur during early and midpregnancy. It was hypothesized that TIDA activity would be reduced during the antepartum surge to allow elevated PRL secretion to occur.
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Materials and Methods |
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5) in plastic
cages under a 14-h light, 10-h dark cycle (lights on at 0500 h).
Temperature was maintained at 22 ± 1 C, and all rats had free
access to food and water. The estrous cycle was monitored by daily
cytological examination of vaginal smears. Proestrous females were
paired with males, and mating was confirmed by the presence of sperm in
vaginal smears the following morning. This was designated day 0 of
pregnancy, and parturition usually occurred on the morning of day 22 in
our colony. All animal experimental protocols were approved by the
University of Otago committee on ethics in the care and use of
laboratory animals.
Pattern of PRL secretion during early and late pregnancy
For serial blood sampling, the right jugular vein was cannulated
under ether anesthesia following the protocol of Harms and Ojeda
(32). The patency of the cannulae was maintained by daily
flushing with heparinized saline. For early pregnancy sample
collection, cannulae were implanted on day 5 (n = 7 rats). Serial
blood samples (300 µl) were collected from each rat at 0900, 1200,
1500, 1700, 1900, and 2200 h
on day 6 and at 0100, 0300, 0500,
and 0900 h on day 7. The underlined times represent the
expected times of the diurnal and nocturnal PRL surges, respectively
(6, 7, 8). Blood samples were centrifuged, and the plasma was
stored at -20 C until PRL RIA. Red blood cells were resuspended in
sterile saline and replaced into the rat after the subsequent sample
collection. For late pregnant sample collection, cannulae were
implanted on day 18 (n = 5 rats), and blood samples were taken
from day 20 of gestation until the day of parturition: at 1200, 1700,
and 2200 h on day 20; 0100, 0300, 0500, 0900, 1200, 1700, and
2200 h on day 21; and 0100, 0300,
0500, and 0900 h on day 22. The underlined
times represent the expected time of the antepartum PRL surge
(22, 26).
Estimation of TIDA activity during pregnancy
Exp 1. Groups of rats were killed by decapitation at 1200
and 0300 h on days 8 and 9 during early pregnancy and on days 21
and 22 during late pregnancy. These times were selected to represent
the trough and peak time points of plasma PRL concentrations,
respectively. For early pregnancy, the animals that had previously been
used for serial sampling were included in the brain collection group,
whereas separate groups of animals were used for the two end points
during late pregnancy. Trunk blood was collected at the time of death
and allowed to clot at 4 C overnight, and serum was stored at -20 C
until PRL RIA. The brains were immediately frozen on dry ice and stored
at -80 C until microdissection of the median eminence using the
micropunch technique (33).
Exp 2. To further characterize the time course of changes during late pregnancy, Exp 1 was repeated with additional time points. Groups of animals were killed at 1200 and 0300 h on days 7 and 8, 11 and 12, 15 and 16, 19 and 20, 20 and 21, and 21 and 22 of pregnancy, respectively. Trunk blood and brain tissue were collected as described above.
Measurement of catecholamines in the median eminence
Frozen coronal brain sections (300 µm) were cut in a cryostat
at -9 C, thaw-mounted onto glass slides, and refrozen. The median
eminence was punched from three consecutive sections (approximately
Bregma -2.30 to -3.30) (33) and placed in 50 µl tissue
buffer (0.05 M
Na2HPO4, 0.03 M
citric acid, and 12% methanol in HPLC grade water, pH 3.0). Samples
were then sonicated in an ultrasonic cell disrupter and centrifuged at
10,000 rpm for 10 min, and the supernatant was collected and stored at
-20 C until measurement of catecholamines and metabolites by HPLC. The
tissue pellet was dissolved in 1 N NaOH, and tissue protein
content was measured using an assay kit (Bio-Rad Laboratories, Inc., Hercules, CA).
Catecholamine and metabolite concentrations were assayed by isocratic HPLC with electrochemical detection. The mobile phase, consisting of tissue buffer, containing 0.1 M EDTA and 0.15 g/liter octane sulfonic acid (pH 3.0), was pumped at a flow rate of 600 µl/min. Catecholamines were separated on a reverse phase C18 column (100 mm; Microsorb-MV, Rainin LC and Supplies, Walnut Creek, CA) and detected using a conditioning cell (+100 mV) and a dual electrode analytical cell (-150 mV and +370 mV) by a Coulochem II detector (ESA, Bedford, MA). Norepinephrine, dopamine, and 3,4-dihydroxyphenylacetic acid (DOPAC) were identified on the basis of their peak retention times (3.7, 8.4, and 10.1 min, respectively) and quantified by comparison with external standards.
RIA for PRL
Plasma or serum concentrations of PRL were measured by RIA using
reagents provided by the NIDDK National Hormone and Pituitary program.
Samples (20- to 100-µl aliquots) or standard preparation (rPRL-RP-3)
were incubated overnight with primary antibody (anti-rPRL-S-9; final
dilution, 1:490 000) and freshly iodinated rPRL-I-6 (10,000 cpm) in a
total volume of 400 µl. Bound counts were precipitated by sheep
antirabbit IgG and 1.5 ml 4% polyethylene glycol at 4 C, followed by
centrifugation at 1,600 x g for 30 min. These
conditions provided approximately 19.5% total binding, with
nonspecific binding less than 2%, and a sensitivity of 1 ng/ml. The
data collected from the assay were analyzed using AssayZap software for
the Macintosh computer (Biosoft, Ferguson, MO). Intraassay
coefficients of variance were less than 10%.
Statistical analysis
All data are presented as the mean and SEM. Plasma
and serum PRL concentrations are presented as nanograms per ml.
Dopamine and DOPAC concentrations in the median eminence are expressed
as picograms per µg protein. Differences between groups were analyzed
using one-way ANOVA, followed by Fisher’s protected least significant
difference post-hoc test. The significance level for all
statistics was set at P < 0.05.
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Results |
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). The peak of the diurnal surge
was approximately 230 ng rPRL-RP-3/ml and occurred at 1700 h,
whereas the peak of the nocturnal surge was approximately 260 ng
rPRL-RP-3/ml and occurred at 0300 h. Between PRL surges were clear
troughs of secretion, with plasma PRL falling to basal levels at
1200 and 2300 h. Plasma PRL concentrations during days 20–22 of
pregnancy show the antepartum PRL surge during the last night of
pregnancy (Fig. 1B). The surge began at approximately 1700 h on
day 21 of gestation, and PRL concentrations were still high at
0900 h on day 22. The antepartum PRL surge was of similar
magnitude to the surges of early pregnancy. Interestingly, the time of
onset of the surge in late pregnancy rats was comparable to that of the
diurnal surge in early pregnancy. Unlike the surge of early pregnancy,
the initial peak of PRL during late pregnancy was not followed by a
trough, and plasma PRL concentrations remained high throughout the
nights of days 21–22. All rats delivered pups between 0800–1200 h on
day 22 of gestation, indicating a normal duration of pregnancy in this
colony.
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) and
were inversely related to serum PRL concentrations at the same time
point (Fig. 2A). During late pregnancy, however, there was no apparent
association between serum PRL levels and TIDA activity, as median
eminence DOPAC content was low at both 1200 h on day 21 and
0300 h on day 22 compared with that at 1200 h during early
pregnancy (P < 0.05; Fig. 2B). By contrast, there were
marked differences in PRL concentrations at these two time points (Fig. 2A). Median eminence dopamine, like DOPAC, was also significantly
reduced at 1200 and 0300 h during late pregnancy compared with
that at 1200 h during early pregnancy (Fig. 2C). No difference was
observed in dopamine concentrations between 1200–0300 h in either late
or early pregnancy. Changes in catecholamines were specific to the
dopaminergic neurons, as norepinephrine concentrations in the median
eminence were not affected by time of day or stage of pregnancy (data
not shown).
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). DOPAC concentrations were
consistently high during the second half of pregnancy, not
significantly different from levels observed at 1200 h during
early pregnancy, associated with low levels of serum PRL. DOPAC
concentrations significantly decreased between 0300–1200 h on day 21,
approximately 36 h before parturition. Despite this large decrease
in DOPAC concentrations, there was no change in serum PRL
concentrations over this time.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The dopamine transporter plays an important role in regulating the amount of dopamine reaching the lactotroph, illustrated by the hypoprolactinemia observed in animals lacking the dopamine transporter (38) or after pharmacological blockade of the transporter (39). Once released from TIDA neurons a proportion of dopamine is recaptured by the dopamine transporter in the median eminence and is metabolized by mitochondrial monoamine oxidase to form DOPAC, which is then rapidly removed from the terminal. The concentration of DOPAC in the median eminence at any time, therefore, reflects current levels of dopamine metabolism within these neurons, providing an accurate index of TIDA neuronal activity (40). Although dopamine concentrations in the median eminence have been shown to be an unreliable measure of TIDA neuronal activity (40) compared with DOPAC content, we also observed changes in dopamine content in the median eminence during late pregnancy. The significant suppression of dopamine concentration observed compared with that in early pregnancy probably reflects an overall reduction in dopamine synthesis in these neurons.
The observation of a broad nocturnal PRL surge in the night preceding parturition is largely consistent with our previous description of PRL secretion during late pregnancy (22), except that in the present study the antepartum PRL surge began earlier than the previously reported 2300 h (22). This difference is likely to be due to the different strains of rat used (Sprague Dawley vs. Wistar) or the different light cycle conditions (14-h light, 10-h dark cycle vs. 12-h light, 12-h dark cycle) under which the animals were housed. In comparing the antepartum surge with the surges of early pregnancy it is apparent that the onset of the surge in the present study was similar to the onset of the diurnal surge of early pregnancy. The major difference between early and late pregnancy was the lack of a trough after the initial peak of PRL secretion during the antepartum PRL surge, which resulted in the formation of a broad peak. This may be explained by the TIDA neurons becoming less responsive to PRL or placental lactogen during late pregnancy (27, 28). During early pregnancy, it seems likely that the diurnal surge of PRL reaches a level where it would begin to stimulate the activity of the TIDA neurons. This increased dopamine tone would then inhibit further PRL release from the anterior pituitary, resulting in the trough between the two surges. During late pregnancy, however, PRL short-loop feedback becomes less effective or nonfunctional (27, 28), implying the TIDA neurons are unresponsive to PRL. This is illustrated in the present studies where, despite prolonged elevations of PRL during the night preceding parturition, TIDA neurons are not activated. Hence, there is a prolonged elevation of PRL levels in the blood during the last night of pregnancy.
These data support and extend previous studies suggesting that TIDA neuronal activity is reduced during late pregnancy compared with earlier times throughout pregnancy (11, 31). The decreased TIDA neuronal activity observed during late pregnancy correlates well with the previously reported reduction in tyrosine hydroxylase gene expression on day 20 (11). In that study, levels of TIDA activity measured neurochemically were not significantly lower on day 20 than during early and midpregnancy. This is consistent with our finding that TIDA activity acutely falls between 0300–1200 h on day 21. It seems likely that the reduction in tyrosine hydroxylase gene expression precedes measurable changes in neurotransmitter release. Interestingly, there are several reports of decreased tyrosine hydroxylase activity in TIDA neurons during lactation (41, 42). This is apparently due to the coordinated effect of suckling-induced suppression of TIDA activity (43, 44), combined with a lack of response to the stimulatory actions of PRL (29, 30). Data from the present study suggest that the decrease in TIDA activity has already occurred by the last day of pregnancy. This neuronal adaptation precedes the onset of the suckling stimulus, suggesting that it is mediated by the hormonal changes of late pregnancy.
The mechanism(s) of suppression of TIDA activity and subsequent increase in PRL secretion during late pregnancy have not been determined. The timing of the observed changes correlates with the increasing estrogen/progesterone ratio brought about by luteolysis during late pregnancy (11). We have demonstrated previously that the changes in ovarian steroids are critical for controlling the timing of the antepartum PRL surge (22). Changes in ovarian steroids during late pregnancy cause activation of the hypothalamic opioid systems (45), and endogenous opioid peptides have been implicated in regulating the antepartum PRL surge (23). Endogenous opioids are also required for the PRL surges of early pregnancy (46) and in response to suckling (43, 44) and apparently act by decreasing the activity of TIDA neurons (47, 48, 49). Hence, it is possible that activation of hypothalamic opioid pathways during late pregnancy might suppress the inhibitory dopaminergic tone and thus account for the decrease in TIDA activity observed in the present study.
The removal of dopaminergic inhibition is clearly necessary for generation of the antepartum PRL surge, as we have previously demonstrated that administration of a dopamine agonist can block this surge (28). The expected consequence of a generalized removal of the inhibitory dopamine tone at the end of pregnancy would be a concurrent increase in PRL secretion. PRL secretion, however, still occurs as a relatively discrete noctural surge. It is possible that some PRL inhibition is maintained by other neuroendocrine dopaminergic neurons via the neurointermediate lobe (2). Alternatively, the data suggest the involvement of other factors in addition to the changes in TIDA neuronal activity. One possibility is that the reduced dopamine tone during late pregnancy unmasks the endogenous stimulatory rhythm postulated to control PRL secretion (50). This would allow the surge pattern of PRL secretion seen during early pregnancy to reoccur. This hypothesis is supported by the observation that vasoactive intestinal polypeptide is involved in the regulation of both the nocturnal component of the endogenous stimulatory rhythm during early pregnancy (51, 52) and the nocturnal antepartum PRL surge (23). In the present study the antepartum PRL surge was shown to begin by 1700 h on day 21, perhaps temporally related to the endogenous diurnal surge. As described above, the lack of the normal response of TIDA neurons to elevated PRL would result in the prolonged antepartum PRL surge without exhibiting the trough that one sees between the diurnal and nocturnal peaks of early pregnancy.
In conclusion, the present study has shown that unlike early pregnancy, in which PRL secretion is tightly linked to TIDA neuronal activity, PRL secretion during late pregnancy is not closely associated with TIDA activity. Hence, the data suggest that dopamine is not the only regulator of the antepartum PRL surge. Moreover, the data provide further evidence that the lack of responsiveness of TIDA neurons to PRL during late pregnancy contributes to the maintenance of the surge. Although the mechanism controlling this adaptation is not known, it is postulated that the suppression of TIDA activity and the lack of responsiveness may be induced by activation of endogenous opioid pathways after steroid hormone changes at the end of pregnancy. The overall reduction of the inhibitory dopamine tone at the end of pregnancy may unmask an endogenous stimulatory rhythm similar to that described in early pregnancy, allowing other factors to increase PRL secretion in a discrete surge pattern. Regardless of the specific mechanism involved, the change in neuroendocrine regulation of PRL represents a physiologically important adaptation of the maternal brain, allowing a state of hyperprolactinemia that is essential for milk production (53) and maternal behavior (54).
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Acknowledgments |
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Footnotes |
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Received August 29, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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opiate receptor subtypes. J
Neuroendocrinol 8:771–776[CrossRef][Medline]
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) and 0300 h (
) during
early pregnancy (1200 h on day 8; 0300 h on day 9) and late
pregnancy (1200 h on day 21; 0300 h on day 22; n =
5–7/group). *, Significant difference at 0300 h compared with
1200 h at the same stage of pregnancy; 
, significant difference
of each time point during late pregnancy compared with 1200 h of
early pregnancy; 
, significant difference compared with 0300 h
during early pregnancy.