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Endocrinology Vol. 140, No. 5 2159-2166
Copyright © 1999 by The Endocrine Society

ARTICLES

Feedback Effects of Placental Lactogens on Prolactin Levels and Fos-Related Antigen Immunoreactivity of Tuberoinfundibular Dopaminergic Neurons in the Arcuate Nucleus during Pregnancy in the Rat1

Youngsoo Lee and James L. Voogt

Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, Kansas 66160-7401

Address all correspondence and requests for reprints to: Dr. James L. Voogt, Department of Molecular and Integrative Physiology, University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, Kansas 66160-7401. E-mail: jvoogt{at}kumc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PRL in the rat exerts its luteotropic action during the first half of pregnancy. After midpregnancy, placental lactogens (PLs) take the place of PRL to stimulate progesterone secretion from the corpus luteum. Simultaneously, PLs trigger a negative feedback on PRL secretion. However, the brain mechanisms for the negative feedback induced by PLs are not fully understood. Here, we report changes in plasma PRL levels, tuberoinfundibular dopaminergic (TIDA) neuronal activity as measured by Fos-related antigen (FRA)/tyrosine hydroxylase (TH) immunoreactivity, and TH catalytic activity as measured by dihydroxyphenylalanine (DOPA) accumulation in the stalk-median eminence (SME) after experimental manipulation of PL levels.

On day 4 of pregnancy, animals received Rcho-1 cells intracerebroventricularly (icv) to increase the level of PLs in the brain or HRP-1 cells as controls. On day 12 of pregnancy, hysterectomy alone or icv HRP-1 injection plus hysterectomy were performed to remove the source of PLs. Rcho-1 icv injection plus hysterectomy were performed to examine the effect of replacement of the PL source. Sham-hysterectomized animals were used as a control group. Animals were killed 2 days after each treatment at 0200 and 1800 h, which represent the peak times of PRL surges, and at 1400 h, which represents the intersurge time, by either transcardial perfusion for FRA/TH immunocytochemistry or decapitation 30 min after NSD 1015 injection to assess DOPA accumulation with HPLC-electrochemical detection.

Rcho-1 cells completely abolished PRL surges on day 6 of pregnancy and increased the percentage of FRA/TH immunoreactivity in the dorsomedial, ventrolateral, and caudal subdivisions of the arcuate nucleus. This change in neuronal activity reflected the amount of DOPA accumulation in the SME, which was high at all time points. On day 14 of pregnancy, removal of the PL source by hysterectomy resulted in increased PRL levels and decreased neuronal activity of TIDA neurons at all three time points. Similar profiles were observed in animals that received icv HRP-1 injection plus hysterectomy. Replacement of the source of PL with Rcho-1 cells in hysterectomized rats resulted in low PRL secretion, high neuronal activity of TIDA neurons, and high TH catalytic activity. These patterns were the same as those in sham-operated animals.

Our results demonstrate that PLs induce an increase in the neuronal activity of dopaminergic neurons, as measured by FRA/TH immunoreactivity and TH catalytic activity in the SME. Removal of the PL source elevates plasma PRL levels at all times during the second half of pregnancy and does not restore PRL surges.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE BIOLOGICAL actions of PRL are extremely diverse. One of its principal functions is luteotropic, maintaining progesterone secretion from the corpora lutea that is indispensable for gestation in the rat (1). During the first half of pregnancy, PRL shows a unique daily secretory pattern. A nocturnal surge occurs in the early morning and is sustained until day 10, whereas a diurnal surge occurs in the late afternoon and is terminated on day 8 of pregnancy (2, 3). However, only the nocturnal surge is fully capable of maintaining pseudopregnancy. When the diurnal surge was blocked, the nocturnal surge was extended so that the amount of PRL released was nearly equivalent to the amount of both surges (4). After termination of PRL surges at midpregnancy, PRL remains at very low levels until just before parturition (3, 5, 6).

The neuronal mechanisms that regulate PRL surges during early pregnancy and terminate these surges at midpregnancy are not fully understood. The temporal coincidence of the appearance of placental lactogens (PLs) and the disappearance of PRL surges at midpregnancy suggests that PLs, especially PL-I and PL-II, may exert a negative feedback on PRL surges. PL-I is expressed by trophoblast giant cells during a window of time between implantation and midgestation, and PL-II is expressed by the same cell types from midgestation until term (7, 8; for review, see Ref. 9). Removal of the PL sources by hysterectomy during the second half of pregnancy resulted in reinitiation of PRL secretion (10). The number of conceptuses and the levels of PLs showed an inverse relationship with the number of days that the PRL surge was present (11). A single conceptus was sufficient to cause termination of PRL surges (12). Administration of conditioned medium containing PLs or placement of purified PL-I into the hypothalamus abolished the nocturnal PRL surge (13, 14). Furthermore, central or systematic implantation of rat choriocarcinoma (Rcho-1) cells, which secrete PLs, completely inhibited the nocturnal surge of PRL in part by increasing the activity of tuberoinfundibular dopaminergic (TIDA) neurons and by blocking the stimulatory effect of serotonin (15, 16). However, it is unknown whether interference of the negative feedback signal during the second half of pregnancy will cause resumption of PRL surges.

It is well documented that dopamine (DA) from the arcuate nucleus (ARC)/median eminence inhibits PRL secretion tonically through D2 receptors found on the lactotrophs (17, 18; for reviews, see Refs. 19, 20). The neuronal activity of dopaminergic neurons is partially regulated by the level of circulating PRL or other lactogens. Either physiological or artificial hyperprolactinemia induced an increase in tyrosine hydroxylase (TH) catalytic activity, the rate-limiting enzyme for DA synthesis, in the stalk-median eminence (SME) and increased TH messenger RNA in the ARC (21, 22). The effect of PRL on DA secretion is strongly supported by the existence of PRL receptors on dopaminergic neurons (23, 24). Some PLs, especially PL-I and PL-II, bind to PRL receptors and display PRL-like bioactivities, including luteotropic functions (for review, see Ref. 25).

Activator protein-1 (AP-1) regulatory elements are located on the TH gene and are important in the regulation of TH expression (26). Hoffman et al. (27) demonstrated that Fos-related antigens (FRAs) can be used as markers for measuring the neuronal activity of TIDA neurons. In the pseudopregnant rat, there is a semicircadian rhythm of FRA expression in the dopaminergic neurons, which results in inverted rhythms of PRL surges (28).

The aims of the present study were 1) to determine the effect of PLs on PRL secretion, and 2) to investigate the effect of PLs on the activity of individual TIDA neurons in the ARC as measured by FRA immunoreactivity and on the TH catalytic activity in the SME as measured by accumulation of dihydroxyphenylalanine (DOPA).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult male and female Sprague Dawley rats (Sasco, Omaha, NE) were housed in an American Association for Laboratory Animal Care-accredited facility and were maintained in accordance with the NIH Guide for the Care and Use of Laboratory Animals. All procedures for animal use were approved by the institutional animal care and use committee at the University of Kansas Medical Center. Animals were acclimated to controlled temperature (22 C) and lighting conditions (lights on, 0600–1800 h), with food and water ad libitum. Daily vaginal smears were obtained by lavage to follow the estrous cycle and pregnancy. Each female was placed into a cage with a single male on the evening of proestrus. The presence of sperm in the vaginal smear was designated day 0 of pregnancy.

Maintenance of Rcho-1 and HRP-1 cells in culture
A cell line (Rcho-1) generated from choriocarcinoma explants and a cell line (HRP-1) from chorioallantoic placental explants were maintained in culture as previously described (29, 30) with some modifications. Briefly, Rcho-1 cells were cultured in NCTC-135 culture medium (Sigma Chemical Co., St. Louis, MO) supplemented with 20% heat-inactivated FBS, 26 mM sodium bicarbonate, 10 mM HEPES, 1 mM sodium pyruvate, 100 µg/ml penicillin, 100 U/ml streptomycin, and 3.5 µl/liter 2-mercaptoethanol. HRP-1 cells were cultured in RPMI 1640 culture medium (Sigma Chemical Co.) supplemented with 10% heat-inactivated FBS, 1 mM sodium pyruvate, 2 mM L-glutamate, 100 µg/ml penicillin, 100 U/ml streptomycin, and 100 µl/liter 2-mercaptoethanol. Both cell lines were placed in a humidified atmosphere of 95% air-5% CO2 at 37 C. Rcho-1 and HRP-1 cells were routinely maintained in 75-cm2 flasks and passaged by brief exposure to 0.25% trypsin-0.02% EDTA, followed by mechanical scraping cells from the culture dish. For intracerebroventricular (icv) injection, the cells were harvested by brief exposure to trypsin-EDTA solution, scraped, centrifuged, and resuspended in culture medium. The Rcho-1 cell line was used for the source of PLs, and the HRP-1 cell line, which does not express known members of PLs, was used as a control cell line.

Experimental design
Exp 1. This experiment was designed to examine the effect of PLs from Rcho-1 cells on the expression of PRL surges during the first half of pregnancy. Animals were anesthetized by ketamine hydrochloride (100 mg/1 kg BW) with 10% acepromazine before stereotaxic surgery. On day 4 of pregnancy, either Rcho-1 cells or HRP-1 cells (100,000 cells/15 µl cell culture medium) were injected into the left lateral ventricle using the coordinates of -0.26 mm from the bregma, 1 mm left, and 4 mm deep. A Hamilton syringe (Hamilton Corp., Reno, NV) with a 23-gauge needle was used for icv injection. Five animals in each group were treated. Intact pregnant animals (n = 4) that received no surgery were used as a second control group. One day after stereotaxic surgery, a SILASTIC brand cannula (Dow Corning Corp., Midland, MI) was inserted through the jugular vein into the right atrium according to the technique of Harms and Ojeda (31). Blood samples for PRL RIA were collected at 3-h intervals for 2 days (from 1400 h on day 6 to 1400 h on day 8 of pregnancy). At each time point, 250 µl blood were collected, and the same volume of heparinized saline (2% heparin) was replaced. Pregnancy was confirmed by examination of the uterus for implantation sites at the end of the experiment. Intracerebroventricular injection sites also were confirmed.

Exp 2. This experiment was designed to examine the effect of PLs on PRL levels, FRA expression in TH-positive neurons in the ARC, and TH catalytic activity in the SME during the first half of pregnancy. Based on the results from Exp 1, Rcho-1 or HRP-1 cells were injected during an intersurge period on day 4 of pregnancy. Treated animals (RCHO and HRP) were killed by either transcardial perfusion for FRA/TH immunocytochemistry (ICC) or decapitation for measurement of DOPA accumulation in the SME at 0200, 1400, and 1800 h on day 6 of pregnancy.

Exp 3. This experiment was designed to examine the effects of placental removal on reinitiation of PRL surges and on TIDA neuronal activity. To extend or reinitiate PRL surges, animals were hysterectomized (HS) or received sham surgery (SHS) on day 12 of pregnancy, according to the protocol of a previous report (10). Two days later, brains or SMEs were collected at 0200, 1400, and 1800 h.

Exp 4. This experiment was designed based on the results of Exp 1 and 3. To test the hypothesis that loss of the source of PLs by hysterectomy caused reinitiation of PRL secretion by an action on TIDA neurons, icv injection of Rcho-1 or HRP-1 cells was combined with hysterectomy on day 12 of pregnancy. Animals (RCHO/HS and HRP/HS) were either perfused with paraformaldehyde or decapitated at three time points on day 14 of pregnancy. Brains or SMEs were removed for analysis.

Immunocytochemistry and microscopy
Immunofluorescence. To demonstrate that Rcho-1 cells were alive and capable of producing PLs in the ventricle, animals were killed 3 days after Rcho-1 icv injection. Brains were quickly frozen after decapitation and stored at -80 C. Brain sections (25 µm thick) were collected in the Reichert-Jung Cryocut 1800 (Cambridge Instruments Co., Buffalo, NY). These sections were washed several times with 10 mM PBS (pH 7.5) containing 0.3% Triton X-100 after fixation in PBS containing 4% paraformaldehyde for 15 min at room temperature and incubated with PL-I antibody raised in rabbits (1:400; gift from Dr. Michael Soares) for 24 h at 4 C. After a series of washings with 10 mM PBS (pH 7.5) containing 0.3% Triton X-100, the brain sections were incubated with indocarbocyanine (CY3)-antirabbit goat serum at a 1:400 dilution (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for 1 h at room temperature in the dark and then coverslipped with Fluoromount G. The CY3 signal was detected using the proper filter (XF34, Omega Optical, Brattleboro, VT) in a Nikon Optiphot microscope (Nikon, Melville, NY; Fig. 1AGo).



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  		Figure 1. Representative photomicrographs of Rcho-1 cells and HRP-1 cells in the ventricle (magnification, x200). A, Rcho-1 cells in the lateral ventricle show PL-I-positive immunoreactivity 3 days after icv injection. Arrows indicate PL-I-immunopositive signals visualized by CY3. Insets show granular staining of PL-I immunoreactivity at high magnification. B, Cluster of Rcho-1 cells in the third ventricle. This cluster is attached to the median eminence, and the giant Rcho-1 cells possessing very large nuclei show strong FRA immunoreactivity. C, HRP-1 cells in the third ventricle.

 
Immunocytochemistry. Two days after treatments, animals were killed at 0200, 1400, and 1800 h with an overdose of sodium pentobarbital. Blood samples for PRL RIA were collected soon after the injection of 0.1 ml heparin (1,000 U). Brains were taken after transcardial perfusion with 4% paraformaldehyde in 50 mM potassium PBS (KPBS), pH 7.6. Every third brain section (25 µm thick) from -2.12 to -4.16 mm postbregma was stained for FRA and TH dual immunoreactivity and analyzed. The details for FRA ICC were described previously (5). Briefly, brain sections were incubated with Fos antibody (K-25, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at a 1:5,000 dilution with KPBS containing 0.4% Triton X-100 for 2 days at 4 C after several washing steps with KPBS. This Fos antibody was raised in rabbits against a peptide corresponding to amino acids 128–152 within a highly conserved domain of the human c-Fos protein and binds to all members of the Fos family. FRA immunoreactivity was developed with diaminobenzidine (0.2 mg/ml), nickel sulfate (25 mg/ml), and hydrogen peroxide (0.83 µl of a 3% solution/ml) in 175 mM sodium acetate buffer (pH 6.5) after consecutively incubating with biotinylated goat antirabbit Ig and avidin DH-biotinylated horseradish peroxidase-H complex (Vectastain Elite Kit, Vector Laboratories, Inc., Burlingame, CA). After these procedures, brain sections were washed with KPBS extensively and treated with 0.6% hydrogen peroxide in methanol to quench a surplus of peroxidase from FRA ICC, followed by 1% normal horse serum in KPBS. These brain sections were incubated with TH monoclonal antibody at 1:50,000 dilution (Chemicon, Temecula, CA) for 2 days at 4 C. Brain sections then were treated with biotinylated horse antimouse Ig and avidin-biotinylated horseradish peroxidase complex. Signals of TH immunoreactivity were developed with diaminobenzidine (0.6 mg/ml) and hydrogen peroxide (2 µl of a 30% solution/ml) in 50 mM Tris buffer (pH 7.25). Finally, brain sections were rinsed with KPBS, dehydrated in an ethanol series, cleaned in Histoclear (National Diagnostics, Atlanta, GA), and coverslipped with DPX mountant (BDH Laboratory Supplies, Poole, UK). FRA-positive signals were consistently confined to the nucleus of the cells as a dark blue color; however, TH signals were located in the soma and the axon as a bright brown color. The ARC was defined according to the rat brain atlas of Paxinos and Watson (32). This nucleus was divided arbitrarily into two groups, the rostral ARC (from -2.1 mm to -3.8 mm from the bregma) and the caudal ARC (from -3.8 mm to -4.1 mm from the bregma). In the rostral ARC, FRA/TH immunoreactivity was measured separately in dorsomedial and ventrolateral subareas subdivided by a 50° angle with the horizontal line (Figs. 5Go and 6Go).



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  		Figure 5. Plasma PRL levels and percentage of FRA/TH-immunopositive cells in the three subareas of the ARC on day 14 of pregnancy. Bars with different letters differ significantly. A, Plasma PRL levels in HS animals (white bar), SHS animals (black bar), RCHO/HS animals (gray bar), and HRP/HS animals (hatched bar) at 0200, 1400, and 1800 h. Bars represent the mean (±SEM) plasma PRL levels. The neuronal activity of dopaminergic neurons in the dorsomedial subdivision of the ARC (B), in the ventrolateral subdivision of the ARC (C), and in the caudal subdivision of the ARC (D) is shown. The neuronal activity of dopaminergic neurons is represented by the number of FRA/TH-immunopositive neurons divided by the total number of TH-immunopositive neurons in the area of interest. There were 5 rats in each experimental group. Nineteen to 22 brain sections in the rostral to mid-ARC and 4–7 brain sections in the caudal ARC were examined. The mean numbers of TH-positive cells per section were: in the dorsomedial subdivision: HS group, 24.6 ± 1.2; SHS group, 22.4 ± 0.8; RCHO/HS group, 24.0 ± 0.8; HRP/HS, 22.6 ± 1.0; in the ventrolateral subdivision: HS group, 8.0 ± 0.6; SHS group, 6.8 ± 0.6; RCHO/HS, 7.4 ± 0.4; HRP/HS group, 8.0 ± 0.6; in the caudal subdivision: HS group, 16.6 ± 1.0; SHS group, 13.8 ± 0.8; RCHO/HS, 16.4 ± 1.2; HRP/HS, 14.2 ± 1.2.

 


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  		Figure 6. Representative photomicrograph of FRA/TH dual immunoreactivity in the ARC at 1400 h on day 14 of pregnancy (magnification, x200). Arrowheads indicate single staining of TH, and arrows indicate dual signals of FRA and TH. 3rd, Third ventricle.

 
HPLC-electrochemical detection for TH activity
Each treated animal was injected with m-hydroxybenzylhydrazine dihydrochloride (NSD 1015; Sigma Chemical Co., 100 mg/kg, ip injection), an L-aromatic amino acid decarboxylase inhibitor, 30 min before death. After decapitation, the brains were quickly removed, and the SME was dissected with fine scissors. After brief exposure to dry ice, the SME was sonicated in 120 µl 0.1 N perchloric acid and centrifuged at 10,000 x g for 3 min. The supernatant was stored at -80 C until assay. The pellet was dissolved in 25 µl 0.5 N sodium hydroxide and analyzed for protein assay by the Bradford method (33). The amount of DOPA accumulation in the supernatant was detected by HPLC-electrochemical detection, as described previously (21) with mobile phase modification. Briefly, the mobile phase was composed of 50 mM dibasic sodium phosphate, 30 mM citric acid, 1 mM EDTA, 250 mg/liter octane sulfonic acid, and 14% methanol. The pH of the mobile phase was adjusted to pH 2.85 with 85% phosphoric acid.

PRL RIA
Plasma PRL levels were determined by the rat PRL RIA material provided by the NIDDK. RP-3 was used as the standard reference, and the limit of sensitivity for the assay was 50 pg. [125I]PRL was purchased from DuPont (Boston, MA). The interassay coefficient of variation was 5.4%.

Statistical analysis
Results are expressed as the mean ± SEM. The number of animals in each experimental group was five for FRA/TH ICC experiments and four for HPLC experiments. Statistical analysis of the data was performed using two-way ANOVA to evaluate the effects of time, treatment, and time vs. treatment interactions, followed by multiple comparisons with Fisher’s protected least significant difference test. These statistical tests were conducted using the StatView program (Abacus Concepts, Berkley, CA) for Macintosh. Comparisons with P < 0.05 were considered significantly different.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of treatments on PRL secretion
To assess the long term effects of PL feedback on PRL surges during the first half of pregnancy, Rcho-1 cells were injected icv to continuously elevate PL levels in the brain. HRP-1 cells were used as controls. Although both cell lines were injected into the left lateral ventricle, large clusters of both cell lines were observed in the third ventricle in close proximity to dopaminergic neurons (Fig. 1Go, B and C), indicating that Rcho-1 and HRP-1 cells could freely migrate throughout the ventricular system in the brain. The differentiated giant cells possessing very large nuclei in the Rcho-1 cells showed strong FRA immunoreactivity (Fig. 1BGo), indicating the genetic viability of the cells 2 days after icv injection (Fig. 1BGo). Shida et al. (34) demonstrated that an AP-1 site is a critical element for promoter activity of the gene encoding mouse PL-I. These giant Rcho-1 cells resembled the differentiated, PL-secreting trophoblast giant cells found in the placenta (29). Furthermore, PL-I-positive immunoreactivity was detected in Rcho-1 cells in situ (Fig. 1AGo).

PLs from Rcho-1 cells completely eliminated PRL surges during early pregnancy, whereas animals that had HRP-1 cells or were intact (no injection) showed two normal daily surges of PRL (Fig. 2Go). These daily surges of PRL in control rats were somewhat lower by day 8, probably due to the effect of surgery and/or blood sampling. Rcho-1 cells exerted a negative feedback effect on PRL surges less than 48 h after injection and continued on day 8 of pregnancy when Rcho-1 cells still produced PL-I in the ventricle (Figs. 1AGo and 2Go). These experiments demonstrate that Rcho-1 cells were viable and produced PL-I in the ventricle for at least 4 days after icv injection and allowed us to design the appropriate time points for blood sampling and brain collection in the rest of experiments.



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  		Figure 2. Time course of plasma PRL levels during early pregnancy after icv injection of Rcho-1 and HRP-1 cells. On day 4 of pregnancy, icv injections of Rcho-1 (n = 5) or HRP-1 (n = 5) cells were given. Blood samples from the jugular vein were taken every 3 h from 1400 h on day 6 to 1400 h on day 8 of pregnancy. PLs from the Rcho-1 cells completely eliminated PRL surges, whereas control groups composed of animals with HRP-1 cells or nonoperated pregnant animals (n = 4) showed two daily surges of PRL. The data for PRL levels in icv HRP-1-injected animals and nonoperated pregnant animals were combined because there was no difference between the two groups.

 
On day 6 of pregnancy, PRL levels in RCHO animals were very low at all time points, comparable to PRL levels at 1400 h in the HRP group (Fig. 3AGo). PRL surges occurred normally in HRP animals, high at 0200 and 1800 h and low at 1400 h. This is similar to the pattern described previously (5). Hysterectomy resulted in substantially elevated PRL levels at all three time points on day 14 of pregnancy (Fig. 5AGo). These elevated PRL levels also occurred in HRP/HS animals at all time points. In fact, PRL was significantly higher at 0200 h in HRP/HS groups than in HS animals. This may reflect a stress effect due to the cranial surgery. However, the high levels of PRL were not observed in SHS groups and RCHO/HS groups, but remained low and unchanged at all three times.



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  		Figure 3. Plasma PRL levels and percentage of TH-immunopositive neurons that express FRAs in the three subareas of the ARC on day 6 of pregnancy. Bars with different lettersdiffer significantly. A, Plasma PRL levels in RCHO animals (white bar) and HRP animals (black bar) at 0200, 1400, and 1800 h. Bars represent the mean (±SEM) of plasma PRL levels. The neuronal activity of dopaminergic neurons in the dorsomedial subdivision of the ARC (B), in the ventrolateral subdivision of the ARC (C), and in the caudal subdivision of the ARC (D) is shown. The neuronal activity of dopaminergic neurons is represented by the number of FRA/TH-immunopositive neurons divided by the total number of TH-immunopositive neurons in the area of interest. There were 5 rats in each experimental group. Nineteen to 22 brain sections in the rostral to mid-ARC and 4–7 brain sections in the caudal ARC were examined. The mean numbers of TH-positive cells per section were: in the dorsomedial subdivision: RCHO group, 21.8 ± 1.0; HRP group, 20.2 ± 1.2; in the ventrolateral subdivision: RCHO group, 6.0 ± 0.8; HRP group, 6.2 ± 0.6; in the caudal subdivision: RCHO group, 14.0 ± 1.2; HRP group, 10.4 ± 1.0.

 
Overall, PLs from either the intact placentas or from Rcho-1 cells blocked PRL secretion, and hysterectomy resulted in an increase in PRL secretion, but not in PRL surges. However, HRP-1 cells showed no effect on PRL secretion at either stage of pregnancy.

Pattern of FRA immunoreactivity in TIDA neurons
The neuronal activity of dopaminergic neurons was measured by FRA-positive immunoreactivity in TH-immunopositive cells in the ARC at several time points before and after midpregnancy. During the first half of pregnancy, HRP animals showed an inverse pattern of neuronal activity in TH neurons, compared with plasma PRL (Figs. 3Go and 4Go, B and D). Elevated neuronal activity of TH neurons was measured in the ARC at 1400 h, especially in the dorsomedial and ventrolateral subdivisions. On the other hand, the percentage of FRA/TH neurons was very low in these hypothalamic areas at 0200 and 1800 h. In the caudal portion of the ARC, the mean value of neuronal activity at 1400 h appeared higher than those at 0200 and 1800 h; however, these values were not statistically different. In contrast, RCHO groups expressed greatly augmented neuronal activity in TH neurons at all three time points when the PRL levels were very low (Figs. 3Go and 4Go, A and C). The level of activation of dopaminergic neurons in RCHO groups was much higher at all three time points than the level of activation of TH-positive neurons at 1400 h in HRP animals, even though PRL levels were similarly low.



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  		Figure 4. Representative photomicrography of FRA/TH dual immunoreactivity in the arcuate nucleus at 0200 and 1400 h on day 6 of pregnancy (magnification, x200). Arrowheads indicate single TH immunoreactivity, and arrows indicate FRA/TH dual staining. The slanted gray line divides the dorsomedial and ventrolateral subdivisions of the ARC. The upper part is the dorsomedial subdivision. 3rd, Third ventricle.

 
During the second half of pregnancy, over 40% of TH neurons in the dorsomedial and ventrolateral subdivisions of the ARC expressed FRA in SHS animals at all three time points, correlated with very low PRL levels (Figs. 5Go and 6BGo). The same pattern of neuronal activity was observed in RCHO/HS animals (Figs. 5Go and 6CGo). PRL levels in these animals were very low, comparable to PRL levels in the SHS group. However, the level of neuronal activity in TH-immunopositive neurons was different, especially in the dorsomedial subdivision in these two treated groups (Fig. 5BGo). The percentage of FRA/TH neurons in RCHO/HS animals was less than those in SHS animals at all three time points, whereas in the ventrolateral subdivision the percentage of FRA-positive TH cells was similar in both treated groups. HS and HRP/HS animals demonstrated a lower percentage of activated TH neurons (Figs. 5Go and 6Go, A and D). In the caudal portion of the ARC, there were no significant differences among groups at 0200 h. However, SHS and RCHO/HS animals showed elevated neuronal activity at 1400 and 1800 h (Fig. 5DGo).

TH catalytic activity
Overall, the level of TH catalytic activity, as measured by DOPA accumulation in the SME, showed an inverse pattern compared with PRL levels in all experimental groups on either day 6 or 14 of pregnancy. The only exception was at 1400 h on day 14, when both hysterectomized groups (HS and HRP/HS) demonstrated high levels of both PRL secretion and DOPA accumulation (Fig. 7Go). Rcho-1 icv injection during the first half of pregnancy induced an increase in TH enzyme activity at three time points, resulting in low levels of PRL secretion. HRP animals showed the expected pattern of DOPA accumulation and PRL surges. At 0200 and 1800 h on day 14 of pregnancy, removal of PLs caused a decrease in DOPA levels. However, sham hysterectomy or Rcho-1 icv injection into hysterectomized rats resulted in an increase in DOPA levels. The differences in DOPA accumulation due to treatments at 1400 h on day 14 of pregnancy were less apparent than those at 0200 and 1800 h.



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  		Figure 7. TH catalytic activity in the SME after treatments on day 6 (A) and day 14 (B) of pregnancy. Bars represent the mean (±SEM) DOPA accumulation per U protein. There were four animals in each group. See Figs. 3Go and 4Go for descriptions of experimental groups.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results of this study demonstrate the following. 1) Rcho-1 cells inhibited both nocturnal and diurnal PRL surges. 2) Rcho-1 cells resulted in an increase in the percentage of dual labeled FRA/TH neurons in the ARC. 3) Hysterectomy after midpregnancy resulted in an elevation of PRL levels at all three times, but not a resumption of PRL surges. 4) Rcho-1 cells prevented this increase in PRL. 5) Hysterectomy resulted in a decreased percentage of FRA/TH-labeled neurons compared with sham-operated controls. 6) Intracerebroventricular injection of Rcho-1 cells mimicked sham hysterectomy in its effect on FRA/TH expression, whereas HRP-1 icv injection with hysterectomy mimicked hysterectomy alone. 7) Rcho-1 cells abolished the circadian rhythm of DOPA accumulation before midpregnancy, whereas hysterectomy after midpregnancy restored the rhythm.

The inhibitory effects of Rcho-1 cells on PRL surges confirm earlier studies from our laboratory (15, 16). This is probably due to the secretion of PLs from the cells acting on hypothalamic structures nearby (14). As demonstrated in Fig. 1Go, these cells are actively expressing PL-I in the ventricle. To answer the question of how the PLs are acting, we used the measurement of FRA as a marker for assessing neuronal activation. It has been well documented that FRAs serve a very useful purpose in measuring changes (both increases and decreases) in neuronal activity, especially that of TIDA neurons (27, 28, 35). Rcho-1 cells increased the percentage of FRA/TH dual stained neurons in three subareas of the ARC at all three time points compared with that in HRP-1-injected controls. Thus, the pattern of neuronal activity, which was reported to correlate inversely with PRL levels before midpregnancy, was disrupted by this treatment. This result is in agreement with our earlier studies, which showed that Rcho-1 cells injected icv resulted in elevation of DOPA accumulation in the SME (16), an indication that PLs indeed increase the activity of TIDA neurons. However, the increment in TIDA neuronal activity induced by Rcho-1 cells did not contribute much to the induction of TH catalytic activity on day 6 of pregnancy, possibly due to posttranslational modifications that can affect the enzyme activity (36).

Given our earlier observation that after midpregnancy, DOPA accumulation as an index of TH activity was uniformly elevated, with a loss in rhythm correlated with a loss in PRL surges (36), we designed an experiment to determine the effect of removal of the source of PLs on FRA expression in TIDA neurons after midpregnancy. As shown in Figs. 5Go and 6Go, hysterectomy decreased the percentage of FRA-labeled TH neurons at all times and in all areas of the ARC. The same results occurred when hysterectomized animals received HRP-1 by icv injection, indicating again the lack of PL secretion by these cells. Plasma PRL levels were elevated at both the intersurge and surge times in these two groups, inversely correlated with the low activity of TIDA neurons. This is slightly different from the results using DOPA accumulation as a measure of TH catalytic activity, in which TH activity is low during the surges but high during the intersurge period in HS and HRP/HS animals. Currently we do not have a good explanation for this discrepancy, except to suggest that using FRA measurement in dopaminergic neurons reflects neuronal activity, whereas the measurement of DOPA accumulation assesses enzyme function only, which may or may not directly reflect DA secretion. A second possibility is that an endogenous stimulatory rhythm (35, 37, 38, 39) wanes during the second half of pregnancy and/or dominant inhibitory rhythm prevails. Removal of the source of PLs might only block the inhibitory inputs, especially dopaminergic inputs (14, 16), and PRL secretion will increase but not show surges.

The rats that received hysterectomy plus Rcho-1 cells or sham hysterectomy showed the expected lack of PRL surges and uniformly elevated dopaminergic activity as measured by either FRA/TH neurons or DOPA accumulation. These rats had high PL levels due to either Rcho-1 cells in the ventricle or secretion from the trophoblast giant cells. This suggests that after midpregnancy, secretions from the placenta are capable of inhibiting the semicircadian rhythm of TIDA neurons and thus block PRL secretion.

The semicircadian PRL rhythm and activity of TH-positive neurons during the first half of pregnancy agree with a study reported by Lerant et al. (28), in which they measured the pattern of FRA/TH dual staining in the ARC and periventricular nucleus of the hypothalamus in pseudopregnant rats. They found there was a semicircadian rhythm in the activity of dopaminergic neurons in the periventricular nucleus and rostral, dorsomedial mid-, and caudal ARC that correlated inversely with plasma PRL levels, whereas TH neurons in the ventrolateral ARC did not. We also found that the dorsomedial subdivision of the ARC had the majority of TH-positive neurons, and TH neurons in the ventrolateral subdivision of the ARC did not stain as intensely for TH ICC (see Figs. 3Go and 5Go for the difference in the number of TH neurons in two subdivisions). They discussed the importance of the decrease in activity of the dopaminergic neurons before initiation of the PRL surge as a critical mechanism for surge development, but they also pointed out that the peak of the PRL surge and its termination may be due to the appearance of an endogenous secretory rhythm of PRL-releasing factors (35, 37, 38, 39).

We cannot completely rule out the possibility that PLs exert their negative feedback on PRL secretion by activating and/or inhibiting other neuronal types separate from TIDA neurons. PRL receptors were visualized in the ARC (40, 41). Only some of them were colocalized with neurons that showed TH immunoreactivity (23, 24), indicating that other neuronal types also possess PRL receptors. More work will be needed to identify the cell types expressing PRL receptors in the ARC, and to investigate the change in neuronal activity in response to PLs.

In summary, our results suggest that manipulation of PL levels results in predictable changes in the activity of DA neurons as measured by FRA/TH ICC and contribute to our understanding of the contribution of DA to PRL surges during the first half of pregnancy and to their loss at midpregnancy.


   Acknowledgments
 
We thank Dr. Michael J. Soares (University of Kansas, Medical Center) for providing the antibody to placental lactogen I and advice on maintaining the cell cultures. We also thank the National Hormone and Pituitary Program, NIDDK, and NICHHD for the gift of PRL RIA materials.


   Footnotes
 
1 This work was supported by NICHHD Grant HD-24190 (to J.L.V.) and Center Grant in Reproductive Sciences HD-33994. Back

Received December 2, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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