J Appl Physiol 119: 61–68, 2015. First published April 30, 2015; doi:10.1152/japplphysiol.00254.2015.
Ventilatory, metabolic, and thermal responses to hypercapnia in female rats: effects of estrous cycle, ovariectomy, and hormonal replacement Danuzia A. Marques,1 Débora de Carvalho,2 Glauber S. F. da Silva,1 Raphael E. Szawka,3 Janete A. Anselmo-Franci,4 Kênia C. Bícego,1 and Luciane H. Gargaglioni1 1
Department of Animal Morphology and Physiology, São Paulo State University, UNESP FCAV at Jaboticabal, São Paulo, Brazil; 2Federal Institute of Para, Para, Brazil; 3Department of Physiology and Biophysics, Institute of Biological Sciences, Federal University of Minas Gerais, Minas Gerais, Brazil; and 4Department of Morphology, Stomatology and Physiology, Dental School of Ribeirao Preto, University of São Paulo, São Paulo, Brazil Submitted 25 March 2015; accepted in final form 23 April 2015
Marques DA, de Carvalho D, da Silva GS, Szawka RE, AnselmoFranci JA, Bícego KC, Gargaglioni LH. Ventilatory, metabolic, and thermal responses to hypercapnia in female rats: effects of estrous cycle, ovariectomy, and hormonal replacement. J Appl Physiol 119: 61–68, 2015. First published April 30, 2015; doi:10.1152/japplphysiol.00254.2015.—The aim of this study was to examine how estrous cycle, ovariectomy, and ˙ E), tidal hormonal replacement affect the respiratory [ventilation (V ˙ O2), and thermoregvolume, and respiratory frequency], metabolic (V ulatory (body temperature) responses to hypercapnia (7% CO2) in female Wistar rats. The parameters were measured in rats during different phases of the estrous cycle, and also in ovariectomized (OVX) rats supplemented with 17-estradiol (OVX⫹E2), with a combination of E2 and progesterone (OVX⫹E2P), or with corn oil (OVX⫹O, vehicle). All experiments were conducted on day 8 after ovariectomy. The intact animals did not present alterations during ˙ E, tidal volume, respiratory normocapnia or under hypercapnia in V ˙ O2, and V ˙ E/V ˙ O2 in the different phases of the estrous frequency, V cycle. However, body temperature was higher in female rats on estrus. Hormonal replacement did not change the ventilatory, thermoregulatory, or metabolic parameters during hypercapnia, compared with the OVX animals. Nevertheless, OVX⫹E2, OVX⫹E2P, and OVX⫹O presented lower hypercapnic ventilatory responses compared with intact females on the day of estrus. Also, rats in estrus showed higher ˙ E and V ˙ E/V ˙ O2 during hypercapnia than OVX animals. The data V suggest that other gonadal factors, besides E2 and P, are possibly involved in these responses. CO2; estrogen; progesterone; cycling rats; breathing SEX HORMONES, INCLUDING
androgens, estrogens, such as 17estradiol (E2), and progestogens, such as progesterone (P), are present in both males and females, although marked differences in the plasma levels occurs throughout the lifespan of the animals (3). In female rats, E2 levels are low on estrus, elevated on metestrus and diestrus, and reach a peak during the proestrus, falling again as the next cycle starts (18). On the other hand, levels of P increase between the night of metestrus and early morning of diestrus, and fall thereafter (18). Then, on the afternoon of proestrus, P levels increase again, reaching a second peak, and then return to baseline levels on estrus (43). These hormones affect neuromodulatory systems that influence the central respiratory network (3), acting directly on respiratory motor or adjacent neurons (5, 13), brain stem Address for reprint requests and other correspondence: L. H. Gargaglioni, 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 14884-900, Brazil (e-mail: [email protected]). http://www.jappl.org
premotor neurons (13), rhythm generating neurons (3). They can also influence peripheral or central chemoreceptors (5), which suggests an important hormonal role in the breathing control (3). Most of the experiments in respiratory control, however, are performed in male rats. When females are used, the studies do not consider the natural hormonal oscillations that occur during the estrous cycle. In women, studies demonstrated that the ventilatory responses to hypoxia and hypercapnia vary in the different phases of the menstrual cycle (34, 48). For example, the ˙ E) and tidal volume (VT) are increased in the ventilation (V luteal phase, compared with the follicular phase (13, 35, 41), and consequently arterial partial pressure of CO2 is reduced. Additionally, central and peripheral chemosensitivity are increased in luteal phase (16, 35, 48), and administration of medroxyprogesterone acetate, a synthetic variant of P, stimulates the ventilatory system (40, 52), suggesting that the increased ventilatory response during the luteal phase is related to the elevated plasmatic concentrations of P. Wenninger et al. (47) reported that female rats present a ˙ E/V ˙ O2 compared with male rats. Variasignificantly higher V ˙ O2 also occur during tions in the body temperature (Tb) and V the menstrual and estrous cycle (11, 28, 33), but this is still a poorly explored issue. The hormonal variations in different stages of the estrous cycle may present additional challenges to data collection and interpretation. To the best of our knowledge, no study has examined the influence of the estrous cycle on ventilatory, metabolic, and thermoregulatory parameters in normocapnic and hypercapnic conditions in rodents. We hypothesized that hormonal fluctuations observed during the estrous cycle have an impact on ˙ E, V ˙ O2, and Tb. We also assessed the possible role of E2 basal V ˙ E, V ˙ O2, and P in those influences. To this end, we measured V and Tb in intact, as well as ovariectomized (OVX) without replacement and OVX with E2 and P replacement female rats. MATERIALS AND METHODS
Animals Experiments were performed in unanesthetized adult female Wistar rats weighting 250 –300 g. The age of the animals ranged from 90 to 120 days. The animals were fed ad libitum and housed in a temperature-controlled chamber, maintained at 24 –26°C (ALE 9902001; Alesco, Monte Mor, Brazil), with a 12:12-h light-dark cycle (lights on at 6:00 AM). All experimental procedures were in compliance with the Brazilian College of Animal Experimentation guidelines and approved by the local Animal Care and Use Committee (protocol no. 007827-09).
8750-7587/15 Copyright © 2015 the American Physiological Society
61
˙ E, V ˙ O2, and Tb Hormonal Influence on V
62
We used cycling female rats on the days of proestrus (n ⫽ 8), estrus (n ⫽ 6), metestrus (n ⫽ 4), and diestrus (n ⫽ 4), and OVX rats treated with corn oil (OVX⫹O; n ⫽ 6), E2 (OVX⫹E2; n ⫽ 6), or E2 and P (OVX⫹E2P; n ⫽ 5). We used 39 animals in total. Ventilatory, Tb, and Metabolic Measurements ˙ E measurements were obtained using the barometric method The V (whole body plethysmography) (2), as previously described by Biancardi et al. (6), de Carvalho et al. (12), and Lopes et al. (25). Tb was recorded by the use of a temperature datalogger (SubCue, Calgary, AB, Canada) implanted in the abdominal cavity, as previously described in the literature (6). The datalogger was programmed to acquire data every 5 min. ˙ O2) The metabolic rate was measured by indirect calorimetry (V using a closed respirometry system, as described by Almeida et al. (1). At the end of the normocapnic and hypercapnic periods, the airflow in the chamber was interrupted for 2 min, and the air was continuously sampled by an O2 analyzer (PowerLab System. ADInstruments/Chart Software, version 7.3, Sydney, Australia). While the chamber was sealed, the oxygen fraction did not drop to ⬍19%, and CO2 did not increase ⬎0.7%. The percentage of oxygen decay inside the chamber was plotted against time, and the slope of the resulting curve corre˙ O2. The values are presented in milliliters per sponded to the rate of V minute per kilogram in STPD (standard conditions of temperature, pressure, and dry air).
•
Marques DA et al.
h later. The regimens of hormonal treatment used yielded physiological levels of plasma E2 and P, as described by Szawka et al. (44). On the day of experiment, vaginal cytology was performed to monitor the effectiveness of the hormonal treatment. Animals in which hormone replacement was effective presented cornified epithelial cells in the vaginal cytology (27). In contrast, when the hormonal replacement was not efficient, the smears displayed predominance of small leukocytes, similarly as found in the cytology of OVX⫹O rats, and the animals were not used in the experiments. Experimental Protocol Each animal was individually placed in a plethysmograph chamber (5 liters), kept at 25°C and allowed to move freely, while the chamber was flushed with humidified room air. The baseline ˙ E and V ˙ O2 were taken after animals acclimameasurements of V tized for ⬃1 h. Subsequently, a hypercapnic gas mixture (7% CO2 in air, White Martins, Sertãozinho, São Paulo, Brazil) was flushed ˙ E was measured at 30 min. through the chamber for 30 min, and V ˙ E was calculated before (time zero) and 30 min after hypercapnia V or normocapnia. The percentage of CO2 and the exposure time were chosen based on pilot experiments and previous studies (6, ˙ O2 was measured once again after the stimuli. The 15). The V experiments were carried out from 900 to 1300. Tb was continuously measured during the experiment.
Vaginal Smears
Statistical Analysis
The animals were subjected to daily vaginal lavages to monitor the progression through the estrous cycle. Vaginal smears were performed daily at 9:00 AM to verify estrous cycle regularity. Only females exhibiting at least four consecutive regular cycles were used in the experiments. The estrous cycle stage was determined using the criteria used by Freeman (18).
Statistical analysis of the blood hormone concentration data was performed by one-way ANOVA followed by Tukey post hoc ˙ E, respiratory frequency (fR), VT, Tb, and V ˙ O2 data were testing. V compared using repeated two-way ANOVA. When interactions between the factors were observed, groups were compared using Bonferroni’s post hoc test. The significance level was set to P ⬍ 0.05. The statistical analysis was performed using computer software (SigmaStat; Systat Software, Point Richmond, CA). Data are presented as group means ⫾ SE for each parameter investigated.
Blood Collection for Plasma Hormone Assays After the hypercapnic challenge, and just before euthanasia, a blood sample of ⬃1 ml was collected from the heart of anesthetized rats into heparinized syringes. The plasma was separated by centrifugation at 3,000 rpm for 20 min at 4°C and stored at ⫺20°C for posterior quantification of P and E2 by radioimmunoassay. Plasma P and E2 concentrations were determined by double-antibody radioimmunoassay with MAIA kits provided by Biochem Immunosystem (Bologna, Italy). The lower limits of detection for E2 and P were 5.0 pg/ml and 0.02 ng/ml, respectively. Ovariectomy and Hormonal Replacement Before the ovariectomy surgery, animals were anesthetized, via intraperitoneal injection of 100 mg/kg of ketamine (Union National Pharmaceutical Chemistry S/A, Embu-Guaçu, São Paulo, Brazil) and 10 mg/kg of xylazine (Laboratories Calier S/A, Barcelona, Spain). A temperature datalogger (SubCue, Calgary, AB, Canada) was also implanted in the abdominal cavity through a midline laparotomy. After this procedure, bilateral ovariectomies were performed using an incision 1.5 cm inferior to the palpated rib cage. Ovaries and surrounding fat tissue were removed; the incision was closed by suturing the muscles and stapling the skin. After surgery, animals were treated with two doses of enrofloxacin (10 mg/kg intramuscular) and flunixin meglumine [2.5 mg/kg subcutaneous (SC)] to prevent infection and postsurgical discomfort, respectively. Eight days after ovariectomy, rats were treated with corn oil (0.2 ml SC) or E2 (10 g/0.2 ml SC, 17-estradiol cypionate; Pfizer, São Paulo, Brazil), at 9 AM for 3 days. On the 4th day, oil-treated rats received a final oil injection (OVX⫹O group), while E2-treated rats received an injection containing corn oil (OVX⫹E2 group) or P (2.5 mg/0.2 ml SC, OVX⫹E2P group; Sigma, St. Louis, MO), and the experiments were performed 2
RESULTS
Effects of Estrous Cycle Stages on V˙E, V˙O2, and Tb Vaginal cytology. Figure 1 shows the cytological observations characteristic of specific stages of the estrous cycle in cycling females. Proestrous vaginal smears showed nucleated epithelial cells (Fig. 1A), whereas estrous smears present densely stained cornified cells (Fig. 1B). Metestrus was characterized by the observation of lightly stained cornified cells in addition to some leukocytes and nucleated cells (Fig. 1C). Finally, diestrous smears showed presence of leukocytes (Fig. 1D). Hormonal profile. Figure 2 shows plasma E2 and P concentrations in cycling female rats. Plasmatic concentration of E2 was significantly higher in proestrus than in estrus, metestrus, and diestrus. The plasma concentration of P was lower on estrus compared with proestrus, metestrus, and diestrus. Pulmonary V˙E and metabolism. In normocapnic conditions, ˙ E were observed in different phases of the no differences in V ˙ E in estrous cycle (Fig. 3). Hypercapnia caused an increase in V all groups [hypercapnia effect: P ⬍ 0.0001; F(3,22) ⫽ 131.7; no interaction] due to an increase in VT [hypercapnia effect: P ⬍ 0.0001; F(3,22)⫽ 84.6; no interaction] and fR [hypercapnia effect: P ⬍ 0.0001; F(3,22) ⫽ 301.1; no interaction]. There was no difference in the hypercapnic ventilatory response between
J Appl Physiol • doi:10.1152/japplphysiol.00254.2015 • www.jappl.org
˙ E, V ˙ O2, and Tb Hormonal Influence on V
•
63
Marques DA et al.
Fig. 1. Evaluation of the stage of the estrous cycle in cycling females. Representative microphotographs are shown of vaginal cytological samples taken from a female rat during proestrus (A), estrus (B), metestrus (C), and diestrus (D).
groups in the different phases of the estrous cycle, as seen in Fig. 3. ˙ O2 during hypercapnia [hypercapAll groups had lower V nia effect: P ⫽ 0.0127; F(3,22) ⫽ 6.8; no interaction], and no differences were observed between the groups in different
A
Proestrus (n=8) Estrus (n=6) Metaestrus (n=4) Diestrus (n=4)
E2 concentration (pg/mL)
250 200
*
150 100 50
40 30 (ng/mL)
B P concentration
0
20 10
*
0 Fig. 2. Changes in plasma concentration of 17-estradiol (E2; A) and progesterone (P; B) during the different phases of the estrous cycle. Values are means ⫾ SE; n, no. of rats. *Difference between estrus group compared with proestrus, metestrus, and diestrus (P ⬍ 0.05).
stages of the estrous cycle. Hypercapnia caused a significant ˙ E/V ˙ O2 [hypercapnia effect: P ⬍ 0.001; increase in the V F(3,22) ⫽ 85.6; no interaction], and, again, no difference was found between groups (Fig. 4). Tb. As seen in Fig. 5, Tb of rats on estrus was higher than that of rats on other stages of the estrous cycle, during both normocapnia and hypercapnia [cycle effect: P ⬍ 0.001; F(3,22) ⫽ 7.8; no interaction]. Hypercapnia caused a decrease in Tb only in estrous and proestrous rats [hypercapnia effect: P ⫽ 0.0003; F(3,22)⫽ 20.2; no interaction]. Effects of Ovariectomy and Hormonal Replacement on V˙E, V˙O2, and Tb Pulmonary V˙E and metabolism. There was no difference in the ventilatory parameters between groups during normo˙ E in all capnia (Fig. 6). Hypercapnia induced an increase in V groups [hypercapnia effect: P ⬍ 0.0001, F(3,19) ⫽ 160.7, interaction: P ⬍ 0.0001; F(3,19) ⫽ 17.1], which was a result of an increase in VT [hypercapnia effect: P ⬍ 0.0001, F(3,19) ⫽ 85.6, interaction: P ⫽ 0.0064; F(3,19) ⫽ 4.8] and fR [hypercapnia effect: P ⬍ 0.001, F(3,19) ⫽ 94.8, no interaction]. OVX animals (OVX⫹O, OVX⫹E2, and OVX⫹E2P) showed an attenuated hypercapnic ventilatory response (43% lower for all groups) compared with rats on estrus [hormone effect: P ⬍ 0.0001, F(3,19) ⫽ 30.1; interaction: P ⬍ 0.0001; F(3,19) ⫽ 17.1] due to lower VT [hormone effect: P ⫽ 0.0001, F(3,19) ⫽ 9.2, interaction: P ⫽ 0.0064; F(3,19) ⫽ 4.8] and fR [hormone effect: P ⫽ 0.0030, F(3,19) ⫽ 5.5, no interaction]. However, the hormonal replacement did not affect the ventilatory responses to CO2, as they were similar across all OVX groups. ˙ O2 of the estrus group was higher comIn normocapnia, V pared with OVX⫹E2P animals [hormone effect: P ⫽ 0.0017, ˙ O2 was F(3,19) ⫽ 6.2, no interaction] (Fig. 7). No difference in V
J Appl Physiol • doi:10.1152/japplphysiol.00254.2015 • www.jappl.org
˙ E, V ˙ O2, and Tb Hormonal Influence on V
64 Proestrus (n=8) Estrus (n=6) Metaestrus (n=4) Diestrus (n=4)
2800 2100
20 15 10
.
700 0
5 0
30
300
25
. .
VE/VO2
VT (mLKg-1)
0
30
250
20 15 10
200 150 100 50
5
0 0 CO2 0%
0 0
30
180
30 2 7% CO
˙ O2) and respiratory equivalent (V ˙ E/V ˙ O2) of rats Fig. 4. Oxygen consumption (V in proestrus, estrus, metestrus, and diestrus during normocapnia (0% CO2) and hypercapnia (7% CO2). Values are means ⫾ SE; n, no. of rats.
160 140
DISCUSSION
120
The present study demonstrates that, despite the hormonal ˙ O2, and V ˙ E, V ˙ E/V ˙ O2 during fluctuations of cycling rats, V normocapnia and hypercapnia were not different in different stages of the estrous cycle. As shown in previous studies, our measurements of Tb were higher in estrous rats in both normocapnic and hypercapnic conditions. Additionally, this study also shows that ovariectomy promotes an attenuation of the ˙ E and V ˙ E/V ˙ O2) compared with the response to hypercapnia (in V response obtained from intact animals in estrus. Moreover, we also saw that hormonal replacement did not reverse these effects, suggesting that this treatment does not restore the ventilatory responses of an intact animal.
100 80 60 0%0 CO2
7%30CO2
˙ E), tidal volume (VT), and respiratory frequency (fR) of Fig. 3. Ventilation (V the rats in proestrus, estrus, metestrus, and diestrus during normocapnia (0% CO2) and hypercapnia (7% CO2). Values are means ⫾ SE; n, no. of rats.
observed between the groups under hypercapnic conditions [hypercapnia effect: P ⫽ 0.9756; F(3,22) ⫽ 0.00095, no inter˙ O2 during action], and hormone replacement did not affect V CO2 exposure (Fig. 7). ˙ E/V ˙ O2 was not different between groups during normocapV ˙ E/V ˙ O2 in all groups nia (Fig. 7). Hypercapnia increased V [hypercapnia effect: P ⬍ 0.001, F(3,22) ⫽ 69.4, interaction P ⫽ 0.0083; F(3,22) ⫽ 5.4] (Fig. 7); however, the effect was greater ˙ E/V ˙ O2 of the estrus group, compared with OVX groups in V [hormone effect: P ⫽ 0.0059, F(3,22) ⫽ 5.9, interaction P ⫽ 0.0083; F(3,22) ⫽ 5.4]. Tb. Tb of rats on estrus was higher than that of the OVX groups during normocapnia [hormone effect: P ⫽ 0.0070, F(3,22) ⫽ 4.8, no interaction], but not under hypercapnia (Fig. 8). Exposure to hypercapnia caused a drop in Tb in all groups, except for the OVX⫹E2 animals [hypercapnia effect: P ⫽ 0.028, F(3,19) ⫽ 5.8, no interaction] (Fig. 8).
Proestrus (n=8) Estrus (n=6) Metaestrus (n=4) Diestrus (n=4)
39
Tb (°C)
fR (breaths.min -1)
Proestrus (n=8) Estrus (n=6) Metaestrus (n=4) Diestrus (n=4)
25
1400
.
Marques DA et al.
VO2 (mL.Kg-1.min-1)
VE (mL Kg-1min-1)
3500
•
38
37
36
0%0CO2
30 2 7% CO
Fig. 5. Body temperature (Tb) of rats in proestrus, estrus, metestrus, and diestrus during normocapnia (0% CO2) and hypercapnia (7% CO2). Values are means ⫾ SE; n, no. of rats.
J Appl Physiol • doi:10.1152/japplphysiol.00254.2015 • www.jappl.org
˙ E, V ˙ O2, and Tb Hormonal Influence on V
OVX+O (n=6) OVX+E2 (n=6) OVX+E2 P (n=5) Estrus (n=6)
2800 2100
*
1400
.
700 0
65
Marques DA et al.
OVX+O (n=6) OVX+E2 (n=6) OVX+E2 P (n=5) Estrus (n=6)
25 VO2 (mL.Kg-1.min-1)
VE (mL Kg -1min-1)
3500
•
.
20 15 10 5
*
20
300
15
250
10
200
VE/VO2
VT (mLKg -1)
25
. .
5 0
150 100 50 0
180 fR (breaths.min -1)
*
160
0%CO2
140
7% CO2
˙ O2 and V ˙ E/V ˙ O2 of intact rats in estrus, and OVX⫹O, OVX⫹E2, and Fig. 7. V OVX⫹E2P rats during normocapnia (0% CO2) and hypercapnia (7% CO2). Values are means ⫾ SE; n, no. of rats. *Difference between estrus group compared with OVX⫹O, OVX⫹E2, and OVX⫹E2P groups (P ⬍ 0.05).
120 100 80 60 0%CO 2
7% CO 2
˙ E, VT, and fR of intact rats in estrus, ovariectomized (OVX) rats with Fig. 6. V corn oil replacement (OVX⫹O), OVX rats replaced with E2 (OVX⫹E2), and OVX rats replaced with a combination of E2 and P (OVX⫹E2P) during normocapnia (0% CO2) and hypercapnia (7% CO2). Values are means ⫾ SE; n, no. of rats. *Difference between estrus group compared with OVX⫹O, OVX⫹E2, and OVX⫹E2P groups (P ⬍ 0.05).
possibly due to augmented levels of P. Hayashi et al. (22) ˙ E and VT in the luteal phase, observed higher values for basal V when comparing it with the follicular phase. ˙ O2 levels were observed under In this study, no changes in V normocapnia in the different phases of the estrous cycle, suggesting that the hormonal fluctuations that occur during the cycle have a small effect (if any) in the metabolic rate under the present conditions. This has also been observed in women during the menstrual cycle (42, 45). Regarding Tb, our results show an increase on the day of estrus, similar to that previously
Estrous Cycle and V˙E, Metabolism, and Tb
OVX+O (n=6) OVX+E2 (n=6) OVX+E2 P (n=5) Estrus (n=6)
39
38 Tb (°C)
˙ E in women is higher Many studies have demonstrated that V during the luteal than the follicular phase (22, 35, 41), and also ˙ E (40, that the administration of P promotes an increase in V ˙ E is related to an 52), which suggests that this increase in V ˙E augment of P levels. In the present study, no difference in V ˙ and VO2 was observed in the different groups under normocapnic conditions, which suggests that hormonal changes during estrous cycle were not sufficient to promote alteration in ˙ E and metabolism. In this regard, Brack et al. (7) resting V performed experiments using anesthetized cycling female rats and reported no differences in respiratory rates in different ˙ E were not stages of the estrous cycle, although VT and V measured. In addition, a previous study demonstrated that phrenic nerve activity is not estrous cycle dependent (51). ˙ E is not Similarly, White et al. (48) showed that resting V different between follicular and luteal phases in women; however, VT and end-tidal PCO2 is higher in the luteal phase,
37
36 0% CO2
7% CO2
Fig. 8. Tb of intact rats in estrus, and OVX⫹O, OVX⫹E2, and OVX⫹E2P rats during normocapnia (0% CO2) and hypercapnia (7% CO2). Values are means ⫾ SE; n, no. of rats.
J Appl Physiol • doi:10.1152/japplphysiol.00254.2015 • www.jappl.org
66
˙ E, V ˙ O2, and Tb Hormonal Influence on V
reported (23, 50). During estrus, Tb was ⬃0.6°C higher than in other phases. In women, Tb can increase up to 0.4°C after ovulation, whereas in rodents there is an elevation in Tb immediately before, rather than after presumed ovulation (33). Despite the fact that Tb was higher during estrus in this study, ˙ E/V ˙ O2 of cycling rats did not vary in different metabolism and V phases of the cycle. We found no difference in ventilatory responses to CO2 during the different stages of the estrous cycle. In women, there is a controversy in the literature about the influence of menstrual cycle in the CO2 drive to breathe. Some studies have demonstrated that alterations of sex hormones during the menstrual cycle have little or no effect on CO2 sensitivity (48), while others have reported that, during the luteal phase, the hypercapnic ventilatory response is more pronounced than that in the follicular phase (16, 35). Shoene et al. (35) suggest that the increased ventilatory response during the luteal phase is related to the higher P concentration. This contradiction may reflect differences in study design and on the data analysis. Additionally, Preston et al. (32) demonstrated that CO2 sensitivity was not different between pre- and postmenopausal women. In anesthetized rats, Zabka et al. (51) demonstrated that the short-term ventilatory response to hypoxia is similar across estrous phases. The present data show that the estrous cycle of rats does not interfere with the ventilatory responses to CO2 in females with a regular cycle. Therefore, our findings, taken in conjunction with those of others, indicate that normally occurring alterations in sex hormones have very little or no effect on the sensitivity to CO2. ˙ O2 during hypercapnic exposure We observed a decrease in V in all phases of the estrous cycle. This was also observed in male rats during 5% CO2 exposure (36). However, CO2 exposure only decreased Tb of rats in estrus and proestrus. In many species, a decrease in Tb is observed during hypercapnia, including rats (19). Nevertheless, previous studies from our laboratory did not observe a Tb alteration during CO2 challenge in male Wistar rats (6, 13, 14, 31). Ovariectomy, Hormonal Replacement, V˙E, Metabolism, and Tb ˙ E were found during In the present study, no differences in V normocapnia and hypercapnia between spayed females treated with corn oil, E2, or P. It has long been recognized that E2 participates in the respiratory adjustments observed during late gestation (9, 29). Nevertheless, the administration of E2 alone ˙ E in male rats (34). Some evidence indicates a did not affect V ˙ E (37). In fact, as synergistic effect of estrogen and P on V estrogen upregulates P receptors (24), this mechanism is likely involved with the respiratory effects of P. E2 and P receptors (ER-␣, ER-; PR-A, PR-B) were identified in many respiratory regions of the brain, including the nucleus of the solitary tract (38, 39), the hypoglossal nuclei (4), and the motor nuclei of the phrenic nerve (4). In fact, some studies have reported an ˙ E (10, 44, inhibitory effect of E2 in some areas important for V 46, 49). Also, studies in OVX rats (8) and castrated cats (20, 21) have demonstrated that administration of P combined with ˙ E and hypercapestrogen promotes an increase in pulmonary V nic ventilatory response through receptor-mediated mechanisms of action involving central and peripheral sites. The fact ˙ E might be related to deficienthat we found no difference in V
•
Marques DA et al.
cies in the hormonal replacement protocol. We choose not to use P treatment without E2, since it is well known that P sensitivity is highly dependent on prior or concurrent estrogen exposure, once estrogens induce P receptor expression in the brain (4, 26, 30). Interestingly, our results demonstrate that OVX rats that underwent hormonal replacement showed a ventilatory response to hypercapnia 43% lower compared with female rats on estrus. This comparison was made because lower levels of sexual hormones are observed during estrus. It is interesting to note that hormonal replacement is used in the literature for several purposes (3). However, at least in the perspective of respiratory control, this approach has its limitations, since hormonal replacement did not revert the effects of ovariectomy in the ventilatory responses to hypercapnia. In this context, when studying female rats, it is important to consider the possibility that replacing hormones using the present protocol might not perfectly mimic the conditions of an intact cycling animal, despite the fact that said protocol yielded physiological plasmatic levels of E2 and P, according to Szawka et al. (44). What is more, OVX⫹E2 and OVX⫹E2P rats presented plasmatic levels of E2 higher than those in OVX⫹O rats. In the present work, we show that hormonal replacement did not restore the ventilatory, metabolic, and thermal variables studied. Even though E2 and P are the main female gonadal ˙ E, hormones, other gonadal hormones might play a role in V metabolism, and temperature. Gonadal hormones include sexual steroids and androgens, and also peptide hormones, such as anti-Müllerian hormone, activin, inhibin, and follistatin, which might be involved. However, there are no studies in the literature reporting the involvement of these hormones. Humans are able to easily manipulate the levels of their sexual hormones (with oral contraceptives and steroids), but the respiratory consequences of this are not fully understood (3). In conclusion, despite the hormonal fluctuations during the estrous cycle, the ventilatory responses to hypercapnia in female rats were similar along the cycle, but OVX rats presented a reduced ventilatory response to CO2. Our data demonstrate that hormonal replacement with E2 or P (the main ˙ E under the present sexual hormones) did not alter the V experimental condition, suggesting that other gonadal factors may be involved in these responses. This study contributes to the understanding of the respiratory and metabolic responses to CO2 exposure in female conscious rats, being an important complement to the existing literature. ACKNOWLEDGMENTS We thank Euclides Roberto Secato and Ruither O. G. Carolino for excellent technical assistance. GRANTS This work was supported by Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP; 2012/19966-0), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; 442560/2014-1), INCT-Fisiologia Comparada. D. A. Marques was the recipient of a CNPq scholarship. G. S. F. da Silva was supported by the Young Investigator Award (FAPESP; 2013/17606-9). DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). AUTHOR CONTRIBUTIONS Author contributions: D.A.M., D.d.C., R.E.S., J.A.A.F., K.C.B., and L.H.G. conception and design of research; D.A.M. and D.d.C. performed experiments;
J Appl Physiol • doi:10.1152/japplphysiol.00254.2015 • www.jappl.org
˙ E, V ˙ O2, and Tb Hormonal Influence on V D.A.M. and D.d.C. analyzed data; D.A.M., G.S.F.d.S., R.E.S., K.C.B., and L.H.G. interpreted results of experiments; D.A.M. prepared figures; D.A.M. and L.H.G. drafted manuscript; D.A.M., D.d.C., G.S.F.d.S., R.E.S., J.A.A.F., K.C.B., and L.H.G. edited and revised manuscript; D.A.M., D.d.C., G.S.F.d.S., R.E.S., J.A.A.F., K.C.B., and L.H.G. approved final version of manuscript. REFERENCES 1. Almeida MC, Steiner AA, Coimbra NC, Branco LG. Thermoeffector neuronal pathways in fever: a study in rats showing a new role of the locus coeruleus. J Physiol 558: 283–294, 2004. 2. Bartlett D Jr, Tenney SM. Control of breathing in experimental anemia. Respir Physiol 10: 384 –395, 1970. 3. Behan M, Kinkead R. Neuronal control of breathing: sex and stress hormones. Compr Physiol 1: 2101–2139, 2011. 4. Behan M, Thomas CF. Sex hormone receptors are expressed in identified respiratory motoneurons in male and female rats. Neuroscience 130: 725–734, 2005. 5. Behan M, Zabka AG, Thomas CF, Mitchell GS. Sex steroid hormones and the neural control of breathing. Respir Physiol Neurobiol 136: 249 – 263, 2003. 6. Biancardi V, Bicego KC, Almeida MC, Gargaglioni LH. Locus coeruleus noradrenergic neurons and CO2 drive to breathing. Pflügers Arch 455: 1119 –1128, 2008. 7. Brack KE, Jeffery SM, Lovick TA. Cardiovascular and respiratory responses to a panicogenic agent in anaesthetised female Wistar rats at different stages of the oestrous cycle. Eur J Neurosci 23: 3309 –3318, 2006. 8. Brodeur P, Mockus M, McCullough R, Moore LG. Progesterone receptors and ventilatory stimulation by progestin. J Appl Physiol 60: 590 –595, 1986. 9. Brownell LG, West P, Kryger MH. Breathing during sleep in normal pregnant women. Am Rev Respir Dis 133: 38 –41, 1986. 10. Cason AM, Kwon B, Smith JC, Houpt TA. c-Fos induction by a 14T magnetic field in visceral and vestibular relays of the female rat brainstem is modulated by estradiol. Brain Res 1347: 48 –57, 2010. 11. Das TK, Jana H. Basal oxygen consumption during different phases of menstrual cycle. Indian J Med Res 94: 16 –19, 1991. 12. de Carvalho D, Bicego KC, de Castro OW, da Silva GS, GarciaCairasco N, Gargaglioni LH. Role of neurokinin-1 expressing neurons in the locus coeruleus on ventilatory and cardiovascular responses to hypercapnia. Respir Physiol Neurobiol 172: 24 –31, 2010. 13. Dempsey JA, Olsen E, Burt JR, Skatrud JB. Hormones and neurochemicals in the regulation of breathing. In: Handbook of Physiology. The Respiratory System. Control of Breathing. Bethesda, MD: Am. Physiol. Soc., 1986, sect. 3, vol. II, pt. 1, chapt. 7, p. 181–221. 14. de Souza Moreno V, Bícego KC, Szawka RE, Anselmo-Franci JA, Gargaglioni LH. Serotonergic mechanisms on breathing modulation in the rat locus coeruleus. Pflügers Arch 459: 357–368, 2010. 15. Dias MB, Nucci TB, Margatho LO, Antunes-Rodrigues J, Gargaglioni LH, Branco LG. Raphe magnus nucleus is involved in ventilatory but not hypothermic response to CO2. J Appl Physiol 103: 1780 –1788, 2007. 16. Dutton K, Blanksby BA, Morton AR. CO2 sensitivity changes during the menstrual cycle. J Appl Physiol 67: 517–522, 1989. 17. Findlay JK. An update on the roles of inhibin, activin, and follistatin as local regulators of folliculogenesis. Biol Reprod 48: 15–23, 1993. 18. Freeman ME. The neuroendocrine control of the ovarian cycle of the rat. In: Knobil and Neill’s Physiology of Reproduction, edited by Neill JD. Philadelphia, PA: Elsevier, 2006, vol. 2, p. 2327–2388. 19. Gautier H, Bonora M, Trinh HC. Ventilatory and metabolic responses to cold and CO2 in intact and carotid body-denervated awake rats. J Appl Physiol 75: 2570 –2579, 1993. 20. Hannhart B, Pickett CK, Weil JV, Moore LG. Influence of pregnancy on ventilatory and carotid body neural output responsiveness to hypoxia in cats. J Appl Physiol 67: 797–803, 1989. 21. Hannhart B, Pickett CK, Moore LG. Effects of estrogen and progesterone on carotid body neural output responsiveness to hypoxia. J Appl Physiol 68: 1909 –1916, 1990. 22. Hayashi K, Kawashima T, Suzuki Y. Effect of menstrual cycle phase on the ventilatory response to rising body temperature during exercise. J Appl Physiol 113: 237–245, 2012. 23. Kent S, Hurd M, Satinoff E. Interactions between body temperature and wheel running over the estrous cycle in rats. Physiol Behav 49: 1079 – 1084, 1991.
•
Marques DA et al.
67
24. Leavitt WW, Blaha GC. An estrogen-stimulated, progesterone-binding system in the hamster uterus and vagina. Steroids 19: 263–274, 1972. 25. Lopes LT, Biancardi V, Vieira EB, Leite-Panissi C, Bícego KC, Gargaglioni LH. Participation of the dorsal periaqueductal grey matter in the hypoxic ventilatory response in unanaesthetized rats. Acta Physiol (Oxf) 211: 528 –537, 2014. 26. Maclusky NJ, Mcewen BS. Progestin receptors in rat brain: distribution and properties of cytoplasmic progestin-binding sites. Endocrinology 106: 192–202, 1980. 27. Markowska AL, Savonenko AV. Effectiveness of estrogen replacement in restoration of cognitive function after long-term estrogen withdrawal in aging rats. J Neurosci 22: 10985–10995, 2002. 28. McLean JH, Coleman WP. Temperature variation during the estrous cycle: active vs. restricted rats. Psychon Sci 22: 179 –180, 1971. 29. Moore LG, McCullough RE, Weil JV. Increased HVR in pregnancy: relationship to hormonal and metabolic changes. J Appl Physiol 62: 158 –163, 1987. 30. Parsons B, Mcewen BS, Pfaff DW. A discontinuous schedule of estradiol treatment is sufficient to activate progesterone-facilitated feminine sexual behavior and to increase cytosol receptors for progestins in the hypothalamus of the rat. Endocrinology 110: 613–619, 1982. 31. Patrone LG, Bícego KC, Hartzler LK, Putnam RW, Gargaglioni LH. Cardiorespiratory effects of gap junction blockade in the locus coeruleus in unanesthetized adult rats. Respir Physiol Neurobiol 190: 86 –95, 2014. 32. Preston ME, Jensen D, Janssen I, Fisher JT. Effect of menopause on the chemical control of breathing and its relationship with acid-base status. Am J Physiol Regul Integr Comp Physiol 296: R722–R727, 2009. 33. Refinetti R, Menaker M. The circadian rhythm of body temperature. Physiol Behav 51: 613–637, 1992. 34. Regensteiner JG, Woodard WD, Hagerman DD, Weil JV, Pickett CK, Bender PR, Moore LG. Combined effects of female hormones and metabolic rate on ventilatory drives in women. J Appl Physiol 66: 808 –813, 1989. 35. Schoene RB, Robertson HT, Pierson DJ, Peterson AP. Respiratory drives and exercise in menstrual cycles of athletic and nonathletic women. J Appl Physiol 50: 1300 –1305, 1981. 36. Seifert EL, Mortola JP. Circadian pattern of ventilation during acute and chronic hypercapnia in conscious adult rats. Am J Physiol Regul Integr Comp Physiol 282: R244 –R251, 2002. 37. Shahar E, Redline S, Young T, Boland LL, Baldwin CM, Nieto FJ, O’Connor GT, Rapoport DM, Robbins JA. Hormone replacement therapy and sleep-disordered breathing. Am J Respir Crit Care Med 167: 1186 –1192, 2003. 38. Shughrue PJ, Lane MV, Merchenthaler I. Comparative distribution of estrogen receptor-alpha and -beta mRNA in the rat central nervous system. J Comp Neurol 388: 507–525, 1997. 39. Simerly RB, Chang C, Muramatsu M, Swanson LW. Distribution of androgen and estrogen receptor mRNA-containing cells in the rat brain: an in situ hybridization study. J Comp Neurol 294: 76 –95, 1990. 40. Skatrud JB, Dempsey JA, Kaiser DG. Ventilatory response to medroxyprogesterone acetate in normal subjects: time course and mechanism. J Appl Physiol 44: 393–344, 1978. 41. Slatkovska L, Jensen D, Davies GA, Wolfe LA. Phasic menstrual cycle effects on the control of breathing in healthy women. Respir Physiol Neurobiol 154: 379 –388, 2006. 42. Smekal G, Von Duvillard SP, Frigo P, Tegelhofer T, Pokan R, Hofmann P, Tschan H, Baron R, Wonisch M, Renezeder K, Bachl N. Menstrual cycle: no effect on exercise cardiorespiratory variables or blood lactate concentration. Med Sci Sports Exerc 39: 1098 –1106, 2007. 43. Smith MS, Freeman ME, Neill JD. The control of progesterone secretion during the estrous cycle and early pseudopregnancy in the rat: prolactin, gonadotropin and steroid levels associated with rescue of the corpus luteum of pseudopregnancy. Endocrinology 96: 219 –226, 1975. 44. Szawka RE, Rodovalho GV, Monteiro PM, Carrer HF, AnselmoFranci JA. Ovarian-steroid modulation of locus coeruleus activity in female rats: involvement in luteinising hormone regulation. J Neuroendocrinol 21: 629 – 639, 2009. 45. Takase K, Nishiyasu T, Asano K. Modulating effects of the menstrual cycle on cardiorespiratory responses to exercise under acute hypobaric hypoxia. Jpn J Physiol 52: 553–560, 2002. 46. Ueyama T, Tanioku T, Nuta J, Kujira K, Ito T, Nakai S, Tsuruo Y. Estrogen alters c-Fos response to immobilization stress in the brain of ovariectomized rats. Brain Res 1084: 67–79, 2006.
J Appl Physiol • doi:10.1152/japplphysiol.00254.2015 • www.jappl.org
68
˙ E, V ˙ O2, and Tb Hormonal Influence on V
47. Wenninger JM, Olson EB Jr, Cotter CJ, Thomas CF, Behan M. Hypoxic and hypercapnic ventilatory responses in aging male vs. aging female rats. J Appl Physiol 106: 1522–1528, 2009. 48. White DP, Douglas NJ, Pickett CK, Zwillich CW, Weil JV. Sleep deprivation and the control of ventilation. Am Rev Respir Dis 128: 984 –986, 1983. 49. Xue B, Hay M. 17-Estradiol inhibits excitatory amino acid-induced activity of neurons of the nucleus tractus solitarius. Brain Res 976: 41–52, 2003.
•
Marques DA et al.
50. Yanase M, Tanaka H, Nakayama T. Effects of estrus cycle on thermoregulatory responses during exercise in rats. Eur J Appl Physiol Occup Physiol 58: 446 –451, 1989. 51. Zabka AG, Behan M, Mitchell GS. Selected contribution: time-dependent hypoxic respiratory responses in female rats are influenced by age and by the estrus cycle. J Appl Physiol 91: 2831–2838, 2001. 52. Zwillich CW, Natalino MR, Sutton FD, Weil JV. Effects of progesterone on chemosensitivity in normal men. J Lab Clin Med 92: 262–269, 1978.
J Appl Physiol • doi:10.1152/japplphysiol.00254.2015 • www.jappl.org
Ventilatory, metabolic, and thermal responses to hypercapnia in female rats: effects of estrous cycle, ovariectomy, and hormonal replacement Danuzia A. Marques,1 Débora de Carvalho,2 Glauber S. F. da Silva,1 Raphael E. Szawka,3 Janete A. Anselmo-Franci,4 Kênia C. Bícego,1 and Luciane H. Gargaglioni1 1
Department of Animal Morphology and Physiology, São Paulo State University, UNESP FCAV at Jaboticabal, São Paulo, Brazil; 2Federal Institute of Para, Para, Brazil; 3Department of Physiology and Biophysics, Institute of Biological Sciences, Federal University of Minas Gerais, Minas Gerais, Brazil; and 4Department of Morphology, Stomatology and Physiology, Dental School of Ribeirao Preto, University of São Paulo, São Paulo, Brazil Submitted 25 March 2015; accepted in final form 23 April 2015
Marques DA, de Carvalho D, da Silva GS, Szawka RE, AnselmoFranci JA, Bícego KC, Gargaglioni LH. Ventilatory, metabolic, and thermal responses to hypercapnia in female rats: effects of estrous cycle, ovariectomy, and hormonal replacement. J Appl Physiol 119: 61–68, 2015. First published April 30, 2015; doi:10.1152/japplphysiol.00254.2015.—The aim of this study was to examine how estrous cycle, ovariectomy, and ˙ E), tidal hormonal replacement affect the respiratory [ventilation (V ˙ O2), and thermoregvolume, and respiratory frequency], metabolic (V ulatory (body temperature) responses to hypercapnia (7% CO2) in female Wistar rats. The parameters were measured in rats during different phases of the estrous cycle, and also in ovariectomized (OVX) rats supplemented with 17-estradiol (OVX⫹E2), with a combination of E2 and progesterone (OVX⫹E2P), or with corn oil (OVX⫹O, vehicle). All experiments were conducted on day 8 after ovariectomy. The intact animals did not present alterations during ˙ E, tidal volume, respiratory normocapnia or under hypercapnia in V ˙ O2, and V ˙ E/V ˙ O2 in the different phases of the estrous frequency, V cycle. However, body temperature was higher in female rats on estrus. Hormonal replacement did not change the ventilatory, thermoregulatory, or metabolic parameters during hypercapnia, compared with the OVX animals. Nevertheless, OVX⫹E2, OVX⫹E2P, and OVX⫹O presented lower hypercapnic ventilatory responses compared with intact females on the day of estrus. Also, rats in estrus showed higher ˙ E and V ˙ E/V ˙ O2 during hypercapnia than OVX animals. The data V suggest that other gonadal factors, besides E2 and P, are possibly involved in these responses. CO2; estrogen; progesterone; cycling rats; breathing SEX HORMONES, INCLUDING
androgens, estrogens, such as 17estradiol (E2), and progestogens, such as progesterone (P), are present in both males and females, although marked differences in the plasma levels occurs throughout the lifespan of the animals (3). In female rats, E2 levels are low on estrus, elevated on metestrus and diestrus, and reach a peak during the proestrus, falling again as the next cycle starts (18). On the other hand, levels of P increase between the night of metestrus and early morning of diestrus, and fall thereafter (18). Then, on the afternoon of proestrus, P levels increase again, reaching a second peak, and then return to baseline levels on estrus (43). These hormones affect neuromodulatory systems that influence the central respiratory network (3), acting directly on respiratory motor or adjacent neurons (5, 13), brain stem Address for reprint requests and other correspondence: L. H. Gargaglioni, 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 14884-900, Brazil (e-mail: [email protected]). http://www.jappl.org
premotor neurons (13), rhythm generating neurons (3). They can also influence peripheral or central chemoreceptors (5), which suggests an important hormonal role in the breathing control (3). Most of the experiments in respiratory control, however, are performed in male rats. When females are used, the studies do not consider the natural hormonal oscillations that occur during the estrous cycle. In women, studies demonstrated that the ventilatory responses to hypoxia and hypercapnia vary in the different phases of the menstrual cycle (34, 48). For example, the ˙ E) and tidal volume (VT) are increased in the ventilation (V luteal phase, compared with the follicular phase (13, 35, 41), and consequently arterial partial pressure of CO2 is reduced. Additionally, central and peripheral chemosensitivity are increased in luteal phase (16, 35, 48), and administration of medroxyprogesterone acetate, a synthetic variant of P, stimulates the ventilatory system (40, 52), suggesting that the increased ventilatory response during the luteal phase is related to the elevated plasmatic concentrations of P. Wenninger et al. (47) reported that female rats present a ˙ E/V ˙ O2 compared with male rats. Variasignificantly higher V ˙ O2 also occur during tions in the body temperature (Tb) and V the menstrual and estrous cycle (11, 28, 33), but this is still a poorly explored issue. The hormonal variations in different stages of the estrous cycle may present additional challenges to data collection and interpretation. To the best of our knowledge, no study has examined the influence of the estrous cycle on ventilatory, metabolic, and thermoregulatory parameters in normocapnic and hypercapnic conditions in rodents. We hypothesized that hormonal fluctuations observed during the estrous cycle have an impact on ˙ E, V ˙ O2, and Tb. We also assessed the possible role of E2 basal V ˙ E, V ˙ O2, and P in those influences. To this end, we measured V and Tb in intact, as well as ovariectomized (OVX) without replacement and OVX with E2 and P replacement female rats. MATERIALS AND METHODS
Animals Experiments were performed in unanesthetized adult female Wistar rats weighting 250 –300 g. The age of the animals ranged from 90 to 120 days. The animals were fed ad libitum and housed in a temperature-controlled chamber, maintained at 24 –26°C (ALE 9902001; Alesco, Monte Mor, Brazil), with a 12:12-h light-dark cycle (lights on at 6:00 AM). All experimental procedures were in compliance with the Brazilian College of Animal Experimentation guidelines and approved by the local Animal Care and Use Committee (protocol no. 007827-09).
8750-7587/15 Copyright © 2015 the American Physiological Society
61
˙ E, V ˙ O2, and Tb Hormonal Influence on V
62
We used cycling female rats on the days of proestrus (n ⫽ 8), estrus (n ⫽ 6), metestrus (n ⫽ 4), and diestrus (n ⫽ 4), and OVX rats treated with corn oil (OVX⫹O; n ⫽ 6), E2 (OVX⫹E2; n ⫽ 6), or E2 and P (OVX⫹E2P; n ⫽ 5). We used 39 animals in total. Ventilatory, Tb, and Metabolic Measurements ˙ E measurements were obtained using the barometric method The V (whole body plethysmography) (2), as previously described by Biancardi et al. (6), de Carvalho et al. (12), and Lopes et al. (25). Tb was recorded by the use of a temperature datalogger (SubCue, Calgary, AB, Canada) implanted in the abdominal cavity, as previously described in the literature (6). The datalogger was programmed to acquire data every 5 min. ˙ O2) The metabolic rate was measured by indirect calorimetry (V using a closed respirometry system, as described by Almeida et al. (1). At the end of the normocapnic and hypercapnic periods, the airflow in the chamber was interrupted for 2 min, and the air was continuously sampled by an O2 analyzer (PowerLab System. ADInstruments/Chart Software, version 7.3, Sydney, Australia). While the chamber was sealed, the oxygen fraction did not drop to ⬍19%, and CO2 did not increase ⬎0.7%. The percentage of oxygen decay inside the chamber was plotted against time, and the slope of the resulting curve corre˙ O2. The values are presented in milliliters per sponded to the rate of V minute per kilogram in STPD (standard conditions of temperature, pressure, and dry air).
•
Marques DA et al.
h later. The regimens of hormonal treatment used yielded physiological levels of plasma E2 and P, as described by Szawka et al. (44). On the day of experiment, vaginal cytology was performed to monitor the effectiveness of the hormonal treatment. Animals in which hormone replacement was effective presented cornified epithelial cells in the vaginal cytology (27). In contrast, when the hormonal replacement was not efficient, the smears displayed predominance of small leukocytes, similarly as found in the cytology of OVX⫹O rats, and the animals were not used in the experiments. Experimental Protocol Each animal was individually placed in a plethysmograph chamber (5 liters), kept at 25°C and allowed to move freely, while the chamber was flushed with humidified room air. The baseline ˙ E and V ˙ O2 were taken after animals acclimameasurements of V tized for ⬃1 h. Subsequently, a hypercapnic gas mixture (7% CO2 in air, White Martins, Sertãozinho, São Paulo, Brazil) was flushed ˙ E was measured at 30 min. through the chamber for 30 min, and V ˙ E was calculated before (time zero) and 30 min after hypercapnia V or normocapnia. The percentage of CO2 and the exposure time were chosen based on pilot experiments and previous studies (6, ˙ O2 was measured once again after the stimuli. The 15). The V experiments were carried out from 900 to 1300. Tb was continuously measured during the experiment.
Vaginal Smears
Statistical Analysis
The animals were subjected to daily vaginal lavages to monitor the progression through the estrous cycle. Vaginal smears were performed daily at 9:00 AM to verify estrous cycle regularity. Only females exhibiting at least four consecutive regular cycles were used in the experiments. The estrous cycle stage was determined using the criteria used by Freeman (18).
Statistical analysis of the blood hormone concentration data was performed by one-way ANOVA followed by Tukey post hoc ˙ E, respiratory frequency (fR), VT, Tb, and V ˙ O2 data were testing. V compared using repeated two-way ANOVA. When interactions between the factors were observed, groups were compared using Bonferroni’s post hoc test. The significance level was set to P ⬍ 0.05. The statistical analysis was performed using computer software (SigmaStat; Systat Software, Point Richmond, CA). Data are presented as group means ⫾ SE for each parameter investigated.
Blood Collection for Plasma Hormone Assays After the hypercapnic challenge, and just before euthanasia, a blood sample of ⬃1 ml was collected from the heart of anesthetized rats into heparinized syringes. The plasma was separated by centrifugation at 3,000 rpm for 20 min at 4°C and stored at ⫺20°C for posterior quantification of P and E2 by radioimmunoassay. Plasma P and E2 concentrations were determined by double-antibody radioimmunoassay with MAIA kits provided by Biochem Immunosystem (Bologna, Italy). The lower limits of detection for E2 and P were 5.0 pg/ml and 0.02 ng/ml, respectively. Ovariectomy and Hormonal Replacement Before the ovariectomy surgery, animals were anesthetized, via intraperitoneal injection of 100 mg/kg of ketamine (Union National Pharmaceutical Chemistry S/A, Embu-Guaçu, São Paulo, Brazil) and 10 mg/kg of xylazine (Laboratories Calier S/A, Barcelona, Spain). A temperature datalogger (SubCue, Calgary, AB, Canada) was also implanted in the abdominal cavity through a midline laparotomy. After this procedure, bilateral ovariectomies were performed using an incision 1.5 cm inferior to the palpated rib cage. Ovaries and surrounding fat tissue were removed; the incision was closed by suturing the muscles and stapling the skin. After surgery, animals were treated with two doses of enrofloxacin (10 mg/kg intramuscular) and flunixin meglumine [2.5 mg/kg subcutaneous (SC)] to prevent infection and postsurgical discomfort, respectively. Eight days after ovariectomy, rats were treated with corn oil (0.2 ml SC) or E2 (10 g/0.2 ml SC, 17-estradiol cypionate; Pfizer, São Paulo, Brazil), at 9 AM for 3 days. On the 4th day, oil-treated rats received a final oil injection (OVX⫹O group), while E2-treated rats received an injection containing corn oil (OVX⫹E2 group) or P (2.5 mg/0.2 ml SC, OVX⫹E2P group; Sigma, St. Louis, MO), and the experiments were performed 2
RESULTS
Effects of Estrous Cycle Stages on V˙E, V˙O2, and Tb Vaginal cytology. Figure 1 shows the cytological observations characteristic of specific stages of the estrous cycle in cycling females. Proestrous vaginal smears showed nucleated epithelial cells (Fig. 1A), whereas estrous smears present densely stained cornified cells (Fig. 1B). Metestrus was characterized by the observation of lightly stained cornified cells in addition to some leukocytes and nucleated cells (Fig. 1C). Finally, diestrous smears showed presence of leukocytes (Fig. 1D). Hormonal profile. Figure 2 shows plasma E2 and P concentrations in cycling female rats. Plasmatic concentration of E2 was significantly higher in proestrus than in estrus, metestrus, and diestrus. The plasma concentration of P was lower on estrus compared with proestrus, metestrus, and diestrus. Pulmonary V˙E and metabolism. In normocapnic conditions, ˙ E were observed in different phases of the no differences in V ˙ E in estrous cycle (Fig. 3). Hypercapnia caused an increase in V all groups [hypercapnia effect: P ⬍ 0.0001; F(3,22) ⫽ 131.7; no interaction] due to an increase in VT [hypercapnia effect: P ⬍ 0.0001; F(3,22)⫽ 84.6; no interaction] and fR [hypercapnia effect: P ⬍ 0.0001; F(3,22) ⫽ 301.1; no interaction]. There was no difference in the hypercapnic ventilatory response between
J Appl Physiol • doi:10.1152/japplphysiol.00254.2015 • www.jappl.org
˙ E, V ˙ O2, and Tb Hormonal Influence on V
•
63
Marques DA et al.
Fig. 1. Evaluation of the stage of the estrous cycle in cycling females. Representative microphotographs are shown of vaginal cytological samples taken from a female rat during proestrus (A), estrus (B), metestrus (C), and diestrus (D).
groups in the different phases of the estrous cycle, as seen in Fig. 3. ˙ O2 during hypercapnia [hypercapAll groups had lower V nia effect: P ⫽ 0.0127; F(3,22) ⫽ 6.8; no interaction], and no differences were observed between the groups in different
A
Proestrus (n=8) Estrus (n=6) Metaestrus (n=4) Diestrus (n=4)
E2 concentration (pg/mL)
250 200
*
150 100 50
40 30 (ng/mL)
B P concentration
0
20 10
*
0 Fig. 2. Changes in plasma concentration of 17-estradiol (E2; A) and progesterone (P; B) during the different phases of the estrous cycle. Values are means ⫾ SE; n, no. of rats. *Difference between estrus group compared with proestrus, metestrus, and diestrus (P ⬍ 0.05).
stages of the estrous cycle. Hypercapnia caused a significant ˙ E/V ˙ O2 [hypercapnia effect: P ⬍ 0.001; increase in the V F(3,22) ⫽ 85.6; no interaction], and, again, no difference was found between groups (Fig. 4). Tb. As seen in Fig. 5, Tb of rats on estrus was higher than that of rats on other stages of the estrous cycle, during both normocapnia and hypercapnia [cycle effect: P ⬍ 0.001; F(3,22) ⫽ 7.8; no interaction]. Hypercapnia caused a decrease in Tb only in estrous and proestrous rats [hypercapnia effect: P ⫽ 0.0003; F(3,22)⫽ 20.2; no interaction]. Effects of Ovariectomy and Hormonal Replacement on V˙E, V˙O2, and Tb Pulmonary V˙E and metabolism. There was no difference in the ventilatory parameters between groups during normo˙ E in all capnia (Fig. 6). Hypercapnia induced an increase in V groups [hypercapnia effect: P ⬍ 0.0001, F(3,19) ⫽ 160.7, interaction: P ⬍ 0.0001; F(3,19) ⫽ 17.1], which was a result of an increase in VT [hypercapnia effect: P ⬍ 0.0001, F(3,19) ⫽ 85.6, interaction: P ⫽ 0.0064; F(3,19) ⫽ 4.8] and fR [hypercapnia effect: P ⬍ 0.001, F(3,19) ⫽ 94.8, no interaction]. OVX animals (OVX⫹O, OVX⫹E2, and OVX⫹E2P) showed an attenuated hypercapnic ventilatory response (43% lower for all groups) compared with rats on estrus [hormone effect: P ⬍ 0.0001, F(3,19) ⫽ 30.1; interaction: P ⬍ 0.0001; F(3,19) ⫽ 17.1] due to lower VT [hormone effect: P ⫽ 0.0001, F(3,19) ⫽ 9.2, interaction: P ⫽ 0.0064; F(3,19) ⫽ 4.8] and fR [hormone effect: P ⫽ 0.0030, F(3,19) ⫽ 5.5, no interaction]. However, the hormonal replacement did not affect the ventilatory responses to CO2, as they were similar across all OVX groups. ˙ O2 of the estrus group was higher comIn normocapnia, V pared with OVX⫹E2P animals [hormone effect: P ⫽ 0.0017, ˙ O2 was F(3,19) ⫽ 6.2, no interaction] (Fig. 7). No difference in V
J Appl Physiol • doi:10.1152/japplphysiol.00254.2015 • www.jappl.org
˙ E, V ˙ O2, and Tb Hormonal Influence on V
64 Proestrus (n=8) Estrus (n=6) Metaestrus (n=4) Diestrus (n=4)
2800 2100
20 15 10
.
700 0
5 0
30
300
25
. .
VE/VO2
VT (mLKg-1)
0
30
250
20 15 10
200 150 100 50
5
0 0 CO2 0%
0 0
30
180
30 2 7% CO
˙ O2) and respiratory equivalent (V ˙ E/V ˙ O2) of rats Fig. 4. Oxygen consumption (V in proestrus, estrus, metestrus, and diestrus during normocapnia (0% CO2) and hypercapnia (7% CO2). Values are means ⫾ SE; n, no. of rats.
160 140
DISCUSSION
120
The present study demonstrates that, despite the hormonal ˙ O2, and V ˙ E, V ˙ E/V ˙ O2 during fluctuations of cycling rats, V normocapnia and hypercapnia were not different in different stages of the estrous cycle. As shown in previous studies, our measurements of Tb were higher in estrous rats in both normocapnic and hypercapnic conditions. Additionally, this study also shows that ovariectomy promotes an attenuation of the ˙ E and V ˙ E/V ˙ O2) compared with the response to hypercapnia (in V response obtained from intact animals in estrus. Moreover, we also saw that hormonal replacement did not reverse these effects, suggesting that this treatment does not restore the ventilatory responses of an intact animal.
100 80 60 0%0 CO2
7%30CO2
˙ E), tidal volume (VT), and respiratory frequency (fR) of Fig. 3. Ventilation (V the rats in proestrus, estrus, metestrus, and diestrus during normocapnia (0% CO2) and hypercapnia (7% CO2). Values are means ⫾ SE; n, no. of rats.
observed between the groups under hypercapnic conditions [hypercapnia effect: P ⫽ 0.9756; F(3,22) ⫽ 0.00095, no inter˙ O2 during action], and hormone replacement did not affect V CO2 exposure (Fig. 7). ˙ E/V ˙ O2 was not different between groups during normocapV ˙ E/V ˙ O2 in all groups nia (Fig. 7). Hypercapnia increased V [hypercapnia effect: P ⬍ 0.001, F(3,22) ⫽ 69.4, interaction P ⫽ 0.0083; F(3,22) ⫽ 5.4] (Fig. 7); however, the effect was greater ˙ E/V ˙ O2 of the estrus group, compared with OVX groups in V [hormone effect: P ⫽ 0.0059, F(3,22) ⫽ 5.9, interaction P ⫽ 0.0083; F(3,22) ⫽ 5.4]. Tb. Tb of rats on estrus was higher than that of the OVX groups during normocapnia [hormone effect: P ⫽ 0.0070, F(3,22) ⫽ 4.8, no interaction], but not under hypercapnia (Fig. 8). Exposure to hypercapnia caused a drop in Tb in all groups, except for the OVX⫹E2 animals [hypercapnia effect: P ⫽ 0.028, F(3,19) ⫽ 5.8, no interaction] (Fig. 8).
Proestrus (n=8) Estrus (n=6) Metaestrus (n=4) Diestrus (n=4)
39
Tb (°C)
fR (breaths.min -1)
Proestrus (n=8) Estrus (n=6) Metaestrus (n=4) Diestrus (n=4)
25
1400
.
Marques DA et al.
VO2 (mL.Kg-1.min-1)
VE (mL Kg-1min-1)
3500
•
38
37
36
0%0CO2
30 2 7% CO
Fig. 5. Body temperature (Tb) of rats in proestrus, estrus, metestrus, and diestrus during normocapnia (0% CO2) and hypercapnia (7% CO2). Values are means ⫾ SE; n, no. of rats.
J Appl Physiol • doi:10.1152/japplphysiol.00254.2015 • www.jappl.org
˙ E, V ˙ O2, and Tb Hormonal Influence on V
OVX+O (n=6) OVX+E2 (n=6) OVX+E2 P (n=5) Estrus (n=6)
2800 2100
*
1400
.
700 0
65
Marques DA et al.
OVX+O (n=6) OVX+E2 (n=6) OVX+E2 P (n=5) Estrus (n=6)
25 VO2 (mL.Kg-1.min-1)
VE (mL Kg -1min-1)
3500
•
.
20 15 10 5
*
20
300
15
250
10
200
VE/VO2
VT (mLKg -1)
25
. .
5 0
150 100 50 0
180 fR (breaths.min -1)
*
160
0%CO2
140
7% CO2
˙ O2 and V ˙ E/V ˙ O2 of intact rats in estrus, and OVX⫹O, OVX⫹E2, and Fig. 7. V OVX⫹E2P rats during normocapnia (0% CO2) and hypercapnia (7% CO2). Values are means ⫾ SE; n, no. of rats. *Difference between estrus group compared with OVX⫹O, OVX⫹E2, and OVX⫹E2P groups (P ⬍ 0.05).
120 100 80 60 0%CO 2
7% CO 2
˙ E, VT, and fR of intact rats in estrus, ovariectomized (OVX) rats with Fig. 6. V corn oil replacement (OVX⫹O), OVX rats replaced with E2 (OVX⫹E2), and OVX rats replaced with a combination of E2 and P (OVX⫹E2P) during normocapnia (0% CO2) and hypercapnia (7% CO2). Values are means ⫾ SE; n, no. of rats. *Difference between estrus group compared with OVX⫹O, OVX⫹E2, and OVX⫹E2P groups (P ⬍ 0.05).
possibly due to augmented levels of P. Hayashi et al. (22) ˙ E and VT in the luteal phase, observed higher values for basal V when comparing it with the follicular phase. ˙ O2 levels were observed under In this study, no changes in V normocapnia in the different phases of the estrous cycle, suggesting that the hormonal fluctuations that occur during the cycle have a small effect (if any) in the metabolic rate under the present conditions. This has also been observed in women during the menstrual cycle (42, 45). Regarding Tb, our results show an increase on the day of estrus, similar to that previously
Estrous Cycle and V˙E, Metabolism, and Tb
OVX+O (n=6) OVX+E2 (n=6) OVX+E2 P (n=5) Estrus (n=6)
39
38 Tb (°C)
˙ E in women is higher Many studies have demonstrated that V during the luteal than the follicular phase (22, 35, 41), and also ˙ E (40, that the administration of P promotes an increase in V ˙ E is related to an 52), which suggests that this increase in V ˙E augment of P levels. In the present study, no difference in V ˙ and VO2 was observed in the different groups under normocapnic conditions, which suggests that hormonal changes during estrous cycle were not sufficient to promote alteration in ˙ E and metabolism. In this regard, Brack et al. (7) resting V performed experiments using anesthetized cycling female rats and reported no differences in respiratory rates in different ˙ E were not stages of the estrous cycle, although VT and V measured. In addition, a previous study demonstrated that phrenic nerve activity is not estrous cycle dependent (51). ˙ E is not Similarly, White et al. (48) showed that resting V different between follicular and luteal phases in women; however, VT and end-tidal PCO2 is higher in the luteal phase,
37
36 0% CO2
7% CO2
Fig. 8. Tb of intact rats in estrus, and OVX⫹O, OVX⫹E2, and OVX⫹E2P rats during normocapnia (0% CO2) and hypercapnia (7% CO2). Values are means ⫾ SE; n, no. of rats.
J Appl Physiol • doi:10.1152/japplphysiol.00254.2015 • www.jappl.org
66
˙ E, V ˙ O2, and Tb Hormonal Influence on V
reported (23, 50). During estrus, Tb was ⬃0.6°C higher than in other phases. In women, Tb can increase up to 0.4°C after ovulation, whereas in rodents there is an elevation in Tb immediately before, rather than after presumed ovulation (33). Despite the fact that Tb was higher during estrus in this study, ˙ E/V ˙ O2 of cycling rats did not vary in different metabolism and V phases of the cycle. We found no difference in ventilatory responses to CO2 during the different stages of the estrous cycle. In women, there is a controversy in the literature about the influence of menstrual cycle in the CO2 drive to breathe. Some studies have demonstrated that alterations of sex hormones during the menstrual cycle have little or no effect on CO2 sensitivity (48), while others have reported that, during the luteal phase, the hypercapnic ventilatory response is more pronounced than that in the follicular phase (16, 35). Shoene et al. (35) suggest that the increased ventilatory response during the luteal phase is related to the higher P concentration. This contradiction may reflect differences in study design and on the data analysis. Additionally, Preston et al. (32) demonstrated that CO2 sensitivity was not different between pre- and postmenopausal women. In anesthetized rats, Zabka et al. (51) demonstrated that the short-term ventilatory response to hypoxia is similar across estrous phases. The present data show that the estrous cycle of rats does not interfere with the ventilatory responses to CO2 in females with a regular cycle. Therefore, our findings, taken in conjunction with those of others, indicate that normally occurring alterations in sex hormones have very little or no effect on the sensitivity to CO2. ˙ O2 during hypercapnic exposure We observed a decrease in V in all phases of the estrous cycle. This was also observed in male rats during 5% CO2 exposure (36). However, CO2 exposure only decreased Tb of rats in estrus and proestrus. In many species, a decrease in Tb is observed during hypercapnia, including rats (19). Nevertheless, previous studies from our laboratory did not observe a Tb alteration during CO2 challenge in male Wistar rats (6, 13, 14, 31). Ovariectomy, Hormonal Replacement, V˙E, Metabolism, and Tb ˙ E were found during In the present study, no differences in V normocapnia and hypercapnia between spayed females treated with corn oil, E2, or P. It has long been recognized that E2 participates in the respiratory adjustments observed during late gestation (9, 29). Nevertheless, the administration of E2 alone ˙ E in male rats (34). Some evidence indicates a did not affect V ˙ E (37). In fact, as synergistic effect of estrogen and P on V estrogen upregulates P receptors (24), this mechanism is likely involved with the respiratory effects of P. E2 and P receptors (ER-␣, ER-; PR-A, PR-B) were identified in many respiratory regions of the brain, including the nucleus of the solitary tract (38, 39), the hypoglossal nuclei (4), and the motor nuclei of the phrenic nerve (4). In fact, some studies have reported an ˙ E (10, 44, inhibitory effect of E2 in some areas important for V 46, 49). Also, studies in OVX rats (8) and castrated cats (20, 21) have demonstrated that administration of P combined with ˙ E and hypercapestrogen promotes an increase in pulmonary V nic ventilatory response through receptor-mediated mechanisms of action involving central and peripheral sites. The fact ˙ E might be related to deficienthat we found no difference in V
•
Marques DA et al.
cies in the hormonal replacement protocol. We choose not to use P treatment without E2, since it is well known that P sensitivity is highly dependent on prior or concurrent estrogen exposure, once estrogens induce P receptor expression in the brain (4, 26, 30). Interestingly, our results demonstrate that OVX rats that underwent hormonal replacement showed a ventilatory response to hypercapnia 43% lower compared with female rats on estrus. This comparison was made because lower levels of sexual hormones are observed during estrus. It is interesting to note that hormonal replacement is used in the literature for several purposes (3). However, at least in the perspective of respiratory control, this approach has its limitations, since hormonal replacement did not revert the effects of ovariectomy in the ventilatory responses to hypercapnia. In this context, when studying female rats, it is important to consider the possibility that replacing hormones using the present protocol might not perfectly mimic the conditions of an intact cycling animal, despite the fact that said protocol yielded physiological plasmatic levels of E2 and P, according to Szawka et al. (44). What is more, OVX⫹E2 and OVX⫹E2P rats presented plasmatic levels of E2 higher than those in OVX⫹O rats. In the present work, we show that hormonal replacement did not restore the ventilatory, metabolic, and thermal variables studied. Even though E2 and P are the main female gonadal ˙ E, hormones, other gonadal hormones might play a role in V metabolism, and temperature. Gonadal hormones include sexual steroids and androgens, and also peptide hormones, such as anti-Müllerian hormone, activin, inhibin, and follistatin, which might be involved. However, there are no studies in the literature reporting the involvement of these hormones. Humans are able to easily manipulate the levels of their sexual hormones (with oral contraceptives and steroids), but the respiratory consequences of this are not fully understood (3). In conclusion, despite the hormonal fluctuations during the estrous cycle, the ventilatory responses to hypercapnia in female rats were similar along the cycle, but OVX rats presented a reduced ventilatory response to CO2. Our data demonstrate that hormonal replacement with E2 or P (the main ˙ E under the present sexual hormones) did not alter the V experimental condition, suggesting that other gonadal factors may be involved in these responses. This study contributes to the understanding of the respiratory and metabolic responses to CO2 exposure in female conscious rats, being an important complement to the existing literature. ACKNOWLEDGMENTS We thank Euclides Roberto Secato and Ruither O. G. Carolino for excellent technical assistance. GRANTS This work was supported by Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP; 2012/19966-0), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; 442560/2014-1), INCT-Fisiologia Comparada. D. A. Marques was the recipient of a CNPq scholarship. G. S. F. da Silva was supported by the Young Investigator Award (FAPESP; 2013/17606-9). DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). AUTHOR CONTRIBUTIONS Author contributions: D.A.M., D.d.C., R.E.S., J.A.A.F., K.C.B., and L.H.G. conception and design of research; D.A.M. and D.d.C. performed experiments;
J Appl Physiol • doi:10.1152/japplphysiol.00254.2015 • www.jappl.org
˙ E, V ˙ O2, and Tb Hormonal Influence on V D.A.M. and D.d.C. analyzed data; D.A.M., G.S.F.d.S., R.E.S., K.C.B., and L.H.G. interpreted results of experiments; D.A.M. prepared figures; D.A.M. and L.H.G. drafted manuscript; D.A.M., D.d.C., G.S.F.d.S., R.E.S., J.A.A.F., K.C.B., and L.H.G. edited and revised manuscript; D.A.M., D.d.C., G.S.F.d.S., R.E.S., J.A.A.F., K.C.B., and L.H.G. approved final version of manuscript. REFERENCES 1. Almeida MC, Steiner AA, Coimbra NC, Branco LG. Thermoeffector neuronal pathways in fever: a study in rats showing a new role of the locus coeruleus. J Physiol 558: 283–294, 2004. 2. Bartlett D Jr, Tenney SM. Control of breathing in experimental anemia. Respir Physiol 10: 384 –395, 1970. 3. Behan M, Kinkead R. Neuronal control of breathing: sex and stress hormones. Compr Physiol 1: 2101–2139, 2011. 4. Behan M, Thomas CF. Sex hormone receptors are expressed in identified respiratory motoneurons in male and female rats. Neuroscience 130: 725–734, 2005. 5. Behan M, Zabka AG, Thomas CF, Mitchell GS. Sex steroid hormones and the neural control of breathing. Respir Physiol Neurobiol 136: 249 – 263, 2003. 6. Biancardi V, Bicego KC, Almeida MC, Gargaglioni LH. Locus coeruleus noradrenergic neurons and CO2 drive to breathing. Pflügers Arch 455: 1119 –1128, 2008. 7. Brack KE, Jeffery SM, Lovick TA. Cardiovascular and respiratory responses to a panicogenic agent in anaesthetised female Wistar rats at different stages of the oestrous cycle. Eur J Neurosci 23: 3309 –3318, 2006. 8. Brodeur P, Mockus M, McCullough R, Moore LG. Progesterone receptors and ventilatory stimulation by progestin. J Appl Physiol 60: 590 –595, 1986. 9. Brownell LG, West P, Kryger MH. Breathing during sleep in normal pregnant women. Am Rev Respir Dis 133: 38 –41, 1986. 10. Cason AM, Kwon B, Smith JC, Houpt TA. c-Fos induction by a 14T magnetic field in visceral and vestibular relays of the female rat brainstem is modulated by estradiol. Brain Res 1347: 48 –57, 2010. 11. Das TK, Jana H. Basal oxygen consumption during different phases of menstrual cycle. Indian J Med Res 94: 16 –19, 1991. 12. de Carvalho D, Bicego KC, de Castro OW, da Silva GS, GarciaCairasco N, Gargaglioni LH. Role of neurokinin-1 expressing neurons in the locus coeruleus on ventilatory and cardiovascular responses to hypercapnia. Respir Physiol Neurobiol 172: 24 –31, 2010. 13. Dempsey JA, Olsen E, Burt JR, Skatrud JB. Hormones and neurochemicals in the regulation of breathing. In: Handbook of Physiology. The Respiratory System. Control of Breathing. Bethesda, MD: Am. Physiol. Soc., 1986, sect. 3, vol. II, pt. 1, chapt. 7, p. 181–221. 14. de Souza Moreno V, Bícego KC, Szawka RE, Anselmo-Franci JA, Gargaglioni LH. Serotonergic mechanisms on breathing modulation in the rat locus coeruleus. Pflügers Arch 459: 357–368, 2010. 15. Dias MB, Nucci TB, Margatho LO, Antunes-Rodrigues J, Gargaglioni LH, Branco LG. Raphe magnus nucleus is involved in ventilatory but not hypothermic response to CO2. J Appl Physiol 103: 1780 –1788, 2007. 16. Dutton K, Blanksby BA, Morton AR. CO2 sensitivity changes during the menstrual cycle. J Appl Physiol 67: 517–522, 1989. 17. Findlay JK. An update on the roles of inhibin, activin, and follistatin as local regulators of folliculogenesis. Biol Reprod 48: 15–23, 1993. 18. Freeman ME. The neuroendocrine control of the ovarian cycle of the rat. In: Knobil and Neill’s Physiology of Reproduction, edited by Neill JD. Philadelphia, PA: Elsevier, 2006, vol. 2, p. 2327–2388. 19. Gautier H, Bonora M, Trinh HC. Ventilatory and metabolic responses to cold and CO2 in intact and carotid body-denervated awake rats. J Appl Physiol 75: 2570 –2579, 1993. 20. Hannhart B, Pickett CK, Weil JV, Moore LG. Influence of pregnancy on ventilatory and carotid body neural output responsiveness to hypoxia in cats. J Appl Physiol 67: 797–803, 1989. 21. Hannhart B, Pickett CK, Moore LG. Effects of estrogen and progesterone on carotid body neural output responsiveness to hypoxia. J Appl Physiol 68: 1909 –1916, 1990. 22. Hayashi K, Kawashima T, Suzuki Y. Effect of menstrual cycle phase on the ventilatory response to rising body temperature during exercise. J Appl Physiol 113: 237–245, 2012. 23. Kent S, Hurd M, Satinoff E. Interactions between body temperature and wheel running over the estrous cycle in rats. Physiol Behav 49: 1079 – 1084, 1991.
•
Marques DA et al.
67
24. Leavitt WW, Blaha GC. An estrogen-stimulated, progesterone-binding system in the hamster uterus and vagina. Steroids 19: 263–274, 1972. 25. Lopes LT, Biancardi V, Vieira EB, Leite-Panissi C, Bícego KC, Gargaglioni LH. Participation of the dorsal periaqueductal grey matter in the hypoxic ventilatory response in unanaesthetized rats. Acta Physiol (Oxf) 211: 528 –537, 2014. 26. Maclusky NJ, Mcewen BS. Progestin receptors in rat brain: distribution and properties of cytoplasmic progestin-binding sites. Endocrinology 106: 192–202, 1980. 27. Markowska AL, Savonenko AV. Effectiveness of estrogen replacement in restoration of cognitive function after long-term estrogen withdrawal in aging rats. J Neurosci 22: 10985–10995, 2002. 28. McLean JH, Coleman WP. Temperature variation during the estrous cycle: active vs. restricted rats. Psychon Sci 22: 179 –180, 1971. 29. Moore LG, McCullough RE, Weil JV. Increased HVR in pregnancy: relationship to hormonal and metabolic changes. J Appl Physiol 62: 158 –163, 1987. 30. Parsons B, Mcewen BS, Pfaff DW. A discontinuous schedule of estradiol treatment is sufficient to activate progesterone-facilitated feminine sexual behavior and to increase cytosol receptors for progestins in the hypothalamus of the rat. Endocrinology 110: 613–619, 1982. 31. Patrone LG, Bícego KC, Hartzler LK, Putnam RW, Gargaglioni LH. Cardiorespiratory effects of gap junction blockade in the locus coeruleus in unanesthetized adult rats. Respir Physiol Neurobiol 190: 86 –95, 2014. 32. Preston ME, Jensen D, Janssen I, Fisher JT. Effect of menopause on the chemical control of breathing and its relationship with acid-base status. Am J Physiol Regul Integr Comp Physiol 296: R722–R727, 2009. 33. Refinetti R, Menaker M. The circadian rhythm of body temperature. Physiol Behav 51: 613–637, 1992. 34. Regensteiner JG, Woodard WD, Hagerman DD, Weil JV, Pickett CK, Bender PR, Moore LG. Combined effects of female hormones and metabolic rate on ventilatory drives in women. J Appl Physiol 66: 808 –813, 1989. 35. Schoene RB, Robertson HT, Pierson DJ, Peterson AP. Respiratory drives and exercise in menstrual cycles of athletic and nonathletic women. J Appl Physiol 50: 1300 –1305, 1981. 36. Seifert EL, Mortola JP. Circadian pattern of ventilation during acute and chronic hypercapnia in conscious adult rats. Am J Physiol Regul Integr Comp Physiol 282: R244 –R251, 2002. 37. Shahar E, Redline S, Young T, Boland LL, Baldwin CM, Nieto FJ, O’Connor GT, Rapoport DM, Robbins JA. Hormone replacement therapy and sleep-disordered breathing. Am J Respir Crit Care Med 167: 1186 –1192, 2003. 38. Shughrue PJ, Lane MV, Merchenthaler I. Comparative distribution of estrogen receptor-alpha and -beta mRNA in the rat central nervous system. J Comp Neurol 388: 507–525, 1997. 39. Simerly RB, Chang C, Muramatsu M, Swanson LW. Distribution of androgen and estrogen receptor mRNA-containing cells in the rat brain: an in situ hybridization study. J Comp Neurol 294: 76 –95, 1990. 40. Skatrud JB, Dempsey JA, Kaiser DG. Ventilatory response to medroxyprogesterone acetate in normal subjects: time course and mechanism. J Appl Physiol 44: 393–344, 1978. 41. Slatkovska L, Jensen D, Davies GA, Wolfe LA. Phasic menstrual cycle effects on the control of breathing in healthy women. Respir Physiol Neurobiol 154: 379 –388, 2006. 42. Smekal G, Von Duvillard SP, Frigo P, Tegelhofer T, Pokan R, Hofmann P, Tschan H, Baron R, Wonisch M, Renezeder K, Bachl N. Menstrual cycle: no effect on exercise cardiorespiratory variables or blood lactate concentration. Med Sci Sports Exerc 39: 1098 –1106, 2007. 43. Smith MS, Freeman ME, Neill JD. The control of progesterone secretion during the estrous cycle and early pseudopregnancy in the rat: prolactin, gonadotropin and steroid levels associated with rescue of the corpus luteum of pseudopregnancy. Endocrinology 96: 219 –226, 1975. 44. Szawka RE, Rodovalho GV, Monteiro PM, Carrer HF, AnselmoFranci JA. Ovarian-steroid modulation of locus coeruleus activity in female rats: involvement in luteinising hormone regulation. J Neuroendocrinol 21: 629 – 639, 2009. 45. Takase K, Nishiyasu T, Asano K. Modulating effects of the menstrual cycle on cardiorespiratory responses to exercise under acute hypobaric hypoxia. Jpn J Physiol 52: 553–560, 2002. 46. Ueyama T, Tanioku T, Nuta J, Kujira K, Ito T, Nakai S, Tsuruo Y. Estrogen alters c-Fos response to immobilization stress in the brain of ovariectomized rats. Brain Res 1084: 67–79, 2006.
J Appl Physiol • doi:10.1152/japplphysiol.00254.2015 • www.jappl.org
68
˙ E, V ˙ O2, and Tb Hormonal Influence on V
47. Wenninger JM, Olson EB Jr, Cotter CJ, Thomas CF, Behan M. Hypoxic and hypercapnic ventilatory responses in aging male vs. aging female rats. J Appl Physiol 106: 1522–1528, 2009. 48. White DP, Douglas NJ, Pickett CK, Zwillich CW, Weil JV. Sleep deprivation and the control of ventilation. Am Rev Respir Dis 128: 984 –986, 1983. 49. Xue B, Hay M. 17-Estradiol inhibits excitatory amino acid-induced activity of neurons of the nucleus tractus solitarius. Brain Res 976: 41–52, 2003.
•
Marques DA et al.
50. Yanase M, Tanaka H, Nakayama T. Effects of estrus cycle on thermoregulatory responses during exercise in rats. Eur J Appl Physiol Occup Physiol 58: 446 –451, 1989. 51. Zabka AG, Behan M, Mitchell GS. Selected contribution: time-dependent hypoxic respiratory responses in female rats are influenced by age and by the estrus cycle. J Appl Physiol 91: 2831–2838, 2001. 52. Zwillich CW, Natalino MR, Sutton FD, Weil JV. Effects of progesterone on chemosensitivity in normal men. J Lab Clin Med 92: 262–269, 1978.
J Appl Physiol • doi:10.1152/japplphysiol.00254.2015 • www.jappl.org
