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Molecular Endocrinology, doi:10.1210/me.2005-0466
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Molecular Endocrinology 20 (6): 1322-1332
Copyright © 2006 by The Endocrine Society

Differential Response of Progesterone Receptor Isoforms in Hormone-Dependent and -Independent Facilitation of Female Sexual Receptivity

Shaila K. Mani, Andrea M. Reyna, Jian Zhong Chen, Biserka Mulac-Jericevic and Orla M. Conneely

Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030

Address all correspondence and requests for reprints to: Shaila Mani, Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030. E-mail: smani{at}bcm.tmc.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Neurobehavioral effects of progesterone are mediated primarily by its interaction with neural progesterone receptors (PRs), expressed as PR-A and PR-B protein isoforms. Whereas the expression of two isoforms in the neural tissues is suggestive of their selective cellular responses and modulation of distinct subsets of PR-induced target genes, the role of individual isoforms in brain and behavior is unknown. We have previously demonstrated a critical role for PRs as transcriptional mediators of progesterone (ligand-dependent), and dopamine (ligand-independent)-facilitated female reproductive behavior in female mice lacking both the isoforms of PR. To further elucidate the selective contribution of the individual PR isoforms in female sexual receptive behavior, we used the recently generated PR-A and PR-B isoform-specific null mutant mice. We present evidence for differential responses of each isoform to progesterone and dopamine agonist, SKF 81297 (SKF), and demonstrate a key role for PR-A isoform in both hormone-dependent and -independent facilitation of sexual receptive behavior. Interestingly, whereas both the isoforms were essential for SKF-facilitated sexual behavior, PR-A appeared to play a more important role in the 8-bromo-cAMP-facilitated lordosis response, raising the possibility of distinct intracellular signaling pathways mediating the responses. Finally, we also demonstrate that antiprogestin, RU38486, was an effective inhibitor of PR-A-mediated, progesterone-dependent, but not SKF or 8-bromo-cAMP-dependent sexual receptivity. The data reveal the selective contributions of individual isoforms to the signaling pathways mediating female reproductive behavior.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
OVARIAN STEROID HORMONE, progesterone (P), modulates cellular functions in the central nervous system to coordinate reproduction and reproductive behavior in female rodents (1). The effects of P on female reproductive behavior are primarily mediated by its interaction with specific estrogen-induced, intracellular progesterone receptors (PRs), which function as ligand-dependent nuclear transcription factors, regulating the expression of genes and genomic networks in the brain (2, 3). In addition to P, neurotransmitter dopamine (DA) can facilitate sexual receptive behavior in female rats and mice by ligand-independent activation of PRs (4, 5). Thus, neural PRs are critical transcriptional mediators for signaling pathways initiated by both P and DA in the regulation of female sexual behavior.

PRs are expressed as two structurally related, but functionally distinct, isoforms, PR-A and PR-B, derived from a single gene as a result of transcription of two alternate estrogen-inducible promoters and translation initiation at two different AUG codons (6, 7). The isoforms differ in that the PR-B protein contains an additional sequence of 164 amino acids in its amino-terminal region encoding a transactivation function that is not present in PR-A (8, 9). This region encoding a transactivation function, activation function 3, allows the efficient recruitment of a specific subset of coactivators to PR-B and not to PR-A (10), conferring differential cell- and promoter-specific transactivation properties to P-bound PR-A and PR-B (11, 12, 13). The isoforms also respond differently to P antagonists (14). Whereas antagonist-bound PR-A remains inactive, antagonist-bound PR-B can function as a strongly active transcription factor by modulating intracellular phosphorylation pathways (15, 16, 17). Furthermore, PR-A and PR-B isoforms can dimerize and bind DNA as three species: A:A or B:B homodimers or A:B heterodimers, the relative complement of which could determine the significant overall physiological responses to P. Recent in vivo studies in mice with selective ablation of PR-A knockout (PRAKO–/–) and PR-B knockout (PRBKO–/–) proteins, provide compelling evidence that the biological activities of the two isoforms are distinct in that they differentially contribute to P-dependent gene expression and physiological responses in the female reproductive tract and mammary gland (18, 19, 20).

As in other P-responsive tissues, the ratio of PR isoforms varies in the brain of rats during development (21) and under different hormonal conditions (22). RT-PCR analyses of neural PR isoforms in rats reveal the expression of both PR-A and PR-B in different regions of the brain in both males and females (21, 22, 23). Whereas these studies indicate an up-regulation of both PR isoforms by estradiol and down-regulation by P in the female hypothalamus, such a regulation was observed only in PR-B isoform in the preoptic area. PR-A expression alone was up-regulated by estradiol, whereas P had no significant effects on either isoforms in the hippocampus (22). Estradiol and P treatments had no effects on PR isoforms in both the cerebellum and frontal cortex (24). PR isoform expression in the brain has also been demonstrated to vary with estrous cycle in a region-specific manner, with PR-B being predominantly expressed in the hypothalamus on the day of proestrus (23). In the male rat brain, estradiol up-regulates PR-A isoform expression in the cerebellum alone, and not in the other regions, demonstrating a sexually dimorphic pattern of PR isoform regulation by steroid hormones (25). In contrast, using ribonuclease protection analyses for the PR isoforms, Scott et al. (26) demonstrated induction of PR-B by estradiol in the hypothalamus of both male and female rats, under circumstances in which PR-A could not be detected. Studies in estradiol-treated rhesus macaques indicate a region-specific regulation of the PR isoforms, with PR-B expression being predominant in the hypothalamus and PR-A being dominant in the pituitary (27).

Although these observations suggest that the differential expression of the PR isoforms may subserve the region-specific cellular responses to P in the brain, their selective role in mediating reproductive behavior has remained unexplored. To evaluate the contribution of the individual isoforms to reproductive behavior, we used the PR-A and PR-B isoform-specific mutants, generated by introduction of point mutation into the PR gene at the ATG codons encoding Met 1 (M1A) and Met 166 (M166A), to selectively ablate expression of PR-B and PR-A proteins, respectively (18, 20). We now report the results of studies examining the selective contributions of PR-A and PR-B isoforms in the modulation of sexual receptivity in these mutant mice. We present evidence for distinct responses of individual isoform to P and DA and demonstrate a key role for PR-A isoform in both ligand-dependent and -independent facilitation of sexual receptivity. We also provide information on the differential effects of PR antagonist, RU38486, on PR-A- and PR-B-mediated sexual receptive behavior by P and DA. Finally, we also demonstrate differences in the ligand-independent activation of PR-A and PR-B, by DA and cyclic nucleotide, 8-Br-cAMP, suggesting the possible involvement of distinct intracellular signaling pathways in their mediation of the female sexual behavior.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Progesterone Receptor Isoforms Are Ablated in Hypothalamus of the Null Mutants
As a prelude to examining the role of PR-A and PR-B isoforms in female sexual behavior, we set out to confirm the completeness of ablation of the isoforms in the medial basal hypothalamus (MBH) of the PRAKO–/– and PRBKO–/–null mutant mice. MBH tissue samples from wild-type littermates (PR +/+) and total (PR-A + PR-B) PR knockout (PRKO–/–) were used as positive and negative controls, respectively. Estradiol benzoate (EB)-induced PR-A and PR-B proteins in the cytosol from MBH from all four groups of ovariectomized mice were analyzed by Western immunoblotting. EB priming caused a robust induction of both PR-A and PR-B isoforms in the hypothalamus of the wild-type littermates, with PR-B isoform being the dominant protein (Fig. 1Go). Both PR-A and PR-B proteins were detected as doublets, most likely representing different phosphorylated forms of each isoform. PR-B isoform was detectable in the MBH of EB-primed PR-A null mutants (PRAKO–/–) whereas PR-A isoform was not detectable (Fig. 1Go). Interestingly, ablation of PR-A isoform in the PR-A null mutants (PRAKO–/–) demonstrated no apparent up-regulation of the remaining PR-B isoform in the MBH, suggesting no developmental compensatory effects of the PR-B isoform in the absence of PR-A. Similarly, PR-A isoform alone was detectable in the EB-primed MBH of PR-B isoform-specific null mutants (PRBKO–/–), demonstrating that the ablation of PR-B was complete. Thus, the null mutants for PR-A and PR-B retain only one of the isoforms, substantiating our interpretation of their role in subsequent behavioral observations. EB priming was unable to induce either of the isoforms, PR-A and PR-B, in MBH of PRKO–/– mice (Fig. 1Go). Normalization of the protein content also confirmed no evidence of up-regulation of the detectable isoform upon ablation of the other.


Figure 1
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  		Fig. 1. EB-Induced PR-A and PR-B Isoforms in the MBH of Isoform-Specific Null Mutant and Wild-Type Female Mice

All female mice were ovariectomized and primed with EB (0.5 µg sc) 2 wk after surgery. The animals were killed under anesthesia 48 h after priming, and MBH was isolated and processed for Western immunoblotting as described in Methods and Materials. Cytosol protein (100 µg) was loaded onto 7.5% polyacrylamide gels and transferred to nitrocellulose membrane, membrane was probed with antibodies to progesterone receptors, and antibody binding was detected by chemiluminescence as detailed in Materials and Methods. The experiment was performed three times for each sample, and a representative autoradiograph is shown. Top panel depicts PR isoforms detected in MBH cytosol from PR wild type (PR+/+; lanes 1–2), PR-A isoform null mutant (PRAKO–/–; lanes 3–4), PR-B isoform null mutant (PRBKO–/–; lanes 5–6), and PR null mutant (PRKO–/–; lanes 7–8). The molecular weight markers are in lane 9, adjacent to lane 8. The bottom panel represents the ß-actin in the same tissues. Each lane represents tissue from individual MBH (n = 8 for each group). The PR-A and PR-B bands were quantified by densitometry and normalized to ß-actin, and the ratios of PR-A and PR-B to ß-actin were determined. PR-B/ß-actin ratios were 0.51 ± 0.01 (PR+/+), 0.50 ± 0.02 (PRAKO–/–), 0 ± 0 (PRBKO–/–), and 0 ± 0 (PRKO). No significant differences were found between the PRAKO–/– and their wild-type littermates in the PR-B/ ß-actin ratios (P > 0.05). PR-A/ß-actin ratios were 0.40 ± 0.03 (PR+/+), 0 ± 0 (PRAKO–/–), 0.33 ± 0.05 (PRBKO–/–), and 0 ± 0 (PRKO). Statistically significant differences were not found between PRBKO–/– and their wild-type littermates in the PR-A/ß-actin ratios (P > 0.05).

 
Hormonal Facilitation of Sexual Receptivity in PR-A and PR-B Null Mutants
Because hormonal priming and testing experience contribute to the induction of female sexual behavior in mice (5, 28), the lordosis response of PR-A and PR-B female null mutants was observed weekly, for a period of 8 wk. As shown in Fig. 2AGo, EB alone was not capable of inducing high levels of lordosis in the null mutants or their wild-type littermates during the entire testing period. Administration of P, 48 h after EB priming, resulted in higher levels of lordosis in the wild-type mice (Fig. 2BGo). This increased responsiveness to P improved with weekly priming and testing experience, with lordosis quotients (LQs) reaching more than 80%, as has been previously demonstrated (5). In contrast, PRAKO–/– mice showed minimal P-facilitated lordosis in the presence of males during the entire testing period, demonstrating that the dominant PR-B isoform alone was incapable of mediating the effects of P on sexual behavior. Notably, whereas PRBKO–/– mice demonstrated improved PR-A-dependent receptivity with experience (Fig. 2BGo), as reflected by the increase in their LQs, the responses were lower compared with their wild-type littermates.


Figure 2
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  		Fig. 2. Hormonal Induction of Sexual Receptivity in Ovariectomized Progesterone Receptor Isoform Null Mutant Mice and Their Wild-Type Littermates

All female mice, progesterone receptor A null mutants (PRAKO–/–), progesterone receptor B null mutants (PRBKO–/–), and progesterone receptor wild-type mice (PR+/+) were administered EB (0.5 µg) alone (panel A) or EB followed by P (100 µg) 48 h later (panel B). The hormones were administered every week for 8 wk and tested weekly for sexual receptivity in the presence of wild-type PR male mice as described in Materials and Methods. C, Representative summary of the sexual receptive behavior on wk 8 of repeated hormonal priming and testing. Background strains C57BL/6 and 129SvEv were similarly treated and tested. Statistically significant (*, P < 0.05) differences were observed in the behavioral response of PRAKO–/– null mutants compared with the wild-type animals upon EB+P treatment. Values are represented as mean LQ (lordosis responses per mounts x 100) ± SEM (n = 10–12 animals for each group). Veh, Vehicle.

 
A summary of the differential response of PR-A and PR-B isoforms to EB+P on wk 8 is depicted in Fig. 2CGo. PRAKO–/– mice showed significantly reduced levels of P-facilitated lordosis compared with their wild-type littermates (P < 0.05; Fig. 2CGo). No significant differences in P-facilitated lordosis response were observed between PRAKO–/– and PRBKO–/– null mutants (P > 0.05). Similar to our earlier observations (5), both the background strains, C57BL/6 and 129SvEv, displayed high levels of P-facilitated lordosis, indicating that the strain variations had no effect on the current behavioral observations (Fig. 2CGo). Thus, PR-B expression alone is insufficient to mediate P-facilitated lordosis response, and its coexpression with PR-A clearly contributes to the overall lordosis response observed in PR+/+ animals, indicating that functional participation of both the isoforms is critical for P-mediated effects on sexual receptivity.

Ligand-Independent Activation of PR-A and PR-B Isoforms in Sexual Receptivity
Our previous studies have demonstrated a critical role for PRs as transcriptional mediators of DA-facilitated sexual receptive behavior in female rats and mice. EB-primed PRKO mice with targeted deletion of PRs exhibited minimal levels of lordosis in response to DA agonist, validating that DA-facilitated lordosis response was PR dependent in mice (5). To evaluate the selective contributions of PR-A and PR-B isoforms in ligand-independent mechanism of activation by DA, we examined the effects of DA agonist, SKF 81297 (SKF), in the facilitation of lordosis response in PRAKO–/– and PRBKO–/– mice. As seen in Fig. 3AGo, EB priming had no significant effect on lordosis response in PRAKO–/–, PRBKO–/–, and PR+/+ mice (P > 0.05). Kruskall-Wallis one-way ANOVA on ranks indicated a statistically significant overall effect of EB + SKF treatment, over EB-treatment alone, in all genotypes (P < 0.0001) examined. Statistically significant differences in SKF-facilitated lordosis response were not observed between the genotypes (EB-primed), although the PR+/+ mice demonstrated higher lordosis response levels (P > 0.05). EB-primed, PRBKO–/– mice responded to SKF treatment by exhibiting higher lordosis quotient compared with PRAKO–/– mice treated identically. These data indicate that PR-A is capable of mediating SKF-facilitated lordosis response, although not to the same levels as seen in the PR+/+ mice containing the full complement of PR-A and PR-B.


Figure 3
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  		Fig. 3. Ligand-Independent Induction of Sexual Receptivity in PR Isoform-Specific Null Mutant Mice by DA Agonist SKF, 8-Br-cAMP, and Serotonin

Ovariectomized PRAKO–/–, PRBKO–/–, and PR+/+ mice were primed weekly with 0.5 µg EB and 100 µg P and tested weekly for 4 consecutive weeks for sexual receptive behavior as described in Materials and Methods. On wk 5, stainless steel cannulae were implanted stereotaxically into the third cerebral ventricle. On wk 6, the cannulated animals were injected sc with 0.5 µg EB, followed by icv administration of (A) dopamine D1 agonist SKF (50 ng) or (B) 8-Br-cAMP (50 ng) or (C) SKF+8-Br-cAMP or (D) serotonin (Ser, 50 ng) 48 h later. Sexual receptive behavior in the presence of PR wild-type male mice was observed 30 min later. Control mice received vehicle (Veh; artificial cerebrospinal fluid) or EB followed by artificial cerebrospinal fluid. Statistically significant differences (P < 0.01) were observed in SKF, 8-Br-cAMP, and serotonin-treated animals compared with EB-treated controls. ANOVA followed by Dunn’s test indicated no significant differences in the lordosis responses between the groups under the same treatment (P > 0.05).

 
A critical role for PR-A isoform as a dynamic mediator of ligand-independent activation of sexual receptivity was also confirmed in studies using cyclic nucleotide, 8-bromo-cAMP (8-Br-cAMP). As shown in Fig. 3BGo, intracerebroventricular (icv) administration of 8-Br-cAMP to EB-primed PRAKO–/–, PRBKO–/–, and their wild-type littermates, facilitated sexual receptive behavior in PRBKO–/– and PR+/+, but not in the PRAKO–/– mice. The reduction in 8-Br-cAMP-facilitated lordosis in PRAKO–/– mice (containing only PR-B isoform) was significant compared with PRBKO–/– and the wild-type littermates (P < 0.05). Although the PRBKO–/– null mutants exhibited lower LQs compared with the wild-type littermates, the decrease was not significant. Thus, PR-A isoform appears to be more ligand-independently active in the presence of 8-Br-cAMP, indicating subtle differences in the response of isoforms to ligand-independent activation. Combined administration of SKF and 8-Br-cAMP resulted in an enhanced response, with PRBKO–/– null mutants demonstrating higher LQs (Fig. 3CGo). PRAKO–/– mice, however, were not able to exhibit more than 30% lordosis response in the presence of PR-B alone, demonstrating that PR-A is both necessary and sufficient for the lordosis response in mice stimulated via the ligand-independent pathway, but not sufficient for the full magnitude of response observed in the presence of both PR-A and PR-B.

Intracerebroventricular administration of serotonin, previously shown to stimulate lordosis in a PR-independent manner (5), facilitated sexual receptive behavior in both PRAKO–/– and PRBKO–/– null mutant mice (Fig. 3DGo), confirming our previous observations that PRs were not involved in serotonin-facilitated lordosis response.

Progesterone Receptor Antagonist RU38486 Effects on PR-A and PR-B Isoform Mutants: Relationship to Sexual Receptivity
Human and chicken PR-A and PR-B proteins respond differentially to PR antagonist RU38486 in vitro. Whereas the antagonist bound PR-A remains inactive, antagonist bound PR-B functions as an active transcription factor and modulates cAMP-mediated signaling pathways (14). We therefore examined the physiological effects of antagonist-bound PR-A and PR-B isoforms on ligand-dependent and -independent mechanisms of activation of sexual receptivity in mice. A dose-response effect for RU38486 inhibition of P-facilitated lordosis in EB-primed wild-type mice indicated an effective inhibition of sexual receptive behavior at a dose of 1 µg (Fig. 4Go). As seen in Fig. 5AGo, P had no effect on lordosis response in EB-primed PRAKO–/– mice, whereas EB-primed PRBKO–/– and wild-type PR+/+ mice responded to P displaying higher levels of lordosis. RU38486 had no effect on P-facilitated lordosis response mediated through PR-B isoform (in PRAKO–/– mice). The PR antagonist, however, significantly inhibited P-facilitated lordosis in the PRBKO–/– mice (P < 0.01) and their wild-type littermates (P < 0.001). RU38486, by itself, had no effect on lordosis response of the three genotypes tested (Fig. 5AGo). In contrast to its effects on P-dependent activation of lordosis, the antagonist RU38486 had no significant inhibitory effects on SKF-facilitated responses mediated by PR-A isoform in the PRBKO–/– mice (Fig. 5BGo). RU3846 did not significantly inhibit SKF-facilitated lordosis response in the PRAKO–/– and the wild-type littermates. The antagonist had no effect on 8-Br-cAMP-facilitated lordosis in PRAKO–/– and PRBKO–/– mice. RU38486, however, significantly reduced 8-Br-cAMP-facilitated lordosis response in wild-type littermates (P < 0.05; Fig. 5CGo). Similar effects of RU 38486 were also observed on SKF+ 8-Br-cAMP-facilitated lordosis (Fig. 5DGo). Thus, both isoforms of PR, probably via heterodimerization, appear to be required in the RU38486 effects on ligand-independent PR-mediated lordosis.


Figure 4
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  		Fig. 4. Dose Response of icv Administered RU38486 on P-Facilitated Lordosis Response of PR Wild-Type Mice in the Presence of Wild-Type PR Male Mice

Ovariectomized wild-type PR mice (PR+/+) were subjected to weekly hormonal priming and behavioral testing as described for Fig. 2Go. Third cerebral ventricle cannulations were performed on wk 5, followed by EB priming on wk 6 and icv administration of RU38486 (RU, 0.1–2µg) 48 h later. P was infused 60 min after RU, and the mice were tested for sexual responsiveness 30 min after icv administration of P. Control groups of animals received vehicle (Veh; artificial cerebrospinal fluid) or EB followed by artificial cerebrospinal fluid (EB) or EB followed by P (EB+P). Statistically significant differences were observed (*, P < 0.05) in EB+ RU+P animals compared with EB+P-treated controls (n = 10 animals for each group).

 

Figure 5
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  		Fig. 5. PR Antagonist, RU38486 (RU) Effects on P-Dependent and -Independent Facilitation of Sexual Receptive Behavior in PR Isoform-Specific Null Mutant Mice

Ovariectomized PRAKO–/–, PRBKO–/–, and PR+/+ mice were primed weekly with 0.5 µg EB and 100 µg P and tested weekly for 4 consecutive weeks for sexual receptive behavior as described in Materials and Methods. On wk 5, stainless steel cannulae were implanted stereotaxically into the third cerebral ventricle. On wk 6, the cannulated animals were injected sc with 0.5 µg EB followed 48 h later by icv administration of RU (1 µg). After RU administration (60 min), the animals were given icv infusions of P (panel A) or SKF (panel B) or 8-Br-cAMP (panel C) or SKF+8-Br-cAMP (panel D) and tested 30 min later for sexual receptive behavior in the presence of wild-type PR male mice. Control mice received EB followed by artificial cerebrospinal fluid, RU, SKF, 8-Br-cAMP, or SKF+8-Br-cAMP. ANOVA followed by Dunn’s test for multiple comparisons indicated statistically significant differences in P-facilitated receptive behavior in PR+/+ (**, P < 0.001) and PRBKO–/– (*, P < 0.01) mice that received RU treatment compared with EB+P treatment alone. No significant differences were observed with or without RU treatment on SKF-facilitated lordosis behavior in any of the three groups examined (P > 0.05). RU significantly inhibited 8-Br-cAMP (***, P < 0.05) and SKF+8-Br-cAMP (**, P < 0.001) facilitation of lordosis in PR+/+ mice.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Using PR null mutant mice (PRKO–/–), we have previously demonstrated a critical requirement of PRs as transcriptional mediators in the cross talk between P and DA-initiated pathways in the facilitation of female sexual receptive behavior (5). In the present study, we have extended these observations to elucidate the distinct and selective contribution of the individual PR isoforms, PR-A and PR-B, in mediating the ligand-dependent and -independent mechanisms of facilitation of female receptive behavior. Using the recently generated PR isoform-specific null mutant mice, PRAKO–/– and PRBKO–/–, we have shown that PR-A isoform plays a key role in mediating hormone-dependent and -independent sexual receptive behavior in female mice. PRAKO–/–mice displayed significantly reduced levels of P-facilitated lordosis response compared with their wild-type littermates. PRBKO–/–mice responded to P, with higher LQ than the PRAKO–/– mice, but lower than the wild-type littermates. Both, PRAKO–/–and PRBKO–/– mice displayed SKF-facilitated lordosis response, although not to the same level as their wild-type littermates. Interestingly, 8-Br-cAMP-facilitated lordosis response was significantly reduced in PRAKO–/– mice compared with their wild-type littermates. Furthermore, whereas PRBKO–/– mice responded to 8-Br-cAMP and displayed lordosis, their response levels were lower than the wild-type mice. These data suggest a predominant role for PR-A isoform, while delegating a contributory role for PR-B, in mediation of female sexual receptive behavior. The studies also indicate that although PR-A expression alone is sufficient for lordosis response, it is not adequate to mediate the full magnitude of the response, suggesting that the functional participation of both the isoforms contributes to the overall lordosis response.

Similar to earlier observations (5, 28), our current data also indicate that sequential hormonal treatment of EB + P and testing experience accentuate a robust receptive response in ovariectomized female mice. Whereas such a paradigm demonstrated increases in lordosis response in hormonally primed mice, the lordosis levels did not substantially increase in PRAKO–/– null mutants, indicating that the PR-B isoform alone was incapable of mediating P-facilitated lordosis responses. Interestingly, PRBKO–/– mice demonstrated increased responsiveness to repeated hormone administration and testing, despite displaying reduced levels of lordosis (< 50% LQ) even at 8 wk of testing, revealing the importance of PR-A in P facilitation of lordosis. In contrast, the wild-type littermates exhibited >80% LQ by wk 8, further implying that whereas PR-A was capable of mediating P-facilitated lordosis response, functional contribution of both PR-A and PR-B, most likely through heterodimerization, appears to be more effective for the display of the full complement of hormone-dependent receptive behavior in mice. Such a cooperative influence of one PR isoform on the other has been demonstrated in the P-binding properties in vitro (14, 29, 30). In this context, it is interesting to note, that whereas our in vivo results are in general agreement with the in vitro observations revealing distinct functional roles for PR-A and PR-B in human and chicken (11, 12, 31), a dominant P-dependent transcriptionally active role for PR-B isoform was not evident as observed in vitro in other cell types (14, 32, 33, 34). An increase in expression of PR-B upon deletion of PR-A and vice versa was not apparent in the hypothalamic tissues, eliminating the possibility of developmental compensation being the causative factor. That a developmental defect could be responsible for the lack of sexual behavioral response in PR-A null mutants is also refuted by our current observations that the neurotransmitter, serotonin, was capable of inducing a lordosis response in both PRAKO–/– and PRBKO–/–, consistent with our previous reports that this response is independent of PRs.

Studies of human PR reveal that, although 8-Br-cAMP potentiates the ability of progestins to induce the transactivation function of the PR in T47 D cells, human PR is not activated in a ligand-independent manner (35). Chicken PR, on the other hand, has been shown to be activated in a ligand-independent manner by modulators of kinases and phosphatases, growth factors and neurotransmitters (36, 37, 38), suggesting distinct species variations. Ligand-independent activation of PR by DA agonists and cAMP-enhancing agents has also been demonstrated in the mouse (5). The contribution of PR-A and PR-B isoforms appears to be distinctive for ligand-independent mechanisms of facilitation of female sexual behavior by DA agonist SKF and protein kinase A activator, 8-Br-cAMP. Intracerebroventricular administration of SKF induced lordosis response in both PRAKO–/– and PRBKO–/– mutant mice, although not to the same level as in their wild-type littermates. These data suggest that whereas PR-A and PR-B are both independently capable of mediating SKF-facilitated receptivity, the individual isoforms are not sufficient to mediate the full magnitude of response contributed by both PR-A and PR-B in wild-type mice. 8-Br-cAMP effects on lordosis were primarily mediated via PR-A isoform, because PRAKO–/– mice failed to display lordosis and PRBKO–/– mice exhibited reduced levels of lordosis, compared with wild-type mice, suggesting that 8-Br-cAMP and DA are perhaps using different mechanisms to induce activation of PRs.

Studies on site-directed mutagenesis of phosphorylation sites of chicken PR (cPR; common to both A and B isoforms) have demonstrated that ligand-independent activation of cPR in response to DA or 8-Br-cAMP is not mediated by receptor phosphorylation per se (39), but rather is a consequence of altered phosphorylation states of accessory proteins/factors that interact with PR. Recent studies indicate that cAMP-induced phosphorylation of steroid receptor coactivator 1 (SRC-1) markedly enhanced functional cooperation with other receptor-associated proteins to elicit target gene transcription (40, 41). Phosphorylation did not require cPR-A expression, indicating that the association of SRC-1 with the receptor was not a prerequisite for phosphorylation of SRC-1. Furthermore, phosphorylation of SRC-1 by 8-Br-cAMP also involved activation of yet another signaling pathway, MAPK pathway in COS-1 cells (41). It is possible that DA and cAMP activation of murine PRs could involve altered phosphorylation of distinct coactivators, several coactivators or other phosphorylation sites on the coactivators in the multicomponent steroid receptor complexes that serve as a sensor(s) for multiple modulatory signals.

Interestingly, an enhanced effect of SKF and 8-Br-cAMP was observed in facilitation of lordosis response in PRBKO–/– mutant mice, suggesting that the enhanced effect was more pronounced on PR-A than on PR-B isoform. Such an increase has also been observed in cAMP enhancement of P-dependent activation of human PR (35), with hPR-A showing greater transcriptional activity than hPR-B in vitro (32). Thus, convergence of multiple signaling pathways activated by ligand-independent mechanisms appears to be a reinforcing mechanism to achieve maximal functional outcome in reproductive behavior.

P antagonist RU38486 represses the physiological actions of P by actively inhibiting PR activation by several mechanisms: by promoting a higher affinity interaction of PR with DNA than the agonist (42), by its ability to heterodimerize with agonist-bound PR (30, 43), and by its ability to recruit corepressors, silencing mediator of retinoid and thyroid hormone receptor and nuclear receptor corepressor (44, 45). It has been shown that RU38486 can function as a partial agonist under certain cellular conditions (46, 47). This antagonist to agonist switch on PR transactivation (48, 49) was demonstrated to be specific to PR-B isoform and selective to cAMP-enhancing agents (14, 17). It has also been reported that cAMP dissociates the interaction of RU38486-bound PR with corepressors, accompanied by facilitation of PR-coactivator interactions to induce partial agonist activity (45). Thus, PR-A and PR-B isoforms respond differentially to RU 38486, not only for ligand-dependent activation, but also for ligand-independent activation in vitro.

The effects of RU38486 in vivo on hormone-dependent and -independent facilitation of sexual receptive behavior in PRAKO–/– and PRBKO–/– mice were distinct. Whereas the antagonist inhibited the P-facilitated lordosis in the wild-type and the PR-B null mutant mice, it had no significant effect on lordosis response of PR-A null mutants. Similarly, the antagonist effects on SKF or 8-Br-cAMP-facilitated lordosis response in PRAKO–/– mice were not significant. However, antagonist-bound PR-A appeared to exhibit agonist properties in the presence of SKF and 8-Br-cAMP, because it did not reduce the facilitatory effects of the compounds in PR-B null mutant mice. Interestingly, the antagonist completely inhibited the facilitatory effects in the wild-type littermates, suggesting that the functional contribution of both the isoforms, perhaps by heterodimerization, was a requisite for the effective inhibition by RU38486. The behavioral phenotypes observed in these studies do not agree with the published in vitro findings, suggesting the possibility that, in addition to the species differences, tissue-specific variations could be contributory factors. It also remains to be determined whether tissue-specific coactivators and corepressors have a role in this isoform-dependent reproductive behavior.

In summary, PRAKO–/– and PRBKO–/– mice have provided valuable insights into the role of individual isoforms in P-dependent and -independent facilitation of reproductive behavior. The studies provide compelling evidence that the isoforms have the differential capacity to regulate tissue-specific responses to distinct signaling mechanisms in mediating the complex reproductive behavior. Cell- and/or tissue-specific responses to steroid hormones and other signaling molecules could be potentially modulated by the interactions of the receptor isoforms with coactivators and corepressors to facilitate PR-dependent reproductive behavior.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Female PRAKO–/– and PRBKO–/– null mutant mice and their wild-type littermates, generated at the breeding colony at Baylor College of medicine, were obtained at 4 wk of age. Wild-type males (8 wk old) belonging to the same mutant strains were also obtained from the same breeding colony and used as stimulus males in the behavior studies. The mice were of mixed background strain (C57Bl6J x 129SvEv). All the animals were maintained on a 12-h light, 12-h dark reversed light cycle with lights off at 0012 h, and food and water available ad libitum. Their care and maintenance were in compliance with Federal guidelines and approved by the Institutional Animal Protocol and Care Committee of Baylor College of Medicine.

All steroids, 8-Br-cAMP, DA agonist SKF, and serotonin were obtained from Sigma-RBI Chemical Co. (St. Louis, MO). Reagents for Western analyses were purchased from Bio-Rad Laboratories (Hercules, CA), and the primary antibodies for PRs, PR (C-19) and PR (C-20), were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Progesterone receptor antagonist, RU38486, was obtained from Roussel-UCLAF (Romainville, France). All other chemicals were of reagent grade and obtained from Fisher Scientific (Pittsburgh, PA) or Sigma Chemical Co. (St. Louis, MO).

Procedures
Female mice used in this study were ovariectomized under anesthesia following approved protocols, 2 wk before hormonal manipulation. All hormones were dissolved in sesame oil and injected sc unless specified. The mice were primed with EB (0.5 µg) 48 h before the administration of P (100 µg), and behavioral testing was performed in the presence of wild-type males 6 h later. The doses of hormones used and the testing methodologies were based on our published studies (5). The female mice were subjected to weekly administration of steroid hormones and testing for their sexual receptive behaviors in the presence of males for 8 consecutive weeks. The behavioral experiments were repeated three times with different generations of mice.

Behavioral Studies
All behavioral tests were conducted under red light illumination during the dark phase of the reversed light-dark cycle. A single test of receptivity involved placing of the female mouse into the home cage of a male, placed on a mirror stand, with the mirror at an inclined angle of 45° to allow ventral viewing. The male mice used were wild-type littermates of the same background strain as the experimental mice. Sexual behavior tests were carried out for 30 min after the introduction of the female or until the male mounted the female 10 times (whichever occurred first) as per published procedures (5). Only the mounts in which the male showed pelvic thrusting were scored. The male was replaced with a different male if he did not mount the female during the testing period. A positive lordosis response of a female was typified by its display of an immobile posture with the arching of the back and elevation of the hindquarters to facilitate male intromission upon mounting. Failure to achieve lordosis was demonstrated by responses such as vocalization and active attempts by the female to escape and/or display of a boxing posture with the hindquarters down. LQ was used as a measure of sexual receptivity and calculated as a percentage of the number of lordosis responses divided by the total number of mounts by the male. The observers were blind to the genotype and treatment conditions until all behavioral measurements were completed.

Stereotaxic Surgery and Central Administration of Compounds
Stereotaxic implantation of the guide cannula into the third cerebral ventricle of the female mice was performed as previously described (5). The cannula was implanted on wk 5 of repeated hormone (EB + P) priming and behavioral testing. Intracerebroventricular administration of compounds, P, DA agonist SKF, serotonin, progesterone receptor antagonist RU38486, and cyclic nucleotide, 8-Br-cAMP, was performed on wk 6. The compounds were administered 48 h after EB priming (sc) of the experimental animals, and behavioral observations were done 30 min later. In studies dealing with antagonist administration, the antagonist was always administered icv, 60 min before the administration of P, SKF, and 8-Br-cAMP. A dose-response curve for effective inhibition of P-facilitated lordosis by RU38486 was generated in wild-type mice, and a suitable dose was then selected for further studies. The doses of SKF and 8-Br-cAMP were based on previous studies (4, 5, 50).

Western Immunoblotting for PR Isoforms in the Hypothalamus
Female PRAKO–/– and PRBKO–/– mice, their wild-type littermates (PR+/+), and PRKO–/– mice (8) were ovariectomized at 4 wk of age. Two weeks after ovariectomies, the mice were primed with EB (0.5 µg sc) for 48 h and killed under anesthesia, after which the tissues were processed at 0–4 C. The MBH was dissected out, bounded caudally by the caudal edge of the mamillary bodies and rostrally by the caudal edge of the optic chiasm. Diagonal cuts were made extending from hypothalamic fissures to the midpoint of corpus callosum to form the lateral boundaries, and a cut below the level of fornix formed the dorsal boundary. Tissues were homogenized in TEGT [10 mM Tris-HCl, 1.5 mM Na2EDTA, 10% glycerol, and 12 mM monothioglycerol (pH 7.4)] containing protease inhibitors (0.5 mM leupeptin, 0.5 mM pepstatin, 1 mM aprotinin, 1 mM bacitracin, 5 mM phenylmethylsulfonylfluoride, and 1 mM sodium orthovanadate) using a Powergen 125 tissue grinder with a 7-mm probe (Fisher Scientific). Homogenates were centrifuged at 48,000 x g for 60 min at 4 C, and high-speed supernatant was collected. Protein concentrations in the supernatant consisting of cytosol were determined using the colorimetric bicinchoninic acid assay kit from Pierce Chemical Co. (Rockford, IL).

Equal amounts of protein (100 µg) from each sample were electrophoresed on SDS-PAGE [7.5% polyacrylamide (51)] gels followed by transfer to a nitrocellulose membrane for Western blotting (52). The membranes were immunoblotted using commercially available polyclonal antibodies to PR from Santa Cruz Biotechnology. The primary antibodies consisted of a combination of polyclonal antibodies, PR C-19 (epitope mapping an internal region; sc538) and PR C-20 (epitope mapping C terminus; sc539) at 1:200 final dilution. Antibody binding was revealed by incubation with donkey antirabbit horseradish peroxidase-conjugated IgG (1:10,000) followed by chemiluminescence detection with the enhanced chemiluminescence reagent ECL (Amersham Pharmacia Biotech, Piscataway, NJ). MagicMarker XP western standards (Invitrogen, Carlsbad, CA) were included for molecular weight determination. The PR-A and PR-B bands were quantified by densitometry with the use of Phosphor Imager:SF (Molecular Dynamics, Sunnyvale, CA). The protein content was normalized to ß-actin, a housekeeping protein, by stripping and reprobing the blots with a goat anti-ß actin polyclonal antibody (Santa Cruz Biotechnology). The linear range of signals for densitometry was obtained by exposing the chemiluminescent membranes to x-ray film for varying periods of time. MBH from each animal was individually processed and subjected to Western immunoblotting. Each group consisted of eight animals.

Data Analysis
Statistical analysis was done using Prism (GraphPad Software, Inc., San Diego, CA). For each significant ANOVA, post hoc comparisons were made using Dunn’s method for comparison of all groups vs. the control group or Tukey-Kramer method for multiple comparisons.


   ACKNOWLEDGMENTS
 
We thank Loc H. Nguyen for technical assistance.


   FOOTNOTES
 
This work was supported by United States Public Health Service Grants MH57442 and MH63954 (to S.K.M.) and HD32007 (to O.M.C.) from the National Institutes of Health.

The authors listed in the manuscript (S.K.M., A.M.R., J.Z.C., B.M.-J., O.M.C.) have nothing to declare.

First Published Online February 16, 2006

Abbreviations: 8-Br-cAMP, 8-Bromo-cAMP; DA, dopamine; EB, estradiol benzoate; icv, intracerebroventricular; LQ, lordosis quotient; MBH, medial basal hypothalamus; P, progesterone; PR, progesterone receptor; PRAKO, PR-A knockout; PRBKO, PR-B knockout; SKF, SKF 81297.

Received for publication November 21, 2005. Accepted for publication February 6, 2006.


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NURSA Molecule Pages Link:

Nuclear Receptors:   PR
Ligands:   Progesterone  |  RU486



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