Ovarian influence on circadian and infradian modulation of nicotine‐seeking in female mice

Thirty‐two female C57BL/6 mice (Laboratorio de Medicina Traslacional, Instituto de Ciencias de la Salud, Universidad Veracruzana, Mexico) were studied at 5 weeks of age. Animals were group‐housed (4 per cage, 29 × 19 × 12 cm) for a 1‐week acclimation period at the Centro de Investigaciones Biomédicas of Universidad Veracruzana. After acclimation, the animals were individually housed in polycarbonate cages (32 × 14 × 12 cm) furnished with pine shavings and a running wheel (11 cm diameter). They had ad libitum access to food and water, maintained at a controlled room temperature (22 ± 2°C) and relative humidity (45 ± 2%). Animals were kept under a light intensity of 300 lux with a 12/12 light/dark cycle (lights on at 05:00 h; zeitgeber time ‐ZT‐ 0), as described by Pittendrigh.25 Light–dark entrainment was confirmed during the one‐week baseline period that preceded all experimental procedures. Animals were kept under a controlled 12:12 h L/D cycle, and their wheel‐running activity was continuously recorded in 15 min bins. Actograms and activity profiles were generated using Lab‐Cib® software, which allowed verification of stable daily activity onsets aligned to lights‐off. Once L/D entrainment was confirmed, experiments were started. All procedures received approval from the Internal Committee Care and Use of Laboratory Animals (CICUAL) of the Centro de Investigaciones Biomédicas, Universidad Veracruzana (Comité Interno para el Cuidado y Uso de Animales de Laboratorio; protocol no. CLCIB2024/3), and were conducted in accordance with international and national ethical guidelines, including the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and the Official Mexican Standard NOM‐062‐ZOO‐1999 for the production, care, and use of laboratory animals. All efforts were made to minimize animal discomfort throughout the study.

Figure 1 illustrates the steps of the experimental design. Animals were randomly assigned to one of four groups: (1) intact (INT‐W), with no surgical procedure; (2) OVX‐W, ovariectomized animals; both groups received water nebulization (n = 12). Additionally, (3) intact (INT‐N) and (4) OVX‐N groups received nicotine nebulization (3 mg/mL nicotine tartrate [N5260‐25G, Sigma‐Aldrich Corp.], dissolved in distilled water; n = 12). At the beginning of the experiment, animals were housed four per cage for 1 week to acclimate, and then individually for another week to record their baseline locomotor activity, before nebulization. For the following 14 days, animals were transferred from their home cages to the nebulization chamber, where they were exposed to either distilled water or a nicotine solution (3 mg/mL) at ZT4 for 20 min.

A separate nicotine dose–response experiment, conducted to determine the experimental concentration used in the main study, is described in detail in the Nicotine nebulization procedure section.

Nebulization was conducted inside custom polycarbonate exposure chambers (37 × 45 × 19 cm), each divided into six equal compartments. At the center of each chamber, a polyvinyl chloride tube (1 inch diameter) was connected to a nebulizer (OMRON NE‐C801KD). The tube contained four evenly spaced perforations along its length to ensure uniform distribution of the aerosolized solution, and its distal end was sealed during treatment delivery (Figure 1).

This system has been previously validated in our laboratory for the effective delivery of stimulant compounds via inhalation.15, 16

Nicotine tartrate (N5260‐25G, Sigma‐Aldrich) was dissolved in distilled water immediately before use. To characterize the behavioral efficacy of nebulized nicotine and to determine the experimental concentration, mice were exposed to nicotine concentrations of 0, 1, 2, or 3 mg/mL via nebulization for 20 min at ZT4. Locomotor activity was recorded continuously, and nicotine‐induced changes in activity were quantified relative to individual baseline levels.

Based on these dose–response experiments, 3 mg/mL nicotine was selected for the main study, as it reliably increased locomotor activity without evidence of overt toxicity, respiratory distress, or behavioral suppression as assessed by daily health monitoring and behavioral observation. As a positive control for nicotine exposure, a separate group of mice received nicotine by subcutaneous injection (3.5 mg/kg). INT‐N and OVX‐N animals received nicotine nebulization once daily at the selected concentration, whereas control groups were exposed to distilled water under identical experimental conditions.

At 6 weeks of age, animals were transferred from their home cage to an anesthesia chamber with 4% isoflurane (Fluriso VETONE®) vaporized with 30% oxygen for 10 s. Following induction, anesthesia was maintained by using 1.5% isoflurane with 30% oxygen via a face mask.26 A small incision (3–5 mm) was made on the disinfected flank, cutting through the skin, subcutaneous fat, and muscle layers. The fat pad was gently lifted outward using forceps to expose the ovary and part of the uterus through the incision. The ovary was removed by dissecting the tissue between the suture and the ovary. The uterine horn was then returned to the abdominal cavity. The same procedure was repeated on the right flank.27

Muscular incisions were closed with two simple interrupted stitches using non‐absorbable sterile sutures (5–0 gauge, colorless, braided, U.S.P.‐coated, cutting reverse 3/8 19 mm, Atramat® PGLA90). Skin closure was achieved with a mattress stitch using an absorbable sterile suture (5–0 gauge, ST‐5½ circle tapered atraumatic needle, 26 mm, Surgical®). The surgical area was cleaned with 0.9% saline and benzalkonium chloride, followed by Nitrofural (Furacin®).

Post‐surgery, metamizole was administered subcutaneously using a 1 mL insulin syringe (31Gx 6 mm) for analgesia. Animals were placed in a recovery chamber (25 ± 2°C) until the anesthesia effects subsided. Upon regaining consciousness, animals were returned to a clean individual cage for monitoring.

Daily observations were conducted to detect any signs of inflammation or infection, with particular attention to the surgical wound. Fourteen days after surgery, successful ovariectomy was confirmed by vaginal cytology, characterized by the continuous presence of leukocytes, indicative of diestrus.26

Animals' locomotor activity was monitored continuously for 4 weeks, recording the number of wheel revolutions every 15 min. Each sensor detected complete wheel rotations using a pair of magnets, ensuring precise measurement of locomotor activity. Data were stored in Excel databases for subsequent analysis, and actograms were generated using the Lab‐Cib® software.

Vaginal cytology samples were collected using a pipette filled with 0.9% saline solution. The pipette was gently inserted into the surface of the vaginal canal, and a wash was performed with the saline solution. The sample was then spread onto a glass slide and examined under a light microscope (Leica®, DM500). The estrous cycle was determined using the vaginal smear method,28 and samples were assessed based on the dominant cell type in each stage, providing an estimate of the animals' circulating hormonal levels.

During proestrus (P), nucleated epithelial cells were predominant, appearing either in clusters or individually. Occasionally, some cornified cells indicated a preovulatory day. In the estrus stage (E), the smear was characterized by a dominance of cornified squamous epithelial cells, typically observed in clusters. These cells lacked a nucleus, had granular cytoplasm, and displayed an irregular shape. A mix of nucleated epithelial cells, cornified epithelial cells, and a predominance of leukocytes identified the metestrus (M) stage. Lastly, in the diestrus (D) stage, leukocytes were the most prominent cell type. For reproductive analysis, estrous cycle data were recorded daily in an Excel database, with one data point per day after nebulization. A pattern‐coded chart was developed to visualize complete cycles.28

After the initial examination, the samples were air‐dried at room temperature. The slides were immersed in a Coplin jar containing methylene blue for 1 min, followed by two consecutive 1‐min rinses with running water.29 After removing the water, the slides were allowed to air‐dry at room temperature. Samples were then fixed with Entellan™ and covered with a coverslip. After 48 h of drying, the smears were analyzed under light microscopy to confirm cell type identification. Representative cell types in vaginal smears were evaluated using microphotographs of cytologically stained samples.

On experimental day 15, the animals were euthanized at ZT4 (time of nebulization) using an intraperitoneal injection of Pentobarbital (Aranda® 75 mg/kg). Perfusion was performed with 0.9% saline solution, followed by 4% paraformaldehyde in phosphate buffer (PB, pH 7.4). Both ovaries were removed, separated, and fixed in 4% paraformaldehyde for 48 h. Right ovaries were used for the Hematoxylin–Eosin Staining protocol (HE), while left ovaries were prepared for transmission electron microscopy (TEM).

For HE staining, the right ovaries were embedded in 10%, 20%, and 30% sucrose solutions. Cryostat sections (18–20 μm) were prepared at −20°C (Thermo Scientific®, HM 525), and five to eight sections per ovary were mounted and stained for follicle counting. The protocol included rehydration in dH₂O (5 min), Hematoxylin staining (8 min), and water rinsing (3 min). Differentiation was performed with acidic alcohol for 2 min, followed by a 3‐min rinse with tap water. Samples were transferred to a 1% saturated lithium carbonate solution for 12 min, rinsed with tap water for 7 min, and stained with Eosinaphloxin for 2 min. The samples were then dehydrated through graded ethanol (80%, 90%, and 100%) for 2 min each. Slides were fixed with Entellan™ and coverslipped.

Follicles were classified according to Myers et al.30 as follows: primordial (oocyte with squamous granulosa cells), primary (cuboidal granulosa cells), secondary (multiple layers of granulosa cells without antrum), early antral (one or two antral cavities), antral (single, large ample antral space), and preovulatory (cumulus cell layer surrounding the oocyte). Photomicrographs were obtained using a Zeiss® Axio Scope A1 microscope at 20× magnification with a Zeiss® Axiocam 101 Monochrome Camera.

Follicle diameters (FD) were measured manually from the histological HE samples. FD was calculated using the sphere formula 4/3πR³, where R is the average radius obtained from four independent measurements.31

Ovaries were fixed in 2.5% glutaraldehyde and 0.1M sodium cacodylate‐buffered picric acid (pH 7.4) for 2 h at room temperature. The ovaries were then rinsed three times with 0.1M sodium cacodylate (pH 7.4) and subsequently stored in the same buffer at 4°C until further processing. Tissues were post‐fixed in 1% osmium tetroxide and 0.1M sodium cacodylate for 1 h at room temperature in the dark.

Tissues were dehydrated through a graded ethanol series (35%, 50%, 70%, 95%, and 100%), then specimens were immersed in propylene oxide and embedded in Epon (812 resin; Electron Microscopy Science, Hatfield, PA). The samples were polymerized at 55°C for 48 h. Ultrathin sections (70 nm) were cut using an EM‐UC7 ultramicrotome (Leica Inc., Karnataka, India) and transferred onto 100‐mesh copper grids. Sections were contrasted with 0.5% uranyl acetate and 0.5% lead citrate. Ultrastructural analysis was performed with a JEM‐JEOL‐2100 transmission electron microscope operated at 200 kV, equipped with a Gatan 4K camera.

Locomotor activity data were recorded as the number of wheel revolutions per subject and compiled in an Excel database. Data were arranged according to experimental phases: habituation phases (baseline), nebulization and perfusion. Activity profiles were initially visualized graphically to illustrate temporal patterns across groups.

The mean percentage of locomotor activity was calculated from 15‐min interval recordings corrected over 24‐h periods for seven consecutive days. A general linear model (GLM; McCullagh32) with factorial ANOVA with repeated‐measures was performed to compare (1) effect of nebulized nicotine on locomotor activity, (2) total locomotor activity over 24 h, and (3) the 2‐h window before and after nebulization among the four experimental groups (INT‐W, INT‐N, OVX‐W, OVX‐N), followed by a Bonferroni post hoc test. Additionally, daily anticipatory locomotor activity was analyzed using (1) model factorial ANOVA with repeated‐measures across 14 time points, followed by a Bonferroni post hoc test to assess differences among groups and days, and (2) a Kruskal–Wallis test was performed to assess changes in the amplitude over estrous cycle stages.33

A complete estrous cycle was defined as the identification of a proestrus phase followed by an estrus phase. Estrous cycle length was determined by counting the number of days between one proestrus and the subsequent proestrus.28, 34

Ovarian analysis using HE staining involved calculating the average number of each follicle type (primordial, primary, secondary, preovulatory and corpus luteum) per ovary. The total follicle count was obtained by summing all follicle types. A double‐masked evaluation was conducted to ensure greater statistical rigor.30, 35

The theoretical ovarian diameter was analyzed using group means and normal error distribution was assessed with the Lilliefors test (n > 50), and differences between the nicotine and water groups were compared using a Mann–Whitney U test.

All response variables were tested for normal error distribution using the Shapiro–Wilk and Lilliefors tests, as well as homogeneity of variances with the Levene test.33 When these assumptions were not met, the non‐parametric Mann–Whitney U test and Kruskal–Wallis test were applied,33 and in the ANOVA models, data were rank‐transformed according to the method of Conover.36 Additionally, all statistical analyses were conducted using GraphPad Prism® version 10 (GraphPad Software, Sand Diego, CA) and JMP® version 18 software (SAS Institute Inc., Cary, NC), and results are presented as medians ± interquartile ranges or means ± standard errors.

To evaluate the behavioral efficacy of nebulized nicotine and determine the experimental dose, mice were exposed to increasing concentrations of nicotine (0–3 mg/mL) using the nebulization system as described in section 2. A separate group receiving nicotine by subcutaneous injection (3.5 mg/kg) served as a positive control.

Nebulized nicotine produced a dose‐dependent increase in locomotor activity (Figure 2). Exposure to low concentrations of nicotine (1 and 2 mg/mL) did not significantly alter wheel‐running activity during the 2‐h period following nebulization compared with water‐treated controls (p < .05). In contrast, exposure to 3 mg/mL nicotine resulted in a significant increase in locomotor activity, comparable to that observed following subcutaneous nicotine administration (3.5 mg/kg) (one‐way ANOVA, F_{(4, 18)} = 9.83, p < .0001).

Representative actograms demonstrate clear anticipatory locomotor activity in INT‐N animals prior to scheduled nicotine exposure, whereas this pattern is not apparent in OVX‐N mice (Figure 3A). These qualitative patterns align with the qualitative analysis of anticipatory activity and support a modulatory role of ovarian hormones in nicotine‐induced anticipatory behavior.

Basal activity profiles averaged over 1 week and over the last 7 days of the nebulization phase are shown in Figure 3B, demonstrating that all animals were entrain to L/D cycle before the experiment began. During nebulization phase (Figure 3C), the activity profiles confirm the patterns observed in the actograms: the INT‐N group developed anticipatory activity, whereas the OVX‐N group showed increased activity only after nebulization, with the pre‐nebulization activity remaining comparable to the OVX‐W group. Despite these temporal differences, total daily (24 h) wheel‐running activity did not differ significantly among groups (INT‐W vs. INT‐N vs. OVX‐W vs. OVX‐N) (F_{(3, 20)} = 0.07; p = .97).

The percentage of wheel revolutions was calculated over a 4 h window (2 h PRE and 2 h POST nebulization) to assess anticipatory behavior and the acute response to nicotine. Only ovary‐intact animals receiving nicotine nebulization (INT‐N) exhibited an increase in locomotor activity during the 2 h preceding nebulization (Figure 4A).

Data were analyzed using a factorial ANOVA with two repeated‐measures (PRE and POST), which showed a contrast effect among groups (F_{(3, 20)} = 32.18, p < .0001). Post hoc multiple comparisons of the interaction between groups and the two repeated‐measure groups specifically highlighted significant differences at the PRE time point between INT‐N and INT‐W groups (p < .0001), and between INT‐N and OVX‐N groups (p < .0001) (Figure 4B). There was no significant main effect of Time (F_{(1, 20)} = 0.90, p = .90). However, a significant Group × Time interaction was observed (F_{(3, 20)} = 7.18, p < .001; Figure 4B), indicating that changes between PRE and POST differed across groups.

We analyzed anticipatory daily activity patterns in intact female mice over the 14‐day nebulization period to evaluate whether the estrous cycle influences nicotine‐seeking behavior. Anticipatory locomotor activity displayed a 4‐day periodic pattern (F_{(13, 130)} = 17.52, p < .001; Figure 5A). When actograms of the INT‐N group were examined in relation to estrous cycle stage (H = 13.95, p = .003; Figure 5B), increases in anticipatory activity were predominantly observed during the transition from proestrus to estrus (p = .015), whereas reduced amplitudes were more frequently associated with non‐receptive phases (metestrus/diestrus) (p = .012; Figure 5B). Given that proestrus is characterized by a brief circadian surge of estradiol followed by a rapid decline at estrus. These observations suggest that nicotine‐induced anticipatory activity is modulated not simply by elevated estrogen levels, but by dynamic ovarian hormonal fluctuations, likely reflecting estrogen‐dependent neural priming and delayed behavioral expression during subsequent hormonal withdrawal.

In contrast, activity in OVX‐N mice remained stable throughout the nebulization phase, with no significant differences across days (F_{(13, 130)} = 1.52, p = .34; Figure 5C).

The number of complete estrous cycles during the nebulization period was evaluated, and a significant reduction in cycle count was observed in the INT‐N group compared to the INT‐W group (t = 3.35, p < .01; Figure 6A). When analyzing weekly cycle duration, no differences were found during the first week (t = 0.44, p = .66). However, during the second week, the nicotine group displayed a significantly longer cycle duration (t = 0.442, p < .0001; Figure 6B).

Figure 6C illustrates typical rhythmicity in control animals, with estrus occurring every 4–5 days. In contrast, the INT‐N group exhibited desynchronization and an extended metestrus phase, lasting 3–4 days. Further analysis of each phase revealed a significant increase in the duration of the metestrus in nicotine‐exposed mice (t = 3.36, p < .001; Figure 6D).

Ovarian morphology analysis revealed that animals exposed to nebulized nicotine had significantly smaller ovaries compared to controls exposed to nebulized water (Figure 7A). This suggests a detrimental effect of nicotine on follicular development and ovarian size. As shown in Figure 7B, the number of primary (U = 105.5, p < .0001), secondary (U = 106, p < .0001), antral (U = 198.5, p = .04), and corpus luteum (U = 109, p < .0001) follicles was significantly reduced in the nicotine group. Consequently, the total number of follicles was lower in the INT‐N group compared to the INT‐W group (t = 4.90, p < .01; Figure 7C).

Histological analysis using HE staining revealed clear structural alterations in the ovaries of nicotine‐treated animals. While water‐treated animals retained standard follicular architecture, the nicotine group exhibited cortical atrophy and a high number of apoptotic cells, particularly in antral and preovulatory follicles (Figure 7D). Additionally, the theoretical ovarian diameter was assessed and found to be significantly smaller in the nicotine group than in the control (t = 3.49, p < .01; Figure 7E).

Representative histological images further confirmed the structural damage. Control follicles showed typical organization, whereas follicles from nicotine‐exposed mice displayed increased apoptosis and disrupted granulosa cell junctions (Figure 7F). Female mice exposed to nebulized water showed a normal ovarian ultrastructure characterized by well‐preserved cellular organization and intact organelles. Primordial and developing follicles displayed oocytes with large, centrally located nuclei, finely dispersed chromatin, and one or more prominent nucleoli. The cytoplasm was rich in organelles, including numerous spherical to elongated mitochondria with intact cristae, often clustered around the nucleus. Smooth endoplasmic reticulum and Golgi complexes were also evident. In mature oocytes, cortical granules were observed near the oolemma. Granulosa cells appeared structurally intact, with clear cell membranes, uniform chromatin distribution, and abundant mitochondria. The zona pellucida appeared as a continuous, electron‐dense layer, into which microvilli from both the oocyte and granulosa cells extended.

In contrast, mice exposed to nebulized nicotine exhibited abnormalities in ovarian structure, indicating cellular stress, damage, or dysfunction. Changes were observed across multiple components, including oocytes, granulosa cells, theca cells, and stroma tissue. The most notable findings included oocyte and follicular degeneration. Oocyte nuclei frequently showed signs of pyknosis, chromatin margination, or karyorrhexis, all suggestive of apoptosis. Cytoplasmic vacuolization was commonly observed, along with swollen or irregular mitochondria showing disrupted cristae and reduced density, indicating impaired metabolic activity. The endoplasmic reticulum appeared dilated or fragmented, and Golgi complexes were often disorganized or diminished. Microvilli extending into the zona pellucida were decreased or missing, weakening communication between oocytes and granulosa cells. The zona pellucida frequently appeared irregular, thickened, or partially disintegrated. Additionally, granulosa cells exhibited signs of degeneration, including shrunken shape, irregular membranes, increased intercellular spaces, cytoplasmic vacuolation, and nuclear condensation or fragmentation. Apoptotic bodies were widespread throughout the tissue. These observations indicate an accelerated pattern of follicular atresia and imply compromised ovarian function related to nicotine exposure.

Nebulization provides a non‐invasive method,15, 16 simulating modern nicotine vaping among young populations,37 while avoiding stress‐related circadian disruptions associated with invasive delivery routes.14, 38, 39, 40, 41 Although direct pharmacokinetic equivalence between nebulized nicotine exposure in mice and human vaping cannot be precisely established, nicotine concentrations equal to or higher than those here are commonly reported in recent preclinical inhalation and vapor studies.42, 43 Importantly, the concentration employed in the present study falls within the lower range of doses used in these models and was sufficient to elicit robust, time‐dependent behavioral effects.

Using this approach, intact C57BL/6 females exhibited anticipatory locomotor activity 1–2 h before nicotine administration, whereas OVX‐N mice did not, highlighting the role of estradiol in mediating behavioral sensitization.44 Anticipatory activity was significantly higher in INT‐N compared to OVX‐N, whereas post‐nebulization responses were similar, indicating that ovarian hormones, most likely estradiol, influence anticipatory rather than post‐exposure activity, as this effect was abolished by ovariectomy. Anticipatory activity observed in our nicotine‐treated females aligns with earlier findings in male mice treated with intraperitoneal nicotine39 or nebulized methamphetamine.15, 16 Psychostimulants such as nicotine and methamphetamine are known to enhance locomotor activity when administered every 24 h,15, 16, 39 whereas irregular dosing prevents animals from predicting subsequent sessions.17 Extensive research exists on the reciprocal interaction between the reward and circadian systems.45 Disruptions in circadian clock function are associated with a wide variety of physical, mental, and emotional disorders, including substance abuse and dependence.46 For instance, disruptions in sleep patterns and changes in circadian clock gene expression correlate with increased alcohol consumption and sensitivity47, 48, 49, 50, 51 and other drugs of abuse such as cocaine.52 Interestingly, polymorphisms in one clock gene, Per2, are also associated with variations in the ability to regulate alcohol intake in humans,47 and alcohol preference varies with chronotype and circadian genotype in mice.53, 54

Notably, anticipatory activity was absent in ovariectomized females, indicating that ovarian hormones, particularly estradiol, are essential for this response. Estrogens have been shown to modulate clock gene expression and activity in the suprachiasmatic nucleus (SCN),55 supporting our observation of hormonal status–dependent circadian reorganization. Moreover, the amplitude of the induced anticipatory activity correlated with the rhythm of the estrous cycle, suggesting that endogenous hormonal oscillations, especially estradiol, modulate nicotine‐seeking motivation. Consistent with this, our group recently reported that ovarian hormones influence the seeking and consumption of psychoactive substances in rodents, with estrogen enhancing drug intake, whereas progesterone may confer protection against vulnerability to addiction.10, 56 This is particularly relevant to the addiction field, where drug consumption has traditionally been reported as higher in men than in women, leading research to focus predominantly on male subjects.6, 7, 10 However, further studies are necessary to explore the mechanisms mediated by ovarian hormones. Recent unpublished findings from our group show greater activation of dopaminergic cells in the ventral tegmental area during the anticipation period in intact compared with ovariectomized female mice, specifically during the acquisition phase of nicotine intake. These results suggest that anticipation may be regulated by the reward system, revealing previously unexplored circuitry linking circadian responses to addiction independently of the suprachiasmatic nucleus. Further research is essential to determine whether nicotine acts as a circadian zeitgeber and to clarify the underlying endocrine pathways involved. Specifically, subsequent studies should (1) determine whether exogenous estradiol replacement in OVX mice restores nicotine‐induced anticipatory activity, (2) perform longitudinal quantification of circulating estradiol levels in intact females across the nebulization period to correlate hormonal peaks with behavioral intensity, (3) assess the persistence and extinction of anticipatory activity following nicotine withdrawal, and (4) confirm the endogenous nature of this rhythm by monitoring behavior under constant conditions (e.g., constant darkness) to exclude masking effects. Together, these approaches will be essential to establish the interplay between the hypothalamic–pituitary–gonadal (HPG) axis and the circadian oscillators driving nicotine‐seeking behavior.

Locomotor activity over 14 days in intact females exhibited a clear infradian pattern (~4‐day periodicity) corresponding to estrous cycles. Activity was notably higher during phases characterized by high estradiol levels, such as proestrus and estrus, and lower during metestrus and diestrus, suggesting an interaction between ovarian hormone levels and the response to nicotine.24, 57, 58 This variation may be mediated by the action of estrogens on receptors present in components of the circadian system,55, 59, 60 thereby influencing the expression of drug‐seeking behaviors.

Nicotine exposure disrupted estrous cyclicity in intact females, reducing the number of complete cycles (t = 3.35, p < .01), prolonging the second‐week cycle (t = 0.44, p < .0001), and extending metestrus duration (t = 3.36, p < .001). These alterations suggest that nicotine can interfere with infradian reproductive rhythms via estradiol‐dependent mechanisms. OVX‐N mice, lacking ovarian hormones, did not show these disruptions, reinforcing the requirement of hormonal regulation for nicotine‐induced reproductive cycle effects.

Beyond behavioral changes, we evaluated the effects of nicotine on ovarian structure and function. Morphological changes were observed in females exposed to nicotine nebulization (INT‐N), related to cortical atrophy, loss of granulosa integrity, and signs of follicular degeneration (e.g., pyknotic, marginal, or fragmented nuclei, altered mitochondria, and cytoplasmic vacuolization). These alterations are consistent with studies reporting ovarian damage induced by tobacco smoke.61, 62, 63 Moreover, a significant reduction in ovarian size and in the number of follicles (primary, secondary, preovulatory), and corpora lutea was also recorded. Even after a relatively brief exposure period, fulliculogenesis was compromised.64, 65, 66 These findings highlight the high sensitivity of ovarian tissue to inhaled nicotine and suggest that nicotine use could negatively affect fertility, even with intermittent use, as nicotine has been shown to reduce endogenous estradiol synthesis.67, 68, 69 The ovarian structural damage, coupled with disruption of the estrous cycle and changes in locomotor activity associated with motivation and drug seeking, provides further evidence of the risks of nicotine use in women of reproductive age.70, 71

Our findings align with previous evidence indicating that exposure to cigarette smoke reduces ovarian follicle number.65, 72, 73, 74 Notably, our study isolates the specific effects of nicotine, independently of other components of tobacco smoke. We observed a similar decrease in follicle count after nicotine exposure, indicating that nicotine alone might directly harm follicles development. Supporting this, earlier studies in female albino rats reported that nicotine altered the ratio of normal to abnormal follicles, significantly reduced the number of healthy follicles, impaired uterine physiological markers, and decreased the frequency of estrous cycles.75 Overall, these observations highlight the reproductive toxicity of nicotine and emphasize the importance of evaluating its isolated impact, separate from other constituents of tobacco smoke. Additionally, administration of cigarette smoke has been shown to reduce ovarian volume, which is associated with a corresponding decline in the total number of follicles.63, 64, 65

Activity‐induced synthesis of brain‐derived estradiol is essential for hippocampal long‐term potentiation (LTP) in ovariectomized female mice, suggesting a compensatory role for locally produced estrogens when peripheral hormone input is absent.76 The persistence of LTP when brain aromatase remains active highlights the importance of neuroestradiol. Moreover, the brain appears to regulate local estrogenic mechanisms differently, depending on whether estrogen loss occurs gradually, as in menopause, or abruptly, as in ovariectomy.77

In this context, our results showed that the OVX‐N group did not exhibit the same level of nicotine‐seeking behavior as the INT‐N group, despite undergoing identical administration protocols. The absence of nicotine‐seeking behavior in ovariectomized females could be attributed to hormonal regulatory changes following ovary removal, particularly the marked decline in circulating estrogens. Several preclinical studies investigating the systemic effects of ovarian hormones on responses to PAS have employed ovariectomy as a model. This approach offers a valuable perspective on how ovarian hormones influence vulnerability and behavioral responses to addictive compounds.78

Our findings suggest that fluctuations in estradiol levels, particularly those induced by nicotine exposure, may have significant consequences for the molecular regulation of circadian rhythms. Estradiol acts directly on the suprachiasmatic nucleus (SCN), the central circadian pacemaker, by binding estrogen receptors and modulating the transcription‐translation feedback loop. This interaction regulates the expression of core clock genes such as Per, Cry, and Bmal1, thereby enhancing the stability and amplitude of the circadian cycles.79 In mouse models, nicotine and related components significantly affect the expression of Clock, Bmal1, Per2, Cry2, and Rev‐erbα/β in peripheral tissues such as the lung, leading to altered phase timing and diminished oscillatory amplitude.80 Together, these findings imply that nicotine interferes with circadian regulation not only through hormonal suppression but also via direct molecular disruption of the clock machinery, potentially compounding the destabilization of systemic rhythmicity.

Several studies have shown that both sex and menstrual cycle phase significantly influence the subjective, physiological, and cognitive responses to nicotine. Controlled intravenous nicotine administration in abstinent smokers revealed that women exhibit a lower subjective response (manifested as diminished feelings of pleasure or reward) but exhibit greater physiological arousal compared to men. Furthermore, women in the luteal phase of their menstrual cycle, characterized by a relative predominance of progesterone over estradiol, exhibit a more pronounced reduction in the subjective effects of nicotine, alongside enhanced cognitive performance, compared to those in the follicular phase.81 These findings suggest that cyclical hormonal fluctuations modulate sensitivity to nicotine reinforcement and may contribute to observed sex differences in vulnerability to nicotine use and relapse. The luteal phase may represent a physiological window less prone to nicotinic reinforcement, possibly due to functional antagonism between progesterone and estradiol‐mediated effects on dopaminergic circuits and nicotinic receptors.