RU486 blocks effects of allopregnanolone on the response to restraint stress

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Abstract

These experiments were designed to provide information about the potential involvement of progesterone receptors in the ability of allopregnanolone (3α-hydroxy-5α-pregnan-20-one) to reduce the lordosis-inhibiting effects of restraint stress. Ovariectomized Fischer rats were hormonally primed with 10 μg estradiol benzoate and 4 mg/kg allopregnanolone or vehicle. One hour before allopregnanolone, rats were injected with the progesterone receptor antagonist, RU486 (11β-(4-dimethylamino)phenyl-17β-hydroxy-17-(1-propynyl)estra-4,9-dien-3-one), or vehicle. Four hours after allopregnanolone or vehicle, sexual behavior was examined before and after a 5-min restraint stress. Lordosis behavior of rats primed only with estradiol benzoate declined after the 5 min of restraint while allopregnanolone prevented this decline. RU486 attenuated the ability of allopregnanolone to prevent the restraint-induced decline in lordosis behavior. These findings are consistent with earlier suggestions that progesterone receptors are involved in allopregnanolone's ability to reduce the effects of restraint stress.

Highlights

► Brief restraint reduced lordosis of female rats primed with 10 µg estradiol benzoate. ► Allopregnanolone reduced the inhibitory effects of restraint on lordosis behavior. ► RU486, attenuated allopregnanolone’s protection against restraint stress.

Introduction

Female rat sexual behavior is temporally coordinated with ovulation to maximize reproductive fitness (Blaustein, 2008, Sodersten, 1981). Both estrogens and progestins influence this reproductive synchrony (Auger, 2004, Mani et al., 1997, Pfaff, 1970) so that reproductive behavior abruptly ceases following ovariectomy. However, sexual behavior can be restored in ovariectomized rats by exogenous treatment with gonadal hormones (Auger, 2004, Pfaff, 2005). The full repertoire of female sexual behavior includes appetitive, precopulatory and copulatory activities and gonadal hormones appear to differentially influence these various events (Blaustein, 2008, Erskine, 1989, Frye, 2007). The lordosis reflex (required for copulation) is thought to depend exclusively on estradiol (Auger, 2004, Blaustein, 2008). Progesterone is not required, but the probability and frequency of lordosis behavior can be increased by progesterone; and progesterone may be required for the occurrence of precopulatory activity such as hopping and darting behavior (Erskine, 1989, Frye and Vongher, 1999, Sodersten, 1981). Progesterone also reduces measures of anxiety (Auger and Forbes-Lorman, 2008, Gomez et al., 2002) and we have previously suggested that progesterone's anxiolytic action contributes to its facilitation of female rat sexual behavior (Truitt et al., 2003, Uphouse et al., 2008, White and Uphouse, 2004).

When ovariectomized Fischer inbred rats are hormonally primed with 10 μg estradiol benzoate, lordosis behavior is comparable to that obtained following hormonal priming with estradiol benzoate and progesterone. However, when subjected to a mild 5-min restraint stress, rats primed only with estradiol benzoate show disruption of lordosis behavior (Hassell et al., 2011, White and Uphouse, 2004). The lordosis disruption is transient, lasting 5–10 min after the restraint experience (Uphouse et al., 1993, White and Uphouse, 2004). The restraint experience amplifies the negative effect of the serotonin 1A (5-HT1A) receptor agonist, 8-OH-DPAT [(+/−)-8-hydroxy-2-(di-n-propylamino)tetralin] on lordosis behavior (Uphouse et al., 2007) but is attenuated by the 5-HT2 receptor agonist DOI [(+/−)-2,5-dimethoxy-4-iodophenyl-2-aminopropane HCl]. We have suggested that this dual regulation by 5-HT is important to the female's continuity of mating in the presence of a mild stressor. Increased 5-HT release, precipitated by stress (Chaouloff, 2000), can activate all 5-HT receptors. Activation of the higher affinity 5-HT1A receptors produces a rapid decline in lordosis behavior; activation of slower acting, lordosis facilitating 5-HT2 receptors attenuates the decline in behavior and allows mating to continue (Uphouse, 1997, Wolf et al., 1998, Wolf et al., 1999). Progesterone attenuates this response to stress by reducing the lordosis-inhibitory effects of 5-HT1A receptor activation, in part by reducing extracellular 5-HT (Farmer et al., 1996, Maswood et al., 1995, Maswood et al., 1999, Truitt et al., 2003, White and Uphouse, 2004). Therefore, one of the effects of progesterone in the modulation of female rat sexual behavior includes its attenuation of the negative effects of mild stress on lordosis behavior. However, the relative roles of the parent molecule and its metabolites in the mechanisms leading to this attenuation are unknown.

Progesterone modulates reproductive and nonreproductive behaviors through classical progesterone-receptor-mediated mechanisms that can be initiated via both ligand-dependent and ligand-independent pathways (Conneely et al., 2003, Dressing et al., 2011, Mani et al., 1997, Pluchino et al., 2009). Progesterone can also be metabolized by 5α-reductase into 5α-dihydroprogesterone and then by 3α-hydroxysteroid dehydrogenase (3α-HSD) into allopregnanolone (Rupprecht, 2003, Schule et al., 2011). Progesterone metabolites, such as allopregnanolone, contribute to the regulation of female rat sexual behavior (Blaustein, 2008, Frye et al., 1998, Mani and Portillo, 2010) and are especially important mediators of progesterone's anxiolytic effects (Dubrovsky, 2006, Eser et al., 2008). However, these progesterone metabolites do not appear to be required for progesterone's attenuation of the negative effects of 5 min restraint on lordosis behavior (Hassell et al., 2011, Miryala et al., 2011).

In previous studies, we questioned if progesterone's reduction of the lordosis-inhibiting effect of restraint stress resulted from the parent molecule, progesterone, or if progesterone metabolites were responsible (Hassell et al., 2011, Miryala et al., 2011). Progesterone's attenuation of the response to restraint stress (a) was mimicked by the nonmetabolizable progestin, medroxyprogesterone; (b) was not attenuated by the 5α-reductase inhibitor, finasteride, or the 3α-HSD inhibitor, indomethacin; but (c) was attenuated by the progesterone/glucocorticoid receptor antagonist, RU486, and by the more selective progesterone receptor antagonist, CDB4124, that is devoid of glucocorticoid receptor action (Hassell et al., 2011, Miryala et al., 2011, Uphouse and An Antiprogestin, submitted for publication). These data, therefore, were consistent with a requirement for progesterone-receptor mediated events in progesterone's attenuation of the lordosis-inhibiting effect of restraint. However, the progesterone metabolite, allopregnanolone, was also effective in reducing the effects of restraint (Miryala et al., 2011). Since inhibition of progesterone metabolism with finasteride or indomethacin did not eliminate progesterone's ability to attenuate the negative response to restraint, allopregnanolone's ability to reduce the response to restraint could mean that redundant mechanisms (e.g. progesterone-receptor mediated or progesterone-metabolite initiated events) were capable of reducing the lordosis-inhibiting effect of restraint. However, this seems unlikely since blocking progesterone receptors blocked progesterone's attenuation of restraint while blocking progesterone metabolism did not (Hassell et al., 2011, Miryala et al., 2011). An alternative explanation is that allopregnanolone is also able to lead to activation of progesterone receptors.

Allopregnanolone does not bind directly to intracellular progesterone receptors (Raynaud et al., 1974, Smith et al., 1974) but may indirectly activate progesterone receptors through a variety of intracellular signaling pathways (Etgen et al., 2006, Frye and Walf, 2008, Gonzalez-Flores et al., 2010, Mani et al., 2000). PKC, MAPK, and Src kinase-dependent signaling are modulated by allopregnanolone (Etgen and Acosta-Martinez, 2003, Frye and Walf, 2008, Gonzalez-Flores et al., 2006, Mani et al., 2000) and each of these mechanisms can contribute to ligand-independent activation of progesterone receptors (Boonyaratanakornkit et al., 2007, Gonzalez-Flores et al., 2006, Li and Shang, 2007, Mani and Portillo, 2010, Tetel, 2009). Such an indirect activation of progesterone receptors by allopregnanolone has been suggested previously (Auger and Forbes-Lorman, 2008, Beyer et al., 1995, Gonzalez-Flores et al., 2006, Gonzalez-Flores et al., 2010, Miryala et al., 2011). Moreover, at least 1 h of priming by allopregnanolone was required for the progesterone metabolite to reduce the lordosis-inhibiting effect of restraint (Miryala et al., 2011). Therefore, the collective data are consistent with the hypothesis that progesterone receptors contribute to allopregnanolone's attenuation of the lordosis-inhibiting effect of restraint. If so, RU486 should be able to block the effect of allopregnanolone. The following experiment was designed to test this hypothesis.

Section snippets

Materials

Estradiol benzoate, allopregnanolone (3α-hydroxy-5α-pregnan-20-one), RU486 (11β-(4-dimethylamino)phenyl-17β-hydroxy-17-(1-propynyl)estra-4,9-dien-3-one), dimethyl sulfoxide (DMSO) and sesame seed oil were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). Propylene glycol was obtained 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).

Behavior before restraint

With the exception of a single rat that had been treated only with estradiol benzoate and vehicles, every rat showed L/M ratios greater than 0.7 during the pretest. This single rat was omitted from data analyses. L/M ratios from the remaining rats are shown in Table 1. There were significant effects of both allopregnanolone (F1,55 = 12.86, p  0.001) and RU486 (F1,55 = 9.12, p  0.004) and their interaction (F1,55 = 8.80, p  0.004) on L/M ratios during the pretest before restraint. These differences were

Discussion

These experiments were designed to provide information relevant to the hypothesis that allopregnanolone's reduction of the lordosis-inhibiting effect of restraint could include an indirect activation of the progesterone receptor. If this hypothesis were correct, then the progesterone receptor antagonist, RU486, should reduce allopregnanolone's effect. The results of this study are consistent with this hypothesis.

Allopregnanolone's neural effects have often been attributed to positive effects at

Conclusions

Consistent with prior research, 5 min restraint reduced lordosis behavior of ovariectomized rats hormonally primed with 10 μg estradiol benzoate. Addition of allopregnanolone priming attenuated the response to restraint and RU486 reduced the effect of allopregnanolone. Estradiol benzoate priming, alone, was sufficient to induce lordosis behavior but did not produce proceptive behavior. Neither allopregnanolone nor RU486 altered estradiol's induction of lordosis behavior before restraint but

Acknowledgments

The authors express appreciation to Mr. Dan Wall and Ms. Karolina Blaha-Black for animal care and to Dr. Jutatip Gutarak for reading a prior version of the manuscript. The research was supported by NIH HD28419 and by TWU REP.

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  • Cited by (0)

    1

    Current address: Department of Biological and Biomedical Sciences Program, University of North Carolina, Chapel Hill, NC 27599, United States.

    2

    Current address: Department of Biology, University of South Dakota, Vermillion, SD, United States.

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