Urine cortisol concentration as a biomarker of stress is unrelated to IVF outcomes in women and men.

PURPOSE: Our primary objective was to assess associations between urine cortisol as a biomarker of psychological stress and in vitro fertilization (IVF) outcomes. A secondary objective was to assess associations between toxic metals and cortisol. METHODS: Urine and blood specimens were collected from 52 women and 28 male partners completing a first IVF procedure, on the day of oocyte retrieval. Urine cortisol was measured with an enzyme-linked immunosorbent assay. Mercury (Hg), cadmium (Cd), and lead (Pb) were determined in blood and Cd in urine by inductively coupled plasma-mass spectrometry. RESULTS: No associations were indicated for cortisol with IVF outcomes in multivariable regression models adjusted for covariates. However, we detected positive linear associations for cortisol and urine Cd (ß = 9.96, 95%CI 1.52, 21.44) and blood Hg (ß = 1.44, 95%CI 0.31, 3.18). An exploratory stratified analysis suggested a potential inverse association between urine cortisol and oocyte fertilization among women with low, but not high blood Hg. CONCLUSION: While limited, these preliminary data suggest that psychological stress may not play a major role in IVF outcomes, which therefore could be one less concern for couples and their clinicians. Our data also raise the possibility for toxic metals to modify associations between cortisol and IVF outcomes among women. However, these preliminary results require corroboration in an experimental animal model and confirmation in a larger, more definitive observational study.

Membrane progestin receptors in the midbrain ventral tegmental area are required for progesterone-facilitated lordosis of rats.

Progesterone (P4) and its metabolites, rapidly facilitate lordosis of rats partly through actions in the ventral tegmental area (VTA). The study of membrane progestin receptors (mPRs), of the Progestin and AdipoQ Receptor (PAQR) superfamily, has been limited to expression and regulation, instead of function. We hypothesized that if mPRs are required for progestin-facilitated lordosis in the VTA, then mPRs will be expressed in this region and knockdown will attenuate lordosis. First, expression of mPR was examined by reverse-transcriptase polymerase chain reaction (RT-PCR) in brain and peripheral tissues of proestrous Long-Evans rats. Expression of mPRα (paqr7) was observed in peripheral tissues and brain areas, including hypothalamus and midbrain. Expression of mPRß (paqr8) was observed in brain tissues and was abundant in the midbrain and hypothalamus. Second, ovariectomized rats were estrogen (E2; 0.09 mg/kg, SC), and P4 (4 mg/kg, SC) or vehicle-primed, and infused with antisense oligodeoxynucleotides (AS-ODNs) targeted against mPRα and/or mPRß intracerebroventricularly or to the VTA. Rats were assessed for motor (open field), anxiety (elevated plus maze), social (social interaction), and sexual (lordosis) behavior. P4-facilitated lordosis was significantly reduced with administration of AS-ODNs for mPRα, mPRß, or co-administration of mPRα and mPRß to the lateral ventricle, compared to vehicle. P4-facilitated lordosis was reduced, compared to vehicle, by administration of mPRß AS-ODNs, or co-administration of mPRα and mPRß AS-ODNs, but not mPRα AS-ODNs alone, to the VTA. No differences were observed for motor, anxiety, or social behaviors. Thus, mPRs in the VTA are targets of progestin-facilitated lordosis of rats.

Motivated behaviors and levels of 3α,5α-THP in the midbrain are attenuated by knocking down expression of pregnane xenobiotic receptor in the midbrain ventral tegmental area of proestrous rats.

INTRODUCTION: Progesterone (P4 ) and its product, 5α-pregnan-3α-ol-20-one (3α,5α-THP), act in the midbrain ventral tegmental area (VTA) to alter motivated behaviors, such as mating, and motor and anxiety behavior. Of interest is whether 3α,5α-THP formation requires the pregnane xenobiotic receptor (PXR), which is expressed in the midbrain of rats. AIM: The role of PXR in the midbrain for 3α,5α-THP formation, which precedes modulation of motivated behaviors, was investigated. METHODS: Rats had estrous cycle phase determined and were assessed when they were in diestrus or proestrus. Diestrous and proestrous rats were infused with control or antisense oligodeoxyribonucleotides (AS-ODNs) targeted against PXR to the VTA. MAIN OUTCOME MEASURES: In pilot studies, PXR gene and protein expression in the midbrain were determined with quantitative reverse transcriptase polymerase chain reaction and Western blotting, respectively. Diestrous and proestrous rats infused with control or AS-ODNs to the VTA were tested for anxiety (open field and plus maze), social (social interaction), and sexual (paced mating) behavior. Expression of PXR in the midbrain was verified with Western blotting. Plasma estradiol, P4 , dihydroprogesterone (DHP), and 3α,5α-THP levels, and brain P4 , DHP, and 3α,5α-THP levels were measured. We predicted that proestrous rats infused with PXR AS-ODNs would have decreased anti-anxiety, social, and sexual behavior, lower midbrain expression of PXR, and lower midbrain levels of 3α,5α-THP compared with controls. RESULTS: Results supported the hypothesis that formation of 3α,5α-THP requires PXR and may be important for motivated behaviors. PXR AS-ODN, compared with control, infusions to the VTA reduced PXR expression and 3α,5α-THP levels in the midbrain and attenuated sexual receptivity of proestrous rats. CONCLUSIONS: Knockdown of PXR in the midbrain reduces 3α,5α-THP levels and sexual receptivity of proestrous rats. Thus, PXR in the midbrain may be required for the observed increase in 3α-5α-THP during proestrus, which has subsequent effects on motivated, reproductive behaviors.

Raloxifene and/or estradiol decrease anxiety-like and depressive-like behavior, whereas only estradiol increases carcinogen-induced tumorigenesis and uterine proliferation among ovariectomized rats.

Controversy surrounds the efficacy and safety of 17beta-estradiol (E2)-mimetic therapies to women for treatment of menopausal symptoms. An important question is the nature of the trophic actions of E2-mimetics in the brain for behavioral processes versus in the periphery for beneficial effects related to osteoporosis, or unwanted proliferative effects in the reproductive tissues, such as mammary glands and uterus. Of recent interest are the effects of selective estrogen receptor modulators, which can have tissue specific actions, for these processes. In the present study, the effects were determined of E2 alone, or co-administered with a selective estrogen receptor modulator, raloxifene, for anxiety-like, depression-like, and trophic peripheral effects in ovariectomized rats that were exposed to a chemical carcinogen (7,12-dimethylbenz(a)anthracene), or not. Once per week, rats were administered vehicle, E2 (0.09 mg/kg) and/or raloxifene (1 mg/kg) subcutaneously 44-48 h before testing in a positive control, E2-dependent behavior (lordosis), depression (forced swim test), and anxiety (elevated plus maze) behavioral assays. In addition to behavioral endpoints, incidence and number of tumors, and tumor, pituitary gland, and uterine weight 14 weeks after carcinogen-exposure, and weekly hormone treatments, were analyzed. Rats administered 7,12-dimethylbenz(a)anthracene had an increased number and size of tumors, compared with vehicle treatment. E2 + raloxifene increased the number of tumors. Administration of E2 or E2 +raloxifene, but not raloxifene alone, increased pituitary and uterine weight, compared with vehicle administration. E2 or E2 + raloxifene, but not raloxifene alone, also increased the incidence of lordosis and reduced the depression-like behavior in the forced swim test (i.e. decreased time spent immobile) compared with vehicle administration. However, administration of E2 or raloxifene reduced anxiety behavior in the elevated plus maze (i.e. increased time spent on the open arms of the maze), compared with vehicle treatment. Together these data show that E2 and/or raloxifene can have some effects to alter the behavior of ovariectomized rodents, depending upon the task. As well, E2, with or without raloxifene, can also have clear trophic actions in peripheral tissues, such as carcinogen-induced tumors, uterus, and pituitary glands.

Conjugated equine estrogen, with medroxyprogesterone acetate, enhances formation of 5alpha-reduced progestogens and reduces anxiety-like behavior of middle-aged rats.

The mechanisms by which progestogens influence affective behaviors in females are poorly understood despite clear changes in mood/affect that are associated with their decline during menopause. Conjugated equine estrogens (CEE), with or without medroxyprogesterone acetate (MPA), are commonly prescribed hormone-replacement, but there is heterogeneity in responses to these pharmacotherapies. One way in which these compounds differ is in their capacity to potentiate metabolism of progesterone to its 5alpha-reduced products, dihydroprogesterone and 5alpha-pregnan-3alpha-ol-20-one (3alpha,5alpha-THP). This study investigated whether responses to CEE and MPA may be related to the capacity to metabolize progesterone. Middle-aged female rats that had maintained reproductive status, or those that had a decline, were administered vehicle, CEE and/or MPA. Effects on anxiety-like (open field, elevated plus maze) and social behaviors (social interaction test), and plasma and hippocampus steroid levels were determined. We hypothesized that CEE, but not MPA, would decrease anxiety-like behavior coincident with increased hippocampal metabolism of progesterone. CEE, or CEE+MPA, increased central entries in the open field and time spent on the open arms of the plus maze, but did not alter social interaction of rats that had maintained reproductive status. CEE and/or CEE+MPA increased E2 and 3alpha,5alpha-THP in plasma and/or hippocampus of rats, but MPA increased levels of dihydroprogesterone in the hippocampus of rats with declining reproductive status. Simple regressions showed that hippocampus 3alpha,5alpha-THP levels accounted for a significant proportion of the variance in anxiety-like behavior. Therefore, effects of CEE to reduce anxiety-like behavior of middle-aged rats may be owing, in part, to its capacity to enhance levels of 3alpha,5alpha-THP in the hippocampus.

Progesterone's Effects on Cognitive Performance of Male Mice Are Independent of Progestin Receptors but Relate to Increases in GABAA Activity in the Hippocampus and Cortex.

Progestogens' (e.g., progesterone and its neuroactive metabolite, allopregnanolone), cognitive effects and mechanisms among males are not well-understood. We hypothesized if progestogen's effects on cognitive performance are through its metabolite allopregnanolone, and not actions via binding to traditional progestin receptors (PRs), then progesterone administration would enhance performance in tasks mediated by the hippocampus and cortex, coincident with increasing allopregnanolone concentrations, brain derived neurotrophic factor (BDNF) and/or muscimol binding of PR knock out (PRKO) and wild-type PR replete mice. Experiment 1: Progesterone (4 mg/kg, subcutaneously (SC; n = 12/grp), or oil vehicle control, was administered to gonadally-intact adult male mice PRKO mice and their wild-type counterparts and cognitive behaviors in object recognition, T-maze and water maze was examined. Progesterone, compared to vehicle, when administered post-training increased time investigating novel objects by the PRKO and wild-type mice in the object recognition task. In the T-maze task, progesterone administration to wild-type and PRKO mice had significantly greater number of spontaneous alternations compared to their vehicle-administered counterparts. In the water maze task, PRKO mice administered vehicle spent significantly fewer seconds in the quadrant associated with the escape platform on testing compared to all other groups. Experiment 2: Progesterone administered to wild-type and PRKO mice increased plasma progesterone and allopregnanolone levels (n = 5/group). PRKO mice had higher allopregnanolone levels in plasma and hippocampus, but not cortex, when administered progesterone and compared to wild-type mice. Experiment 3: Assessment of PR binding revealed progesterone administered wild-type mice had significantly greater levels of PRs in the hippocampus and cortex, compared to all other groups (n = 5/group). Wild-type mice administered progesterone, but not vehicle, had increased BDNF levels in the hippocampus, but not the cortex, compared to PRKOs. Wild-type as well as PRKO mice administered progesterone experienced significant increases in maximal GABAA agonist, muscimol, binding in hippocampus and cortex, compared to their vehicle-administered counterparts. Thus, adult male mice can be responsive to progesterone for cognitive performance, and such effects may be independent of PRs trophic actions of BDNF levels in the hippocampus and/or increases in GABAA activity in the hippocampus and cortex.

Effects of two estradiol regimens on anxiety and depressive behaviors and trophic effects in peripheral tissues in a rodent model.

BACKGROUND: With aging and menopause, which are associated with decreases in ovarian steroids such as 17beta-estradiol (E(2)), women might experience negative psychological symptoms, including anxiety and depression. Some women use E(2)-based therapies to alleviate these symptoms, but E(2) has been associated with trophic effects that might increase vulnerability to some steroid-sensitive cancers, such as breast cancer, in both premenopausal and postmenopausal women. OBJECTIVE: This study investigated the relationships between the possible beneficial effects of E(2) on anxiety and depressive behaviors concurrent with trophic effects using an animal model of E(2) decline and replacement. METHODS: Dose-dependent effects of E(2) on affective, sexual, and motor behavior of young adult rats were studied. Ovariectomized (OVX) rats were administered the chemical carcinogen 7,12-dimethylbenz(a) anthracene (DMBA) 1.25 mg or inactive vehicle (vegetable oil; control) by gavage. E(2) (0.03 or 0.09 mg/kg) or vehicle was administered subcutaneously 44 to 48 hours before assessments of anxiety (light-dark transition), depression (forced swim test), sexual (lordosis), and motor (activity monitor) behaviors. Fourteen weeks after carcinogen exposure, E(2) concentrations in plasma and brain regions (cortex, hippocampus, and hypothalamus) were determined. Incidences and numbers of tumors and uterine weight were analyzed. RESULTS: Administration of E(2) (0.09 mg/kg) was associated with significant increases in antianxiety-like behavior in the light-dark transition task, antidepressant-like behavior in the forced swim test, and physiologic circulating and central E(2) concentrations compared with E(2) (0.03 mg/kg) and vehicle. Compared with vehicle, E(2) (0.9 > 0.3 mg/kg) was associated with significant increases in lordosis and uterine weight. Administration of DMBA was associated with significant increases in the incidences and numbers of tumors; this effect was augmented by E(2)administration. CONCLUSIONS: Based on the findings in this rat model, the hypothesis that E(2) may be effective in reducing anxiety and depressive behaviors and enhance sexual behavior in OVX rats, concurrent with trophic effects in the periphery, was supported. Moderate physiologic levels of E(2) might have beneficial effects on affective and sexual behaviors in female rodents, but regimens including E(2) might increase tumorigenic capacity.

Effects of non-contingent cocaine on 3alpha-androstanediol. I. Disruption of male sexual behavior.

One of the hallmarks of drug abuse is a reduction in the salience of, and motivation for, natural rewards, such as mating. The effects of psychostimulants on male sexual interest and performance are conflicting; use of psychostimulants can produce increases in risky sexual behaviors but have detrimental effects on sexual ability. We hypothesize that these conflicting effects on sexual behavior are due to interactions between cocaine and androgens, such as testosterone and its neuroactive metabolite, 3α-androstanediol (3α-diol). Male rats were administered saline or cocaine (5, 10, or 20mg/kg, i.p.). Motor behavior was observed in the first 30min following drug-administration, and then sexual responding was assessed for 15min. Levels of androgens (testosterone, 3ɑ-diol, and testosterone's aromatized metabolite, estradiol) were measured in circulation and brain regions (frontal cortex, hippocampus, hypothalamus/striatum (hypo/str), and midbrain). Cocaine had no effect on measures of sexual interest (i.e. anogenital investigation). However, cocaine had substantial effects on consummatory sexual behaviors, such as the latency to mount/intromit and the number of sexual contacts. Frontal cortex and hypo/str 3α-diol levels were strongly correlated with consummatory behaviors in saline administered rats; however, this relationship was disrupted by cocaine at all dosages, concomitant with impaired sexual behaviors. Additionally, there was a shift in metabolism at low dosages of cocaine to push testosterone metabolism in the midbrain towards 3α-diol. On the contrary, moderate and high dosages of cocaine shifted testosterone metabolism towards estradiol. These data demonstrate that the association between cortical and hypo/str 3α-diol levels and sexual behavior of male rats is disrupted by non-contingent cocaine and that there may be dose-dependent effects of acute cocaine on androgen metabolism.

Effects of non-contingent cocaine on 3 alpha-androstanediol. II. Disruption of lordosis of proestrous rats.

Drug use influences sexual behavior, performance, and can be associated with increased sexual risk-taking. Our prior results using an animal model indicate that progestogens contribute to hormonally-mediated changes in sexual behavior of female rodents during acute cocaine exposure. Androgens, such as testosterone, and its metabolite 3ɑ-androstanediol (3α-diol), and estradiol, are known to influence male sexual behavior, but can also alter the expression of sexual behavior of female rodents. As such, we investigated the influence of endogenous androgen and estradiol fluctuations on cocaine-mediated changes in motor behavior and sexual receptivity of rats during diestrous or proestrous phases of the estrous cycle. Female rats were administered saline or cocaine (5, 10, or 20mg/kg, i.p.). Motor behavior was observed in the first 30min following drug administration, and then sexual responding was assessed for 15min. Cocaine decreased aggressive behavior in response to attempted mounts by a male among non-receptive (diestrous) rats and inhibited sexual behavior among sexually receptive (proestrous) rats. Cocaine dose-dependently altered concentrations of testosterone metabolites (estradiol and 3α-diol), but not testosterone, which correlated to motor and sexual behaviors of diestrous and proestrous rats, respectively. These data suggest that actions of 3α-diol may be involved in female sexual and motor behavior in response to cocaine, in a cycle-dependent manner.

The Steroidogenesis Inhibitor Finasteride Reduces the Response to Both Stressful and Rewarding Stimuli.

Finasteride (FIN) is the prototypical inhibitor of steroid 5α-reductase (5αR), the enzyme that catalyzes the rate-limiting step of the conversion of progesterone and testosterone into their main neuroactive metabolites. FIN is clinically approved for the treatment of benign prostatic hyperplasia and male baldness; while often well-tolerated, FIN has also been shown to cause or exacerbate psychological problems in vulnerable subjects. Evidence on the psychological effects of FIN, however, remains controversial, in view of inconsistent clinical reports. Here, we tested the effects of FIN in a battery of tests aimed at capturing complementary aspects of mood regulation and stress reactivity in rats. FIN reduced exploratory, incentive, prosocial, and risk-taking behavior; furthermore, it decreased stress coping, as revealed by increased immobility in the forced-swim test (FST). This last effect was also observed in female and orchiectomized male rats, suggesting that the mechanism of action of FIN does not primarily reflect changes in gonadal steroids. The effects of FIN on FST responses were associated with a dramatic decrease in corticotropin release hormone (CRH) mRNA and adrenocorticotropic hormone (ACTH) levels. These results suggest that FIN impairs stress reactivity and reduces behavioral activation and impulsive behavior by altering the function of the hypothalamus-pituitary-adrenal (HPA) axis.

Antisense oligodeoxynucleotides for estrogen receptor-beta and alpha attenuate estradiol's modulation of affective and sexual behavior, respectively.

Estradiol (E(2)) modulates affective and socio-sexual behavior of female rodents. E(2)'s functional effects may involve actions through alpha and beta isoforms of estrogen receptor (ERs). The importance of E(2)'s actions at these isoforms for anxiety (open field, elevated plus maze), depression (forced swim test), and sexual behavior (lordosis) was investigated using an antisense oligonucleotide (AS-ODN) strategy. If ERbeta is required for anti-anxiety and antidepressant-like effects, and ERalpha is required for sexual receptivity, of E(2), then intracerebroventricular administration of AS-ODNs against these ERs should attenuate these effects and reduce immunoreactivity of ERs in brain regions that mediate these behaviors, such as the hippocampus and ventral medial hypothalamus (VMH). Ovariectomized rats were primed with 17beta-E(2) (10 microg) 48 h before testing (hour 0). At hours 0, 24, and 47.5, rats were infused with saline vehicle, scrambled control AS-ODNs, or AS-ODNs targeted against ERalpha and/or ERbeta, and were tested at hour 48. Rats infused with ERbeta AS-ODNs, alone, or with ERalpha AS-ODNs had significantly decreased open field central entries, decreased plus maze open arm time and entries, increased time spent immobile, and decreased time spent swimming in the forced swim test, and decreased ERbeta immunoreactivity in the brain than did rats administered ERalpha AS-ODNs, vehicle, or scrambled AS-ODNs. Rats that were administered ERalpha AS-ODNs, alone, or with ERbeta AS-ODNs had significantly decreased lordosis and decreased ERalpha immunoreactivity in the brain compared to rats administered ERbeta AS-ODNs, vehicle, or scrambled AS-ODNs. Thus, ERbeta and ERalpha may be required for E(2)'s modulation of affective and sexual behavior, respectively.

Progesterone to ovariectomized mice enhances cognitive performance in the spontaneous alternation, object recognition, but not placement, water maze, and contextual and cued conditioned fear tasks.

Research on how steroid hormones mediate mnemonic processes have focused on effects of 17beta-estradiol (E(2)); yet, progesterone (P(4)) co-varies with E(2) across endogenous hormonal milieu, and itself may influence cognitive processes. We investigated the hypothesis that acute P(4) treatment enhances cognitive performance compared to vehicle. Ovariectomized (OVX) c57/BL6J mice were randomly assigned to be subcutaneously injected with oil vehicle or P(4) (10mg/kg). Mice were trained in the spontaneous alternation, object recognition, object placement, water maze, or fear conditioning tasks, and injected with vehicle or P(4) before training or immediately post-training, and then were tested 1, 4, or 24h later. The data obtained from these experiments supported our hypothesis. P(4) increased the percentage of spontaneous alterations made in a T-maze more so than did vehicle. P(4), compared to vehicle, increased the percentage of time spent exploring the novel object in the object recognition task, but did not alter performance in the object placement task. P(4), compared to vehicle, decreased latencies to reach the location in the water maze where the platform had been during training in a probe trial, but did not alter performance in the control, cued trial. Compared to vehicle, P(4) treatment increased freezing in contextual and cued fear testing. Thus, acute P(4) treatment to OVX mice can improve cognitive performance across a variety of tasks.

Estradiol or diarylpropionitrile administration to wild type, but not estrogen receptor beta knockout, mice enhances performance in the object recognition and object placement tasks.

Cognitive processes mediated by the hippocampus and cortex are influenced by estradiol (E(2)); however, the mechanisms by which E(2) has these effects are not entirely clear. As such, studies were conducted to begin to address the role of actions at the beta form of the intracellular estrogen receptor (ERbeta) for E(2)'s cognitive effects in adult female mice. We investigated whether E(2) improved performance of wild type (WT) and ERbeta knockout (betaERKO) mice in tasks considered to be mediated by the cortex and hippocampus, the object recognition and object placement tasks. WT and betaERKO mice were ovariectomized (ovx) and E(2) (0.1 mg/kg), an ERbeta selective ER modulator (SERM), diarylpropionitrile (DPN; 0.1 mg/kg), or oil vehicle was administered to mice following training in these tasks. We hypothesized that if E(2) has mnemonic effects, in part, due to its actions at ERbeta, then WT mice administered E(2) or DPN would have improved performance compared to vehicle WT controls, which would not be different from betaERKO mice administered vehicle, E(2) or DPN. Alternatively, activation of ERalpha (with E(2), which is a ligand for both ERalpha and ERbeta) may produce opposing effects on cognition and/or the activation of ERalpha and ERbeta vs. either receptor isoform alone may produce a different pattern of effects. Results obtained supported the hypothesis that ERbeta activation is important for mnemonic effects. Ovx WT, but not betaERKO, mice administered E(2) or DPN had a greater percentage of time exploring a novel object in the object recognition task and a displaced object in the object placement task. Thus, actions at ERbeta may be important for E(2) or SERMs to enhance cognitive performance of female mice in the object recognition and placement tasks.

Estradiol or diarylpropionitrile decrease anxiety-like behavior of wildtype, but not estrogen receptor beta knockout, mice.

Clinical and basic studies demonstrate that estrogen (E-sub-2)-based therapies influence anxiety and mood, but the receptor targets (e.g., a or ss isoform of the estrogen receptor, ER) for these effects requires further investigation. To address the specificity of E2's anxiolytic-like effects through ERss, anxiety, motor, and nociceptive behavior of ovariectomized, wildtype (WT), and ERss knockout (ssERKO) mice was examined. Mice were administered oil vehicle or ER agonists, 17ss-E2 (E2; 0.1 mg/kg; similar affinity for ERa and ERss), and a selective ER modulator, diarylpropionitrile (DPN; 0.1 mg/kg; greater affinity for ERss than ERa). Performance of mice in anxiety (open field, elevated plus maze, elevated zero maze, social interaction), motor activity (activity monitor) and nociception (tailflick, pawlick) measures was compared. Results supported our hypothesis that ERss is important in modulation of anxiety-like behavior by E2 in some tasks. Administration of E2 or DPN to WT, but not ssERKO, mice increased open field central entries, plus maze open arm time, zero maze open quadrant time, and social interaction. This pattern was neither seen in motor activity nor pain threshold measures. Thus, actions of ERss may be important for modulating anxiety-like behavior of mice.

Conjugated equine estrogen enhances rats' cognitive, anxiety, and social behavior.

The ovarian hormone, 17beta-estradiol (E2), has numerous targets in the body and brain, and can influence cognitive, affective, and social behavior. However, functional effects of commonly prescribed E2-based hormone therapies are less known. The effects of conjugated equine estrogen (CEE) on middle-aged female rats for cognitive (object recognition), anxiety (open field, plus maze), and social (social interaction, lordosis) behavior were compared with vehicle. Our hypothesis that CEE would enhance cognitive, anxiety, and social behavior was supported. CEE improved object recognition, increased time spent on the open arms of the plus maze, and time spent interacting with a conspecific, but did not alter open field behavior or lordosis. Thus, CEE can enhance cognitive, antianxiety, and social behavior of middle-aged rats.

Androgens with activity at estrogen receptor beta have anxiolytic and cognitive-enhancing effects in male rats and mice.

Testosterone (T) and its metabolites may underlie some beneficial effects for anxiety and cognition, but the mechanisms for these effects are unclear. T is reduced to dihydrotestosterone (DHT), which can be converted to 5alpha-androstane,3alpha,17beta-diol (3alpha-diol) and/or 5alpha-androstane-3beta,17beta-diol (3beta-diol). Additionally, T can be converted to androstenedione, and then to androsterone. These metabolites bind with varying affinity to androgen receptors (ARs; T and DHT), estrogen receptors (ERbeta; 3alpha-diol, 3beta-diol), or GABA(A)/benzodiazepine receptors (GBRs; 3alpha-diol, androsterone). Three experiments were performed to investigate the hypothesis that reduced anxiety-like and enhanced cognitive performance may be due in part to actions of T metabolites at ERbeta. Experiment 1: Gonadectomized (GDX) wildtype and ERbeta knockout mice (betaERKO) were subcutaneously (SC) administered 3alpha-diol, 3beta-diol, androsterone, or oil vehicle at weekly intervals, and tested in anxiety tasks (open field, elevated plus maze, light-dark transition) or for cognitive performance in the object recognition task. Experiment 2: GDX rats were administered SC 3alpha-diol, 3beta-diol, androsterone, or oil vehicle, and tested in the same tasks. Experiment 3: GDX rats were androsterone- or vehicle-primed and administered an antagonist of ARs (flutamide), ERs (tamoxifen), or GBRs (flumazenil), or vehicle and then tested in the elevated plus maze. Both rats and wildtype mice, but not betaERKO mice, consistently had reduced anxiety and improved performance in the object recognition task. Androsterone was only effective at reducing anxiety-like behavior in the elevated plus maze and this effect was modestly reduced by flumazenil administration. Thus, actions at ERbeta may be required for T's anxiety-reducing and cognitive-enhancing effects.

In the ventral tegmental area, progestogens' membrane-mediated actions for lordosis of rats involve the second-messenger phospholipase C.

Steroid hormones have pervasive functional effects. Although steroids are generally known to have actions via binding to their cognate steroid receptors, it is becoming clearer that steroids can have non-traditional actions that do not require activation of cognate steroid receptors. We have found that progestogen-facilitated lordosis of rodents is enhanced by activation of dopamine type 1 (D1) or GABA(A) receptors and their downstream effectors, such as second messengers, in the ventral tegmental area (VTA). The role of phospholipase C in these effects is not clear. If progestins' actions through D1 and GABA(A) receptors in the VTA are mediated through PLC, then inhibiting PLC formation in the VTA, via infusions of U73122 (400nM/side), should reduce progestin (5alpha-pregnan-3alpha-ol-20-one; 3alpha,5alpha-THP; 100 or 200ng/side)-facilitated lordosis and its enhancement by D1 (SKF38393; 100ng/side) or GABA(A) (muscimol; 100ng/side) receptor agonists in ovariectomized, estradiol-primed rats. We found that 3alpha,5alpha-THP-, SKF38393-, and muscimol-facilitated lordosis was attenuated by infusions of the PLC inhibitor, U73122, but not vehicle, to the VTA. Thus, progestogens' non-traditional actions in the VTA to enhance lordosis through D1 and/or GABA(A) include activity of PLC.

Progesterone enhances performance of aged mice in cortical or hippocampal tasks.

Ovarian steroids alter cognitive performance of young individuals. Whether progesterone enhances learning and memory in tasks involving the prefrontal cortex and/or hippocampus in aged mice was investigated. Aged mice received progesterone (10 mg/kg, s.c.) or vehicle and were tested for cortical and/or hippocampal learning and memory. Progesterone increased spontaneous alterations in the T-maze and time spent exploring novel objects in the object recognition task. Progesterone increased the time mice spent in the quadrant of the water maze where the hidden platform had been during training, increased latencies to crossover to the shock-associated side of the inhibitory avoidance chamber, and increased freezing in the contextual fear conditioning task. Progesterone did not enhance performance in tasks mediated by the amygdala (cued conditioning), striatum (conditioned place preference), or cerebellum (rotarod) in these aged mice. Thus, progesterone improved learning and memory in tasks mediated by the prefrontal cortex and/or hippocampus of aged mice.

Rapid and estrogen receptor beta mediated actions in the hippocampus mediate some functional effects of estrogen.

The steroid hormone, estradiol (E(2)), has numerous targets in the central nervous system, including the hippocampus, which plays a key role in cognition and affective behavior. This review focuses on our evidence from studies in rodents that E(2) has diverse mechanisms in the hippocampus for its functional effects. E(2) has rapid, membrane-mediated effects in the hippocampus to enhance cognitive performance. Administration of E(2) to the hippocampus of rats for 10 min following training enhances performance in a hippocampus-mediated task. Increased cell firing in the hippocampus occurs within this short-time frame. Furthermore, administration of free E(2) or an E(2) conjugate, E(2):bovine serum albumin (BSA), to the hippocampus produces similar performance-enhancing effects in this task, suggesting that E(2) has membrane actions in the hippocampus for these effects. Further evidence that E(2) has rapid, membrane-mediated effects is that co-administration of E(2) and inhibitors of mitogen-activated protein kinase (MAPK), rather than intracellular E(2) receptors (ERs) or protein synthesis, attenuate the enhancing effects of E(2) in this task. Despite these data that demonstrate E(2) can have rapid and/or membrane-mediated effects in the hippocampus, there is clear evidence to suggest that intracellular ERs, particularly the beta (rather than alpha) isoform of ERs, may be important targets for E(2)'s functional effects for hippocampal processes. Administration of ligands that are specific for ERbeta, but not ERalpha, have enhancing effects on hippocampal processes similar to that of E(2) (which has similar affinity for ERalpha and ERbeta). These effects are attenuated when ERbeta expression is knocked down in transgenic models or with central administration of antisense oligonucleotides. Thus, there may be a convergence of E(2)'s actions through rapid, membrane-mediated effects and intracellular ERs in the hippocampus for these functional effects.

Membrane actions of progestins at dopamine type 1-like and GABAA receptors involve downstream signal transduction pathways.

In the ventral tegmental area (VTA), progestins facilitate lordosis via rapid actions at membrane dopamine Type 1-like (D(1)) and/or GABA(A) receptors (GBRs), rather than via cognate, intracellular progestin receptors (PRs). Downstream signal transduction pathways involved in these effects were investigated using lordosis as a bioassay. If progestins' actions at D(1) and/or GBRs in the VTA require activation of G-proteins, adenylyl cyclase, cyclic AMP-dependent protein kinase A (PKA), phospholipase C (PLC), and/or PKC, then pharmacologically blocking these pathways would be expected to attenuate progestin-facilitated lordosis and its enhancement by D(1) and GBR activity. Ovariectomized, estradiol-primed rats were infused first with vehicle or signal transduction inhibitor, and second with vehicle, a D(1) or GBR agonist, and then with vehicle or progestins to the VTA. Rats were tested for lordosis following infusions. Results indicated that initiation of G-proteins, adenylyl cyclase, PKA, PLC, or PKC in the VTA is required for rapid effects of progestins through D(1) and/or GBRs to facilitate lordosis. As well, progestins' actions at n-methyl-d-aspartate receptors (NMDARs) may modulate activity at D(1) and/or GBRs and mitogen activated protein kinase (MAPK) may be a common signaling pathway. Findings from a microarray study demonstrated that there was upregulation of genes associated with steroid metabolism, GBRs, D(1), NMDARs and signal transduction factors in the midbrain VTA of naturally receptive mated compared to non-mated rats. Thus, in the VTA, progestins have rapid membrane-mediated actions via D(1), GBRs, NMDARs and their downstream signal transduction pathways.