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There are sex differences in free-running rhythms, activity level and activity distribution that are attributed, in part, to the action of gonadal hormones. We tested the hypothesis that non-classical estrogenic signaling pathways at estrogen receptor subtype 1 (ESR1) modify the amplitude and phase of activity. We used ESR1 knock-out mice (ERKO) and non-classical estrogen receptor knock-in mice (NERKI). ERKO animals are unable to respond to estrogen at the ESR1 and NERKI animals lack the ability to respond to estrogens via the estrogen response element-mediated pathway, but can still respond via non-classical mechanisms. We compared intact male and female ERKO, NERKI and wildtype (WT) mice with respect to total wheel-running activity, activity distribution across the 24-h day, phase angle of activity onset and free-running period (τ) and the duration of activity in constant conditions. WT females had significantly greater activity than WT males, and this activity was more consolidated to the dark phase of the light:dark cycle. These sex differences were absent in the NERKI and ERKO animals. Among females, NERKI and ERKO animals had greater activity during the light phase than WT counterparts. Additionally, we have identified a novel contribution of non-classical estrogen signaling pathways on the distribution of activity. Our data suggest that total activity is ESR1-dependent and daily activity patterns depend on both classical and non-classical actions of estrogens. These data will aid in identifying the mechanisms underlying sex differences in sleep–wake cycles and the influence of steroid hormones on circadian patterns.
In humans there are sex differences in circadian rhythms and the epidemiology of healthy sleep and sleep disorders. Objective measures of healthy sleep find sex differences in the total amount of sleep, sleep onset latency and sleep efficiency; there are also sex differences in the prevalence of some sleep disorders, including insomnia, restless leg syndrome and sleep apnea, reviewed in (Krishnan & Collop 2006). The intrinsic circadian period of melatonin secretion and body temperature is shorter in women than in men even when the same bedtimes and wake times are maintained; women tend to wake up earlier and have a greater preference for morning activities than men (Duffy et al. 2011). Further, in women, sleep parameters and the amplitude of body temperature rhythms change from the follicular to luteal phases of the menstrual cycle and change in post-menopausal women following hormone replacement therapy. Together, these data suggest that ovarian hormones modify these daily and circadian rhythms (Leibenluft 1993, Parry et al. 1997, Shibui et al. 2000).
Animal studies provide strong evidence that the exposure to steroid hormones during the development and circulating steroid hormones in adults results in sex differences in the expression of biological rhythms, including sleep–wake patterns. In female rodents, total daily wheel-running activity depends on the stage of the reproductive cycle; when estrogens are elevated, there is an increase in both total wheel-running activity and phase advance in the timing of the daily activity onset in hamsters (Morin et al. 1977), degus (Labyak & Lee 1995, Mahoney et al. 2011) and rats (Wollnik & Turek 1988). In rats, the duration of the active period (α) in constant conditions and in a light:dark (LD) cycle varies throughout the estrous cycle with the greatest duration of the active period occurring on the day of estrus (Albers et al. 1981). Ovariectomy results in reduced total daily activity in both rats and mice (Albers 1981, Ogawa et al. 2003). Intact and estradiol-treated ovariectomized female mice have a longer duration of wake time than males (Paul et al., 2006, 2009). Ovariectomy lengthens free-running period (τ) and estradiol replacement shortens free-running period in female rats (Albers 1981), hamsters (Morin et al. 1977) and degus (Labyak & Lee 1995). There are species differences in the expression of circadian rhythms, for example, in the phase response to light pulse (Daan & Pittendrigh 1976), it is equally possible that the effects of steroid hormones on the expression of circadian rhythms are also dependent upon species.
While the role of estradiol regulation of circadian rhythms in females has been researched, its function with respect to rhythms in males is less clear. Testosterone is the primary gonadal hormone in males and is converted to estradiol and dihydrotestosterone (DHT). Gonadectomy of male mice decreases total wheel-running activity, changes the distribution of activity across the day and increases τ (Daan et al. 1975). Testosterone replacement restores activity and decreases free-running period to that of intact animals. Interestingly, DHT replacement also results in a partial restoration of activity (Karatsoreos et al. 2007), indicating that biological rhythms are regulated by both androgenic and estrogenic actions, at least in males.
The actions of estrogens are primarily mediated through two distinct nuclear receptors: estrogen receptor 1 (ESR1, also known as estrogen receptor α) and ESR2 (also known as estrogen receptor β). To exert action via nuclear receptor-mediated transcriptional activation, estrogens bind to nuclear receptors, which undergo a conformational change promoting dimerization. The hormone-receptor dimer then binds to the estrogen response element (ERE) on regulatory regions of target genes to alter gene transcription. In addition to these ‘classical’ nuclear receptor-mediated mechanisms, there are more recently characterized ‘non-classical’ pathways (Hall et al. 2001, Kousteni et al. 2001). These alternative pathways include the action of the hormone-receptor dimer on non-ERE transcription factors, membrane estrogen receptor-initiated action via protein kinases and ligand-independent ER signaling via second messenger pathways (McDevitt et al. 2008).
Previous studies in this and other laboratories have shown that a lack of estrogens, in gonadectomized animals and in transgenic models, influences the expression of circadian rhythms compared with control animals with endogenous estradiol. Aromatase knock-out mice are unable to synthesize estradiol and show increased activity during the light phase, decreased overall activity levels and increased free-running periods relative to wild-type littermates (Brockman et al. 2011). While these data provide evidence for a role of estrogens in the regulation of circadian patterns, the underlying mechanisms of their action remain relatively unknown. Estrogen receptor knock-out models indicate that the total daily activity level is regulated largely via estrogenic action at ESR1, but other circadian parameters have not been examined (Ogawa et al. 2003). To better characterize the interaction of estrogens and daily and circadian rhythmicity, we are using two transgenic mouse models with alterations in estrogen responsiveness at different points in their response pathway: ESR1 knock-out (ERKO) and ‘non-classical’ estrogen receptor knock-in (NERKI) mice. ERKO mice cannot respond to estrogen via ESR1, but retain the ability to respond at ESR2. NERKI mice have a modified ESR1 receptor knocked into their genome that has a mutation in the ERE-binding domain. In NERKI mice, therefore, estradiol action via ERE-mediated transcription is eliminated but estrogens can still produce effects via other transcriptional pathways (Jakacka et al. 2002). NERKI mice maintain intact ESR2 signaling. This transgenic model allows us to explore effects of estrogens on rhythms that are not mediated by ERE-DNA binding via ESR1.
These transgenic mice provide insight into the mechanism of estrogenic organization of circadian rhythms that differs from gonadectomized mice because they retain circulating gonadally produced steroid hormones, yet have specific mutations in signaling pathways. Further, these mice are uniquely suited for discerning how estrogenic actions at ESR1 organize the expression of circadian rhythms across hormone-sensitive periods of development, rather than an acute disruption with ovariectomy/gonadectomy or estrogen antagonist administration. We will test the hypothesis that estrogens modify the expression of daily and circadian rhythms via ESR1 and via ‘non-classical’ estrogen mechanisms by analyzing male and female wildtype (WT), NERKI and ERKO mice. We are interested in identifying the mechanisms underlying the expression of sex differences under normal physiological conditions thus we used gonadally intact animals throughout these studies.
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We tested the hypothesis that estradiol modifies daily and circadian rhythms via its actions on ESR1 and non-classical signaling pathways. We examined activity parameters in intact animals, as we wanted to understand the mechanisms underlying sex differences in rhythms under physiological conditions as well as identify functional consequences of disrupted estrogenic response on activity patterns. We found significant sex differences in total activity, LD ratio and activity levels during the dark phase of the day in WT animals that are abolished in the NERKI and ERKO genotypes. Our data also indicate that impairments in estrogenic signaling pathways result in changes in the distribution of activity during LD and constant conditions. Lastly, data from NERKI animals showed a novel contribution of non-ERE mediated transcription and estrogen-activated second messenger signaling pathways on LD ratio, and activity levels during the dark phase. Our data suggest that the total daily activity is ESR1-dependent and daily activity patterns (distribution of activity across the day, LD ratio and α) are modulated by both classical and non-classical actions at ESR1. These results provide insight to the mechanisms that underlie sex differences in the hormonal regulation of daily and circadian rhythms.
The most striking effects of altered estrogenic signaling were observed in the distribution and amount of daily wheel-running activity. Compared with males, WT females had sustained and elevated activity across the day (Fig. 1), had more daily activity and were more nocturnal (Fig. 2). Ogawa et al. (2003) described total locomotor activity as dependent on ESR1 in both male and female animals, and did not find sex differences in total wheel-running activity in WT mice. In that study gonadectomized animals were treated with oil or estradiol. We find sex differences in total activity that may be present in intact animals only. Both NERKI and ERKO females have decreased wheel-running activity relative to WT females. These data support the hypothesis that total wheel-running activity is regulated through ERE-mediated actions at ESR1 and provides evidence that there is no contribution from non-classical pathways. Both NERKI and ERKO females have increased LD ratios relative to WT animals. However, NERKI males had an intermediate LD ratio relative to WT and ERKO males. This suggests that there is a sex-specific contribution of non-classical signaling pathways on activity patterns.
Among females, there were significant differences in activity levels between ERKO and WT animals throughout the dark phase. In this measure, NERKI females appeared to have intermediate activity as they did not differ from ERKO or WT females at most of the time points examined. This provides evidence that non-classical signaling, independent of ERE-mediated transcription, contributes to activity distribution in females. Identifying how the independent but related estrogen signaling pathways mediate activity patterns and amplitude, and whether there are sex differences in these mechanisms will provide a foundation for understanding sleep disturbances in humans.
We measured the duration of τ in constant dark and light conditions. In other species, there is an effect of estradiol on the free-running rhythm when animals are housed in constant darkness; ovariectomized female hamsters have a lengthening of τ and administration of estradiol shortens this free-running period (Morin et al. 1977). We predicted that ERKO females, which cannot respond to circulating estradiol via ESR1, would be similar to ovariectomized rodents and thus have a longer τ in DD than WT animals. In DD and LL, however, we did not detect any significant differences in τ among females. In DD, we found a sex difference between NERKI males and females and in LL there were sex differences in τ in all genotypes. Additionally, the change in the duration of τ in LL relative to DD was greater in females than males and did not differ among genotypes within a sex despite differences in the ability to respond to estrogens at ESR1. It is possible that the duration of τ and the change in τ under different lighting conditions are not modified by developmental exposure to estradiol. In contrast, circulating hormones, and the ability to respond to the activational effects of these hormones, may have a modulatory role on the free-running period.
Our data indicate that the contributions of non-ERE mediated transcription and estrogenic regulation of second messenger pathways may not always be additive with the classical pathways. For example, in males, we saw an ‘over-compensation’ in the phase angle of activity onset and the period of the free-running rhythm in DD (Fig. 3 and 4). These effects may be due to compensatory mechanisms in the animal as a result of the impaired ESR1 signaling and also indicate that non-classical signaling pathways can modify the expression of circadian rhythms.
The differences in the expression of circadian activity patterns in adulthood may be due, in part, to a role of estrogens in the regulation of response to photic cues. Estrogens may strengthen the coupling between endogenous behavioral rhythms and the external light environment, as we see more pronounced genotype differences in entrainment (LD) than in free-running conditions. In mammals, the suprachiasmatic nucleus (SCN) of the hypothalamus generates circadian rhythms in activity and estrous cyclicity and entrains activity to the LD cues (Stephan & Nunez 1977). In addition to the ability to respond to light, the SCN is also regulated by internal cues, such as hormonal secretion. The influence of gonadal hormones on daily rhythms may be mediated by acting both centrally on the SCN and peripherally on downstream targets. In humans, there are estrogen receptors (ESR1 and ESR2) in the SCN, with significantly greater ESR1 expression in females relative to males (Kruijver & Swaab 2002). The distribution of estrogen receptors in the mouse brain is less clear, although there is evidence for both ESR1 and ESR2 in the SCN with ESR2 being relatively more abundant (Shughrue 1998, Mitra et al. 2003, Vida et al. 2008). The function of these SCN estrogen receptors remains unknown, although it has been hypothesized that they may not play a major role because of their relatively low expression levels (Karatsoreos et al. 2007). It is probable that estrogens modify behavioral patterns by acting on a number of targets including those downstream from the SCN.
We have identified sex differences in daily and circadian activity patterns and characterized how these differences are modified by the ability to respond to estrogens at ESR1. This initial study sets the foundation for future work, which will identify the contribution of organizational and activational hormone effects on the expression of rhythmic patterns. ERKO and NERKI mice are useful in identifying the organizational role of hormones on circadian rhythms, as these animals have had an impaired ability to respond to endogenous estrogen over their lifespan, and they shed light on the estrogenic mediation of sex differences in circadian behaviors. Future studies will determine if there is a functional consequence of estrogen signaling impairment on the ability to entrain to the LD cycle or respond to circadian disruptions.