Gonadal Steroid Modulation of Sleep and Wakefulness in Male and Female Rats Is Sexually Differentiated and Neonatally Organized by Steroid Exposure

Abstract

The paucity of clinical and preclinical studies investigating sex differences in sleep has resulted in mixed findings as to the exact nature of these differences. Although gonadal steroids are known to modulate sleep in females, less is known about males. Moreover, little evidence exists concerning the origin of these sex differences in sleep behavior. Thus, the goal of this study was to directly compare the sensitivity of sleep behavior in male and female Sprague Dawley rats to changes in the gonadal steroid milieu and to test whether the sex differences in sleep are the result of brain sexual differentiation or differences in circulating gonadal steroids. Here we report the magnitude of change in sleep behavior induced by either estradiol (E2) or testosterone (T) was greater in females compared with males, suggesting that sleep behavior in females is more sensitive to the suppressive effects of gonadal steroids. Furthermore, we demonstrated that the organizational effects of early gonadal steroid exposure result in male-like responsivity to gonadal steroids and directly alter the activity of the ventrolateral preoptic area (VLPO), an established sleep-promoting nucleus, in adult masculinized females. Moreover, the nonaromatizable androgen dihydrotestosterone did not suppress sleep in either males or females, suggesting that the T-mediated effect in females was due to the aromatization of T into E2. Together our data suggest that, like sex behavior, sex differences in sleep follow the classical organizational/activational effects of gonadal steroids.

Men and women sleep differently, with women reporting poorer quality and more disrupted sleep across various stages of life (for review see References 1–5). The reason for these sex differences in sleep is unknown; however, findings from clinical and basic research studies strongly implicate a role for gonadal steroids, like estradiol (E2) and progesterone (P4), in sleep modulation in females (for review see Reference 1). Sleep disruptions and complaints in women typically coincide with marked changes in the gonadal steroidal profile during puberty, the menstrual cycle, pregnancy, and menopause (for review see References 1–10). Likewise, fluctuations in the ovarian hormonal milieu across the estrous cycle correlate with changes in sleep and wakefulness in female rodents (11–17). On the night of proestrus, when the ovarian steroids E2 and P4 are elevated, both non-rapid eye movement (NREM) and rapid eye movement (REM) sleep are significantly reduced compared with other phases of the estrous cycle. Ovariectomy (OVX) eliminates these fluctuations in time spent asleep or awake typically observed throughout the estrous cycle, and hormone replacement reinstates the suppression of sleep (both NREM and REM) seen on proestrus (18–25).

In our established treatment paradigm of acute injections that mimics the natural cyclic change in E2, sleep is markedly reduced by E2 alone (23). However, in one study using chronic exposure to E2 and P4, the addition of P4 to low levels of E2 moderately enhances E2-mediated suppression of REM sleep (18). Taken together, these studies strongly implicate E2 as the primary steroid eliciting changes in sleep-wake behavior. In male rodents, castration does not significantly alter sleep and wakefulness compared to gonadally intact males (26, 27). Curiously, in castrated mice T replacement results in a small but significant increase in the amount of NREM sleep during the dark phase (22). These studies suggest that sleep behavior in males may be less sensitive to the effects of gonadal steroids.

Previous work in our laboratory suggests that there are sex differences in the hormonal modulation of both the activity of known sleep nuclei and the expression of the enzymes responsible for producing known somnogens (16). In the ventrolateral preoptic area (VLPO), an established sleep-promoting nucleus, we have demonstrated sex differences in gonadal steroid-mediated suppression of sleep-active neurons and protein levels of the lipocalin-type prostaglandin D synthase (L-PGDS), the synthesizing enzyme for the somnogen prostaglandin D2. Female rats but not male rats respond to changes in the hormonal milieu (via E2 replacement after OVX or castration, respectively). Taken together with the behavioral data, these findings raise the possibility that there are sex differences in the activational effects of gonadal steroids on organized sleep circuitry.

Few comparative studies of sleep in males and females have been done (26, 28, 29); therefore, our understanding of the underlying cause of these sex differences in sleep is still unclear. Here we hypothesize that the sex differences observed in sleep and wakefulness are due to the activational effects of E2 on differentially organized brain circuitry. In this study, we set out to address the origin of the sex differences observed in sleep by systematically testing the basic principles defining sex differences in the brain as recently outlined by McCarthy et al (30). These sex differences may be due to activational effects (transient actions after exposure to gonadal steroids), organizational effects (permanent changes in the neural substrate induced by exposure to gonadal steroids during perinatal development or puberty that can alter how the brain responds), or both. To test these possible outcomes, we first gonadectomized (GDX) adult male and female rats and analyzed their sleep-wake behaviors after sex-specific steroidal manipulations. We also tested whether pubertal events in females sensitize their sleep behavior to E2's effects. Then, to test whether organization of the brain circuitry by early exposure to gonadal steroids contributes to sex differences in sleep, female pups were masculinized via neonatal exposure to T. These manipulations allowed for a direct comparison in the magnitude of change after steroid replacement in males and females as well as a test of whether sex differences in the sleep-wake cycle are due to organizational and/or activational effects of gonadal steroids, a significant gap in our knowledge.

Materials and Methods

Animals

All experiments were performed in adult Sprague Dawley female (250–300 g) and male (350–400 g) rats. Adult females were mated in our animal facility to allow for manipulations immediately after birth when necessary. Due to a limited number of transmitters, recording slots, and animals per litter, experiments were run in multiple cohorts, containing a minimum of two experimental groups per cohort. The animals were housed under a 12-hour light, 12-hour dark cycle with free access to food and water for the duration of the study. All procedures were performed in accordance with the National Institutes of Health guide for care and use of laboratory animals. All experiments were approved by and were in accordance with the guidelines of the University of Maryland Institutional Animal Care and Use Committee.

Surgeries

In adulthood, all animals were GDX and simultaneously implanted with TL11M2-F40-EET transmitters (Data Sciences International) under isoflurane anesthesia as previously described (16, 23, 24, 31). Briefly, immediately after GDX, a bipotential-lead transmitter was implanted sc through a dorsal incision of the abdominal region. Another incision was made along the midline of the head and neck to expose the skull and neck muscle. Two burr holes were drilled asymmetrically and stainless steel screws (Plastics One) were implanted at 3 mm anterior/+1.5 mm lateral and 7 mm posterior/−1.5 mm lateral relative to the bregma. The four transmitter leads were threaded sc to the head incision. Electroencephalographic (EEG) leads were wrapped around the screws and secured with dental cement. Electromyogram (EMG) leads were implanted directly in the dorsal cervical neck muscle, approximately 1.0 mm apart, and sutured in place. The skin along the head was sutured closed, and the dorsal incision was stapled closed with wound clips. All animals were treated with antibiotic ointment and topical lidocaine as well as carpofen (5 mg/kg) postoperatively and then allowed 7 days to recover before the start of the experiments.

Experiment 1

Female (n = 8) and male (n = 4) rats were used to determine whether there are sex differences in the effects of sex-specific gonadal steroid replacement on sleep. A subset (n = 4) of control females was OVX on postnatal day 22, prior to puberty. Vaginal opening, a commonly used marker of puberty, did not occur in any of these females, based on visual inspection. Prepubertal OVX effectively eliminates the onset of adult ovarian hormones and estrus cyclicity and thus was used to determine whether females' responsivity to the modulatory effects of E2 on sleep and wakefulness in adulthood is set during development or whether further modification at the time of puberty is required.

All animals received an oil injection for baseline data recordings (Figure 1A). Then females received two injections, 24 hours apart of estradiol benzoate (EB; 5 μg, then 10 μg; Sigma-Aldrich), whereas males received two injections 500 μg of testosterone propionate (TP) (Figure 1A). EB is a synthetic ester of E2, which nonspecific steroidal esterases deesterify to produce biologically active E2 in vivo (32, 33). Our EB replacement paradigm mimics the gradual rise in E2 that occurs during proestrus as we have previously shown (23). The dosage of TP has been reported to reinstate physiological levels of T in males within 24 hours (34).

Figure 1.

Sex differences in sleep and wakefulness are hormonally modulated. A, Gonadal steroid replacement and analysis paradigm-within animal design. B, There are no differences in sleep and wakefulness in females and males after GDX under baseline conditions in either the light or dark phase. C, E2 significantly increased wake and decreased both NREM and REM sleep in females, whereas T does not significantly alter sleep and wake in males. *, P < .05 vs oil. D, The magnitude of change by gonadal steroids was significantly different for REM sleep. E2 suppressed sleep by 64%, whereas T reduced it by 25%. *, P < .05 vs female. E, Sleep/wake modulation by E2 in females is determined during development and does not require further hormonal events during puberty. Prepubertal OVX did not significantly affect female's responsivity to E2's arousing and sleep suppressive effects. *, P < .05 vs female. Data are represented as mean ± SEM.

Experiment 2

A subset of female pups (n = 5) received sc injections of 100 μg of TP (Sigma-Aldrich) dissolved in 100 μL sesame oil on the day of birth and postnatal day 1 to masculinize the neural substrates, as previously described (35, 36). These females are referred to as masculinized females throughout the manuscript and represent genetic females with male-like brain organization. Control female (n = 6) and male littermates (n = 7) were treated with 100 μL sesame oil on the same treatment days. All animals were raised to adulthood (∼80 d old) and used for the sleep studies described below.

These cohorts of animals were used to test whether exposure to gonadal steroids neonatally organizes the sleep circuitry in such a way, which alters the effects of E2 and T on sleep behavior. The order of hormone replacement (either EB or TP) was randomized in each group to control for timing. Exogenous steroids were allowed 7 days to wash out before the next round of injections were given (Figure 2A).

Figure 2.

The responsivity of the sleep/wake circuitry to the modulatory effects of gonadal steroids is developmentally organized. A, Gonadal steroid replacement and analysis paradigm-within animal design. The order for EB or TP replacement was randomized to control for the order of exposure. B, E2-mediated suppression of REM sleep was dependent on brain organization. E2 significantly reduced REM sleep in females only. Similarly, T also suppressed REM sleep in females only. *, P < .05 vs oil. C, The magnitude of REM sleep suppression by E2 is significantly different based on brain organization. There is a significant difference between females and males. There are no differences between T's effects on REM sleep. *, P < .05 vs females. Data are represented as mean ± SEM.

Neonatal androgen treatment has been reported to result in an anovulatory condition characterized by lack of estrous cyclicity and reduced ovarian weight that has been attributed to a reduced number of ovulatory follicles (37–40), traits indicative of a masculinized hypothalamic-pituitary-gonadal axis. Paired ovarian weights were recorded at the time of adult ovariectomies, as an independent measure of the masculinizing effects of neonatal TP exposure in females. Neonatal TP exposure significantly reduced the paired ovarian weight by 31% compared with females treated with oil (two tailed t test; t₁₀ = 3.96, P < .003; mean weights ± SEM in grams were: masculinized females, 0.142 ± 0.01; and females, 0.206 ± 0.01). These control females and masculinized females were used in the Fos immunoreactivity experiment described below.

Experiment 3

Dihydrotestosterone (DHT) benzoate (DHTB; Sigma-Aldrich), a nonaromatizable androgen, was administered to GDX male and female rats (n = 4 per group) to determine whether T exerts its effects through androgens or via aromatization to E2. Similarly to the EB and TP injection paradigm, two injections of 500 μg DHTB were given 24 hours apart. This dose of DHTB has been reported to effectively restore copulation in GDX males (41).

Data acquisition

EEG and EMG data were collected using Dataquest ART 4.0 software (DSI). For each animal, data collected during the baseline and hormone days were scored in 10-second epochs with Neuroscore 2.0 (DSI). Each epoch was scored as wake (low amplitude, high frequency EEG with high amplitude EMG), NREM sleep (high amplitude, low frequency EEG with low amplitude EMG), or REM sleep (low amplitude, high frequency EEG with muscle atonia or periodic muscle twitches). Transitions into a new vigilance state were considered only if two epochs (20 sec) of the new vigilance state was observed. The total duration (in minutes) for each vigilance state was analyzed for the light and dark phase for each scoring period (oil, EB, TP, or DHTB treatment). Table 1 includes data from both the light phase and dark phase. Only the dark phase data are represented in Table 2, Table 3, Figure 1E, and Figure 2, as it is when we see the most prominent effects of gonadal steroids on sleep.

Table 1.

Mean Time Spent in Each Vigilance State During the Light Phase After Oil and Sex-Specific Gonadal Steroid Replacement

Sex	Wake, min		NREM Sleep, min		REM Sleep, min
	Oil	Sex-Steroid	Oil	Sex-Steroid	Oil	Sex-Steroid
Females	176.2 ± 9.7	159.1 ± 11.3a	447.7 ± 16.4	456.0 ± 14.0	95.8 ± 13.6	104.9 ± 10.4
Males	170.8 ± 16.1	168.2 ± 8.5	457.6 ± 16.1	460.6 ± 12.8	89.3 ± 10.1	91.2 ± 8.1

Data are represented as mean ± SEM.

^aP < .05 vs oil.

Table 2.

Mean Time Spent in Each Vigilance State During the Dark Phase After Oil, E2, and T Replacement

	Wake, min			NREM Sleep, min
	Oil	E2	T	Oil	E2	T
Females	488.6 ± 14.7	551.1 ± 15.3a	532.2 ± 16.8	178.7 ± 10.8	146.5 ± 12.6a	156.7 ± 12.4
Males	517.3 ± 17.5	500.6 ± 10.5	515.4 ± 8.4	156.7 ± 13.5	179.7 ± 10.0	164.8 ± 8.1
Masculinized Females	486.7 ± 21.5	489.1 ± 12.0	479.5 ± 10.9	187.7 ± 17.2	195.6 ± 9.3	199.8 ± 9.8

Data are represented as mean ± SEM.

^aP < .05 vs oil.

Table 3.

Mean Time Spent in Each Vigilance State During the Dark Phase After Oil and DHT Replacement in Females and Males

Sex	Wake, min		NREM Sleep, min		REM Sleep, min
	Oil	DHT	Oil	DHT	Oil	DHT
Females	510.8 ± 43.3	523.4 ± 36.5	173.9 ± 35.3	165.3 ± 28.7	35.3 ± 8.1	30.8 ± 8.2
Males	541.8 ± 13.8	506.2 ± 17.1	146.4 ± 6.4	174.0 ± 16.0	31.4 ± 7.9	39.8 ± 5.6

Data are represented as mean ± SEM.

Fos immunoreactivity (Fos-ir)

In a separate cohort of masculinized females (n = 5), control females (n = 6), and males (n = 6), animals were overdosed with pentobarbital before being transcardially perfused with 0.1 M PBS followed by 4% paraformaldehyde in PBS. The brains were removed, postfixed in 2.5% acrolein/4% paraformaldehyde in PBS, cryoprotected in 30% sucrose in PBS, frozen on dry ice, and stored at −80°C before being sectioned in a cryostat into 30-μm-thick sections along the coronal plane. The sections were placed into an ethylene glycol-based storage solution at −20°C until processed for Fos-ir. Cohorts containing animals from all treatment groups were immunocytochemically processed in the same tray. Briefly, sections were rinsed free of the cryoprotectant in 0.1 M PBS, reacted with 1% sodium borohydride in PBS, and rinsed. Sections were then incubated in 0.015% phenylhydrazine to release endogenous peroxidase activity, rinsed, and incubated for 48 hours at 4°C with a rabbit polyclonal Fos antibody (Ab 4191; Oncogene Science) at a dilution of 1:250 000 in 10% normal goat serum and 0.3% Triton X-100 in PBS. After primary incubation, the sections were rinsed in PBS and incubated in biotinylated secondary antibody (goat antirabbit; Vector Laboratories) for 1 hour, followed by washes in PBS. Finally, the samples were incubated with an avidin-biotin horseradish-peroxidase complex (Vectastain ABC Elite kit; Vector Laboratories) for 1 hour at room temperature and washed in PBS and then in 0.175 M sodium acetate solution. The sections were visualized with a nickel sulfate (25 mg/mL) and 3,3′-diaminobenzidine tetrahydrochloride (0.2 mg/mL; Polysciences) in sodium acetate solution containing 0.005% H₂O₂, rinsed in acetate solution, and transferred to PBS. After visualization, the sections were mounted serially on 2% gelatin-coated glass slides and coverslipped.

Fos-ir quantification and analysis

We systematically counted the number of Fos-ir cells in the VLPO with the aid of the Neurolucida software, an image-combining computer microscopy program (MicroBrightField) as previously described (16). Briefly, slides were anatomically matched and numerically coded so that the investigator conducting the analysis was blind to the experimental group. Sections analyzed were from one in three series (adjacent sections were separated by 90 μm). Three brain sections corresponding to plates 19 and 20 of the rat brain atlas of Paxinos and Watson (42) were used in the analysis. The placement and size of the contours were in accordance with previously defined parameters (43). The contour began approximately 1 mm from the ipsilateral ventricular wall and extended 0.7 mm laterally and 0.3 mm dorsally. Six contours per bilateral section of the VLPO were counted and an average count of Fos-ir was derived.

Statistical Analysis

All data are represented as mean ± SEM, and all statistical tests were conducted using GraphPad Prism 4 on a Macintosh computer.

Two-way, repeated-measures ANOVAs were run on the mean times spent in each vigilance state between GDX, oil treated males, and females during the light and dark phase of the 24-hour baseline day. Data were binned into 3-hour blocks across the light-dark cycle. Paired t tests were used to determine whether E2 in females and T in males alter the mean time in wake or NREM or REM sleep in both phases.

The mean times in each vigilance state were analyzed using two-way, repeated-measures ANOVAs followed by Bonferroni post hoc tests with the adult hormone status (oil baseline vs gonadal steroid replacement) and brain organization (feminization vs masculinization) as independent variables. To compare the magnitude of change induced by either E2 or T between females, males, and masculinized females, the percent change from oil baseline for each gonadal steroid was calculated [((hormone-oil)/oil) 100]. Mann-Whitney U nonparametric tests were used to compare the percent change of a vigilance state induced by either sex-specific gonadal steroids between males and females. Mann-Whitney U nonparametric tests were also used to determine whether undergoing puberty is required for E2's effects on sleep in adulthood. A Kruskal-Wallis test was used to compare the percent change between females, males, and masculinized females for each steroid treatment.

One-way ANOVA was performed on the mean number of Fos-ir cells in the VLPO and a Tukey's multiple comparison post hoc test was used to determine significance between groups.

Results

Steroid replacement results in sexually differentiated sleep behavior in gonadectomized adult male and female rats

Quantitative analysis of the EEG/EMG traces demonstrated no significant differences in the total time spent in wake (F_1,42 = 0.11, P > .05), NREM sleep (F_1,42 = 0.18, P > .05) and REM sleep (F_1,42 = 0.31, P > .05) between GDX males and females in either the dark or light phase (Figure 1B). For each vigilance state, there was a main effect of time (wake: F_7,42 = 61.42 P < .0001; NREM: F_7,42 = 87.53 P < .0001; REM: F_7,42 = 8.34 P < .0001). Both males and females showed a circadian rhythm in sleep and wake behaviors. During the dark (or active) phase, both sexes are awake more, whereas they acquire more NREM and REM sleep during the light (or quiescent) phase. From this point, sleep in the absence of gonadal steroids is referred to as baseline sleep.

E2 and T replacement in females and males, respectively, was used to examine the effects of gonadal steroids on time spent in each vigilance state. Similar to our previous findings (16, 23, 24), E2 in females increased wake at the expense of sleep, during the 12-hour dark phase. On average, E2 significantly increased wakefulness (t₃ = 11.51, P < .001; Figure 1C) by about 12.5% or 63.5 minutes. NREM sleep (t₃ = 6.698, P < .01; Figure 1C) and REM sleep (t₃ = 20.86, P < .001; Figure 2C) are both significantly suppressed by E2 by about 21% (33.7 min) and 64% (28.6 min), respectively. In males, T did not significantly change any vigilance state (Figure 1C); however, there were trends for T-mediated increased wakefulness (t₃ = 2.925, P = .06; Figure 1C) and decreased REM sleep (t₃ = 3.039, P = .06; Figure 1C). In the light phase, gonadal steroid replacement had fewer effects on behavior (Table 1). In females, there was a small but significant suppression of wake by E2 (∼17 min; t₃ = 3.189, P < .05; Table 1). There were no additional effects on either sleep phase or any significant effects of T in males during the light phase; therefore, only dark-phase data will be represented in subsequent results.

Analysis of the percent change from baseline for each vigilance state demonstrates a sex difference in the magnitude of change induced by the steroid replacement for REM sleep only (U = 0.00, P < .05, Figure 1D). E2 significantly suppresses REM sleep in females by about 64%, whereas T suppresses REM sleep in males by about 25%.

Furthermore, neither the timing of the OVX (before or after puberty) nor the timing of the first exposure to E2 significantly affected the magnitude of E2's increase in wake or suppression of sleep (Figure 1E).

E2 and T affect sleep and wake in females but not males or masculinized females

Sleep and wake analysis after E2 and T replacement (randomized) in females, males, and masculinized females was used to determine whether brain organization (eg, masculinized vs feminized) underlies the responsivity of the sleep circuitry to the modulatory effects of gonadal steroids. All groups were compared back to their oil baseline after E2 replacement. There was a significant interaction between E2 and brain organization for wake (F_2,15 = 5.277, P < .05, Table 2) and REM sleep (F_2,15 = 5.708, P < .05; Figure 2B). E2 in females significantly increased wakefulness (t = 3.406, P < .05) and decreased REM sleep (t = 5.507, P < .001); however, E2 was ineffective at modulating sleep and wakefulness in animals with masculinized brain circuitry. There was a significant interaction between E2 and brain organization for NREM sleep (F_2,15 = 3.967, P < .05); however, due to the stringency of the Bonferroni post hoc analysis and the increase in comparisons, no significant effects were found. Because E2 has been previously shown to suppress NREM sleep in females in our laboratory (16, 23, 24), an uncorrected Fisher's least significant differences test was applied and demonstrated that E2 significantly suppressed NREM sleep (t = 2.196, P < .05; Table 2). Next, all groups were compared back to their oil baseline after T replacement. T also had a significant main effect on REM sleep (F_1,15 = 10.60, P < .01; Figure 2B). T suppressed REM in females (t = 3.819, P < .01) but not masculinized females or males. There were no significant effects of T or brain organization on wake (Table 2) or NREM sleep (Table 2). The significant interactions between E2 and brain organization for wake (Table 2) and REM sleep (Figure 2B) as well as the significant effect of T on REM sleep in females only (Figure 2B) indicate the importance of brain organization for gonadal steroid mediated effects on wake and REM sleep. Masculinization of the brain during early development renders males and masculinized females unresponsive to E2.

Analysis of the percentage change in REM sleep induced by gonadal steroid replacement further supports the notion that brain organization underlies the responsivity to the modulatory effects of gonadal steroids on sleep. The magnitude of E2's suppression of REM sleep is significantly different (H₂ = 7.630, P < .05; Figure 2C). Dunn's multiple comparison test revealed a significant difference between females and males (P < .05) but not between females and masculinized females or males and masculinized females. There was not a significant difference in the percent change in REM sleep induced by T replacement among the three groups (H₂ = 4.841, P > .05; Figure 2C).

Fos expression in the VLPO is sensitive to the organizing effects of gonadal steroids

We have previously shown that there is a sex difference in the number of sleep-active cells (via the expression of c-Fos) in the VLPO. Females have more Fos-ir cells at baseline than males (16). Here Fos-ir cells in the VLPO of females, males, and masculinized females collected 8 hours into their sleep (light) phase were quantified to examine whether this sleep-associated brain nucleus is sensitive to the organizing effects of gonadal steroids. Males and masculinized females possessed 58% and 46% less Fos-ir cells than control females, respectively (F_2,14 = 7.176, P < .01; Figure 3).

Figure 3.

Fos expression in the VLPO is developmentally organized. A, Photomicrographs of Fos-ir in females, males, and masculinized females in the VLPO. B, Males and masculinized females have 58% and 46% less Fos-ir cells, respectively, compared with females. *, P < .05 vs male and masculinized female. Data are represented as mean ± SEM.

Estradiol, not androgens, mediate changes in sleep and wakefulness

DHT, a nonaromatizable T, was used to determine whether T-mediated effects on sleep are mediated by androgens or through the aromatization of T to E2. DHT does not significantly alter female or male wake, NREM sleep, or REM sleep (Table 3), suggesting androgens do not play a role in sleep/wake modulation.

Discussion

In this study, the role of gonadal steroids was tested in the organization and the modulation of sleep and wakefulness in male and female rodents. Our first major finding is that the magnitude of E2-mediated changes in sleep differs between males and females. Such a finding indicates a sex difference in the sensitivity of the circuitry to the modulatory effects of E2. Our data strongly suggest that our T-mediated effects are due to its aromatization to E2 because DHT did not affect sleep and wakefulness. Our second novel finding is that a component of the hypothalamic neural circuitry involved in sleep is organized by early gonadal steroid exposure. These experiments have systematically shown that the sex differences observed in sleep and wakefulness are due to the activational effects of E2 on the brain circuitry organized in early life.

Developmental influences

In rodents, sexual differentiation of the brain begins around day 18 of embryonic development and continues until postnatal day 10 (for review see References 44 and 45). During this critical period, the processes of masculinization, defeminization, or feminization take place so that each animal displays the appropriate behavior for its sex when exposed to gonadal steroids in adulthood, which is referred to as the organization/activational hypothesis (for review see Reference 46). Prior to the current study, few studies provided a thorough investigation into the organization of the sleep circadian rhythm and behavior. Branchey et al (47) reported that sleep behavior is suppressed by E2 and P4 in adult males that were neonatally castrated compared with males castrated in adulthood. Yamaoka (21) showed that the circadian NREM and REM sleep rhythms in females masculinized at birth did not change after a single injection of 20 μg EB. Together these studies suggest a role for sexual differentiation of the brain mediating E2's effects on sleep rhythm and behavior; however, neither study directly compared the effects of E2 with control females nor did they investigate which brain areas may underlie the sex difference in the sensitivity of sleep to E2. Our present study demonstrates that the critical period for brain sexual differentiation contributes to the foundations of sex differences in the physiological processes mediating sleep and wake. For the first time, clear evidence is provided in support of the hypothesis that sex differences in sleep are established by the organizational actions of gonadal steroids early in life. Furthermore, we clearly show that the VLPO is sexually differentiated during development.

Both the circadian and sleep systems undergo significant alterations during adolescence (for review see References 48 and 49), a transitional developmental period characterized by the maturation of adult social and cognitive behaviors (for review see Reference 50). Puberty demarks the activation of the hypothalamic-pituitary-gonadal axis and gonadal maturation (for review see Reference 50) and typically occurs during adolescence. It is unclear whether the development of adult sleep-wake patterns during adolescence is due to age-related maturation or hormone-dependent events that occur during puberty. Hagenauer et al showed in both the rat (51) and the degu (52) that a shift in chronotype (timing of activity) occurs during puberty, and this phase shift depends on gonadal steroids. By adulthood, most activity is consolidated to the beginning of the active phase (51, 52). This shift in chronotype during the adolescent transition is similar across multiple species, including humans (for review see References 49 and 53). The role of gonadal steroids in adolescent sleep changes is less clear. Few studies have investigated the role of ovarian steroids on sleep during puberty. Sieck et al (54, 55) assessed sleep and wakefulness before and after either natural or precociously induced puberty. After their designated pubertal event, vaginal opening, there is an increase in wake at the expense of both NREM and REM sleep, suggesting that these changes in sleep and wake are mediated by the hormonal events occurring during puberty, not age-related changes (54, 55). What remains unclear, however, is whether the hormone-dependent events during puberty are necessary for the adult-like suppression of sleep induced by E2. Our data suggest that the responsivity of the circuitry underlying E2's effects on sleep in females is set early during development and that hormone-dependent changes that occur during puberty have no further effects on adult sleep organization.

Sexual differentiation of sleep circuitry

The neural circuitry and mechanisms underlying sleep and wakefulness have been extensively studied (for review see Reference 56). However, the precise targets of E2 action in the sleep circuitry have not been fully elucidated. Our previous work suggests that the VLPO, a major player in sleep initiation and maintenance (for review see Reference 57), is one likely target. In female rodents, E2 down-regulates the mRNA expression and protein levels of L-PGDS (16, 58), the enzyme responsible for the production of prostaglandin D2, which potently promotes sleep (for review see Reference 59). In conjunction with the decrease in L-PGDS, E2 decreases the activation of sleep-active VLPO neurons while increasing wake-associated Fos expression in the histaminergic tuberomammillary nuclei (16). Similarly, the wake-promoting hypocretin system is highly sensitive to fluctuations in endogenous and exogenous ovarian steroids (60–63). Taken together, these findings suggest that E2 may influence sleep-wake states via coordinated actions in arousal- and sleep-active cells via distinct mechanisms.

Unlike the females, fluctuations of gonadal steroids in adult males do not influence L-PGDS protein levels in the VLPO or the activation states of sleep-wake neurons, suggesting that the male sleep-wake circuitry is less responsive to gonadal steroids (16). Indeed, our current behavioral findings that neither T nor E2 induces significant changes in the sleep-wake behaviors in male rats correlate with our previous anatomical and biochemical findings. Moreover, the current findings demonstrate that the sleep circuitry involving the VLPO as well as the sleep-wake behaviors are organized and established by early exposure to gonadal steroids.

Hormonal modulation of sleep

It is quite clear that sex differences in sleep and wakefulness in rodents are hormonally modulated. As demonstrated here and previously reported by others, exogenous E2 in females significantly increases wakefulness at the expense of both NREM and REM sleep (18–25). Males, on the other hand, seem insensitive to changes in gonadal steroids. Castration does not significantly change sleep or wakefulness in males (26, 27), suggesting a resilience of the male sleep circuitry to changes in T levels. T replacement, however, increases NREM sleep during the dark phase in mice (22). In the current findings, acute administration of T in males resulted in a minor increase in wake and concomitant suppression of REM that did not reach statistical significance, and unlike the robust and reproducible changes in female sleep-wake patterns induced by E2, the effects of gonadal steroids in males were not reproducible (compare Figures 1 and 2). In contrast, a recent finding in adult male rats suggests that chronic E2 replacement [via SILASTIC brand (Dow Corning Corp) capsule implants] induces arousal at the expense of sleep (27). Comparatively, long-term vs short-term exposure to E2 may account for the differences. However, the magnitude of change in males induced by chronic E2 exposure compared with females is uncertain because only males were examined in that particular study.

Although it is clear that E2 in female rats induces robust and reproducible changes in sleep and wakefulness, the mechanism underlying E2 action is unclear and unaddressed by current studies. Our current finding of sex differences in the sensitivity of sleep behavior to E2 and circuitry can help direct more mechanistic research to understand how E2 is suppressing sleep in females. Rodent studies investigating how and why E2 can modulate sleep are crucial for furthering our understanding of sex differences in sleep in the clinical population.

It has been well documented that women report poorer sleep quality and more sleep complaints than men (64) (for review see References 1, 2, 4, and 5). Typically these sleep complaints are reported during the luteal phase, when E2 and P4 are elevated. Some polysomnographic studies also show that REM sleep is reduced during this phase (65–70). Additionally, women are at a 2-fold greater risk for developing insomnia throughout their lifetime compared with men (71–73). This difference in risk emerges at puberty (74) and increases with age (71–73), further implicating a role of gonadal steroidal modulation of sleep in women. Furthermore, women suffering from endocrine disorders like polycystic ovarian syndrome (75, 76) and premenstrual dysphoric disorder (77, 78) experience significant sleep disruptions as well. Because female rodents are sensitive to the sleep-suppressive effects of E2, they provide a good model for furthering mechanistic investigation into E2's actions on sleep.

The nature of the relationship between T and sleep in men remains unclear. T levels closely correspond to the sleep cycle (79, 80), and disruptions in sleep decrease the amount of circulating T (81, 82). Lower levels of T, which decline naturally with age, are correlated with less consolidated sleep (83). High-dose T replacement in older men decreases sleep efficiency and total sleep time and worsens sleep apnea (84). Others have also shown that T administration is associated with sleep apnea (84–87); however, blocking androgen action, via flutamide administration, does not affect sleep architecture or breathing parameters in men with sleep apnea (88). Interestingly, total sleep times in males undergoing male-to-female transgender therapy, which consists of high levels of estrogens and antiandrogens remain relatively unchanged (89). Stage 1 sleep and β-activity during NREM sleep significantly increased in individuals during the hormone replacement period, but all other sleep parameters remained the same (89). Together these data suggest that the male brain in humans, as in rodents, is relatively resilient to changes in gonadal steroids.

Conclusion

Modulation of sleep and wakefulness by E2 in females but not males is a classic example of the organizational/activational model for sex differences. Early exposure of gonadal steroids during development renders the male sleep/wake circuitry insensitive to the suppressive effects of E2. This finding in rodents may potentially translate in some way to the human population. Further investigation into inherent sex differences, like Fos-ir in the VLPO, and the mechanisms underlying E2 modulation throughout the sleep circuitry may aid in our understanding of why women are at a greater risk for sleep disruptions and insomnia. Because most sleep studies are done in men or male rodents, treatment generalized to the male physiology may not effectively alleviate sleep disruptions in women. Advancing our understanding of the mechanisms underlying hormonal modulation of sleep is imperative for uncovering novel drug targets for the treatment of sleep disruption in women.