α-Estrogen and Progesterone Receptors Modulate Kisspeptin Effects on Prolactin: Role in Estradiol-Induced Prolactin Surge in Female Rats

Abstract

Kisspeptin (Kp) regulates prolactin (PRL) in an estradiol-dependent manner. We investigated the interaction between ovarian steroid receptors and Kp in the control of PRL secretion. Intracerebroventricular injections of Kp-10 or Kp-234 were performed in ovariectomized (OVX) rats under different hormonal treatments. Kp-10 increased PRL release and decreased 3,4-dihydroxyphenylacetic acid levels in the median eminence (ME) of OVX rats treated with estradiol (OVX+E), which was prevented by tamoxifen. Whereas these effects of Kp-10 were absent in OVX rats, they were replicated in OVX rats treated with selective agonist of estrogen receptor (ER)α, propylpyrazole triol, but not of ERβ, diarylpropionitrile. Furthermore, the Kp-10–induced increase in PRL was two times higher in OVX+E rats also treated with progesterone (OVX+EP), which was associated with a reduced expression of both tyrosine hydroxylase (TH) and Ser⁴⁰-phosphorylated TH in the ME. Kp-10 also reduced dopamine levels in the ME of OVX+EP rats, an effect blocked by the progesterone receptor (PR) antagonist RU486. We also determined the effect of Kp antagonism with Kp-234 on the estradiol-induced surges of PRL and luteinizing hormone (LH), using tail-tip blood sampling combined with ultrasensitive enzyme-linked immunosorbent assay. Kp-234 impaired the early phase of the PRL surge and prevented the LH surge in OVX+E rats. Thus, we provide evidence that Kp stimulation of PRL release requires ERα and is potentiated by progesterone via PR activation. Moreover, alongside its essential role in the LH surge, Kp seems to play a role in the peak phase of the estradiol-induced PRL surge.

Prolactin (PRL) is a pituitary hormone recognized mainly for its critical role in promoting lactation (1). However, a wide range of reproductive and nonreproductive functions are also attributed to PRL. In rodents, for example, PRL acts as a luteotrophic hormone, essential for proper luteal function and fertility (2), besides stimulating parental behavior (3). On the other hand, chronic hyperprolactinemia is a frequent cause of infertility in humans of both sexes (4). PRL secretion is tonically inhibited by dopamine released in the portal circulation from neuroendocrine dopaminergic neurons. Among them, the tuberoinfundibular dopaminergic (TIDA) neurons, located in the arcuate nucleus (ARC) and projecting to the external zone of the median eminence (ME), provide the main dopaminergic input to the anterior pituitary (5, 6). PRL secretion in females is known to be strongly influenced by ovarian steroids. In rodents, estradiol induces an afternoon surge of PRL concurrent with the preovulatory luteinizing hormone (LH) surge on proestrus (7). Progesterone, in turn, modulates PRL secretion in a bimodal way: It induces an acute stimulation followed by an inhibition over the next day when administered to ovariectomized (OVX), estradiol-treated (OVX+E) rats (8). These actions of estradiol and progesterone seem to require activation of classic receptors for estrogen (ER) and progesterone (PR), because they are prevented by the selective ER modulator tamoxifen and the PR antagonist RU486, respectively (9,–11).

Concerning the neuroendocrine regulation of PRL release, TIDA neurons have been shown to contain ERα and PR, thus being targets for the central actions of ovarian steroids (12, 13). Accordingly, estradiol decreases the expression and activity of tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine biosynthesis, in the ARC (11). This effect is consistent with the lower dopaminergic activity in the ME during the estradiol-induced PRL surge (10). Progesterone amplifies PRL release induced by estradiol through inhibition of TIDA neurons, as has been demonstrated by its effects in further reducing dopaminergic activity and TH phosphorylation in the ME (10, 14). Although dopamine is considered the main hypothalamic output controlling PRL secretion, the neuronal inputs regulating the activity of TIDA neurons are still poorly understood. It has been shown recently that TIDA perikarya receive synaptic inputs from kisspeptin (Kp) nerve terminals, which are also regulated by estradiol (15, 16). Thus, Kp emerges as a new player in the control of dopamine-PRL axis.

The Kps belong to a family of peptides encoded by the gene Kiss1 and are essential to reproduction. The binding of Kp to the Kiss1 receptor (Kiss1r), also referred as G protein–coupled receptor 54, is critical to the onset of puberty and fertility (17). Animals lacking Kiss1r are infertile because of suppression of gonadotropin secretion (18). Accordingly, Kp potently stimulates LH secretion (19, 20) through the activation of gonadotropin-releasing hormone neurons (21). In the hypothalamus, Kp neurons are located in the anteroventral periventricular nucleus (AVPV) and ARC. Moreover, Kp messenger RNA (mRNA) and protein levels are increased by estradiol in the AVPV but suppressed in the ARC (22, 23), suggesting differential roles for these neuronal populations in the positive and negative feedback regulation of LH secretion.

Besides its essential role in LH secretion, Kp is also involved in the control of PRL secretion. Associated with Kp inputs onto TIDA neurons, intracerebroventricular (ICV) injection of Kp-10 increases PRL release through inhibition of dopamine release. However, this effect requires high levels of estradiol because it was found to happen only in proestrous and OVX+E rats (13, 15). It seems therefore that kisspeptin regulation of PRL release is physiologically relevant in females under conditions of high levels of ovarian steroids, such as the preovulatory period and late pregnancy.

Thus, we aimed to investigate the interaction between ovarian steroid receptors and Kp in the control of PRL secretion. The results here reported demonstrate that Kp stimulation of PRL release requires ERα, because it is blocked by tamoxifen and restored by propylpyrazole triol (PPT) but not diarylpropionitrile (DPN). Progesterone, in turn, acts through PR to potentiate the Kp effects on the activity of TIDA neurons. Moreover, the Kp antagonist Kp-234 impaired the early peak phase of the PRL surge in OVX+E rats, revealing a stimulatory role for Kp in the estradiol-induced PRL surge.

Materials and Methods

Animals

Adult female Wistar rats weighing 200–250 g were house grouped four per cage under conditions of controlled lighting (lights on 07:00 to 19:00 hours) and temperature (22°C ± 1°C). Vaginal smears were taken daily, and only rats showing at least three consecutive regular estrous cycles were included in the studies. Experimental protocols were approved by the Ethics Committee on the Use of Experimental Animals of the University of Minas Gerais. In all experiments, food and water were provided ad libitum. After all surgeries and during the experiments, rats were maintained in individual cages. All experiments were conducted between 10:00 and 12:00 hours.

Experiment one: effect of ER blockade on Kp stimulation of PRL release

Because we have found in previous studies that ICV injections of Kp-10 increased PRL secretion in OVX+E but not OVX rats (15), this experiment aimed to determine the role of ER in the effect of Kp-10 on PRL release. Tamoxifen was used to block ER, as we previously described (9). Seven to 10 days before the experiment, rats were OVX and implanted with a guide cannula into the right lateral cerebral ventricle. Rats were treated with subcutaneous (SC) injections of estradiol (OVX+E; 17β-estradiol cypionate 10 µg/0.2 mL per rat; n = 5–6 per group) or estradiol plus tamoxifen (OVX+ET; 17β-estradiol cypionate 10 µg/0.2 mL and tamoxifen 3 mg/0.2 mL per rat; n = 8 per group) daily for three consecutive days, and the experiments were conducted on the fourth day (9). The jugular vein was cannulated 1 day before the experiment. Between 10:00 and 12:00 hours, blood samples were withdrawn through the jugular vein catheter 5 minutes before and 5, 10, 20, 30, and 60 minutes after the ICV injection of Kp-10 (3 nmol/3 µL) or vehicle (Veh) (15). Blood samples were evaluated for plasma PRL levels by radioimmunoassay (RIA). After the last sample, OVX+E rats were perfused for immunofluorescence analysis of TH and Ser⁴⁰-phosphorylated TH (S⁴⁰pTH) expression in the ME.

Experiment two: effect of ER blockade on Kp inhibition of TIDA neurons

Using 3,4-dihydroxyphenylacetic acid (DOPAC) levels as an index of dopaminergic activity (24), we have shown that Kp-10 inhibits the activity of TIDA terminals in the ME in an estradiol-dependent manner (13). Here we tested whether tamoxifen would block this effect of Kp. For that, OVX rats implanted with an ICV guide cannula were treated with estradiol (OVX+E; n = 6 per group) or estradiol and tamoxifen (OVX+ET; n = 4–8 per group) daily for three consecutive days, as described in experiment one. On the fourth day, rats were decapitated 10 minutes after the ICV injection of 3 nmol Kp-10 or Veh. Trunk blood was collected for plasma PRL measurement. The brains were rapidly removed, and the ME was dissected under a stereoscopic microscope, frozen in liquid nitrogen, and stored at –80°C. Concentrations of dopamine and DOPAC were determined in the ME via high-performance liquid chromatography with electrochemical detection (HPLC-ED).

Experiment three: role of ER subtypes in Kp regulation of TIDA neurons and PRL

Because results of experiments one and two showed that tamoxifen blocked the effects of Kp-10 on TIDA neurons and PRL, we then investigated the involvement of ERα and ERβ in this action of Kp. Seven to 10 days before experiment, rats were OVX and implanted with an ICV guide cannula. The animals were treated with SC injections of the selective ERα agonist PPT (OVX+PPT; 1.5 mg/0.2 mL per rat; n = 6–9 per group), the selective ERβ agonist DPN (OVX+DPN; 1.5 mg/0.2 mL per rat; n = 3–6 per group), or oil (OVX; 0.2 mL per rat; n = 6–8 per group) for three consecutive days, and the experiment was conducted on the fourth day, as described in experiment two. Rats were decapitated 10 minutes after an ICV injection of Kp-10 or Veh for measurement of plasma PRL levels and dopamine and DOPAC concentrations in the ME.

Experiment four: effect of progesterone on Kp stimulation of PRL release

In this experiment, we evaluated whether progesterone would modulate the Kp-induced increase in PRL secretion, as it does for the estradiol-induced PRL surge (8, 25). OVX rats implanted with an ICV guide cannula were treated with estradiol for three consecutive days as described in experiment one. The jugular vein was cannulated 1 day before the experiment. On the fourth day, rats received an injection of progesterone at 08:00 hours (OVX+EP; progesterone 2.5 mg/0.2 mL per rat, SC; n = 6 per group). Two hours later, blood samples were withdrawn from the jugular vein catheter at –5, 5, 10, 20, 30, and 60 minutes after the ICV injection of Kp-10 or Veh, and plasma PRL levels were determined. After the last blood sample, rats were transcardially perfused. The brains were processed for immunofluorescence double labeling of S⁴⁰pTH and TH in the ME. Plasma and brain samples of OVX+E rats injected with Kp-10 in experiment one (n = 6) were analyzed in the same assays and used as reference of Kp-10 effects on rats treated with estradiol only.

Experiment five: effect of progesterone on Kp inhibition of TIDA neurons

Given the results of experiment four showing progesterone amplification of PRL release in response to Kp, we next evaluated whether progesterone modulates the effect of Kp-10 on the activity of TIDA neurons. The involvement of PR was also investigated by using RU486 to block this receptor activation. OVX rats implanted with an ICV guide cannula were treated with estradiol for three consecutive days as described in experiment one. On the fourth day, rats were treated SC with RU486 (1.5 mg/0.2 mL per rat) or oil at 07:00 hours, followed by an injection of progesterone (2.5 mg/0.2 mL per rat; OVX+EP+RU, n = 4–5 per group; OVX+EP, n = 6 per group) at 08:00 hours. Two hours later, rats were decapitated 10 minutes after ICV injection of Kp-10 or Veh. Plasma PRL levels and dopamine and DOPAC concentrations in the ME were determined.

Experiment six: effect of Kp-234 on the estradiol-induced surges of PRL and LH

The Kp antagonist Kp-234 (26) was used to investigate whether activation of Kiss1r is necessary for generation of PRL and LH surges in the positive feedback model of OVX+E rats. In this experiment, we performed tail-tip blood sampling in rats for measurement of whole blood PRL and LH levels by ultrasensitive enzyme-linked immunosorbent assay (ELISA), as recently described (27, 28). Rats were handled daily for ~30 days to acclimate to the procedure of tail-tip blood sampling. Seven to 10 days before the experiment, rats were OVX and implanted with an ICV cannula. Then they were treated with estradiol for three consecutive days as described in experiment one. On the fourth day, rats received three ICV injections of Kp-234 (6 nmol/1.2 µL; n = 6) or Veh (n = 6) at 10:00, 12:00, and 14:00 hours. Tail-tip blood samples were withdrawn at 30-minute intervals from 13:00 to 18:00 hours for PRL and LH measurement by ELISA.

Surgeries, ICV injection, and blood sampling

Ovariectomy, stereotaxy, and transcardial perfusion were performed under ketamine (80 mg/kg, IP) and xylazine (10 mg/kg, IP) anesthesia. Ovariectomy was performed through bilateral incisions in the skin and muscle layers. Rats were positioned in a stereotaxic instrument, and a 22-gauge stainless-steel guide cannula was implanted into the right cerebral lateral ventricle (coordinates: 1.0 mm posterior to bregma, 1.6 lateral to midline, and 3.2–3.6 mm below the skull) as previously described (15). For jugular vein cannulation, rats were anesthetized with tribromoethanol (250 mg/kg, IP), because ketamine and xylazine interfere with hormonal secretion, and a Silastic cannula (Dow Corning Corp., Midland, MI) was inserted through the external jugular vein into the right atrium (29). After surgeries, animals were treated with pentabiotic (24,000 UI/kg, IM; Fort Dodge) and analgesic (flunixin meglumine, 2.5 mg/kg, SC). For ICV injections, a 30-gauge stainless-steel needle was inserted through the guide cannula and connected to an injection pump set to inject 3 µL of solution per minute. The injection needle was left in place for 1 minute after injections to prevent solution reflux. For serial blood sampling from the jugular vein (experiments one and four), polyethylene tubing (PE-50) filled with heparinized saline (0.9% NaCl, 30 IU heparin/mL) was connected to the jugular vein catheter. Blood samples of 400 µL were withdrawn into plastic heparinized syringes, and an equal volume of sterile saline was replaced after removal of each blood sample. We have previously reported that removal of similar blood volume does not alter PRL secretion (9). In decapitated rats (experiments two, three, and five), samples of trunk blood were collected immediately after decapitation. Plasma was obtained by centrifugation and stored at – 20°C until hormonal assay. For tail-tip blood sampling (experiment six), the distal 1 to 2 mm of the tail was clipped and 10-µL tail-tip blood samples were collected at 30-minute intervals via micropipette. Whole blood samples were immediately suspended in phosphate-buffered saline (PBS) with 0.05% Tween-20 (PBS-T) at a 1:20 dilution, placed on ice, and stored at –20°C until hormonal assay.

Hormonal and drug treatment

17β-Estradiol cypionate (10 µg/0.2 mL per rat, SC; Pfizer, São Paulo, Brazil), progesterone (2.5 mg/0.2 mL per rat, SC; Sigma-Aldrich, St. Louis, MO), tamoxifen (3 mg/0.2 mL per rat, SC; Sigma-Aldrich), and RU486 (1.5 mg/0.2 mL per rat, SC; Sigma-Aldrich) were diluted in corn oil. The regimens of hormonal treatment used were found to yield physiological levels of plasma 17β-estradiol and progesterone (30) and generate preovulatory-like surges of LH and PRL (9, 30). The dosage of RU486 used has been reported to prevent progesterone effects on PRL and dopamine (10). PPT and DPN (1.5 mg/0.2 mL per rat, SC; Tocris Bioscience, Bristol, UK) were diluted in corn oil with 10% ethanol. The dosage used for PPT and DPN was based on a previous study reporting effects on LH and at the pituitary for this treatment regimen (31). PPT is 410 times more selective for ERα than for ERβ, whereas DPN is 70 times more selective for ERβ than for ERα (32, 33). PPT and tamoxifen but not DPN can also act as G protein–coupled estrogen receptor agonists (34). Kp-10 (3 nmol/3 µL, ICV; Metastin 45-54-amide, human; Phoenix Pharmaceuticals, Burlingame, CA) was dissolved in 0.01 M PBS, pH 7.4 (15). Kp-234 (6 nmol/1.2 µL, ICV; Tocris Bioscience) was dissolved in 20% dimethyl sulfoxide (DMSO) in PBS. The dosage used of Kp-234 was based on the previous report in rats and mice of effective ICV dosages between 1 and 15 nmol per injection (26).

Immunofluorescence

Rats were anesthetized and transcardially perfused with PBS followed by 4% paraformaldehyde. Frontal sections of 30 µm were cut in four series throughout the rostrocaudal extension of the hypothalamus according to the rat brain atlas (35) and stored at –20°C in cryoprotectant solution. All steps were performed at 22°C, except for incubation with the primary antibodies, performed at 4°C for 40 hours. Sections of the ME were double labeled for S⁴⁰pTH and TH with immunofluorescence as previously described (36). Briefly, sections were incubated with a cocktail containing the rabbit anti-rat S⁴⁰pTH (36-8600 Zymed Laboratories; Invitrogen, Camarillo, CA) at 1:10,000 and mouse anti-rat TH (T1299; Sigma-Aldrich) at 1:10,000, followed by a cocktail containing Alexa Fluor 488-coupled anti-rabbit donkey immunoglobulin G (IgG) and Alexa Fluor 568-coupled anti-mouse goat IgG (Molecular Probes, Eugene, OR) at 1:1,000 for 2 hours. Images were taken with a fluorescent microscope (Axiovert 40 CFL, Carl Zeiss, Hallbergmoos, Germany) using a digital camera with an image analysis system (Axiocam MRC and Axiovision SE64, Carl Zeiss). As controls, omission of the primary antibodies resulted in no labeling (see Results), and the emissions of the fluorophores Alexa Fluor 488 and Alexa Fluor 568 were confirmed to be selectively detected by their respective filter sets. The mouse anti-TH antibody has been extensively used, and its labeling corresponds to previous reports of TH staining in the rat brain (5). The rabbit anti-S⁴⁰pTH antibody has been reported to yield only a single band in western blots of murine brains (14, 37). Moreover, previous studies using this antibody have shown that the S⁴⁰pTH labeling differs from those of anti-TH antibodies in terms of functional responses, attesting to its specificity (9, 14, 37). All sections analyzed from the three groups evaluated (OVX+EP+Veh, OVX+EP+Kp-10, and OVX+E+Kp-10) were processed in the same immunohistochemistry. The optical density of S⁴⁰pTH and TH in external layer of the ME was measured in four sections per rat via Image J software (National Institutes of Health, Bethesda, MD). Images were captured with a 20-x objective. A region of interest (width, 280 µm; length, 140 µm) was used to delimit the area in which analyses were performed. The integrated optical density (IOD) of S⁴⁰pTH and TH in the ME was calculated and subtracted from the respective backgrounds. The S⁴⁰pTH and TH IODs were expressed as a percentage of the control group (OVX+EP-Veh; considered as 100%) and as the S⁴⁰pTH/TH ratio.

HPLC-ED

The ME was homogenized in 50 µL of a solution containing 0.15 M perchloric acid, 0.1 mM EDTA, and 1.7 µM 3,4-dihydroxybenzylamine as the internal standard. The homogenates were centrifuged for 20 minutes at 12,000 g and 4°C. Protein content in the pellet was determined via the Bradford method (38). In the supernatant, dopamine and DOPAC concentrations were determined as previously described (39). Briefly, 20 µL of the supernatant was injected into an HPLC-ED system equipped with a C-18 column (250 × 4 mm, Purospher, 5 µm; Merck, Darmstadt, Germany), preceded by a 4- × 4-mm guard column with the same constitution. A mobile phase containing 100 mM sodium dihydrogen phosphate monohydrate, 10 mM sodium chloride, 0.1 mM EDTA, 0.38 M sodium 1-octanesulfonic acid, and 10% methanol in ultrapure water (pH 3.5) was pumped through the system at a flow rate of 1.0 mL/min. The potential in the electrochemical detector (L-ECD-6A, Shimadzu, Kyoto, Japan) was set to 0.85 V. Chromatography data were plotted with Class-VP software (Shimadzu, Kyoto, Japan). Quantification was performed via the internal standard method based on the peak height. All samples from the same experiment were measured in one assay. The intraassay coefficient of variation was 1.4% for dopamine and 1.6% for DOPAC. Dopamine levels were considered to reflect neurotransmitters in synaptic vesicles, whereas DOPAC was used as an index of dopamine release (24). DOPAC/DA ratio was calculated as a measure of neurotransmitter turnover.

RIA

Plasma PRL levels (experiments one through five) were assayed by double-antibody RIA with kits provided by the National Institute of Diabetes and Digestive and Kidney Diseases–National Hormone and Pituitary Program (NIDDK-NHPP; Harbor-UCLA, St. Torrance, CA). The antiserum was the anti-rat PRL-S9, and the reference preparation was PRL-RP3. Iodination was performed with chloramine-T (1 mg/mL in 0.05 M phosphate buffer) for 1 minute. The ¹²⁵I-hormones were eluted in a Sephadex-G75 column with 0.01 M phosphate buffer (pH 7.4). All samples from the same experiment were assayed in one RIA, and the same applies to the three groups in experiment four (OVX+EP+Veh, OVX+EP+Kp-10, and OVX+E+Kp-10). The lower limit of detection was 0.7 ng/mL, and the intraassay coefficient of variation was 2.5%.

ELISA

Whole blood PRL and LH levels (experiment six) were assayed by ultrasensitive ELISAs, adapted from previously described methods (27, 28). For PRL and LH assays, respectively, the reference preparations used were rat PRL (rPRL) RP-3 [AFP-4459B, National Institute of Diabetes and Digestive and Kidney Diseases–National Hormone and Pituitary Program (NIDDK-NHPP)] and rat LH (rLH) RP-3 (AFP718B, NIDDK-NHPP), the capture antibodies used were guinea pig anti-rPRL (anti-rPRL-IC; AFP65191, NIDDK-NHPP) at 1:1,500 and monoclonal anti-bovine LH-β subunit (518B7, University of California) at 1:2,500, and the detection antibodies used were rabbit anti-mouse PRL (F. Talamantes; University of Santa Cruz, CA) at 1:25,000 and rabbit anti-rLH (AFP240580Rb, NIDDK-NHPP) at 1:40,000. The secondary antibody used was horseradish peroxidase-conjugated goat anti-rabbit IgG (P044801-2, Dako Pathology Solutions, Santa Clara, CA) at 1:2,000 in both assays. A 96-well high-binding plate (9018, Corning, Kennebunk, ME) was coated with 50 µL of capture antibody diluted in PBS overnight at 4°C. The capture antibody was decanted, and wells were incubated with 200 µL of blocking buffer (5% skim milk powder in PBS-T) for 2 hours at room temperature (RT). A standard curve was generated via a twofold serial dilution of the reference preparation. The plate was washed with PBS-T, and wells were incubated with 50 µL of standards or samples for 24 hours at RT. After washing, wells were incubated with 50 µL of detection antibody diluted in blocking buffer for 24 hours at 4°C. After washing, wells were incubated with 50 µL of secondary antibody diluted in 50% PBS, 50% blocking buffer for 1.5 hours at RT. After a final wash, wells were incubated with 100 µL of 2 mg/mL o-phenylenediamine dihydrochloride (P1526, Sigma-Aldrich) diluted in citrate–phosphate buffer (pH 5.0) containing 0.02% hydrogen peroxide for 45 minutes at RT. The reaction was stopped with 50 µL of 3 M HCl. The absorbance was determined at 490 nm with a microplate reader, and the wavelength of 650 nm was used for background correction. The concentrations of PRL and LH were obtained by interpolating the optical density of samples against a nonlinear regression of the respective standard curves. All samples from the experiment were assayed in one assay. For PRL ELISA, the lower limit of detection was 0.20 ng/mL, and the intraassay and interassay coefficients of variation were 2.1% and 11.3%, respectively. Supplemental Fig. 1 shows a standard curve ranging from 0.03 to 15 ng/mL of rPRL (r² = 0.996) and a dilution test of plasma samples from diestrous, lactating, and OVX+E rats. Diestrous and lactating rats were euthanized between 10:00 and 12:00 hours. The OVX+E rat was treated with estradiol, as described in experiment one, and euthanized at 18:00 hours by the time of LH and PRL surges. A 1:100 dilution was used for measuring PRL in blood samples from experiment six. For LH ELISA, the lower limit of detection was 0.07 ng/mL, and the intraassay and interassay coefficients of variation were 3.3% and 10.4%, respectively. Supplemental Fig. 2 shows a standard curve ranging from 0.01 to 2.5 ng/mL of rLH (r² = 0.998) and a dilution test of samples from male and OVX+E rats. Whole blood and plasma were collected from intact males euthanized between 10:00 and 12:00 hours, and the plasma of the OVX+E rat was the same used for PRL ELISA. A 1:20 dilution was used for measuring LH in whole blood samples.

Statistical analysis

Data are presented as the mean ± standard error of the mean (SEM). For hormonal data, comparisons between time points within the same experimental group were made by one-way analysis of variance (ANOVA) for repeated measures. The integrated hormonal responses were expressed as the area under curve with respect to increase over baseline (AUCi). Differences in the AUCi were determined by two-way ANOVA (experiment one), one-way ANOVA (experiment four), or Student t test (experiment six). For hormonal and neurochemical data, comparisons between groups were performed by two-way ANOVA. Immunofluorescence results were analyzed by one-way ANOVA. In all analyses, ANOVA was followed by the Bonferroni post hoc test. P < 0.05 was considered statistically significant.

Results

Effect of ER blockade on Kp stimulation of PRL release

Estradiol induces afternoon surges of PRL in OVX+E rats (8), and Kp-10 increases PRL secretion in an estradiol-dependent manner (15). We therefore used tamoxifen to investigate the participation of ER in Kp-induced increase in PRL release. The uterine weight in OVX+ET rats was significantly lower than that of OVX+E rats (174.2 ± 5.0 and 469.3 ± 33.3 mg/100 g body weight, respectively; P < 0.001, Student t test), as previously reported (9). Figure 1 shows the effect of ICV Kp-10 on PRL release in OVX+E and OVX+ET rats. Two-way-ANOVA revealed a significant effect of Kp-10 on OVX+E rats (P < 0.05), which was prevented by tamoxifen in OVX+ET rats (P = 0.27). Compared with –5 minutes and Veh, plasma PRL was significantly higher 5 minutes after treatment with Kp-10 [P < 0.05; Fig. 1(a)]. On the other hand, Kp-10 was unable to increase plasma PRL levels in OVX+ET rats at any time point evaluated [Fig. 1(b)]. The AUCi, reflecting the integrated secretory response of PRL, was significantly altered by Kp-10 (P < 0.05) and tamoxifen (P < 0.01), with significant interaction between them (P < 0.05). The AUCi of PRL was higher in OVX+E rats injected with Kp-10 compared with Veh (P < 0.05) and with OVX+ET rats injected with Kp-10 [Fig. 1(b)].

Figure 1.

Tamoxifen prevents the stimulatory effect of Kp-10 on PRL secretion. OVX rats were treated with SC estradiol (OVX+E; n = 5–6 per group) or estradiol plus tamoxifen (OVX+ET; n = 8 per group) daily for 3 days, and experiments were conducted on the fourth day. Between 10:00 and 12:00 hours, blood samples were withdrawn at –5, 5, 10, 20, 30, and 60 minutes, related to the ICV injection of 3 nmol Kp-10 or Veh. (a, b) Plasma PRL levels in (a) OVX+E and (b) OVX+ET rats. *P < 0.05 compared with –5 minutes in the same group; #P < 0.05 compared with Veh. Inset, AUCi of PRL levels. *P < 0.05 Veh vs. Kp-10; ##P < 0.01 compared with OVX+ET-Kp10. Data shown as mean ± SEM.

Effect of ER blockade on Kp inhibition of TIDA neurons

We have previously reported that Kp-10 reduces the dopaminergic activity in the ME in an estradiol-dependent manner (13). This experiment evaluated whether the ER blockade with tamoxifen would affect Kp-10 action on TIDA neurons. Figure 2 shows plasma PRL, DOPAC, dopamine, and DOPAC/dopamine ratio in the ME of OVX+E and OVX+ET rats injected with ICV Kp-10 or Veh. Confirming the findings in experiment one, Kp-10 increased plasma PRL in OVX+E (P < 0.05) but not in OVX+ET rats. As determined by two-way ANOVA, DOPAC levels in the ME were changed by Kp-10 (P < 0.05) but not tamoxifen, and there was a trend toward interaction between these two factors (P = 0.09). Accordingly, Kp-10 reduced DOPAC levels in OVX+E (P < 0.05), whereas it was ineffective in OVX+ET rats. There were no detectable effects of either Kp-10 or tamoxifen on dopamine levels and DOPAC/dopamine ratio in the ME.

Figure 2.

Tamoxifen prevents the inhibitory effect of Kp-10 on the activity of TIDA neurons. OVX rats were treated with estradiol (OVX+E; n = 6 per group) or estradiol and tamoxifen (OVX+ET; n = 4–8 per group) daily for 3 days, and experiments were conducted on the fourth day. Between 10:00 and 12:00 hours, rats were euthanized 10 minutes after ICV injection of 3 nmol Kp-10 or Veh. Trunk blood was collected for PRL measurements, and the ME was immediately dissected. Mean ± SEM levels of plasma (a) PRL, (b) DOPAC, (c) dopamine, and (d) DOPAC/dopamine ratio in the ME of OVX+E and OVX+ET rats treated with Kp-10 or Veh. *P < 0.05 Veh vs. Kp-10. #P < 0.05 compared with OVX+ET-Kp10.

Role of ER subtypes in Kp regulation of TIDA neurons and PRL

Because activation of ER was found to be necessary for Kp-10 action on TIDA neurons, we used selective agonists of ERα (PPT) and ERβ (DPN) to determine the ER subtype involved in Kp regulation of PRL. PPT treatment increased (P < 0.001), whereas DPN had no effect on uterine weight compared with OVX rats (OVX+PPT: 527.6 ± 61.8; OVX+DPN: 144.5 ± 21.3; OVX: 116.3 ± 10.1 mg/100 g body weight), consistent with previous reports (31). Figure 3 shows the effect of ICV Kp-10 on PRL levels and dopamine parameters in the ME of OVX, OVX+PPT, and OVX+DPN rats. As determined by two-way ANOVA, there were main effects for both Kp-10 (P < 0.01) and ER agonists (P < 0.05) on plasma PRL [Fig. 3(a)]. However, compared with Veh-treated rats, Kp-10 increased plasma PRL only in OVX+PPT rats (P < 0.05). DOPAC levels were higher in OVX+PPT than in OVX rats (P < 0.05) and even higher in OVX+DPN rats compared with the other groups (P < 0.05). Kp-10, in turn, reduced DOPAC levels in the ME of OVX+PPT rats (P < 0.05) but had no effect in OVX or OVX+DPN rats [Fig. 3(b)]. ME dopamine was significantly increased by DPN regardless of Kp-10 [Fig. 3(c)]. Similar to DOPAC, the DOPAC/dopamine ratio was higher in OVX+PPT and OVX+DPN rats compared with the OVX group (P < 0.05). However, the DOPAC/dopamine ratio was reduced by Kp-10 in both OVX+PPT and OVX+DPN rats (P < 0.05) as opposed to no change in OVX rats [Fig. 3(d)].

Figure 3.

Effects of Kp-10 on PRL and TIDA neuron activity require ERα activation. OVX rats were treated with PPT (OVX+PPT; n = 6–9 per group), DPN (OVX+DPN; n = 3–6 per group), or oil (OVX; n = 6–8 per group) daily for 3 days, and experiments were conducted on the fourth day. Between 10:00 and 12:00 hours, rats were euthanized 10 minutes after ICV injection of 3 nmol Kp-10 or Veh. Trunk blood was collected for PRL measurements, and the ME was dissected. Mean ± SEM levels of plasma (a) PRL, (b) DOPAC, (c) dopamine, and (d) DOPAC/dopamine ratio in the ME of OVX, OVX+PPT, and OVX+DPN rats treated with Kp-10 or Veh. *P < 0.05 Veh vs. Kp-10. ##P < 0.01 and #P < 0.05 compared with OVX for the same ICV treatment. ++P < 0.01 and +P < 0.05 compared with OVX and OVX+PPT for the same ICV treatment.

Effect of progesterone on Kp stimulation of PRL release

Figure 4 shows the effect of ICV Kp-10 on PRL release in OVX+EP rats exposed to high physiological levels of progesterone (30). OVX+E rats injected with Kp-10 from experiment one were used as a reference of the response of rats treated with estradiol only (gray line and column), with plasma and brain samples of this group being analyzed together in the same assays of RIA and immunofluorescence. Progesterone amplified the PRL increase induced by Kp-10, as demonstrated by the response of OVX+EP rats compared with OVX+E rats [Fig. 4(a)]. Accordingly, PRL levels were significantly increased at 5, 10, and 20 minutes after Kp-10 compared with –5 minutes and compared with OVX+EP rats treated with Veh (P < 0.05). Additionally, the AUCi of PRL in OVX+EP rats treated with Kp-10 was six times higher than in OVX+EP rats receiving Veh (P < 0.01) and two times higher than in OVX+E rats treated with Kp-10 [P < 0.05; Fig. 4(b)]. The phosphorylation of TH is a limiting step in the synthesis of dopamine, known to be modulated by progesterone (14). We therefore investigated the effect of Kp-10 on expression of S⁴⁰pTH and TH in the ME of OVX+E and OVX+EP rats by immunofluorescence (Fig. 5). Sixty minutes after ICV treatments, OVX+EP rats injected with Kp-10 displayed lower expression of S⁴⁰pTH in the ME, which was statistically significant compared with OVX+EP rats treated with Veh (P < 0.05) and tended to reach significance in comparison with OVX+E rats treated with Kp-10 (P = 0.08; Fig. 5(k)]. The expression of TH was also reduced in OVX+EP rats receiving Kp-10 compared with Veh [P < 0.05; Fig. 5(l)], and consequently the S⁴⁰pTH/TH in the ME did not differ between groups [Fig. 5(m)].

Figure 4.

Progesterone amplifies the Kp-10–induced increase in PRL secretion. OVX rats were treated with estradiol for 3 days and received an injection of progesterone at 08:00 hours on the fourth day (OVX+EP; n = 6 per group). Between 10:00 and 12:00 hours, blood samples were withdrawn at –5, 5, 10, 20, 30, and 60 minutes, related to the ICV injection of 3 nmol Kp-10 or Veh. OVX+E rats injected with Kp-10 from experiment one were used as reference of the response of rats treated with estradiol only (gray line and column; n = 6). (a) Plasma PRL levels. ***P < 0.001, **P < 0.01, and *P < 0.05 compared with –5 minutes in the OVX+EP-Kp10 group; #P < 0.05, ##P < 0.01 compared with OVX+EP+Veh. (b) AUCi of PRL levels. **P < 0.01 compared with OVX+EP-Veh and *P < 0.05 compared with OVX+E-Kp10.

Figure 5.

Kp-10 reduces expression of TH and S⁴⁰pTH in the ME of progesterone-treated rats. OVX rats were treated with estradiol for 3 days and received an injection of progesterone at 08:00 hours on the fourth day (OVX+EP). Between 10:00 and 12:00 hours, blood samples were withdrawn at –5, 5, 10, 20, 30, and 60 minutes, related to the ICV injection of 3 nmol Kp-10 or Veh. Rats were perfused after the last blood sample, and the brains were processed for immunofluorescence double labeling of S⁴⁰pTH and TH (n = 5 per group). OVX+E rats injected with Kp-10 (experiment one) were used as reference of the response of rats treated with estradiol only (gray column; n = 5). (a–d) Low-power photomicrographs of (a) S⁴⁰pTH and (b) TH double labeling in the ME, (c) overlapping images, and (d) omission of primary antibodies control in an OVX+EP+Veh rat. (e–j) Representative S⁴⁰pTH and TH double labeling in the ME of (e–g) OVX+EP-Veh and (h–j) OVX+EP-Kp10 rats. (k–m) IOD of S⁴⁰pTH and TH immunoreactivity in the ME, (k, l) expressed as percentage control, and (m) as the S⁴⁰pTH/TH ratio. *P < 0.05 OVX+EP-Veh vs. OVX+EP-Kp10. 3V, third ventricle. Scale bars, (a–d) 200 µm and (e–j) 100 µm.

Effect of progesterone on Kp inhibition of TIDA neurons

Next, we evaluated whether progesterone would alter the dopaminergic response to Kp and whether this effect could be blocked by RU486. Figure 6 shows the effects of ICV Kp-10 on PRL release and dopaminergic parameters in the ME of OVX+EP and OVX+EP+RU rats. Kp-10 increased PRL levels in OVX+EP rats (P < 0.05), whereas this response was abolished in OVX+EP+RU rats [Fig. 6(a)]. Nevertheless, Kp-10 lowered DOPAC levels equally in OVX+EP and OVX+EP+RU rats [P < 0.05; Fig. 6(b)]. On the other hand, the effect of Kp-10 on dopamine was associated with the PRL response, because Kp-10 decreased dopamine levels in OVX+EP (P < 0.05) but not in OVX+EP+RU rats. In addition, regarding Veh-treated rats, dopamine was reduced (P < 0.05) and DOPAC/dopamine ratio increased (P < 0.01) in OVX+EP+RU rats compared with OVX+EP [Fig. 6(c) and 6(d)]. Interestingly, this Kp-10–induced suppression of ME dopamine is progesterone dependent, because it was not found in OVX+E [Fig. 2(c)] or OVX+PPT [Fig. 3(c)] rats. And it seems to reflect a decrease in dopamine biosynthesis, which is corroborated by the effect of Kp-10 in reducing the expression of S⁴⁰pTH and TH in the ME of OVX+EP rats (Fig. 5).

Figure 6.

Kp-10 inhibition of ME dopamine requires activation of progesterone receptor. OVX rats were treated with estradiol for 3 days. On the fourth day, rats were treated with RU486 or oil at 07:00 hours, followed by an injection of progesterone (OVX+EP+RU, n = 4–5 per group; OVX+EP, n = 6 per group) at 08:00 hours. Between 10:00 and 12:00 hours, rats were euthanized 10 minutes after ICV injection of 3 nmol Kp-10 or Veh. Trunk blood was collected for PRL measurements, and the ME was dissected. Mean ± SEM levels of plasma (a) PRL, (b) DOPAC, (c) dopamine, and (d) DOPAC/dopamine ratio in the ME of OVX+EP and OVX+EP+RU rats treated with Kp-10 or Veh. *P < 0.05 Veh vs. Kp-10. ##P < 0.01 and #P < 0.05 compared with OVX+EP-Veh.

Effect of Kp-234 on the Estradiol-Induced Surges of PRL and LH

Because our data showed that Kp stimulation of PRL release depends on ERα and is modulated by PR, we determined the effect of Kp-234 on the generation of PRL and LH surges in the positive feedback model of OVX+E rats (Fig. 7). As determined by two-way ANOVA, PRL levels in OVX+E rats changed as a function of time (P < 0.001) and Kp-234 (P < 0.001), with significant interaction between these two factors (P < 0.05). Both Veh- and Kp-234–treated OVX+E rats displayed afternoon increases in PRL secretion. However, PRL levels were higher from 14:30 to 16:00 hours in Veh rats compared with those injected with Kp-234 (P < 0.05). Interestingly, PRL levels displayed a sharp increase at 14:30 hours and remained elevated until 18:00 hours in Veh rats (P < 0.01), whereas the increase was slow and gradual in Kp-234 rats, being significantly higher at 16:30–18:00 hours [P < 0.01; Fig. 7(a)]. The AUCi of PRL was ~55% lower in Kp-234–treated rats [P < 0.01; Fig. 7(b)]. LH levels in OVX+E rats also changed as a function of time (P < 0.01) and Kp-234 (P < 0.001) but with no interaction between these two factors. OVX+E rats receiving Veh displayed a significant increase in LH levels at 17:30–18:00 hours (P < 0.05), whereas the LH surge was completely absent in Kp-234–treated rats [Fig. 7(c)]. The AUCi of LH was suppressed in ~80% by Kp-234 [P < 0.05; Fig. 7(d)].

Figure 7.

Kp-234 impairs the initial phase of the PRL surge and prevents the LH surge in OVX+E rats. OVX rats were treated with estradiol for 3 days. On the fourth day, rats received three ICV injections of Kp-234 (n = 6) or Veh (Veh; n = 6) at 10:00, 12:00, and 14:00 hours. Tail-tip blood samples were withdrawn at 30-minute intervals from 13:00 hours to 18:00 hours for PRL and LH measurement in whole blood by ELISA. (a) PRL levels. ***P < 0.001, **P < 0.01 compared with 13:00 hours in OVX+E-Veh (14:30–18:00 hours) and OVX+E-Kp234 (16:30–18:00 hours) rats; #P < 0.05 compared with OVX+E-Kp234. (b) AUCi of PRL levels. *P < 0.05 compared with OVX+E-Kp234. (c) LH levels. **P < 0.01, *P < 0.05 compared with 13:00 hours in OVX+E-Veh rats; ##P < 0.01 and #P < 0.05 compared with OVX+E-Kp234. (d) AUCi of LH levels. *P < 0.05 compared with OVX+E-Kp234. Data shown as mean ± SEM.

Discussion

The current study reports findings on the essential role played by the ovarian steroids and their receptors in the Kp regulation of PRL secretion, with possible implication to a Kp effect on the estradiol-induced PRL surge. Kp-10 injected ICV increased plasma PRL and reduced DOPAC levels in the ME of OVX+E rats, and these effects were abolished by the ER blockade with tamoxifen. Accordingly, effects of Kp-10 were absent in OVX rats and fully restored by the selective activation of ERα with PPT. Conversely, ERβ activation in OVX+DPN rats was ineffective in restoring PRL responsiveness to Kp-10. Our results also revealed that progesterone treatment in OVX+EP rats doubled the PRL response to Kp-10, and this response was associated with a Kp-10–induced reduction of S⁴⁰pTH and TH expression in the ME. Consistent with the inhibition of TH, Kp-10 also reduced dopamine and DOPAC levels in the ME of OVX+EP rats, which was prevented by PR blockade with RU486. It seems, therefore, that activation of ERα is necessary, whereas PR amplifies the effects of Kp on TIDA neurons and PRL secretion in female rats. Moreover, Kp-234 attenuated the early peak phase of the PRL surge and blocked the LH surge in OVX+E rats, revealing a role for Kp in the estradiol-induced PRL surge.

We have previously described that Kp fibers are in close apposition to TIDA neuron perikarya in female rats (15). These projections have also been reported to be synaptic inputs negatively regulated by estradiol (16). Moreover, ICV injections of Kp-10 have been found to suppress dopamine release in the ME and to increase PRL secretion in vivo, whereas there was no effect of Kp-10 on PRL released from pituitary cells in vitro (15). In this previous study, as in the experiments here reported, DOPAC levels in the ME were taken as an index of dopamine release (24). Of note, the Kp effect on PRL requires higher dosages than those for LH stimulation and appears to be markedly dependent on estrogen (13, 15). Taking these findings together, it seems that this regulation takes place in the brain and is tuned to work under high levels of estradiol, which may explain why Kp effects on PRL were not detected in circumstances of systemic administration or low estrogen background (40,–42). Moreover, Kp neurons have been shown to express PRL receptors (43), and high PRL levels suppress Kiss1 mRNA and peptide levels in the AVPV and ARC of rodents (23, 44, 45). Thus, Kp regulation of PRL axis seems to involve, on one hand, the Kp stimulation of PRL through inhibition of TIDA neurons and, on the other hand, a negative feedback effect of PRL on Kp neurons, revealing a mechanism that theoretically can result in autoregulation of PRL release and suppression of LH secretion. Thus, the interaction between PRL and Kp may exert substantial impacts on fertility and reproduction that are still not completely understood.

We have recently reported that tamoxifen acts as an ER antagonist with respect to the hypothalamic effects of estradiol on the dopamine–PRL axis, resulting in the blockade of the estradiol-induced PRL surge (9). The present results show that tamoxifen also prevents the effect of Kp-10 on TIDA neurons and PRL secretion. This confirms our previous report of a lack of effect of Kp-10 on OVX rats (13) and demonstrates that ER activation is critical for Kp stimulation of PRL. Moreover, as a selective ER modulator tamoxifen has mixed estrogenic and antiestrogenic effects and binds to both ERα and ERβ (46), which raises the question of the specific roles played by ER subtypes in this regulatory pathway. In line with this idea, PRL and dopamine responses to Kp-10 were fully restored in OVX rats treated with the selective agonist of ERα, PPT, but not of ERβ, DPN. Thus, these results suggest that activation of ERα is necessary and sufficient to make the dopamine–PRL axis responsive to Kp. This is in agreement with the predominant expression of ERα over ERβ in both Kp (47) and TIDA (12, 48) neurons. Nevertheless, OVX+DPN rats displayed higher DOPAC and dopamine levels in the ME and responded to Kp-10 with a reduction in the DOPAC/dopamine ratio, but this effect did not result in significant changes in PRL secretion. Thus, activation of ERβ also interferes in the activity of TIDA neurons and seems to render them responsive to the action of Kp-10, although these effects are not linked to an increase in PRL secretion. With regard to the Kp regulation of gonadotropin axis, LH response to Kp-10 has been shown to be potentiated by ERα and negatively modulated by ERβ (49), whereas follicle-stimulating hormone response is stimulated by both ER subtypes (50). Thus, the roles played by ERs in the Kp effect on PRL differ from those in gonadotropin regulation, reflecting the distinct nature of the neuroendocrine systems involved.

The mechanisms whereby estradiol and ERα activation make TIDA neurons responsive to Kp remain unclear. Conceivably, estradiol might alter expression of Kiss1r in TIDA neurons or modulate intracellular pathways associated with Kp signaling. Although controversies remain about the presence of Kiss1r in the ARC of mice (51), different laboratories have reported expression of Kiss1r in the ARC of rats (52, 53). Indeed, in a recent collaboration study of double in situ hybridization, we also identified Kiss1r-mRNA containing neurons in the ARC of female rats. However, TH mRNA-expressing neurons were not found to coexpress Kiss1r in the ARC, periventricular hypothalamic nucleus, or AVPV (54). This finding suggests at least two possibilities for the Kp regulation of TIDA neurons. One possibility is an indirect action through neuronal afferents to TIDA neurons. For instance, oxytocin neurons could fulfill this role. Oxytocin has been shown to be regulated by Kp and to modulate the activity TIDA neurons as well (53, 55, 56). On the other hand, in line with its synaptic inputs onto TIDA neurons, Kp might act via other receptors to directly regulate the activity of these cells. In fact, the inhibitory effects of Kp on dopamine contrast with the well-described stimulatory effects of Kiss1r on gonadotropin-releasing hormone neurons (57). Studies in cell lines expressing different RFamide receptors have shown that Kp binds with high affinity and activates neuropeptide FF (NPFF) receptors, which are Gi/o protein-coupled receptors and therefore could account for the inhibitory effects of Kp (58). Accordingly, a recent electrophysiology study has shown both stimulatory and inhibitory effects of Kp-10 on the excitability of unidentified ARC neurons. Interestingly, these effects were similar in wild-type and G protein–coupled receptor 54 knockout mice and could be mimicked by the agonist of NPFF receptor, RFamide-related peptide 3 (59). In fact, in our double insitu hybridization evaluation in the female rat brain, there was marked expression of the NPFF1 receptor in discrete hypothalamic nuclei including the ARC. However, neurons double-labeled to TH and NPFF1 were found to be very few in the ARC, as opposed to a high level of coexpression in the periventricular hypothalamic nucleus (54). This suggests that a Kp action via NPFF1 receptor is more likely to take place on the periventricular population of neuroendocrine dopaminergic neurons rather than on TIDA neurons (5). Overall, the mode of action of Kp on dopaminergic neurons remains an open question and reflects a neuroendocrine system that begins to be unraveled. Nevertheless, the strong estrogen dependency is key feature that should be taken into consideration when investigating possible receptors and pathways involved in the Kp regulation of TIDA neurons.

Progesterone has long been shown to modulate the estradiol-induced increase in PRL secretion. The day of proestrus in rats is characterized by higher circulating levels of estradiol and progesterone, which generate the preovulatory surges of LH and PRL (60). On the afternoon of proestrus and in the positive feedback model of OVX+E rats, progesterone anticipates and amplifies the estradiol-induced surge of PRL (8, 25, 61). By the time of the PRL surge, estradiol elicits an afternoon decrease in the inhibitory dopaminergic tonus, and progesterone anticipates this response, as demonstrated by the reduction in dopamine activity and TH phosphorylation in the ME (10, 14). In line with this action of progesterone, the present results showed that progesterone is also able to amplify and prolong the Kp-10–induced increase in PRL release. Moreover, this magnification was associated with inhibitory effects of Kp-10 on both TH and S⁴⁰pTH expression and dopamine content in the ME OVX+EP rats, which was not found in OVX+E rats. Thus, our findings suggest that in the presence of progesterone, Kp reduces not only dopamine release (reflected by lower DOPAC levels) but also dopamine biosynthesis, which is probably responsible for the greater response of PRL in OVX+EP rats. Our double-labeled immunofluorescence showed no change in the S⁴⁰pTH/TH ratio, indicating no effect of Kp-10 on the phosphorylation state of TH but rather on the content of phosphorylated TH in the ME. However, because rats were perfused at 60 minutes after the ICV injections, we cannot exclude the possibility that a decrease in the rate of TH phosphorylation may have occurred concurrently with the suppression in ME dopamine found at 10 minutes after Kp-10 injection. Furthermore, our results also showed that the cotreatment with RU486 in OVX+EP rats abolished the effects of Kp-10 on PRL release and dopamine levels in the ME, suggesting that progesterone modulation of TIDA neuron and PRL responses to Kp requires PR activation. This finding is in agreement with the expression of PR in TIDA neurons, which is greatly influenced by estradiol (62), and with previous evidence that RU486 prevents the facilitatory effects of progesterone on the estradiol-induced PRL surge (10).

The roles played by ERα and PR in Kp stimulation of PRL suggest that Kp may be involved in estradiol-induced PRL secretion. We investigated this question by adapting to the rat a method of tail-tip blood sampling combined with ultrasensitive ELISAs for PRL and LH, previously described in mice (27, 28). The ELISA assays reliably detected rat PRL and LH in reference preparations and samples containing different endogenous levels (Supplemental Figs. 1 and 2). The surges of PRL and LH reported here were compatible with those found in our laboratory when we used plasma samples derived from jugular vein catheters measured by RIA (9), evidencing the efficacy of the present method. Moreover, this method has the advantages of avoiding surgery and requiring very small blood volume. The proestrous PRL surge is composed of an early peak phase followed by a plateau (61). Our results showed that, in the model of OVX+E rats, ICV Kp-234 impaired the early peak but did not interfere in the late phase of the PRL surge. Although a shift in the surge time cannot be excluded, it is unlikely because the regular shape of the PRL surge was altered by Kp-234, with a marked attenuation in the early sharp increase. Interestingly, a similar effect has been reported for posterior pituitary lobectomy (63). Thus, our finding suggests a role for Kp-Kiss1r signaling in the estradiol-induced PRL surge. It is of interest to determine in future studies whether this effect involves a dopaminergic or nondopaminergic mechanism. Additionally, Kp-234 completely prevented the LH surge in OVX+E rats. This description of blockade of the estradiol-induced LH surge by the Kp antagonist adds information to the already described inhibition of LH pulsatility and postcastration rise (26). Moreover, these results confirm the efficacy of the ICV Kp-234 treatment used and provide further evidence of the key role of Kp–Kiss1r signaling in the positive feedback effect of estradiol (17, 57).

The current study provides evidence on the interaction between ovarian steroids and Kp in the control of PRL secretion. These findings should guide futures studies aiming to elucidate the neuroanatomy and physiology of the interaction between Kp and the PRL axis and the impact of this neuroendocrine system in female reproduction.

Appendix.

Antibody Table

Peptide/Protein Target	Name of Antibody	Manufacturer, Catalog #, or Name of Person Providing the Antibody	Species Raised in; Monoclonal or Polyclonal	Dilution Used	RRID
TH	Monoclonal anti–tyrosine hydroxylase antibody produced in mouse	Sigma-Aldrich, T1299	Mouse; monoclonal	1:10,000	AB_477560
TH phosphorylated at Ser40	Rabbit antiphospho-TH (pS40)	Invitrogen, 36-8600	Rabbit; polyclonal	1:10,000	AB_138590
Rabbit IgG	Donkey anti-rabbit IgG (H+L) secondary antibody, Alexa Fluor® 488	Invitrogen, A-21206	Donkey; polyclonal	1:1000	AB_141708
Mouse IgG	Goat anti-mouse IgG (H+L) secondary antibody, Alexa Fluor 568	Invitrogen, A-11004	Goat; polyclonal	1:1000	AB_141371

Abbreviation: RRID, research resource identifier.

Abbreviations:

 
• ANOVA

  analysis of variance

 
• ARC

  arcuate nucleus

 
• AUCi

  area under curve with respect to increase over baseline

 
• AVPV

  anteroventral periventricular nucleus

 
• DOPAC

  3,4-dihydroxyphenylacetic acid

 
• DPN

  diarylpropionitrile

 
• ELISA

  enzyme-linked immunosorbent assay

 
• ER

  estrogen receptor

 
• HPLC-ED

  high-performance liquid chromatography with electrochemical detection

 
• ICV

  intracerebroventricular

 
• IgG

  immunoglobulin G

 
• IOD

  integrated optical density

 
• Kp

  kisspeptin

 
• LH

  luteinizing hormone

 
• ME

  median eminence

 
• mRNA

  messenger RNA

 
• NIDDK-NHPP

  National Institute of Diabetes and Digestive and Kidney Diseases–National Hormone and Pituitary Program

 
• NPFF

  neuropeptide FF

 
• OVX

  ovariectomized

 
• OVX+E

  ovariectomized rats treated with estradiol

 
• OVX+EP

  ovariectomized rats treated with estradiol and progesterone

 
• PBS

  phosphate-buffered saline

 
• PPT

  propylpyrazole triol

 
• PR

  progesterone receptor

 
• PRL

  prolactin

 
• RIA

  radioimmunoassay

 
• rLH

  rat luteinizing hormone

 
• rPRL

  rat prolactin

 
• RT

  room temperature

 
• S⁴⁰pTH

  Ser⁴⁰-phosphorylated tyrosine hydroxylase

 
• SC

  subcutaneous(ly)

 
• SEM

  standard error of the mean

 
• TH

  tyrosine hydroxylase

 
• TIDA

  tuberoinfundibular dopaminergic

 
• Veh

  vehicle.

Acknowledgments

We thank Simone F. Pio (Universidade Federal de Minas Gerais, Belo Horizonte, Brazil) for technical support in the laboratory. We also thank Dr. Agnès O. Martin (Institut de Génomique Fonctionnelle, Montpellier, France) for the generous gift of the rabbit anti-mouse PRL antibody (F. Talamantes).

This work was supported by Fundação de Amparo à Pesquisa do Estado de Minas Gerais, Conselho Nacional de Desenvolvimento Científico e Tecnológico and Fundação de Amparo à Pesquisa do Estado de São Paulo.

Disclosure Summary: The authors have nothing to disclose.

Author notes

Address all correspondence and requests for reprints to: Raphael E. Szawka, PhD, Departamento de Fisiologia e Biofísica, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Avenida Antônio Carlos, 6627, 31270-901, Belo Horizonte, MG, Brazil. E-mail: [email protected].