Physiology&Behavior,Vol. 52, pp. 1113-1116, 1992
0031-9384/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd.
Printed in the USA.
Perinatal Estradiol Benzoate Administration Affects Control of Ventilation in Adult Male Rats EVELYN
H . S C H L E N K E R , *l M A X
GOLDMANt
AND
SUSAN
WALSH~f
Departments of*Physiology and Pharmacology and tBiology, The University of South Dakota, Vermillion, SD 5 7069 R e c e i v e d 13 A p r i l 1992 SCHLENKER, E. H., M. GOLDMAN AND S. WALSH. Perinatalestradiolbenzoate administration affects controlof ventilation in adult male rats. PHYSIOL BEHAV 52(6) I 113-1116, 1992.--One injection of estradiol benzoate (EB) (100/~g) or vehicle was administered to male rat pups 5 days after birth. Two months later ventilation, ventilatory responses to 7% carbon dioxide, and to 580 mg/kg aspartic acid (an agent used as a marker of sexually dimorphism in the control of ventilation) were evaluated and body weight, testes weight, and nose-anus length were measured in animals in each group. The EB-treated rats had similar tidal volumes, frequency of breathing, and minute ventilation as did control male rats. The ventilatory responses of EB-treated rats to hypercapnia were markedly less than those of control animals. Aspartic acid administration depressed ventilation in control animals, but had no effect on ventilation in EB-trcated males. Body and testes weights, as well as nose-anus length, were less in EB-treated compared with control rats. However, when body weight was normalized by nose-anus length and testes weight was normalized by body weight, the values were comparable between the two groups. Thus, perinatal EB treatment of male rat pups results in small, hypogonadal adult animals whose ventilation in response to hypercapnia was diminished and whose response to aspartic acid was female-like relative to those of control rats. Estradiol benzoate
Control of ventilation
Critical periods
E S T R A D I O L benzoate (EB) administered to neonatal male rats affects subsequent development of sexual behavior and peripheral sex organs, causes a reduction of growth hormone and testicular hormones, and decreases gonadotropin synthesis and release in the hypothalamus (1,6-9,11,19,22). The effect this treatment has on control of ventilation has not been documented. Recently we showed that hypogonadism induced by neonatal administration of testosterone propionate (TP) to male rat pups resulted in adult animals whose ventilation in response to hypercapnic gas challenges was markedly diminished compared to those of control males (17). Furthermore, when both groups were given a dose of 580 mg/kg aspartic acid subcutaneously, ventilation was depressed to a similar extent in both groups. Aspartic acid administered as a bolus causes a long-lasting depression of ventilation in intact and also in postpubertal castrated adult male rats, but not in intact female rats (16). Thus, we have used aspartic acid administration as marker of sexual dimorphism in the control of ventilation. The purpose of this study was to test the hypothesis that male rats treated perinatally with EB would exhibit as adults a female-like ventilatory response to acute aspartic acid administration and that their ventilation
1Requests for reprints should be addressed to Evelyn H. Schlenker.
1113
Male rats
Aspartic acid
in response to hypercapnia would be decreased in a similar manner to that noted in hypogonadal male rats ( 15,17). METHOD Sprague-Dawley rats (Sasco, Omaha, NE) were bred in our laboratory. Day 5 after birth (where the day of birth is considered day l), male rat pups received one subcutaneous injection of either EB (100 #g per rat) or an equal volume of sesame oil, the vehicle. All rats were studied at 2 months of age. Eight control (vehicle-treated) and eight EB-treated male rats were utilized in these experiments. Rats were selected for this experiment so that EB-treated and control animals were chosen from the same litters. Animals were housed in groups of two in steel mesh cages in a temperature-controlled animal facility on a 12-h on/12-h off lighting regime. Food and water were available ad lib. Ventilation was determined in awake rats placed into a cylindrical 30 cm long by 14 diameter Plexiglas chamber utilizing the principle of Boyle's Law. One side of the chamber contained ports to: 1. measure the flow rate of air or carbon dioxide going through the chamber using a G i l m o n t rotameter;
1114
S ( ' H L E N K E R , (}()LDMAN !\NI) W,\I.S[t
FABLE 1
1ABLf! 2
BODY WEIGHTS, "IESTES WEIGHTS, NOSE-ANUS LENGTH
VEN ['ILA IORY PARAMETERS IN CONI RO] AND EB-TREATED MALE RAJS
IN CONTROLAND EB-TREATEDMALE RATS Control Body weight (g) Nose-anus length (cm) Lee lndext Testes weight (g) Testes/body weight
332.5 23.6 29.3 1.62 0.46
_+ 9,5 _+ 0.2 _+ 0.3 _+0.07 _+0.04
EB 264.0 _~ 10.6" 21.4 + 0.1" 29.9 _+ 0.4 0.93 + 0.23:~ (I.40 + 0.03
Values are mean _+ SEM. * Significant differences between groups at p < 0.001. ? The Lee Index is the cubed root of body weight divided by the noseanus length multiplied by 100. ~t Significant differences between groups at p < 0.01.
2. measure the chamber temperature and relative humidity using a Digitec digital thermometer and a Cole-Parmer hygrometer; and 3. measure the fractional content of carbon dioxide exiting the chamber using a Beckman LB-2 carbon dioxide analyzer. The other side of the chamber was sealed with a No. 15 rubber stopper that contained ports to: 1. allow air or carbon dioxide to flow through the chamber; 2. measure pressure changes associated with ventilation in the rats using a low pressure transducer (Statham) coupled with a Grass polygraph recording system; and 3. calibrate the system using a l-ml glass syringe. Additional characteristics of this system have been previously described (20).
Procedures Each rat was weighed and then injected subcutaneously with 0.2-0.3 ml of saline one week and 580 mg]kg aspartic acid the following week. This dose of aspartic acid was chosen based upon an extensive dose-response time study (16). After injection, the rat was placed into the chamber and ventilation was evaluated 45 rain later. Rats that had received saline were then exposed to 7% carbon dioxide in oxygen, and their ventilatory responses were measured. Subsequently, they were removed from the chamber to measure body temperature using a Sensortek thermometer and small animal thermocouple. In addition, noseanus length was measured. This variable was used with the body weight to calculate the Lee Index (the cubed root of body weight divided by the nose-anus length). The Lee Index normalizes body weight by body length and has been used extensively to evaluate obesity in animals (15). Ventilatory parameters that were evaluated in this study included tidal volume, frequency of breathing, and minute ventilation. Following testing, the rats were sacrificed by an overdose of sodium pentobarbital and their testes removed and immediately weighed. The organ data were expressed both in terms of absolute weights and as relative weights by normalizing them by body weight. Data were analyzed using Student's unpaired t-tests to determine if ventilatory data and body and organ weights were different between the EB and control groups. A two-way analysis of variance (ANOVA) with a posteriori least means square test was utilized to compare ventilatory parameters of rats in each group in response to saline versus aspartic acid treatment. Significance was accepted at p < 0.05.
Tidal volume (ml) Frequency (bpm) Minute ventilation (ml/min)
('ontrol
EB
1.09 -t 0. I 0 1~5 + 10 225.6 + 13.0
1.43 : 0.07 140 -: 8 197.7 ± 8.4
Values are mean _+ SEM.
RESULTS
Rats treated perinatally with EB weighed less, were shorter, and had lighter testes (Table l). When body weight was normalized by body length (the Lee Index) and testes weight was normalized by body weight, there were no significant differences noted between the two groups in these variables. Ventilatory parameters determined 45 min after saline injection, including tidal volume, frequency of breathing, and minute ventilation, were not different between the two groups (Table 2). Whereas both groups increased their ventilation in response to the hypercapnic challenge, the minute ventilation of the control group was 144% greater than that of the EB group due to larger tidal volumes and the frequency of breathing in the former group (Table 3). Control rats decreased their minute ventilation in response to the administration of 580 mg/kg aspartic acid compared to their ventilation after saline administration (Fig. 1). The frequency of breathing dropped from 135 _+ 10 breaths per min to 105 _ 7 breaths per min (p < 0.001). In contrast, the EB group showed no significant drop in either minute ventilation (Fig. l) or in the frequency of breathing (140 _+ 8 to 129 _+ 7 breaths per min, p > 0.5). DISCUSSION The major findings in this study are that EB affects body size and weight, and testicular growth, as well as control of ventilation. The EB-treated animals responded to a hypercapnic challenge with markedly lower minute ventilation compared with control animals' response. In addition, the male-like minute ventilation response to aspartic acid was not seen in the EB group. Hypogonadism has been associated with a diminished ventilatory response to hypercapnia in both rats and men (14,15,20,21). Treatment of male rat pups with TP 1 day after birth induces hypogonadism in adult rats (13). The ventilation
TABLE 3 VENT1LATORYPARAMETERSOF CONTROL AND EB-TREATED MALE RATSIN RESPONSETO 7% CARBONDIOXIDE
Tidal volume (ml) Frequency (bpm) Minute ventilation (ml/min)
Control
EB
2.46 _+ 0.1 l 175 _+ 8 427.8 _+ 21.1
2.09 _+_0.10" 143 _ 7t 296.8 +- 14.5~
Values are mean _+ SEM. * Significant differences between groups at p < 0.01. # Significant differences between groups at p < 0.05. Significant differences between groups at p < 0.00 l.
VENTILATION IN ESTROGEN-TREATED MALE RATS
~'E (ml/mln) 25O
-i-
[]
Saline
[]
Aspartic Acid
-I-
20O
150
100 / / / / / J / / /
////////i /////////
/ / / / / / / / /
Y//////A
50 //////.-.-/1
/J/J/J/J/ / / / / / / / / / //J/J//Jl IIiiiii1~ FI/////11
0
Control
EB
FIG. 1. The minute ventilation (VE)response of control and EB-treated male rats to saline (white bars) and to aspartic acid (hatched bars). The asterisk indicates a significantdecrease (p < 0.01) in minute ventilation in the control rats after administration of 580 mg/kg aspartic acid versus saline. of TP animals in response to a hypercapnic challenge was significantly less than that of vehicle-treated control rats (17). Schlenker and Goldman (l 5) showed that male rats treated neonatally with high doses of aspartic acid resulted in adult animals who were hypogonadal, obese, and stunted in growth. Furthermore, these males hypoventilated and exhibited a markedly diminished ventilatory response to hypercapnia compared to controls. Interestingly, female rats also treated neonatally with aspartic acid did not exhibit any of the ventilatory abnormalities seen in male rats. Moreover, White and his colleagues (20) noted that the response of hypogonadal patients to hypoxic and to hypercapnic challenges was lower than those in normal adult males. Testosterone replacement increased the oxygen consumption and the ventilatory responses of the patients to hypoxia, but had no effect on their responses to hypercapnia. The EB-treated rats exhibited a smaller tidal volume and lower frequency of breathing in response to hypercapnia. Re-
1115 spiratory muscles and/or central nervous system characteristics may have been altered in EB-treated animals. In castrated male rats, obese male Zucker rats, and neonatally aspartic acid-treated male rats, skeletal muscles are lighter and have smaller fiber diameters than do their respective controls (2-4,10). Structural and functional studies of EB-treated rat diaphragms may be useful to determine to what extent alterations in these structures may contribute to diminished minute ventilation in response to hypercapnia. Moreover, the decreased frequency of breathing exhibited by EB-treated rats in response to hypercapnia suggests that alterations in the central nervous system perception of elevated levels of carbon dioxide or drive in response to carbon dioxide contribute to the diminished minute ventilation noted in these animals. Electrophysiological characterization of cells in the respiratory control centers from EB-treated and control rats with and without exposure to hypercapnia may help explain some very fundamental differences noted in this study. Another new finding in this study is that male rats treated with EB perinatally exhibited a female-like minute ventilation in response to aspartic acid. Neither postpubertal castration nor neonatal treatment of male rats with TP (which results in hypogonadal adult males) altered their minute ventilation response to aspartic acid compared with that of intact males (l 5,16). Importantly, neonatal treatment of female rats with TP results in adult rats that show a male-like ventilatory response to aspartic acid (18). These results, taken together with those in the present study, suggest that steroid hormone perturbations during critical periods of brain development alter sexual dimorphic characteristics associated with the control of ventilation in rats. Once the organization of brain regions has been designated, additional perturbations may not alter the ventilatory response of rats to aspartic acid or hypercapnia. To test this hypothesis, the effects of testosterone replacement at different ages in EB-treated male rats on control of ventilation needs to be investigated. In conclusion, EB treatment of male rat pups alters their normal, adult male-like ventilatory responses both to hypercapnia and to aspartic acid, suggesting that brain reorganization occurring at a critical period in development results in longterm perturbations in their control of ventilation.
REFERENCES 1. Arnold, A. P.; Gorski, R. A. Gonadal steroid induction of structural sex differencesin the central nervous system. Annu. Rev. Neurosci. 35:413-442; 1984. 2. Burbach, J. A.; Schlenker, E. H.; Goldman, M. Characterization of muscles from aspartic acid obese rats. Am. J. Physiol. 249:R106110; 1985. 3. Campion, D. R.; Purchas, R. W.; Merkel, R. W.; Romsos, D. R. Genetic obesity and muscle satellitecell. Proc. Soc. Exp. Biol. Med. 176:143-147; 1984. 4. Durschlag, R. P.; Layman, D. K. Skeletal muscle growth in lean and obese Zucker rats. Growth 47:282-291; 1983. 5. Dux, L.; Dux, E.; Guba, F. Further data on the androgenic dependency of skeletal musculature: The effect of prepubertal castration on the structuraldevelopmentof the skeletal muscles. Horm. Metab. Res. 14:191-194; 1982. 6. Farber, K. A.; Basham, K.; Hughes, C. L., Jr. The effect of neonatal exposure to DES and O, P'-DDT on pituitary responsiveness to GnRH in adult castrated rats. Reprod. Toxicol. 5:363-369; 199 I. 7. Frick, J.; Chand, C. C.; Kind, FI A. Testosterone plasma levels in adult male rats injected with oestradiol benzoate or testosterone propionate. Steroids 13:21-27; 1969.
8. Hendricks, S. E.; Gerall, A. A. Effect of neonatally administered estrogen on development of male and female rats. Endocrinology 87:435-439; 1970. 9. Jansson, J-O.; Eden, S.; Isaksson, O. Sexual dimorphism in the control of growth hormone secretion. Endocr. Rev. 6:128-150; 1985. 10. Jiang, B.; Klueber, M. K. Structural and functional analysis of murine skeletal muscle after castration. Muscle Nerve 12:67-77; 1989. 11. Kind, F. A.; Maqueco, M.; Folch-Pi, A. Recoveryof gonadal function in male rats treated neonatally with 17 B-oestradiolbenzoate. Acta Endocrinol. 47:200-208; 1964. 12. Maiter, D. J.; Koenig, I.; Kaplan, L. M. Sexuallydimorphic expression of growth hormone releasinghormone gene is not mediated by circulatinggonadal hormonesin adult rats. Endocrinology128:17091716; 1991. 13. Piacsek, B. E.; Hostetter, M. W. Neonatal androgenization in the male rat: Evidence for central and peripheral defects. Biol. Reprod. 30:344-351; 1984. 14. Schlenker, E. H. Ventilatory control in lean and obese Zucker rats. FASEB J. 4:A1100; 1990.
1116
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. Acute effects of aspartic acid on ventilation of male and female rats. Physiol. Behav. 42:313-318; 1988. 17. Schlenker, E. H.: Goldman, M.; Holman, G. Control of ventilation in hypogonadal, androgenized male rats. Physiol. Behav. Physiol. Behav. 51:581-584; 1992. 18. Schlenker, E. H.; Goldman, M.; Holman, G. The effects ofaspartic acid on ventilation in androgenized and ovariectomized female rats. J. Appl. Physiol. 76:2255-2258~ 1992.
SCHLENKER,
(iOI,DMAN
ANI) WAI,S[I
19. Werner, H . Koch, F.; Baldine, t.: (iozes, 1. Steroid regulation ol somatostatin mRNA in the rat hypothalamus. J. Biol. (7hem, 2¢'~: 7666-7671: 1988. 20. White, D. P.; Scheinder, B. K.: Santen, R. J.; McDermotl, M.; I>ickctl. C. K.: Zwillich, C. W.; Weil, J. V. Influence of testosterone on ventilation and chemosensitivity in male subjects..1. A,ppl, Physiol 5c): 1452-1457: 1985. 21. Wittels, E. H. Obesity and hormonal factors in sleep and sleep apnea. Med. Clin. North Am. 69:1265-1280; 1985. 22. Zadina, J. E.; Dunlap, J. L.; Geall, A. A. Modifications induced by neonatal steroids in reproductive organs and behavior of male rats. J. Comp. Physiol. Psychol. 93:314-322: 1979.
0031-9384/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd.
Printed in the USA.
Perinatal Estradiol Benzoate Administration Affects Control of Ventilation in Adult Male Rats EVELYN
H . S C H L E N K E R , *l M A X
GOLDMANt
AND
SUSAN
WALSH~f
Departments of*Physiology and Pharmacology and tBiology, The University of South Dakota, Vermillion, SD 5 7069 R e c e i v e d 13 A p r i l 1992 SCHLENKER, E. H., M. GOLDMAN AND S. WALSH. Perinatalestradiolbenzoate administration affects controlof ventilation in adult male rats. PHYSIOL BEHAV 52(6) I 113-1116, 1992.--One injection of estradiol benzoate (EB) (100/~g) or vehicle was administered to male rat pups 5 days after birth. Two months later ventilation, ventilatory responses to 7% carbon dioxide, and to 580 mg/kg aspartic acid (an agent used as a marker of sexually dimorphism in the control of ventilation) were evaluated and body weight, testes weight, and nose-anus length were measured in animals in each group. The EB-treated rats had similar tidal volumes, frequency of breathing, and minute ventilation as did control male rats. The ventilatory responses of EB-treated rats to hypercapnia were markedly less than those of control animals. Aspartic acid administration depressed ventilation in control animals, but had no effect on ventilation in EB-trcated males. Body and testes weights, as well as nose-anus length, were less in EB-treated compared with control rats. However, when body weight was normalized by nose-anus length and testes weight was normalized by body weight, the values were comparable between the two groups. Thus, perinatal EB treatment of male rat pups results in small, hypogonadal adult animals whose ventilation in response to hypercapnia was diminished and whose response to aspartic acid was female-like relative to those of control rats. Estradiol benzoate
Control of ventilation
Critical periods
E S T R A D I O L benzoate (EB) administered to neonatal male rats affects subsequent development of sexual behavior and peripheral sex organs, causes a reduction of growth hormone and testicular hormones, and decreases gonadotropin synthesis and release in the hypothalamus (1,6-9,11,19,22). The effect this treatment has on control of ventilation has not been documented. Recently we showed that hypogonadism induced by neonatal administration of testosterone propionate (TP) to male rat pups resulted in adult animals whose ventilation in response to hypercapnic gas challenges was markedly diminished compared to those of control males (17). Furthermore, when both groups were given a dose of 580 mg/kg aspartic acid subcutaneously, ventilation was depressed to a similar extent in both groups. Aspartic acid administered as a bolus causes a long-lasting depression of ventilation in intact and also in postpubertal castrated adult male rats, but not in intact female rats (16). Thus, we have used aspartic acid administration as marker of sexual dimorphism in the control of ventilation. The purpose of this study was to test the hypothesis that male rats treated perinatally with EB would exhibit as adults a female-like ventilatory response to acute aspartic acid administration and that their ventilation
1Requests for reprints should be addressed to Evelyn H. Schlenker.
1113
Male rats
Aspartic acid
in response to hypercapnia would be decreased in a similar manner to that noted in hypogonadal male rats ( 15,17). METHOD Sprague-Dawley rats (Sasco, Omaha, NE) were bred in our laboratory. Day 5 after birth (where the day of birth is considered day l), male rat pups received one subcutaneous injection of either EB (100 #g per rat) or an equal volume of sesame oil, the vehicle. All rats were studied at 2 months of age. Eight control (vehicle-treated) and eight EB-treated male rats were utilized in these experiments. Rats were selected for this experiment so that EB-treated and control animals were chosen from the same litters. Animals were housed in groups of two in steel mesh cages in a temperature-controlled animal facility on a 12-h on/12-h off lighting regime. Food and water were available ad lib. Ventilation was determined in awake rats placed into a cylindrical 30 cm long by 14 diameter Plexiglas chamber utilizing the principle of Boyle's Law. One side of the chamber contained ports to: 1. measure the flow rate of air or carbon dioxide going through the chamber using a G i l m o n t rotameter;
1114
S ( ' H L E N K E R , (}()LDMAN !\NI) W,\I.S[t
FABLE 1
1ABLf! 2
BODY WEIGHTS, "IESTES WEIGHTS, NOSE-ANUS LENGTH
VEN ['ILA IORY PARAMETERS IN CONI RO] AND EB-TREATED MALE RAJS
IN CONTROLAND EB-TREATEDMALE RATS Control Body weight (g) Nose-anus length (cm) Lee lndext Testes weight (g) Testes/body weight
332.5 23.6 29.3 1.62 0.46
_+ 9,5 _+ 0.2 _+ 0.3 _+0.07 _+0.04
EB 264.0 _~ 10.6" 21.4 + 0.1" 29.9 _+ 0.4 0.93 + 0.23:~ (I.40 + 0.03
Values are mean _+ SEM. * Significant differences between groups at p < 0.001. ? The Lee Index is the cubed root of body weight divided by the noseanus length multiplied by 100. ~t Significant differences between groups at p < 0.01.
2. measure the chamber temperature and relative humidity using a Digitec digital thermometer and a Cole-Parmer hygrometer; and 3. measure the fractional content of carbon dioxide exiting the chamber using a Beckman LB-2 carbon dioxide analyzer. The other side of the chamber was sealed with a No. 15 rubber stopper that contained ports to: 1. allow air or carbon dioxide to flow through the chamber; 2. measure pressure changes associated with ventilation in the rats using a low pressure transducer (Statham) coupled with a Grass polygraph recording system; and 3. calibrate the system using a l-ml glass syringe. Additional characteristics of this system have been previously described (20).
Procedures Each rat was weighed and then injected subcutaneously with 0.2-0.3 ml of saline one week and 580 mg]kg aspartic acid the following week. This dose of aspartic acid was chosen based upon an extensive dose-response time study (16). After injection, the rat was placed into the chamber and ventilation was evaluated 45 rain later. Rats that had received saline were then exposed to 7% carbon dioxide in oxygen, and their ventilatory responses were measured. Subsequently, they were removed from the chamber to measure body temperature using a Sensortek thermometer and small animal thermocouple. In addition, noseanus length was measured. This variable was used with the body weight to calculate the Lee Index (the cubed root of body weight divided by the nose-anus length). The Lee Index normalizes body weight by body length and has been used extensively to evaluate obesity in animals (15). Ventilatory parameters that were evaluated in this study included tidal volume, frequency of breathing, and minute ventilation. Following testing, the rats were sacrificed by an overdose of sodium pentobarbital and their testes removed and immediately weighed. The organ data were expressed both in terms of absolute weights and as relative weights by normalizing them by body weight. Data were analyzed using Student's unpaired t-tests to determine if ventilatory data and body and organ weights were different between the EB and control groups. A two-way analysis of variance (ANOVA) with a posteriori least means square test was utilized to compare ventilatory parameters of rats in each group in response to saline versus aspartic acid treatment. Significance was accepted at p < 0.05.
Tidal volume (ml) Frequency (bpm) Minute ventilation (ml/min)
('ontrol
EB
1.09 -t 0. I 0 1~5 + 10 225.6 + 13.0
1.43 : 0.07 140 -: 8 197.7 ± 8.4
Values are mean _+ SEM.
RESULTS
Rats treated perinatally with EB weighed less, were shorter, and had lighter testes (Table l). When body weight was normalized by body length (the Lee Index) and testes weight was normalized by body weight, there were no significant differences noted between the two groups in these variables. Ventilatory parameters determined 45 min after saline injection, including tidal volume, frequency of breathing, and minute ventilation, were not different between the two groups (Table 2). Whereas both groups increased their ventilation in response to the hypercapnic challenge, the minute ventilation of the control group was 144% greater than that of the EB group due to larger tidal volumes and the frequency of breathing in the former group (Table 3). Control rats decreased their minute ventilation in response to the administration of 580 mg/kg aspartic acid compared to their ventilation after saline administration (Fig. 1). The frequency of breathing dropped from 135 _+ 10 breaths per min to 105 _ 7 breaths per min (p < 0.001). In contrast, the EB group showed no significant drop in either minute ventilation (Fig. l) or in the frequency of breathing (140 _+ 8 to 129 _+ 7 breaths per min, p > 0.5). DISCUSSION The major findings in this study are that EB affects body size and weight, and testicular growth, as well as control of ventilation. The EB-treated animals responded to a hypercapnic challenge with markedly lower minute ventilation compared with control animals' response. In addition, the male-like minute ventilation response to aspartic acid was not seen in the EB group. Hypogonadism has been associated with a diminished ventilatory response to hypercapnia in both rats and men (14,15,20,21). Treatment of male rat pups with TP 1 day after birth induces hypogonadism in adult rats (13). The ventilation
TABLE 3 VENT1LATORYPARAMETERSOF CONTROL AND EB-TREATED MALE RATSIN RESPONSETO 7% CARBONDIOXIDE
Tidal volume (ml) Frequency (bpm) Minute ventilation (ml/min)
Control
EB
2.46 _+ 0.1 l 175 _+ 8 427.8 _+ 21.1
2.09 _+_0.10" 143 _ 7t 296.8 +- 14.5~
Values are mean _+ SEM. * Significant differences between groups at p < 0.01. # Significant differences between groups at p < 0.05. Significant differences between groups at p < 0.00 l.
VENTILATION IN ESTROGEN-TREATED MALE RATS
~'E (ml/mln) 25O
-i-
[]
Saline
[]
Aspartic Acid
-I-
20O
150
100 / / / / / J / / /
////////i /////////
/ / / / / / / / /
Y//////A
50 //////.-.-/1
/J/J/J/J/ / / / / / / / / / //J/J//Jl IIiiiii1~ FI/////11
0
Control
EB
FIG. 1. The minute ventilation (VE)response of control and EB-treated male rats to saline (white bars) and to aspartic acid (hatched bars). The asterisk indicates a significantdecrease (p < 0.01) in minute ventilation in the control rats after administration of 580 mg/kg aspartic acid versus saline. of TP animals in response to a hypercapnic challenge was significantly less than that of vehicle-treated control rats (17). Schlenker and Goldman (l 5) showed that male rats treated neonatally with high doses of aspartic acid resulted in adult animals who were hypogonadal, obese, and stunted in growth. Furthermore, these males hypoventilated and exhibited a markedly diminished ventilatory response to hypercapnia compared to controls. Interestingly, female rats also treated neonatally with aspartic acid did not exhibit any of the ventilatory abnormalities seen in male rats. Moreover, White and his colleagues (20) noted that the response of hypogonadal patients to hypoxic and to hypercapnic challenges was lower than those in normal adult males. Testosterone replacement increased the oxygen consumption and the ventilatory responses of the patients to hypoxia, but had no effect on their responses to hypercapnia. The EB-treated rats exhibited a smaller tidal volume and lower frequency of breathing in response to hypercapnia. Re-
1115 spiratory muscles and/or central nervous system characteristics may have been altered in EB-treated animals. In castrated male rats, obese male Zucker rats, and neonatally aspartic acid-treated male rats, skeletal muscles are lighter and have smaller fiber diameters than do their respective controls (2-4,10). Structural and functional studies of EB-treated rat diaphragms may be useful to determine to what extent alterations in these structures may contribute to diminished minute ventilation in response to hypercapnia. Moreover, the decreased frequency of breathing exhibited by EB-treated rats in response to hypercapnia suggests that alterations in the central nervous system perception of elevated levels of carbon dioxide or drive in response to carbon dioxide contribute to the diminished minute ventilation noted in these animals. Electrophysiological characterization of cells in the respiratory control centers from EB-treated and control rats with and without exposure to hypercapnia may help explain some very fundamental differences noted in this study. Another new finding in this study is that male rats treated with EB perinatally exhibited a female-like minute ventilation in response to aspartic acid. Neither postpubertal castration nor neonatal treatment of male rats with TP (which results in hypogonadal adult males) altered their minute ventilation response to aspartic acid compared with that of intact males (l 5,16). Importantly, neonatal treatment of female rats with TP results in adult rats that show a male-like ventilatory response to aspartic acid (18). These results, taken together with those in the present study, suggest that steroid hormone perturbations during critical periods of brain development alter sexual dimorphic characteristics associated with the control of ventilation in rats. Once the organization of brain regions has been designated, additional perturbations may not alter the ventilatory response of rats to aspartic acid or hypercapnia. To test this hypothesis, the effects of testosterone replacement at different ages in EB-treated male rats on control of ventilation needs to be investigated. In conclusion, EB treatment of male rat pups alters their normal, adult male-like ventilatory responses both to hypercapnia and to aspartic acid, suggesting that brain reorganization occurring at a critical period in development results in longterm perturbations in their control of ventilation.
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