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The 26S Proteasome Participates in the Sequential Inhibition of Estrous Behavior Induced by Progesterone in Rats

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

Address all correspondence and requests for reprints to: Dr. Anne M. Etgen, Department of Neuroscience, Albert Einstein College of Medicine, 1300 Morris Park Avenue, F113, Bronx, New York 10461. E-mail: etgen{at}aecom.yu.edu.

	Abstract

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Estrous behavior induced by progesterone (P) treatment of estradiol-primed rats is followed by a period in which females do not respond behaviorally to a second administration of P [sequential inhibition (SI)]. SI is thought to involve P-dependent down-regulation of hypothalamic P receptor (PR) content. This study tested the hypothesis that the 26S proteasome participates in the regulation of SI and brain PR content in female rats. Ovariectomized, estrogen-primed (estradiol benzoate, 2 µg sc) adult rats were injected with P (1 mg sc) alone or P with the proteasome inhibitors Z-Ile-Glu (OBu¹)-Ala-Leu-H (PSI, 300 µg/100 g sc) or N-tosyl-lysyl chloromethyl ketone (TLCK, 200 µg ip) administered 48 h after estradiol priming. Sexual behavior was assessed in all animals 4 h later. These two agents inhibit 26S proteasome-mediated protein degradation by different mechanisms. To explore SI, the animals received a second P injection 24 h after the first, and a second sexual behavior test was performed 4 h later. After this test, brains were excised, and proteins were extracted from the preoptic area and the hypothalamus and processed for semiquantitative immunoblotting. In the first sexual behavior test (facilitation test), all animals treated with estradiol + P exhibited intense lordosis behavior. In the second sexual behavior test (inhibition test), both lordosis and proceptivity were significantly reduced in response to the second administration of P (SI). The magnitude of SI was significantly attenuated by the administration of either PSI or TLCK concurrently with the first P injection. The first P injection reduced PR content in the hypothalamus but not in the preoptic area. In contrast, PSI and TLCK significantly increased PR content in both structures. Our results suggest that PR degradation by the 26S proteasome participates in the expression of P-induced SI in female rats.

	Introduction

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PROGESTERONE (P) PLAYS a key role in the regulation of female sexual behavior in mammals (1, 2). P has a biphasic role in the expression of estrous behavior in ovariectomized, estrogen-primed rats. An initial administration of P facilitates sexual behavior but also induces a refractoriness of the brain for a period of 24–36 h, during which additional administration of P does not stimulate lordosis. This action of P is termed sequential inhibition (SI) (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14).

Considerable evidence supports the interpretation that many of the facilitatory effects of P on estrous behavior, particularly those that involve the ventromedial hypothalamus, are mediated by the intracellular P receptor (PR) (15, 16, 17). For example, the PR antagonist RU 486 inhibits P-facilitated lordosis in guinea pigs and rats (18, 19, 20), even when administered directly into the ventromedial hypothalamus (19, 20). Intracerebral infusion of antisense oligonucleotides to PR also blocks the lordosis induced by P (21, 22, 23). Moreover, female mice with targeted deletion of the PR gene do not show P-facilitated sexual behavior (24). More limited evidence suggests that P-dependent SI also involves ligand-receptor interaction. Progestins with a higher affinity for the PR induce SI more potently than do progestins with a low affinity (5, 14, 25). Interestingly, the PR antagonist RU 486 does not block the SI induced by P (18).

The precise molecular mechanisms underlying P-induced SI have not been elucidated. However, P-dependent down-regulation of PR in the hypothalamus and the preoptic area is highly correlated with the expression of SI (26, 27). Moreover, the duration of sexual receptivity correlates with the retention time of PR in hypothalamic cell nuclei (28, 29). That is, a longer duration of the receptivity is positively correlated with a more prolonged elevation of PR concentration in hypothalamic cell nuclei (29). However, experiments to date have not assessed the possibility that the reduction of sexual receptivity characteristic of SI is due to the proteolytic degradation of PR.

In several species, PR is expressed as two isoforms, a full-length form (PR-B, 110–120 kDa) and the N-terminally truncated form (PR-A, 72–86 kDa) (30, 31, 32), that are functionally different (32, 33). In brain areas thought to mediate P regulation of female sexual behavior, such as the hypothalamus and preoptic area, PR is up-regulated by estrogen and down-regulated by P (15, 16, 34, 35, 36). PR up-regulation by estradiol is mediated by estrogen-responsive elements located in the PR promoter (37). PR down-regulation by P is associated with ligand-dependent proteolysis; P induces PR phosphorylation, which in turn targets these receptors for degradation by the ubiquitin-proteasome pathway (38, 39). Recent evidence implicates the 26S proteasome in the degradation of PR in some areas of the rat brain in vivo (40). Therefore, this study tested the hypothesis that the 26S proteasome participates in the regulation of both hypothalamic PR content and of SI in female rats by using two proteasome inhibitors with different mechanisms of action, Z-Ile-Glu (OBu¹)-Ala-Leu-H (PSI) and N-tosyl-lysyl chloromethyl ketone (TLCK). PSI is a reversible inhibitor of the chymotrypsin-like activity of the 20S catalytic subunit of the proteasome (41, 42), whereas TLCK is an irreversible inhibitor of trypsin-like serine proteases (43, 44).

	Materials and Methods

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Animals
Adult female Sprague Dawley rats (175–200 g) were purchased from Taconic Farm (Germantown, NY) for the PSI experiments or from Bioterio de Centro de Neurobiología (Universidad Nacional Autónoma de México, Juriquilla, Queretaro, Mexico) for the TLCK experiments. All rats were maintained under a 14-h light, 10-h dark reverse light-dark cycle (lights on 2100–1100), with food and water available ad libitum. One week after arrival, animals underwent bilateral ovariectomy (OVX) under xylazine (4 mg/kg) and ketamine (80 mg/kg) anesthesia. Rats were then housed singly in plastic cages and randomly assigned to the different treatments. All procedures used in these experiments followed the National Institutes of Health 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.

Treatments
Hormone administration was initiated 1 wk after OVX. Steroid hormones were injected sc in oil vehicle (sesame oil/10% ethanol). Rats were injected either with oil vehicle (n = 8) or 2 µg of estradiol benzoate (EB; n = 40). After 48 h, the animals treated with oil received PSI vehicle [dimethylsulfoxide (DMSO) sc] and oil. The animals primed with EB were divided into three groups that received the following treatments: group 1, oil + DMSO (n = 16); Group 2, P (1 mg) + PSI (300 µg/100 g, sc; n = 16); and group 3, P + DMSO (n = 8). Female sexual behavior was determined 4 h later in all groups (facilitation test). Twenty hours later (72 h after EB priming or the first oil injection), the group without EB priming received oil. Animals in groups 1 and 2 were divided into two subgroups (n = 8 each), one which received oil and one which received 1 mg of P. Animals in group 3 received P. Sexual behavior was determined 4 h later (SI test). In other groups of rats, TLCK (200 µg ip, n = 14) or physiological saline (n = 13) were administered instead of PSI and DMSO.

EB and P were obtained from Steraloids, Inc. (Wilton, NH). PSI and TLCK were purchased from Sigma (St. Louis, MO) or Affiniti Research Products Ltd (Exeter, UK). The doses of PSI and TLCK that we administered did not detectably modify rat motor behavior (data not shown).

Behavioral testing
Female rats were placed in 20-gallon glass tanks until they received 10 mounts with pelvic thrusting from an experienced 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 to 3, according to the lordosis score proposed by Hardy and DeBold (45). Proceptivity was analyzed by determining the incidence of hopping, darting, and ear wiggling across the whole receptivity test. The proportion of animals displaying any of these behaviors was used as a measure of proceptivity. Immediately after the second sexual behavioral test for assessing SI, the animals were anesthetized with halothane and decapitated, and the preoptic area and the hypothalamus were excised from four animals in each treatment group (46) and processed for protein extraction.

Protein extraction and immunoblotting
The preoptic area and the hypothalamus were homogenized separately in lysis buffer with protease inhibitors (10 mM Tris-HCl, 1 mM dithiothreitol, 30% glycerol, 1% Triton X-100, 15 mM sodium azide, 1 mM EDTA, 4 µg/ml leupeptin, 22 µg/ml aprotinin, 1 mM phenylmethylsulfonylfluoride, and 1 mM sodium orthovanadate). Proteins were obtained by centrifugation for 15 min at 20,000 x g at 4 C and quantified by the method of Bradford (Bio-Rad Laboratories, Hercules, CA). Proteins (80 µg) were separated by electrophoresis on 7.5% SDS-PAGE at 80 V. Colored and enhanced chemiluminescence markers (Bio-Rad and Invitrogen Life Technologies, Inc., Carlsbad, CA) were included for size determination. Hypothalamus and preoptic area samples were always loaded on separate gels. Four gels were run for each tissue for each of the two proteasome inhibitors. Proteins were transferred at 75 V for 4 h at 4 C to nitrocellulose membranes (Amersham, Piscataway, NJ), which were blocked for 2 h at room temperature with 5% nonfat dry milk and 0.5% BSA. Membranes were then incubated with 1 µg/ml of mouse anti-PR monoclonal antibody (AB-52; sc-810, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), which recognizes both PR isoforms (PR-A and PR-B) in rats (47), for 2 h at room temperature. Blots were then incubated for 1 h with a 1:1500 dilution of goat antimouse IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology). Signals were detected by chemiluminescence (Amersham).

To correct for differences in the amount of total protein loaded in each lane, PR protein content was normalized to that of ß-actin. Blots were stripped with glycine (0.1 M, pH 2.5; 0.5% sodium dodecyl sulfate) overnight at 4 C and for 30 min at 37 C and reprobed with 1 µg/ml of goat anti-ß-actin polyclonal antibody (Santa Cruz) for 2 h at room temperature. Blots were then incubated for 1 h with a 1:1500 dilution of donkey antigoat IgG conjugated to horseradish peroxidase (Santa Cruz). Signals were detected by chemiluminescence. The intensity of PR isoform and ß-actin signals was quantified by densitometry using a Scan Primax Colorado 600p apparatus (Primax, Utrecht, The Netherlands) and Scion Image software (Scion Corp., Frederick, MD). Based on the band intensity of the ß-actin loading control, we had very little variation among gels.

Statistical analysis
LQ and lordosis score data were analyzed by a Mann-Whitney U test, and the proportion of proceptive animals was analyzed using the Fisher’s exact probability test. PR content was analyzed by one-way ANOVA followed by a Student’s t test. Differences were considered statistically significant at P < 0.05.

	Results

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Sexual behavior
The administration of P to OVX, EB-primed rats produced high levels of lordosis and proceptivity during the first behavioral test (facilitation test) in the PSI experiments (Figs. 1 and 2; Table 1). The TLCK experiments used rats from a different vendor, and the EB + P-treated females in these experiments showed somewhat lower LQ (Fig. 3) and lordosis intensity scores (Table 2) and virtually no proceptive behavior (data not shown) during the facilitation test. On the facilitation test, the administration of PSI or TLCK along with P did not modify sexual behavior (Figs. 1–3; Tables 1 and 2). As expected, the animals that were not primed with EB but which were treated with vehicle did not exhibit lordosis behavior (Figs. 1 and 3, Tables 1 and 2) or proceptivity (Fig. 2). In the second behavioral test (SI test), the animals injected with oil vehicle 4 h earlier did not show sexual behavior, whereas animals treated for the first time with P exhibited high levels of lordosis (Figs. 1–3; Tables 1 and 2). In contrast, the second administration of P to animals injected with P alone on the previous day failed to induce high levels of these behaviors. Therefore, the first administration of P induced SI, as expected. However, in EB-primed animals treated with PSI or TLCK plus P before the facilitation test, the second administration of P induced high levels of lordosis behavior again, indicating that PSI and TLCK blocked the SI induced by P (Figs. 1–3; Tables 1 and 2).

View larger version (16K): [in this window] [in a new window]   FIG. 1. Effect of PSI on the facilitation and sequential inhibition of lordosis induced by P. OVX animals, treated as described in Materials and Methods, were assessed for lordosis behavior (n = 8/group). *, P < 0.001 vs. inhibition test of animals treated with PSI.

View larger version (13K): [in this window] [in a new window]   FIG. 2. Effect of PSI on proceptive behaviors induced by P in the facilitation and SI tests. Proceptivity was assessed in OVX animals (n = 8/group) according to the criteria described in Materials and Methods. *, P < 0.01 vs. inhibition test of animals treated with PSI.

View larger version (16K): [in this window] [in a new window]   FIG. 3. Effect of the proteasome inhibitor TLCK on the facilitation and sequential inhibition of lordosis induced by P. OVX animals, treated as described in Materials and Methods, were assessed for lordosis behavior (n = 6–7/group). *, P < 0.001 vs. inhibition test of animals treated with TLCK.

View larger version (35K): [in this window] [in a new window]   FIG. 4. PSI effects on PR isoform content in the preoptic area of female rats after the SI test. A representative immunoblot (upper panel) and the densitometric analysis (lower panel) are shown. OVX rats were treated as described in Materials and Methods. Proteins (80 µg) were separated by electrophoresis on 7.5% SDS-PAGE, transferred to nitrocellulose membranes, and incubated with anti-PR antibody. The densitometric analysis was performed using the ß-actin signal to correct for differences in the amount of total loaded protein. Results are expressed as mean ± SEM (n = 4). &, P < 0.05 vs. no hormone, vehicle-infused controls; *, P < 0.05 vs. EB-treated rats given P at 72 h; +, P < 0.05 vs. EB-treated rats given P at 48 and 72 h.

View larger version (30K): [in this window] [in a new window]   FIG. 5. PSI effects on PR isoform content in the hypothalamus of female rats after the SI test. A representative immunoblot (upper panel) and the densitometric analysis (lower panel) are shown. OVX rats were treated as described in Materials and Methods. Proteins (80 µg) were analyzed as described in Fig. 4. Results are expressed as mean ± SEM (n = 4). &, P < 0.05 vs. no hormone, vehicle-infused controls; *, P < 0.05 vs. EB-treated rats given P at 72 h; **, P < 0.05 vs. EB-treated animals with no P; +, P < 0.05 vs. EB-treated rats given P at 48 and 72 h.

View larger version (23K): [in this window] [in a new window]   FIG. 6. TLCK effects on PR isoform content in the preoptic area of female rats after the SI test. A representative immunoblot (upper panel) and the densitometric analysis (lower panel) are shown. OVX rats were treated as described in Materials and Methods. Proteins (80 µg) were analyzed as described in Fig. 4. Results are expressed as mean ± SEM (n = 4). &, P < 0.05 vs. no hormone, vehicle-infused controls; *, P < 0.05 vs. EB-treated rats given P at 72 h; +, P < 0.05 vs. EB-treated rats given P at 48 and 72 h.

View larger version (26K): [in this window] [in a new window]   FIG. 7. TLCK effects on PR isoform content in the hypothalamus of female rats after the SI test. A representative immunoblot (upper panel) and the densitometric analysis (lower panel) are shown. OVX rats were treated as described in Materials and Methods. Proteins (80 µg) were analyzed as described in Fig. 4. Results are expressed as mean ± SEM (n = 4). &, P < 0.05 vs. no hormone, vehicle-infused controls; *, P < 0.05 vs. EB-treated rats given P at 72 h; **, P < 0.05 vs. EB-treated animals with no P; +, P < 0.05 vs. EB-treated rats given P at 48 and 72 h.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The period of P-induced SI in rats and guinea pigs is characterized by a decreased behavioral sensitivity to P and is correlated with decreased levels of PR in brain areas that mediate female sexual behavior, such as the mediobasal hypothalamus and the preoptic area (12, 15, 26). These observations have led to the hypothesis that SI of reproductive behavior is causally related to P-induced down-regulation of brain PRs (7, 12, 15, 48, 49, 50). Our new findings clearly show that pharmacological agents that inhibit proteolysis mediated by the 26S proteasome prevent SI of lordosis behavior when they are administered to estrogen-primed rats concurrently with the first administration of P. This was true regardless of whether the agent reversibly inhibits the chymotrypsin-like activity of the 26S proteasome (PSI) or irreversibly inhibits the serine-like protease activity of the proteasome (TLCK) (41, 44). Both PSI and TLCK also increase PR content in the hypothalamus and preoptic area of female rats, resulting in significantly higher levels of PR in both brain areas in rats that were still highly receptive during the inhibition test than in rats exhibiting P-induced SI. Hence, the present data support the hypothesis that PR down-regulation in the brain is causally related to P-induced SI and suggest that the 26S proteasome is an important mediator of PR degradation.

It is notable that the two proteasome inhibitors completely blocked P-induced SI of lordosis, whereas their effects on PR content were more modest. It could be argued that this reflects the inherent technical limitations associated with quantitative analysis of immunoblots containing protein from a heterogeneous tissue. Alternatively, these findings may point to a key role of PR in the regulation of female sexual behavior such that even subtle changes in total hypothalamic PR content, of either the A or B isoform, significantly alter P facilitation of lordosis. Nonetheless, the participation of proteins other than PR in the expression of SI cannot be dismissed because inhibition of the 26S proteasome also increases the content of estrogen receptors, which are critically involved in the regulation of estrous behavior (51, 52).

The involvement of the 26S proteasome in PR down-regulation may explain why SI is a ligand-dependent event. Recent evidence suggests that P-induced down-regulation of PR is associated with ligand-dependent PR phosphorylation, which in turn targets the phosphorylated receptors for degradation by the ubiquitin-proteasome pathway (38, 39). Progestins with a higher binding affinity for PR than P itself, such as R5020 and norgestrel, are more potent inducers of SI than P (5, 14, 25). In contrast, progestins with a low PR binding affinity, including norethynodrel and some ring-A reduced progestins, such as 5-pregnan-3-ol-20-one, 20-hydroxypregnenone, and desoxycorticosterone, do not induce SI even though they facilitate lordosis behavior (5, 8, 48). It is not yet known whether progestins with a higher PR affinity produce efficient PR phosphorylation or induce a higher rate or degree of PR degradation.

Two of the main factors that regulate PR expression are estradiol and P. In many tissues, estradiol increases PR content, whereas P reduces it (36, 47). As expected, our estrogen-priming regimen increased the content of PR both in the hypothalamus and the preoptic area of OVX animals. However, a detectable P-induced reduction of PR content was only observed in the hypothalamus. The failure of P to down-regulate PR in the preoptic area has been reported previously (40). Interestingly, PSI and TLCK administration increased PR content in the preoptic area despite the failure of P to induce down-regulation of PR in this brain region. This observation confirms previous data indicating that the 26S proteasome can degrade PR independent of the effects of P on its receptor. Indeed, even in the absence of ligand, the 26S proteasome participates in the regulation of PR turnover (38, 40).

Although the content of both PR isoforms was increased by PSI, a relatively greater effect was observed on PR-B both in the hypothalamus and the preoptic area. One interpretation of this observation is that PR-B has a greater involvement than PR-A in the facilitation of reproductive behavior. However, TLCK increased the content of both PR isoforms to the same extent. Thus, a specific role for the two PR isoforms in the regulation of sexual behavior remains to be clarified. PSI and TLCK clearly inhibit different protease activities within the proteasome (41, 44), but the mechanisms involved in their differential effects on the content of PR isoforms in the hypothalamus and the preoptic area are not completely understood.

In summary, we demonstrate that PR degradation by the 26S proteasome in the hypothalamus and the preoptic area is essential for the expression of P-dependent SI of female reproductive behavior in rats. These findings support the hypothesis that the behavioral refractoriness to P is causally related to ligand-dependent PR down-regulation in brain regions that govern reproductive function. Moreover, they implicate the 26S proteasome as an important molecular mediator of P-induced down-regulation of brain PRs in vivo.

	Acknowledgments

 
The authors gratefully acknowledge the excellent technical assistance of Dr. Jun (Alice) Shu and Francisco Camacho.

	Footnotes

 
This work was supported by Department of Health and Human Services Grant R37 MH41414, by the Department of Neuroscience, Albert Einstein College of Medicine, and by Consejo Nacional de Ciencia y Tecnologia, Mexico (Project No. 35025-N).

Abbreviations: EB, Estradiol benzoate; DMSO, dimethylsulfoxide; LQ, lordosis quotient; OVX, ovariectomy; P, progesterone; PR, progesterone receptor; PSI, proteasome inhibitor Z-Ile-Glu (OBu¹)-Ala-Leu-H; SI, sequential inhibition; TLCK, N-tosyl-lysyl chloromethyl ketone.

Received September 4, 2003.

Accepted for publication January 28, 2004.

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