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BOR - Papers in Press, published online ahead of print May 25, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.041624
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BIOLOGY OF REPRODUCTION 73, 491–499 (2005)
DOI: 10.1095/biolreprod.105.041624
© 2005 by the Society for the Study of Reproduction, Inc.

Pituitary Adenylate Cyclase Activating Polypeptide Messenger RNA in the Paraventricular Nucleus and Anterior Pituitary During the Rat Estrous Cycle1

Joseph P. Moore, Jr 2, 3, Laura L. Burger 4, Alan C. Dalkin 4, and Stephen J. Winters 3

Division of Endocrinology and Metabolism,3 Department of Medicine, University of Louisville, Louisville, Kentucky 40202 Department of Internal Medicine,4 University of Virginia, Charlottesville, Virginia 22908


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The neuropeptide pituitary adenylate cyclase activating polypeptide (ADCYAP 1, or PACAP) has been demonstrated to enhance gonadotropin-releasing hormone (GnRH)-induced gonadotropin secretion and regulate gonadotropin subunit gene expression in cultures of anterior pituitary cells. In the present study, we used in situ hybridization and real-time polymerase chain reaction to examine the expression of Pacap mRNA within the paraventricular nucleus (PVN) and anterior pituitary throughout the estrous cycle of the rat. Levels of luteinizing hormone in serum and pituitary gonadotropin subunit mRNAs were evaluated and displayed cyclic fluctuations similar to those reported previously. Pacap mRNA expression in the PVN and pituitary varied significantly during the estrous cycle, with the greatest changes occurring on the day of proestrus. Pacap mRNA levels in the PVN declined significantly on the morning of diestrus. During proestrus, PVN Pacap mRNA levels significantly increased 3 h before the gonadotropin surge and then declined. Pituitary expression of Pacap mRNA also varied on the afternoon of proestrus with a moderate decline at the time of the gonadotropin surge and a significant increase later in the evening. Expression of the mRNA species encoding the 288 amino acid form of follistatin increased significantly following the rise in pituitary Pacap mRNA, at the termination of the secondary surge in follicle-stimulating hormone beta (Fshb) gene expression. These results suggest that PACAP is involved in events before and following the gonadotropin surge, perhaps through increased gonadotroph sensitivity to GnRH and suppression of Fshb subunit expression through increased follistatin, as previously observed in vitro.

anterior pituitary, estrous cycle, follicle-stimulating hormone, gonadotropin, luteinizing hormone, neuroendocrinology, PACAP, pituitary hormones, PVN


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Pituitary adenylate cyclase activating polypeptide (ADCYAP 1, or PACAP) is a ubiquitously expressed neuropeptide that was originally isolated from preparations of ovine hypothalami and was named for its ability to stimulate cAMP production in rat anterior pituitary cells [1]. Neurons containing Pacap mRNA are widely distributed throughout the central nervous system with the highest concentration within the diencephalon in the lateral habenular nucleus, the paraventricular nucleus (PVN), the preoptic nucleus (POA), and the ventromedial hypothalamic nucleus [2]. Many PACAP immunoreactive nerve terminals are found within the median eminence, and the plasma concentration of PACAP in the pituitary stalk in rats is 2- to 4-fold higher than in the peripheral circulation [3, 4]. Binding studies identified PACAP-specific receptors on anterior pituitary cell membranes of rats [5], and experiments using biotinylated PACAP ligand localized PACAP receptor binding to each anterior pituitary cell type [6]. These data suggest that PACAP has a role as a hypophysiotropic factor.

There is strong evidence that PACAP plays a role in the synthesis and secretion of the gonadotropins. In primary cultures of rat pituitary cells, PACAP stimulates the activation of both adenylate cyclase and phospholipase C [7, 8] and stimulates the secretion of each of the gonadotropins in a dose-dependent manner [9–11]. In vitro, PACAP stimulates transcription of gonadotropin glycoprotein hormones and alpha subunit (Cga, or a-subunit) mRNA, lengthens luteinizing hormone beta (Lhb) mRNA and presumably prolongs its half-life, but reduces follicle-stimulating hormone beta (Fshb) mRNA levels [3]. The decreased levels of Fshb mRNA are likely due to decreased activin stimulation of Fshb transcription, as PACAP significantly increases pituitary expression of the potent activin sequestering peptide follistatin [3, 12]. PACAP also enhances GnRH-stimulated gonadotropin secretion from cultures of primary rat pituitaries as well as from {alpha}T3 cells [3, 8, 10]. This may be partly due to stimulation of GnRH receptors [13, 14] or to increasing the number of gonadotrophs that respond to the GnRH signal [15], among other mechanisms. Thus, PACAP may have an in vivo role in gonadotropin regulation by acting synergistically with GnRH to stimulate gonadotropin secretion and regulate the expression of the gonadotropin subunits; however, studies demonstrating modulation of PACAP signaling to the pituitary under physiological conditions in vivo have been, for the most part, inconclusive.

Systemic administration of PACAP to conscious male rats significantly increased circulating levels of LH [16, 17], while PACAP injection into the lateral ventricle of the rat brain produced little or no rise in circulating LH [16]. These variable effects suggest that PACAP acts directly on the pituitary gland in vivo to stimulate LH release. On the other hand, administration of the full-length PACAP (1–38) peptide into the third ventricle on the afternoon of proestrus abolished the ensuing LH surge [18, 19], while the same treatment using the naturally occurring, truncated PACAP (1–27) peptide enhanced the LH surge [18]. Systemic administration of PACAP before the estrous surge had no enhancing or detrimental effects on LH secretion or subsequent ovulation [20].

Studies have shown that steroids influence PACAP expression in various tissues. In preovulatory ovarian follicles, progesterone stimulates PACAP synthesis [21–23]. In ovariectomized mice and rats, progesterone increased PACAP peptide and mRNA levels, respectively, in the medial basal hypothalamus (MBH) [24, 25]. Progesterone and estrogen both increased Pacap mRNA expression within the ventromedial nucleus of ovariectomized rats. A preliminary report in cycling female rats suggested that expression of Pacap mRNA in the anterior pituitary is increased on the afternoon of proestrus (W. Wuttke, abstract, Neuroendocrinology 1994; 60[suppl 1]:17). Furthermore, Pacap mRNA expression in the MBH and preoptic area declines several days before the first ovulation in prepubertal rats and subsequently increases in the MBH on the first day of proestrus [26]. These results suggest that changing concentrations of circulating steroid hormones during the reproductive cycle may regulate PACAP expression. However, there have been no published reports relating PACAP expression within the hypothalamus or pituitary to the events of the natural estrous cycle in mammals.

The present experiments were performed to characterize changes in Pacap mRNA expression within the hypothalamus and anterior pituitary gland during the estrous cycle in rats. We reasoned that if PACAP enhances responsiveness to GnRH or plays a role in the differential regulation of Fshb mRNA through follistatin, a rise in PACAP expression should precede these events in the estrous cycle. In a pilot investigation, we observed a significant increase in Pacap mRNA expression in the paraventricular nucleus (PVN) following ovariectomy in rats (85th annual meeting of the Endocrine Society, Philadelphia, PA, June 19–22, 2003, abstract P2-647). In addition, we documented a reciprocal relationship between pituitary Fshb expression levels and Pacap mRNA expression within the PVN of maturing male rats [27]. Therefore, we chose to examine Pacap mRNA expression in the PVN of cycling rats by employing in situ hybridization. Dynamic changes in expression levels of the gonadotropin subunits and pituitary follistatin were evaluated and compared to the expression of PACAP mRNA within the PVN and anterior pituitary gland.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Female Sprague-Dawley rats (8 wk old), obtained from Charles River Laboratories (Wilmington, MA), were maintained in a temperature-controlled environment with a 14L:10D photoperiod (0400–1800 lights-on) and provided laboratory chow and water ad libitum. All experiments were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals according to a protocol approved by the Animal Care and Use Committee of the University of Louisville. Female Sprague-Dawley rats were monitored daily for stage of the estrous cycle by vaginal cytology. Animals were studied further only if two consecutive 4-day cycles were documented. Groups of cycling female rats (six per group) were killed at 0900 h on each day of the cycle and at 1200, 1500, 1800, 2100, and 2400 h on the day of proestrus. Animal subjects were decapitated after lethal exposure to carbon dioxide, and trunk blood was collected. The pituitaries were harvested and rapidly frozen in RNA extraction buffer, and the brains were removed and rapidly frozen in mounting medium on ethanol/dry-ice slurry. The pituitaries and brains were then stored at –80°C.

Immunoassays

An enzyme immunoassay developed by Amersham Pharmacia Biotech (Peapack, NJ) was used to measure LH protein in serum. The within-assay coefficient of variation of replicates in the standard curve was <7.6%. The range of the assay standards was 0.41–100 ng/ml.

RNA Extraction and Northern Analysis

RNA was extracted using the Perfect RNA total RNA Isolation kit (5 Prime-3 Prime, Boulder, CO). The concentration of total RNA was determined by reading the optical density at 260 nm. Sample purity was determined by calculating the ratio of sample absorbance at 260:280 nm, and samples were rejected if the ratio was less than 1.8. For Northern analysis, aliquots of pituitary RNA samples were subjected to electrophoresis on 1.2% agarose-formaldehyde gels. The RNAs were transferred to Nytran membranes (Schleicher & Schuell, Keene, NH) and cross-linked to the membranes by baking for 2 h at 80–90°C, followed by irradiation for 2 min with ultraviolet light. Purified cDNAs for rat Fshb (Dr. Richard Maurer, Oregon Health Sciences University, Portland, OR) and Lhb (Dr. James Roberts, Mount Sinai School of Medicine, New York, NY) were labeled by the random primer method with [32P]dCTP (3000 Ci/mmol; New England Nuclear Research Products, Boston, MA) to a specific activity of 6–8 x 108 dpm/µg, as described previously [27]. Labeled probes were added to the hybridization solutions at a concentration of approximately 5 ng/ml for 48–72 h. Membranes were washed, autoradiographed, and analyzed using a Bio-Rad GS-700 Imaging Densitometer (Hercules, CA). Membranes were rehybridized without stripping for normalization to cyclophilin cDNA.

In Situ Hybridization

Preparation of tissues Cryostat-cut, fresh-frozen coronal sections (thickness, 14 µm) were saved at levels from the optic chiasm to the mamillary bodies. Sections were thaw-mounted onto chrom-alum/gelatin subbed slides, dried, and stored at –80°C before processing. Tissue sections were fixed by immersion in 4% paraformaldehyde in 0.1 M PBS (pH 7.2) for 5 min and then rinsed with 0.1 M PBS for 3 min. Slides were then immersed for 10 min in a solution of 0.25% acetic anhydride containing 0.1 M triethanolamine (pH 8.0). Sections were rinsed with 0.2x SSC (30 mM sodium chloride and 3 mM sodium citrate) for 10 min, dehydrated through a graded series of ethanols, and dried in a desiccator.

Preparation of radiolabeled probes Pacap mRNA in the PVN was detected using a 33P-labeled riboprobe [27]. A 670-base pair (bp) Pacap cDNA subcloned into pGEM-3Zf(–) was received from Dr. A. Arimura (Tulane University, Belle Chasse, LA). The plasmid was linearized with BamH1 and transcribed with T7 RNA polymerase using the MAXIscript kit (Ambion, Austin, TX) to produce a 670-bp, 33P-labeled antisense riboprobe. Labeling was accomplished by adding [{alpha}-33P] uridine 5'-triphosphate to produce probes with a specific activity of approximately 2 x 107 cpm/pmol. A sense probe was used to document background hybridization.

Hybridization Groups of slides for hybridization were from six animals per group, with three slides from each animal containing three 14-µm sections. The specific slides for each animal correspond to 1) 42–84 µm posterior to the optic chiasm, 2) 28–56 µm anterior to the start of the arcuate nucleus, and 3) the slide containing sections midway between the first two. Matched sections through the hypothalamus were hybridized with 100 µl of heat-denatured hybridization buffer containing 0.2 pmol of sense or antisense probe pipetted onto each section at 55°C for 16 h. Following hybridization, sections were rinsed with 2x SSC for 30 min and then incubated with 5 µg/ml of ribonuclease A (RNase A, Sigma type X-A; Sigma, St. Louis, MO) in RNase buffer (10 mM Tris, 0.5 M NaCl, and 1 mM EDTA, pH 8.0) for 30 min at 37°C followed by a 30-min rinse with RNase buffer. Sections were then rinsed with 0.1x SSC at 60°C for 1 h, dehydrated through ethanol, and dried in a dessicator.

Slides were then dipped into Kodak NTB-3 emulsion at 44°C and stored in light-tight boxes at 4°C. Slides were developed after 21–28 days with Kodak D-19 developer for 4 min at 15°C, fixed with Kodak fixer for 6 min, stained with hematoxylin, coverslipped with DePeX (Gurr-BDH Chemicals Ltd, Poole, UK), and examined with a Nikon photomicroscope (Nikon Corporation, Tokyo, Japan).

Analysis Pacap labeling within the PVN was quantified at the population and single-cell levels. Digital images of the PVN on each section were produced using a Nikon microscope and image-analysis software. Pacap labeling in the PVN was evaluated by analyzing the optical densities of the areas corresponding to the PVN in each section using Scion Image analysis software (Scion Corp., Frederick, MD). Numbers of Pacap mRNA-containing cells were determined by counting all labeled cells in each of three sections per animal. Individual labeled cells were identified under high power using bright-field microscopy. Only labeled cells with an identifiable nucleus were included in the analysis. The number of pixels covered by reduced silver grains overlying each labeled cell was counted for all labeled cells in each of three regions per PVN per animal using a Scion Image analysis system. Extreme care was taken to ensure that lighting was constant for all sections analyzed within a given series. Background labeling per unit area was estimated for each section by averaging the number of pixels covered by silver grains in each of 10 fields without labeled cells across the cortex. Background was calculated and subtracted from each cell. Only those cells for which the number of overlying pixels exceeded the background by 4 SD were considered labeled and were included in the analysis. In this way, the sections served as their own internal controls.

Measurement of Pituitary Follistatin mRNA

Quantitative reverse transcriptase-polymerase chain reaction A previously described competitive template quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assay was used to measure total follistatin (Fst) mRNA levels [28]. Fst cDNA (Dr. Kelly Mayo, Northwestern University, Evanston, IL) was size-altered by substituting a 163-bp fragment of unrelated DNA for a 72-bp SplI/AccIII segment to create a competitive template that was used as an internal standard. The method therefore allows the same oligonucleotide primers to be used to amplify the native and competitive template cDNAs. Samples from each experimental group were analyzed simultaneously.

Real-Time PCR for mRNA Encoding 288 Amino Acid Form of Follistatin

Follistatin mRNA is subject to alternative splicing resulting in two peptides consisting of 314 or 288 amino acids. We evaluated expression of the mRNA species encoding for the 288 amino acid form of FST (Fst-288, or Fst-344) that encodes for a precursor peptide referred to as follistatin 344 (FST-344). FST-288 binds heparin sulfate proteoglycans as well as activin and has a 10-fold greater FSH-suppressing activity than FST-315 [29]. We have shown previously that expression of Fst-288 varies independently of total Fst mRNA during sexual maturation [27].

Construction of cRNA standard curves For quantitative real-time PCR (qRT-PCR), a standard curve of known amounts of Fst-288 mRNA was prepared. Briefly, Fst-288 specific primers (forward-5'-GAGGCCCAAAAGACAAAACA-3') and (reverse-5'-ATGGGGGAATACAGGGAGAG-3') were synthesized with and without a T7-promoter sequence (5'-GGATCCTAATACGACTCACTATAGGGAGG-3') at the 5' end of the forward primer and with an oligo-dT(T12) at the 5' end of the reverse primer. PCR was performed to produce cDNA containing the T7 promoter sequence. The PCR product (1 µg) was then used as template in an in vitro transcription reaction (MAXIscript T7 kit; Ambion, Austin, TX). The subsequent cRNA was quantified using a spectrophotometer and serial diluted to make a standard of known starting material containing 1011–104 molecules cRNA. These standards were processed in parallel with experimental RNA samples for quantitative real-time PCR using the primers lacking the T7 and dT sequences. The same technique was used for analysis of PACAP mRNA within the anterior pituitary gland. The specific primers used were (forward-5'-CCTACCGCAAAGTCTTGGAC-3') and (reverse-5'-TTGACAGCCATTTGTTTTCG-3').

Quantitative real-time PCR RNA (2 µg) isolated from pituitary samples was reverse transcribed in parallel with cRNA standards using an oligo dT(12–24) as the primer. Reverse-transcribed cRNA standards and samples were amplified in parallel by PCR on a Stratagene MX4000 Multiplex Quantitative PCR System (Stratagene, La Jolla, CA) using the Brilliant SYBR Green QPCR Master Mix (Stratagene) and the primers designed for use in the cRNA standard preparation but without the T7 and oligo-dT sequences. Accumulation of PCR product was monitored in real time (Mx4000; Stratagene), and the crossing threshold (Ct) was determined using the Mx4000 software. For each set of primers, a no-template control and a no-reverse amplification control were included. Postamplification dissociation curves were performed to verify the presence of a single amplification product in the absence of DNA contamination. Concentrations of mRNAs were determined using the change in crossover time method with normalization to glyceraldehyde-3-phosphate dehydrogenase mRNA and interpolation using the standard curve of known starting mRNA concentrations.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Verification of Estrous Cycles in Female Rats

Serum levels of LH were measured to verify the stage of the estrous cycle. As depicted in Figure 1, the female rats studied demonstrated a significant surge in LH concentrations at 1500 h on the day of proestrus. These results are consistent with those of other investigations in which cycling female rats were maintained on a similar light:dark schedule with a peak in LH occurring between 3 and 2 h before lights-off [30–32].



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  		FIG. 1. Serum LH, Lhb, and Fshb mRNA levels across the estrous cycle. Serum LH levels confirm cycle stage. MET, Metestrus; DI, diestrus; PRO, proestrus; EST, estrus. Results are shown as the mean ± SEM from six animals per time point. Lhb and Fshb mRNA levels were determined by Northern hybridization with normalization to cyclophilin mRNA levels. Data are expressed relative to levels at 0900 h on proestrus. *, significantly different (P < 0.05) from preceding data point as determined by ANOVA and post hoc Fisher PLSD analysis

Changes in Pituitary Steady-State Levels of Lhb and Fshb mRNAs During the Rat Estrous Cycle

Significant changes in Lhb expression were found throughout the rat estrous cycle (Fig. 1). We observed a significant decline in Lhb mRNA levels between 0900 h on diestrus and 0900 h on proestrus followed by a significant increase between 1500 and 1800 h on the afternoon of proestrus. Lhb mRNA levels demonstrated a second significant decline between 2400 h proestrus and 0900 h on the morning of estrus. Fshb mRNA levels also demonstrated significant variation across the estrous cycle. Fshb mRNA levels rose throughout the morning of proestrus to a peak at 1200 h. A decline in Fshb mRNA was observed between 1500 and 1800 h on the afternoon of proestrus, followed by an abrupt and significant increase between 2100 and 2400 h in the evening of proestrus. This second rise in Fshb mRNA levels was short lived, and a significant decline was observed between 2400 h proestrus and 0900 on the morning of estrus.

PACAP mRNA Expression Levels Within the PVN During the Rat Estrous Cycle

Analysis of Pacap mRNA levels in the PVN summarized in Figure 2 revealed significant variations throughout the estrous cycle, with the most striking change in expression occurring on the afternoon of proestrus. Measurements of the mean overall intensity of Pacap mRNA labeling within the midpoint of the PVN of each group were evaluated for statistical differences. A significant decrease in Pacap mRNA labeling was observed between the mornings of metestrus and diestrus. Furthermore, a dramatic and significant (P < 0.0001) rise in Pacap mRNA labeling was observed between 0900 and 1200 h on the morning of proestrus followed by a significant (P = 0.0006) decline between 1200 and 1500 h. The intensity of PVN Pacap mRNA labeling at proestrus 1200 was significantly higher than at all other times examined. The timing of this surge in PVN Pacap mRNA labeling (Fig. 2A) was 3 h before the previously mentioned surge in circulating LH (bars, Fig. 2B). Analysis of Pacap labeling at the level of the individual cell in the PVN revealed a slight but nonsignificant increase in the number of Pacap mRNA-positive cells at 1200 h on the day of proestrus (Fig. 2B). Instead, the observed fluctuations in overall PVN Pacap mRNA labeling intensity during the estrous cycle were due primarily to corresponding significant changes in Pacap expression within individual cells, as determined by the number of reduced silver grains per labeled cell (Fig. 2C). Using nomenclature described by Wiegand and Price [33], Pacap mRNA was found in individual cells in the lateral and dorsal subdivisions of the PVN. Pacap mRNA-positive cells were infrequently observed within the medial, or parvocellular, part of the PVN. In sections collected from the midpoint of the PVN, Pacap mRNA was localized predominantly within the lateral PVN with a few positive cells within the dorsal division. Increased Pacap mRNA expression at 1200 h on the day of proestrus was observed in both the lateral and the dorsal divisions of the PVN.



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  		FIG. 2. Expression of Pacap mRNA within PVN throughout the estrous cycle. Values in the graphs represent the mean ± SEM of the (A) optical density of silver grain accumulation, (B) number of Pacap-labeled cells per PVN section (line graph), and (C) the number of reduced silver grains per labeled cell in the PVN of six animals per group. For reference, the mean serum concentration of LH (ng/ml) for each group is presented as a bar graph in B. *, P < 0.05 vs. preceding data point as determined by ANOVA and post hoc Fisher PLSD analysis. MET, Metestrus; DI, diestrus; PRO, proestrus; EST, estrus

The Afternoon Rise in Pacap mRNA Is Specific to the Day of Proestrus

To determine if the midday rise in PVN Pacap mRNA was unique to the day of proestrus, a second experiment was performed using coronal sections from the midpoint of the PVN from female rats at 0900, 1200, and 1500 h on the days of diestrus as well as proestrus. As in the previous experiment, a significant rise in PVN Pacap mRNA labeling was observed between 0900 and 1200 h on the day of proestrus (Fig. 3). In contrast, there was no significant change in the level of Pacap mRNA labeling at these times on the day of diestrus.



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  		FIG. 3. The afternoon increase in PVN Pacap mRNA expression is specific to proestrus. Photomicrographs are dark-field (A and B) and merged bright-field/dark-field images (C and D) of Pacap mRNA labeling in the PVN at 1200 h on diestrus (A and C) or proestrus (B and D). Arrows in C and D indicate Pacap mRNA-labeled cells. 3V, Third ventricle; Fx, fornix. Bars = 100 µm. Values in the graph represent the mean ± SEM of the optical density of silver grain accumulation in the PVN of six animals per group. *, significantly (P < 0.05) greater than all other data points as determined by ANOVA and post hoc Fisher PLSD analysis

Regional Distribution of Pacap mRNA Expression Within the PVN

Quantitative analyses were performed to evaluate the regional distribution of Pacap mRNA expression throughout the PVN as depicted in Figure 4. In coronal sections of the PVN, Pacap mRNA labeling was localized to the lateral and medial divisions in the anterior (rostral) portion of the PVN, and labeling was predominant in the posterior division of the PVN in sections from the posterior (caudal) PVN. As summarized in Figure 5, PACAP mRNA-positive cells were localized throughout the PVN; however, a greater number of cells per section were observed in the middle portion of the PVN, primarily within the lateral division. Analysis of the overall density of Pacap labeling in individual coronal sections through the anterior, middle, and posterior extent of the PVN reflected the predominance of Pacap mRNA-labeled cells in the middle portion of the PVN. Furthermore, the rise in PVN Pacap mRNA labeling observed at midday on proestrus was significant only on coronal sections from the middle region of the PVN (Fig. 5).



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  		FIG. 4. Analysis of Pacap mRNA labeling throughout the PVN. Diagrammatic illustrations overlying photomicrographs of Pacap mRNA in situ hybridization demonstrate the analyzed subdivisions of the PVN and the distribution of Pacap labeling. The different panels are representative sections through the (A) anterior, (B) midline, and (C) posterior regions of the PVN. 3V, Third ventricle; Fx, fornix; PVNd, PVN dorsal; PVNm, PVN medial; PVNl, PVN lateral; PVNp, PVN posterior. Bars = 100 µm



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  		FIG. 5. Distribution of Pacap mRNA-positive cells in the PVN on proestrus. Values represent the mean ± SEM (n = 6 per column) of the (A) optical density of silver grain accumulation and (B) number of Pacap-labeled cells per PVN section in the anterior, midline, and posterior extent of the PVN at the indicated times on the day of proestrus. *, P < 0.05 vs. preceding data point as determined by ANOVA and post hoc Fisher PLSD analysis

Pituitary FST Expression During the Estrous Cycle

Competitive template-quantitative PCR was performed to analyze pituitary Fst mRNA levels throughout the estrous cycle. Our initial analysis revealed no significant change in total pituitary Fst mRNA levels during the estrous cycle (Table 1). We also examined the levels of pituitary expression of the mRNA species encoding for the 288 amino acid isoform of FST peptide. Real-time PCR analysis revealed a significant increase in Fst-288 mRNA levels between 2400 h on proestrus and 0900 h on the morning of estrus (Table 1).


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  		TABLE 1. Pituitary follistatin mRNA expression throughout the estrous cycle.*

Pituitary PACAP Expression During the Estrous Cycle

Quantitative real-time PCR analysis of pituitary Pacap mRNA levels revealed significant changes in expression in the evening hours of proestrus (Fig. 6). A modest decline in the amount of pituitary Pacap mRNA was observed between 1500 and 1800 h on proestrus following the LH surge. The amount of Pacap mRNA remained lower between 1800 and 2100 h, followed by a significant increase between 2100 and 2400 h. Interestingly, this pattern was similar to that observed for the expression of Fshb (Fig. 1); however, the levels of Fshb declined significantly during the early hours on the day of estrus (Fig. 6), whereas Pacap mRNA levels did not decline significantly.



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  		FIG. 6. Pituitary expression of Pacap mRNA throughout the estrous cycle. Results are expressed as the mean ± SEM of the number of copies (x106) of Pacap mRNA per 1 µg of total pituitary RNA. Each value represents the mean of five animals per group. * P < 0.05 vs. prior time point determined by ANOVA and post hoc Fisher PLSD analysis. MET, Metestrus; DI, diestrus; PRO, proestrus; EST, estrus


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Gonadotropin synthesis and secretion are regulated by GnRH, gonadal steroids, and the gonadal-derived peptides inhibin, activin, and follistatin [34]. The present experiments add anatomical and temporal data in support of the hypothesis that the neuropeptide PACAP may also play a role in gonadotropin regulation during the rat estrous cycle. We observed significant variations in Pacap mRNA expression within the PVN and anterior pituitary gland. Pacap expression in the PVN rose significantly 3 h before the LH surge on the afternoon of proestrus. Pituitary expression of Pacap rose significantly on the evening of proestrus and was followed by a significant rise in Fst-288 mRNA expression and a decline in Fshb mRNA expression. These data strengthen our hypothesis that PACAP acts as a hypophysiotropic factor to modulate gonadotroph responsiveness to GnRH as well as an intrapituitary factor involved in selective suppression of FSH levels through stimulation of follistatin expression.

Pacap mRNA levels in the PVN rose more than 2-fold between 0900 and 1200 h on proestrus approximately 3 h preceding the LH surge. This finding is consistent with the hypothesis that PACAP is involved in the physiological regulation of the gonadotropins during the estrous cycle. Although we did not measure PACAP protein levels or secretion, other investigations have demonstrated that newly synthesized hormones are preferentially secreted following stimulatory input [35–37]. The changes in PVN Pacap mRNA were due to increased expression in a relatively stable population of cells. In addition, the midday rise in Pacap mRNA expression within the PVN was found to be specific to the day of proestrus. This finding eliminates the possibility of a daily variation in Pacap expression in the PVN because of the daylight cycle but does not preclude the involvement of PACAP of a different origin in diurnal regulation of the pituitary or any other system.

The distribution of Pacap mRNA within the PVN of the female rat suggests that PACAP is synthesized within a subset of magnocellular neurons. The majority of magnocellular neurons extend terminal processes to the posterior pituitary gland. Therefore, PACAP secreted from the magnocellular neurons could reach gonadotrophs via the short portal vessels from the posterior lobe to the anterior lobe [38] or via the small number of neurons within the lateral magnocellular division of the PVN that extend axons that terminate in the median eminence [33]. Pacap mRNA-labeled neurons were also observed within the posterior subdivision of the PVN that is comprised of parvocellular neurons based on size and efferent projections. Therefore, PACAP could also be delivered to gonadotrophs in the anterior pituitary via the hypothalamic-pituitary portal system of capillaries. Indeed, PACAP peptide levels in the portal vasculature in rats were 2- to 4-fold higher than in the circulation; however, the source(s) of portal PACAP is not known [39].

The observation that Pacap mRNA-labeled neurons are predominant within the magnocellular neurons within the lateral subdivision of the PVN suggests colocalization with oxytocin and/or vasopressin. Other neuropeptides known to colocalize in magnocellular neurons include cholecystokinin, dynorphin, corticotropin-releasing factor, angiotensin II-, dynorphin, and galanin [40]. In ovariectomized rats, peptides that are localized to vasopressinergic cells showed evidence of enhanced expression in response to exogenous estrogen, while at least two peptides that colocalized with oxytocin appeared driven in the opposite direction [40]. Furthermore, the regional distribution of estrogen receptor beta immunoreactivity in the PVN [41] is very similar to that observed for Pacap mRNA in this investigation. Thus, the rise in Pacap mRNA on 1200 h of proestrus may have been induced by the antecedent increase in circulating estradiol [42].

Steroid hormone regulation of Pacap expression has previously been established. Within the rat ovary, cAMP signaling stimulates Pacap mRNA expression, and that stimulation is enhanced by progesterone receptor activation [21]. In ovariectomized rats, estrogen priming, progesterone, and combined estrogen with progesterone treatment each induced a significant increase in Pacap mRNA expression within the ventromedial nucleus of the hypothalamus [24]. In a similar model, progesterone, progesterone together with estrogen, but not estrogen alone induced a significant increase in Pacap and PACAP receptor mRNA expression in the medial basal hypothalamus as well as PACAP receptor mRNA in the preoptic area of the hypothalamus [25]. Because intracerebroventricular (i.c.v.) injection of PACAP stimulates Gnrh mRNA expression [43] and i.c.v. injection of PACAP receptor antisense oligodeoxynucleotide decreased Gnrh mRNA expression [26], estrogen-sensitive PACAP neurons within the PVN may influence GnRH neurons during the estrous cycle. PACAP neurons within the PVN could directly contact GnRH neurons through specific efferent pathways into the organum vasculosum laminae terminalis [44] or through modulation of GnRH release from axon terminals within the median eminence.

It is also possible that the rise in PVN Pacap mRNA expression at midday on proestrus is coincidentally related to the gonadotropin surge. It is well known that reproductive behavior in rats is tightly coupled to gonadotropin and GnRH release. Therefore, it is entirely possible that the increase in PVN Pacap expression is more directly related to female receptivity to mating following the gonadotropin surge. Apostolakis et al. [24] demonstrated that i.c.v. administration of PACAP to estrogen-primed ovariectomized rats resulted in a dose-dependent increase in female receptivity to mating in a manner similar to that invoked by progesterone administration. This increase in reproductive behavior was blocked dose dependently by preadministration of a PACAP antagonist. Furthermore, the PACAP antagonist also blocked the progesterone-stimulated increase in reproductive behaviors. Other evidence linking PACAP expression to reproductive behavior is the observation that female mice that are genetically deficient in PACAP expression are infertile and display an almost complete lack of receptivity [45].

Another potential role for PACAP is to regulate expression of GnRH receptors and thereby GnRH responsiveness. PACAP stimulates GnRH receptor transcription in immortalized LßT-2 gonadotroph cells [13, 14]. Since GnRH receptor levels increase before the gonadotropin surge on the afternoon of proestrus [46], increased PACAP signaling in the pituitary could increase GnRH receptor expression and increase gonadotroph responsiveness.

While PACAP has been viewed as a hypophysiotropic factor, Pacap mRNA and protein have also been detected in the pituitary, and the peptide may also function as a paracrine regulator of gonadotrophs. An increase in the expression of pituitary Pacap mRNA on the afternoon of proestrus was previously described in a preliminary report by Wuttke and colleagues (abstract, Neuroendocrinology 1994; 60: S1:17). Koves and colleagues [47] reported that PACAP immunoreactive cells are detectable within the rat anterior pituitary on proestrus but not during other times of the cycle and that PACAP was localized within cells that were immunoreactive for LH and FSH peptide. The secretion of PACAP by rat pituitary cultures from various time points on the day of proestrus was also observed [48], and the number of PACAP-secreting pituitary cells was increased at 1000 and 2000 h on proestrus but nearly absent at 1600 h. There were 12 times as many cells secreting PACAP at 2000 h as compared to 1000 h. Conversely, there were very few PACAP-secreting cells derived from the same time points of diestrous rats.

In primary cultures of rat pituitary cells, PACAP stimulates significant accumulation of follistatin mRNA [3, 49, 50]. In this investigation, we did not detect a significant change in pituitary total follistatin mRNA levels during the rat estrous cycle. Previously it was reported that follistatin expression increased markedly at the time of the gonadotropin surge on the afternoon of proestrus [51]. The disparity in results may be due to differences in assay techniques or by a mistiming in sample collection that, despite our sampling every 3 h, resulted in our failure to observe the previously reported short-lived increase in follistatin mRNA [51]. However, our analysis of the mRNA species encoding the 288 amino acid isoform of FST did show an increase in expression on the morning of estrus. Furthermore, the timing of increased Fst-288 mRNA expression suggests that this event may be responsible for the decrease in Fshb mRNA expression that effectively ends the secondary surge of FSH by abolishing pituitary activin signaling. Interestingly, the rise in Fst-288 mRNA on the morning of estrus is preceded by a significant increase in the level of Pacap mRNA within the anterior pituitary. PACAP expression on the evening of proestrus may therefore play a role in regulating pituitary follistatin and thereby terminating the secondary surge of FSH that follows ovulation in the rat.

In cultures of primary pituitary cells, the mode of delivery of PACAP, that is, static vs. perfused or pulsatile vs. continuous, appears to partly determine its effect on gonadotropin synthesis and secretion. Rat pituitary cultures perifused with hourly pulses of PACAP demonstrate episodes of LH, FSH, and {alpha}-subunit secretion that decrease in amplitude over time, while continuous exposure to PACAP results in a rapid but transient release of the gonadotropins [3]. Furthermore, hourly pulses of PACAP increased Lhb and {alpha}-subunit gene expression with no effect on Fshb mRNA levels, while continuous delivery of PACAP stimulated {alpha}-subunit expression lengthened Lhb transcripts but decreased Fshb mRNA levels [3]. As such, the significant rise in pituitary Pacap mRNA at proestrus 2400, causing continuous exposure of pituitary cells to elevated levels of PACAP, may produce the subsequent decline in Fshb mRNA (Fig. 2) because of increased expression of pituitary follistatin on the morning of estrus (Table 1). The brief surge in PVN Pacap mRNA levels at midday on the afternoon of proestrus, on the other hand, may increase pulsatile PACAP release and produce a short-lived increase in PACAP delivery to the pituitary, allowing for increased GnRH responsiveness with little or no effect on follistatin or Fshb expression.

In conclusion, the results presented in this report suggest that PACAP plays a role in the events preceding and following ovulation during the estrous cycle in rats. We hypothesize that increased PACAP expression in the PVN before the gonadotropin surge leads to increased responsiveness of gonadotrophs to GnRH by increasing GnRH receptor expression and/or acting synergistically with GnRH to increase gonadotropin release. In addition, we propose that increased pituitary expression of PACAP following ovulation stimulates follistatin expression and thereby contributes to the cessation of the secondary FSH surge.


   ACKNOWLEDGMENTS
 
The authors would like to acknowledge the expert technical assistance of Mr. Dushan Ghooray and Mr. Alan Icard.


   FOOTNOTES
 
1 Supported in part by NIH grants RR-P20 RR17702-SPID 0005 from the Institutional Development Award (IdeA) Program of the NCRR (J.P.M.) and R01-HD-036034 (S.J.W.) by the Walter F. and Avis Jacobs Foundation and by the Commonwealth of Kentucky Research Challenge Fund. A portion of this work was presented at the 85th annual meeting of The Endocrine Society, Philadelphia, Pennsylvania, June 19–22, 2003 (abstract P2-647). Back

2 Correspondence: Joseph P. Moore, Jr., University of Louisville, School of Medicine, Division of Endocrinology and Metabolism, ACB, Third Floor, Room A3G11, 530 South Jackson St., Louisville, KY 40292. FAX: 502 852 2492; jpmoor03{at}gwise.louisville.edu Back

Received: 8 March 2005.

First decision: 6 April 2005.

Accepted: 9 May 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Kimura C, Ohkubo S, Ogi K, Hosoya M, Itoh Y, Onda H, Miyata A, Jiang L, Dahl RR, Stibbs HH, Arimura A, Fujino M. A novel peptide which stimulates adenylate cyclase: molecular cloning and characterization of the ovine and human cDNAs. Biochem Biophys Res Commun 1990 166:81-89[CrossRef][Medline]
  2. McArdle CA. Pituitary adenylate cyclase-activating polypeptide: a key player in reproduction?. Endocrinology 1994 135:815-817[CrossRef][Medline]
  3. Tsujii T, Ishizaka K, Winters SJ. Effects of pituitary adenylate cyclase-activating polypeptide on gonadotropin secretion and subunit messenger ribonucleic acids in perifused rat pituitary cells. Endocrinology 1994 135:826-833[Abstract]
  4. Ackland JF, Schwartz NB. Changes in serum immunoreactive inhibin and follicle-stimulating hormone during gonadal development in male and female rats. Biol Reprod 1991 45:295-300[Abstract]
  5. Gottschall PE, Tatsuno I, Miyata A, Arimura A. Characterization and distribution of binding sites for the hypothalamic peptide, pituitary adenylate cyclase-activating polypeptide. Endocrinology 1990 127:272-277[Abstract]
  6. Vigh S, Arimura A, Gottschall PE, Kitada C, Somogyvari-Vigh A, Childs GV. Cytochemical characterization of anterior pituitary target cells for the neuropeptide, pituitary adenylate cyclase activating polypeptide (PACAP), using biotinylated ligands. Peptides 1993 14:59-65[CrossRef][Medline]
  7. Rawlings SR, Demaurex N, Schlegel W. Pituitary adenylate cyclase-activating polypeptide increases [Ca2]i in rat gonadotrophs through an inositol trisphosphate-dependent mechanism. J Biol Chem 1994 269:5680-5686[Abstract/Free Full Text]
  8. Schomerus E, Poch A, Bunting R, Mason WT, McArdle CA. Effects of pituitary adenylate cyclase-activating polypeptide in the pituitary: activation of two signal transduction pathways in the gonadotrope-derived alpha T3-1 cell line. Endocrinology 1994 134:315-323[Abstract]
  9. Hart GR, Gowing H, Burrin JM. Effects of a novel hypothalamic peptide, pituitary adenylate cyclase-activating polypeptide, on pituitary hormone release in rats. J Endocrinol 1992 134:33-41[Abstract]
  10. Culler MD, Negro-Vilar A. Passive immunoneutralization of endogenous inhibin: sex-related differences in the role of inhibin during development. Mol Cell Endocrinol 1988 58:263-273[CrossRef][Medline]
  11. Perrin D, Soling HD, Wuttke W, Jarry H. The stimulatory effect of pituitary adenylate cyclase activating polypeptide (PACAP) on LH release from rat pituitary cells in vitro does not involve calcium mobilization. Exp Clin Endocrinol 1993 101:290-296[Medline]
  12. Carroll RS, Corrigan AZ, Gharib SD, Vale W, Chin WW. Inhibin, activin, and follistatin: regulation of follicle-stimulating hormone messenger ribonucleic acid levels. Mol Endocrinol 1989 3:1969-1976[Abstract]
  13. Ngan ES, Leung PC, Chow BK. Interplay of pituitary adenylate cyclase-activating polypeptide with a silencer element to regulate the upstream promoter of the human gonadotropin-releasing hormone receptor gene. Mol Cell Endocrinol 2001 176:135-144[CrossRef][Medline]
  14. Sadie H, Styger G, Hapgood J. Expression of the mouse gonadotropin-releasing hormone receptor gene in alpha T3-1 gonadotrope cells is stimulated by cyclic 3',5'-adenosine monophosphate and protein kinase A, and is modulated by Steroidogenic factor-1 and Nur77. Endocrinology 2003 144:1958-1971[Abstract/Free Full Text]
  15. Cassina P, Sellers J, Neill JD. Effect of cAMP on GnRH stimulated LH secretion from individual pituitary gonadotropes. Mol Cell Endocrinol 1995 114:127-135[CrossRef][Medline]
  16. Osuga Y, Mitsuhashi N, Mizuno M. In vivo effect of pituitary adenylate cyclase activating polypeptide 38 (PACAP 38) on the secretion of luteinizing hormone (LH) in male rats. Endocrinol Jpn 1992 39:153-156[Medline]
  17. Jarry H, Leonhardt S, Schmidt WE, Creutzfeldt W, Wuttke W. Contrasting effects of pituitary adenylate cyclase activating polypeptide (PACAP) on in vivo and in vitro prolactin and growth hormone release in male rats. Life Sci 1992 51:823-830[CrossRef][Medline]
  18. Kantora O, Molnar J, Arimura A, Koves K. PACAP38 and PACAP27 administered intracerebroventricularly have an opposite effect on LH secretion. Peptides 2000 21:817-820[CrossRef][Medline]
  19. Koves K, Molnar J, Kantor O, Lakatos A, Fogel K, Kausz M, Vandermeers-Piret MC, Somogyvari-Vigh A, Arimura A. Role of PACAP in the regulation of gonadotroph hormone secretion during ontogenesis: a single neonatal injection of PACAP delays puberty and its intracerebroventricular administration before the critical period of proestrous stage blocks ovulation in adulthood. Ann N Y Acad Sci 1998 865:590-594[Free Full Text]
  20. Koves K, Molnar J, Kantor O, Lakatos A, Gorcs TJ, Somogyvari-Vigh A, Furst Z, Arimura A. PACAP participates in the regulation of the hormonal events preceeding the ovulation. Acta Biol Hung 1996 47:239-249[Medline]
  21. Ko C, In YH, Park-Sarge OK. Role of progesterone receptor activation in pituitary adenylate cyclase activating polypeptide gene expression in rat ovary. Endocrinology 1999 140:5185-5194[Abstract/Free Full Text]
  22. Ko C, Park-Sarge OK. Progesterone receptor activation mediates LH-induced type-I pituitary adenylate cyclase activating polypeptide receptor (PAC(1)) gene expression in rat granulosa cells. Biochem Biophys Res Commun 2000 277:270-279[CrossRef][Medline]
  23. Park JI, Kim WJ, Wang L, Park HJ, Lee J, Park JH, Kwon HB, Tsafriri A, Chun SY. Involvement of progesterone in gonadotrophin-induced pituitary adenylate cyclase-activating polypeptide gene expression in pre-ovulatory follicles of rat ovary. Mol Hum Reprod 2000 6:238-245[Abstract/Free Full Text]
  24. Apostolakis EM, Lanz R, O'Malley BW. Pituitary adenylate cyclase-activating peptide: a pivotal modulator of steroid-induced reproductive behavior in female rodents. Mol Endocrinol 2004 18:173-183[Abstract/Free Full Text]
  25. Ha CM, Kang JH, Choi EJ, Kim MS, Park JW, Kim Y, Choi WS, Chun SY, Kwon HB, Lee BJ. Progesterone increases mRNA levels of pituitary adenylate cyclase-activating polypeptide (PACAP) and type I PACAP receptor (PAC(1)) in the rat hypothalamus. Brain Res Mol Brain Res 2000 78:59-68[Medline]
  26. Choi EJ, Ha CM, Kim MS, Kang JH, Park SK, Choi WS, Kang SG, Lee BJ. Central administration of an antisense oligodeoxynucleotide against type I pituitary adenylate cyclase-activating polypeptide receptor suppresses synthetic activities of LHRH-LH axis during the pubertal process. Brain Res Mol Brain Res 2000 80:35-45[Medline]
  27. Moore JP Jr, Wilson L, Dalkin AC, Winters SJ. Differential expression of the pituitary gonadotropin subunit genes during male rat sexual maturation: reciprocal relationship between hypothalamic pituitary adenylate cyclase-activating polypeptide and follicle-stimulating hormone beta expression. Biol Reprod 2003 69:234-241[Abstract/Free Full Text]
  28. Kirk SE, Dalkin AC, Yasin M, Haisenleder DJ, Marshall JC. Gonadotropin-releasing hormone pulse frequency regulates expression of pituitary follistatin messenger ribonucleic acid: a mechanism for differential gonadotrope function. Endocrinology 1994 135:876-880[Abstract]
  29. Inouye S, Guo Y, DePaolo L, Shimonaka M, Ling N, Shimasaki S. Recombinant expression of human follistatin with 315 and 288 amino acids: chemical and biological comparison with native porcine follistatin. Endocrinology 1991 129:815-822[Abstract]
  30. Lloyd JM, Hoffman GE, Wise PM. Decline in immediate early gene expression in gonadotropin-releasing hormone neurons during proestrus in regularly cycling, middle-aged rats. Endocrinology 1994 134:1800-1805[Abstract]
  31. Moss RL, Cooper KJ. Temporal relationship of spontaneous and coitus-induced release of luteinizing hormone in the normal cyclic rat. Endocrinology 1973 92:1748-1753[Medline]
  32. Eyigor O, Jennes L. Kainate receptor subunit-positive gonadotropin-releasing hormone neurons express c-Fos during the steroid-induced luteinizing hormone surge in the female rat. Endocrinology 2000 141:779-786[Abstract/Free Full Text]
  33. Wiegand SJ, Price JL. Cells of origin of the afferent fibers to the median eminence in the rat. J Comp Neurol 1980 192:1-19[CrossRef][Medline]
  34. Winters SJ, Moore JP. Intra-pituitary regulation of gonadotrophs in male rodents and primates. Reproduction 2004 128:13-23[Abstract/Free Full Text]
  35. Kagabu Y, Mishiba T, Okino T, Yanagisawa T. Effects of thyrotropin-releasing hormone and its metabolites, Cyclo(His-Pro) and TRH-OH, on growth hormone and prolactin synthesis in primary cultured pituitary cells of the common carp, Cyprinus carpio. Gen Comp Endocrinol 1998 111:395-403[CrossRef][Medline]
  36. Van Schravendijk CF, Kiekens R, Pipeleers DG. Pancreatic beta cell heterogeneity in glucose-induced insulin secretion. J Biol Chem 1992 267:21344-21348[Abstract/Free Full Text]
  37. Mangat H, de Bold AJ. Stretch-induced atrial natriuretic factor release utilizes a rapidly depleting pool of newly synthesized hormone. Endocrinology 1993 133:1398-1403[Abstract]
  38. Murakami T, Miyake T, Ohtsuka A, Kikuta A, Taguchi T. Microcirculatory patterns in adult rat cerebral hypophysis: a scanning electron microscope study of replicated specimens. Arch Histol Cytol 1993 56:243-260[Medline]
  39. Dow RC, Bennie J, Fink G. Pituitary adenylate cyclase-activating peptide-38 (PACAP)-38 is released into hypophysial portal blood in the normal male and female rat. J Endocrinol 1994 142:R1-R4[Abstract]
  40. Levin MC, Sawchenko PE. Neuropeptide co-expression in the magnocellular neurosecretory system of the female rat: evidence for differential modulation by estrogen. Neuroscience 1993 54:1001-1018[CrossRef][Medline]
  41. Alves SE, Lopez V, McEwen BS, Weiland NG. Differential colocalization of estrogen receptor beta (ERbeta) with oxytocin and vasopressin in the paraventricular and supraoptic nuclei of the female rat brain: an immunocytochemical study. Proc Natl Acad Sci U S A 1998 95:3281-3286[Abstract/Free Full Text]
  42. Mahesh VB. The dynamic interaction between steroids and gonadotropins in the mammalian ovulatory cycle. Neurosci Biobehav Rev 1985 9:245-260[CrossRef][Medline]
  43. Li S, Grinevich V, Fournier A, Pelletier G. Effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on gonadotropin-releasing hormone and somatostatin gene expression in the rat brain. Brain Res Mol Brain Res 1996 41:157-162[Medline]
  44. Larsen PJ, Moller M, Mikkelsen JD. Efferent projections from the periventricular and medial parvicellular subnuclei of the hypothalamic paraventricular nucleus to circumventricular organs of the rat: a Phaseolus vulgaris-leucoagglutinin (PHA-L) tracing study. J Comp Neurol 1991 306:462-479[CrossRef][Medline]
  45. Shintani N, Mori W, Hashimoto H, Imai M, Tanaka K, Tomimoto S, Hirose M, Kawaguchi C, Baba A. Defects in reproductive functions in PACAP-deficient female mice. Regul Pept 2002 109:45-48[CrossRef][Medline]
  46. Bauer-Dantoin AC, Hollenberg AN, Jameson JL. Dynamic regulation of gonadotropin-releasing hormone receptor mRNA levels in the anterior pituitary gland during the rat estrous cycle. Endocrinology 1993 133:1911-1914[Abstract]
  47. Koves K, Kantor O, Scammell JG, Arimura A. PACAP colocalizes with luteinizing and follicle-stimulating hormone immunoreactivities in the anterior lobe of the pituitary gland. Peptides 1998 19:1069-1072[CrossRef][Medline]
  48. Koves K, Kantor O, Molnar J, Heinzlmann A, Szabo E, Szabo F, Nemeskeri A, Horvath J, Arimura A. The role of PACAP in gonadotropic hormone secretion at hypothalamic and pituitary levels. J Mol Neurosci 2003 20:141-152[CrossRef][Medline]
  49. Fujii Y, Okada Y, Moore JP, Dalkin AC, Winters SJ. Evidence that PACAP and GnRH downregulate follicle-stimulating hormone-beta mRNA levels by stimulating follistatin gene expression: effects on folliculostellate cells, gonadotrophs and LbetaT2 gonadotroph cells. Mol Cell Endocrinol 2002 192:55-64[CrossRef][Medline]
  50. Winters SJ, Dalkin AC, Tsujii T. Evidence that pituitary adenylate cyclase activating polypeptide suppresses follicle-stimulating hormone-beta messenger ribonucleic acid levels by stimulating follistatin gene transcription. Endocrinology 1997 138:4324-4329[Abstract/Free Full Text]
  51. Halvorson LM, Weiss J, Bauer-Dantoin AC, Jameson JL. Dynamic regulation of pituitary follistatin messenger ribonucleic acids during the rat estrous cycle. Endocrinology 1994 134:1247-1253[Abstract]



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