Abstract
Many diseases of the respiratory system occur differently in males and females, indicating a possible role of gonadal hormones in respiratory control. We hypothesized that testosterone (T) is important for the ventilatory chemosensitivity responses in males. To test this hypothesis, we evaluated ventilation (V̇E), metabolic rate and body temperature (Tb) under normoxia/normocapnia, hypercapnia and hypoxia in orchiectomized (ORX), ORX with testosterone replacement (ORX+T) or flutamide (FL, androgen receptor blocker)-treated rats. We also performed immunohistochemistry to evaluate the presence of androgen receptor (AR) in the carotid body (CB) of intact males. Orchiectomy promoted a reduction V̇E and ventilatory equivalent (V̇E/V̇O2) under room-air conditions, which was restored with testosterone treatment. Moreover, during hypoxia or hypercapnia, animals that received testosterone replacement had a higher V̇E and V̇E/V̇O2 than control and ORX, without changes in metabolic and thermal variables. Flutamide decreased the hypoxic ventilatory response without changing the CO2-drive to breathe, suggesting that the testosterone effect on hypercapnic hyperventilation does not appear to involve the AR. We also determined the presence of AR in the CB of intact animals. Our findings demonstrate that testosterone seems to be important for maintaining resting V̇E in males. In addition, the influence of testosterone on V̇E, either during resting conditions or under hypoxia and hypercapnia, seems to be a direct and specific effect, as no changes in metabolic rate or Tb were observed during any treatment. Finally, a putative site of testosterone action during hypoxia is the CB, since we detected the presence of AR in this structure.
Introduction
Many breathing disorders, such as sleep apnea and sudden infant death syndrome (SIDS), have a higher occurrence in males than in females, which may indicate the existence of sex differences in respiratory control (Kapsimalis & Kryger 2002, Chahrour & Zoghbi 2007, Behan & Wenninger 2008). A central issue that may play an important role in the differences between males and females is the presence of sex hormones, for example, progesterone, estradiol and testosterone (T), produced by both sexes in different concentrations throughout life (Behan & Kinkead 2011). Many studies have suggested testosterone as a potential cause of the higher prevalence of sleep disorders in men, causing a destabilization in ventilatory control during sleep (Mateika et al. 2004, Lozo et al. 2017). This idea was reinforced by studies reporting that the apnea/hypopnea index increased in hypogonadal men after testosterone replacement (Matsumoto et al. 1985, Schneider et al. 1986). Further, inhibition of testosterone action via the 5α-reductase pathway alleviates breathing instability during sleep (Chowdhuri et al. 2013). On the other hand, it has been shown that lower testosterone levels are correlated with chronic obstructive pulmonary disease (COPD) (Kaparianos et al. 2011) and anabolic steroid supplementation improved respiratory muscle strength and decreases hypoxemia (Schols et al. 1995). Furthermore, asthma prevails in boys before puberty and this sex preference reverses after puberty and in adulthood (Canguven & Albayrak 2011). Indeed, testosterone has a protective role in asthmatic patients, maintaining the physiological balance of autoimmunity and protective immunity by preserving the number of immune cells (Canguven & Albayrak 2011).
Data regarding the effect of testosterone on breathing control is still controversial, probably due to differences in experimental designs. For instance, some authors have shown that ventilation rises after testosterone replacement in both humans (White et al. 1985, Fogel et al. 2001) and animals (Tatsumi et al. 1994), whereas others found no change (Koepchen 1953, Tenorio-Lopes et al. 2017) or even inhibition of ventilation (Bayliss et al. 1987). In fact, it has been suggested that the impact of testosterone on breathing regulation in animals and humans requires its aromatization to estradiol. Thus, the role of testosterone in breathing function may ultimately be mediated by estradiol, the availability of which is controlled by aromatase (Simpson et al. 2002, Zabka et al. 2006). Likewise, there is no agreement regarding the role of testosterone in the ventilatory responses to hypercapnia and hypoxia (Matsumoto et al. 1985, White et al. 1985, Tatsumi et al. 1994, Joseph et al. 2002, Fournier et al. 2014, Boukari et al. 2016, Tenorio-Lopes et al. 2017).
Sex hormones receptors are widely distributed in brain regions important for respiratory control, such as the retrotrapezoid nucleus (RTN), nucleus of the solitary tract (NTS), locus coeruleus (LC), caudal medullary raphe and hypothalamus (Pascual et al. 2002, Szawka et al. 2009, Behan & Kinkead 2011W). Estradiol and progesterone receptors were also described in the carotid body (CB), a main peripheral chemosensitive site involved in the regulation of ventilation (Joseph et al. 2006). Although the presence of androgen receptor (AR) in brain chemosensitive sites is well known (Simerly et al. 1990), there are no data in the literature regarding the presence of AR in peripheral sites, such as the CB. A study by Tatsumi et al. (1994) demonstrated that testosterone replacement increased the carotid sinus nerve response to hypoxia, and this greater chemosensitivity was eliminated by the transection of the carotid sinus nerve. In contrast, there are no reports so far on the presence of AR in the cells of the CB.
Besides breathing control, testosterone can also regulate metabolic rate and body temperature (Tb). For instance, replacement of testosterone in hypogonadal men causes an increase in oxygen consumption (V̇O2) (White et al. 1985). Additionally, it was demonstrated that the pre-optic area of the hypothalamus (POA), an important region for Tb regulation, has a large expression of AR (Simerly et al. 1990) and testosterone affects the activity of temperature-sensitive and insensitive neurons in the POA (Silva & Boulant 1986). Thus, it is reasonable to consider a certain degree of interaction between metabolic, thermoregulatory and reproductive functions. Therefore, we further investigate the participation of testosterone and AR in the ventilatory, metabolic and thermal responses to hypoxia and hypercapnia in adult male rats.
Materials and methods
Animals
Experiments were performed on unanesthetized, adult male Wistar rats obtained from UNESP (Botucatu, Brazil; body mass, 250–310 g). The animals had free access to water and food, and were housed in a temperature-controlled room (25 ± 1°C) with a 12 h light:12 h darkness cycle (lights on at 06:30 h). The experimental protocols were run between 09:00 and 13:00h. to avoid daily variations in hormone levels, and were in agreement with the guidelines of the National Council of Control in Animal Experimentation (CONCEA, Brazil) and approved by the local Animal Care and Use Committee (CEUA # 8.129/16).
Surgery
Animals were anesthetized with ketamine-xylazine (100 and 10 mg/kg, i.p., respectively) and the body area designated for the procedure was shaved and cleaned with 2% chlorhexidine digluconate (Rioquimica, SP, Brazil). The animals were prophylactically treated with antibiotic (5% enrofloxacin, s.c.; 0.5 mg/kg, Schering-Plough) and, immediately after surgery, with analgesic anti-inflammatory (flunixin meglumine, i.m.; 2.5 mg/kg, Schering-Plough). Ten days before the experiments, the animals were submitted to the following surgical procedures.
Implantation of a temperature-measuring device
All animals were implanted with a miniature temperature datalogger (SubCue Dataloggers, Calgary, Canada) in the peritoneal cavity via midline laparotomy to record Tb. After implantation of the device, the abdominal muscles and skin were sutured in layers.
Orchiectomy, hormone and androgen receptor antagonist treatments
Three groups of rats (ORX, and ORX+T, ORX+FL) were submitted to bilateral orchiectomy by incision of the scrotum and ligation of the spermatic cords; the skin was then sutured. Three days after the surgeries, the animals were treated at 10:00 h daily for 7 consecutive days prior to the experiment with (1) vehicle (corn oil, ORX group; 0.2 mL/rat, s.c., Liza; Cargill, Sao Paulo, Brazil) or (2) testosterone propionate (ORX+T group; 0.25 mg/0.2 mL/rat, s.c.; dose: 830 µg/kg; Organon, Sao Paulo, Brazil). This dose has been reported to yield physiological testosterone levels in the plasma (Kalil et al. 2013). For controls, we used gonad-intact rats (intact group) and sham operation, which involved exposure of the testicles without excision (SHAM group). For the antagonist treatment, 3 days after the datalogger implantation, orchiectomized (ORX) or SHAM animals received the AR blocker flutamide (FL, 8 mg/kg/day, s.c.; Sigma) daily for 7 days prior to the experiment. The FL dose was determined according to previously published experiments (Reckelhoff et al. 1999, Davis et al. 2019).
After the experiments, animals were euthanized with tribromoethanol (0.3 mL/100 g, i.p.), the seminal vesicle was removed and its wet mass was measured. The mass of the seminal vesicle was used to confirm the effectiveness of the hormone treatment, as testosterone causes trophic effects on the seminal vesicle (de Carvalho et al. 2016).
Ventilatory, Tb, and metabolic measurements
Ventilation (V̇E) measurements were obtained using the barometric method (whole-body plethysmography), as previously described by Drorbaugh & Fenn (1955) and performed in our laboratory (Biancardi et al. 2008, Marques et al. 2015).
Body temperature was recorded every 5 min by a temperature datalogger, as described in the ‘Surgery’ section. Metabolic rate was measured by indirect calorimetry (V̇O2) using a closed respirometry system, as described previously (Almeida et al. 2004, Marques et al. 2015). At the end of the normoxic/normocapnic, hypoxic and hypercapnic intervals, the airflow in the chamber was interrupted for 5 min, and the air was continuously sampled with an O2 analyzer (PowerLab System, ADInstruments, Bella Vista, Australia). The percentage of oxygen decline inside the chamber was plotted against time, and the slope of the resulting curve was corrected for by the chamber volume and body mass of the animals in order to estimate the V̇O2 using the closed-circuit indirect calorimetry measures, as were used in our previous studies (Marques et al. 2015, 2017). V̇O2 was measured, and the ratio of carbon dioxide released to oxygen consumed (V̇CO2/V̇O2), called the respiratory quotient, was estimated in 0.85, as with previous studies in the literature (Raurich et al. 1989). The values are presented in mL of oxygen/min/kg in STPD (standard conditions of temperature, pressure, and dry air).
Before the experiments, the O2 analyzer was calibrated with two known gas mixtures. Over the 5 min that the chamber was sealed, the reduction of the O2 fraction inside was 0.5−1.0%, which is not considered significant to the animal (Marques et al. 2017). According to our previous measurements, the level of CO2 inside the chamber at the end of a 5 min period with the rat breathing inside the box showed a small change (initial mean value of 0.033% CO2; final mean value 0.2824% CO2).
Experimental protocol
Each animal was individually placed inside a plethysmograph chamber (5 L), maintained at 25°C, and allowed to move while the chamber was flushed with humidified room air. The basal measurements (time zero) of V̇E and V̇O2 were taken after the animal had acclimated for approximately 1 h. Subsequently, a hypercapnic gas mixture (7% CO2, 21% O2 with N2 balance; White Martins, Sao Paulo, Brazil) or a hypoxic mixture (7% O2 with N2 balance; White Martins) was flushed through the chamber for 30 min, and V̇E and V̇O2 were measured at 30 min of exposure. The chamber was then flushed with room air for 90 min, so that the ventilation could return to basal values. After that, a hypoxic or hypercapnic gas mixture was flushed into the chamber for 30 min, and V̇E and V̇O2 were again measured at 30 min. The order of gas exposures (hypercapnic and hypoxic) was randomly chosen, and the CO2 and O2 percentages and exposure times were chosen based on pilot experiments and previous studies (Biancardi et al. 2008, Scarpellini et al. 2009).
CB removal and immunohistochemistry
We performed immunohistochemistry to verify the presence or absence of AR at the CB. This protocol was used for qualitative purposes only. Adult males were anesthetized with ketamine-xylazine (100 and 10 mg/kg, i.p., respectively), an incision in the ventral surface of the neck was made and, using forceps, the carotid bifurcations were removed en bloc. The dissected tissue was 24-h post-fixed in 4% PFA and maintained in a 30% sucrose solution at 4°C, as described by Joseph et al. (2006). The tissue was fixed in Tissue-Tek OCT (Sakura Finetek, Torrance, CA, USA), cut into 40-µm sections using a cryostat (CM1860 – Ag Protect; Leica), and mounted on glass slides for subsequent analysis.
Immunofluorescence
Glass slides containing CB sections were treated for 30 min in an antigenic retrieval solution (Dako) at 70°C. The slides were then cooled at room temperature for 20 min. Subsequently, the sections were washed with phosphate-buffered saline (PBS) five times for 5 min each, and then incubated for 1 h in a solution of PBS with 0.4% triton X-100 (TX-100) and 10% goat serum to prevent non-specific labeling, followed by 24 h of incubation with a mix of mouse monoclonal anti-tyrosine hydroxylase (TH) antibody (1:400; T1299, Sigma-Aldrich) and rabbit anti-AR antibody (1:200; ab133273, Abcam) diluted in a solution of PBS with 0.4% TX-100 and 3% goat serum. On the next day, the slides were washed five times for 5 min each with PBS, and then incubated for 4 h at room temperature with a cocktail containing the AlexaFluor 594-coupled anti-mouse goat IgG and the AlexaFluor 488-coupled anti-rabbit goat IgG (1:1000; A11032 and A11015, Life Technologies) in PBS with 0.4% TX-100 and 3% goat serum. After 4 h, the slides were washed five times for 5 min in PBS, mounted with ProLong™ Gold (Molecular Probes) and sealed with a coverslip to be examined by confocal microscopy. We also included a negative (without primary antibody) control to test the specificity of the immunohistochemistry protocol (Torlakovic et al. 2014). In addition, we simultaneously performed immunohistochemistry in the rat testis, a tissue well known to express AR-positive labeling (positive control) (Vornberger et al. 1994, Mayerhofer et al. 1996).
Statistical analyses
Data are presented as means ± s.e.m. Seminal vesicle mass, V̇E, fR, VT, Tb, V̇O2 and V̇E/V̇O2 data obtained during normoxia were analyzed using one-way ANOVA. The effects of treatments and gas exposure (hypoxia or hypercapnia) were analyzed using two-way ANOVA for repeated measures. Whenever ANOVA resulted in significant main effects or interactions, a Bonferroni post hoc test was performed to verify where the differences existed. Differences were considered to be significant when P < 0.05. Symbols (*) were used only when the post-test detected differences between treatments; P-values are shown in the graphs where pairs were analyzed.
Results
All data for ventilatory, metabolic and thermal variables tested during normoxic/normocapnic, hypoxic and hypercapnic gas exposures among groups are described in Tables 1 and 2. Two-way ANOVA values for the effects of treatments and gas exposure (hypoxia or hypercapnia) for all parameters of intact, ORX and ORX+T groups are described in Table 3. Table 4 presents the two-way ANOVA values for SHAM, SHAM+FL and ORX+FL animals. One unique aspect to this study was the use of a physiological dose of testosterone (a dose at which the resulting plasma concentration of testosterone following replacement was similar to levels found in intact rats) (Kalil et al. 2013), as well as the comparison of castrated with intact animals. No difference was observed between the intact and SHAM groups.
Seminal vesicle mass, total ventilation (V̇E), tidal volume (VT), breathing frequency (fR) oxygen consumption (V̇O2), respiratory equivalent (V̇E/V̇O2), and body temperature (Tb) data of intact, ORX (orchidectomized) and ORX+T (orchidectomized+testosterone) groups, during normoxic normocapnia (21% O2; 0% CO2), hypercapnia (7% CO2) and hypoxia (7% O2) exposure.
| INTACT (n=11) | ORX (n=11) | ORX+T (n=6) | |
|---|---|---|---|
| Seminal vesicle mass (mg/100 g) | 336.6 ± 19.2 | 89.0a,b ± 16.0 | 627.9a ± 35.3 |
| Normoxia | |||
| V̇E (mL/kg/min) | 552.6 ± 23.8 | 471.5a,b ± 19.1 | 612.1 ± 25.5 |
| VT (mL/kg) | 6.2 ± 0.3 | 5.5b ± 0.3 | 7.1 ± 0.4 |
| fR (breaths/min) | 90.6 ± 3.4 | 86.0 ± 2.5 | 87.0 ± 5.4 |
| V̇O2 (mL/kg/min) | 18.0 ± 0.6 | 18.0 ± 0.6 | 19.7 ± 0.4 |
| V̇E/V̇O2 |
30.7 ± 1.3 | 26.3a,b ± 1.1 | 31.2 ± 1.6 |
| Tb (°C) | 37.1 ± 0.1 | 37.0 ± 0.1 | 37.2 ± 0.1 |
| Hypoxia | |||
| V̇E (mL/kg/min) | 997.9 ± 54.6 | 950.0 ± 57.0 | 1341.8a ± 109.5 |
| VT (mL/kg) | 8.6 ± 0.1 | 8.7 ± 0.4 | 10.2 ± 0.6 |
| fR (breaths/min) | 121.2 ± 5.7 | 109.0 ± 6.0 | 131.0a ± 5.0 |
| V̇O2 (mL/kg/min) | 10.6 ± 0.6 | 11.5 ± 0.4 | 11.2 ± 0.3 |
| V̇E/V̇O2 | 96.7 ± 5.8 | 85.1b ± 4.8 | 119.4a ± 7.4 |
| Tb (°C) | 35.9 ± 0.1 | 35.9 ± 0.2 | 36.2 ± 0.3 |
| Hypercapnia | |||
| V̇E (mL/kg/min) | 1277.1 ± 46.1 | 1247.0b ± 59.6 | 2002.1a ± 188.7 |
| VT (mL/kg) | 9.4 ± 0.3 | 9.4 ± 0.5 | 13.1a ± 1.2 |
| fR (breaths/min) | 136.7 ± 2.8 | 130.1b ± 2.0 | 153.3a ± 3.7 |
| V̇O2 (mL/kg/min) | 15.2 ± 0.4 | 15.7 ± 0.4 | 16.9 ± 0.4 |
| V̇E/V̇O 2 | 87.1 ± 2.9 | 78.9b ± 4.0 | 124.3a ± 8.8 |
| Tb (°C) | 36.9 ± 0.1 | 37.0 ± 0.1 | 37.0 ± 0.2 |
Values are mean ± s.e.
aSignificant difference from intact; bSignificant difference from ORX+T.
Seminal vesicle mass, total ventilation (V̇E), tidal volume (VT), breathing frequency (fR) oxygen consumption (V̇O2), respiratory equivalent (V̇E/V̇O2), and body temperature (Tb) of SHAM, SHAM+flutamide (SHAM+FL) and orchidectomized+flutamide (ORX+FL) groups, during normoxic normocapnia (21% O2; 0% CO2), hypercapnia (7% CO2) and hypoxia (7% O2) exposure.
| SHAM (n=6) | SHAM+FL (n=6) | ORX+FL (n=6) | |
|---|---|---|---|
| Seminal vesicle mass (mg/100 g) | 322.4 ± 18.9 | 212.8a,b ± 41.9 | 18.83a ± 0.8 |
| Normoxia | |||
| V̇E (mL/kg/min) | 719.6 ± 25.2 | 727.4 ± 15.9 | 674.23 ± 34.5 |
| VT (mL/kg) | 6.3 ± 0.2 | 6.7 ± 0.3 | 7.12 ± 0.2 |
| fR (breaths/min) | 113.6 ± 3.9 | 109.5 ± 5.7 | 94.67a ± 4.4 |
| V̇O2 (mL/kg/min) | 18.8 ± 0.7 | 17.4 ± 0.7 | 17.2 ± 1.0 |
| V̇E/V̇O 2 | 38.8 ± 1.8 | 42.1 ± 2.2 | 39.5 ± 1.8 |
| Tb (°C) | 37.2 ± 0.2 | 37.1 ± 0.1 | 36.93 ± 0.2 |
| Hypoxia | |||
| V̇E (mL/kg/min) | 1719.7 ± 77.8 | 1273.5a ± 43.6 | 1341.5 ± 61.5 |
| VT (mL/kg) | 9.2 ± 0.4 | 9.12 ± 0.26 | 10.0 ± 0.4 |
| fR (breaths/min) | 188.0 ± 5.4 | 131.5a ± 8.92 | 134.5 ± 4.4 |
| V̇O2 (mL/kg/min) | 16.8 ± 0.6 | 14.9 ± 1.2 | 14.8 ± 1.2 |
| V̇E/V̇O2 | 102.6 ± 2.1 | 85.4a ± 4.3 | 92.3 ± 6.0 |
| Tb (°C) | 36.4 ± 0.1 | 36.3 ± 0.2 | 36.5 ± 0.1 |
| Hypercapnia | |||
| V̇E (mL/kg/min) | 1875.9 ± 97.9 | 1908.7 ± 128.4 | 1939.9 ± 112.4 |
| VT (mL/kg) | 12.4 ± 0.5 | 13.5 ± 0.6 | 14.8 ± 0.7 |
| fR (breaths/min) | 151.0 ± 6.9 | 140.5 ± 5.2 | 131.5 ± 6.2 |
| V̇O2 (mL/kg/min) | 19.7 ± 0.9 | 20.1 ± 1.1 | 19.4 ± 0.9 |
| V̇E/V̇O 2 | 96.6 ± 7.8 | 96.4 ± 7.9 | 98.4 ± 5.5 |
| Tb (°C) | 37.2 ± 0.2 | 37.2 ± 0.1 | 37.0 ± 0.2 |
Values are mean ± s.e.
aSignificant difference from SHAM; bSignificant difference from ORX+FL.
Two-way ANOVA results for total ventilation (V̇E), tidal volume (VT), breathing frequency (fR) oxygen consumption (V̇O2), respiratory equivalent (V̇E/V̇O2), and body temperature (Tb) of intact, ORX (orchidectomized), and ORX+T (orchidectomized+testosterone) groups during hypoxia (7% O2) and hypercapnia (7% CO2) exposures compared with normoxic normocapnic condition.
| ANOVA results | |||
|---|---|---|---|
| Gas effect | Treatment effect | Factorial interaction | |
| Hypoxia | |||
| V̇E (mL/kg/min) | P < 0.0001 F(1,25)=241.70 | P=0.0005 F(2,25)=8.01 | P=0.04 F(2,25)=2.98 |
| VT (mL/kg) | P < 0.0001 F(1,25)=132.80 | P=0.06 F(2,25)=2.69 | NS |
| fR (breaths/min) | P < 0.0001 F(1,25)=68.28 | P=0.003 F(2,25)=5.59 | NS |
| V̇O2 (mL/kg/min) | P < 0.0001 F(1,25)=578.90 | P=0.16 F(2,25)=1.99 | NS |
| V̇E/V̇O 2 | P < 0.0001 F(1,25)=602.50 | P=0.0003 F(2,25)=12.14 | P=0.0002 F(2,25)=12.29 |
| Tb (°C) | P < 0.0001 F(1,25)=102.00 | P=0.12 F(2,25)=2.078 | NS |
| Hypercapnia | |||
| V̇E (mL/kg/min) | P < 0.0001 F(1,25)=523.30 | P < 0.0001 F(2,25)=17.47 | P < 0.0001 F(2,25)=12.24 |
| VT (mL/kg) | P < 0.0001 F(1,25)=271.70 | P=0.0007 F(2,25)=7.51 | P=0.002 F(2,25)=6.40 |
| fR (breaths/min) | P < 0.0001 F(1,25)=592.10 | P=0.004 F(2,25)=5.53 | P=0.0001 F(2,25)=9.91 |
| V̇O2 (mL/kg/min) | P < 0.0001 F(1,25)=84.10 | P=0.08 F(2,25)=2.77 | NS |
| V̇E/V̇O 2 | P < 0.0001 F(1,25)=439.70 | P < 0.0001 F(2,25)=14.47 | P=0.0004 F(2,25)=10.73 |
| Tb (°C) | P=0.11 F(1,25)=2.75 | P=0.52 F(2,25)=0.76 | NS |
Two-way ANOVA results for total ventilation (V̇E), tidal volume (VT), breathing frequency (fR) oxygen consumption (V̇O2), respiratory equivalent (V̇E/V̇O2), and body temperature (Tb) data of SHAM, SHAM+flutamide (SHAM+FL) and orchidectomized+flutamide (ORX+FL) groups, during hypoxia (7% O2) and hypercapnia (7% CO2) exposures compared with normoxic normocapnic condition.
| ANOVA results | |||
|---|---|---|---|
| Gas effect | Treatment effect | Factorial interaction | |
| Hypoxia | |||
| V̇E (mL/kg/min) | P < 0.0001 F(1,15)=39.10 | P=0.00057 F(2,15)=12.27 | P=0.0006 F(2,15)=12.86 |
| VT (mL/kg) | P < 0.0001 F(1,15)=188.30 | P=0.092 F(2,15)=2.80 | NS |
| fR (breaths/min) | P < 0.0001 F(1,15)=103.00 | P < 0.0001 F(2,15)=25.24 | P=0.0004 F(2,15)=13.83 |
| V̇O2 (mL/kg/min) | P=0.0007 F(1,15)=17.83 | P=0.25 F(2,15)=1.51 | NS |
| V̇E/V̇O 2 | P < 0.0001 F(1,15)=511.70 | P=0.22 F(2,15)=1.65 | P=0.01 F(2,15)=6.28 |
| Tb (°C) | P < 0.0001 F(1,15)=69.10 | P=0.83 F(2,15)=0.19 | NS |
| Hypercapnia | |||
| V̇E (mL/kg/min) | P < 0.0001 F(1,15)=461.70 | P=0.97 F(2,15)=0.023 | NS |
| VT (mL/kg) | P=0.0001 F(1,15)=330.20 | P=0.0103 F(2,15)=6.30 | NS |
| fR (breaths/min) | P < 0.0001 F(1,15)=161.60 | P=0.044 F(2,15)=3.88 | NS |
| V̇O2 (mL/kg/min) | P=0.004 F(1,15)=11.52 | P=0.69 F(2,15)=0.37 | NS |
| V̇E/V̇O 2 | P < 0.0001 F(1,15)=197.40 | P=0.95 F(2,15)=0.04 | NS |
| Tb (°C) | P=0.77 F(1,15)=0.09 | P=0.52 F(2,15)=0.67 | NS |
Effect of orchiectomy, testosterone replacement and antagonist treatment on seminal vesicle mass
Orchiectomy significantly reduced the seminal vesicle mass, compared to control animals (Table 1). On the other hand, testosterone replacement in ORX animals not only reversed the effect of removing the testicles, but caused a further increase in seminal vesicle mass (Table 1). Treatment with FL in SHAM rats caused a reduction in the seminal vesicle mass compared to the untreated SHAM group. ORX+FL rats displayed an even lower seminal vesicle mass than SHAM+FL rats, further attesting to the antiandrogenic efficacy of FL (Table 2).
Effect of orchiectomy, testosterone replacement and antagonist treatment on thermal and metabolic responses to hypercapnia and hypoxia
Neither hypercapnia nor orchiectomy with or without testosterone replacement affected the Tb of rats. Regarding hypoxia, 7% O2 decreased similarly Tb in all animals (Tables 1 and 2). The same response pattern was observed in animals that received FL treatment (Tables 3 and 4).
During room-air conditions, V̇O2 was not affected by orchiectomy, testosterone replacement or FL treatment (Tables 1 and 2). Hypoxia caused a reduction in V̇O2, with no difference among treatments (Tables 3 and 4). Hypercapnia affected V̇O2 in all groups with no differences among them (Tables 3 and 4).
Effect of orchiectomy, testosterone replacement and antagonist treatment on ventilation
Orchiectomy reduced V̇E by 15% compared to the intact and ORX+T groups under room-air conditions, mainly via an effect on VT. Testosterone replacement restored the baseline V̇E and VT (Table 1). Flutamide treatment did not cause changes in V̇E or VT in either SHAM or ORX rats; however, FL promoted a decrease in the fR of ORX+FL animals compared to the SHAM group (Table 2).
Hypoxia caused an increase in the ventilatory variables in all groups (Figs 1 and 2). Orchiectomy did not affect V̇E, VT or fR under hypoxia, compared to intact animals. On the other hand, testosterone replacement caused a further rise of V̇E, compared to the intact group, due to a higher fR. Flutamide reduced the hypoxic ventilatory response in the SHAM group, compared to the untreated SHAM group, due to a decrease in fR (Fig. 2).
Effect of orchiectomy and testosterone replacement on V̇E, VT and fR of rats exposed to hypoxia (7% O2). Values are presented as % from baseline. *Significant statistical difference (P < 0.05) using one-way ANOVA with the Bonferroni post hoc test. Differences between pairs are represented and P-values are included when significant. In the graph, each point represents one animal’s data; gross values are described in Table 1.
Citation: Journal of Endocrinology 247, 1; 10.1530/JOE-20-0257
Effect of orchiectomy and testosterone replacement on V̇E, VT and fR of rats exposed to hypoxia (7% O2). Values are presented as % from baseline. *Significant statistical difference (P < 0.05) using one-way ANOVA with the Bonferroni post hoc test. Differences between pairs are represented and P-values are included when significant. In the graph, each point represents one animal’s data; gross values are described in Table 1.
Citation: Journal of Endocrinology 247, 1; 10.1530/JOE-20-0257
Effect of orchiectomy and testosterone replacement on V̇E, VT and fR of rats exposed to hypoxia (7% O2). Values are presented as % from baseline. *Significant statistical difference (P < 0.05) using one-way ANOVA with the Bonferroni post hoc test. Differences between pairs are represented and P-values are included when significant. In the graph, each point represents one animal’s data; gross values are described in Table 1.
Citation: Journal of Endocrinology 247, 1; 10.1530/JOE-20-0257
Effect of flutamide (FL) on V̇E, VT and fR of SHAM and orchiectomized (ORX) rats exposed to hypoxia (7% O2). Values are presented as % from baseline. *Significant statistical difference (P < 0.05) using one-way ANOVA with the Bonferroni post hoc test. Differences between pairs are represented and P-values are included when significant. In the graph, each point represents one animal’s data; gross values are described in Table 2.
Citation: Journal of Endocrinology 247, 1; 10.1530/JOE-20-0257
Effect of flutamide (FL) on V̇E, VT and fR of SHAM and orchiectomized (ORX) rats exposed to hypoxia (7% O2). Values are presented as % from baseline. *Significant statistical difference (P < 0.05) using one-way ANOVA with the Bonferroni post hoc test. Differences between pairs are represented and P-values are included when significant. In the graph, each point represents one animal’s data; gross values are described in Table 2.
Citation: Journal of Endocrinology 247, 1; 10.1530/JOE-20-0257
Effect of flutamide (FL) on V̇E, VT and fR of SHAM and orchiectomized (ORX) rats exposed to hypoxia (7% O2). Values are presented as % from baseline. *Significant statistical difference (P < 0.05) using one-way ANOVA with the Bonferroni post hoc test. Differences between pairs are represented and P-values are included when significant. In the graph, each point represents one animal’s data; gross values are described in Table 2.
Citation: Journal of Endocrinology 247, 1; 10.1530/JOE-20-0257
Hypercapnia caused an increase in the ventilatory variables in all groups (Figs 3 and 4). CO2 exposure increased all respiratory variables similarly in intact and ORX animals (Fig. 3). Nonetheless, a further increase in V̇E and fR was observed in ORX animals that received testosterone, compared to the intact and ORX groups (Fig. 3). Flutamide did not affect hypercapnic ventilatory response in either the ORX or SHAM groups (Fig. 4).
Effect of orchiectomy and testosterone replacement on V̇E, VT and fR of rats exposed to hypercapnia (7% CO2 and 21% O2). Values are presented as % from baseline. *Significant statistical difference (P < 0.05) using one-way ANOVA with the Bonferroni post hoc test. Differences between pairs are represented and P-values are included when significant. In the graph, each point represents one animal’s data; gross values are described in Table 1.
Citation: Journal of Endocrinology 247, 1; 10.1530/JOE-20-0257
Effect of orchiectomy and testosterone replacement on V̇E, VT and fR of rats exposed to hypercapnia (7% CO2 and 21% O2). Values are presented as % from baseline. *Significant statistical difference (P < 0.05) using one-way ANOVA with the Bonferroni post hoc test. Differences between pairs are represented and P-values are included when significant. In the graph, each point represents one animal’s data; gross values are described in Table 1.
Citation: Journal of Endocrinology 247, 1; 10.1530/JOE-20-0257
Effect of orchiectomy and testosterone replacement on V̇E, VT and fR of rats exposed to hypercapnia (7% CO2 and 21% O2). Values are presented as % from baseline. *Significant statistical difference (P < 0.05) using one-way ANOVA with the Bonferroni post hoc test. Differences between pairs are represented and P-values are included when significant. In the graph, each point represents one animal’s data; gross values are described in Table 1.
Citation: Journal of Endocrinology 247, 1; 10.1530/JOE-20-0257
Effect of flutamide (FL) on V̇E, VT and fR of SHAM and orchiectomized (ORX) rats exposed to hypercapnia (7% CO2 and 21% O2). Values are presented as % from baseline. *Significant statistical difference (P < 0.05) using one-way ANOVA with the Bonferroni post hoc test. Differences between pairs are represented and P-values are included when significant. In the graph, each point represents one animal’s data; gross values are described in Table 2.
Citation: Journal of Endocrinology 247, 1; 10.1530/JOE-20-0257
Effect of flutamide (FL) on V̇E, VT and fR of SHAM and orchiectomized (ORX) rats exposed to hypercapnia (7% CO2 and 21% O2). Values are presented as % from baseline. *Significant statistical difference (P < 0.05) using one-way ANOVA with the Bonferroni post hoc test. Differences between pairs are represented and P-values are included when significant. In the graph, each point represents one animal’s data; gross values are described in Table 2.
Citation: Journal of Endocrinology 247, 1; 10.1530/JOE-20-0257
Effect of flutamide (FL) on V̇E, VT and fR of SHAM and orchiectomized (ORX) rats exposed to hypercapnia (7% CO2 and 21% O2). Values are presented as % from baseline. *Significant statistical difference (P < 0.05) using one-way ANOVA with the Bonferroni post hoc test. Differences between pairs are represented and P-values are included when significant. In the graph, each point represents one animal’s data; gross values are described in Table 2.
Citation: Journal of Endocrinology 247, 1; 10.1530/JOE-20-0257
Regarding the air convection requirement (V̇E/V̇O2), Fig. 5 shows measurements of V̇E/V̇O2 for intact, ORX and ORX with testosterone replacement. Orchiectomy decreased the V̇E/V̇O2 ratio under normoxia by 16%, and testosterone replacement restored it to baseline values (Fig. 5, top). Under hypoxia and hypercapnia, the V̇E/V̇O2 ratio increased in all groups, with a further increase observed in ORX+T rats (Fig. 5, middle and bottom).
Effect of orchiectomy and testosterone replacement on respiratory equivalent (V̇E/V̇O2) of rats exposed to normocapnia (0% CO2 and 21% O2; top), hypoxia (7% O2 and 0% CO2; middle) and hypercapnia (7% CO2 and 21% O2; bottom). Values are presented as % from baseline. *Significant statistical difference (P < 0.05) using one-way ANOVA with the Bonferroni post hoc test. Differences between pairs are represented and P-values are included when significant. In the graph, each point represents one animal’s data; gross values are described in Table 1.
Citation: Journal of Endocrinology 247, 1; 10.1530/JOE-20-0257
Effect of orchiectomy and testosterone replacement on respiratory equivalent (V̇E/V̇O2) of rats exposed to normocapnia (0% CO2 and 21% O2; top), hypoxia (7% O2 and 0% CO2; middle) and hypercapnia (7% CO2 and 21% O2; bottom). Values are presented as % from baseline. *Significant statistical difference (P < 0.05) using one-way ANOVA with the Bonferroni post hoc test. Differences between pairs are represented and P-values are included when significant. In the graph, each point represents one animal’s data; gross values are described in Table 1.
Citation: Journal of Endocrinology 247, 1; 10.1530/JOE-20-0257
Effect of orchiectomy and testosterone replacement on respiratory equivalent (V̇E/V̇O2) of rats exposed to normocapnia (0% CO2 and 21% O2; top), hypoxia (7% O2 and 0% CO2; middle) and hypercapnia (7% CO2 and 21% O2; bottom). Values are presented as % from baseline. *Significant statistical difference (P < 0.05) using one-way ANOVA with the Bonferroni post hoc test. Differences between pairs are represented and P-values are included when significant. In the graph, each point represents one animal’s data; gross values are described in Table 1.
Citation: Journal of Endocrinology 247, 1; 10.1530/JOE-20-0257
Flutamide treatment did not change V̇E/V̇O2 during normoxia or hypercapnia (Fig. 6, top and bottom). However, under hypoxia, the V̇E/V̇O2 ratio was lower after FL treatment in the SHAM+FL group (Fig. 6, middle).
Effect of flutamide (FL) on respiratory equivalent (V̇E/V̇O2) of SHAM and orchiectomized (ORX) rats exposed to normocapnia (0% CO2 and 21% O2; top), hypoxia (7% O2 and 0% CO2; middle) and hypercapnia (7% CO2 and 21% O2; bottom). Values are presented as % from baseline. *Significant statistical difference (P < 0.05) using one-way ANOVA with the Bonferroni post hoc test. Differences between pairs are represented and P-values are included when significant. In the graph, each point represents one animal’s data; gross values are described in Table 2.
Citation: Journal of Endocrinology 247, 1; 10.1530/JOE-20-0257
Effect of flutamide (FL) on respiratory equivalent (V̇E/V̇O2) of SHAM and orchiectomized (ORX) rats exposed to normocapnia (0% CO2 and 21% O2; top), hypoxia (7% O2 and 0% CO2; middle) and hypercapnia (7% CO2 and 21% O2; bottom). Values are presented as % from baseline. *Significant statistical difference (P < 0.05) using one-way ANOVA with the Bonferroni post hoc test. Differences between pairs are represented and P-values are included when significant. In the graph, each point represents one animal’s data; gross values are described in Table 2.
Citation: Journal of Endocrinology 247, 1; 10.1530/JOE-20-0257
Effect of flutamide (FL) on respiratory equivalent (V̇E/V̇O2) of SHAM and orchiectomized (ORX) rats exposed to normocapnia (0% CO2 and 21% O2; top), hypoxia (7% O2 and 0% CO2; middle) and hypercapnia (7% CO2 and 21% O2; bottom). Values are presented as % from baseline. *Significant statistical difference (P < 0.05) using one-way ANOVA with the Bonferroni post hoc test. Differences between pairs are represented and P-values are included when significant. In the graph, each point represents one animal’s data; gross values are described in Table 2.
Citation: Journal of Endocrinology 247, 1; 10.1530/JOE-20-0257
CB immunohistochemistry
Immunohistochemistry revealed that the CB (labeled for TH in red) is richly endowed with AR (labeled in green) (Fig. 7A, B and C). A negative (without primary antibody) labeling control can be seen in Fig. 7A and B, demonstrating the specificity of TH and AR immunoreactivity. At the same time, we performed the same double labeling in tissue from the rat testis, known to contain a high expression of AR, as a positive control, which further supports the specificity of AR labeling in the CB. This result is presented in Fig. 7D, E, F and G.
Immunofluorescence for (A) tyrosine hydroxylase (TH-red) and (B) androgen receptor (AR-green) in the carotid body of an adult male rat. Photomicrographs are presented as positive staining in the left side and negative (without primary antibody) in the right side. (C) Detail of overlapping TH+AR photomicrograph. Immunohistology of the rat testis. (D) AR positive labeling in green. (E) Detail of AR labeling. (F) AR-negative photomicrograph (without primary antibody) with green background, and (G) detail of AR negative. A full colour version of this figure is available at https://doi.org/10.1530/JOE-20-0257.
Citation: Journal of Endocrinology 247, 1; 10.1530/JOE-20-0257
Immunofluorescence for (A) tyrosine hydroxylase (TH-red) and (B) androgen receptor (AR-green) in the carotid body of an adult male rat. Photomicrographs are presented as positive staining in the left side and negative (without primary antibody) in the right side. (C) Detail of overlapping TH+AR photomicrograph. Immunohistology of the rat testis. (D) AR positive labeling in green. (E) Detail of AR labeling. (F) AR-negative photomicrograph (without primary antibody) with green background, and (G) detail of AR negative. A full colour version of this figure is available at https://doi.org/10.1530/JOE-20-0257.
Citation: Journal of Endocrinology 247, 1; 10.1530/JOE-20-0257
Immunofluorescence for (A) tyrosine hydroxylase (TH-red) and (B) androgen receptor (AR-green) in the carotid body of an adult male rat. Photomicrographs are presented as positive staining in the left side and negative (without primary antibody) in the right side. (C) Detail of overlapping TH+AR photomicrograph. Immunohistology of the rat testis. (D) AR positive labeling in green. (E) Detail of AR labeling. (F) AR-negative photomicrograph (without primary antibody) with green background, and (G) detail of AR negative. A full colour version of this figure is available at https://doi.org/10.1530/JOE-20-0257.
Citation: Journal of Endocrinology 247, 1; 10.1530/JOE-20-0257
Discussion
The present study shows that orchiectomy promoted a reduction in V̇E and V̇E/V̇O2 during room-air conditions, and that testosterone replacement was able to restore it. Testosterone increased the ventilatory response to hypoxia and hypercapnia, without changing V̇O2 and Tb, suggesting that this hormone may act directly on the peripheral and/or central chemoreflex. Treatment with the antiandrogenic agent FL reduces the hypoxia-induced hyperventilation, independently of metabolic changes, suggesting that testosterone provides an excitatory drive to breathe in hypoxic conditions. Accordingly, we demonstrate the presence of AR in the CB, indicating a putative site for testosterone action, which seems to be particularly relevant during hypoxia. The efficacy of orchiectomy and testosterone replacement was confirmed by the respective effects in decreasing and increasing the mass of the seminal vesicles (de Carvalho et al. 2016). Flutamide treatment decreased the relative seminal vesicle weights in both SHAM and ORX rats, indicating the efficacy of the antiandrogenic effect in our model.
ORX rats experienced a decrease in ventilation during room-air conditions due to a lower VT, and testosterone replacement restored it to control values. These data are in contrast with previous studies, in which castration did not affect V̇E (Bairam et al. 2009, Fournier et al. 2014, Tenorio-Lopes et al. 2017). The reason for this discrepancy might be related to the fact that we performed experiments 7 days after orchiectomy, whereas these previous studies performed experiments 14 days after castration. Interestingly, FL treatment did not affect basal V̇E, suggesting that testosterone does act through AR to stimulate breathing under basal conditions. Because FL does not interfere with estrogenic actions or rapid androgen signaling, our present findings suggest that testosterone modulation of basal V̇E may depend on the activation of membrane AR (Thomas 2019) or estrogen receptors, after aromatization to estradiol (Russell & Grossmann 2019). Thus, the diversity of cellular pathways through which testosterone may act adds further complexity to the understanding of its role in respiratory control.
It is known that elevated testosterone concentrations in the plasma have an impact on respiratory parameters (Fogel et al. 2001, Gonzales et al. 2011), and that AR is expressed in brainstem nuclei involved in breathing (Simerly et al. 1990). For instance, studies have demonstrated moderate labeling of AR in brain areas involved in the hypoxic and hypercapnic ventilatory response, such as the NTS, LC, parabrachial nucleus, periaqueductal gray (PAG) and C1 noradrenergic neurons (Hamson et al. 2004, Fournier et al. 2014). In fact, brainstem areas such as LC and ventrolateral PAG have the strongest AR staining compared to other brain regions (Hamson et al. 2004). Interestingly, LC neurons are considered central CO2/H+ chemoreceptors and modulate hypercapnic ventilatory response (Gargaglioni et al. 2010). In addition, a previous study of our laboratory showed that chemical lesion of the ventral PAG reduced the ventilatory response to hypercapnia, which demonstrates that this area also contributes to the CO2-drive to breathe (Lopes et al. 2012). Furthermore, AR is expressed not only in neurons, but also in glial cells, including astrocytes, oligodendrocytes and microglia, which play a key role in breathing control and chemosensitivity (Swift-Gallant et al. 2016). In fact, White et al. (1985) showed that V̇E increases following testosterone administration in hypogonadal men, and that this increase is associated with comparable increases in metabolic rate. In women, testosterone also increases baseline ventilation during wakefulness (Fogel et al. 2001). On the other hand, Koepchen (1953) reported that administration of testosterone has no acute effects on ventilation in men under resting conditions, while in male cats, testosterone substantially inhibits phrenic activity (Bayliss et al. 1987).
In contrast to to our previous studies in female rats, in which ovariectomy reduced the hypoxic and hypercapnic ventilatory response (Marques et al. 2015, 2017), no changes in the hypoxic and hypercapnic chemoreflexes were observed in ORX males in the present study (compared to intact rats). In addition, we show here that V̇O2 and Tb under hypercapnia or hypoxia were not affected by gonadectomy. More recently, Tenorio-Lopes et al. (2017) demonstrated that hyperventilation caused by 5% CO2 exposure was not influenced by castration. However, at higher CO2 levels (10%), ORX rats displayed a greater increase in the hypercapnic ventilatory response, compared to intact rats. Since we used 7% CO2, it is possible that higher levels of CO2 are needed to observe the castration effect on the response to hypercapnia; however, future experiments must be performed to verify this possibility.
According to the present study, supplementation with testosterone in ORX rats caused an increase in V̇E under hypoxia and hypercapnia, without changing V̇O2, indicating a primary effect on respiratory chemosensitivity. The findings regarding the effect of testosterone treatment and the CO2-drive to breathe are contradictory. For instance, the hypercapnic ventilatory drive does not change significantly after testosterone treatment in hypogonodal men (Matsumoto et al. 1985, White et al. 1985). On the other hand, manipulation of testosterone levels in healthy women increases ventilatory sensitivity to CO2 (Ahuja et al. 2007). The same was observed in castrated male cats, in which the administration of testosterone promoted a 47% increase in the hypercapnic ventilatory response (Tatsumi et al. 1994). Additionally, testosterone has also been shown to depress the hypercapnic ventilatory drive in infant monkeys during sleep (Emery et al. 1994). In the present study, we used a paradigm of hormone treatment aimed to yield physiological levels of testosterone in the plasma (Kalil et al. 2013); therefore, the differential responses found in the literature may be related to the differences among species, experimental protocols, or even doses and regimens of testosterone treatment. Moreover, as observed for basal ventilation, FL treatment did not change the hypercapnic ventilatory response. This suggests that the effect of testosterone on the CO2-drive to breath is not mediated by AR. As discussed previously, this is possibly another situation where testosterone effects may be due to its enzymatic conversation to estradiol (Samy et al. 2003) and/or through activation of nongenomic signaling mechanisms. Further studies are needed to clarify this issue.
We demonstrated that the hypoxic ventilatory response was also increased by testosterone replacement, as was observed in the study by Tatsumi et al. (1994), in which testosterone-treated male cats had a greater CB chemosensitivity to hypoxia. Regarding humans, one study found a decrease (Matsumoto et al. 1985), whereas another observed an increase (White et al. 1985) in the hypoxic ventilatory response after testosterone treatment in hypogonadal males. Our data showed that the androgen antagonist in SHAM rats decreased the hypoxic ventilatory response acting on breathing frequency and ventilatory equivalent (V̇E/V̇O2 - without changing V̇O2), suggesting a direct effect on hypoxic chemosensitivity. Furthermore, we performed immunohistochemistry to confirm the presence of AR in the CB, suggesting a cellular site for the effect of testosterone on hyperventilation during hypoxic conditions. Our findings provide evidence that testosterone leads to a higher V̇E under low-O2 partial pressure acting through AR that are, at least in part, located in peripheral chemoreceptors.
Regarding Tb, treatment with testosterone did not affect the changes induced by high levels of CO2 or low levels of O2. It is important to point out that, the effect of testosterone replacement was specific to the ventilatory measurements, without affecting thermal or metabolic variables, demonstrating a specific physiological effect.
Our study demonstrated that castrated animals that received testosterone had V̇E increases of 57% under hypercapnia and 35% during hypoxia, compared to intact animals. Our study used adult, middle-aged animals that were gonad-intact, ORX or ORX+T. As far as we know, previous studies that evaluated the effects of testosterone supplementation on ventilatory control in adult, middle-aged men or animals (the model for our study) did not use an intact group with normal physiological testosterone levels as a reference for comparison, which might have led them to underestimate the effects of testosterone on ventilation. Some studies evaluated ventilatory chemosensitivity changes in hypogonadal men with or without testosterone supplementation (Matsumoto et al. 1985, White et al. 1985, Schneider et al. 1986). Similarly, an animal study performed by Tatsumi et al. (1994) used castrated male cats with or without testosterone replacement. Thus, a unique factor in our study was the comparison of ORX animals with ORX+T replacement (a model similar to that found in the current literature) with gonad-intact subjects. The differences found in the present study, among others, demonstrated that the ORX+T group did not mimic the responses observed in the intact group, even when a physiological dose was used for supplementation, which sheds new light on the role of testosterone replacement in chemosensitivity. We also contributed more specific data to the literature about the specific action of testosterone using a specific androgen antagonist.
The different effects observed between endogenously produced testosterone and testosterone supplementation on ventilation might be related to the ultradian rhythm of testosterone release, or even because the testicles produce other hormones, such as anti-Mullerian hormone, inhibin, activin, and follistatin, among others (Ying 1988, Anderson et al. 1998, Matuszczak et al. 2013), which might also affect respiratory control. Therefore, future studies are needed to verify whether other testicular hormones are important for breathing control. Another possibility is that the testicles are also responsible for converting part of the testosterone produced into the potent metabolite, dihydrotestosterone (DHT) (Folman et al. 1972, Dorrington & Fritz 1975, Steers 2001, Liu & Veldhuis 2018). Thus, the role of DHT in ventilation remains unknown.
Androgens, such as testosterone, are used by people with few restrictions (Behan & Kinkead 2011). In our work, we demonstrated that testosterone can change ventilatory chemosensitivity. In 2017, the Forum of International Respiratory Societies (2017) published a report which asserts that more than one billion people in the world suffer from acute or chronic respiratory diseases. Interestingly, men are more likely than women to be affected, which brings us to the discussion at hand, where we highlight the role of sex hormones in the control of the respiratory drive (for a review, see Gargaglioni et al. 2019). Here we suggest that androgenic treatments, as well as the development of new synthetic androgenic drugs, should take into account the impacts they can have on the respiratory system in men.
In summary, the present study demonstrated that orchiectomy decreases ventilation in rats during room-air conditions, and that testosterone is able to restore it. In addition, testosterone replacement promotes an increase in the ventilatory chemosensitivity under hypoxia without changing metabolism or Tb, suggesting that androgens stimulate the respiratory response to low-O2 levels. A putative site of testosterone action is the CB, since we detected the presence of AR in this structure. Nevertheless, testosterone could also be acting on the CNS, since AR is present in respiratory regions. Testosterone administration also increased the hypercapnic ventilatory response; however, FL did not affect the CO2-drive to breathe, suggesting that the testosterone effect does not appear to involve the AR. The present findings contribute to elucidate the modulation of androgens on the control of ventilation, metabolism, and Tb adult male rats.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work received financial support from the Sao Paulo Research Foundation (FAPESP; 2019/09469-8) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq – 407490/2018-3). D A M was the recipient of a FAPESP scholarship 2016/04276-9 and CNPq 140715/2015-0.
Acknowledgements
We thank Dr Vincent Joseph for the kind donation of the anti-AR antibody and discussions, Dr Richard Kinkead for the suggestions and Dr Elaine da Silva for CB removal assistance.
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