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Endocrinology, doi:10.1210/en.2007-1623
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Endocrinology Vol. 149, No. 6 3206-3214
Copyright © 2008 by The Endocrine Society

Hormonal Regulation of Suppressors of Cytokine Signaling (SOCS) Messenger Ribonucleic Acid in the Arcuate Nucleus during Late Pregnancy

Frederik J. Steyn, Greg M. Anderson and David R. Grattan

Centre for Neuroendocrinology and Department of Anatomy and Structural Biology, University of Otago School of Medical Sciences, Dunedin 9054, New Zealand

Address all correspondence and requests for reprints to: Assoc. Prof. Dave Grattan, Centre for Neuroendocrinology and Department of Anatomy and Structural Biology, University of Otago School of Medical Sciences, P.O. Box 913, Dunedin 9054, New Zealand. E-mail: dave.grattan{at}anatomy.otago.ac.nz.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Prolactin stimulates tuberoinfundibular dopamine neurons in the arcuate nucleus of the hypothalamus, mediated by signal transducer and activator of transcription 5b (STAT5b). During late pregnancy, these neurons become unresponsive to prolactin, with a loss of prolactin-induced activation of STAT5b and decreased dopamine secretion. Suppressors of cytokine signaling (SOCS) proteins inhibit STAT-mediated signaling, and SOCS mRNAs are specifically elevated in the arcuate nucleus during late pregnancy. We hypothesized that changes in circulating ovarian steroids during late pregnancy might induce expression of SOCS mRNAs, thus disrupting STAT5b-mediated prolactin signaling. Rats were ovariectomized on d 18 of pregnancy and treated with ovarian steroids to simulate an advanced, normal, or delayed decline in progesterone. Early progesterone withdrawal caused an early increase in prolactin secretion, and increased SOCS-1 and -3 and cytokine-inducible SH2-containing protein (CIS) mRNA levels in the arcuate nucleus. Prolonged progesterone treatment prevented these changes. To determine whether ovarian steroids directly alter SOCS mRNA levels, estrogen- and/or progesterone-treated ovariectomized nonpregnant rats were acutely injected with prolactin (300 µg sc) or vehicle. SOCS-1 and -3 and CIS mRNA levels in the arcuate nucleus were significantly increased by estrogen or prolactin, whereas progesterone treatment reversed the effect of estrogen. Results demonstrate that estrogen and prolactin can independently induce SOCS mRNA in the arcuate nucleus and that this effect is negatively regulated by progesterone. This is consistent with the hypothesis that declining progesterone and high levels of estrogen during late pregnancy induce SOCS in the tuberoinfundibular dopamine neurons, thus contributing to their insensitivity to prolactin at this time.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PROLACTIN (PRL) SECRETION is tonically inhibited by dopamine secreted into pituitary portal blood from the tuberoinfundibular dopamine (TIDA) neurons in the hypothalamus. Periventricular-hypophyseal and tuberohypophyseal dopamine neurons also contribute to the regulation of PRL secretion, reaching the anterior pituitary gland from the neurointermediate lobe via short portal vessels (1). PRL stimulates dopamine secretion from all three populations of neuroendocrine dopamine neurons, thereby inhibiting its own secretion by a short-loop negative-feedback system. Thus, under most conditions, an increase in PRL secretion results in an increase in dopamine activity and a subsequent return of basal PRL levels (1). For example, a pattern of twice-daily PRL surges is initiated after mating, each surge associated with a short-term decrease in dopamine secretion followed by an increase in dopamine that is involved in returning PRL concentrations to basal levels before the subsequent surge (2, 3). The onset of placental lactogen (PL) secretion at midpregnancy results in the termination of the PRL surges. Because PL are structurally homologous to PRL, they bind to PRL receptors (PRL-R) on hypothalamic dopamine neurons and stimulate dopamine release, thereby inhibiting pituitary PRL secretion by negative feedback (4, 5). Consequently, pituitary PRL secretion declines at midpregnancy and remains at basal levels for most of the remainder of pregnancy (6, 7). Despite continued secretion of PL throughout the second half of pregnancy, activity of TIDA neurons decreases during late pregnancy (8). The reduced dopamine secretion allows an antepartum PRL surge, probably also involving one or more PRL-releasing factors (9). Multiple factors have been implicated as potential PRL-releasing factors (see Ref. 1 for review), but the specific factors involved in stimulation of PRL secretion during late pregnancy have not been determined. The antepartum PRL surge is sustained over a prolonged period because the TIDA neurons fail to respond to the elevated PRL (or PL) at this time (8, 10, 11). This change in PRL regulation during late pregnancy persists into lactation (12, 13), despite continued expression of PRL-R on the TIDA neurons (14).

PRL’s stimulatory action on dopamine synthesis and secretion is mediated by the transcription factor, signal transducer and activator of transcription 5b (STAT5b) (15, 16). The level of PRL-induced STAT5b activation within TIDA neurons is markedly reduced during lactation (15), suggesting that the loss of response to PRL during late pregnancy may be mediated by changes in PRL signal transduction. A family of cytokine signaling inhibitors, the suppressors of cytokine signaling (SOCS) proteins, have been shown to act as endogenous inhibitors of the Janus kinase (JAK)/STAT pathways (17). We, and others, have recently demonstrated an increase in SOCS mRNA levels in the arcuate nucleus during late pregnancy (18) and lactation (18, 19). It seems likely that elevated SOCS proteins might contribute to the changes in PRL signal transduction in TIDA neurons during late pregnancy by disrupting PRL-induced STAT5b signaling.

The elevated SOCS mRNA in the arcuate nucleus appears to be dependent on high PRL levels characteristic of late pregnancy and lactation (18), but it does not occur at other times when serum PRL levels are elevated. For example, the PRL surges of early pregnancy do not appear to induce an increase in SOCS mRNA expression (18). Similarly, elevated levels of PL observed throughout the second half of pregnancy are not associated with increased SOCS expression. Thus, some additional factors, specific to late pregnancy, must be involved in regulating SOCS expression at this time. Because PRL readily induces SOCS mRNA in ovariectomized animals (18), it is possible that ovarian factors present during pregnancy suppress PRL induction of SOCS. In this regard, plasma concentrations of progesterone are known to decline markedly about 1 d before the antepartum PRL surge (20) and concurrent increase in SOCS mRNA levels, whereas estrogen concentrations remain high throughout late pregnancy (21). Furthermore, the presence of both estrogen and progesterone receptors in TIDA neurons throughout pregnancy (22) suggests a direct effect of steroid hormones on TIDA neurons. The aim of this study was to test the hypothesis that the changing levels of these ovarian steroid hormones during late pregnancy alter the ability of PRL to activate SOCS in the arcuate nucleus.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal care and treatment
Animals were obtained from the University of Otago animal facility. All experiments were performed on female Sprague Dawley rats (11–14 wk old, 250–300 g), housed individually after surgery in a 12-h light, 12-h dark cycle (lights on at 0600 h). Room temperature was maintained at 21 ± 1 C, and free access to food and water was provided. Regular 4-d estrous cycles were evaluated by daily vaginal smears. For late pregnancy experiments, animals were mated during the night of proestrus. Mating was confirmed by observing sperm in vaginal smears on the morning of mating. This day was designated as d 0 of pregnancy (parturition normally occurs on the morning of d 22 in our colony). For experiment 2, animals were observed half-hourly to monitor the onset and timing of parturition. The University of Otago Committee on Ethics in the Care and Use of Laboratory Animals approved all experimental procedures and protocols.

Experiment 1: plasma estrogen, progesterone, and PRL concentrations during late pregnancy
To characterize temporal changes in plasma estrogen and progesterone concentrations in relation to the antepartum PRL surge during late pregnancy in our colony, animals (n = 8) were fitted with indwelling arterial cannulas under halothane anesthesia on d 18 of pregnancy. After 48 h recovery, serial blood samples were collected for analysis of plasma PRL, progesterone, and estrogen concentrations via RIA (see below). For PRL, blood samples were collected at 1100, 1800, and 2300 h on d 20 of pregnancy; 0100, 0300, 0500, 1100, 1700, and 2300 h on d 21 of pregnancy; and 0100, 0300, 0500, and 1100 h on d 22 of pregnancy. For estrogen and progesterone, the same samples were assayed, except that the 2300-h samples were pooled with the following 0100-h samples and the 0500-h samples were pooled with the 1100-h samples.

Experiment 2: effects of estrogen and progesterone on PRL secretion during late pregnancy
To examine the effects of ovarian steroids on PRL and TIDA neuronal activity during late pregnancy, animals were ovariectomized between 0900 and 1100 h on d 18 of pregnancy and treated with an estrogen and/or progesterone replacement regimen. To replace estrogen, an estrogen-containing implant [1.57 mm inner diameter, 3.18 mm outer diameter Silclear Tubing, Degonia Silicone; 30 mm length; filled with 200 µg/ml 17β-estradiol (Sigma-Aldrich, St. Louis, MO) dissolved in 10% ethanol in sesame oil] was implanted sc at the time of ovariectomy. To replace progesterone, three progesterone implants were implanted sc before ovariectomy (dimensions as for estrogen implants except that the length was 40 mm, packed with crystalline progesterone; Sigma-Aldrich). To prevent a fall in estrogen and progesterone levels at the time of ovariectomy, it was necessary to ensure that steroid capsules were fully primed before the surgery. To do this, estradiol capsules were incubated at 37 C in saline the night before ovariectomy and inserted immediately before ovariectomy surgery. Progesterone capsules were implanted at 1700 h on d 17 of pregnancy, approximately 18 h before ovariectomy on d 18. The duration of progesterone exposure was experimentally altered by removing progesterone implants under brief halothane anesthesia. Initial attempts to induce a PRL surge after the withdrawal of progesterone early (0900 h) on d 21 of pregnancy resulted in an approximate advance of 12 h in the onset of parturition (preliminary experiments, data not shown). It appeared that the withdrawal of progesterone implants, unlike the physiological fall in progesterone levels after luteolysis, resulted in the immediate decrease of plasma progesterone and thus the rapid onset of parturition. It was found that removal of progesterone at 1700 h on d 21 of pregnancy simulated the normal decline in progesterone (Normal P Withdrawal, n = 6) in terms of the time of parturition. In other groups, progesterone implants were removed at the time of ovariectomy, i.e. estrogen replacement only (Advanced P Withdrawal, n = 7), or at 1700 h on d 22 of pregnancy (Delayed P Withdrawal, n = 6). An additional group received progesterone replacement only with progesterone withdrawal occurring at 1700 h on d 21 of pregnancy (No Estrogen, n = 6).

To establish the effect of ovarian hormone manipulations on PRL secretion, groups of animals were fitted with indwelling jugular cannulas at the time of ovariectomy. Blood samples were collected at regular intervals until the time of parturition. For Normal P Withdrawal (n = 6) and No Estrogen animals (n = 6), blood samples were collected at 1100, 1800, and 2300 h on d 20 of pregnancy; 0100, 0300, 0500, 1100, 1700, and 2300 h on d 21 of pregnancy; and 0100, 0300, 0500, and 1100 h on d 22 of pregnancy. For Advanced P Withdrawal animals (n = 7), blood samples were collected at 1800 and 2300 h on d 18 of pregnancy and 0100, 0300, 0500, and 1100 h on d 19 of pregnancy. For Delayed P Withdrawal animals (n = 6), blood samples were collected at 1100, 1800, and 2300 h on d 21 of pregnancy; 0100, 0300, 0500, 1100, 1700, and 2300 h on d 22 of pregnancy; and 0100, 0300, and 0500 h on d 23 of pregnancy. To determine plasma PRL levels, blood samples were analyzed by RIA (see below). Animals were observed half-hourly after the removal of progesterone to monitor the time of onset of parturition.

Experiment 3: effects of estrogen and progesterone on SOCS-1 and -3 and cytokine-inducible SH2-containing protein (CIS) mRNA levels in the arcuate nucleus and TIDA neuronal activity during late pregnancy
To test the hypothesis that the late pregnancy increase in SOCS mRNA levels within the arcuate nucleus is determined by the timing of withdrawal of progesterone and the antepartum PRL surge, SOCS-1 and -3 and CIS mRNA levels were quantified in additional groups of late pregnant animals after steroid manipulation (as outlined in experiment 2). These three members of the SOCS family were chosen for analysis based on previous work showing that they are regulated by PRL in the arcuate nucleus (18). Advanced P Withdrawal animals were killed before (1700 h on d 18 of pregnancy, n = 8) and during (1100 h on d 18 of pregnancy, n = 8) the time of the expected advanced antepartum PRL surge. Normal P Withdrawal (n = 8) and Delayed P Withdrawal (n = 7) animals were killed at the time of the expected antepartum PRL surge as observed in intact animals (0300 h of d 22 of pregnancy). As a control, sham ovariectomized late pregnant animals were killed at 1700 h (n = 8) and 2300 h (n = 8) on d 18 of pregnancy and at 0300 h (n = 8) on d 22 of pregnancy. The No Estrogen group was not included in this part of the experiment because the antepartum PRL surge was not observed in the absence of estrogen (Results, experiment 2). All animals were killed by decapitation, and at the time of killing, trunk blood samples and brains were collected. Efficacy of hormone replacement on the serum levels of estradiol, progesterone, and PRL was measured by RIA (see below). Brains were microdissected, as described previously (18), to collect the arcuate nucleus for measurement of SOCS-1 and -3 and CIS mRNA and the median eminence to assess TIDA neuronal activity (see below).

Experiment 4: effects of estrogen and progesterone on SOCS-1 and -3 and CIS mRNA levels in response to PRL
To test the hypothesis that ovarian steroids alter the levels of SOCS mRNA in the arcuate nucleus, ovarian steroid levels were manipulated in nonpregnant rats. All animals were ovariectomized on d 1 of the experiment and treated with one of three hormone replacement regimes: blank implants (ovariectomy only, n = 12), estrogen-containing implants (as in experiment 2, but estrogen implants were filled with 100 µg/ml 17β-estradiol) (E, n = 12), or estrogen and progesterone implants (progesterone as in experiment 2) (E+P, n = 12). Previous work confirmed that this estrogen dose was sufficient to induce physiological effects of estrogen in the hypothalamus (23). Additional animals treated with progesterone alone (experiment 2) were not included because earlier work showed that in the absence of estrogen animals do not express progesterone receptors within the arcuate nucleus (23). To eliminate possible effects of endogenous PRL, all animals received four sc injections of the dopamine receptor agonist bromocriptine methanesulfonate (200 µg in 200 µl 10% ethanol; Research Biochemicals, Inc., Natick, MA) at 8-hourly intervals starting at 0900 h on d 7 of the experiment. At the time of the final bromocriptine injection, half of the animals were injected sc with ovine PRL (oPRL; 300 µg in 300 µl saline; Sigma) to induce an acute PRL surge (PRL, E+PRL, and E+P+PRL, n = 6 per treatment), or vehicle only (ovariectomy only, E, and E+P, n = 6 per treatment). A similar dose of oPRL was previously shown to be sufficient to induce SOCS mRNA within the arcuate nucleus in ovariectomized animals (18). All animals were killed by decapitation 2 h after oPRL or vehicle injections. Brains and trunk blood samples were collected at the time of killing for microdissection of the arcuate nucleus and serum hormone analysis, respectively. Levels of SOCS-1 and -3 and CIS mRNA within the arcuate nucleus were determined by real time RT-PCR (see below). To confirm the efficacy of hormone treatments, serum was analyzed by RIA to measure estrogen, progesterone, and rat PRL levels (see below).

Real-time quantitative PCR measurement of SOCS mRNA
TaqMan probes and forward (sense) and reverse (antisense) primers to the rat genes encoding SOCS-1 and -3 and CIS were designed using Primer Express software (see Ref. 18 for details). Real-time PCR was performed using a protocol similar to that described previously (18), except that it was preceded by RNA extraction using commercial solid-phase spin columns (RNeasy Mini kit; QIAGEN, Inc., Valencia, CA). Tissue punches were collected into a lysis buffer, and total RNA was extracted following the protocol supplied with the kit. The total amount of RNA in each sample was quantified using an ND-1000 spectrophotometer (NanoDrop, Biolab, Auckland, New Zealand), and 100 ng total RNA was added to the RT reaction for all samples. A 22.5-µl reaction mixture was used, containing Absolute qPCR Rox master mix (ABgene; Epsom, Surrey, UK), template cDNA (2.5 µl), and primers and probes at optimized concentrations (see Ref. 18 for details). Pooling multiple punches of cortex created a stock of cDNA containing relatively high levels of SOCS-1 and -3 and CIS mRNA. A 4-fold dilution series of this was run on each plate as external standards. An ABI PRISM 7300 Sequence Detection Systems (Applied Biosystems) was used to detect fluorescence during each PCR cycle. A standard curve of threshold cycle (CT) vs. dilution factor (r2 < 0.97) was used to calculate relative levels of SOCS mRNA. CT values obtained from the serially diluted cortex punches were plotted on a graph where the y-axis represented the CT value of the cortex sample and the x-axis an arbitrary 4-fold unit assigned to each dilution. CT values for each sample were calculated against this curve to generate a standardized arbitrary unit. Results are expressed as relative levels of SOCS mRNA per nanogram of total RNA. Samples and standards were run in duplicate, and coefficients of variation were almost always less than 1%.

Assessment of TIDA neuronal activity
As an estimate of TIDA neuronal activity (24), dihydroxyphenylacetic acid (DOPAC) levels within the median eminence were measured by isocratic HPLC with electrochemical detection, as described previously (8). The mobile phase consisted of tissue buffer containing 0.1 mM EDTA and 0.4 mM sodium octyl sulfate (pH 3.0) and was pumped at a flow rate of 600 µl/min. Catecholamines were separated on a reverse-phase Luna 3-µm C18 column (150 mm; Phenomenex, Auckland, New Zealand) and detected using a conditioning cell (+350 mV) and a dual-electrode analytical cell (–150 and +350 mV) using a Coulochem II detector (ESA, Bedford, MA). DOPAC was quantified by comparison with external standards, and values were corrected for total protein content in the tissue punches as measured by a Bio-Rad DC protein Assay kit (Bio-Rad Laboratories Inc., Hercules, CA).

RIA of rat PRL, estradiol, and progesterone
Serum and plasma rat PRL concentrations were determined by RIA using reagents provided by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) National Hormone and Pituitary program. This assay does not cross-react with oPRL. Radioactive iodinated tracer was prepared from NIDDK-rat PRL-I-9 using the chloramine-T method. Samples (10-µl aliquots) and a series of standards (NIDDK rPRL-RP-3) were incubated in primary antibody (final dilution 1:75,000, NIDDK rabbit anti-rPRL-RIA9) and tracer (15,000 cpm) containing 0.25% normal rabbit serum. A secondary antibody (sheep antirabbit serum, SAR 10 AB2; AgResearch, Invermay, New Zealand) was used to precipitate the bound fraction. The sensitivity of the PRL assay was 6.3 ng/ml, with intra- and interassay coefficients of variation of 10.2 and 14.6%, respectively.

Commercial RIA kits were used to measure serum and plasma estradiol (3rd Generation Estradiol RIA DSL-39100; Diagnostic Systems Laboratories, Webster, TX) and progesterone (Active Progesterone RIA DSL-39100; Diagnostic Systems Laboratories) levels, using protocols outlined in the kits. Serial dilutions of a high standard in rat serum provided a parallel profile to the standard curve, confirming that the assays were suitable for the analysis of ovarian steroids in rat serum. Inter- and intraassay coefficients of variation for estradiol assays were 22.1 and 4.6%, and for progesterone assays were 4.9 and 3.9%, respectively.

Statistical analysis
All data are presented as means ± SEM. Differences between groups were identified by one-way ANOVA. Where significant differences occurred, these were localized to groups using the Tukey post hoc test. The threshold level for statistical significance was set at 5% (P < 0.05).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Experiment 1: plasma estrogen, progesterone, and PRL concentrations during late pregnancy
Changes in plasma PRL, estrogen, and progesterone levels during late pregnancy are shown in Fig. 1Go. Plasma PRL levels remained low until around 1700 h on d 21 of pregnancy when values increased dramatically to peak by 0300 h on d 22 before returning to basal values after parturition. Plasma progesterone levels were high on d 20 of pregnancy and then decreased markedly by 0600 h on d 21 of pregnancy and remained low for the duration of pregnancy. Estrogen levels were also elevated on d 20 of pregnancy (compared with d 18 in our colony, unpublished results), and remained high throughout the duration of sampling. Parturition occurred between 0900 and 1100 h on d 22 of pregnancy.


Figure 1
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  		FIG. 1. Mean (±SEM) plasma estrogen, progesterone and PRL levels during d 20–22 of pregnancy (n = 6). Thick black bars along the x-axis represent the dark period of the light/dark cycle. Parturition occurred during the morning of d 22 of pregnancy. (Peak PRL values on d 22 of pregnancy are significantly elevated compared with d 20, whereas plasma progesterone levels throughout d 21 and 22 were significantly reduced compared with d 20, P < 0.05.)

 
Experiment 2: effects of estrogen and progesterone on PRL secretion during late pregnancy
Alterations in steroid hormone levels markedly changed the timing of the onset of parturition (Table 1Go). Intact animals in the colony normally give birth between 0900 and 1100 h on d 22 of pregnancy. During preliminary experiments, withdrawal of progesterone at 0600 h on d 21 of pregnancy, to mimic the fall in levels of progesterone observed at this time in intact animals, advanced the timing of parturition (data not shown). Withdrawal of progesterone at 1700 h on d 21 of pregnancy, however, resulted in parturition on the morning of d 22 (similar to that observed in intact animals). Hence, this was selected as our basic model (Normal P Withdrawal). Parturition was advanced by about 72 h or delayed by about 18 h after advance or delayed progesterone withdrawal, respectively. The timing of parturition after the normal withdrawal of progesterone in the absence of estrogen (No Estrogen) was similar to that observed in intact animals. These animals had difficulty in giving birth and were unable to successfully deliver pups. Thus, for the No Estrogen group, the time at which animals started to display behaviors associated with labor (uterine discharge and contractions) was noted, and the experiment was terminated by humanely killing the rat.


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  		TABLE 1. Timing of parturition after steroid manipulation in late pregnant animals

 
Changes in antepartum PRL secretion after hormone treatments in ovariectomized late pregnant animals are illustrated in Fig. 2Go. Withdrawal of progesterone at 1700 h on d 21 of pregnancy (Normal P Withdrawal) resulted in an antepartum PRL surge that resembled that seen in intact animals, peaking around 0300 h of d 22 of pregnancy. This surge was advanced to 2300 h on d 18 of pregnancy in the Advanced P Withdrawal animals. The antepartum PRL surge was not observed after the delayed withdrawal of progesterone on d 22 of pregnancy (Delayed P Withdrawal) or in animals that did not receive estrogen (No Estrogen). In addition, the tightly regulated suppression of PRL during late pregnancy was not observed in the absence of estrogen (No Estrogen). Although the timing of the PRL surge in the Normal P Withdrawal group was similar to that observed in intact animals, the concentration at the peak of this surge was decreased to about 50% of intact values (compare Figs. 1Go and 2Go, P < 0.05).


Figure 2
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  		FIG. 2. Mean (±SEM) plasma PRL profile during late pregnancy after normal (Normal P Withdrawal, n = 6), advanced (Advanced P Withdrawal, n = 7), and delayed (Delayed P Withdrawal, n = 6) withdrawal of progesterone (P) in the presence of estrogen (E) and normal withdrawal of progesterone in the absence of estrogen (No Estrogen, n = 6). Peak values for Normal and Advanced P Withdrawal animals are significantly different compared with preceding samples. *, P < 0.05. Thick black bars along the x-axis represent the dark period of the light/dark cycle. Dark and light gray bars along the top of each graph represent the duration of estrogen and progesterone treatment, respectively.

 
Experiment 3: effects of estrogen and progesterone on SOCS-1 and -3 and CIS mRNA levels in the arcuate nucleus and TIDA neuronal activity during late pregnancy
As described above, untreated pregnant animals exhibited a marked PRL surge at 0300 h on d 22 of pregnancy. This was accompanied by significantly reduced DOPAC concentrations in the median eminence, indicative of reduced activity of TIDA neurons (Fig. 3Go, left panel, P < 0.05). In association with the increase in PRL concentrations, there was a significant increase in mRNA for SOCS-1 and -3 and CIS in the arcuate nucleus (P < 0.05). After the advanced withdrawal of progesterone on d 18 of pregnancy, PRL levels were significantly elevated at both 1700 and 2300 h on d 18 of pregnancy compared with intact animals, representing an advanced antepartum PRL surge (Fig. 3Go, left panel, P < 0.05). At times of elevated serum PRL concentrations, median eminence DOPAC concentrations were significantly reduced compared with intact animals (P < 0.05). An increase in SOCS-1 and -3 and CIS mRNA was observed at 1700 and 2300 h on d 18 of pregnancy, associated with the elevated PRL concentrations. This, however, did not reach significance for SOCS-1 until 2300 h (P < 0.05).


Figure 3
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  		FIG. 3. Changes in serum PRL, median eminence DOPAC concentration, and SOCS mRNA expression in the arcuate nucleus during late pregnancy and after advanced progesterone withdrawal (left panel) and after normal and delayed progesterone withdrawal (right panel). Compared with controls (Intact, n = 8), an increase in serum PRL concentrations, decrease in median eminence DOPAC concentrations, and an increase in SOCS mRNA within the arcuate nucleus was measured after advanced progesterone withdrawal (Advanced P Withdrawal 1700 and 2300 h, n = 8). *, Significantly different from Intact 1700 h, d 18, P < 0.05. After the delayed withdrawal of progesterone (Delayed P Withdrawal, n = 8), a decrease in serum PRL concentrations, increase in median eminence DOPAC concentrations, and a decrease in SOCS mRNA within the arcuate nucleus were measured compared with controls (Normal P Withdrawal, n = 8). {dagger}, Significantly different from Normal P Withdrawal, P < 0.05.

 
Withdrawal of progesterone at 1700 h on d 21 resulted in the generation of a PRL surge at 0300 h on d 22 of pregnancy, similar to that which occurs in intact animals. Again, this PRL surge was accompanied by a significantly reduced DOPAC concentration in the median eminence and increased SOCS-1 and -3 and CIS mRNA levels in the arcuate nucleus (Fig. 3Go, right panel, P < 0.05). In animals where progesterone remained intact (Delayed P Withdrawal), PRL levels were significantly reduced compared with that observed after progesterone withdrawal (Fig. 3Go, right panel, P < 0.05). Decreased PRL levels were associated with high DOPAC concentrations in the median eminence. Compared with the Normal P Withdrawal group, SOCS-1 and -3 and CIS mRNA levels measured within the arcuate nucleus were significantly reduced in the Delayed P Withdrawal group (P < 0.05).

Experiment 4: effects of estrogen and progesterone on SOCS-1 and -3 and CIS mRNA levels in response to PRL
Serum levels of estradiol and progesterone concentrations after hormone treatments in ovariectomized nonpregnant animals are illustrated in Fig. 4Go. Low levels of hormone (less than 5.0 pg/ml estradiol and 5.0 ng/ml progesterone) were observed in ovariectomized animals. Prolonged estrogen exposure increased serum estradiol levels to about 15 pg/ml (P < 0.05), whereas prolonged progesterone exposure significantly increased serum progesterone levels to about 65 ng/ml at the time of killing (P < 0.05). Serum rat PRL levels, as measured by RIA, were below the level of detection of the assay in all samples (data not shown), confirming the effectiveness of bromocriptine treatment to block endogenous PRL secretion.


Figure 4
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  		FIG. 4. Mean (±SEM) levels of serum estrogen and progesterone in nonpregnant ovariectomized animals (OVX only) treated with a regime of chronic exposure to estrogen with (OVX+E+P) or without progesterone (OVX+E) and estrogen (E+PRL) or estrogen and progesterone (OVX+E+PRL) and a single ip PRL injection (oPRL, 300 µg in 300 µl saline, black bars) (all groups, n = 6). Bars not identified with the same letter are significantly different from each other, P < 0.05.

 
Changes in SOCS mRNA levels in ovariectomized animals treated with estrogen with or without progesterone are illustrated in Fig. 5Go. Compared with levels in ovariectomized only animals, a significant increase in SOCS-1 and -3 and CIS mRNA expression was observed after prolonged estrogen exposure (E, P < 0.05), even though endogenous PRL was suppressed by bromocriptine. Prolonged progesterone treatment (E+P) completely prevented the estrogen-induced increase in levels of SOCS-1 and -3 and CIS mRNA. As described previously (18), oPRL treatment induced an increase in levels of SOCS-1 and -3 and CIS mRNA within the arcuate nucleus compared with that observed in untreated control animals (OVX only, P < 0.05). For SOCS-1 and CIS, the PRL-induced increase in SOCS mRNA expression was at least partially prevented after either prolonged estrogen (E+PRL) or estrogen combined with progesterone treatment (E+P+PRL). In contrast, PRL-induced SOCS-3 mRNA expression did not change after either steroid hormone treatment.


Figure 5
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  		FIG. 5. Changes in SOCS mRNA levels within the arcuate nucleus after ovarian steroid and/or PRL treatments in nonpregnant ovariectomized rats. PRL induced a significant increase in SOCS-1 and -3 and CIS mRNA, whereas estrogen (E+PRL) or estrogen and progesterone (E+P+PRL) partially suppressed SOCS-1 and CIS in response to PRL (oPRL, 300 µg in 300 µl saline, black bars). Estrogen (E), by itself, significantly increased SOCS-1 and -3 and CIS compared with controls (OVX only), and this effect was completely blocked by progesterone (E+P) (all groups, n = 6). Bars not identified with the same letter are significantly different from each other, P < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It is well established that changes in ovarian steroid hormone levels during late pregnancy induce an antepartum PRL surge (25), associated with a loss of responsiveness of TIDA neurons to PRL, such that elevated levels of PRL do not result in a compensatory increase in dopamine secretion. The present data support the hypothesis that increased SOCS expression in the arcuate nucleus during late pregnancy and lactation (18, 19) might impair PRL activation of STAT5b, resulting in a failure of activation of TIDA neurons. Moreover, the data suggest that ovarian steroid hormones play a critical role in this response, with estrogen facilitating the effect of PRL to activate SOCS expression and thereby inhibit negative feedback, but only after the decline in progesterone during late pregnancy. Although estrogen has been shown to activate specific members of the SOCS family of proteins in the liver, and in some cell lines (26, 27, 28), this has not been observed in the brain previously. Moreover, the inhibitory effect of progesterone on estrogen or PRL-induced SOCS has not previously been reported. The data suggest a novel interaction between ovarian steroids and cytokine signaling pathways. This interaction may play an important role in the adaptations of PRL negative feedback during late pregnancy and lactation.

To investigate the specific roles of ovarian steroids in controlling PRL secretion during late pregnancy, we developed a model in which these steroids could be experimentally manipulated. As reported previously (25), ovariectomy dramatically affected the progression of pregnancy. To maintain pregnancy in the absence of the ovaries, it was necessary to ensure continuous progesterone exposure, and then timed withdrawal of progesterone rapidly induced parturition and the associated changes in PRL secretion. The withdrawal of progesterone at 1700 h on d 21 of pregnancy (Normal P Withdrawal) resulted in the onset and timing of parturition at a similar time as observed in intact animals. In this model, parturition was preceded by a nocturnal PRL surge at the expected time, although this was smaller (~50%) in magnitude than that seen in intact animals. The reduction in magnitude of the surge was not related to a change in absolute levels of estrogen after steroid manipulation, because higher doses of estrogen had no effect (data not shown). It is possible that some other ovarian factor is involved, or that the timing of hormonal changes was not completely replicated in the experimental model.

Early withdrawal of progesterone, in the presence of maintained estrogen, resulted in the rapid onset of an antepartum PRL surge similar in magnitude to that normally observed during d 22 of pregnancy. This was followed by parturition early on d 19 of pregnancy. In contrast, the delayed withdrawal of progesterone resulted in the absence of the antepartum PRL surge and a significant delay in the onset of parturition. In the absence of estrogen, serum PRL levels remained slightly elevated, and the withdrawal of progesterone on d 21 of pregnancy did not induce a PRL surge. This may be due to a reduction in the level of progesterone receptors expressed in the hypothalamus, because this is dependent on the presence of estrogen (29, 30), resulting in a lack of steroid hormone action to regulate TIDA neurons. Overall, these results confirm that the timing of withdrawal of progesterone is critical for the normal induction of the antepartum PRL surge and parturition and that this requires exposure to estrogen.

The PRL surge during late pregnancy is associated with decreased activity of TIDA neurons (8), at least partially due to a loss of responsiveness to stimulation by PRL (11). In the present study, the PRL surges that followed either normal or advanced withdrawal of progesterone during late pregnancy were always associated with decreased concentrations of median eminence DOPAC (an established marker for the rate of dopamine secretion from TIDA neurons). This suggests that after withdrawal of progesterone, TIDA neurons rapidly ceased to produce dopamine, even in the continued presence of PL that would normally act to stimulate these neurons. In contrast, after delayed progesterone withdrawal, the antepartum PRL surge was abolished, associated with maintained high levels of DOPAC in the median eminence. Together, these results suggest that the altered PRL feedback that normally results in suppression of dopamine secretion and the induction of an antepartum PRL surge is dependent upon the withdrawal of progesterone in the presence of elevated levels of estrogen.

It has been hypothesized that elevated SOCS proteins might contribute to the change in TIDA responsiveness to lactogens during late pregnancy (18) and in response to suckling during lactation (19). As was the case in intact animals, the PRL surge observed after the withdrawal of progesterone on d 21 of pregnancy was associated with increased levels of SOCS-1 and -3 and CIS mRNA within the arcuate nucleus. Elevated levels of all three SOCS mRNA were also observed within the arcuate nucleus at the time of the advanced PRL surge, whereas levels of SOCS mRNA expression did not change after prolonged progesterone exposure. Thus, elevated SOCS expression during late pregnancy required progesterone withdrawal, although it was not clear after experiment 3 whether the increase in SOCS mRNA was driven by progesterone withdrawal independently or was induced by the PRL surge that followed progesterone withdrawal. This question was directly examined in experiment 4, with the data suggesting that SOCS expression is induced by PRL but that this increase can be inhibited by previous exposure to high levels of progesterone.

Although the micropunch technique can specifically isolate the arcuate nucleus for analysis, this nucleus contains multiple cell types (31) and is involved in numerous diverse functions (32). Thus, we cannot assume that results are specifically isolated to TIDA neurons; they may represent the integrated response of multiple PRL-responsive cell types, including the TIDA neurons. Approximately 80–90% of all PRL-R-expressing cells within the arcuate nucleus are TIDA neurons (14), and over 90% of TIDA neurons contain PRL-R mRNA (14) and protein (32). Furthermore, most phospho-STAT5 immunoreactivity observed within the arcuate nucleus after treatment with PRL occurs within TIDA neurons (18). Therefore, it is likely that the majority of PRL-mediated changes in SOCS mRNA would have occurred within TIDA neurons. Similarly, estrogen and progesterone receptors are expressed in TIDA neurons (22), consistent with the hypothesis that ovarian hormone-induced changes in SOCS proteins may also occur directly in TIDA neurons. Additional work will, however, be required to establish the location within the arcuate nucleus of the changes in SOCS protein expression as observed by this study. Furthermore, loss-of-function experiments (for example, using transgenic SOCS knockout mouse lines) will be required to confirm the role of SOCS proteins in modulating TIDA neuron responsiveness to PRL.

SOCS gene expression is induced by multiple cytokines acting through the JAK/STAT pathway, forming an intracellular feedback pathway to regulate the tone of these pathways (33). Our previous work suggested that the increase in SOCS mRNA expression within the arcuate nucleus during late pregnancy was dependent on elevated levels of PRL (18). During late pregnancy, however, PRL fails to activate STAT5b in TIDA neurons (18). This presents something of a paradox, because PRL-induced SOCS expression mediated by the JAK/STAT pathway should be self-limiting by effectively suppressing PRL signal transduction through this pathway. Thus, it seems likely that the induction of SOCS mRNA, although mediated in part by PRL, requires the involvement of other factors. The present data implicate elevated levels of estrogen as a key factor that contributes to the stimulation of SOCS mRNA during late pregnancy, after the decline of progesterone.

Estrogen has been reported to induce SOCS mRNAs in liver and in certain cell lines (26, 27, 28). We have shown here that, in ovariectomized rats, both PRL and estrogen can induce the expression of SOCS mRNA within the arcuate nucleus. The effect of estrogen was not dependent on PRL secretion, because all animals received bromocriptine to suppress endogenous PRL secretion. Because the effects of estrogen and PRL on the expression of SOCS mRNAs were not additive in nonpregnant ovariectomized animals, it is possible that both hormones are acting through a common mechanism. Alternatively, transcription of SOCS may have already been maximal in response to either estrogen or PRL in our experiment. Importantly, the stimulatory effect of either estrogen or PRL was largely attenuated by previous administration of progesterone. Progesterone is known to prevent estrogen-mediated gene induction (34) and signaling in other tissues (35). Similarly, progesterone has been shown to induce signal transduction through the JAK/STAT5 pathway in mammary tissue, including regulatory effects on PRL signaling, and both stimulatory (36, 37, 38) and inhibitory (39) effects have been described. The present data suggest that progesterone exerts similar negative effects on PRL and estrogen signaling in the brain, meaning that estrogen and/or PRL can induce SOCS mRNA only during late pregnancy after the withdrawal of progesterone. This would explain the low level of SOCS mRNA within the arcuate nucleus on d 18 of pregnancy, when progesterone levels are elevated, and the marked rise in SOCS mRNA expression on d 22 of pregnancy, when progesterone levels are low (18).

Although the stimulation of all three SOCS mRNA by PRL and by estrogen individually, and the prevention of the latter by progesterone was unambiguous in experiment 4, the combined effects of PRL and estrogen were less consistent in two ways. First, the two hormones together did not cause a significant increase in SOCS-1 and CIS mRNA when compared with basal levels, raising the possibility that competitive effects might in some way prevent the full response seen after either hormone alone. Second, progesterone was unable to block the stimulatory effect of estrogen and PRL on SOCS-3 mRNA levels. Thus, estrogen and PRL may interact differently with SOCS-3 compared with SOCS-1 and CIS. Further research will be needed to elucidate the nature of this interaction and its relevance to late pregnancy when both PRL/lactogens and estrogen are present at high concentrations.

We propose that withdrawal of progesterone during late pregnancy is the key event allowing the induction of SOCS mRNA within the arcuate nucleus, and this induction can be independently stimulated by either PRL or estrogen. Despite the potential role for estrogen to induce SOCS mRNA in the arcuate nucleus during late pregnancy, our previous data suggested that the sustained elevation in SOCS mRNA required the continued presence of PRL (19). As outlined earlier, however, PRL-induced activation of STAT5b is impaired during lactation (16). The continued ability of PRL to activate SOCS in the absence of STAT5b activation suggests that alternative signaling pathways must be involved. PRL binding to its receptor may activate a range of signaling pathways. Likely candidates for mediating these actions are the MAPK family of signal transduction proteins (ERK1 and -2, p38, and JNK) (40, 41, 42). We have shown that PRL acutely activates ERK1 and -2 in dopamine neurons in vitro and that this activation is not required for induction of dopamine synthesis (43). Interestingly, p38 MAPK has been reported to activate SOCS3 (44). PRL activates p38 (45) MAPK and JNK (46) in other systems, but this has never been evaluated in TIDA neurons. Interestingly, estrogen can also activate MAPK (47), and thus, both estrogen and PRL might be acting through a similar pathway. Importantly, however, for either estrogen or PRL to sustain elevated SOCS mRNA during late pregnancy via MAPK signaling, this signaling itself should be insensitive to inhibition by SOCS proteins. This appears to be true for MAPK signaling, because in macrophages overexpressing SOCS1 or -3 or CIS, MAPK signaling was not impaired (48).

In conclusion, this study has demonstrated that the withdrawal of progesterone allowing luteolysis during late pregnancy is the key trigger necessary to induce suppression of negative feedback regulation of PRL. The dramatically increased estrogen/progesterone ratio during late pregnancy may favor activation of alternative pathways in response to PRL, leading to a MAPK-induced induction of SOCS in TIDA neurons. Elevated SOCS mRNA would prevent PRL-induced activation of STAT5b, effectively inhibiting dopamine production in TIDA neurons, resulting in a decrease in dopamine release with the consequent induction of the antepartum PRL surge. Because TIDA neurons do not then respond to PRL with an increase in dopamine production, this allows a period of sustained hyperprolactinemia to be maintained during late pregnancy and lactation, unencumbered by a regulatory feedback system. This state of hyperprolactinemia is essential to milk production and maternal behavior and potentially may be involved in a range of other brain functions during lactation. Thus, these data demonstrate a novel interaction between steroidal and cytokine signaling pathways in the brain, which mediates an important maternal adaptation to pregnancy.


   Footnotes
 
Disclosure Statement: The authors have nothing to disclose.

First Published Online March 6, 2008

Abbreviations: CIS, Cytokine-inducible SH2-containing protein; CT, cycle threshold; DOPAC, dihydroxyphenylacetic acid; JAK, Janus kinase; oPRL, ovine PRL; PL, placental lactogen; PRL, prolactin; PRL-R, PRL receptor; SOCS, suppressors of cytokine signaling; STAT5b, signal transducer and activator of transcription 5b; TIDA, tuberoinfundibular dopamine.

Received November 27, 2007.

Accepted for publication February 26, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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