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Endocrinology Vol. 145, No. 12 5560-5567
Copyright © 2004 by The Endocrine Society

Regulation of Lordosis by Cyclic 3',5'-Guanosine Monophosphate, Progesterone, and Its 5{alpha}-Reduced Metabolites Involves Mitogen-Activated Protein Kinase

Oscar González-Flores, Jun Shu, Ignacio Camacho-Arroyo and Anne M. Etgen

Department of Neuroscience, Albert Einstein College of Medicine (O.G.-F., J.S., A.M.E.), Bronx, New York 10461; Centro de Investigación en Reproducción Animal, Centro de Investigacion y de Estudios Avanzados-Universidad Autonoma de Tlaxcala (O.G.-F.), Tlaxcala 90140, Mexico; and Departamento de Biología, Facultad de Quimica, Universidad Nacional Autonoma de Mexico (I.C.-A.), Coyoacan 04510, Mexico

Address all correspondence and requests for reprints to: Dr. Anne M. Etgen, Department of Neuroscience, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461. E-mail: etgen{at}aecom.yu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Progesterone (P) and its ring A-reduced metabolites regulate sexual behavior in ovariectomized, estrogen-primed female rats when they are administered intracerebrally and systemically. The present study tested the hypothesis that the MAPK pathway participates in P facilitation and sequential inhibition of sexual behavior. The role of MAPK in lordosis facilitation by two ring A-reduced metabolites of P, 5{alpha}-dihydroprogesterone (5{alpha}-DHP) and 5{alpha},3{alpha}-pregnanolone (5{alpha},3{alpha}-Pgl), was also assessed. In Experiment 1, the MAPK inhibitor PD98059 was infused intracerebroventricularly before progestin administration. Lordosis behavior induced by P, 5{alpha}-DHP, and 5{alpha},3{alpha}-Pgl was abolished 2 h after progestin ad-ministration by PD98059. P and 5{alpha},3{alpha}-Pgl facilitation of proceptive behaviors was also decreased by the MAPK inhibitor. Experiment 2 examined the effects of MAPK inhibition on P sequential inhibition. Estrogen-primed females received intracerebroventricular infusions of PD98059 or vehicle 30 min before systemic administration of P and were tested for lordosis 4 h later. Animals received a second injection of P 24 h later and were retested for lordosis. The MAPK inhibitor blocked both lordosis facilitation and sequential inhibition produced by systemic administration of P. Because cGMP can also facilitate lordosis behavior, and cGMP-dependent protein kinase can activate MAPK, experiment 3 determined whether interference with MAPK would affect cGMP enhancement of lordosis. The icv infusion of PD98059 significantly inhibited lordosis behavior induced by 8-bromo-cGMP, a cell-permeable cGMP analog, at both 2 and 4 h. These data support the hypothesis that the MAPK pathway is involved in lordosis regulation by P and some of its ring A-reduced metabolites as well as by the second messenger, cGMP.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MANY HORMONES AND neurotransmitters facilitate female sexual behavior when administered to estrogen-primed rodents (for review, see Refs.1 and 2). Among these are progesterone (P) and several of its ring A-reduced metabolites, which promote the expression of intense lordosis behavior when administered intracerebrally or systemically (3, 4, 5, 6, 7). The cellular mechanisms underlying the sexual behavior induced by ring A-reduced progestins are not completely understood. It is well known that many ring A-reduced progestins allosterically modulate {gamma}-aminobutyric acid A (GABAA) receptors (8, 9, 10), and this property may partly account for their actions on female reproductive behavior (11, 12, 13, 14, 15). We have recently implicated the second messenger cGMP and the cGMP-dependent protein kinase (PKG) in the facilitation of lordosis by ring A-reduced metabolites of P (16). Other evidence suggests that the P receptor (PR) could be an important molecular effector. Despite the low PR binding affinity of some behaviorally active, ring A-reduced progestins (17, 18, 19, 20, 21), the PR antagonist RU 486 attenuates the facilitatory effects of these progestins on lordosis behavior (3, 5).

Protein phosphorylation is one major mechanism by which a variety of hormones and neurotransmitters regulate intracellular events in mammalian cells (22). Protein kinases, which catalyze the phosphorylation of proteins and are often activated by second messengers, may participate in the facilitation of lordosis by P and its ring A-reduced metabolites. Analogs of cAMP and cGMP facilitate lordosis in estrogen-primed rats (1, 23, 24, 25, 26, 27, 28). Likewise, inhibitors of PKG and the cAMP-dependent protein kinase abolish or attenuate progestin-facilitated receptivity (27, 29). Cyclic nucleotide enhancement of female reproductive behavior also appears to involve PR, because RU 486 inhibits the lordosis induced by these agents (25, 27).

The PR undergoes phosphorylation upon agonist binding, and this is thought to be essential for PR activation (30, 31, 32). Thus, it might be hypothesized that cyclic nucleotides facilitate lordosis by cAMP-dependent protein kinase- and PKG-dependent phosphorylation of the PR. However, available evidence indicates that the PR is not phosphorylated by either of these kinases (31, 32, 33). The PR is phosphorylated by MAPK and cyclin-dependent protein kinases (34, 35, 36, 37, 38). Although these kinases are generally associated with growth factor signaling, recent evidence implicates both growth factors and MAPK in estrogen priming of estrous behavior in rats (39, 40). Moreover, MAPK phosphorylation can target PRs for degradation by the 26S proteasome, and we have shown that this pathway may mediate P sequential inhibition of lordosis (41). Therefore, the purpose of the present study was to test the hypothesis that MAPK participates in the facilitation and sequential inhibition of lordosis behavior by P. We also tested the role of MAPK in lordosis facilitation by two ring A-reduced metabolites of P, 5{alpha}-dihydroprogesterone (5{alpha}-DHP) and 5{alpha},3{alpha}-pregnanolone (5{alpha},3{alpha}-Pgl), in estrogen-primed rats. Because there is evidence that PKG can activate MAPK (42, 43, 44, 45, 46), we also determined whether interference with MAPK would affect cGMP enhancement of receptive behavior.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal treatments
Adult female Sprague Dawley rats (175–200 g) were purchased from Taconic Farms (Germantown, NY) and maintained on a 14-h light, 10-h dark cycle, with lights off at 1100 h. One week after arrival, animals were anesthetized with xylazine (4 mg/kg) and ketamine (80 mg/kg), placed into a stereotaxic apparatus, and secured with ear bars and nose piece set at +5 mm. A 26-gauge guide cannula (Plastics One, Roanoke, VA) was implanted into the third ventricle using coordinates from the atlas of Pellegrino et al. (47). Animals were bilaterally ovariectomized (OVX) at the same time as stereotaxic surgery. Then rats were housed singly in plastic cages, with food and water available ad libitum. All procedures used in these experiments followed the NIH Guide for the Care and Use of Laboratory Animals and were approved by the institutional animal care and use committee at Albert Einstein College of Medicine (Bronx, NY).

Drug administration and behavior testing
Behavioral testing for the response to intracerebroventricularly (icv) infused progestins or drugs was initiated 1 wk after stereotaxic surgery and OVX, after estradiol benzoate (EB) treatment (2 µg/0.1 ml oil, 24 and 48 h before behavioral testing). Progestins or the cell-permeable cGMP analog 8-bromo-cGMP (8Br-cGMP) were administered icv at the following doses 20.5 h after the second EB injection: 130 ng for P, 13 ng for 5{alpha}-DHP and 5{alpha},3{alpha}-Pgl, and 0.9 ng for 8Br-cGMP. The progestin doses were selected based on a prior report (16) and more extensive dose-response curves (Gonzalez-Flores, O., and C. Beyer, unpublished observations) to produce intermediate to high levels of lordosis in EB-primed rats. An 8Br-cGMP dose that reliably facilitates lordosis was selected based on previous work in the Etgen laboratory (26, 48). Animals (n = 8/group) were assigned to receive one of the three progestins or 8Br-cGMP alone or in combination with the specific MAPK inhibitor PD98059 (3 µg; dose selected from Ref.49). PD98059 inhibits the p42/44 MAPKs, which have been implicated in estrogen priming of lordosis (40) and in MAPK phosphorylation of PRs (34, 35, 36, 37, 38). For 3 consecutive weeks, each animal was infused with PD98059 plus progestin or 8Br-cGMP, vehicle plus progestin or 8Br-cGMP, or vehicle plus vehicle in a counterbalanced order.

Progestins, 8Br-cGMP, and appropriate vehicle were administered 30 min after PD98059 or vehicle, which was infused approximately 20 h after the second EB injection. Progestins were dissolved in peanut oil and injected in a volume of 1 µl. The cell-permeable cGMP analog, 8Br-cGMP, was dissolved in sterile saline, and PD98059 was prepared in 10% dimethylsulfoxide; these agents were infused in a volume of 2 µl. All drugs were administered into the third ventricle through the guide cannula over 1.5 min, and another 1.5 min were allowed for drug diffusion before removal of the infusion needle. All steroids were obtained from Steraloids, Inc. (Newport, RI); 8Br-cGMP and PD98059 were purchased from Calbiochem (La Jolla, CA). Two and 4 h after progestin or 8Br-cGMP administration, females were placed in 20-gallon glass tanks until they received 10 mounts with pelvic thrusting from an experienced stimulus male. The lordosis quotient [LQ = (number of lordosis/10 mounts) x 100] was used to assess receptive behavior. The intensity of lordosis was quantified on a scale of 0–3 according to the lordosis score proposed by Hardy and DeBold (50). Proceptivity was analyzed by determining the incidence of hopping, darting, and ear-wiggling across the entire receptivity test. The proportion of animals displaying any of these behaviors was used as a measure of proceptivity. In a separate group of animals, we evaluated whether the p42/44 MAPKs are specifically involved in lordosis by infusing estrogen-primed rats with 8 µg SB203850 (Tocris, Ellisville, MO), an inhibitor of the p38 MAPKs, before systemic administration of 200 µg P. Lordosis was tested 4 h after P administration.

After the final lordosis test, the animals were anesthetized with halothane, and 1% methylene blue was administered into the cannula. The brain was removed and sectioned in the transverse plane to check the cannula position in the third ventricle. Animals whose cannulae were not in the ventricle were discarded from the experiment.

A separate group of animals was used to test for P facilitation and sequential inhibition of lordosis after systemic administration of P. Hormone administration was initiated 1 wk after OVX. Rats were injected with 2 µg EB (n = 24), and 44 h later were divided into three groups that received the following treatments: group 1, vehicle icv, followed 30 min later by oil, sc (n = 8); group 2, PD98059 icv, followed 30 min later by 1 mg P, sc (n = 8); and group 3, vehicle icv, followed 30 min later by 1 mg P, sc (n = 8). Female sexual behavior was determined 4 h later in all groups (facilitation test). Twenty-four hours later (72 h after EB priming), all animals received 1 mg P, sc. Sexual behavior was determined 4 h later (sequential inhibition test).

Statistical analysis
A Friedman test, followed by a Wilcoxon signed-rank test, were used to determine significant differences between mean LQ scores in the different treatment groups. The proportion of proceptive animals observed in groups that received progestins or 8Br-cGMP plus vehicle were compared with that in groups treated with progestins or 8Br-cGMP plus PD98059 using the Fisher exact test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PD98059 inhibits progestin-facilitated reproductive behavior
Figure 1Go shows the LQ values obtained 2 and 4 h after icv injection of P, 5{alpha}-DHP, and 5{alpha},3{alpha}-Pgl in OVX, estrogen-primed rats. At the 2 h test, all three progestins significantly facilitated lordosis compared with EB-primed rats that received no progestin plus PD98059 (P < 0.001). Animals receiving this treatment displayed weak lordosis behavior. LQ values at 4 h after injection of the three progestins were similar to those observed at 2 h. It is important to note that observation of rats infused icv with doses of PD98059 ranging from 0.2–3 µg revealed no evidence of locomotor activation or inhibition (data not shown).



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  		FIG. 1. Facilitation of lordosis behavior in OVX, estrogen-primed rats 2 and 4 h after icv infusion of P (130 ng), 5{alpha}-DHP (13 ng), and 5{alpha},3{alpha}-Pgl (13 ng). The facilitation of lordosis produced by these progestins was antagonized by infusing the MAPK inhibitor, PD98059 (3 µg), 30 min before progestin. Each value is the mean ± SE of eight animals. *, P < 0.01; **, P < 0.001; +, P < 0.003 (vs. corresponding group receiving progestin plus vehicle).

 
Figure 1Go also shows that the lordosis behavior induced by P, 5{alpha}-DHP, and 5{alpha},3{alpha}-Pgl was completely blocked at the 2 h test by the previous injection of 3 µg PD98059 (P < 0.001 for P and 5{alpha},3{alpha}-Pgl; P < 0.01 for 5{alpha}-DHP). LQ values in the 2 h test of the three progestin-infused groups pretreated with the MAPK inhibitor did not differ significantly from those observed in EB-primed rats infused with oil vehicle plus PD98059. At the 4 h test, PD98059 continued to reduce lordosis induced by only 5{alpha},3{alpha}-Pgl (P < 0.003). This may reflect the fact that 5{alpha},3{alpha}-Pgl was more effective than the other progestins in maintaining high levels of receptivity at 4 h postinfusion.

Figure 2Go shows the effect of MAPK inhibition on proceptive behaviors induced by P, 5{alpha}-DHP, and 5{alpha},3{alpha}-Pgl at 2 and 4 h after hormone infusion. P and 5{alpha},3{alpha}-Pgl induced clear proceptivity in most females at 2 h (75% and 87%, respectively) and 4 h (62% for both P and 5{alpha},3{alpha}-Pgl) after hormone injection. In this experiment, 5{alpha}-DHP did not induce high proceptivity. The Fisher exact test showed that PD98059 significantly reduced (P < 0.001) the display of proceptive behaviors at 2 h in P- and 5{alpha},3{alpha}-Pgl-treated rats. At 4 h, PD98059 was less effective in reducing proceptive behavior induced by both progestins.



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  		FIG. 2. Facilitation of proceptivity in OVX, estrogen-primed rats 2 and 4 h after icv infusion of P, 5{alpha}-DHP, and 5{alpha},3{alpha}-Pgl. The proceptive action of P and 5{alpha},3{alpha}-Pgl at 2 h was antagonized by the infusion of PD98059. Drug and hormone doses are as in Fig. 1Go (n = 8/group). **, P < 0.001 vs. corresponding group receiving progestin plus vehicle.

 
PD98059 also prevents P sequential inhibition of lordosis
In agreement with the results obtained when P was administered icv, Fig. 3Go shows that inhibition of MAPK by icv infusion of PD98059 30 min before sc injection of P also inhibits lordosis behavior during the facilitation test. In contrast, icv infusion of 8 µg SB203850, an inhibitor of the p38 MAPKs, had very little effect on receptivity (mean LQ after vehicle, 84 ± 0.6; mean LQ after SB203850, 61 ± 1.0; n = 8). During the sequential inhibition test, animals that received vehicle infusion before P showed low levels of lordosis (P < 0.001 vs. facilitation test), confirming that P induces sequential inhibition. However, if rats were infused with PD98059 30 min before the first systemic P injection, they continued to show high levels of lordosis behavior after the second injection of P 24 h later.



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  		FIG. 3. Effect of the MAPK inhibitor, PD98059, on the facilitation and sequential inhibition of lordosis and proceptivity induced by systemically administered P. Each value is the mean ± SE of eight animals. *, P< 0.001 vs. facilitation test of other groups; **, P < 0.01 vs. inhibition test of animals treated with PD98059.

 
PD98059 also inhibits cGMP-facilitated reproductive behavior
Figure 4Go shows the effect of PD98059 on lordosis behavior induced by icv administration of 8Br-cGMP. The cyclic nucleotide induced clear lordosis behavior 2 and 4 h after its administration; however, this compound did not induce intense proceptive behavior. The MAPK inhibitor PD98059 significantly decreased the lordosis behavior induced by 8Br-cGMP at 2 (P < 0.01) and 4 h (P < 0.05).



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  		FIG. 4. Lordosis and proceptive behavior induced in OVX, estrogen-treated rats by icv injection of 8Br-cGMP (0.9 ng). The facilitation of lordosis produced by this agent was antagonized by infusing PD98059 (3 µg). Each value is the mean ± SE of eight animals. +, P < 0.05; *, P < 0.01 (vs. corresponding group receiving 8Br-cGMP plus vehicle).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Protein phosphorylation is thought to be involved in the facilitation of sexual behavior in female rodents by a number of pharmacological compounds, because nonsteroidal agents such as GnRH (51) and prostaglandin E2 (52) that act on G protein-coupled receptors associated with protein kinase activation potently facilitate lordosis behavior in OVX rats primed with estradiol. Our results show that the MAPK inhibitor, PD98059, significantly inhibits estrous behavior induced by P, by two of its ring A-reduced metabolites, and by 8Br-cGMP in estrogen-primed rats. These findings raise the intriguing possibility that MAPK, which is known to phosphorylate PRs (34, 35, 36, 37, 38), may be a final mediator of lordosis facilitation by a variety of hormonal and pharmacological agents. Because PKG can activate MAPK (42, 43, 44, 46, 53), this mechanism is compatible with the previous proposal by Whalen and Lauber (54) that the enhancement of lordosis by a variety of structurally unrelated compounds may involve cGMP. Levels of cGMP are high in the hypothalamus of female rats during proestrus (55), and guanine nucleotides facilitate lordosis behavior in estrogen-primed rats (26, 28).

cGMP is a key mediator of cellular responses to nitric oxide (NO), which also plays an important role in the activation of sexual behavior in female rats treated with estradiol and P (26, 27, 56). We recently found that an inhibitor of NO synthesis, a specific inhibitor of NO-stimulated guanylyl cyclase, and the PKG inhibitor, KT5823, significantly attenuate lordosis behavior induced by P and the two ring A-reduced metabolites examined in the current study. These data support the hypothesis that the NO-cGMP-PKG pathway is involved in lordosis induced by progestins, even those that have little or no direct binding to PRs (16). The present results also show that lordosis behavior induced by cGMP is inhibited by PD98059, suggesting that cGMP-induced sexual behavior may be mediated through activation of the MAPK pathway. Activation of MAPK varies with hormonal condition; OVX reduces, and estradiol treatment increases, MAPK signaling in the hypothalamus of female rats (57). Moreover, ovarian steroids rapidly activate MAPK in cortical neurons (58). In rat pinealocytes, treatment with dibutyryl cGMP dose-dependently increased the phosphorylation of both p44 and p42 isoforms of MAPK, and this effect was abolished by PD98059 and the PKG inhibitor KT5823 (42). Taken together, these observations indicate that the cGMP/PKG signaling pathway is an important mechanism used by hormones and neurotransmitters in activating MAPK.

We do not know the mechanism by which P or its 5{alpha}-reduced metabolites activate PKG or MAPK in the brain, but these actions could be mediated at least partially through their membrane effects (59, 60). It is clearly established that membrane effects of 3{alpha}-reduced progestins, such as 5{alpha},3{alpha}-Pgl, involve modulation of the GABAA receptor, an integral membrane protein that forms a ligand-gated chloride channel, and that this mechanism is important in the facilitation of lordosis by these progestins (14, 60, 61, 62). Thus, it is possible that activation of GABAA receptors by progestins modulates MAPK activity. To date, we have been unable to detect an increase in p42/p44 MAPK phosphorylation in hypothalamic extracts at 15 or 60 min after icv administration of progestins to OVX, estrogen-primed rats using immunoblotting techniques (data not shown). This is likely to reflect the fact that only a small proportion of hypothalamic cells respond to progestins, making it difficult to detect changes in grossly dissected tissue samples. Thus, we have not been able to determine whether progestin-induced MAPK activation would be blocked by GABAA antagonists.

A variety of G protein-coupled receptors that facilitate lordosis behavior in estrogen-primed rats can also activate MAPK signaling (63). For example, GnRH receptors in pituitary gonadotropes can couple to several different G proteins to activate the MAPK pathway, including extracellular signal-regulated kinase, c-Jun NH2-terminal kinase, and p38 MAPK (45). Cross-talk between the cAMP and Ras/MAPK signaling pathways is also well established, and cAMP can either stimulate or inhibit MAPK activity (64). Theoretically, it is possible that P and its ring A-reduced metabolites stimulate estrous behavior through the release of GnRH, which then activates MAPK.

A likely molecular target of MAPK in lordosis facilitation is the PR. PRs are phosphorylated at several serine residues by MAPK, and at least two phosphorylation sites, serine 294 and serine 345, are common to the A and B isoforms of PR (35, 36, 37). Phosphorylation by MAPK is a positive signal for ligand-dependent and independent transcriptional activation of PR and for the ubiquitination and targeting of several proteins to the 26S proteasome for proteolytic degradation. PD98059 has been used to test whether progestin-induced PR down-regulation depends on MAPK activation. PD98059 blocked PR protein loss by approximately 70% in T47D-YB cells treated with the synthetic progestin R5020.

Based on these results, we recently explored the hypothesis that the 26S proteasome participates in the regulation of sequential inhibition, a period in which estrogen-primed females do not respond behaviorally to a second administration of P (41). OVX, estrogen-primed female rats were injected with P alone or with P and one of two different proteasome inhibitors. Our findings clearly showed that inhibition of proteolysis mediated by the 26S proteasome prevents P-dependent sequential inhibition of lordosis behavior. This was true regardless of whether the agent reversibly inhibits the chymotrypsin-like activity of the 26S proteasome or irreversibly inhibits the serine-like protease activity of the proteasome (65, 66). In addition to remaining sexually receptive, female rats treated with proteasome inhibitors did not exhibit the decrement in PR content in the hypothalamus that normally accompanies sequential inhibition in control females. These data support the hypothesis that PR down-regulation in the brain is causally related to P-induced sequential inhibition and suggest that the 26S proteasome is an important mediator of PR degradation. We also found that icv administration of PD98059 to OVX, estrogen-primed rats blocks P-induced sequential inhibition (Fig. 3Go). These observations support the idea that MAPK may first facilitate female sexual behavior through PR phosphorylation, then promote termination of behavioral estrus by targeting PR for degradation by the 26S proteasome.

Figure 5Go suggests a molecular model of intracellular pathways that may underlie the enhancement of estrous behavior by progestins. PR activation, which may be ligand dependent or independent, is proposed to be a key molecular event for the final expression of lordosis. This is because facilitation of estrous behavior by a large variety of agents, such as progestins, peptides, and prostaglandins (3, 25), as well as neurotransmitters and second messengers (25, 27) is inhibited by administration of the PR antagonist RU 486 or antisense oligonucleotides against the PR (25, 27, 67, 68). As shown on the left of Fig. 5Go, the stimulatory effect of progestins may result from their interaction with a membrane component, such as a G protein-coupled receptor, linked to the NO pathway. The idea that progestins stimulate lordosis through activation of the NO pathway has been tested in our laboratory by studying the effect of blockers that act at different levels of this pathway (Fig. 5Go) (16, 26, 27). This pathway could induce PR phosphorylation indirectly via PKG activation of MAPK, which, in turn, phosphorylates PR. In support of this model, we found that the MAPK inhibitor PD 98059 blocked the lordosis induced by 8Br-cGMP. An alternative pathway is illustrated on the right of the Fig. 5Go. Progestins could bind to some component of the cell membrane that activates the MAPK pathway, which, in turn, activates PR. Although not illustrated in our model, it is also important to recognize that activated PRs could facilitate reproductive behavior independently of binding to DNA and modulation of gene expression, e.g. by interacting with membrane proteins or intracellular signaling molecules.



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  		FIG. 5. Schematic representation of the NO and MAPK pathways involved in sexual behavior induced by P and its ring A-reduced progestins in female rats and the sites of pharmacological inhibition. Activation of NO synthase by progestins (left) may lead to the formation of NO, which activates soluble guanylyl cyclase and the generation of cGMP. In turn, cGMP activates PKG, which indirectly phosphorylates PR through MAPK, thereby inducing the expression of female sexual behavior. Theoretically, progestins could also induce sexual behavior by activating the Ras and Rap proteins to directly phosphorylate PR via MAPK (right). Inhibitors of the NO pathway (NG-nitro-L-arginine methyl ester, ODQ, and KT5823) or MAPK (PD98059) blocked the sexual behavior induced by P and its ring A-reduced metabolites (16 26 27 40 ).

 
Finally, it is interesting to speculate about the molecular events that might occur subsequent to PR phosphorylation to increase the probability that mating stimuli will produce robust lordosis responses. Vagino-cervical stimulation, either from an intromitting male or applied by an experimenter, increases the expression of lordosis in estradiol-primed rats in the absence of circulating P (69) and is required for the mating-induced release of norepinephrine in the anterior ventromedial hypothalamus (70). Meredith et al. (71) found that vagino-cervical stimulation also increases the phosphorylation of DARPP-32 (dopamine and cAMP-regulated phosphoprotein), which is a substrate for both PKG and protein kinase A and is involved P facilitation of lordosis (29), in the rat forebrain. Perhaps phosphorylation of hypothalamic PRs is one of the points of convergence of hormonal and neurotransmitter signals that lead to phosphorylation of other key downstream mediators, such as DARPP-32, and thus maximize the likelihood that female rats will exhibit lordosis in response to relevant copulatory stimuli (72).

In conclusion, the facilitation of female reproductive behavior by ovarian steroids may not depend on the action of a single intracellular signal, but, rather, on the coordination of multiple signals, both intra- and extracellular, that impinge on a complex neural substrate. These multiple signals operate through a variety of molecular pathways and on a large number of molecules, including the PR, and are modulated by processes such as protein phosphorylation.


   Acknowledgments
 
We gratefully acknowledge the excellent technical assistance of Marcos Garcia-Juarez.


   Footnotes
 
This work was supported by Grants MH-41414 and HD-29856, the Department of Neuroscience, Albert Einstein College of Medicine, and Consejo Nacional de Ciencia y Tecnologia-Mexico.

Abbreviations: 5{alpha}-DHP, 5{alpha}-Dihydroprogesterone; EB, estradiol benzoate; GABAA, {gamma}-aminobutyric acid A; icv, intracerebroventricularly; LQ, lordosis quotient; NO, nitric oxide; OVX, ovariectomized, ovariectomy; P, progesterone; 5{alpha},3{alpha}-Pgl, 5{alpha},3{alpha}-pregnanolone; PKG, cGMP-dependent protein kinase; PR, progesterone receptor.

Received June 30, 2004.

Accepted for publication August 12, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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