Physiology&Behavior.Vol. 51, pp. 581-584, 1992
0031-9384/92 $5.00 + .00 Copyright © 1992PergamonPressLtd.
Printedin the USA.
Control of Ventilation in Androgenized Hypogonadal Male Rats E V E L Y N H. S C H L E N K E R , *1 M A X G O L D M A N A N D G E R A L D H O L M A N
Departments of Physiology, *Pharmacology, and Biology, University of South Dakota, Vermillion, SD 57069 Received 13 M a y 1991 SCHLENKER, E. H., M. GOLDMAN AND G. "HOLMAN. Control of ventilation in androgenized hypogonadal male rats. PHYSIOL BEHAV 51(3) 581-584, 1992.--We have previously shown that subcutaneous administration of aspartic acid (a dicarboxylicacidic amino acid) at a dose of 580 mg/kg causeslong lastingdepressionof ventilationin adult intact and postpubertally castrated male rats, but not in intact female rats. The purpose of the present study was to determine if hypogonadism induced by perinatal administration of testosterone propionate (TP) will alter ventilation,oxygen consumption, and the ventilatoryresponse to aspartic acid and to hypercapnia in adult males. TP treatment resulted in adult males who had lower body, prostate, heart, and testes weights than those of control male rats. Ventilation in air and oxygen consumption were comparable between the two groups as was the ventilatory response to aspartic acid. In contrast, TP-treated rats exhibited a significantlydecreased ventilatory response to hypercapnia due predominantly to lower tidal volumes compared to control animals. Aspartic acid treatment did not affect oxygen consumption in either group. Thus, TP treatment results in the development of adult male rats who, although hypogonadal, retain a male-like ventilatory response to aspartic acid, but whose response to hypercapnia is more like that of hypogonadal men and rats. Sexual dimorphism
Aspartica c i d
Testosteronepropionate
Development
Male rats
Hypercapnia
hypogonadism will cause a diminished ventilatory response similar to that noted in hypogonadal rats and men (15,20,21).
TESTOSTERONE propionate (TP) administered to neonatal male rat pups results in a hypogonadal syndrome in adults that is dependent on the dose of TP and the time of treatment after birth (19). The hormonal status, sexual behavior, and the morphology and functional characteristics of sexual organs have been investigated in these animals ( 1,6,9, l l, 12,13); however, the impact such treatment has on control of ventilation has not been studied. We reported that treatment of neonatal male rat pups with large levels of aspartic acid over a critical period of development resulted in hypogonadal, obese adult animals who exhibited alveolar hypoventilation and diminished responses to hypercapnic challenges (15). Whether these venfilatory responses were the consequence of hypogonadism, obesity, a central nervous system lesion caused by aspartic acid, or combinations of these factors is not clear. In contrast, adult female rats treated in a similar manner as neonates did not show such abnormalities in the control of ventilation. In addition, we acutely challenged adult intact and postpubescently castrated male and intact female rats with lower doses of aspartic acid, that have been shown not to cause CNS lesions nor convulsions, and demonstrated a reproducible, sexually dimorphic ventilatory response (17). Both intact and castrated male rats, but not intact females, showed a long-lasting depression of ventilation. The purpose of the present study was to use TP treatment in neonatal male rat pups to create an adult hypogonadal male and to determine if such animals retained a male-like ventilatory response to acute administration of aspartic acid. We also exposed these rats to a hypercapnic gas challenge to determine if
METHODS Sprague-Dawley rats (Sasco, Omaha) were bred in our laboratory. On day one after birth, male rat pups received subcutaneous injections of either TP (1 mg/rat) or an equal volume of sesame oil, the vehicle. All rats were studied at 2 months of age. Nine intact (vehicle treated) and 10 TP-treated male rats were utilized in these experiments. Animals were housed in groups of two to three animals in steel mesh cages on a 12 h on/12 h offlighting schedule in a temperature-controlled animal facility. Food and water were available ad lib. Ventilation and oxygen consumption were determined in awake rats placed into a cylindrical 30 cm long × 14 cm diameter Plexiglas chamber. One side of the chamber contained ports to measure: (1) the fractional content of carbon dioxide and oxygen exiting the chamber using a Beckman LB-2 carbon dioxide analyzer and a Beckman OM-14 oxygen analyzer; (2) the flow rate of gas passing through the chamber using a Gilmont rotameter; and (3) the chamber temperature and relative humidity using a Digitec digital thermometer and a Cole-Parmer hygrometer. The other side of the chamber was sealed with a no. 15 rubber stopper and contained a port that allowed air or carbon dioxide to enter the chamber. Another port through the stopper was connected to a low level pressure transducer (Statham) coupled to a low level Grass preamplifier, an amplifier driver, and a Grass poly-
1Requests for reprints should be addressed to Evelyn H. Schlenker.
581
582
SCHLENKER, GOLDMAN AND HOLMAN TABLE 1
TABLE 2
BODY AND ORGAN WEIGHTS OF TESTOSTERONE PROPIONATE (TP) AND C O N T R O L RATS
VENTILATORY PARAMETERS IN TESTOSTERONE PROPIONATE (TP) A N D C O N T R O L RATS AFTER SALINE AND ASPARTIC ACID TREATMENT
Control
TP
358.8 _+29.0 11.52 _+ 1.02 3.52 _+0.29 2.96 +_0.84 0.19 _+0.05 10.44 + 2.08 4.47 _+0.65 7.33 _+2.64
317.7 +_ 1.88" 8.13 _ 1.55"? 3.75 _+ 1.19 3.74 + 1.19~ 0.16 _+0.03 8.84 +_ 1.39 3.71 _+0.56~ 5.75 ___1.89
Control
Body weight (g) Testes Seminal vesicles Prostate Adrenal glands Kidney Heart Lung
Saline Tidal volume Frequency Minute ventilation Aspartic acid Tidal volume Frequency Minute ventilation
Organ weights were multiplied by the body weight equivalent (1000 g/b. wt). Values are means _ SD. *%~tSignificantdifferences: *p < 0.01, tP < 0.001. ~ p < 0.05.
graph recorder. A "Y" tube attached to a 1 mm glass syringe connected in parallel to a pressure transducer port was used to calibrate the system. The same technique has been used in many experiments in our laboratory (14-17). We measured oxygen consumption using the flow-through method that consists of subtracting the fractional content of oxygen in the air exiting from the chamber from the fractional content of the air entering the chamber and multiplying the difference by the flow rate of air going through the chamber (15). Values were corrected to STPD conditions. The ventilatory parameters calculated included tidal volume, the frequency of breathing, and the product of these two parameters--minute ventilation. Both tidal volume and minute ventilation were normalized by multiplying each parameter by the kilogram body weight equivalent (1000 g/b. wt. in g). Oxygen consumption was expressed as milliliters of oxygen consumed per hour divided by the body weight equivalent. PROCEDURES The rat was first weighed and then injected subcutaneously with 0.2-0.3 ml of either saline or 580 mg/kg aspartic acid. This dose was selected because previously we showed that it depressed ventilation in male rats in an extensive dose-response-time experiment (17). After the rat was injected, he was placed into the chamber and ventilation and oxygen consumption were evaluated 45 rain later. All TP-treated and control rats who had received saline were then exposed to 3, 5, and 7% carbon dioxide in oxygen in a cumulative manner and ventilation evaluated at each level. After the rat was removed from the chamber, his body temperature was measured using a Sensottek thermometer with a thermocouple. To further confirm that TP treatment was effective, the rats were euthanized by an overdose of sodium pentobarbital and their testes, seminal vesicles, prostate gland, heart, lungs, and adrenal glands removed and weighed. The organ data were multiplied by the body weight equivalent. Data were analyzed using student's unpaired t-tests to determine if ventilatory and oxygen consumption values and organ and body weights were different between the TP and the control groups. A two-way analysis of variance with post hoc least means test was done to compare ventilatory and oxygen consumption responses of rats in each group to saline versus aspartic acid treatment. Significance was accepted at p < 0.05.
[P
2.23 +_0.50 83 +_ 17 179.7 +_34.8
1.95 +_0.29 90 _+9 174.6 +_23.0
1.77 _+0.32 80 _+ 16 139.4 _+23.0
1.70 _+0.35 78 +__18 129.0 _ 32.4
Values are means +_ SD. Units: tidal volume, ml; frequency, breaths per minute; minute ventilation,ml per minute. Both tidal volume and minute ventilation were corrected for body weight equivalent (see Table l). Significant decreases of tidal volume and minute ventilation at p < 0.01 were found comparing saline and aspartic acid values within each group, but not between groups.
RESULTS
Rats treated neonatally with TP weighed less and had lighter testes, prostate glands, and heart weights than did control animals (Table 1). Ventilation of the two groups breathing air were similar (Table 2). When rats were exposed to hypercapnia, ventilation was lower in the TP group at 5 and 7% carbon dioxide compared to those of control rats (Fig. l). These differences were predominantly due to a lower tidal volume rather than to a decrease in the frequency of breathing (Table 3). Unlike the dissimilar ventilatory responses of the two groups to hypercapnia, their response to aspartic acid was the same (Table 2). In both groups aspartic acid caused a marked depression of ventilation compared to their saline responses. The depression of ventilation was due to a lower tidal volume. Aspartic acid administration had no effect on oxygen consumption in either group relative to values obtained after saline injection (Table 4).
c~ O 4-~ cO -,-8
-,-4
(13 "~. ~> q_~
v
v
3
5
7
Percent Carbon Dioxide FIG. 1. Minute ventilation (VE) of control and testosterone propionate (TP) treated ratsexposed to increasinglevelsof carbon dioxide. S'~ffacant differenceswere noted at 5% (p < 0.01) and 7% (p < 0.05) carbon dioxide between animals in each group.
VENTILATION IN ANDROGENIZED MALE RATS TABLE 3 VENTILATORY RESPONSESOF TESTOSTERONE PROPIONATE (TP) AND CONTROL RATS TO HYPERCAPNIA Control Carbon dioxide,3% Tidal volume Frequency Carbon dioxide, 5% Tidal volume Frequency Carbon dioxide, 7% Tidal volume Frequency
TP
2.42 _+0.58 108 + 27
2.02 + 0.35 111 + 10
2.84 + 0.26 121 + 17
2.30 + 0.47* 126 ___15
3.30 + 0.41 128 + 18
2.63 + 0.54* 138 + 21
Values are means + SD. See Table 2 for units for each parameter. * Significant group difference ofp < 0.01 at each level ofhypercapnia.
DISCUSSION The major findings of this study are that TP treatment of male rat pups 1 day after birth results in hypogonadal adults who show a diminished ventilatory response to hypercapnia, but a similar response to aspartic acid compared to saline-treated animals. Schlenker and Goldman (17) reported that castration of postpubertal male rats did not affect their ventilatory response to aspartic acid compared to that of intact males. In conjunction with the results of the present study, we suggest that this response is not dependent upon the level of circulating steroids. In contrast, we have shown that treatment of female rat pups shortly after birth with TP caused a male-like ventilatory response to aspartic acid (l 8) suggesting that neonatal alterations in the organization of the brain appear necessary to elicit the sexually dimorphic ventilatory response to aspartic acid. Consequently, we can ask the question--will feminization of male brains with neonatal treatment of male rats with estradiol benzoate alter their ventilation in response to aspartic acid? In addition, this study has shown that the depression of ventilation in response to aspartic acid is not the consequence of a depression of oxygen consumption, since this parameter was not altered by aspartic acid in either group. Thus, aspartic acid appears to dissociate ventilation and oxygen consumption in male rats. The diminished ventilatory response of TP-treated male rats to hypercapnia may be related to their hypogonadal state. Schlenker and Goldman (15) reported that male rats made hypogonadal by the administration of high doses of aspartic acid shortly after birth also resulted in adult animals who exhibited decreased responses to hypercapnia. In another study, Young and his coworkers (22) suggested that incomplete expression of sexually dimorphic cellular characteristics of the preoptic-anterior hypothalamus of obese Zucker rats, due perhaps to an impaired process of perinatal brain androgenization, may contribute to the abnormal sexual behaviors noted in these animals (4). We found that adult, obese, male Zucker rats displayed significantly lower minute ventilation responses to hypercapnia than did their age-matched lean counterparts (14). The underlying mechanisms responsible for the diminished ventilatory response of obese male Zucker rats to hypercapnia are not known, but may be associated with altered chestwall mechanics due to abdominal fat loading, weakened respiratory muscles, and/or changes in their ability to sense carbon dioxide. The problems
583 using male, obese, aspartic-acid-treated rats or obese Zucker rats to elucidate the contribution hypogonadism makes to the control of ventilation include a number of confounding factors including altered hormone levels (such as insulin, thyroid, and growth hormones) as well as mechanical factors not seen in TP-treated male rats ( 15,21,22). The inability of TP-treated male rats to increase tidal volume in response to hypercapnia may reflect changes in central chemoreceptor characteristics and/or decrease the ability of respiratory muscles to generate the called-for tension. Evidence for the second possibility comes from a study by Jiang and Klueber (7) who evaluated the effect of postnatal castration of mice on muscle morphometry and contractility. The extensor digitorum longus muscles of castrated male rats were smaller, showed atrophic myofibers, necrosis, disruption of normal myofilament arrangement and also developed less tension than did muscles from intact animals. Burbach et al. (2) noted that the heart, diaphragm, and gastrocnemius from hypngonadal male rats who had been treated neonataUy with aspartic acid also weighed less and the two skeletal muscles had smaller fiber diameters than muscles from vehicle-treated animals. Moreover, the age at which castration occurs appears to influence greatly both the structural and functional development of skeletal and cardiac muscles (5,10,23). White and his colleagues (20) reported that their hypogonadal male patients displayed diminished ventilatory responses to both hypoxic and hypercapnic challenges. Following testosterone replacement the patients increased their metabolic rate and ventilatory response to hypoxia but not to hypercapnia. Neither the magnitude of the tidal volume nor frequency of breathing responses to either challenge were reported in this study. Were central chemoreceptors for carbon dioxide and/or respiratory muscle function altered by low testosterone levels in these patients? Additional evidence that the level of plasma testosterone (2,3) can influence ventilatory responses comes from studies on the control of ventilation in awake, aged male rats who exhibit decrements in ventilatory responses to hypercapnia compared to younger males (16). Recently Lawler et al. reported that the diaphragm/body weight ratios in 9-month-old Spague-Dawley rats were significantly lower than in age-matched female rats (8). Thus, it is possible that in male rats decreased levels of testosterone may alter control of ventilation by the effect on respiratory muscle function. CONCLUSION How neonatal androgenization of male rats leads to decreased ventilatory responses to hypercapnia clearly needs to be investigated further. Studies of respiratory muscle structure and func-
TABLE 4 OXYGEN CONSUMPTION VALUESOF TESTOSTERONE PROPIONATE (TP) AND CONTROL RATS GIVEN SALINE AND ASPARTICACID
Saline Aspartic acid
Control
TP
138.0 ___35.1 142.7 _+23.1
132.5 ___37.9 136.1 _+32.9
Values are means _+SD and were corrected by the body weight equivalent. Units are ml of oxygen consumed per hour. There were no significant differences between the groups as a consequence of treatment.
584
SCHLENKER, GOLDMAN AND HOLMAN
tion in neonatal TP-treated male rats at various ages may be instrumental in determining whether the diminished ventilatory response to hypercapnia is due to weakened muscles and/or to changes in central control mechanisms modulating ventilation.
Finally, testosterone replacement at different doses may allow us to ascertain the reversibility these early neuroendocrine perturbations have on the control of ventilation in TP-treated male rats.
REFERENCES 1. Arnold, A. P.; Gorski, R. A. Gonadal steroid induction of structural sex differences in the central nervous system. Ann. Rev. Neurosci. 7:413-442; 1984. 2. Burbach, J. A.; Schlenker, E. H.; Goldman, M. Characterization of muscles from aspartic acid obese rats. Am. J. Physiol. 249:RI06R110; 1985. 3. Chatterjee B.; Fernandes, G.; Yu, B. P.; Song, C.; Kim, J. M.; Demyan, M.; Roy, A. K. Calorie restriction delays age-dependent loss of androgen responsiveness of the rat liver. FASEB J. 3:169-173; 1989. 4. Doherty, P. C.; Baum, M. J.; Finkelstein, J. A. Evidence of incomplete behavioral sexual differentiation in obese male Zucker rats. Physiol. Behav. 34:177-179; 1985. 5. Dux, L.; Dux, E.; Guba, F. Further data on the androgenic dependency of skeletal musculature: The effect of prepubertal castration on the structural development of the skeletal muscles. Horm. Metab. Res. 14:191-194; 1982. 6. Feigelson, M. Suppression oftesticular maturation and fertility following androgen administration to neonatal male rats. Biol. Reprod. 35:1321-1332; 1986. 7. Jiang, B.; Klueber, M. K. Structural and functional analysis ofmurine skeletal muscle after castration. Muscle Nerve 12:67-77; 1989. 8. Lawler, J. M.; Powers, S. K.; Criswell, D. Gender differences in the diaphragm of the adult Sprague-Dawley rat. FASEB J. 5:A1745; 1991. 9. Moguilevsky, J. A.; Scacchi, P.; Rubenstein, L. Effect of neonatal androgenization on luteinizing hormone, follicle-stimulating hormone, prolactin, and testosterone levels in male rats. J. Endocr. 74: 143-148; 1977. 10. Mooradian, A. D.; Morley, J. E.; Korenman, S. G. Biological actions of androgens. Endocrine Rev. 8:1-28; 1987. 11. Naumenko, E. V.; Serova, L. I.; Pack, V. Ch. Brain noradrenaline in the control of the pituitary-gonadal system of adult rats after prenatal androgenization. Biogenic Amines 3:115-123; 1985.
12. Piacsek, B. E.; Histetter, M. W. Neonatal androgenization in the male rat: Evidence for central and peripheral defects. Biol. Reprod. 30:344-351; 1984. 13. Sachs, B.; Thomas, D. A. Differential effects of perinatal androgen treatment on sexually dimorphic characteristics in rats. Physiol. Behay. 34:735-742; 1985. 14. Sehlenker, E. H. Ventilatory control in lean and obese Zucker rats. FASEB J. 4:A1100; 1990. 15. Schlenker, E. H.; Goldman, M. Aspartic acid administered neonatally affects ventilation of male and female rats differently. J. Appl. Physiol. 61:780-784; 1986. 16. Schlenker, E. H.; Goldman, M. Ventilatory responses of aged male and female rats to hypercapnia and to hypoxia. Gerontology 31: 301-308; 1988. 17. Schlenker, E. H.; Goldman, M. Acute effects of aspartic acid on ventilation of male and female rats. Physiol. Behav. 42:313-318; 1988. 18. Schlenker, E. H.; Goldman, M.; Holman, G. No effects of aspartic acid on ventilation in androgenized and ovariectomized female rats. J. Appl. Physiol. (in press). 19. Swanson, H. E.; van der Werfften, B. The "early-androgen" syndrome: Difference in the responses to prenatal and postnatal administration of various doses of testosterone to female and male rats. Acta. Endocin. 47:37-50; 1964. 20. White, D. P.; Seheinder, B. K.; Santen, R. J.; McDermott, M.; Pickett, C. K.; Zwillich, C. W.; Weil, J. V. Influence of testosterone on ventilation and chemosensitivity in male subjects. J. Appl. Physiol. 59: 1452-1457; 1985. 21. Wittels, E. H. Obesity and hormonal factors in sleep and sleep apnea. Med. Clin. N. Am. 69:1265-1280; 1985. 22. Young, J. K.; Hemming, M. W.; Matsumoto, D. E. Sexual behavior and the sexually dimorphic hypothalamic nucleus of the male Zucker rats. Physiol. Behav. 36:881-886; 1986. 23. Yu, W. A. Sex differences in neuronal loss induced by axotomy in the rat brainstem motor nuclei. Exp. Neuol. 102:230-235; 1988.
0031-9384/92 $5.00 + .00 Copyright © 1992PergamonPressLtd.
Printedin the USA.
Control of Ventilation in Androgenized Hypogonadal Male Rats E V E L Y N H. S C H L E N K E R , *1 M A X G O L D M A N A N D G E R A L D H O L M A N
Departments of Physiology, *Pharmacology, and Biology, University of South Dakota, Vermillion, SD 57069 Received 13 M a y 1991 SCHLENKER, E. H., M. GOLDMAN AND G. "HOLMAN. Control of ventilation in androgenized hypogonadal male rats. PHYSIOL BEHAV 51(3) 581-584, 1992.--We have previously shown that subcutaneous administration of aspartic acid (a dicarboxylicacidic amino acid) at a dose of 580 mg/kg causeslong lastingdepressionof ventilationin adult intact and postpubertally castrated male rats, but not in intact female rats. The purpose of the present study was to determine if hypogonadism induced by perinatal administration of testosterone propionate (TP) will alter ventilation,oxygen consumption, and the ventilatoryresponse to aspartic acid and to hypercapnia in adult males. TP treatment resulted in adult males who had lower body, prostate, heart, and testes weights than those of control male rats. Ventilation in air and oxygen consumption were comparable between the two groups as was the ventilatory response to aspartic acid. In contrast, TP-treated rats exhibited a significantlydecreased ventilatory response to hypercapnia due predominantly to lower tidal volumes compared to control animals. Aspartic acid treatment did not affect oxygen consumption in either group. Thus, TP treatment results in the development of adult male rats who, although hypogonadal, retain a male-like ventilatory response to aspartic acid, but whose response to hypercapnia is more like that of hypogonadal men and rats. Sexual dimorphism
Aspartica c i d
Testosteronepropionate
Development
Male rats
Hypercapnia
hypogonadism will cause a diminished ventilatory response similar to that noted in hypogonadal rats and men (15,20,21).
TESTOSTERONE propionate (TP) administered to neonatal male rat pups results in a hypogonadal syndrome in adults that is dependent on the dose of TP and the time of treatment after birth (19). The hormonal status, sexual behavior, and the morphology and functional characteristics of sexual organs have been investigated in these animals ( 1,6,9, l l, 12,13); however, the impact such treatment has on control of ventilation has not been studied. We reported that treatment of neonatal male rat pups with large levels of aspartic acid over a critical period of development resulted in hypogonadal, obese adult animals who exhibited alveolar hypoventilation and diminished responses to hypercapnic challenges (15). Whether these venfilatory responses were the consequence of hypogonadism, obesity, a central nervous system lesion caused by aspartic acid, or combinations of these factors is not clear. In contrast, adult female rats treated in a similar manner as neonates did not show such abnormalities in the control of ventilation. In addition, we acutely challenged adult intact and postpubescently castrated male and intact female rats with lower doses of aspartic acid, that have been shown not to cause CNS lesions nor convulsions, and demonstrated a reproducible, sexually dimorphic ventilatory response (17). Both intact and castrated male rats, but not intact females, showed a long-lasting depression of ventilation. The purpose of the present study was to use TP treatment in neonatal male rat pups to create an adult hypogonadal male and to determine if such animals retained a male-like ventilatory response to acute administration of aspartic acid. We also exposed these rats to a hypercapnic gas challenge to determine if
METHODS Sprague-Dawley rats (Sasco, Omaha) were bred in our laboratory. On day one after birth, male rat pups received subcutaneous injections of either TP (1 mg/rat) or an equal volume of sesame oil, the vehicle. All rats were studied at 2 months of age. Nine intact (vehicle treated) and 10 TP-treated male rats were utilized in these experiments. Animals were housed in groups of two to three animals in steel mesh cages on a 12 h on/12 h offlighting schedule in a temperature-controlled animal facility. Food and water were available ad lib. Ventilation and oxygen consumption were determined in awake rats placed into a cylindrical 30 cm long × 14 cm diameter Plexiglas chamber. One side of the chamber contained ports to measure: (1) the fractional content of carbon dioxide and oxygen exiting the chamber using a Beckman LB-2 carbon dioxide analyzer and a Beckman OM-14 oxygen analyzer; (2) the flow rate of gas passing through the chamber using a Gilmont rotameter; and (3) the chamber temperature and relative humidity using a Digitec digital thermometer and a Cole-Parmer hygrometer. The other side of the chamber was sealed with a no. 15 rubber stopper and contained a port that allowed air or carbon dioxide to enter the chamber. Another port through the stopper was connected to a low level pressure transducer (Statham) coupled to a low level Grass preamplifier, an amplifier driver, and a Grass poly-
1Requests for reprints should be addressed to Evelyn H. Schlenker.
581
582
SCHLENKER, GOLDMAN AND HOLMAN TABLE 1
TABLE 2
BODY AND ORGAN WEIGHTS OF TESTOSTERONE PROPIONATE (TP) AND C O N T R O L RATS
VENTILATORY PARAMETERS IN TESTOSTERONE PROPIONATE (TP) A N D C O N T R O L RATS AFTER SALINE AND ASPARTIC ACID TREATMENT
Control
TP
358.8 _+29.0 11.52 _+ 1.02 3.52 _+0.29 2.96 +_0.84 0.19 _+0.05 10.44 + 2.08 4.47 _+0.65 7.33 _+2.64
317.7 +_ 1.88" 8.13 _ 1.55"? 3.75 _+ 1.19 3.74 + 1.19~ 0.16 _+0.03 8.84 +_ 1.39 3.71 _+0.56~ 5.75 ___1.89
Control
Body weight (g) Testes Seminal vesicles Prostate Adrenal glands Kidney Heart Lung
Saline Tidal volume Frequency Minute ventilation Aspartic acid Tidal volume Frequency Minute ventilation
Organ weights were multiplied by the body weight equivalent (1000 g/b. wt). Values are means _ SD. *%~tSignificantdifferences: *p < 0.01, tP < 0.001. ~ p < 0.05.
graph recorder. A "Y" tube attached to a 1 mm glass syringe connected in parallel to a pressure transducer port was used to calibrate the system. The same technique has been used in many experiments in our laboratory (14-17). We measured oxygen consumption using the flow-through method that consists of subtracting the fractional content of oxygen in the air exiting from the chamber from the fractional content of the air entering the chamber and multiplying the difference by the flow rate of air going through the chamber (15). Values were corrected to STPD conditions. The ventilatory parameters calculated included tidal volume, the frequency of breathing, and the product of these two parameters--minute ventilation. Both tidal volume and minute ventilation were normalized by multiplying each parameter by the kilogram body weight equivalent (1000 g/b. wt. in g). Oxygen consumption was expressed as milliliters of oxygen consumed per hour divided by the body weight equivalent. PROCEDURES The rat was first weighed and then injected subcutaneously with 0.2-0.3 ml of either saline or 580 mg/kg aspartic acid. This dose was selected because previously we showed that it depressed ventilation in male rats in an extensive dose-response-time experiment (17). After the rat was injected, he was placed into the chamber and ventilation and oxygen consumption were evaluated 45 rain later. All TP-treated and control rats who had received saline were then exposed to 3, 5, and 7% carbon dioxide in oxygen in a cumulative manner and ventilation evaluated at each level. After the rat was removed from the chamber, his body temperature was measured using a Sensottek thermometer with a thermocouple. To further confirm that TP treatment was effective, the rats were euthanized by an overdose of sodium pentobarbital and their testes, seminal vesicles, prostate gland, heart, lungs, and adrenal glands removed and weighed. The organ data were multiplied by the body weight equivalent. Data were analyzed using student's unpaired t-tests to determine if ventilatory and oxygen consumption values and organ and body weights were different between the TP and the control groups. A two-way analysis of variance with post hoc least means test was done to compare ventilatory and oxygen consumption responses of rats in each group to saline versus aspartic acid treatment. Significance was accepted at p < 0.05.
[P
2.23 +_0.50 83 +_ 17 179.7 +_34.8
1.95 +_0.29 90 _+9 174.6 +_23.0
1.77 _+0.32 80 _+ 16 139.4 _+23.0
1.70 _+0.35 78 +__18 129.0 _ 32.4
Values are means +_ SD. Units: tidal volume, ml; frequency, breaths per minute; minute ventilation,ml per minute. Both tidal volume and minute ventilation were corrected for body weight equivalent (see Table l). Significant decreases of tidal volume and minute ventilation at p < 0.01 were found comparing saline and aspartic acid values within each group, but not between groups.
RESULTS
Rats treated neonatally with TP weighed less and had lighter testes, prostate glands, and heart weights than did control animals (Table 1). Ventilation of the two groups breathing air were similar (Table 2). When rats were exposed to hypercapnia, ventilation was lower in the TP group at 5 and 7% carbon dioxide compared to those of control rats (Fig. l). These differences were predominantly due to a lower tidal volume rather than to a decrease in the frequency of breathing (Table 3). Unlike the dissimilar ventilatory responses of the two groups to hypercapnia, their response to aspartic acid was the same (Table 2). In both groups aspartic acid caused a marked depression of ventilation compared to their saline responses. The depression of ventilation was due to a lower tidal volume. Aspartic acid administration had no effect on oxygen consumption in either group relative to values obtained after saline injection (Table 4).
c~ O 4-~ cO -,-8
-,-4
(13 "~. ~> q_~
v
v
3
5
7
Percent Carbon Dioxide FIG. 1. Minute ventilation (VE) of control and testosterone propionate (TP) treated ratsexposed to increasinglevelsof carbon dioxide. S'~ffacant differenceswere noted at 5% (p < 0.01) and 7% (p < 0.05) carbon dioxide between animals in each group.
VENTILATION IN ANDROGENIZED MALE RATS TABLE 3 VENTILATORY RESPONSESOF TESTOSTERONE PROPIONATE (TP) AND CONTROL RATS TO HYPERCAPNIA Control Carbon dioxide,3% Tidal volume Frequency Carbon dioxide, 5% Tidal volume Frequency Carbon dioxide, 7% Tidal volume Frequency
TP
2.42 _+0.58 108 + 27
2.02 + 0.35 111 + 10
2.84 + 0.26 121 + 17
2.30 + 0.47* 126 ___15
3.30 + 0.41 128 + 18
2.63 + 0.54* 138 + 21
Values are means + SD. See Table 2 for units for each parameter. * Significant group difference ofp < 0.01 at each level ofhypercapnia.
DISCUSSION The major findings of this study are that TP treatment of male rat pups 1 day after birth results in hypogonadal adults who show a diminished ventilatory response to hypercapnia, but a similar response to aspartic acid compared to saline-treated animals. Schlenker and Goldman (17) reported that castration of postpubertal male rats did not affect their ventilatory response to aspartic acid compared to that of intact males. In conjunction with the results of the present study, we suggest that this response is not dependent upon the level of circulating steroids. In contrast, we have shown that treatment of female rat pups shortly after birth with TP caused a male-like ventilatory response to aspartic acid (l 8) suggesting that neonatal alterations in the organization of the brain appear necessary to elicit the sexually dimorphic ventilatory response to aspartic acid. Consequently, we can ask the question--will feminization of male brains with neonatal treatment of male rats with estradiol benzoate alter their ventilation in response to aspartic acid? In addition, this study has shown that the depression of ventilation in response to aspartic acid is not the consequence of a depression of oxygen consumption, since this parameter was not altered by aspartic acid in either group. Thus, aspartic acid appears to dissociate ventilation and oxygen consumption in male rats. The diminished ventilatory response of TP-treated male rats to hypercapnia may be related to their hypogonadal state. Schlenker and Goldman (15) reported that male rats made hypogonadal by the administration of high doses of aspartic acid shortly after birth also resulted in adult animals who exhibited decreased responses to hypercapnia. In another study, Young and his coworkers (22) suggested that incomplete expression of sexually dimorphic cellular characteristics of the preoptic-anterior hypothalamus of obese Zucker rats, due perhaps to an impaired process of perinatal brain androgenization, may contribute to the abnormal sexual behaviors noted in these animals (4). We found that adult, obese, male Zucker rats displayed significantly lower minute ventilation responses to hypercapnia than did their age-matched lean counterparts (14). The underlying mechanisms responsible for the diminished ventilatory response of obese male Zucker rats to hypercapnia are not known, but may be associated with altered chestwall mechanics due to abdominal fat loading, weakened respiratory muscles, and/or changes in their ability to sense carbon dioxide. The problems
583 using male, obese, aspartic-acid-treated rats or obese Zucker rats to elucidate the contribution hypogonadism makes to the control of ventilation include a number of confounding factors including altered hormone levels (such as insulin, thyroid, and growth hormones) as well as mechanical factors not seen in TP-treated male rats ( 15,21,22). The inability of TP-treated male rats to increase tidal volume in response to hypercapnia may reflect changes in central chemoreceptor characteristics and/or decrease the ability of respiratory muscles to generate the called-for tension. Evidence for the second possibility comes from a study by Jiang and Klueber (7) who evaluated the effect of postnatal castration of mice on muscle morphometry and contractility. The extensor digitorum longus muscles of castrated male rats were smaller, showed atrophic myofibers, necrosis, disruption of normal myofilament arrangement and also developed less tension than did muscles from intact animals. Burbach et al. (2) noted that the heart, diaphragm, and gastrocnemius from hypngonadal male rats who had been treated neonataUy with aspartic acid also weighed less and the two skeletal muscles had smaller fiber diameters than muscles from vehicle-treated animals. Moreover, the age at which castration occurs appears to influence greatly both the structural and functional development of skeletal and cardiac muscles (5,10,23). White and his colleagues (20) reported that their hypogonadal male patients displayed diminished ventilatory responses to both hypoxic and hypercapnic challenges. Following testosterone replacement the patients increased their metabolic rate and ventilatory response to hypoxia but not to hypercapnia. Neither the magnitude of the tidal volume nor frequency of breathing responses to either challenge were reported in this study. Were central chemoreceptors for carbon dioxide and/or respiratory muscle function altered by low testosterone levels in these patients? Additional evidence that the level of plasma testosterone (2,3) can influence ventilatory responses comes from studies on the control of ventilation in awake, aged male rats who exhibit decrements in ventilatory responses to hypercapnia compared to younger males (16). Recently Lawler et al. reported that the diaphragm/body weight ratios in 9-month-old Spague-Dawley rats were significantly lower than in age-matched female rats (8). Thus, it is possible that in male rats decreased levels of testosterone may alter control of ventilation by the effect on respiratory muscle function. CONCLUSION How neonatal androgenization of male rats leads to decreased ventilatory responses to hypercapnia clearly needs to be investigated further. Studies of respiratory muscle structure and func-
TABLE 4 OXYGEN CONSUMPTION VALUESOF TESTOSTERONE PROPIONATE (TP) AND CONTROL RATS GIVEN SALINE AND ASPARTICACID
Saline Aspartic acid
Control
TP
138.0 ___35.1 142.7 _+23.1
132.5 ___37.9 136.1 _+32.9
Values are means _+SD and were corrected by the body weight equivalent. Units are ml of oxygen consumed per hour. There were no significant differences between the groups as a consequence of treatment.
584
SCHLENKER, GOLDMAN AND HOLMAN
tion in neonatal TP-treated male rats at various ages may be instrumental in determining whether the diminished ventilatory response to hypercapnia is due to weakened muscles and/or to changes in central control mechanisms modulating ventilation.
Finally, testosterone replacement at different doses may allow us to ascertain the reversibility these early neuroendocrine perturbations have on the control of ventilation in TP-treated male rats.
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12. Piacsek, B. E.; Histetter, M. W. Neonatal androgenization in the male rat: Evidence for central and peripheral defects. Biol. Reprod. 30:344-351; 1984. 13. Sachs, B.; Thomas, D. A. Differential effects of perinatal androgen treatment on sexually dimorphic characteristics in rats. Physiol. Behay. 34:735-742; 1985. 14. Sehlenker, E. H. Ventilatory control in lean and obese Zucker rats. FASEB J. 4:A1100; 1990. 15. Schlenker, E. H.; Goldman, M. Aspartic acid administered neonatally affects ventilation of male and female rats differently. J. Appl. Physiol. 61:780-784; 1986. 16. Schlenker, E. H.; Goldman, M. Ventilatory responses of aged male and female rats to hypercapnia and to hypoxia. Gerontology 31: 301-308; 1988. 17. Schlenker, E. H.; Goldman, M. Acute effects of aspartic acid on ventilation of male and female rats. Physiol. Behav. 42:313-318; 1988. 18. Schlenker, E. H.; Goldman, M.; Holman, G. No effects of aspartic acid on ventilation in androgenized and ovariectomized female rats. J. Appl. Physiol. (in press). 19. Swanson, H. E.; van der Werfften, B. The "early-androgen" syndrome: Difference in the responses to prenatal and postnatal administration of various doses of testosterone to female and male rats. Acta. Endocin. 47:37-50; 1964. 20. White, D. P.; Seheinder, B. K.; Santen, R. J.; McDermott, M.; Pickett, C. K.; Zwillich, C. W.; Weil, J. V. Influence of testosterone on ventilation and chemosensitivity in male subjects. J. Appl. Physiol. 59: 1452-1457; 1985. 21. Wittels, E. H. Obesity and hormonal factors in sleep and sleep apnea. Med. Clin. N. Am. 69:1265-1280; 1985. 22. Young, J. K.; Hemming, M. W.; Matsumoto, D. E. Sexual behavior and the sexually dimorphic hypothalamic nucleus of the male Zucker rats. Physiol. Behav. 36:881-886; 1986. 23. Yu, W. A. Sex differences in neuronal loss induced by axotomy in the rat brainstem motor nuclei. Exp. Neuol. 102:230-235; 1988.
