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Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030
Address all correspondence and requests for reprints to: Ede Marie Apostolakis, Ph.D., Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030.
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ABSTRACT |
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INTRODUCTION |
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, PR isoforms A and B]. In the brain, the mechanisms of hormone action are more complex than previously thought and include cross-talk between nuclear receptors and intracellular signaling pathways in the absence of hormones (1, 2, 3, 4, 5), regulation of steroid receptor-mediated gene transcription by steroid receptor coactivators (SRCs) (6, 7), differential physiological effects of P mediated by PR isoforms (8), and nongenomic hormone activation of signaling pathways (9, 10). In the rodent brain, E and P regulate cellular functions that exquisitely control sexual behavior (11). Priming with E for 48 h increases the synthesis of PR in the female hypothalamic ventromedial nucleus (VMN) (12), an event that coincides with the ability of P to facilitate PR-dependent sexual receptivity (11, 13, 14). Of significance, dopaminergic activation of adenylate cyclase transduction pathways in the VMN facilitates lordosis in the absence of P via E-induced PR (3, 4, 5). In addition, E induces changes in local microcircuitry within the VMN (15), an effect that also requires activation of cAMP signaling (16). Indeed, the cAMP transduction pathway may be essential for efficient PR-dependent reproductive behavior (17).
The pleiotropic neuropeptide PACAP, a member of the secretin/glucagons/vasoactive intestinal peptide family (18), is a 38-amino acid peptide that potently increases cAMP levels (19, 20, 21, 22, 23). Anatomically, PACAP is widely distributed in reproductive tissue (18) including many regions of the hypothalamus (24, 25). PACAP exerts its biological action by binding to its cognate membrane receptors, which can be divided into at least three subtypes. As the dominant receptor in the hypothalamus (19, 20, 21, 22, 24), type 1 PACAP receptor (PAC1) is PACAP selective and is coupled to both adenylate cyclase and phospholipase C (21, 22, 23). In contrast, vasoactive intestinal peptide/PACAP receptor type 1 (VPAC1) and type 2 (VPAC2) are more modestly expressed in the hypothalamus, exhibit similar affinities for PACAP and vasoactive intestinal peptide, and are coupled only to adenylate cyclase (25). Although the influence of PACAP has been extensively studied (18, 19, 20, 21, 22, 23, 24, 25, 26), only the most recent studies have examined its role in reproductive physiology. The present study was undertaken to determine whether PACAP in the VMN plays a role in steroid-dependent receptivity. Collectively, our current study supports the hypothesis that endogenous PACAP biologically acts as a positive, feed-forward, autocrine, and/or paracrine factor that mediates steroid receptor function in the female VMN.
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RESULTS |
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and 2, ANOVA P > 0.001). Receptivity was displayed with E + P and E + SKF38393 (Figs. 1 and 3, P < 0.001). In those experiments using oligonucleotides, both randomized antisense sequence (RS) and RS + E had no effect on behavior whereas sexual behavior was exhibited with RS + E + agonist challenge (Fig. 2, B–D, P > 0.001). Likewise, those females treated with RS and E + agonist had comparable levels of receptive behavior (Fig. 2, B–D, P > 0.05). Neither antisense sequence (AS) nor AS + E had an effect on behavior (Fig. 2, B–D, P > 0.001). In experiments with PR knockout (PRKO) mice, serotonin (50 ng icv) induced receptivity in both wild type and null PRKO females (Fig. 2B and Refs.2, 7), demonstrating an intact PR-independent pathway for reproductive behavior in both genotypes. When given P, E-primed wild-type females exhibited receptivity and their PRKO counterparts failed to display lordosis as expected (Fig. 2B). The data from above control conditions confirm the absence of any nonspecific effect of steroid, agonist, and/or oligonucleotide treatment on behavior.
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, bars 11–14; P < 0.05). Likewise, PACAP given icv or bilaterally onto the VMN (1–10 ng/side) also facilitated sex behavior in E-primed rats (Fig. 1A, bars 11–14 and 19–22, respectively, P < 0.05). Behavioral responses to a lower dose (0.2 ng; Fig. 1A, bars 9–10) were statistically insignificant (P > 0.05). A repertoire of proceptive behaviors (hopping, darting, ear wiggling) was observed in conjunction with PACAP-induced lordosis, whereas in its absence, rejection behaviors (kicking, biting, standing, rolling over, and running away) were noted. Finally, no significant weight loss was seen 24–48 h after PACAP treatment in those animals given 2–20 ng PACAP; the more optimal dose of 2 ng was chosen for all subsequent testing in rats. The data raise the possibility that, like dopamine, PACAP may serve as a ligand-independent activator of steroid receptor function in the intact animal.
Inhibition of PACAP Attenuates Facilitated Receptivity
When given 15–30 min before the administration of PACAP, the inhibitor PACAP6–38 (2–20 ng icv) significantly suppressed PACAP38-facilitated receptivity in E-primed females (Fig. 1B, bars 6 and 7, P < 0.05 and P < 0.01, respectively). No receptivity was observed in association with PACAP6–38 (2–20 ng) treatment alone (Fig. 1B, bar 8, P > 0.05) or E + PACAP6–38 (data not shown, P > 0.01). Because PACAP6–38 is an inhibitory fragment of the PACAP neuropeptide that competitively binds to the same membrane receptors as PACAP (18), the data demonstrate specificity of the behavioral response for PACAP-stimulated events in E-primed females.
PR-Dependent Receptivity Is Suppressed by PACAP6–38
To determine whether PACAP initiated an intracellular signal that converges on PR in the VMN for initiation of sexual receptivity, two additional behavioral experiments were undertaken. First, PR oligonucleotides [4 nM, icv or 2 nM bilateral onto (i.n.) VMN] were given concurrent with E. Importantly, those E-primed individuals pretreated with PR AS and challenged with PACAP failed to exhibit a significant lordosis response (Fig. 2A, bar 10), suggesting that PACAP-induced behavior is dependent upon PR. Second, mice with a targeted disruption of PR (PRKO) were tested for PACAP-initiated behavior. Unlike ovx wild-type counterparts with the full complement of PRs (Fig. 2B, bar 15), E-primed PRKO females showed minimal receptivity in response to PACAP (bar 16). Moreover, PACAP6–38 again inhibited PACAP induction of sex behavior in wild-type mice (Fig. 2B, bar 21), demonstrating the specificity of the response for PACAP receptor stimulation. Collectively, the data demonstrate the dependency of PACAP on PR, supporting the idea that PACAP initiates an intracellular signaling pathway that includes the nuclear transcription factor PR for ligand-independent function.
PACAP-Dependent Behavior Requires SRC-1 and SRC-2
Because transcriptional activation of steroid receptors (ER and nuclear PR) in the VMN (11) and subsequent receptivity (8) necessitates the recruitment of SRC-1 and SRC-2 in both rats and mice (6, 7), we questioned whether PACAP-facilitated behavior required SRC-1 and/or SRC-2 by using C57BL/6 mice and antisense oligonucleotides to SRC-1 and SRC-2. As with P (8), PACAP failed to induce receptivity in SRC-1 AS-treated, E-primed females (Fig. 2C, bar 10). Likewise, E-primed mice pretreated with SRC-2 AS failed to exhibit lordosis when challenged with PACAP38 (Fig. 2C, bar 16). Because the SRC oligonucleotides were given concurrently with E, these data may indicate that exogenous PACAP cannot overcome the loss of ER-mediated events within the VMN, a finding consistent with those for dopamine (7). Hence, the requirement for intact nuclear receptor coactivators suggests that steroid receptor genomic activity is required for PACAP initiation of sex behavior in mice.
PACAP-Facilitated Behavior Requires Protein Synthesis
It has long been known that P-facilitated sex behavior in E-primed female rodents requires the synthesis of new proteins in the VMN (27, 28). To determine whether PACAP promotes estrous behavior via a genomic mechanism requiring protein synthesis, the protein synthesis inhibitor anisomycin was administered 1 h before PACAP. PACAP-induced receptivity in E-primed rats was attenuated significantly by anisomycin (Fig. 2D, bar 6). Taken together with the finding that PACAP is required for P-induced behavior (below), the data support the hypothesis that PACAP initiates cell signaling for genomic activation of PR and receptivity.
P-Facilitated Behavior Is Inhibited by PACAP6–38
Next, we addressed the question of whether PACAP plays a role in mediating P-dependent behavior. The results show that P failed to induce lordosis in the experimental E-primed females pretreated with 2 ng of the PACAP inhibitor PACAP6–38 (Fig. 3A, bar 6). Likewise, PACAP6–38 inhibited the display of lordosis in E-primed rats when the D1 dopamine receptor agonist SKF38393 was substituted for P (Fig. 3, bar 9; P > 0.01). Because SKF38393 also has been demonstrated to induce lordosis by a PR-dependent mechanism, the combined data implicate activation of the PACAP receptor-signaling pathway in the facilitation of PR-dependent receptivity.
Steroids Regulate PACAP Levels in the Female Hypothalamic VMN
To assess mRNA expression in micropunch VMN, real-time RT-PCR [quantitative RT-PCR (qRT-PCR)] was performed and the results are shown in Fig. 3B. Quantitative PCR is a system that detects PCR products as they accumulate rather than the final product after a fixed number of cycles. For all studies regardless of PCR methodology, E-priming, P-only and coadministration of E and P induced moderate changes in relative expression of PACAP mRNA (Fig. 3B, bars 2–4) compared with basal expression in VMN tissue (bar 1). Basal expression levels in the VMN were similar to relative levels in whole brain tissue from the same animals (Fig. 3B, bar 5). These findings are consistent with steroid induction of PACAP synthesis in the female VMN.
RIA was used to quantitate the effects of steroids on endogenous PACAP levels in tissue and cerebral spinal fluid (CSF) from ovx PRKO mice and/or rats. PACAP tissue content in the medial basal hypothalamus (MBH) is a sum concentration of PACAP in the VMN and arcuate nucleus as well as that contained within the terminal ends of projections from other related cell groups [such as the medial preoptic area (MPOA)] that participate in reproductive behavior and ovulation. PACAP tissue concentrations in MBH varied in association with steroid treatment. Compared with vehicle treatment, E-priming enhanced PACAP concentrations in MBH from wild-type and PRKO mice by 117 ± 11% and 125 ± 12%, respectively (Fig. 3C, bars 1 and 2; P < 0.05), whereas P-only led to a significant increase in PACAP concentration only in wild-type but not PRKO MBH (bars 3 and 4, respectively). Surprisingly, PACAP levels in the wild-type MBH were reduced by E + P treatment (Fig. 3C, bar 5, P < 0.05) and unchanged in PRKO MBH (bar 6, P > 0.05).
To ascertain whether the E + P reduction in tissue levels might be due to concomitant secretion of immunoreactive (ir) PACAP, the above experiment was repeated in rats and CSF was collected as well as MBH. CSF PACAP is a general reflection of changes in extracellular neuropeptide levels from diffusion out of synaptic endings. As in mice, E induced statistically significant increases in irPACAP in the rat MBH compared with no treatment (Fig. 3D, bar 1). There was a strong statistical trend (P < 0.07) toward enhanced irPACAP levels in P-treated rat MBH (Fig. 3D, bar 3) when compared with no treatment controls. There was no statistical difference between irPACAP levels induced by P compared with E (Fig. 3D, bar 3 vs. bar 1; P > 0.05). In the MBH from rats treated with E + P, irPACAP failed to change compared with no treatment controls (Fig. 3D, bar 5; P > 0.05). Thus, both rodent models demonstrate a similar trend toward enhanced irPACAP concentrations in MBH tissue after steroid-alone treatment but not when treated in combination. Importantly, E + P was associated with a 136 ± 5% increase in irPACAP in the CSF (Fig. 3D, bar 6), whereas neither E nor P only increased irPACAP levels in the CSF (bars 2 and 4). Together with the findings of enhanced PACAP mRNA synthesis, the data are consistent with extracellular secretion of PACAP being induced only by coadministration of E and P.
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DISCUSSION |
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and PR mediate the genomic effects of E and P for the display of sexual behavior in the female rodent (11). In our previous studies, we have shown that dopamine acting on dopamine (DA) type 1 (D1)-like receptors and DA transporters in the VMN induce PR-facilitated receptivity in the absence of P in E-primed rodents (2, 3, 4, 5, 7). Further, the canonical signaling cassette for cAMP/protein kinase A (PKA) mediates the ligand-independent effect of DA and several D1 agonists (3, 4, 17). It is also apparent that SRC-1 and SRC-2 are essential for the functional effect of ER activation and receptivity (7). In the course of our work, we were intrigued with PACAP, a novel first messenger that acts to amplify cAMP signaling in several tissues including the hippocampus and paraventricular and supraoptic nuclei (18, 29). Thus, we wondered if steroids could induce the synthesis and release of this novel messenger for paracrine and/or autocrine modulation of intracellular signaling events within the irPR neurons of the VMN that mediate receptivity. The present study provides strong evidence that PACAP plays a fundamental role in mediating the effect of steroids in the VMN for reproductive behavior. PACAP induced sex behavior in E-primed females, an effect that was blocked by the competitive, inhibitory fragment of PACAP (PACAP6–38). In addition, nuclear coactivator proteins (SRC-1, SRC-2) and steroid receptor activation appear to be downstream of PACAP as evidenced by experiments using AS oligonucleotides and knockout mice. Together, these data indicate that exogenous PACAP may induce ligand-independent steroid receptor-dependent behavior in the rodents. Importantly, the facilitatory effect of P (and the D1-like agonist SKF38393) was blocked by PACAP6–38, thus implicating endogenous PACAP and its hypothalamic receptors as critical components of the cellular mechanism whereby P activate steroid receptor-dependent sex behavior. In experiments to better characterize the mechanisms by which PACAP mediated lordosis, E-enhanced irPACAP levels in the VMN and arcuate nucleus of wild-type and PRKO mice and rats. As in the rat, E also induced PACAP mRNA expression in the VMN from wild-type and PRKO mice by 68% and 59% respectively (our unpublished data), Moreover, P alone statistically enhanced MBH irPACAP (and VMN mRNA expression, our unpublished data), whereas MBH tissue concentrations in irPACAP achieved a strong but statistical insignificant (P < 0.07) level. Notably, irPACAP was significantly increased in CSF and near basal levels in tissue only after coadministration of E and P, thus indicating E + P-induced the secretion of PACAP from the MBH. Taken together, the findings support the hypothesis that a steroid-dependent autocrine/paracrine loop exists within the female hypothalamus for P-facilitated receptivity.
Here we have shown that E and P enhance mRNA and protein levels of the paracrine/autocrine neuropeptide PACAP in the female hypothalamus, likely through regulation of cAMP response element (CRE) [and perhaps P response element (PRE)] motifs in the PACAP gene (18, 29, 30). This is consistent with our previous observations (Ref. 17 and our unpublished data) and those of others (11), whereby E + P enhance activity of the cAMP canonical cassette in the MBH from ovx rodents, a finding also observed on the evening of proestrous in intact rats (31). While behavior is dependent upon E-priming and nuclear PR, it is conceivable that P alone induces the synthesis of PACAP by stimulating cAMP or itself (19, 20, 21, 22, 23). Others (11) have shown that changes in cAMP alone are insufficient to induce receptivity, whereas in the presence of E-priming, cAMP facilitates reproductive behavior. Indeed, as suggested by the rise of irPACAP in P only treated wild-type females, the effect of P alone on synthesis of PACAP may not be related to the nuclear PR. Perhaps P interacts with a novel membrane PR that stimulates cAMP (32). Biologically, our present results suggest that dynamic changes in intracellular signaling within relevant hypothalamic cells set the stage for a switch that will allow initiation of sexual behavior.
It is clear that behavior requires both PACAP and E-induced nuclear PR in the VMN. In contrast to E or P alone, when P was given to E-primed rats, cellular secretion of PACAP in the MBH was detected, concomitant with a dramatic increase in receptivity. Because both receptivity and MBH irPACAP content in PRKO but not wild-type mice were significantly reduced, our data indicate that PR influenced receptivity, in part, through the secretion of PACAP within the MBH. In addition, PACAP-receptor blockade of endogenously secreted PACAP inhibited PR-dependent sexual behavior in rats (and mice, data not shown) treated with either P or SKF38393, thus providing a timing mechanism that rapidly amplifies intracellular signaling. Intracellular stimulation of the PACAP pathway may serve to activate the transcription of specific downstream targets that serve as part of a trigger mechanism required for onset of behavior. Indeed, PR-mediated PACAP may serve a number of functions in addition to being a requisite component of the behavioral response including termination of receptivity. We also take note that, in the MBH of E-primed PRKO mice treated with P, irPACAP was not enhanced when compared with E-primed PRKO MBH. Perhaps the nongenomic activity of P induces an inhibitory influence on irPACAP that is not regulated by its genomic action on the nuclear PR. Regardless, our results demonstrate in the VMN that activation of a novel feed-forward intracellular signaling pathway is intrinsically linked to the more conventional genomic activity of PR and is an essential endogenous component for modulation of sex steroid function and reproductive behavior.
Others have reported the physiological relevance of steroid-induced PACAP in several reproductive tissues. For example, in the preovulatory follicle of the ovary, P stimulated PACAP synthesis via PR (33, 34). In turn, PACAP acted as an auto- or paracrine regulator of acute P production in granulose/lutein cells via its activation of PAC1- and VPAC2-mediated cAMP accumulation (35). The effect of P on the ovary was in contrast to its effect in the female pituitary where E-induced PACAP appeared to be a key player in the complex mechanism by which bidirectional intracellular signaling (via para- and/or autocrine activity of PACAP) regulated sensitization of gonadotropes to GnRH for the initiation of the LH surge (36, 37, 38). Koves and colleagues (39) have shown that pituitary release of PACAP depended on the stage of estrous cycle and on the time of day the animals were killed. On the day of proestrous, the number of PACAP-releasing cells peaked in the morning and in the evening toward the end of the LH surge. These data provide further support for the hypothesis that PACAP may play a bidirectional role in timing of reproduction.
In the female brain, immunoreactive protein and mRNA for PACAP and their receptors have been detected within neurons and glial cells in those regions of the adult hypothalamus associated with receptivity and ovulation, e.g. MPOA, arcuate nucleus, midbrain gray, lateral hypothalamus, ventral tegmental area, and both the external and internal zone of the median eminence and the pituitary stalk (24). In the median eminence, PACAP stimulated the synthesis and release of epidermal growth factor receptor ligands that, in turn, stimulated the release of GnRH into the hypophyseal portal vessels (31). In the MPOA, P enhanced mRNA levels of PACAP and PAC-1 receptors, whereas AS to the PACAP-1 receptor reduced GnRH mRNA, concomitant with delayed onset of prepuberty GnRH surge (40). Some irGnRH fibers project to the VMN and arcuate nucleus (11), thus providing an opportunity for PACAP to enhance a low GnRH signal in the VMN for synchronization of sex behavior with ovulation. However, to our knowledge, this is the first examination of PACAP and its relationship to the dynamic role of steroids and their activity in the VMN cells that mediate receptivity. We have provided evidence that PACAP plays a pivotal role in mediating the steroid receptor-induced activity of the VMN coordinate with reproductive behavior. Indeed, given the complexity of PACAP signaling and the multiple functions that PACAP and steroid receptors for E and P play in the hypothalamus, it is likely that PACAP and these receptors play mutually exclusive as well as overlapping roles in reproduction.
The effect of PACAP in the VMN appears complex and may involve several target genes. The present and published data implicate PACAP and cAMP/PKA signaling in the reproductive function of VMN cells. Both inhibition of protein synthesis and reduction of cofactors for steroid receptor transcription eliminated the display of receptivity in E-primed females challenged with PACAP. Moreover, several other genes induced by PACAP are critical for receptivity including preproenkephalin (41, 42). Indeed, E shares several other functions with PACAP. For example, E and PACAP stimulate morphological spine formation in the brain, a process in the female VMN that is associated with reproductive behavior and ovulation (15, 43, 45, 46). Likewise, both E and PACAP mediate neuroprotective actions after ischemic insult to the brain (47). Although we believe PKA and other kinases play an important role for steroid receptor-mediated events, our results again confirm the requirement for receptor-mediated transcription as a component of steroid-induced reproductive behavior and argue against a direct and sufficient membrane or nongenomic explanation for the response. All in all, the putative ability of PR to modulate the release of PACAP concomitant with receptivity suggests PACAP to be an integral upstream participant in neuroendocrine function and synchronization of reproductive behavior with ovulation.
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MATERIALS AND METHODS |
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Behavioral Testing and Stereotaxic Surgery
For all experiments regardless of animal model, cannula position was confirmed by histology at the end of each experiment. Only data from those animals with appropriate cannula placement were assessed and presented. For each experiment, a standard reproductive behavior paradigm for rats was used (3, 4, 5, 7). That is, E-primed [10 µg sc (sc)] females were screened for steroid-dependent behavior after P [1 µg, icv or onto the VMN (i.n.)] 7–10 d after cannulation. Sexual behavior was measured within 15–120 min after P challenge for LQ [LQ = (total no. of positive responses divided by total no. of mounts for a series of four males) ÷ 100]. Results are expressed as percent positive responses for all females mounted by males ± SEM. Animals served as their own control for each experiment. Each experiment also included control groups of E + P and non-E-primed females. Each group consisted of a minimum of six females and each experiment was repeated two to three times.
In our mouse paradigm (5, 7, 8, 14, 17), each experiment consisted of a minimum of wild-type and mutant cannulated mice (n = 12–16 for each genotype) under identical treatments. All mice were primed with E (0.5 µg sc) and screened for P-induced receptivity as described in our standard mouse paradigm (5, 7, 8, 17). Only those wild-type mice exhibiting 90% or greater LQ and those PRKO mice exhibiting less than 8% LQ were used in experiments. Animals served as their own control for each experiment. Each group consisted of a minimum of four females and each experiment was repeated three to four times. For all rodents, cannula placement was confirmed by histology at the end of the experiments.
RIA for PACAP
Ovx rodents were treated as in the above behavioral studies with decapitation at the time of behavioral testing. Tissue was excised and protein was extracted and purified on LH-20 Sephedex columns as described previously (5, 7). All samples were stored at -70 C until RIA by a commercially prepared kit using I125 as tracer and 100 µg supernatant as recommended by manufacturer (Phoenix Pharmaceuticals, Mountain View, CA). The detection limit of the assay was 2 pg/tube and the working range was 2–256 pg/tube. The intra- and interassay coefficients of variation were below 6%. For each animal, extracts of MBH were assayed in duplicate. Samples for each in vivo treatment group were from three animals per treatment group and the study was replicated once. In a separate group of anesthetized animals treated as described above, CSF was collected using a double barrel, third ventricle cannula and slow infusion of artificial CSF (10 µl over 30 min) into one barrel with simultaneous passive collection from the other barrel. MBH were then collected for protein analysis. Because PACAP content in the MBH of these animals were similar to those for animals in which no CSF was collected, data from extracted tissue were collated according to treatment group for final statistical analysis.
Real-Time qRT-PCR Analysis and Two-Step RT-PCR
Rat brain tissue samples (n =3 animals per treatment group) were collected by micropunch technique using anatomical markers as described previously (4, 7). RNA was extracted using Triazol (Invitrogen, Carlsbad, CA) after the manufacturer’s protocol. Approximately 2 µg of total RNA were reverse transcribed with a random hexamer primer and reverse transcription polymerase (Invitrogen) for 50 min at 42 C in 20 µl reaction volume. One microliter of reverse transcriptase product was analyzed by real-time PCR using ABI Prism PE7700 Sequence Analyzer (PE Applied Biosystems, Foster City, CA) and TaqMan Universal PCR chemistry (PE Applied Biosystems) for PACAP and 18S RNA expression. PACAP-specific primers and probe were designed using Primer Express software (PE Applied Biosystems) and after Applied Biosystems guidelines. The amplicon size is 85 bp and extends over exon 2 and exon 3 of rat PACAP. Primers (600 nM) (5'-GTCTCCTGTTCACCTGCCG, Tm = 58 C, 63% GC, and 5'-TGCAGCGGGTTTCCGT, Tm = 58 C, 63% GC) and 200 nM probe (6-carboxy-fluorescein 5'-CTCAGCTTCCCTGGGATCAGACCAGA-3' 6-carboxy-tetramethyl-rhodamine, Tm = 68 C, 58% GC) were used in 50-µl reaction volume in MicroAmp 96-well plates. Thermal cycling conditions included 2 min. AmpErase UNG activity at 50 C, 10 min AmpliTaq Gold DNA polymerase activation at 95 C and 40 cycles of 15 sec at 95 C and 1 min at 60 C. Singleplex PACAP quantities were normalized against 18S RNA amplification (primer/probe set by PE Applied Biosystems), for which input cDNA was diluted 50-fold. Cycle threshold values (Ct) were analyzed using the SDS1.9 software (PE Applied Biosystems), and relative quantification of PACAP expression was determined using the comparative Ct method (ABI Prism 7700 SDS User Bulletin No. 2, Applied Biosystems). The slope of log input amount vs. 
Ct was -0.0623, indicating similar amplification efficiency of PACAP and 18S RNA reference. Thus, 2-Ct gives the amount of PACAP, normalized to endogenous 18S RNA reference and relative to vehicle treated tissue as calibrator. Before qRT-PCR, studies were performed by semiquantitative two-step RT-PCR (Invitrogen Life Technologies, Carlsbad, CA) using micropunched VMN from additional females (three animals per treatment group, replicated twice for a total of nine independent measures per treatment).
Compounds and Rat PR Oligonucleotides
All injections were prepared immediately before administration unless otherwise stated. Whenever possible, doses were based on published studies for effective concentrations for reproductive behavior or verified when appropriate. Steroids were dissolved in sesame oil. Lyophilized phosphothiolated oligonucleotides (Invitrogen Life Technologies), PACAP38 (Calbiochem Laboratories, La Jolla, CA), PACAP6–38 (Pennsula Laboratories, San Carlos, CA), serotonin, anisomycin, and SKF38393 (Sigma Chemical Co., St. Louis, MO) were dissolved in sterile water; aliquots were stored at -70 C until day of use. The AS oligonucleotide sequence for rat PRa (from the rat mRNA sequence kindly provided by K. E. Mayo, Northwestern University, Evanston, IL) was GC TCA TGA GCG GGG ACA ACA and the PRa RS was TG TTG TCC CCG CTC ATG AGC). The mouse SRC-1 (mSRC-1) (48) was GCC ACT GAG GAA AGA CAC CA (accession code U64828, amino acids 3449–3468) and the mSRC-1 RS was GGG GGG TTT TTT TTA CCC CC TTT GAG TGC ATA GTT ACT. For mSRC-2 (49), the AS was TTT GAG TGC ATA GTT ACT (accession code U64828, amino acids 1584–1601) and mSRC-2 RS was CAG ATA ATG ACT ACA TAC. The in vivo specificities for each oligonucleotide have been reported elsewhere (3, 7).
Statistics
Statistical analyses were performed using one-way ANOVA. Dunnett’s test was used for individual comparisons. For behavioral tests, data are presented together for all figures except Fig. 1A because Dunnett’s test for individual comparisons failed to detect differences in LQ due to method of agonist administration (icv vs. i.n.).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: AS, Antisense sequence; CRE, cAMP response element; CSF, cerebral spinal fluid; Ct, cycle threshold; DA, dopamine; D1, DA receptor type 1; E, estrogen; ER, E receptor; icv, intracerebroventricular; i.n., onto VMN; ir, immunoreactive; LQ, lordosis quotient; MBH, medial basal hypothalamus; mSRC-1, mouse SRC-1; ovx, ovariectomized; P, progesterone; PAC1, type 1 PACAP receptor; PACAP, pituitary adenylate cyclase-activating polypepide; PKA, protein kinase A; PR, P receptor; PRE, P response element; PRKO, PR knockout; qRT-PCR, quantitative RT-PCR; RS, randomized antisense sequence; SRC, steroid receptor coactivator; VMN, ventromedial nucleus; VPAC, vasoactive intestinal polypeptide/PACAP receptor.
Received for publication November 19, 2002. Accepted for publication October 21, 2003.
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