Hypoxic and hypercapnic ventilatory responses in rats with polycystic ovaries

Respiratory Physiology & Neurobiology 217 (2015) 17–24

Contents lists available at ScienceDirect

Respiratory Physiology & Neurobiology journal homepage: www.elsevier.com/locate/resphysiol

Hypoxic and hypercapnic ventilatory responses in rats with polycystic ovaries Luis Henrique Montrezor a,1 , Débora de Carvalho b,1 , Mirela B. Dias c , Janete A. Anselmo-Franci d , Kênia C. Bícego b , Luciane H. Gargaglioni b,∗ a

Department of Medicine, University Center of Araraquara-UNIARA, Araraquara, SP, Brazil Department of Animal Morphology and Physiology, Sao Paulo State University – UNESP, Jaboticabal, SP, Brazil c Department of Physiology, Institute of Bioscience, Sao Paulo State University – UNESP, Botucatu, SP, Brazil d Department of Morphology, Stomatology and Physiology, Dental School of Ribeirao Preto, University of Sao Paulo, Brazil b

a r t i c l e

i n f o

Article history: Received 20 May 2015 Received in revised form 20 June 2015 Accepted 21 June 2015 Available online 25 June 2015 Keywords: Chemosensitivity Hypercapnia Hypoxia Ventilation Sex hormones

a b s t r a c t In female rats, a single injection of estradiol valerate (EV) results in effects that are similar to those observed in women with polycystic ovary syndrome (PCOS). We hypothesized that EV-induced PCOS affects breathing control based on evidence showing an influence of sex hormones on ventilation. To test this hypothesis, we studied the effects of EV treatment on the ventilation of female rats in air, in 7% CO2 and in 7% O2 , at 30, 45 and 60 days after EV injection. The group examined 30 days after EV treatment showed a 61% reduction in the hypercapnic ventilatory response compared to the control group. Basal ventilation, hypoxic ventilatory response, and body temperature were not affected. These results, suggest that the hormonal changes observed in PCOS may result in a temporary inhibition of the central chemoreflex but do not influence basal ventilation or the hypoxic peripheral chemoreflex. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Polycystic ovary syndrome (PCOS) is a clinical disorder that affects up to 10% of women of reproductive age (Goodarzi et al., 2011; Teede and Norman, 2006), and it is the most common endocrine disorder among pre-menopausal women in the United States (Tasali et al., 2008; Knochenhauer et al., 1998). PCOS is characterized by impairment of ovulation, reduced fertility, miscarriage, and imbalance of reproductive hormones (Goodarzi et al., 2011; Franks, 1995). In addition, women with PCOS often exhibit obesity, metabolic syndrome, hyperinsulinemia, insulin resistance, and dyslipidemia, which lead to increased risks of cardiovascular disease and type 2 diabetes (Pasquali et al., 2011; Lobo and Carmina, 2000). However, the mechanisms underlying such disorders remain unclear and may involve an array of factors including hormonal imbalances, epigenetic changes in fetal life, genetic

∗ Correspondence to. Via de acesso Paulo Donato Castellane s/n, 14870-000, Departamento de Morfologia e Fisiologia Animal, Faculdade de Ciências Agrárias e Veterinárias, Universidade Estadual Paulista Júlio de Mesquita Filho, Jaboticabal, SP, Brazil. Fax: +55 16 32024275. E-mail address: [email protected] (L.H. Gargaglioni). 1 L.H.M. and D.C. contributed equally to this study. http://dx.doi.org/10.1016/j.resp.2015.06.009 1569-9048/© 2015 Elsevier B.V. All rights reserved.

abnormalities, lifestyle, and environmental factors (Goodarzi et al., 2011; Pasquali et al., 2011). Previous studies have demonstrated that women affected by PCOS show respiratory disorders such as sleep disordered breathing (Vgontzas et al., 2001; Chatterjee et al., 2014). For instance, obstructive sleep apnea (OSA) has been reported to be higher in women with PCOS in comparison to the general population (Chatterjee et al., 2014; Vgontzas et al., 2001). It is possible that hormonal changes in women affected by PCOS such as hyperandrogenism, hyperestrogenism and variable levels of gonadotropins in the blood (Goodarzi et al., 2011), may be involved in the development of OSA. In fact, a previous study suggested that high free testosterone levels in women with PCOS may be a predisposing factor leading to OSA (Tock et al., 2014). Additionally, evidences indicate that steroidal sex hormones such as progesterone (P4), testosterone (T) and estrogen (E2) are involved in the neural control of respiration (Behan and Kinkead, 2011). Not surprisingly, evidence also suggests that sex hormones influence the peripheral and central chemoreflex. For example, it has been demonstrated that hypoxic and hypercapnic responses differ between the sexes (Behan et al., 2003) and acute administration of testosterone in castrated cats increases the hypoxic and the hypercapnic ventilatory response and promotes enhanced sensitivity of the carotid body (Tatsumi et al., 1994). In addition,

18

L.H. Montrezor et al. / Respiratory Physiology & Neurobiology 217 (2015) 17–24

hypoxic and hypercapnic ventilatory responses increase during pregnancy (Moore et al., 1987). More recently, we have demonstrated that despite the hormonal fluctuations during the estrous cycle, the CO2 ventilatory responses in female rats are similar along the cycle; however ovariectomized rats presented a reduced hypercapnic ventilatory response (Marques et al., 2015). Furthermore, hormonal replacement with estradiol or progesterone in those ovariectomized females did not restore the CO2 -drive to breathing, suggesting that other gonadal factors are possibly involved in this response. Estradiol valerate (EV) treatment in pre-pubertal or adult female rats has been used as an experimental model to induce PCOS and promotes similar effects to those observed in women with this syndrome (Linares et al., 2013). Additionally, rodent models of PCOS have shown many characteristics of the human disorder, including elevated LH, disrupted cyclicity, the presence of follicular cysts/polycystic ovaries, and altered insulin sensitivity, which closely parallels the human condition (Walters et al., 2012). The aim of the present study was to determine whether, PCOS affects breathing control. To this end, we induced PCOS in female rats by administering EV, and we examined the effects of EV on ventilation in air and in conditions of hypoxia and hypercapnia at 30, 45 and 60 days after EV treatment.

2. Materials and methods 2.1. Animals Experiments were performed on unanesthetized adult female Wistar rats (4 month old) weighing 280 ± 9.8 g in control group, 281 ± 5 g in 30 days, 252 ± 7 g in 45 days and 259 ± 11 g in 60 days after estradiol valerate treatment groups (mean ± SEM) on the day of experiment. Six animals were housed in each cage in a temperature-controlled chamber at 24–26 ◦ C (ALE 9902,001; Alesco, Monte Mor, SP, Brazil) with a 12:12 h light/dark cycle (lights on at 7:00 A.M.), and rats had free access to water and food. This study was conducted in compliance with the guidelines of the National Council of Control in Animal Experimentation (CONCEA-MCT-Brazil) and with the approval of the local Animal Care and Use Committee (CEUA-FCAV-UNESP, n# 000222-09). 2.1.1. Induction of polycystic ovary syndrome Estradiol valerate (EV) (Sigma–Aldrich, MO, USA) was dissolved in mineral oil and administered (2 mg/0.2 mL/rat; intramuscular) to induce polycystic ovary syndrome through the formation of follicular cysts (Brawer et al., 1986; Pereira et al., 2014). EV was administered in a single dose at 30, 45 and 60 days before the experiments. The control group received no treatment and only those which were in diestrus at the time of the experiment were used because this phase of the cycle is longer lasting. Estrous cycle regularity was assessed, and only rats showing at least three consecutive regular 4-day cycles were included in the control group or received EV and then used in the experiment. 2.1.2. Surgery For body temperature (Tb) measurements, a datalogger (SubCue, Calgary, AB, Canada) was implanted in all animals by abdominal cavity through a midline laparotomy 24 h prior to the experiment. The surgical procedure was performed under anesthesia using intraperitoneal ketamine (100 mg/kg, Agener, São Paulo, Brazil) and xylazine (10 mg/kg, Coopers, São Paulo, Brazil). After surgery, the rats were treated with subcutaneous antibiotic (10 mg/kg, Enrofloxacina, Flotril® , Schering-Plough, São Paulo, Brazil) and analgesic (2.5 mg/kg, Flunixina meglumina, Banamine® ,

Schering-Plough, São Paulo, Brazil) agents. The dataloggers were programmed to acquire data every 5 min. 2.1.3. Determination of pulmonary ventilation Measurements of pulmonary ventilation (VE ) were performed using the whole body plethysmography method based on the Bartlett and Tenney, 1970 study, which has been described in detail previously (Biancardi et al., 2008; De Carvalho et al., 2010; Patrone et al., 2014). The flow rate of the inflow gas into the animal chamber was controlled by a flowmeter (model 822-13-OV1-PV2-V4, Sierra Instruments, Monterey, CA) and flow was maintained at 0.8 to 1 L/min. 2.1.4. Experimental protocol At 30, 45 and 60 days after the EV injection each animal, including control groups, was individually placed in a Plexiglas chamber (5 L) and allowed to move freely while the chamber was flushed with humidified room air. After the animals remained calm (∼30 min), control VE was measured. A hypercapnic gas mixture (7% CO2 in air, White Martins, Sertãozinho, São Paulo, Brazil) was flushed through the chamber for 30 min, and VE was measured at the end of exposure. Subsequently, the chamber was ventilated for 60 min with humidified room air, and VE was measured again after 60 min. A hypoxic gas mixture (7% O2 and N2 balance, White Martins, Sertãozinho, São Paulo, Brazil) was flushed through the chamber for 30 min, and VE was measured at 30 min. 2.1.5. Hormone assay After measurements of pulmonary ventilation rats were anesthetized and a blood sample of approximately 1 mL was collected from the heart into heparinized syringes. Plasma was separated by centrifugation at 3000 rpm for 20 min at 4 ◦ C and stored at −20 ◦ C for posterior analysis of progesterone, testosterone and estradiol levels by radioimmunoassay (RIA). Plasma progesterone, testosterone and estradiol concentrations were determined by double-antibody RIA with MAIA kits provided by Biochem Immunosystem (Bologna, Italy). The lower limits of detection for estradiol, progesterone and testosterone were 5.0 pg/mL, 0.02 ng/mL and 5.0 pg/mL, respectively. The intra-assay coefficient of variation was 4.3% for estradiol, 7.5% for progesterone and 4% for testosterone. 2.1.6. Ovarian morphology The left ovary was removed, cleaned of adherent connective fat tissue, and fixed in 4% formaldehyde buffer for at least 24 h for use in morphological analysis. Thereafter, the samples were dehydrated and embedded in paraffin. The ovarian morphology characterization of the PCO was performed by histological analysis of 9-␮m serial sections stained with hematoxylin and eosin (HE), and photographs were analyzed with Motic Live Imaging Module (format 1024 × 768) microscopy software. The number of follicles with hyperthecosis, type III follicles and cystic follicles were counted in every section of the ovary as described previously by Brawer et al. (1986). Briefly, antral follicles with hyperthecosis were defined as those medium-sized antral follicles that presented hypertrophied differentiated theca interna cells with increased thickness of the theca layer and a normal granulosa cell layer. Type III follicles were larger and contained four or five layers of small densely packed granulosa cells surrounding a very large antrum and thickened theca interna cell layer. Cystic follicles were defined as those follicles devoid of oocytes and displaying a large antral cavity, a thin granulosa cell layer and a thickened theca interna cell layer (Fig. 1). 2.1.7. Statistical analysis The results are reported as the means ± SEM. The data were analyzed using two-way ANOVA for repeated measures, and Tukey’s

L.H. Montrezor et al. / Respiratory Physiology & Neurobiology 217 (2015) 17–24

19

Fig. 1. Morphologic aspects of ovaries from rats EV-induced PCOS. Control (A), 30 (B), 45 (C) and 60 (D) days after administration of EV (×40). A cluster of degenerated granulosa cells occurs in the antrum of the follicle in a 60 days postreatment ovary (E) (×200). Abnormally thickened theca layer at 60 days EV-treated PCOS (×200). Arrows show healthy follicle; C = follicular cyst; GC = granulosa cell; TC = theca cell.

test was used for multiple comparisons. Statistical analyses were performed using a software program (Sigma Stat, Systat Software, Point Richmond, CA, USA). Values of P < 0.05 were considered to be significant. Only the differences between the groups are presented in the graphs. 3. Results 3.1. Ovarian morphology Fig. 1 shows the morphology of the ovaries of the control group and of the experimental groups at 30, 45 and 60 days after EV treatment. Follicles are present in the ovaries of the control group (Fig. 1A) and in the ovaries of the PCOS groups at 30 (Fig. 1B) and 60 (Fig. 1D) days after EV treatment. The number of follicles at 60 days in the EV-treated group was reduced compared to the control

group. Follicular cysts were observed at 30 (Fig. 1B), 45 (Fig. 1C) and 60 days (Fig. 1D) after EV treatment in the PCOS groups. The ovaries of the PCOS group at 60 days post-injection were small compared with the control group and contained various follicular cysts (data not show). In addition, periantral granulosa cells were degenerated. A cluster of degenerated granulosa cells occurred in the antrum of the follicle in an ovary at 60 days post-treatment (Fig. 1E), and the follicle was typical cystic exhibiting a large central cavity, an attenuated granulosa membrane, and an abnormally thickened theca layer (Fig. 1F). 3.1.1. Effect of EV-induced PCOS on plasma progesterone, testosterone and estradiol concentration Fig. 2 shows plasma progesterone, testosterone and estradiol concentrations of the control group and at 30, 45 and 60 days after EV treatments. In the control group, the plasma concentrations of

20

L.H. Montrezor et al. / Respiratory Physiology & Neurobiology 217 (2015) 17–24

Fig. 2. Plasma steroid hormone levels of the control group (n = 6) and the groups at 30 (n = 5), 45 (n = 5) and 60 days after EV treatment (n = 5). Data are shown as the mean ± SEM of plasma progesterone, estradiol and testosterone concentrations. * indicates significant differences compared to the control group.

progesterone, estradiol and testosterone were 9.86 ± 3.3, 35.8 ± 3.1 and 22.6 ± 1.9 pg/mL, respectively. Plasma estradiol (Fig. 2) was significantly increased at 30, 45 and 60 days in the EV-treated groups compared to the control group (P < 0.05). Testosterone concentration (Fig. 2) was significantly increased at 45 and 60 days post-injection compared with the control group (P < 0.05) and progesterone was increased in 60 days post-injection group compared with control animals (Fig. 2) 3.1.2. Effects of EV administration on VE and Tb under normoxic and normocapnic conditions Under normoxia normocapnic conditions, EV treatment (30, 45 and 60 days) had no effect on VE , VT and fR compared to the control group (Fig. 3). Body temperatures for the control group and for the groups at 30, 45 and 60 days post-treatment were 37.8 ± 0.1,

Fig. 3. Ventilation (VE ), tidal volume (VT ) and respiratory frequency (fR) of the control group (n = 6) and the groups at 30 (n = 5), 45 (n = 5) and 60 days after EV treatment (n = 5) during conditions of normoxia normocarbic. No significant differences were observed.

37.4 ± 0.2, 37.8 ± 0.2 and 37.6 ± 0.1, respectively, and no differences among the groups were observed.

3.1.3. Effects of EV administration on VE and Tb under hypercapnic conditions Hypercapnia caused a significant increase in pulmonary ventilation in all groups, which resulted in increases of both fR and VT (P < 0.0001, Fig. 4A). In contrast, the animals treated with EV 30 days before the experiment showed an attenuation of the respiratory response to CO2 compared to all group (P < 0.001 for control and 60 days group and P < 0.05 for 45 days group). This response was due to a decrease in VT (P < 0.001 for control and P < 0.05 for 60 days group). Neither hypercapnia nor EV treatment changed the Tb of rats (Fig. 4B).

L.H. Montrezor et al. / Respiratory Physiology & Neurobiology 217 (2015) 17–24

21

Fig. 5. (A) Relative changes in ventilation (VE ), tidal volume (VT ) and respiratory frequency (fR) and (B) changes in body temperature () of the control group (n = 6) and the groups at 30 (n = 5), 45 (n = 5) and 60 days after EV treatment (n = 6) during hypoxia (7% O2 ). Values are expressed as the mean ± SEM.

3.1.4. Effects of EV administration on VE and Tb under hypoxic conditions Hypoxia increased pulmonary ventilation in all groups due to increases of both fR and VT (P < 0.0001) (Fig. 5). Nevertheless, EV treatment did not change the ventilatory response to 7% O2 . Hypoxia reduced Tb to a similar extent in all groups (P < 0.0001). EV treatment did not affect this response. 4. Discussion Fig. 4. (A) Relative changes in ventilation (VE ), tidal volume (VT ) and respiratory frequency (fR) and (B) changes in body temperature () of the control group (n = 6) and the groups at 30 (n = 5), 45 (n = 5) and 60 days after EV treatment (n = 6) during hypercapnia (7% CO2 ). * indicates a significant difference among 30 days after EV treatment and control and groups at 45 and 60 days after EV treatment. Values are expressed as the mean ± SEM.

The present study provides evidence that the hormonal alterations observed in PCOS result in a temporary attenuation of the central chemoreflex in unanesthetized rats, as 30 days after EV treatment, the animals showed a reduction of the hypercapnic

22

L.H. Montrezor et al. / Respiratory Physiology & Neurobiology 217 (2015) 17–24

chemoreflex. This chemoreflex was restored by 45 and 60 days after the injection. The etiology of PCOS is unknown, which reflects the involvement of multiple pathophysiological mechanisms, and since the 1960s, a range of animal models have been used to study this condition (Walters et al., 2012). Estradiol valerate (EV) is a long-acting estrogen, and it has been shown that a single dose to pre-pubertal female rats results in ovarian morphological and hormonal alterations that induce the formation of follicular cysts (Rosa-E-Silva et al., 2003). Why and how ovaries become polycystic has been the subject of considerable speculation. Farookhi et al. (1985) proposed that the occurrence of a polycystic ovary may primarily occur as a response to the prolonged absence of gonadotropin surges. In the present study, microscopic examinations of the ovaries obtained from all groups of EV-treated rats revealed the presence of follicular cysts (Fig. 1). Although this response occurred in follicles of all diameters, follicular atresia was selectively enhanced in the large secondary follicles at 45 and 60 days post-injection. Furthermore, at 60 days, the cysts exhibited features such as degenerating patches of granulosa cells occurring at points along the healthy-looking inner cell layer, suggesting that the cysts may be a product of follicular arrest atresia (Schulster et al., 1984). Furthermore, hyperthecosis, which is characteristic of cystic follicles, is also typical of atretic secondary follicles. Granulosa cells from anovulatory women with PCOS hypersecrete estradiol and progesterone compared with size-matched follicles from normal ovaries or polycystic ovaries from ovulatory women (Willis et al., 1998). This phenomenon appears to reflect a condition of advanced maturation of medium-sized antral follicles, though it may also be associated with enhanced activity of P450 arom (aromatase), which is the enzyme of granulosa cells that is hyper-responsive to follicle-stimulating hormone (FSH) in terms of estradiol production (Franks et al., 2000). Corroborating this data, our results also show an increase in plasma concentration of estradiol in all EV-treated groups and progesterone in rats 60 days after EV treatment. Bernuci et al. (2008), using a longterm cold exposure model to induce PCOS, also showed increased secretion of estradiol and testosterone in cold-stressed rats. In addition to morphological alterations, a second important criterion for PCOS diagnosis, hyperandrogenemia (Rotterdam, 2004), was observed in our animals at 30, 45 and 60 days after EV treatment. This hypersecretion of androgens reflects an intrinsic abnormality of theca cell function. In our study, hyperandrogenemia was characterized by an increase in plasma testosterone concentration. Under normoxia normocapnic conditions, no difference in pulmonary ventilation was observed among the groups, suggesting that hormonal changes in EV-treated rats were not sufficient to promote alteration in resting ventilation. Sex hormones easily cross the blood–brain barrier, and the receptors have been mapped in central and peripheral structures that regulate breathing (Kastrup et al., 1999; Haywood et al., 1999; Romeo et al., 2005; Brinton et al., 2008; Hamson et al., 2004; Helena et al., 2009; Fournier et al., 2014). Peripherally, dense immunostaining for progesterone receptors in the peripheral chemoreceptors of fetal, newborn and adult rats has been described (Joseph et al., 2006). One of the earliest suggestions that steroid hormones may have an impact on breathing was a report of decreased alveolar PCO2 during pregnancy (Hasselbach, 1912). Hasselbach suggested that this response may be due to a strongly increased excitability of the brain regions involved in respiratory regulation. In fact, the respiratory stimulant effect of progesterone is well documented (Saaresranta and Polo, 2002; Soliz and Joseph, 2005; Smith et al., 2007; Behan and Wenninger, 2008). One of the implications of the progesterone effect on breathing regulation occurs during pregnancy when progesterone levels are high, which may be important to ensure adequate outflow of

CO2 from the fetus to the maternal circulation to avoid potentially harmful fetal acidosis. Actually, progesterone has been used as a pharmaceutical tool for treating sleep-breathing disorders because it is a potent respiratory stimulant that induces sleep (Andersen et al., 2006). The role of the other sex hormones in breathing regulation is less understood. Some evidence indicates a synergistic effect of estrogen and progesterone on ventilation (Shahar et al., 2003). In fact, as estrogen up-regulates progesterone receptors (Leavitt and Blaha, 1972), this mechanism is likely involved with the respiratory effects of progesterone. Recently, we have demonstrated that hormonal replacement with estradiol and progesterone in ovariectomized rats does not change ventilation (Marques et al., 2015). There is compelling evidence indicating that testosterone also affects ventilation. Indeed, it has been shown that testosterone administration and gonadectomy alter respiratory parameters and the hypoxic and hypercapnic ventilatory responses (Tatsumi et al., 1994; Behan et al., 2003; Zabka et al., 2006; Fournier et al., 2014). However, it has been suggested that the impact of testosterone on breathing regulation in animals and humans requires the conversion of testosterone to estrogen by aromatase. Thus, the role of testosterone in breathing function may ultimately be mediated by estrogen, the availability of which is controlled by aromatase (Simpson et al., 2002; Zabka et al., 2006). At 30 days post-injection, EV-treated rats exhibited a reduced ventilatory response to 7% CO2 , but no effect was observed on the hypoxic ventilatory response. These results suggest that hormonal alterations caused by EV-induced PCOS result in a primary inhibitory modulation of the hypercapnic ventilatory response. We were surprised to find an inhibitory effect on the central chemoreflex considering that the reproductive hormones have mainly been known to stimulate breathing. Sexual hormones act in different central areas involved in ventilatory control including the nucleus of the solitary tract (Simerly et al., 1990; Shughrue et al., 1997), the hypoglossal nuclei (Behan and Thomas, 2005), the motor nuclei of the phrenic nerve (Behan and Thomas, 2005) and the Locus coeruleus (LC) (Pendergast et al., 2008). It is well established that LC is involved in the compensatory responses to hypercapnia (Biancardi et al., 2008; De Carvalho et al., 2010; Gargaglioni et al., 2010) and LC-noradrenergic system integrity is essential for the development of PCO induced by cold stress (Bernuci et al., 2008, 2013). Since, estrogen exerts inhibitory effect on LC neurons (Szawka et al., 2009), it is possible that higher estrogen level in 30 days post-injection EV-treated rats promote the reduction in the central chemoreflex due to an action on LC neurons. Since, LC noradrenergic neurons are not involved in respiratory responses during hypoxic conditions (Biancardi et al., 2010), the fact that we did not observe a difference in hypoxic ventilatory response in EV treated rats reinforces the idea that estrogen may be acting on LC neurons. The inhibitory effect on the CO2 /pH chemoreflex was observed only in the group examined at 30 days after treatment. The fact that this attenuation was not observed at 45 or 60 days post-treatment suggests that, the effect of EV treatment on the CO2 /pH chemoreflex is not long-lasting. The transitory effect may reflect subsequent compensatory mechanisms that are activated to restore this function. In addition, the level of testosterone is not elevated in the group examined at 30 days post-treatment, which might contribute to the difference in CO2 ventilatory response compared to the groups examined at 45 or 60 days post-treatment. In fact, Bernuci et al. (2008) found a long-term adaptation of stress circuitry at the LC level. The authors found that Fos expression in LC neurons induced by cold stress is less pronounced after 8 weeks of chronic cold stress compared with a single acute session, suggesting an attenuation of the LC responsiveness in long-term exposure to cold stress. However more studies are needed to confirm this hypothesis in the present study.

L.H. Montrezor et al. / Respiratory Physiology & Neurobiology 217 (2015) 17–24

The lack of change in CO2 ventilatory response of 45 and 60 days EV-treated rats indicate that respiratory effects of PCOS do not correlate well with the severity of the disease. We do not know if this is the same pattern occurring in humans. At least for sleep-disordered breathing, a strong association with hyperandrogenemia is observed in patients with PCOS (Chatterjee et al., 2014). In this case, elevated testosterone levels may predispose the development of sleep-disordered breathing by enhancing soft tissue deposition in the pharynx, making it more collapsible during sleep and acting centrally promoting the obstruction of the pharynx during sleep (Chatterjee et al., 2014). Additionally, a positive correlation between severity of sleep-disordered breathing and androgens levels is also observed in men (Liu et al., 2003). Considering all three conditions, normoxic normocapnia, hypoxia and hypercapnia, and Tb was not affected by the induction of PCOS in these female rats. This is an interesting result because all three hormones affected by EV-treatment in the present study, i.e., estradiol, progesterone and testosterone, are reported to influence thermoregulation. There is evidence of estrogens being involved in Tb regulation in female animals and humans (Kobayashi et al., 2000; Berendsen and Kloosterboer, 2003; Nelson, 2008), and therapeutic estrogen replacement is used for minimizing hot flashes in peri and post-menopausal women, although this hormone may not be the unique factor involved in these events. The presence of estradiol receptors in hypothalamic nuclei has been reported to be involved in thermoregulation, including in the medial preoptic area (MPO) (Laflamme et al., 1998). Estradiol injection in the MPO of female rats intensifies tail vasoconstriction (heat conservation response) and spontaneous activity during cold exposure (Uchida et al., 2010). Moreover, systemic estradiol alone or in combination with progesterone inhibits Tb reduction during hypoxia in male rat pups (Lefter et al., 2008). Testosterone and estradiol were demonstrated to stimulate warmsensitive neurons in the preoptic region (Silva and Boulant, 1986). In contrast, testosterone can attenuate or even counteract some effects of estrogens in mice (Tuomela et al., 1990). According to Stachenfeld et al. (2010) total sweat losses, but not Tb, during exercise in warm conditions were higher among obese women with PCOS than in the obese control group. These differences in sweat losses are observed in women, whose hypothalamuspituitary axes are blocked by GNRH-antagonists and in those who received replacement of estrogen and testosterone, and no differences were seen in those who received reposition of testosterone only. Thus, the results of the present study indicate that the multiple sex hormone alterations induced by EV treatment either do not affect the central network for thermoregulation or that the net result of the combination of these hormones masks their individual effects. We cannot exclude the possibility of the treatments affecting the thermoeffectors for heat loss (salivation, peripheral vasodilation) or heat gain (thermogenesis) as they were not measured, though this constitutes an interesting focus for future studies. In summary, our results suggest that EV-induced PCOS results in an attenuation of the CO2 /pH chemoreflex, though this effect is not long-lasting. Further investigations are needed to improve the current view of the ventilatory effects of PCOS and to elucidate the compensatory mechanisms involved with the subsequent restoration of the CO2 ventilatory responses.

Conflict of interest The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

23

Funding This work was supported by the Fundac¸ão de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). Acknowledgments We thank Dr. César Augusto Sangaletti Terc¸ariol for the statistical analysis, Dr. Roberto Cuan Ravinal for the ovarian morphology analysis and Ruither O.G. Carolino for technical support. References Andersen, M.L., Bittencourt, L.R., Antunes, I.B., Tufik, S., 2006. Effects of progesterone on sleep: a possible pharmacological treatment for sleep-breathing disorders? Curr. Med Chem. 13 (29), 3575–3582. Bartlett Jr., D., Tenney, S.M., 1970. Control of breathing in experimental anemia. Respir. Physiol. 10, 384–395. Behan, M., Kinkead, R., 2011. Neuronal control of breathing: sex and stress hormones. Compreh. Physiol. 1 (4), 2101–2139. Behan, M., Thomas, C.F., 2005. Sex hormone receptors are expressed in identified respiratory motoneurons in male and female rats. Neuroscience 130 (3), 725–734. Behan, M., Wenninger, J.M., 2008. Sex steroidal hormones and respiratory control. Respir. Physiol. Neurobiol. 164, 213–221. Behan, M., Zabka, A.G., Thomas, C.F., Mitchell, G.S., 2003. Sex steroid hormones and the neural control of breathing. Respir. Physiol. 136, 249–263. Berendsen, H.H., Kloosterboer, H.J., 2003. Oestradiol and mirtazapine restore the disturbed tail-temperature of oestrogen deficient rats. Eur. J. Pharmacol. 482, 329–333. Bernuci, M.P., Szawka, R.E., Helena, C.V., Leite, C.M., Lara, H.E., Anselmo-Franci, J.A., 2008. Locus coeruleus mediates cold stress-induced polycystic ovary in rats. Endocrinology 149 (6), 2907–2916. Bernuci, M.P., Leite, C.M., Barros, P., Kalil, B., Leoni, G.B., Del Bianco-Borges, B., Franci, C.R., Szawka, R.E., Lara, H.E., Anselmo-Franci, J.A., 2013. Transitory activation of the central and ovarian norepinephrine systems during cold stress-induced polycystic ovary in rats. J. Neuroendocrinol. 25, 23–33. Biancardi, V., Bícego, K.C., Almeida, M.C., Gargaglioni, L.H., 2008. Locus coeruleus noradrenergic neurons and CO2 chemosensitivity. Pflügers Archiv. Eur. J. Physiol. 455 (6), 1119–1128. Biancardi, V., da Silva, L.T., Bícego, K.C., Gargaglioni, L.H., 2010. Role of locus coeruleus noradrenergic neurons in cardiorespiratory and thermal control during hypoxia. Respir. Physiol. 170 (2), 150–156. Brawer, J.R., Munoz, M., Farookhi, R., 1986. Development of the polycystic ovarian condition (PCO) in the estradiol valerate-treated rat. Biol. Reprod. 35 (3), 647–655. Brinton, R.D., Thompson, R.F., Foy, M.R., Baudry, M., Wang, J., Finch, C.E., Morgan, T.E., Pike, C.J., Mack, W.J., Stanczyk, F.Z., Nilsen, J., 2008. Progesterone receptors: form and function in brain. Front. Neuroendocrinol. 29 (2), 313–339. Chatterjee, B., Suri, J., Suri, J.C., Mittal, P., Adhikari, T., 2014. Impact of sleep-disordered breathing on metabolic dysfunctions in patients with polycystic ovary syndrome. Sleep Med. 15 (12), 1547–1553. De Carvalho, D., Bícego, K.C., Castro, O.W., Silva, G.S.F., Garcia-Cairasco, N., Gargaglioni, L.H., 2010. Role of neurokinin-1 expressing neurons in the locus coeruleus on ventilatory and cardiovascular responses to hypercapnia. Respir. Physiol. Neurobiol. 172, 24–31. Farookhi, R., Hemmings, R., Brawer, J.R., 1985. Unilateral ovariectomy restores ovulatory cyclicity in rats with a polycystic ovarian condition. Biol. Reprod. 32 (3), 530–540. Fournier, S., Gulemetova, R., Joseph, V., Kinkead, R., 2014. Testosterone potentiates the hypoxic ventilatory response of adult male rats subjected to neonatal stress. Exp. Physiol. 99 (5), 824–834. Franks, S., Mason, H., Willis, D., 2000. Follicular dynamics in the polycystic ovary syndrome. Mol. Cel. Endocrinol. 163, 49–52. Franks, S., 1995. Polycystic ovary syndrome. N. Engl. J. Med. 333 (13), 853–861. Gargaglioni, L.H., Hartzler, L.K., Putnam, R.W., 2010. The Locus coeruleus and central chemosensitivity. Respir. Physiol. Neurobiol. 173 (3), 264–273. Goodarzi, M.O., Dumesic, D.A., Chazenbalk, G., Azziz, R., 2011. Polycystic ovary syndrome: etiology, pathogenesis and diagnosis. Nat. Rev. Endocrinol. 7, 219–231. Hamson, D.K., Jones, B.A., Watson, N.V., 2004. Distribution of androgen receptorimmunoreactivity in the brainstem of male rats. Neuroscience 127 (4), 797–803. Hasselbach, K.A., 1912. Ein Betrag zur Respirationsphysiologie der Gravidität. Scand. Arch. Physiol. 27, 1–12. Haywood, S.A., Simonian, S.X., van der Beek, E.M., Bicknell, R.J., Herbison, A.E., 1999. Fluctuating estrogen and progesterone receptor expression in brainstem norepinephrine neurons through the rat estrous cycle. Endocrinology 140 (7), 3255–3263. Helena, C.V., Gustafsson, J.A., Korach, K., Pfaff, D., Anselmo-Franci, J.A., Ogawa, S., 2009. Effects of estrogen receptor alpha and beta gene deletion on estrogenic

24

L.H. Montrezor et al. / Respiratory Physiology & Neurobiology 217 (2015) 17–24

induction of progesterone receptors in the locus coeruleus in female mice. Endocrine. 36 (1), 169–177. Joseph, V., Doan, V.D., Morency, C.E., Lajeunesse, Y., Bairam, A., 2006. Expression of sex-steroid receptors and steroidogenic enzymes in the carotid body of adult and newborn male rats. Brain. Res. 1073-1074, 71–82. Kastrup, Y., Hallbeck, M., Amandusson, A., Hirata, S., Hermanson, O., Blomqvist, A., 1999. Progesterone receptor expression in the brainstem of the female rat. Neurosci. Lett. 275, 85–88. Knochenhauer, E.S., Key, T.J., Kahsar-Miller, M., Waggoner, W., Boots, L.R., Azziz, R., 1998. Prevalence of the polycystic ovary syndrome in unselected black and white women of the southeastern United States: a prospective study. J. Clin. Endocrinol. Metab. 83 (9), 3078–3082. Kobayashi, T., Tamura, M., Hayashi, M., Katsuura, Y., Tanabe, H., Ohta, T., Komoriy, K., 2000. Elevation of tail skin temperature in ovariectomized rats in relation to menopausal hot flushes. Am. J. Physiol. Regul. Integr. Comp. Physiol. 278, 863–869. Laflamme, N., Nappi, R.E., Drolet, G., Labrie, C., Rivest, S., 1998. Expression and neuropeptidergic characterization of estrogen receptors (ERalpha and ERbeta) throughout the rat brain: Anatomical evidence of distinct roles of each subtype. J. Neurobiol. 36, 357–378. Leavitt, W.W., Blaha, G.C., 1972. An estrogen-stimulated, progesterone-binding system in the hamster uterus and vagina. Steroids 19 (2), 263–274. Lefter, R., Doan, V.D., Joseph, V., 2008. Contrasting effects of estradiol and progesterone on respiratory pattern and hypoxic ventilatory response in newborn male rats. Respir. Physiol. Neurobiol 164, 312–318. Linares, R., Hernández, D., Morán, C., Chavira, R., Cárdenas, M., Domínguez, R., Morales-Ledesma, L., 2013. Unilateral or bilateral vagotomy induces ovulation in both ovaries of rats with polycystic ovarian syndrome. Reprod. Biol. Endocrinol. 11, 68. Liu, P.Y., Yee, B., Wishart, S.M., Jimenez, M., Jung, D.G., Grunstein, R.R., Handelsman, D.J., 2003. The short term effects of high dose testosterone on sleep, breathing and functions in older men. J. Clin. Endocrinol. Metab. 88, 3605–3613. Lobo, R.A., Carmina, E., 2000. The importance of diagnosing the polycystic ovary syndrome. Ann. Intern. Med. 132, 989–993. Marques, D.A., Carvalho, D., da Silva, G.S., Szawka, R.E., Anselmo-Franci, J.A., Bicego, K.C., Gargaglioni, L.H., 2015. Ventilatory, metabolic and thermal responses to hypercapnia in female rats: effects of estrous cycle, ovariectomy and hormonal replacement. J. Appl. Physiol. 1985, http://dx.doi.org/10.1152/japplphysiol. 00254.2015 Moore, L.G., McCullough, R.E., Weil, J.V., 1987. Increased HVR in pregnancy: relationship to hormonal and metabolic changes. J. Appl. Physiol. 62 (1), 158–163. Nelson, H.D., 2008. Menopause. Lancet 371, 760–770. Pasquali, R., Stener-Victorin, E., Yildiz, B.O., Duleba, A.J., Hoeger, K., Mason, H., Homburg, R., Hickey, T., Franks, S., Tapanainen, J., Balen, A., Abbott, D.H., 2011. Forum: research in polycystic ovary syndrome today and tomorrow. Clin. Endocrinol. (Oxf.) 74, 424–433. Patrone, L.G.A., Bícego, K.C., Hartzler, L.K., Putnam, R.W., Gargaglioni, L.H., 2014. Cardiorespiratory effects of gap junction blockade in the locus coeruleus in unanesthetized adult rats. Respir. Physiol. Neurobiol. 190, 86–95. Pendergast, J.S., Tuesta, L.M., Bethea, J.R., 2008. Oestrogen receptor beta contributes to the transient sex difference in tyrosine hydroxylase expression in the mouse locus coeruleus. J. Neuroendocrinol. 20 (10), 1155–1164. Pereira, V.M., Honorato-Sampaio, K., Martins, A.S., Reis, F.M., Reis, A.M., 2014. Downregulation of natriuretic peptide system and increased steroidogenesis in rat polycystic ovary. Peptides 60, 80–85. Romeo, R.D., Bellani, R., McEwen, B.S., 2005. Stress-induced progesterone secretion and progesterone receptor immunoreactivity in the paraventricular nucleus are modulated by pubertal development in male rats. Stress 8 (4), 265–271. Rosa-E-Silva, A., Guimaraes, M.A., Padmanabhan, V., Lara, H.E., 2003. Prepubertal administration of estradiol valerate disrupts cyclicity and leads to cystic ovarian morphology during adult life in the rat: role of sympathetic innervations. Endocrinology 144 (10), 4289–4297.

Rotterdam ESHRE/ASRM-Sponsored PCOS Consensus Worshop Group, 2004. Revised 2003 consensus on diagnostic criteria and long-term health risk related to polycystic ovary syndrome (PCOS). Hum. Reprod. 19 (1), 41–47. Saaresranta, T., Polo, O., 2002. Hormones and breathing. Chest 122, 2165–2182. Schulster, A., Farookhi, R., Brawer, J.R., 1984. Polycystic ovarian condition in estradiol valerate-treated rats: spontaneous changes in characteristic endocrine features. Biol. Reprod. 31, 587–593. Shahar, E., Redline, S., Young, T., Boland, L.L., Baldwin, C.M., Nieto, F.J., O’Connor, G.T., Rapoport, D.M., Robbins, J.A., 2003. Hormone replacement therapy and sleep-disordered breathing. Am. J. Respir. Crit. Care Med. 167 (9), 1186–1192. Shughrue, P.J., Lane, M.V., Merchenthaler, I., 1997. Comparative distribution of estrogen receptor-alpha and -beta mRNA in the rat central nervous system. J. Comp. Neurol. 388, 507–525. Silva, N.L., Boulant, J.A., 1986. Effects of testosterone, estradiol, and temperature on neurons in preoptic tissue slices. Am. J. Physiol. 250 (4), 625–632. Simerly, R.B., Chang, C., Muramatsu, M., Swanson, L.W., 1990. Distribution of androgen and estrogen receptor mRNA-containing cells in the rat brain: an in situ hybridization study. J. Comp. Neurol. 294, 76–95. Simpson, E.R., Clyne, C., Rubin, G., Boon, W.C., Robertson, K., Britt, K., Speed, C., Jones, M., 2002. Aromatase-a brief overview. Ann. Rev. Physiol. 64, 93–127. Smith, S.S., Shen, H., Gong, Q.H., Zhou, X., 2007. Neurosteroid regulation of GABA(A) receptors: focus on the alpha4 and delta subunits. Pharmacol. Ther. 116, 58–76. Soliz, J., Joseph, V., 2005. Perinatal steroid exposure and respiratory control during early postnatal life. Respir. Physiol. Neurobiol. 149 (1–3), 111–122. Stachenfeld, N.S., Yeckel, C.W., Taylor, H.S., 2010. Greater exercise sweating in obese women with polycystic ovary syndrome compared with obese controls. Med. Sci. Sports. Exerc. 42 (9), 1660–1668. Szawka, R.E., Rodovalho, G.V., Monteiro, P.M., Carrer, H.F., Anselmo-Franci, J.A., 2009. Ovarian-steroid modulation of locus coeruleus activity in female rats: involvement in luteinising hormone regulation. J. Neuroendocrinol. 21 (7), 629–639. Tasali, E., Van Cauter, E., Ehrmann, D.A., 2008. Polycystic ovary syndrome and obstructive sleep apnea. Sleep Med. Clin. 3 (1), 37–46. Tatsumi, K., Hannhart, B., Pickett, C.K., Weil, J.V., Moore, L.G., 1994. Effects of testosterone on hypoxic ventilatory and carotid body neural responsiveness. Am. J. Respir. Crit. Care. Med. 149, 1248–1253. Teede, H.J., Norman, R., 2006. Polycystic ovarian syndrome: insights into the enigma that is PCOS today. Endocrine 30 (1), 1–2. Tock, L., Carneiro, G., Togeiro, S.M., Hachul, H., Pereira, A.Z., Tufik, S., Zanella, M.T., 2014. Obstructive sleep apnea predisposes to nonalcoholic Fatty liver disease in patients with polycystic ovary syndrome. Endocr. Pract. 20 (3), 244–251. Tuomela, T., Miettinen, P., Viinikka, L., Perheentupa, J., 1990. Estrogen–androgen antagonism in the regulation of epidermal growth factor in mouse submandibular salivary gland and kidneys. Life Sci. 47 (21), 1925–1932. Uchida, Y., Tokizawa, K., Nakamura, M., Mori, H., Nagashima, K., 2010. Estrogen in the medial preoptic nucleus of the hypothalamus modulates cold responses in female rats. Brain Res. 1339, 49–59. Vgontzas, A.N., Legro, R.S., Bixler, E.O., Grayev, A., Kales, A., Chrousos, G.P., 2001. Polycystic ovary syndrome is associated with obstructive sleep apnea and daytime sleepiness: role of insulin resistance. J. Clin. Endocrinol Metab. 86 (2), 517–520. Walters, K.A., Allan, C.M., Handelsman, D.J., 2012. Rodent models for human polycystic ovary syndrome. Biol. Reprod. 86 (5), 1–12. Willis, D.S., Watson, H., Mason, H.D., Galea, R., Brincat, M., Franks, S., 1998. Premature response to luteinizing hormone of granulosa cells from anovulatory women with polycystic ovary syndrome: relevance to mechanism of anovulation. J. Clin. Endocrinol Metab. 83 (11), 3984–3991. Zabka, A.G., Mitchell, G.S., Behan, M., 2006. Conversion from testosterone to estradiol is required to modulate respiratory long-term facilitation in male rats. J. Physiol. 576, 903–912.