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Allopregnanolone's attenuation of the lordosis-inhibiting effects of restraint is blocked by the antiprogestin, CDB-4124
Pharmacology, Biochemistry and Behavior 122 (2014) 16–19
Contents lists available at ScienceDirect
Pharmacology, Biochemistry and Behavior journal homepage: www.elsevier.com/locate/pharmbiochembeh
Allopregnanolone's attenuation of the lordosis-inhibiting effects of restraint is blocked by the antiprogestin, CDB-4124 Lynda Uphouse ⁎, Cindy Hiegel Department of Biology, Texas Woman's University, Denton, TX 76204, United States
a r t i c l e
i n f o
Article history: Received 21 January 2014 Received in revised form 23 February 2014 Accepted 12 March 2014 Available online 18 March 2014 Keywords: Sexual behavior Antiprogestin Stress Female rats Ovariectomized Progesterone receptor
a b s t r a c t A brief restraint experience reduces lordosis behavior in ovariectomized females that have been hormonally primed with estradiol benzoate. The addition of progesterone to the priming prevents the lordosis inhibition. Based on prior studies with an inhibitor of progesterone metabolism, we have implicated the intracellular progesterone receptor, rather than progesterone metabolites, as responsible for this protection. However, the progesterone metabolite, allopregnanolone (3α-hydroxy-5α-pregnan-20-one), also prevents lordosis inhibition after restraint. In a prior study, we reported that the progestin receptor antagonist, RU486 (11β-(4dimethylamino)phenyl-17β-hydroxy-17-(1-propynyl)estra-4,9-dien-3-one), attenuated the effect of allopregnanolone. Because RU486 can also block the glucocorticoid receptor, in the current studies, we evaluated the effect of the progestin receptor antagonist, CDB-4124 (17α-acetoxy-21-methoxy-11β-[4-N,Ndimethyaminopheny]-19-norpregna-4,9-dione-3,20-dione), which is relatively devoid of antiglucocorticoid activity. Ovariectomized, Fischer rats were injected with 10 μg estradiol benzoate. Two days later, rats received either 60 mg/kg CDB-4124 or 20% DMSO/propylene glycol vehicle 1 h before injection with 4 mg/kg allopregnanolone. After a pretest to confirm sexual receptivity, rats were restrained for 5 min and immediately tested for sexual behavior. Lordosis behavior was reduced by the restraint and attenuated by allopregnanolone. Pretreatment with CDB-4124 reduced allopregnanolone's effect. These findings support prior suggestions that allopreganolone reduces the response to restraint by mechanisms that require activation of the intracellular progesterone receptor. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Female rats have a 4–5 day estrous cycle that is regulated by the hypothalamic–pituitary–gonadal axis. Sexual receptivity is limited to the day of proestrous and is controlled by the gonadal hormones, estradiol and progesterone (Blaustein, 2008). Although only estradiol is required for sexual receptivity, during the estrous cycle, both estradiol and progesterone are thought to participate (Blaustein, 2008; Pfaff, 1970). Progesterone facilitates estradiol-induced copulatory responses (e.g. lordosis behavior) and is thought to be essential for females to show precopulatory behavior such as solicitation and proceptivity (Erskine, 1989; Frye, 2007; Sodersten, 1981). In addition, progesterone plays an important role in changes during the estrous cycle in the female's response to anxiogenic stimuli (Frye et al., 2000; Lovick, 2012). Previously we have suggested that progesterone's facilitation of female rat sexual behavior may, in part, result from such an attenuation of the stress associated with the mating experience.
⁎ Corresponding author. Tel.: +1 940 898 2356; fax: +1 940 898 2382. E-mail addresses: [email protected] (L. Uphouse), [email protected] (C. Hiegel).
http://dx.doi.org/10.1016/j.pbb.2014.03.012 0091-3057/© 2014 Elsevier Inc. All rights reserved.
Progesterone modulates reproductive and nonreproductive behaviors through multiple intracellular mechanisms, including the classical intracellular progesterone receptor and a variety of membrane progesterone receptors (Conneely et al., 2003; Cooke et al., 2013; Dressing et al., 2011; Mani et al., 1997, 2006; Pluchino et al., 2009; Thomas and Pang, 2012). Progesterone can also be metabolized by 5α-reductase into 5α-dihydroprogesterone and then by 3α-hydroxysteroid dehydrogenase into allopregnanolone (3α-hydroxy-5α-pregnan-20-one) (Rupprecht, 2003; Schule et al., 2011). Progesterone metabolites, such as allopregnanolone, are thought to be responsible for many of progesterone's anxiolytic effects (Dubrovsky, 2006; Eser et al., 2008; Frye et al., 2008, 2012). When Fischer female rats are hormonally primed with 10 μg estradiol benzoate, they show high levels of lordosis behavior (Hassell et al., 2011; Miryala et al., 2011). Such rats are, however, vulnerable to the effects of a 5 min restraint stress and show a robust restraint-induced decline in lordosis behavior (White and Uphouse, 2004). When progesterone is added to the hormonal priming, females are relatively unaffected by the restraint stress (Hassell et al., 2011; White and Uphouse, 2004). Although the anxiolytic effects of progesterone have generally been attributed to the ability of the progesterone metabolite,
L. Uphouse, C. Hiegel / Pharmacology, Biochemistry and Behavior 122 (2014) 16–19
allopregnanolone, to enhance the effects of GABA at the GABAA receptor (Barbaccia et al., 2001; Frye et al., 2000, 2012; Girdler and Klatzkin, 2007), the results of our prior studies argue against that possibility for the hormone's attenuation of the response to restraint. The effects of progesterone were mimicked by the non metabolizable progestin and medroxyprogesterone (Hassell et al., 2011); the effects were not attenuated when progesterone metabolism was blocked with the 5αreductase inhibitor, finasteride (Miryala et al., 2011); but the effects were attenuated by the progesterone receptor antagonists, RU486 (11β-(4-dimethylamino)phenyl-17β-hydroxy-17-(1-propynyl)estra4,9-dien-3-one) and CDB-4124 (17α-acetoxy-21-methoxy-11β-[4-N, N-dimethyaminopheny]-19-norpregna-4,9-dione-3,20-dione) (Hassell et al., 2011; Uphouse and Hiegel, 2013). These findings implicated the intracellular progesterone receptor rather than the progesterone metabolite in mediating progesterone's attenuation of restraint-induced lordosis inhibition. Nevertheless, allopregnanolone also protected against the effects of the 5 min restraint experience (Miryala et al., 2011) and allopregnanolone's ability to reduce the lordosis-inhibiting effect of 5 min restraint was attenuated by the progestin receptor antagonist, RU486, (Uphouse et al., 2013). Since allopregnanolone does not interact with the classical intracellular progesterone receptor (Raynaud et al., 1974), the effect of RU486 allows the suggestion that allopregnanolone may indirectly lead to activation of the intracellular progesterone receptor. However, since RU486 also antagonizes glucocorticoid receptors (Lee et al., 2009), it is possible that RU486 attenuated the effects of allopregnanolone through interaction with the glucocorticoid receptor. To assess this possibility, in the following experiment, we investigated the effects of the progestin receptor antagonist, CDB-4124, which has minimal antiglucocorticoid activity (Attardi et al., 2002; Wiehle et al., 2007). 2. Materials and methods 2.1. Materials Estradiol benzoate, allopregnanolone (3α-hydroxy-5α-pregnan-20one), dimethyl sulfoxide (DMSO) and sesame seed oil were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO). Propylene glycol came from Eastman Kodak Company (Rochester, NY). Isoflurane (AErrane®) was purchased from Butler Schein Animal Health (Dublin, OH). Decapicone® restrainers were from Braintree Scientific, Inc. (Braintree, MA). CDB-4124 (17α-acetoxy-21-methoxy-11β-[4-N, N-dimethyaminopheny]-19-norpregna-4,9-dione-3,20-dione) was a generous gift from Dr. Ronald Wiehle and Repros Therapeutics Inc. (The Woodlands, TX). Other supplies came from Fisher Scientific (Houston, TX). 2.2. Animals, housing and surgical procedures Adult Fischer (F-344) female rats were obtained from Charles River Laboratories (Wilmington, MA). Housing was in polycarbonate shoebox cages with food and water available ad lib. The light cycle was a 12 h light/dark cycle with lights on at 12 midnight. Rats were given 2 weeks to acclimate to the facility before use in the experiment. When 90 to 110 days of age, females were anesthetized with AErrane® and ovariectomized as previously described (White and Uphouse, 2004). Two weeks later, rats were injected subcutaneously (sc) with 10 μg estradiol benzoate. Two days later, rats were injected sc with the progesterone receptor antagonist, CDB-4124 (60 mg/kg), or vehicle (20% DMSO + propylene glycol). This dose of CDB-4124 was chosen on the basis of a prior experiment demonstrating attenuation of the effects of progesterone (Uphouse and Hiegel, 2013). One hour later, rats were injected with 4 mg/kg allopregnanolone or vehicle (sesame seed oil). Rats were assigned to one of the three groups: (1) estradiol benzoate, vehicle, and vehicle (EO); (2) estradiol benzoate, vehicle, and allopregnanolone (E-ALLO); and (3) estradiol benzoate,
17
CDB-4124, and allopregnanolone (E-CDB-ALLO). Testing occurred 4 h after allopregnanolone or vehicle. All procedures were in accordance with the PHS Guide and were approved by the TWU IACUC. 2.3. Testing for sexual behavior and restraint procedures Sexual behavior pretesting began 4 h after the sesame seed oil or allopregnanolone injection. Pretesting occurred in the home cage of a sexually active Sprague–Dawley male and behavior was monitored until the male had achieved 10 mounts or for a maximum of 10 min. Immediately after the pretest, females were restrained for 5 min. Thereafter, females were returned to the male's cage for 15 min. Sexual receptivity (L/M ratio; number of lordosis responses divided by number of male mounts) and lordosis quality (measured by the magnitude of the lordosis response) were scored as previously described (White and Uphouse, 2004). Proceptivity (defined as the presence of hopping and darting) and resistance (defined as fighting, boxing, rolling over, trying to escape the cage) were measured as present or absent. When females showed at least one hop/dart sequence or one fighting/boxing sequence, that behavior was categorized as present. Restraint procedures were as previously described (White and Uphouse, 2004). The female was placed head first into a Decapicone® so that her nose was flush with the small opening at the tip of the cone. The base of the cone was gathered around the female's tail and secured tightly with tape. Females were restrained for 5 min, after which they were returned to the male's cage for sexual behavioral testing. 2.4. Statistical procedures Data for L/M ratios and lordosis quality were grouped into the pretest and 3 consecutive 5 min intervals after restraint and were evaluated by repeated measures ANOVA with time relative to restraint as the repeated factor and type of treatment as the independent factor. Post-hoc comparisons were made with Tukey's test. Proceptivity and resistance were compared by Chi-Square procedures. Data were analyzed with SPSS and the statistical reference was Zar (2010). 3. Results A total of 39 rats (14 EO, 11 E-CDB-ALLO, and 14 E-ALLO) were used in the experiment. Of these, 2 EO, 3 E-CDB-ALLO, and 2 E-ALLO were not receptive (as defined by an L/M ratio of at least 0.7) in the pretest and were not restrained. One additional EO and 3 E-ALLO rats did not receive mounts after restraint and were also excluded. L/M ratios before and after restraint are shown in Fig. 1. Only rats showing L/M ratios ≥0.7 in the pretest were included in the ANOVA. There was a significant effect of treatment (F2,25 = 6.72, p ≤ 0.005), due to higher overall L/M ratios in E-ALLO rats, and a significant effect of time relative to restraint (F3,75 = 10.63, p ≤ 0.001). Since all groups showed some decline in L/M ratios after restraint, the time by treatment interaction was not statistically significant (F6,75 = 0.09, p N 0.05). However, rats given E-ALLO showed a smaller decline in L/M ratios than the other two groups. EO rats were different from their pretest at 5 and 15 min after restraint (q 75, 4 = 6.51 and 4.81, respectively, p ≤ 0.05). Rats given E-CDB-ALLO were different from their pretest at 5 and 10 min after restraint (respectively, q75, 4 = 7.05 and 6.03, p ≤ 0.05). Rats given E-ALLO were never significantly different from their pretest. E-CDB-ALLO rats were different from E-ALLO rats at 5 and 10 min after restraint (q75,3 = 5.78 and 5.0, p ≤ 0.05) and EO rats were different from E-ALLO rats at 5 and 15 min after restraint (q75,3 = 4.54 and 4.24, respectively, p ≤ 0.05). EO and E-CDB-ALLO rats did not differ. Lordosis quality remained relatively high after restraint and there was no significant effect of restraint among treatments (p N 0.05) (Table 1). There were few instances of proceptive behavior before or
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L. Uphouse, C. Hiegel / Pharmacology, Biochemistry and Behavior 122 (2014) 16–19
LORDOSIS / MOUNT RATIO
1.0
0.8
0.6
** *
** *
** ** * *
0.4
0.2
0.0 PRE 5 10 15
EO
PRE 5 10 15
PRE 5 10 15
E-ALLO
E-CDB-ALLO
Fig. 1. CDB-4124 attenuates the protective effects of allopregnanolone after restraint stress. Data are the mean ± S.E. L/M ratios before and for consecutive 5 min intervals after a 5 min restraint experience. Ovariectomized rats were hormonally primed with 10 μg estradiol benzoate two days before treatment with vehicles (EO), allopregnanolone (E-ALLO) or CDB-4124 and allopregnanolone (E-CDB-ALLO). Ns for EO, E-ALLO, and ECDB-ALLO were 11, 9, and 8. * indicates a significant decline, within treatment, relative to the pretest. ** indicates a significant difference from E-ALLO rats, within time interval.
after restraint and resistance was present throughout the experiment. There were no differences among groups (all p N 0.05). 4. Discussion The results of this experiment are consistent with prior findings with the progesterone/glucocorticoid receptor antagonist, RU486, and provide additional evidence that the classical intracellular progesterone receptor is important in progesterone's attenuation of the lordosis-inhibiting effects of restraint stress. Allopregnanolone does not bind to this intracellular receptor (Raynaud et al., 1974; Smith et al., 1974). However, the intracellular progesterone receptor can be activated by both ligand-dependent and ligand-independent mechanisms (Dressing et al., 2011; Mani, 2006; Mani et al., 1997; Pluchino et al., 2009). Ligand-independent mechanisms can lead to progesterone receptor activation through a variety of intracellular signaling cascades (Blaustein, 2003; Boonyaratanakornkit et al., 2008; Boonyaratanakornkit et al., 2001; Mani and Portillo, 2010; Mani et al., 2009). Many of these signaling pathways (e.g. cAMP, PKC, PKA, MAPK, Src kinase) are also altered by allopregnanolone (Blackmore, 2008; Etgen and Acosta-Martinez, 2003; Frye and Walf, 2008; Gonzalez-Flores et al., 2006; Mani et al., 2000; Pluchino et al., 2006; Thomas and Pang, 2012) and allopregnanolone's facilitation of lordosis can be blocked by PKC inhibitors (Gonzalez-Flores et al., 2006) or inhibitors of MAPK (Gonzalez-Flores et al., 2004). Therefore, allopregnanolone may indirectly activate intracellular progesterone by activation of intracellular signaling pathways. In addition to the classical intracellular progesterone receptor, there are several membrane progesterone receptors through which Table 1 Characteristics of rats before and after restraint. Group
N
EO Before restraint After restraint E-ALLO Before restraint After restraint E-CDB-ALLO Before restraint After restraint
11
Lordosis quality
% proceptive
% resistive
2.9 ± 0.06 2.3 ± 0.17
14.3% 0%
71.4% 100%
2.8 ± 0.13 2.6 ± 0.06
14.3% 0%
71.4% 100
2.6 ± 0.22 2.6 ± 0.20
9.1% 0%
90.9% 100%
9
8
progesterone exerts its reproductive and nonreproductive effects by activation of intracellular signaling cascades (Pang et al., 2013; Thomas and Pang, 2012; Thomas et al., 2007). Not only the parent molecule, progesterone, but also progesterone metabolites such as allopregnanolone can bind to membrane progesterone receptors (Kelder et al., 2010; Thomas et al., 2007) and activate the relevant signaling cascades (Thomas and Pang, 2012). To our knowledge, it is not known if CDB4124 can interact with these membrane receptors, but RU486 does not appear to block the effects of membrane progesterone receptors (Bashour and Wray, 2012) but does block rapid extranuclear signaling that is mediated through the intracellular progesterone receptor (Hsu and Lee, 2011). Therefore, it is not unreasonable to suggest that allopregnanolone, via binding to membrane progesterone receptors, initiates intracellular signaling pathways to produce ligandindependent activation of intracellular progesterone receptors. Nuclear progesterone receptor antagonists such as RU486 and CDB-4124 would therefore block consequent behavioral effects. In summary, the lordosis behavior of female rats hormonally primed with estradiol benzoate was reduced by 5 min restraint stress. Allopregnanolone attenuated the decline in lordosis behavior and the progestin receptor antagonist, CDB-4124, reduced this attenuation. These findings are consistent with prior studies that implicated the classical intracellular progesterone receptor in the protective effects of progesterone and its metabolite, allopregnanolone, against the lordosisinhibiting effects of restraint stress.
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Contents lists available at ScienceDirect
Pharmacology, Biochemistry and Behavior journal homepage: www.elsevier.com/locate/pharmbiochembeh
Allopregnanolone's attenuation of the lordosis-inhibiting effects of restraint is blocked by the antiprogestin, CDB-4124 Lynda Uphouse ⁎, Cindy Hiegel Department of Biology, Texas Woman's University, Denton, TX 76204, United States
a r t i c l e
i n f o
Article history: Received 21 January 2014 Received in revised form 23 February 2014 Accepted 12 March 2014 Available online 18 March 2014 Keywords: Sexual behavior Antiprogestin Stress Female rats Ovariectomized Progesterone receptor
a b s t r a c t A brief restraint experience reduces lordosis behavior in ovariectomized females that have been hormonally primed with estradiol benzoate. The addition of progesterone to the priming prevents the lordosis inhibition. Based on prior studies with an inhibitor of progesterone metabolism, we have implicated the intracellular progesterone receptor, rather than progesterone metabolites, as responsible for this protection. However, the progesterone metabolite, allopregnanolone (3α-hydroxy-5α-pregnan-20-one), also prevents lordosis inhibition after restraint. In a prior study, we reported that the progestin receptor antagonist, RU486 (11β-(4dimethylamino)phenyl-17β-hydroxy-17-(1-propynyl)estra-4,9-dien-3-one), attenuated the effect of allopregnanolone. Because RU486 can also block the glucocorticoid receptor, in the current studies, we evaluated the effect of the progestin receptor antagonist, CDB-4124 (17α-acetoxy-21-methoxy-11β-[4-N,Ndimethyaminopheny]-19-norpregna-4,9-dione-3,20-dione), which is relatively devoid of antiglucocorticoid activity. Ovariectomized, Fischer rats were injected with 10 μg estradiol benzoate. Two days later, rats received either 60 mg/kg CDB-4124 or 20% DMSO/propylene glycol vehicle 1 h before injection with 4 mg/kg allopregnanolone. After a pretest to confirm sexual receptivity, rats were restrained for 5 min and immediately tested for sexual behavior. Lordosis behavior was reduced by the restraint and attenuated by allopregnanolone. Pretreatment with CDB-4124 reduced allopregnanolone's effect. These findings support prior suggestions that allopreganolone reduces the response to restraint by mechanisms that require activation of the intracellular progesterone receptor. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Female rats have a 4–5 day estrous cycle that is regulated by the hypothalamic–pituitary–gonadal axis. Sexual receptivity is limited to the day of proestrous and is controlled by the gonadal hormones, estradiol and progesterone (Blaustein, 2008). Although only estradiol is required for sexual receptivity, during the estrous cycle, both estradiol and progesterone are thought to participate (Blaustein, 2008; Pfaff, 1970). Progesterone facilitates estradiol-induced copulatory responses (e.g. lordosis behavior) and is thought to be essential for females to show precopulatory behavior such as solicitation and proceptivity (Erskine, 1989; Frye, 2007; Sodersten, 1981). In addition, progesterone plays an important role in changes during the estrous cycle in the female's response to anxiogenic stimuli (Frye et al., 2000; Lovick, 2012). Previously we have suggested that progesterone's facilitation of female rat sexual behavior may, in part, result from such an attenuation of the stress associated with the mating experience.
⁎ Corresponding author. Tel.: +1 940 898 2356; fax: +1 940 898 2382. E-mail addresses: [email protected] (L. Uphouse), [email protected] (C. Hiegel).
http://dx.doi.org/10.1016/j.pbb.2014.03.012 0091-3057/© 2014 Elsevier Inc. All rights reserved.
Progesterone modulates reproductive and nonreproductive behaviors through multiple intracellular mechanisms, including the classical intracellular progesterone receptor and a variety of membrane progesterone receptors (Conneely et al., 2003; Cooke et al., 2013; Dressing et al., 2011; Mani et al., 1997, 2006; Pluchino et al., 2009; Thomas and Pang, 2012). Progesterone can also be metabolized by 5α-reductase into 5α-dihydroprogesterone and then by 3α-hydroxysteroid dehydrogenase into allopregnanolone (3α-hydroxy-5α-pregnan-20-one) (Rupprecht, 2003; Schule et al., 2011). Progesterone metabolites, such as allopregnanolone, are thought to be responsible for many of progesterone's anxiolytic effects (Dubrovsky, 2006; Eser et al., 2008; Frye et al., 2008, 2012). When Fischer female rats are hormonally primed with 10 μg estradiol benzoate, they show high levels of lordosis behavior (Hassell et al., 2011; Miryala et al., 2011). Such rats are, however, vulnerable to the effects of a 5 min restraint stress and show a robust restraint-induced decline in lordosis behavior (White and Uphouse, 2004). When progesterone is added to the hormonal priming, females are relatively unaffected by the restraint stress (Hassell et al., 2011; White and Uphouse, 2004). Although the anxiolytic effects of progesterone have generally been attributed to the ability of the progesterone metabolite,
L. Uphouse, C. Hiegel / Pharmacology, Biochemistry and Behavior 122 (2014) 16–19
allopregnanolone, to enhance the effects of GABA at the GABAA receptor (Barbaccia et al., 2001; Frye et al., 2000, 2012; Girdler and Klatzkin, 2007), the results of our prior studies argue against that possibility for the hormone's attenuation of the response to restraint. The effects of progesterone were mimicked by the non metabolizable progestin and medroxyprogesterone (Hassell et al., 2011); the effects were not attenuated when progesterone metabolism was blocked with the 5αreductase inhibitor, finasteride (Miryala et al., 2011); but the effects were attenuated by the progesterone receptor antagonists, RU486 (11β-(4-dimethylamino)phenyl-17β-hydroxy-17-(1-propynyl)estra4,9-dien-3-one) and CDB-4124 (17α-acetoxy-21-methoxy-11β-[4-N, N-dimethyaminopheny]-19-norpregna-4,9-dione-3,20-dione) (Hassell et al., 2011; Uphouse and Hiegel, 2013). These findings implicated the intracellular progesterone receptor rather than the progesterone metabolite in mediating progesterone's attenuation of restraint-induced lordosis inhibition. Nevertheless, allopregnanolone also protected against the effects of the 5 min restraint experience (Miryala et al., 2011) and allopregnanolone's ability to reduce the lordosis-inhibiting effect of 5 min restraint was attenuated by the progestin receptor antagonist, RU486, (Uphouse et al., 2013). Since allopregnanolone does not interact with the classical intracellular progesterone receptor (Raynaud et al., 1974), the effect of RU486 allows the suggestion that allopregnanolone may indirectly lead to activation of the intracellular progesterone receptor. However, since RU486 also antagonizes glucocorticoid receptors (Lee et al., 2009), it is possible that RU486 attenuated the effects of allopregnanolone through interaction with the glucocorticoid receptor. To assess this possibility, in the following experiment, we investigated the effects of the progestin receptor antagonist, CDB-4124, which has minimal antiglucocorticoid activity (Attardi et al., 2002; Wiehle et al., 2007). 2. Materials and methods 2.1. Materials Estradiol benzoate, allopregnanolone (3α-hydroxy-5α-pregnan-20one), dimethyl sulfoxide (DMSO) and sesame seed oil were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO). Propylene glycol came from Eastman Kodak Company (Rochester, NY). Isoflurane (AErrane®) was purchased from Butler Schein Animal Health (Dublin, OH). Decapicone® restrainers were from Braintree Scientific, Inc. (Braintree, MA). CDB-4124 (17α-acetoxy-21-methoxy-11β-[4-N, N-dimethyaminopheny]-19-norpregna-4,9-dione-3,20-dione) was a generous gift from Dr. Ronald Wiehle and Repros Therapeutics Inc. (The Woodlands, TX). Other supplies came from Fisher Scientific (Houston, TX). 2.2. Animals, housing and surgical procedures Adult Fischer (F-344) female rats were obtained from Charles River Laboratories (Wilmington, MA). Housing was in polycarbonate shoebox cages with food and water available ad lib. The light cycle was a 12 h light/dark cycle with lights on at 12 midnight. Rats were given 2 weeks to acclimate to the facility before use in the experiment. When 90 to 110 days of age, females were anesthetized with AErrane® and ovariectomized as previously described (White and Uphouse, 2004). Two weeks later, rats were injected subcutaneously (sc) with 10 μg estradiol benzoate. Two days later, rats were injected sc with the progesterone receptor antagonist, CDB-4124 (60 mg/kg), or vehicle (20% DMSO + propylene glycol). This dose of CDB-4124 was chosen on the basis of a prior experiment demonstrating attenuation of the effects of progesterone (Uphouse and Hiegel, 2013). One hour later, rats were injected with 4 mg/kg allopregnanolone or vehicle (sesame seed oil). Rats were assigned to one of the three groups: (1) estradiol benzoate, vehicle, and vehicle (EO); (2) estradiol benzoate, vehicle, and allopregnanolone (E-ALLO); and (3) estradiol benzoate,
17
CDB-4124, and allopregnanolone (E-CDB-ALLO). Testing occurred 4 h after allopregnanolone or vehicle. All procedures were in accordance with the PHS Guide and were approved by the TWU IACUC. 2.3. Testing for sexual behavior and restraint procedures Sexual behavior pretesting began 4 h after the sesame seed oil or allopregnanolone injection. Pretesting occurred in the home cage of a sexually active Sprague–Dawley male and behavior was monitored until the male had achieved 10 mounts or for a maximum of 10 min. Immediately after the pretest, females were restrained for 5 min. Thereafter, females were returned to the male's cage for 15 min. Sexual receptivity (L/M ratio; number of lordosis responses divided by number of male mounts) and lordosis quality (measured by the magnitude of the lordosis response) were scored as previously described (White and Uphouse, 2004). Proceptivity (defined as the presence of hopping and darting) and resistance (defined as fighting, boxing, rolling over, trying to escape the cage) were measured as present or absent. When females showed at least one hop/dart sequence or one fighting/boxing sequence, that behavior was categorized as present. Restraint procedures were as previously described (White and Uphouse, 2004). The female was placed head first into a Decapicone® so that her nose was flush with the small opening at the tip of the cone. The base of the cone was gathered around the female's tail and secured tightly with tape. Females were restrained for 5 min, after which they were returned to the male's cage for sexual behavioral testing. 2.4. Statistical procedures Data for L/M ratios and lordosis quality were grouped into the pretest and 3 consecutive 5 min intervals after restraint and were evaluated by repeated measures ANOVA with time relative to restraint as the repeated factor and type of treatment as the independent factor. Post-hoc comparisons were made with Tukey's test. Proceptivity and resistance were compared by Chi-Square procedures. Data were analyzed with SPSS and the statistical reference was Zar (2010). 3. Results A total of 39 rats (14 EO, 11 E-CDB-ALLO, and 14 E-ALLO) were used in the experiment. Of these, 2 EO, 3 E-CDB-ALLO, and 2 E-ALLO were not receptive (as defined by an L/M ratio of at least 0.7) in the pretest and were not restrained. One additional EO and 3 E-ALLO rats did not receive mounts after restraint and were also excluded. L/M ratios before and after restraint are shown in Fig. 1. Only rats showing L/M ratios ≥0.7 in the pretest were included in the ANOVA. There was a significant effect of treatment (F2,25 = 6.72, p ≤ 0.005), due to higher overall L/M ratios in E-ALLO rats, and a significant effect of time relative to restraint (F3,75 = 10.63, p ≤ 0.001). Since all groups showed some decline in L/M ratios after restraint, the time by treatment interaction was not statistically significant (F6,75 = 0.09, p N 0.05). However, rats given E-ALLO showed a smaller decline in L/M ratios than the other two groups. EO rats were different from their pretest at 5 and 15 min after restraint (q 75, 4 = 6.51 and 4.81, respectively, p ≤ 0.05). Rats given E-CDB-ALLO were different from their pretest at 5 and 10 min after restraint (respectively, q75, 4 = 7.05 and 6.03, p ≤ 0.05). Rats given E-ALLO were never significantly different from their pretest. E-CDB-ALLO rats were different from E-ALLO rats at 5 and 10 min after restraint (q75,3 = 5.78 and 5.0, p ≤ 0.05) and EO rats were different from E-ALLO rats at 5 and 15 min after restraint (q75,3 = 4.54 and 4.24, respectively, p ≤ 0.05). EO and E-CDB-ALLO rats did not differ. Lordosis quality remained relatively high after restraint and there was no significant effect of restraint among treatments (p N 0.05) (Table 1). There were few instances of proceptive behavior before or
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L. Uphouse, C. Hiegel / Pharmacology, Biochemistry and Behavior 122 (2014) 16–19
LORDOSIS / MOUNT RATIO
1.0
0.8
0.6
** *
** *
** ** * *
0.4
0.2
0.0 PRE 5 10 15
EO
PRE 5 10 15
PRE 5 10 15
E-ALLO
E-CDB-ALLO
Fig. 1. CDB-4124 attenuates the protective effects of allopregnanolone after restraint stress. Data are the mean ± S.E. L/M ratios before and for consecutive 5 min intervals after a 5 min restraint experience. Ovariectomized rats were hormonally primed with 10 μg estradiol benzoate two days before treatment with vehicles (EO), allopregnanolone (E-ALLO) or CDB-4124 and allopregnanolone (E-CDB-ALLO). Ns for EO, E-ALLO, and ECDB-ALLO were 11, 9, and 8. * indicates a significant decline, within treatment, relative to the pretest. ** indicates a significant difference from E-ALLO rats, within time interval.
after restraint and resistance was present throughout the experiment. There were no differences among groups (all p N 0.05). 4. Discussion The results of this experiment are consistent with prior findings with the progesterone/glucocorticoid receptor antagonist, RU486, and provide additional evidence that the classical intracellular progesterone receptor is important in progesterone's attenuation of the lordosis-inhibiting effects of restraint stress. Allopregnanolone does not bind to this intracellular receptor (Raynaud et al., 1974; Smith et al., 1974). However, the intracellular progesterone receptor can be activated by both ligand-dependent and ligand-independent mechanisms (Dressing et al., 2011; Mani, 2006; Mani et al., 1997; Pluchino et al., 2009). Ligand-independent mechanisms can lead to progesterone receptor activation through a variety of intracellular signaling cascades (Blaustein, 2003; Boonyaratanakornkit et al., 2008; Boonyaratanakornkit et al., 2001; Mani and Portillo, 2010; Mani et al., 2009). Many of these signaling pathways (e.g. cAMP, PKC, PKA, MAPK, Src kinase) are also altered by allopregnanolone (Blackmore, 2008; Etgen and Acosta-Martinez, 2003; Frye and Walf, 2008; Gonzalez-Flores et al., 2006; Mani et al., 2000; Pluchino et al., 2006; Thomas and Pang, 2012) and allopregnanolone's facilitation of lordosis can be blocked by PKC inhibitors (Gonzalez-Flores et al., 2006) or inhibitors of MAPK (Gonzalez-Flores et al., 2004). Therefore, allopregnanolone may indirectly activate intracellular progesterone by activation of intracellular signaling pathways. In addition to the classical intracellular progesterone receptor, there are several membrane progesterone receptors through which Table 1 Characteristics of rats before and after restraint. Group
N
EO Before restraint After restraint E-ALLO Before restraint After restraint E-CDB-ALLO Before restraint After restraint
11
Lordosis quality
% proceptive
% resistive
2.9 ± 0.06 2.3 ± 0.17
14.3% 0%
71.4% 100%
2.8 ± 0.13 2.6 ± 0.06
14.3% 0%
71.4% 100
2.6 ± 0.22 2.6 ± 0.20
9.1% 0%
90.9% 100%
9
8
progesterone exerts its reproductive and nonreproductive effects by activation of intracellular signaling cascades (Pang et al., 2013; Thomas and Pang, 2012; Thomas et al., 2007). Not only the parent molecule, progesterone, but also progesterone metabolites such as allopregnanolone can bind to membrane progesterone receptors (Kelder et al., 2010; Thomas et al., 2007) and activate the relevant signaling cascades (Thomas and Pang, 2012). To our knowledge, it is not known if CDB4124 can interact with these membrane receptors, but RU486 does not appear to block the effects of membrane progesterone receptors (Bashour and Wray, 2012) but does block rapid extranuclear signaling that is mediated through the intracellular progesterone receptor (Hsu and Lee, 2011). Therefore, it is not unreasonable to suggest that allopregnanolone, via binding to membrane progesterone receptors, initiates intracellular signaling pathways to produce ligandindependent activation of intracellular progesterone receptors. Nuclear progesterone receptor antagonists such as RU486 and CDB-4124 would therefore block consequent behavioral effects. In summary, the lordosis behavior of female rats hormonally primed with estradiol benzoate was reduced by 5 min restraint stress. Allopregnanolone attenuated the decline in lordosis behavior and the progestin receptor antagonist, CDB-4124, reduced this attenuation. These findings are consistent with prior studies that implicated the classical intracellular progesterone receptor in the protective effects of progesterone and its metabolite, allopregnanolone, against the lordosisinhibiting effects of restraint stress.
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