eon-7227/92/1301-0023$03.00/0 Endocrinology Copyright 0 1992 by The Endocrine
Vol. 130, No. 1
Printed
Society
in U.S.A.
Cells Inhibit Late Pregnancy and Rat Choriocarcinoma Nocturnal Prolactin Surges and Serotonin-Induced Prolactin Release* HIROSHI
TOMOGANE,
Department
of
Physiology,
ANAHITA University
M. MISTRY,
of Kansas
Medical
AND
JAMES
L. VOOGT
Center, Kansas City, Kansas 66103
ABSTRACT.
injected with the cells had Rcho tumors at the site of injection when analyzed on day 9. These rats also had significantly (P < 0.05) reduced nocturnal PRL surges on days 7 and 8 of pregnancy compared to the control animals, and on day 9, the PRL surge was completely blocked. In another group of day 9 pregnant rats containing Rcho tumors, DOI-induced PRL release was blocked by Rcho cells, whereas in controls, plasma PRL increased from 5 to 47 rig/ml. The final experiment tested whether the presence of Rcho cells affected serotonergicor TRH (1 rg/rat)-induced PRL release in cyclic rats that were ovariectomized 1 day before drug injection. Injection of Rcho cells 8 days earlier completely inhibited 5-hydroxytryptophanor DOI-induced PRL release, but did not affect TRH-induced PRL release. These results indicate that the absence of PRL surges after midpregnancy may be due in part to the inability of serotonin to stimulate PRL at this time compared to early pregnancy. Secretion of placental lactogens or other PRL-like peptides from the placenta in the pregnant rat may be antagonistic to the normal stimuli that cause the PRL surges of early pregnancy, resulting in a loss of surges. (Endocrinology 130: 23-28, 1992)
The purpose of the present study was to determine the effect of hormonal secretion by trophoblast cells on serotonin-induced release of PRL in both pregnant and nonpregnant rats. In the first experiment, three compounds that effectively lead to stimulation of serotonin receptors were injected ip or intraarterially between 0900-1200 h on day 8 or 16 of pregnancy. These included the serotonin precursor 5-hydroxytryptophan (20 mg/kg BW); a releaser of serotonin, fenfluramine (10 mg/kg BW); and a serotonin S2 receptor agonist, DOI (2,5-dimethoxy-4-iodophenyl-2-aminopropane-HCk 500 pg/kg BW). When injected on day 8, these treatments significantly (P < 0.01) increased the level of plasma PRL within 30 min after the injection. However, on day 16 the same treatments could not induce any change in the plasma PRL level. In the second experiment, rat choriocarcinoma (Rcho) cells, which secrete placental lactogen I in uiuo, were injected beneath the kidney capsule on day 1 of pregnancy. Control pregnant rats injected with the cell culture medium RPMI-1640 containing 20% FBS continued to have a nocturnal PRL surges on days 7, 8, and 9, with the peak value of plasma PRL occurring at 0400 h. Rats
T
levels remained low during the time when each surge was expected after treatment with serotonin receptor antagonists or with an inhibitor of serotonin synthesis. Another physiological stimulatory factor for PRL, TRH, which acts directly on the lactotropes, clearly induced PRL release in the first half of pregnancy, but the level induced by TRH declined remarkably in the last half of pregnancy (9, 10). There are no reports describing the responsiveness of PRL to the serotonergic stimulus between different stages of pregnancy. However, the fact that the responsiveness of PRL release to TRH varies with the day of pregnancy suggests that there may also be a change in responsiveness to serotonergic input. If so, this will provide some insight into the hypothalamic components that participate in the lack of PRL surges during the second half of pregnancy, since serotonin stimulates PRL release indirectly by acting in the hypothalamus (11, 12). Termination of PRL surges in pregnant rats occurs at the same time that the level of circulating placental lactogen-I (PL-I) increases, and many reports suggest
HE PATTERN of PRL secretion in pregnant rats is characterized by two daily surges during the first half of pregnancy (l-3). These consist of a nocturnal PRL surge, in which the peak level occurs between 03000500 h, and diurnal PRL surge, with peak PRL levels between 1700-2100 h, under a 12-h light, 12-h dark cycle, with lights on at 0600 h. These surges abruptly terminate at midpregnancy, and PRL secretion remains low until shortly before parturition (4,5). The decline of tuberoinfundibular dopaminergic neuronal activity is thought to be partially responsible for the occurrence of each surge (6). In addition, a stimulatory action of the serotonergic neuronal system on PRL release has been shown to contribute to the generation of these surges (7, 8). PRL Received August 5, 1991. Address all correspondence and requests for reprints to: Dr. James L. Voogt, Department of Physiology, University of Kansas Medical Center, 39th and Rainbow Boulevard, Kansas City, Kansas 66103. * This work was supported by NIH Grant HD-24190 (to J.L.V.) and was presented in part at the 73rd Annual Meeting of The Endocrine Society, Washington DC., 1991.
23
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24
PRL INHIBITION
DURING
that PL-I may cause the termination of the surge (1317). Purified rat PL-I is unavailable. However, recently, rat choriocarcinoma (Rcho) cells transplanted beneath the kidney capsule have been shown to contain PL-I mRNA, as evidenced by Northern blot and in situ hybridization (18). These cells also synthesize PL-I protein, determined by Western blot and immunocytochemical analyses. The Rcho cells have been shown not to produce placental lactogen-II or PRL-like protein-A or -B in ho. Rats containing Rcho tumors develop extensive mammary glands, indicating that the PL-I secreted is biologically active. Therefore, Rcho cells may be useful in uiuo to examine the relationship between PL-I and PRL secretion. The objectives of this study were 1) to compare the responsiveness of PRL to serotonergic stimuli in early pregnancy to late pregnancy, 2) to determine the effect of injection of Rcho cells under the kidney capsule on the occurrence of the nocturnal PRL surge, and 3) to examine the effect of Rcho cells on PRL release induced by TRH and serotonergic stimuli. Results from these experiments contribute to an understanding of which factors are responsible for inhibiting PRL surges during the second half of pregnancy. Materials
and Methods
Animals
Female Sprague-Dawley rats (Sasco Co., Omaha, NE) were used under controlled temperature and lighting conditions, with lights on between 0600-1800h. Rat food and water were supplied ad l&turn. Daily vaginal smearswere taken before and after Rcho cell injection in cycling rats. To induce pregnancy, a single female was placed with a male on the afternoon of proestrus. The presenceof sperm in the vaginal smear was designatedday 0 of pregnancy. Blood sampleswere obtained via a cannula placed in the right commoncarotid artery, which extended outsidethe cage.Each cannulated animal washoused in an individual cageat all times. Approximately 150 ~1blood were withdrawn at each sampling time, and an equivalent amount of saline was replacedvia the cannula. When samples were taken during the dark period, a 25-watt red light wasused. Ovariectomy, cannulation, and Rcho cell injection were performed under ether anesthesia.Cannulation was performed 1 day before blood samplecollection began.All materials injected intraarterially (ia) wereinjected via the carotid cannula. Animal procedureswere carried out as approved by the University of KansasInstitutional Animal Care and Use Committee. Experiments
PREGNANCY
Endo. 1992 Vol 130 - No 1
fluramine (10 mg/kg BW, ip; ResearchBiochemicals)during the morning (0900-1200h) on day 8 or 16 of pregnancy. Rats were injected with saline or distilled water as controls. Blood sampleswere withdrawn just before the injection and at 5- to 60-min intervals after the injection. The secondexperiment determined the effect of Rcho cells on the nocturnal PRL surge in the pregnant rat. Rcho cells (1 x 106)were injected beneath the kidney capsuleon day 0 or 1 of pregnancy using a 26-gaugeneedle in a volume of 50 ~1 medium. Control rats were implanted with medium alone. Blood sampleswere collected at 2400, 0200, 0400, and 0600h on days 7,8, and 9 of pregnancy. Some of these pregnant rats were used to determine the effects of Rcho cells on DOI-induced PRL release.Blood samples were collected before DO1 or vehicle injection and 5, 10, 15,30, and 60 min after the injection. In the third experiment, cyclic rats were injected with Rcho cells or mediumbeneath the kidney capsuleon random daysof the cycle. Seven days after cell injection, the animals were ovariectomized and the next day injected via the carotid cannula with 5-HTP (20 mg/kg BW), DO1 (500 rg/kg BW), or TRH (1 pg/rat; SigmaChemical Co.). Each control animal was injected with the vehicle. Blood sampleswere collected before the injection and 5, 10, 15,30,60, or 90 min after the injection. Rcho cells
Rcho cells were maintained in RPMI-1640 culture medium supplementedwith 20% heat-inactivated fetal bovine serum (FBS; Hazelton, Lenexa, KS) to which were added 50 pM @mercaptoethanol, 1 mM sodiumpyruvate, 100 U/ml penicillin, and 100 rg/ml streptomycin. For cell maintenance, approximately 1.5-2 x lo6 cells were added to each incubation flask containing 12 ml medium and allowedto grow in a humidified atmosphereof 95% air-5% CO,. Cultures were split every 3 days. Rcho cells were collected by brief exposure to 0.25% trypsin-0.02% EDTA, followed by mechanical scrappingwith the aid of a rubber policeman. The number of Rcho cells injected under the kidney capsulewas 1 x 10scells/50 ~1.rat. Hormone
assay
The blood collectedwasplacedin a heparinized tube. Plasma separatedafter centrifugation waskept at -20 C until assayed. Each sample was assayedfor PRL at two different plasma volumesby RIA. The assaymaterials for RIA wereprovided by the NIDDK Hormone Distribution Program, and the reference preparation usedwasNIDDK rat PRL RP-1. Rat PRL labeled with 1251 waspurchasedfrom DuPont (Boston, MA). The limit of sensitivity was 50 pg. Statistics
The first experiment determined the effect of serotonergic stimuli on the level of plasmaPRL in day 8 and day 16pregnant rats. Animals were injected with 5-hydroxytryptophan (5HTP; 50 mg/kg BW, ip; Sigma Chemical Co., St. Louis, MO), 2,5dimethoxy-4-iodophenyl-2-aminopropane-HCl (DOI; 500 pg/ kg BW, ia; ResearchBiochemicals, Inc., Natick, MA), or fen-
Results are expressedas the mean f SE. Two-way analysis of variance for repeated measureswas carried out to compare different treatments over time. Scheffe’s F test or Student’s t test was used to compare means between groups at the same time after one-way analysisof variance.
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PRL
INHIBITION
DURING
Results
Rats were injected with three different serotonergic drugs on either day 8 or 16 of pregnancy between 09001200 h (Fig. 1). Saline injected as a control vehicle on day 8 or 16 did not alter PRL levels from preinjection values, and the data were combined. Injection of the serotonin precursor 5-HTP resulted in peak plasma PRL levels 30 min later in day 8 rats, whereas day 16 rats showed no response. Fenfluramine, which increases extraneuronal serotonin levels by stimulating serotonin release and inhibiting serotonin reuptake, also was effective in causing PRL release in day 8 rats, but was totally ineffective on day 16. DOI, a serotonin S2 agonist, increased PRL immediately in day 8 rats, reaching a peak in 5 min, followed by a decline to baseline levels at 30 min. DO1 had no significant effect in day 16 rats. Thus, all three compounds that increase serotonin input to the hypothalamus significantly increased PRL above preinjection levels on day 8, and these levels were much higher than those after drug injection on day 16. The level of plasma PRL on day 16 of pregnancy did not increase after any of the serotonergic stimuli used and was the same as that in the saline-treated controls.
Rcho-induced inhibition of PRL responseto a serotonin agonist in pregnant rats
PRL release after serotonin receptor agonist (DOI) injection on day 9 of pregnancy was compared between control rats and rats injected with Rcho cells on day 1 of pregnancy (Fig. 3). In the control rats, DO1 injection induced a significant increase in the plasma PRL level 5 min later, which returned to baseline 30 min after injection. This PRL change in the control pregnant rat was not observed in the rats containing Rcho cells. Injection of vehicle into either pregnant control (Fig. 3) or pregnant Rcho rats (data not shown) did not affect PRL levels. Effect of Rcho cells on PRL response to serotonergic agonists or TRH in ovariectomized rats
Cyclic rats with Rcho cells showed continuous diestrous vaginal smears within a few days after cell injection. After a minimum of 4 days of diestrus and 7 days after cell injection, all rats were ovariectomized and received a carotid cannula. Between 0800-1200 h on the next day, two serotonergic stimuli, DO1 and 5-HTP, as well as TRH were injected into separate groups of rats containing either Rcho cells or control rats (Fig. 4). Saline injection did not alter PRL levels. Both 5-HTP and DO1 caused rapid and significant increases in PRL in control rats, whereas these compounds were totally
Rcho-induced inhibition of nocturnal PRL surges
Control pregnant rats injected with medium had nocturnal PRL surges that peaked at 0400 h on days 7, 8, and 9 (Fig. 2). Rats with Rcho cells injected under their kidney capsule on day 1 of pregnancy had significantly lower PRL surges on days 7 and 8, and by day 9, the nocturnal PRL surge was absent. Also, plasma PRL at
FIG. 1. Effects of serotonergic drugs on plasma PRL levels on days 8 and 16 of pregnancy. The serotonin precursor 5HTP (20 mg/kg BW), a releasor of serotonin; fenfluramine (10 mg/kg BW); or the serotonin receptor agonist DO1 (500 @g/kg BW) was injected on day 8 or 16 of pregnancy between 0900-1200 h. All serotonergic drugs increased plasma PRL levels on day 8 (P < 0.05) compared to the preinjection values and did not affect PRL on day 16. Each drug also increased PRL on day 8 compared to levels on day 16. This is indicated by an asterisk at each time where significant (P < 0.05). Each point and its uertical line represent the mean and SE from 511 rats/group.
25
midnight, before the initiation of the nocturnal PRL surge, was significantly lower in rats with Rcho cells than in control pregnant rats. There was no evidence of gross fetal or placental abnormalities in either of these two groups of rats.
Serotonergically induced PRL release in pregnant rats
SALINE
PREGNANCY
,75, 5.HYDROXYTRYPTOPHAN
CONTROLS
0 TIME
(MINUTES)
Ii0
30
TIME
&JTES;
’
FENFLURAMINE 175 z6
130
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125
p!
a= a.-
DOI
'00 -
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i 0
30 TIME
(:lklTES;”
120
0
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(&JTES;
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’
120
PRL INHIBITION
26
DURING
400
+ z 4 0’
aE
300
200
at% a= 5
100
2
0 8-97”“”&,oo.s.s~ 2202 SWS!2SRSSZSS NOOONOOONOO
FIG. 2. Effect of the presence of Rcho cell tumors on the nocturnal PRL surge in pregnant rats. Rcho cells (1 x 106) were implanted beneath the kidney capsule on day 1 of pregnancy. Rats bearing Rcho tumors had significantly (P < 0.05) lower plasma PRL levels on all 3 days compared to controls. *, Times when PRL values are significantly different from controls. Each point and its vertical line represent the mean and SE from seven rats in the control group and five rats in the Rcho group.
01
. 0
I 15
TIME
.
-
CONTROLIDOI
-
RCHO/DOI CONTROL/SALINE
I
’
I
f
60
(?iiiJ”TES‘j
5
FIG. 3. Effect of the presence of Rcho cell tumors on serotonergic receptor agonist (DOI)-induced PRL release in pregnant rats. DO1 (500 rg/kg BW) was injected on day 9 of pregnancy between 0900-1200 h. DO1 significantly increased plasma PRL (P < 0.01 compared to the preinjection value) in control, but not in Rcho, rats. *, Times when PRL values are significantly different from values in controls also injected with DOI. Each point and its uertical line represent the mean and SE from seven rats per group.
ineffective in Rcho rats. Interestingly, the responses to DO1 and 5-HTP were much greater in these ovariectomized control rats than in pregnant rats on day 8 or 9 (Figs. 1 and 3). Injection of TRH caused a significant increase in PRL in both groups of rats compared to preinjection levels or values in vehicle-treated rats. There was no difference in the response to TRH in the two groups. Discussion Previous reports have shown that serotonin receptor antagonists or synthesis inhibitors block the nocturnal and diurnal PRL surges in pregnant rats (7, S), supporting the hypothesis that serotonin is an essential component in the generation of PRL surges. The present data demonstrate that an increase in serotonergic input to the hypothalamus is a very potent mechanism to increase PRL release during early pregnancy, when PRL surges
PREGNANCY
Endo. Voll30.
1992 No 1
are still present. The serotonin precursor 5-HTP; a blocker of serotonin reuptake, fenfluramine; and DOI, a serotonin receptor agonist specific for S2 receptors, were effective in inducing PRL release on day 8 of pregnancy. PRL release in response to the same serotonergic stimuli did not occur on day 16, a time when nocturnal PRL surges are no longer present. This suggests that part of the mechanism for the loss of PRL surges at midpregnancy may be due to a failure of the hypothalamus to transfer the serotonin signal into a signal for PRL release. There is probably a significantly reduced serotonin signal during the last half of pregnancy as well (19). The second half of pregnancy is characterized by the appearance of a number of PLs and PRL-like peptides (20, 21). Numerous reports have shown an inverse relationship between the level of PLs and the occurrence of PRL surges (13-16). It has been reported that when conditioned medium containing PL obtained from incubation of day 11 placentas was infused via the jugular vein of pregnant rats, the nocturnal PRL surge was completely inhibited (17). Furthermore, implantation of human PL into the median eminence region on day 8 of pregnancy completely inhibit the nocturnal PRL surge (22). Placental extracts, maternal serum, or conditioned medium from incubation of placental tissue inhibited PRL release in vitro (23, 24). Rcho cells induced the premature termination of nocturnal PRL surges after their injection on day 1 of pregnancy. Thus, the tumors that formed as a result of injecting these cells acted similarly to developing trophoblasts in utero in inhibiting PRL secretion. The Rcho cells used in the present study originate from the trophoblast cells of rat placenta (25). The tumors formed from the implanted Rcho cells consist of small basophilic cells and large giant cells (18). The giant cells were found to produce PL-I and not to produce PL-II or PRL-like protein-A or -B. In the pregnant rat, PL-I first appears in the circulation by day 6 or 7; the circulating level increases until day 9 or 10 and then begins to decrease (15, 21). On the basis of several reports, it has been suggested that PL-I is the primary factor that terminates the PRL surge in pregnant rats (14-17,21). The present study using Rcho cells supports this conclusion, but at present, one cannot eliminate other secretions from Rcho cells in addition to PL-I as the cause of termination of PRL surges. Injection of Rcho cells into either pregnant or cycling rats that were ovariectomized 1 day before drug injection completely inhibited the serotonergic neuronal stimulusinduced PRL release. The ovariectomized rats with Rcho tumors had extensive mammary development compared to controls. This inhibitory effect of the Rcho cells correlates very well with the loss of PRL responsiveness to serotonin agonists in day 16 pregnant rats. Although
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PRL INHIBITION ‘001
DURING
SALINE
PREGNANCY
27
CONTROLS
FIG. 4. Effects of the presence of Echo cell tumors on PRL release in response to saline, 5-HTP (20 mg/kg BW), DO1 (500 rg/kg BW), or TRH (1 pg/kg BW) in ovariectomized rats. Cycling rats were injected with Rcho cells, ovariectomized 7 days later, and tested the next day between 0900-1200 h. Rcho cells inhibited 5-HTP- or DOI-induced PRL release, but did not affect TRH-induced PRL release. *, Significantly different (P c 0.05) from control values measured at the same time after the injection. Saline had no effect on PRL levels. Each point and its uertical line represent the mean and SE from 5-10 rats/group.
40gE
;
1I;= l!IL!hL 5HYDROXYTRYPTOPHAN
ml 2 5
u: I2i
25
,
500 400 300 200 100
1
+a*
0
15
30
TIME
THYROTROPIN
45
60
75
0
90
0
(MINUTES)
RELEASING
l
15
l
30
TIME
HORMONE
45
60
75
90
75
90
(MINUTES)
DOI
70
~1-1
5z 500 5, g &oo z 400 360
loo
i
0 0
15
30
TIME
PL-2 is the major PL present at this time of pregnancy (20, 21), and Rcho cells secrete primarily PL-I in uiuo (18), there is little evidence that PL-I and PL-II differ in their PRL-like biological properties. Thus, it is quite likely that PLs secreted during the last half of pregnancy act in the hypothalamus to inhibit the control mechanisms responsible for signaling the lactotroph to release PRL. PRL secretion remains low when sufficient amounts of dopamine are released from the tuberoinfundibular neurons (26-29). It is possible that serotonin acts on these neurons to remove or decrease this tonic inhibitory effect of dopamine and, in turn, increase PRL levels. Serotonin neurons have been reported to have receptors on dopaminergic neurons, and this neuronal connection depresses the activity of dopaminergic neurons in midbrain (30). Whether this is the case for tuberoinfundibular dopaminergic neurons is not clear. The present results, in which secretions from the implanted Rcho cells inhibited serotonin stimulus-induced PRL release in pregnant and ovariectomized rats, suggest that PLs may work by blocking the action of serotonin on dopamine neurons. It has been shown that a uterineplacental factor (31), human PL (32), or PRL (33, 34) can stimulate increased activity of tuberoinfundibular dopaminergic neurons, leading to dopamine release and PRL inhibition. The Rcho cells did not inhibit the ability of TRH to stimulate PRL release in cycling rats ovariectomized 1 day earlier. This was unexpected, because it was previously shown that PRL responsiveness to TRH decreased during the second half of pregnancy (9,lO). This suggests that during pregnancy, factors other than those secreted
45
60
(MINUTES)
by Rcho cells may be responsible for this inhibitory effect on TRH-induced PRL release. Indeed, the ovariectomized rat has a significantly different hormonal profile compared to that of the pregnant rat. In conclusion, serotonin-induced PRL release is totally blocked during late pregnancy and by secretions from Rcho cells. This strongly supports the hypothesis that the loss of PRL surges during the second half of pregnancy is due to secretion of PLs acting in the hypothalamus to interfere with the neurohormonal signal. Part of the signal must involve serotonin. Acknowledgments We wish to thank Dr. Michael Soares (University of Kansas Medical Center) for help in establishing the Rcho cell culture in our laboratory, and the NIDDK and the National Hormone
and Pituitary Program (University of Maryland School of Medicine) for the gift of PRL RIA materials. References 1. Butcher RL, Fugo WW, Collins WE 1972 Semicircadian rhythm in plasma levels of prolactin during early gestation in the rat. Endocrinology 90:1125-1127 2. Smith MS, Neil1 JD 1976 Termination at mid-pregnancy of the two daily surges of plasma prolactin initiated by mating in the rat. Endocrinology 98:696-701 3. Voogt JL 1980 Regulation of nocturnal prolactin surges during pregnancy in the rat. Endocrinology 1061670-1676 4. Bridges RS, Goldman BD 1975 Ovarian control of prolactin secretion during late pregnancy in the rat. Endocrinology 97:496-498 5. Grattan DR, Averill RLW 1990 Effect of ovarian steroids on a nocturnal surge of prolactin secretion that precedes parturition in the rat. Endocrinology 126:1199-1205 6. McKay DW, Pasieka CA, Moore KE, Riegle GD, Demarest KT 1982 Semicircadian rhythm of tuberoinfundibular dopamine neu-
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PRL
28
7. 8. 9.
10. 11. 12. 13. 14. 15. 16. 17. 18.
19. 20.
INHIBITION
ronal activity during early pregnancy and pseudopregnancy in the rat. Neuroendocrinology 34:229-233 Mistrv A. Vooat JL 1989 Role of serotonin in nocturnal and diurnal surges of pro&tin in the pregnant rat. Endocrinology 125:28752880 Mistry AM, Voogt JL 1990 Serotonin synthesis inhibition or receptor antagonism reduces pregnancy-induced nocturnal prolactin secretion. Ljfe Sci 47:693-761 Bridaes RS. Terkel J. Sawver CH 1983 Thvroid stimulatina hormone and prolactin secretion: reduced sensitivity to TRH-itimulated prolactin release after midpregnancy in rats. Proc Sot Exp Biol Med 173:527-532 Voogt JL 1986 Changes in pituitary prolactin responsiveness to TRH during pregnancy. Proc Sot Exp Biol Med 182:58-62 Garthwaite TL. Hagen TC 1979 Evidence that serotonin stimulates a prolactin releasing factor in the rat. Neuroendocrinology 29:215220 Pilotte NS, Porter JC 1981 Dopamine in hypophysial portal plasma and prolactin in systemic plasma of rats treated with B-hydroxyt&amine. Endocrinology 108:2137-2141 Yogev L, Terkel J 1980 Timing of termination of nocturnal prolactin surges in pregnant rats as determined by the number of fetuses. J Endocrinol84:421-424 Vooat JL Robertson M. Friesen H 1982 Inverse relationshiu of prolictin and rat placental lactogen during pregnancy. Biol Reprod 26:800-805 Tonkowicz PA, Voogt JL 1983 Examination of rat placental lactogen and prolactin at 6 hr intervals during midpregnancy. Proc Sot Exp Biol Med 173:583-587 Tonkowicz PA, Voogt JL 1983 Termination of prolactin surges with development of placental lactogen secretion in the pregnant rat. Endocrinology 113:1314-1318 Voogt JL, de Greef WJ 1989 Inhibition of nocturnal prolactin surges in the pregnant rat by incubation medium containing placental lactogen. Proc Sot Exp Biol Med 191:403-407 Faria TN, Deb S, Kwok SCM, Vandeputte M, Talamantes F, Soares MJ 1990 Transplantable rat choriocarcinoma cells express placental lactogen: identification of placental lactogen-I immunoreactive protein and messenger ribonucleic acid. Endocrinology 127:3131-3137 Mistry AM, Vidal G, Voogt JL 1991 Dopaminergic and serotonergic activity in the hypothalamus during early and late pregnancy. Brain Res 550:239-246 Duckworth ML, Pelen LM, Schroedter L, Shan P, Friesen HG .,
I
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DURING
21. 22. 23. 24. 25.
26. 27. 28.
29.
30. 31.
32. 33. 34.
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1988 Placental lactogens and extra-hypophyseal prolactin gene family. In: Hoshino K (ed) Prolactin Gene Family and Its Receptors. Elsevier, New York, pp 79-88 Faria TN, Deb S, Kwok SCM, Talamantes F, Soares MJ 1990 Ontogeny of placental lactogen-I and placental lactogen-II expression in the developing rat placenta. Dev Biol 141:279-291 Voogt JL 1980 Regulation of nocturnal prolactin surge during pregnancy in the rat. Endocrinology 106~1670-1676 Goronse WC. Freeman ME 1985 Effects of nlacenta and maternal serum on prdlactin secretion in u&o. Biol Reprod 32:279-283 Voogt JL 1984 Evidence for an inhibitory influence of rat placental lactogen on prolactin release in uitro. Biol Reprod 31:141-147 Shintani S, Glass LE, Page EW 1966 Studies of induced malignant tumors of placental and uterine origin in the rat. III. Identification of experimentally induced choriocarcinoma by detection of placental hormone. Am J Obstet Gynecol95:559-563 Ben-Jonathan N 1985 Dopamine: a prolactin inhibiting hormone. Endocr Rev 6564-589 Ben-Jonathan N, Arbogast LA, Hyde JF 1989 Neuroendocrine regulation of prolactin release. Prog-Neurobiol33:399-447 Pan JJ. Wana PS 1989 Effect of transient douamine antaeonism on thyrotropin-releasing-hormone induced prolactin secretion in the serotonin-blocked, estrogen treated rat. Neuroendocrinology 49:281-289 Haisenleder DJ, Moy JA, Gala RR, Lawson DM 1986 The effect of transient dopamine antagonism on thyrotropin-releasing hormone-induced prolactin release in pregnant rats. Endocrinology 119:1980-1988 Ugedo L, Grenhoff J, Svensson TH 1989 Ritanserin, a 5-HT, receptor antagonist, activates midbrain dopamine neurons by blocking serotonergic inhibition. Psychopharmacology 98:45-50 Demarest KT, Moore KE, Riegle GD 1983 Role of a uterineplacental factor in the cessation of the semicircadian rhythm of tuberoinfundibular dopaminergic neuronal activity at midpregnancy in the rat. Neuroendocrinology 36:409-414 Demarest KT, Duda NJ Riegle GD, Moore KE 1983 Placental lactogen mimics prolactin in activating tuberoinfundibular dopamine neurons. Brain Res 272:175-178 Gudelsky GA, Porter JC 1980 Release of dopamine from tuberoinfundibular neurons into pituitary stalk blood after prolactin or haloperidol administration. Endocrinology 106526-529 Voogt JL 1987 Actions of nrolactin in the brain. In: Rilleman JA (ed)-Actions of Prolactin on Molecular Processes. CRC Press, Boca Raton, pp 27-40 L
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Vol. 130, No. 1
Printed
Society
in U.S.A.
Cells Inhibit Late Pregnancy and Rat Choriocarcinoma Nocturnal Prolactin Surges and Serotonin-Induced Prolactin Release* HIROSHI
TOMOGANE,
Department
of
Physiology,
ANAHITA University
M. MISTRY,
of Kansas
Medical
AND
JAMES
L. VOOGT
Center, Kansas City, Kansas 66103
ABSTRACT.
injected with the cells had Rcho tumors at the site of injection when analyzed on day 9. These rats also had significantly (P < 0.05) reduced nocturnal PRL surges on days 7 and 8 of pregnancy compared to the control animals, and on day 9, the PRL surge was completely blocked. In another group of day 9 pregnant rats containing Rcho tumors, DOI-induced PRL release was blocked by Rcho cells, whereas in controls, plasma PRL increased from 5 to 47 rig/ml. The final experiment tested whether the presence of Rcho cells affected serotonergicor TRH (1 rg/rat)-induced PRL release in cyclic rats that were ovariectomized 1 day before drug injection. Injection of Rcho cells 8 days earlier completely inhibited 5-hydroxytryptophanor DOI-induced PRL release, but did not affect TRH-induced PRL release. These results indicate that the absence of PRL surges after midpregnancy may be due in part to the inability of serotonin to stimulate PRL at this time compared to early pregnancy. Secretion of placental lactogens or other PRL-like peptides from the placenta in the pregnant rat may be antagonistic to the normal stimuli that cause the PRL surges of early pregnancy, resulting in a loss of surges. (Endocrinology 130: 23-28, 1992)
The purpose of the present study was to determine the effect of hormonal secretion by trophoblast cells on serotonin-induced release of PRL in both pregnant and nonpregnant rats. In the first experiment, three compounds that effectively lead to stimulation of serotonin receptors were injected ip or intraarterially between 0900-1200 h on day 8 or 16 of pregnancy. These included the serotonin precursor 5-hydroxytryptophan (20 mg/kg BW); a releaser of serotonin, fenfluramine (10 mg/kg BW); and a serotonin S2 receptor agonist, DOI (2,5-dimethoxy-4-iodophenyl-2-aminopropane-HCk 500 pg/kg BW). When injected on day 8, these treatments significantly (P < 0.01) increased the level of plasma PRL within 30 min after the injection. However, on day 16 the same treatments could not induce any change in the plasma PRL level. In the second experiment, rat choriocarcinoma (Rcho) cells, which secrete placental lactogen I in uiuo, were injected beneath the kidney capsule on day 1 of pregnancy. Control pregnant rats injected with the cell culture medium RPMI-1640 containing 20% FBS continued to have a nocturnal PRL surges on days 7, 8, and 9, with the peak value of plasma PRL occurring at 0400 h. Rats
T
levels remained low during the time when each surge was expected after treatment with serotonin receptor antagonists or with an inhibitor of serotonin synthesis. Another physiological stimulatory factor for PRL, TRH, which acts directly on the lactotropes, clearly induced PRL release in the first half of pregnancy, but the level induced by TRH declined remarkably in the last half of pregnancy (9, 10). There are no reports describing the responsiveness of PRL to the serotonergic stimulus between different stages of pregnancy. However, the fact that the responsiveness of PRL release to TRH varies with the day of pregnancy suggests that there may also be a change in responsiveness to serotonergic input. If so, this will provide some insight into the hypothalamic components that participate in the lack of PRL surges during the second half of pregnancy, since serotonin stimulates PRL release indirectly by acting in the hypothalamus (11, 12). Termination of PRL surges in pregnant rats occurs at the same time that the level of circulating placental lactogen-I (PL-I) increases, and many reports suggest
HE PATTERN of PRL secretion in pregnant rats is characterized by two daily surges during the first half of pregnancy (l-3). These consist of a nocturnal PRL surge, in which the peak level occurs between 03000500 h, and diurnal PRL surge, with peak PRL levels between 1700-2100 h, under a 12-h light, 12-h dark cycle, with lights on at 0600 h. These surges abruptly terminate at midpregnancy, and PRL secretion remains low until shortly before parturition (4,5). The decline of tuberoinfundibular dopaminergic neuronal activity is thought to be partially responsible for the occurrence of each surge (6). In addition, a stimulatory action of the serotonergic neuronal system on PRL release has been shown to contribute to the generation of these surges (7, 8). PRL Received August 5, 1991. Address all correspondence and requests for reprints to: Dr. James L. Voogt, Department of Physiology, University of Kansas Medical Center, 39th and Rainbow Boulevard, Kansas City, Kansas 66103. * This work was supported by NIH Grant HD-24190 (to J.L.V.) and was presented in part at the 73rd Annual Meeting of The Endocrine Society, Washington DC., 1991.
23
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24
PRL INHIBITION
DURING
that PL-I may cause the termination of the surge (1317). Purified rat PL-I is unavailable. However, recently, rat choriocarcinoma (Rcho) cells transplanted beneath the kidney capsule have been shown to contain PL-I mRNA, as evidenced by Northern blot and in situ hybridization (18). These cells also synthesize PL-I protein, determined by Western blot and immunocytochemical analyses. The Rcho cells have been shown not to produce placental lactogen-II or PRL-like protein-A or -B in ho. Rats containing Rcho tumors develop extensive mammary glands, indicating that the PL-I secreted is biologically active. Therefore, Rcho cells may be useful in uiuo to examine the relationship between PL-I and PRL secretion. The objectives of this study were 1) to compare the responsiveness of PRL to serotonergic stimuli in early pregnancy to late pregnancy, 2) to determine the effect of injection of Rcho cells under the kidney capsule on the occurrence of the nocturnal PRL surge, and 3) to examine the effect of Rcho cells on PRL release induced by TRH and serotonergic stimuli. Results from these experiments contribute to an understanding of which factors are responsible for inhibiting PRL surges during the second half of pregnancy. Materials
and Methods
Animals
Female Sprague-Dawley rats (Sasco Co., Omaha, NE) were used under controlled temperature and lighting conditions, with lights on between 0600-1800h. Rat food and water were supplied ad l&turn. Daily vaginal smearswere taken before and after Rcho cell injection in cycling rats. To induce pregnancy, a single female was placed with a male on the afternoon of proestrus. The presenceof sperm in the vaginal smear was designatedday 0 of pregnancy. Blood sampleswere obtained via a cannula placed in the right commoncarotid artery, which extended outsidethe cage.Each cannulated animal washoused in an individual cageat all times. Approximately 150 ~1blood were withdrawn at each sampling time, and an equivalent amount of saline was replacedvia the cannula. When samples were taken during the dark period, a 25-watt red light wasused. Ovariectomy, cannulation, and Rcho cell injection were performed under ether anesthesia.Cannulation was performed 1 day before blood samplecollection began.All materials injected intraarterially (ia) wereinjected via the carotid cannula. Animal procedureswere carried out as approved by the University of KansasInstitutional Animal Care and Use Committee. Experiments
PREGNANCY
Endo. 1992 Vol 130 - No 1
fluramine (10 mg/kg BW, ip; ResearchBiochemicals)during the morning (0900-1200h) on day 8 or 16 of pregnancy. Rats were injected with saline or distilled water as controls. Blood sampleswere withdrawn just before the injection and at 5- to 60-min intervals after the injection. The secondexperiment determined the effect of Rcho cells on the nocturnal PRL surge in the pregnant rat. Rcho cells (1 x 106)were injected beneath the kidney capsuleon day 0 or 1 of pregnancy using a 26-gaugeneedle in a volume of 50 ~1 medium. Control rats were implanted with medium alone. Blood sampleswere collected at 2400, 0200, 0400, and 0600h on days 7,8, and 9 of pregnancy. Some of these pregnant rats were used to determine the effects of Rcho cells on DOI-induced PRL release.Blood samples were collected before DO1 or vehicle injection and 5, 10, 15,30, and 60 min after the injection. In the third experiment, cyclic rats were injected with Rcho cells or mediumbeneath the kidney capsuleon random daysof the cycle. Seven days after cell injection, the animals were ovariectomized and the next day injected via the carotid cannula with 5-HTP (20 mg/kg BW), DO1 (500 rg/kg BW), or TRH (1 pg/rat; SigmaChemical Co.). Each control animal was injected with the vehicle. Blood sampleswere collected before the injection and 5, 10, 15,30,60, or 90 min after the injection. Rcho cells
Rcho cells were maintained in RPMI-1640 culture medium supplementedwith 20% heat-inactivated fetal bovine serum (FBS; Hazelton, Lenexa, KS) to which were added 50 pM @mercaptoethanol, 1 mM sodiumpyruvate, 100 U/ml penicillin, and 100 rg/ml streptomycin. For cell maintenance, approximately 1.5-2 x lo6 cells were added to each incubation flask containing 12 ml medium and allowedto grow in a humidified atmosphereof 95% air-5% CO,. Cultures were split every 3 days. Rcho cells were collected by brief exposure to 0.25% trypsin-0.02% EDTA, followed by mechanical scrappingwith the aid of a rubber policeman. The number of Rcho cells injected under the kidney capsulewas 1 x 10scells/50 ~1.rat. Hormone
assay
The blood collectedwasplacedin a heparinized tube. Plasma separatedafter centrifugation waskept at -20 C until assayed. Each sample was assayedfor PRL at two different plasma volumesby RIA. The assaymaterials for RIA wereprovided by the NIDDK Hormone Distribution Program, and the reference preparation usedwasNIDDK rat PRL RP-1. Rat PRL labeled with 1251 waspurchasedfrom DuPont (Boston, MA). The limit of sensitivity was 50 pg. Statistics
The first experiment determined the effect of serotonergic stimuli on the level of plasmaPRL in day 8 and day 16pregnant rats. Animals were injected with 5-hydroxytryptophan (5HTP; 50 mg/kg BW, ip; Sigma Chemical Co., St. Louis, MO), 2,5dimethoxy-4-iodophenyl-2-aminopropane-HCl (DOI; 500 pg/ kg BW, ia; ResearchBiochemicals, Inc., Natick, MA), or fen-
Results are expressedas the mean f SE. Two-way analysis of variance for repeated measureswas carried out to compare different treatments over time. Scheffe’s F test or Student’s t test was used to compare means between groups at the same time after one-way analysisof variance.
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PRL
INHIBITION
DURING
Results
Rats were injected with three different serotonergic drugs on either day 8 or 16 of pregnancy between 09001200 h (Fig. 1). Saline injected as a control vehicle on day 8 or 16 did not alter PRL levels from preinjection values, and the data were combined. Injection of the serotonin precursor 5-HTP resulted in peak plasma PRL levels 30 min later in day 8 rats, whereas day 16 rats showed no response. Fenfluramine, which increases extraneuronal serotonin levels by stimulating serotonin release and inhibiting serotonin reuptake, also was effective in causing PRL release in day 8 rats, but was totally ineffective on day 16. DOI, a serotonin S2 agonist, increased PRL immediately in day 8 rats, reaching a peak in 5 min, followed by a decline to baseline levels at 30 min. DO1 had no significant effect in day 16 rats. Thus, all three compounds that increase serotonin input to the hypothalamus significantly increased PRL above preinjection levels on day 8, and these levels were much higher than those after drug injection on day 16. The level of plasma PRL on day 16 of pregnancy did not increase after any of the serotonergic stimuli used and was the same as that in the saline-treated controls.
Rcho-induced inhibition of PRL responseto a serotonin agonist in pregnant rats
PRL release after serotonin receptor agonist (DOI) injection on day 9 of pregnancy was compared between control rats and rats injected with Rcho cells on day 1 of pregnancy (Fig. 3). In the control rats, DO1 injection induced a significant increase in the plasma PRL level 5 min later, which returned to baseline 30 min after injection. This PRL change in the control pregnant rat was not observed in the rats containing Rcho cells. Injection of vehicle into either pregnant control (Fig. 3) or pregnant Rcho rats (data not shown) did not affect PRL levels. Effect of Rcho cells on PRL response to serotonergic agonists or TRH in ovariectomized rats
Cyclic rats with Rcho cells showed continuous diestrous vaginal smears within a few days after cell injection. After a minimum of 4 days of diestrus and 7 days after cell injection, all rats were ovariectomized and received a carotid cannula. Between 0800-1200 h on the next day, two serotonergic stimuli, DO1 and 5-HTP, as well as TRH were injected into separate groups of rats containing either Rcho cells or control rats (Fig. 4). Saline injection did not alter PRL levels. Both 5-HTP and DO1 caused rapid and significant increases in PRL in control rats, whereas these compounds were totally
Rcho-induced inhibition of nocturnal PRL surges
Control pregnant rats injected with medium had nocturnal PRL surges that peaked at 0400 h on days 7, 8, and 9 (Fig. 2). Rats with Rcho cells injected under their kidney capsule on day 1 of pregnancy had significantly lower PRL surges on days 7 and 8, and by day 9, the nocturnal PRL surge was absent. Also, plasma PRL at
FIG. 1. Effects of serotonergic drugs on plasma PRL levels on days 8 and 16 of pregnancy. The serotonin precursor 5HTP (20 mg/kg BW), a releasor of serotonin; fenfluramine (10 mg/kg BW); or the serotonin receptor agonist DO1 (500 @g/kg BW) was injected on day 8 or 16 of pregnancy between 0900-1200 h. All serotonergic drugs increased plasma PRL levels on day 8 (P < 0.05) compared to the preinjection values and did not affect PRL on day 16. Each drug also increased PRL on day 8 compared to levels on day 16. This is indicated by an asterisk at each time where significant (P < 0.05). Each point and its uertical line represent the mean and SE from 511 rats/group.
25
midnight, before the initiation of the nocturnal PRL surge, was significantly lower in rats with Rcho cells than in control pregnant rats. There was no evidence of gross fetal or placental abnormalities in either of these two groups of rats.
Serotonergically induced PRL release in pregnant rats
SALINE
PREGNANCY
,75, 5.HYDROXYTRYPTOPHAN
CONTROLS
0 TIME
(MINUTES)
Ii0
30
TIME
&JTES;
’
FENFLURAMINE 175 z6
130
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a= a.-
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0
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(&JTES;
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’
120
PRL INHIBITION
26
DURING
400
+ z 4 0’
aE
300
200
at% a= 5
100
2
0 8-97”“”&,oo.s.s~ 2202 SWS!2SRSSZSS NOOONOOONOO
FIG. 2. Effect of the presence of Rcho cell tumors on the nocturnal PRL surge in pregnant rats. Rcho cells (1 x 106) were implanted beneath the kidney capsule on day 1 of pregnancy. Rats bearing Rcho tumors had significantly (P < 0.05) lower plasma PRL levels on all 3 days compared to controls. *, Times when PRL values are significantly different from controls. Each point and its vertical line represent the mean and SE from seven rats in the control group and five rats in the Rcho group.
01
. 0
I 15
TIME
.
-
CONTROLIDOI
-
RCHO/DOI CONTROL/SALINE
I
’
I
f
60
(?iiiJ”TES‘j
5
FIG. 3. Effect of the presence of Rcho cell tumors on serotonergic receptor agonist (DOI)-induced PRL release in pregnant rats. DO1 (500 rg/kg BW) was injected on day 9 of pregnancy between 0900-1200 h. DO1 significantly increased plasma PRL (P < 0.01 compared to the preinjection value) in control, but not in Rcho, rats. *, Times when PRL values are significantly different from values in controls also injected with DOI. Each point and its uertical line represent the mean and SE from seven rats per group.
ineffective in Rcho rats. Interestingly, the responses to DO1 and 5-HTP were much greater in these ovariectomized control rats than in pregnant rats on day 8 or 9 (Figs. 1 and 3). Injection of TRH caused a significant increase in PRL in both groups of rats compared to preinjection levels or values in vehicle-treated rats. There was no difference in the response to TRH in the two groups. Discussion Previous reports have shown that serotonin receptor antagonists or synthesis inhibitors block the nocturnal and diurnal PRL surges in pregnant rats (7, S), supporting the hypothesis that serotonin is an essential component in the generation of PRL surges. The present data demonstrate that an increase in serotonergic input to the hypothalamus is a very potent mechanism to increase PRL release during early pregnancy, when PRL surges
PREGNANCY
Endo. Voll30.
1992 No 1
are still present. The serotonin precursor 5-HTP; a blocker of serotonin reuptake, fenfluramine; and DOI, a serotonin receptor agonist specific for S2 receptors, were effective in inducing PRL release on day 8 of pregnancy. PRL release in response to the same serotonergic stimuli did not occur on day 16, a time when nocturnal PRL surges are no longer present. This suggests that part of the mechanism for the loss of PRL surges at midpregnancy may be due to a failure of the hypothalamus to transfer the serotonin signal into a signal for PRL release. There is probably a significantly reduced serotonin signal during the last half of pregnancy as well (19). The second half of pregnancy is characterized by the appearance of a number of PLs and PRL-like peptides (20, 21). Numerous reports have shown an inverse relationship between the level of PLs and the occurrence of PRL surges (13-16). It has been reported that when conditioned medium containing PL obtained from incubation of day 11 placentas was infused via the jugular vein of pregnant rats, the nocturnal PRL surge was completely inhibited (17). Furthermore, implantation of human PL into the median eminence region on day 8 of pregnancy completely inhibit the nocturnal PRL surge (22). Placental extracts, maternal serum, or conditioned medium from incubation of placental tissue inhibited PRL release in vitro (23, 24). Rcho cells induced the premature termination of nocturnal PRL surges after their injection on day 1 of pregnancy. Thus, the tumors that formed as a result of injecting these cells acted similarly to developing trophoblasts in utero in inhibiting PRL secretion. The Rcho cells used in the present study originate from the trophoblast cells of rat placenta (25). The tumors formed from the implanted Rcho cells consist of small basophilic cells and large giant cells (18). The giant cells were found to produce PL-I and not to produce PL-II or PRL-like protein-A or -B. In the pregnant rat, PL-I first appears in the circulation by day 6 or 7; the circulating level increases until day 9 or 10 and then begins to decrease (15, 21). On the basis of several reports, it has been suggested that PL-I is the primary factor that terminates the PRL surge in pregnant rats (14-17,21). The present study using Rcho cells supports this conclusion, but at present, one cannot eliminate other secretions from Rcho cells in addition to PL-I as the cause of termination of PRL surges. Injection of Rcho cells into either pregnant or cycling rats that were ovariectomized 1 day before drug injection completely inhibited the serotonergic neuronal stimulusinduced PRL release. The ovariectomized rats with Rcho tumors had extensive mammary development compared to controls. This inhibitory effect of the Rcho cells correlates very well with the loss of PRL responsiveness to serotonin agonists in day 16 pregnant rats. Although
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PRL INHIBITION ‘001
DURING
SALINE
PREGNANCY
27
CONTROLS
FIG. 4. Effects of the presence of Echo cell tumors on PRL release in response to saline, 5-HTP (20 mg/kg BW), DO1 (500 rg/kg BW), or TRH (1 pg/kg BW) in ovariectomized rats. Cycling rats were injected with Rcho cells, ovariectomized 7 days later, and tested the next day between 0900-1200 h. Rcho cells inhibited 5-HTP- or DOI-induced PRL release, but did not affect TRH-induced PRL release. *, Significantly different (P c 0.05) from control values measured at the same time after the injection. Saline had no effect on PRL levels. Each point and its uertical line represent the mean and SE from 5-10 rats/group.
40gE
;
1I;= l!IL!hL 5HYDROXYTRYPTOPHAN
ml 2 5
u: I2i
25
,
500 400 300 200 100
1
+a*
0
15
30
TIME
THYROTROPIN
45
60
75
0
90
0
(MINUTES)
RELEASING
l
15
l
30
TIME
HORMONE
45
60
75
90
75
90
(MINUTES)
DOI
70
~1-1
5z 500 5, g &oo z 400 360
loo
i
0 0
15
30
TIME
PL-2 is the major PL present at this time of pregnancy (20, 21), and Rcho cells secrete primarily PL-I in uiuo (18), there is little evidence that PL-I and PL-II differ in their PRL-like biological properties. Thus, it is quite likely that PLs secreted during the last half of pregnancy act in the hypothalamus to inhibit the control mechanisms responsible for signaling the lactotroph to release PRL. PRL secretion remains low when sufficient amounts of dopamine are released from the tuberoinfundibular neurons (26-29). It is possible that serotonin acts on these neurons to remove or decrease this tonic inhibitory effect of dopamine and, in turn, increase PRL levels. Serotonin neurons have been reported to have receptors on dopaminergic neurons, and this neuronal connection depresses the activity of dopaminergic neurons in midbrain (30). Whether this is the case for tuberoinfundibular dopaminergic neurons is not clear. The present results, in which secretions from the implanted Rcho cells inhibited serotonin stimulus-induced PRL release in pregnant and ovariectomized rats, suggest that PLs may work by blocking the action of serotonin on dopamine neurons. It has been shown that a uterineplacental factor (31), human PL (32), or PRL (33, 34) can stimulate increased activity of tuberoinfundibular dopaminergic neurons, leading to dopamine release and PRL inhibition. The Rcho cells did not inhibit the ability of TRH to stimulate PRL release in cycling rats ovariectomized 1 day earlier. This was unexpected, because it was previously shown that PRL responsiveness to TRH decreased during the second half of pregnancy (9,lO). This suggests that during pregnancy, factors other than those secreted
45
60
(MINUTES)
by Rcho cells may be responsible for this inhibitory effect on TRH-induced PRL release. Indeed, the ovariectomized rat has a significantly different hormonal profile compared to that of the pregnant rat. In conclusion, serotonin-induced PRL release is totally blocked during late pregnancy and by secretions from Rcho cells. This strongly supports the hypothesis that the loss of PRL surges during the second half of pregnancy is due to secretion of PLs acting in the hypothalamus to interfere with the neurohormonal signal. Part of the signal must involve serotonin. Acknowledgments We wish to thank Dr. Michael Soares (University of Kansas Medical Center) for help in establishing the Rcho cell culture in our laboratory, and the NIDDK and the National Hormone
and Pituitary Program (University of Maryland School of Medicine) for the gift of PRL RIA materials. References 1. Butcher RL, Fugo WW, Collins WE 1972 Semicircadian rhythm in plasma levels of prolactin during early gestation in the rat. Endocrinology 90:1125-1127 2. Smith MS, Neil1 JD 1976 Termination at mid-pregnancy of the two daily surges of plasma prolactin initiated by mating in the rat. Endocrinology 98:696-701 3. Voogt JL 1980 Regulation of nocturnal prolactin surges during pregnancy in the rat. Endocrinology 1061670-1676 4. Bridges RS, Goldman BD 1975 Ovarian control of prolactin secretion during late pregnancy in the rat. Endocrinology 97:496-498 5. Grattan DR, Averill RLW 1990 Effect of ovarian steroids on a nocturnal surge of prolactin secretion that precedes parturition in the rat. Endocrinology 126:1199-1205 6. McKay DW, Pasieka CA, Moore KE, Riegle GD, Demarest KT 1982 Semicircadian rhythm of tuberoinfundibular dopamine neu-
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PRL
28
7. 8. 9.
10. 11. 12. 13. 14. 15. 16. 17. 18.
19. 20.
INHIBITION
ronal activity during early pregnancy and pseudopregnancy in the rat. Neuroendocrinology 34:229-233 Mistrv A. Vooat JL 1989 Role of serotonin in nocturnal and diurnal surges of pro&tin in the pregnant rat. Endocrinology 125:28752880 Mistry AM, Voogt JL 1990 Serotonin synthesis inhibition or receptor antagonism reduces pregnancy-induced nocturnal prolactin secretion. Ljfe Sci 47:693-761 Bridaes RS. Terkel J. Sawver CH 1983 Thvroid stimulatina hormone and prolactin secretion: reduced sensitivity to TRH-itimulated prolactin release after midpregnancy in rats. Proc Sot Exp Biol Med 173:527-532 Voogt JL 1986 Changes in pituitary prolactin responsiveness to TRH during pregnancy. Proc Sot Exp Biol Med 182:58-62 Garthwaite TL. Hagen TC 1979 Evidence that serotonin stimulates a prolactin releasing factor in the rat. Neuroendocrinology 29:215220 Pilotte NS, Porter JC 1981 Dopamine in hypophysial portal plasma and prolactin in systemic plasma of rats treated with B-hydroxyt&amine. Endocrinology 108:2137-2141 Yogev L, Terkel J 1980 Timing of termination of nocturnal prolactin surges in pregnant rats as determined by the number of fetuses. J Endocrinol84:421-424 Vooat JL Robertson M. Friesen H 1982 Inverse relationshiu of prolictin and rat placental lactogen during pregnancy. Biol Reprod 26:800-805 Tonkowicz PA, Voogt JL 1983 Examination of rat placental lactogen and prolactin at 6 hr intervals during midpregnancy. Proc Sot Exp Biol Med 173:583-587 Tonkowicz PA, Voogt JL 1983 Termination of prolactin surges with development of placental lactogen secretion in the pregnant rat. Endocrinology 113:1314-1318 Voogt JL, de Greef WJ 1989 Inhibition of nocturnal prolactin surges in the pregnant rat by incubation medium containing placental lactogen. Proc Sot Exp Biol Med 191:403-407 Faria TN, Deb S, Kwok SCM, Vandeputte M, Talamantes F, Soares MJ 1990 Transplantable rat choriocarcinoma cells express placental lactogen: identification of placental lactogen-I immunoreactive protein and messenger ribonucleic acid. Endocrinology 127:3131-3137 Mistry AM, Vidal G, Voogt JL 1991 Dopaminergic and serotonergic activity in the hypothalamus during early and late pregnancy. Brain Res 550:239-246 Duckworth ML, Pelen LM, Schroedter L, Shan P, Friesen HG .,
I
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21. 22. 23. 24. 25.
26. 27. 28.
29.
30. 31.
32. 33. 34.
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1988 Placental lactogens and extra-hypophyseal prolactin gene family. In: Hoshino K (ed) Prolactin Gene Family and Its Receptors. Elsevier, New York, pp 79-88 Faria TN, Deb S, Kwok SCM, Talamantes F, Soares MJ 1990 Ontogeny of placental lactogen-I and placental lactogen-II expression in the developing rat placenta. Dev Biol 141:279-291 Voogt JL 1980 Regulation of nocturnal prolactin surge during pregnancy in the rat. Endocrinology 106~1670-1676 Goronse WC. Freeman ME 1985 Effects of nlacenta and maternal serum on prdlactin secretion in u&o. Biol Reprod 32:279-283 Voogt JL 1984 Evidence for an inhibitory influence of rat placental lactogen on prolactin release in uitro. Biol Reprod 31:141-147 Shintani S, Glass LE, Page EW 1966 Studies of induced malignant tumors of placental and uterine origin in the rat. III. Identification of experimentally induced choriocarcinoma by detection of placental hormone. Am J Obstet Gynecol95:559-563 Ben-Jonathan N 1985 Dopamine: a prolactin inhibiting hormone. Endocr Rev 6564-589 Ben-Jonathan N, Arbogast LA, Hyde JF 1989 Neuroendocrine regulation of prolactin release. Prog-Neurobiol33:399-447 Pan JJ. Wana PS 1989 Effect of transient douamine antaeonism on thyrotropin-releasing-hormone induced prolactin secretion in the serotonin-blocked, estrogen treated rat. Neuroendocrinology 49:281-289 Haisenleder DJ, Moy JA, Gala RR, Lawson DM 1986 The effect of transient dopamine antagonism on thyrotropin-releasing hormone-induced prolactin release in pregnant rats. Endocrinology 119:1980-1988 Ugedo L, Grenhoff J, Svensson TH 1989 Ritanserin, a 5-HT, receptor antagonist, activates midbrain dopamine neurons by blocking serotonergic inhibition. Psychopharmacology 98:45-50 Demarest KT, Moore KE, Riegle GD 1983 Role of a uterineplacental factor in the cessation of the semicircadian rhythm of tuberoinfundibular dopaminergic neuronal activity at midpregnancy in the rat. Neuroendocrinology 36:409-414 Demarest KT, Duda NJ Riegle GD, Moore KE 1983 Placental lactogen mimics prolactin in activating tuberoinfundibular dopamine neurons. Brain Res 272:175-178 Gudelsky GA, Porter JC 1980 Release of dopamine from tuberoinfundibular neurons into pituitary stalk blood after prolactin or haloperidol administration. Endocrinology 106526-529 Voogt JL 1987 Actions of nrolactin in the brain. In: Rilleman JA (ed)-Actions of Prolactin on Molecular Processes. CRC Press, Boca Raton, pp 27-40 L
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