Journal of the Autonomic Nervous System, 17 (1986) 85-100 85 Elsevier

JAN 00645

Testosterone application influences sympathetic activity of intracardiac nerves in non-trained and

trained mice Georg Har tmann 1, Klaus Addicks 2, Manfred Donike 3

and Wilhelm Sch~inzer 3 I Institutff~r Experimentelle Morphologie der Deutschen Sporthochschule K~ln (FR.G.) 2 Anatomisches lnstitut der Universitht K~ln (F. R. G.) and ~ lnstitut fftr Biochemie der Deutschen Sporthochschule K'61n

(F.R.G.)

(Received 28 May 1985 (Revised version received 21 April 1986)

(Accepted 5 May 1986)

K e y words." Testos te rone - Tra in ing - Sympathe t i c nerve fiber - M y o c a r d i u m Norad rena l i ne - N e m a t o s o m e

Abstract

Appl i ca t ion of tes tos terone a n d / o r physical exercise causes degenera t ive and then regenerat ive pa t te rns of in t raca rd iac sympathe t i c neurons. Observa t ions in 3 stages (1, 3 and 6 weeks) i l lustrate the adap ta t ive changes of sympathe t i c neurons as a response to these st imuli and show that the effects fol lowing tes tos terone appl ica- t ion or physical exercise are comparab le . Ul t ras t ruc tu ra l invest igat ions indicate that the sympathe t i c neurons are more sensit ive to tes tos terone than to physical exercise. The combina t ion of tes tos terone plus t ra ining indicates over lapp ing effects of these two stimuli. The system of adrenergic nerve fibers seems to be overs t imula ted . Its react ion pa t t e rn is found not only to depend on t ime but also on the in tensi ty of the st imuli .

Introduction

The admin i s t r a t ion of tes tos terone increases the number and the size of gan- gl ionic cells in neona ta l female rats [15,23,50] and modif ies nervous t ransmiss ion

Correspondence: G. Hartmann, Bayer AG, Pharma EP-V, PMA 1, Zentrales Marketing, D-5090 Leverku- sen, Bayerwerk, F.R.G.

0165-1838/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

86

[18,34,47]. Furthermore, it has a marked effect on the total number of acetylcholine receptors [9] and interacts with catecholamine receptors [41]. The application of exogenous steroid hormones influences metabolism [30,31], and an additional treadmill training reveals enhancing effects of these steroids [4,5,21,32,35]. As yet no findings have been published on the effect of steroid hormone on the autonomic nervous system in combination with physical exercise. The discussion of the effects of physical training on the catecholamine content of the hearts of laboratory animals has been quite controversial. Some investigators report an increase in catecholamine content [3,38,39], others a degrease [13,19,25]. All these investigations have been carried out on the basis of just one training period within a well-defined period. As it is important to demonstrate the time-dependent reactions of the terminal nerve fibers we defined 3 s tages-- to study the effects of (1) training, (2) testosterone and (3) testosterone and training on cardiac adrenergic nerve fibers.

Materials and Methods

This study was carried out on 119 female mice (NMRI) which, at the beginning of the experiments, were 10 weeks old. The mice lived under standard laboratory conditions with a constant l ight-dark cycle of 12 h, constant temperature of 21°C and humidity (50 + 5%). The animals were divided into 4 groups: a control group (C), a group that underwent a training program (T), one that received testosterone proprionate (TP), and finally one that was trained under simultaneous influence of testosterone propionate (TPT). Twice a week the animal groups TP and TPT received i.m. injections of testosterone propionate (Eifelfango, Bad Neuenahr, 3 mg/kg /week) dissolved in sesame oil. The control groups (C) were given sesame oil only. There were no differences in the nocturnal activity of the 4 groups. The numbers of animals used in each experiment are shown in Figs. 3 and 4.

Training program For about 1 week the exercise groups (T, TPT) had to undergo special training

programs to get accustomed to (slow) treadmill running. Within the training period itself the speed of treadmill running was increased to a maximum of 33 m / m i n (constant slopes 3 %). The exercise program--carr ied out 5 times a week--lasted for 1, 3 and 6 weeks. The mice ran at the same time each day and were killed the day after the last exercise.

Electron microscopy After heparinization with Liquemin 5000 (Hof fmann-La Roche, Grenzach-

Whylen), the animals were anesthetized with Nembutal (Abbott, Ingelheim) and perfused through the right ventricle with a solution of 2.5% glutaraldehyde in 0.1 M cacodylate buffer at pH 7.4 for 30 rain. After sufficient fixation of the heart, small pieces of the right ventricle were removed and postfixed in 1% osmium tetroxide, dehydrated in graded alcohol, contrasted in 1% uranyl acetate and 0.5% phos- photungstic acid and embedded in Epon 812. Ultrathin sections were examined in a

87

Zeiss EM 10A electron microscope. From each animal, 100 nerve fiber profiles were examined on the ultrastructural level. Nerve fibers having a diameter of more than 0.5 ~m, containing synaptic vesicles, were characterized as varicosities, smaller ones as axons. The amount of degenerated axons was determined on the basis of 100 (%).

Fluorescence microscopy After cervical dislocation and thoracotomy the hearts of the mice were removed

and freeze-stopped with a Wollenberger clamp (precooled with liquid nitrogen) and placed into liquid nitrogen. The hearts were kept in a cryostat (temperature - 3 0 ° C ) and cut into 16 ~m cryostat serial sections. Cardiac adrenergic nerve fibers of the right ventricle (100/.tm subepicardial parallel to the surface) were made visible with glyoxylic acid-induced fluorescence [6] using the method of de la Torre [12]. These sections were examined under a Leitz orthoplan microscope equipped for fluores- cence with epi-illumination. The primary filter was BG 12 (3 mm), the secondary filter a Leitz K 490.

A residual light amplifying TV Caesicon-Camera (Kranz PIC 762) was used to guarantee the exact proportion of the fluorescing nerve fibers in relation to the visible field of the slide preparation within the microscope. After the inversion of the picture with a picture analyzing system (Artek-counter 982), Fisher Sci., F.R.G.), the nerve fibers were visible on a screen (Hitachi 982, Hitachi Denshi) as dark fibers and were easily detectable against the bright background. Concerning a standard grey-scale display, areas of higher grey scale values were filled up in brightness with the help of a specially designed measuring mask. With its aid only the areas of fluorescencing nerve fibers were measured (Fig. 2a,b). A hundred separate measure- ments were made per section. They comprise a total tissue area of 9 mm 2. Within this standardized area ( = 100%) the proportion of fluorescing nerve fibers was expressed on a percentage basis. This means that not only was the length but also the thickness (e.g. the developing of varicosities) of the nerve fibers recorded. It was not necessary to record any fading of the picture as a highly sophisticated TV-camera was used with a picture-analyzing procedure in 1 /10 s per measurement.

Biochemical analysis After weighing the cryosections of the right ventricles, the samples were exposed

to 2 ml of 0.06 N HC1 which contained isoprenaline as internal standard and homogenized using an Ultra-Turrax apparatus (TP 10 N Janke und Kunkel, KG Staufen) for 1 rain. Eighty mg of act ivated aluminium oxide and 3 ml of 1 M ammonia buffer pH = 8.6 (containing 0.3% glycine) were added to the homogenized samples. The pH value of the samples was controlled by an indicator paper.

The samples were shaken on IKA-VIBRAX-VXR-mixer for 5 min to adsorb the catecholamines. The supernatant of the aluminium oxide was removed by suction. Afterwards the samples were rinsed 3 times with 2 ml of bidistilled water and twice with 2 ml of methanol (HPLC-quality).

The catecholamines were eluted from the aluminium oxide with a 1 ml mixture of 30 ~1 trifluoroacetic acid-methyl orange (1 mg methyl orange in 20 ml trifluoro- acetic acid)/0.7 ml methanol (HPLC-grade) /1 mg glycine by a 5-min-shake. The

88

eluates were transferred into test tubes, concentrated and dried in a vacuum-exsicca- tor over P2Os/KOH.

The analysis was carried out by gas chromatography/mass spectrometry ( GC /MS ) determination. The sensitivity of the high performance liquid chromatog- raphy with electrochemical detection (HPLC-EC) was not high enough to guarantee the reproducibility of the low catecholamine levels in the ventricles. Referring to G C / M S analysis the isolated catecholamines were derivatized according to a method first described by Donike [16]. The residue was dissolved in a 40 ~tl MSTFA (N-methyl-N-trimethylsilyl-trifluoroacetamide)/acetonitrile (60 : 40, v : v) and treated for 5 min on a heating block at 60°C. When the samples were cooled to room temperature, 5 jag of MBTFA (N-methyl-bis-trifluoroacetamide) were added, and the samples were heated for another 5 rain.

GC / MS conditions Equipment: G C / M S 5995 Hewlett Packard. Column: 17 fused silicon capillary

column, FS-ov1701-Cb-0.25 (ID) (Macherey & Nagel). Carrier gas: 20 ml /min helium. Injection temperature 12°C. Transfer-line temperature: 300°C. Source temperature: 200°C. Analyzing temperature: 180°C. SEV: 2200 V. Emission: 0.27 A. Electron energy: 70e V. Purge off time: 0.5 rain. Capillary time before ramp: 0.3 min. Capillary ramp rate: maximum, Temperature profile: 1 min 220°C isotherm, heating rate 5 °C/ra in . Recording with SIM (selected ion monitoring): m / e = 355.3.

All experimental results (concerning electron microscopy, fluorescence mi- croscopy and biochemistry) were analyzed statistically by Student's t-test.

Results

There are no significant changes in the number of fluorescing fibers in the right ventricular myocardium throughout the experimental period after the injections of sesame oil (group C, Figs. 2c, 3a). These results correspond to the noradrenaline concentrations in the heart (Fig. 3b). The ultrastructural features in the control groups show a constant amount of degenerated (3%) adrenergic nerve fibers (Fig. 3c). The quotient of axons/varicosities differs slightly throughout (Figs. la, 5).

The morphometric examinations of t h e fluorescent areas of both group T (training group) and group TP (testosterone propionated group) reveal a significant increase (about 53%, P < 0.001) in comparison to the control group after 1 week (Figs. 2d, 3a). These results are reflected by the biochemical findings: in group TP the noradrenaline concentration increases to 471 + 65 n g /g wet wt. (P < 0.05) and after physical training to 497 ± 66 ng /g (P_< 0.05 Fig. 3b). The ultrastructural features between groups TP and T correspond to each other. The number of degenerated axons and the quantity of varicosities increase slightly under these experimental conditions (Fig. 5).

After 3 weeks a highly significant reduction (P < 0.001) of the fluorescent areas of groups T and TP is observed which Corresponds to the biochemical analysis. Testosterone causes a fall in noradrenaline concentration from 471 + 75 ng /g after

~9

Fig. 1, Electron micrographs of autonomic nerve fibers in murine myocardium, a: axon bundle with variocosities (V) within the fight ventricular interstitium, (M) heart muscle cells, control group; × 11.250. b: degenerated axon as demonstrated by the occurrence of myelin figures (arrow), e.g. group TP, 3 weeks; × 10.000. c: extrusion of axons (arrow) from the cytoplasm of Schwann;s cell (S), group TPT, one week; ×11.250. d: regenerating axon containing nematosomes (arrows) and lysosomes (L), a portion of capillary endothelial cell (E) is located in the left corner of the figure, (M) heart muscle cell, group TP, 6 weeks; × 17.500.

90

Fig. 2. Fluorescence microscopic demonstration of the adrenergic nerve fibers; all pictures × 100. a: nerve fibers after inversion on the monitor, b: black areas (nerve fibers) filled up with the whole frame, c: control group, d: note the increased number of nerve fibers, e.g. group T, one week. e: reduction of nerve fibers, e.g. group TP, 3 weeks, f: increased number of nerve fibers, note dichotomous arborization pattern (arrow head), e.g. group TPT, 6 weeks.

one week to 318___ 80 n g / g we t wt. (P_< 0.05, Fig. 3b). As a consequence of physical exercise the catecholamine concentration drops from 452 + 112 n g / g to 368 + 32 n g / g ( P < 0.05). In relation to group C there is a decrease in fluorescent nerve fibers in groups T ( P _< 0.01) and TP ( P < 0.001) (Fig. 3a), the noradrenaline concentration differs only in group TP ( p _< 0.01, Fig. 3b). Although the histo- and biochemical results of groups TP and T are almost similar, significant differences in the nerve fibers can be found on the ultrastructural level. The triple amount of pathological changes (P _< 0.01) of the axons is demonstrable in group TP (Fig. 3c). The alterations extend to the occurrence of myelin figures (Fig. lb) or axoplasmic lysis.

91

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92

Biochemical analysis reveals a significant rise in catecholamine concentration to 607 _+ 101 n g / g (group TP, P _< 0.01) and 400 + 61 n g / g (group T), respectively. In relation to group C there is only a significant difference in group TP (P _< 0.001, Fig. 3b). The rate of degeneration of adrenergic nerve fibers is 6% (P ~< 0.05) in group T and 18% (P < 0.001) in group TP (Fig. 3c); the proportion of axons to varicosities is 1 : 1.4 in group T ( P _< 0.05, Fig. 5) and 1 : 1 in group TP ( P _< 0.01, Fig. 5).

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Fig. 4. Schematic representation of the area of fluorescing nerve fibers (a), NA-concea~tration in myocardium (b) and the number of degenerated axons (c) during different periods of time. Group T is shown as cross-striped, group TPT as checkered columns; for comparison the groups C are shown left as white columns. For significance see chapter 'Results ' .

93

Axonal varicosities having a diameter of more than 4 m m exhibit signs of regeneration such as accumulat ion of smooth endoplasmic reticulum, vesicle abnormalit ies , concentrat ions of lysosome- l ike bodies as well as concentric lamellar bodies and nematosomes . N e m a t o s o m e s are of spherical or ovoid shape without a l imiting membrane. They are c o m p o s e d of 5 0 - 9 0 nm thick convoluted or concentric short strands of high electron density which are embedded in a fine granular matrix (Fig. ld) . Concentric lamellar bodies consist of whorls or elongated parallel stacks of cisternae, and they are c losely packed in the intervening cytoplasmic matrix.

In comparison to the control group, group TPT (testosterone application and training) shows a similar amount of f luorescing fibers and tissue catecholamines after one week (Figs. 4a, b). In relation to group T there is a remarkable decrease ( P _< 0.01) in the number of f luorescing fibers as well as in noradrenaline concentra- t ion ( p < 0.01) at the same stage (Figs. 4a, 4b).

Due to the retraction of the cytoplasm of Schwann's cells the axons are located on the surface. Somet imes they are extruded from the cytoplasm of Schwann's cells and they are hardly or only slightly in contact with Schwann's cells (Fig. lc) . The quotient of axons /var icos i t i e s increases to 1.1 : 1 ( P < 0.05, Fig. 5).

After 3 weeks group TPT shows a significant loss of noradrenaline (217 +_ 75 n g / g wet wt.). This value is significantly lower in comparison to group C (353 _+ 80 n g / g , P < 0.001) and T (368 _+ 32 n g / g , P < 0.001). The morphometr ic examina- tion of the area of fluorescing fibers conf irms the biochemical results, as there is a 57% reduction of the fibers in relation to group C ( P < 0.001, Fig. 2e) and one of 41% in comparison to group T ( P < 0.001, Fig. 4a).

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After the 6th week group TPT shows an increased number of fluorescing fibers (44%, P < 0.001) and of noradrenaline content (439 _+ 100 ng/g) in comparison to group C (Fig. 4a, b.), in relation to group T the area of fluorescing nerve fibers increased by about 27% (p < 0.01, Fig. 4a). Occasionally, the fluorescing nerve fibers show arborizations (Fig. 2f).

Ultrastructurally, there is an extremely high amount of varicosities (56%, P < 0.001, Fig. 5) and only a small number of degenerated axons (Fig. 4c). Regenerating axons can be differentiated by the following features: polymorphic shape of the granular endoplasmic reticulum, vesicular elements varying in size, and great numbers of mitochondria and longitudinally oriented neurotubules and neurofila- merits in an electron-lucent cytoplasm. Signs of cytoplasmic activation--dilated granular endoplasmic reticulum, enlarged Golgi f ields--are recognizable in the cytoplasm of Schwann's cells after the 6th week of testosterone application and training.

Discussion

Application of testosterone or physical exercise induces a sympathetic plasticity with degenerative and regenerative signs depending on time and on the intensity of stimuli. The reaction patterns illustrate the adaptive potential of the sympathetic neurons and fit well into the idea of Selye described in his 'General Adaptive System [46].

Many investigations have been carried out relative to the effects of training as well as of testosterone application on sympathetic neurons. They show controversial data, which mainly depend on different running speed (m/min) , time intervals (h/days/weeks) , or concentration of hormones (mg/kg/days) : De Schryver et al. [13,14], for example, found a decline in the noradrenaline concentration of cardiac tissue after a 3-month treadmill training (12 m/min) . In contrast, Winckler [49] observed no alterations in the catecholamine content of the adrenergic nerves of the heart following a strenuous 1-h swimming exercise program. Ahlo et al. [3] noticed a hyperactivity of sympathetic nerve fibers in the myocardium following two years of physical exercise (20-25 in/min). They furnished evidence for a t rophic stimulation, growing, and arborization of autonomic nerve fibers. Acute testosterone application (5 mg /kg /day ) causes a 2-fold increase in the noradrenaline concentration in relation to the control group after castration of the female internal reproductive organs [48]. Chronic treatment with testosterone (10 m g / k g / d a y ) produces a decrease in fluorescing fibers in the seminal vesicles of the guinea pig [19]. These results correspond to the biochemically analyzed reduction in the neurotransmitter content following chronic testosterone application [44]. Extended examinations at different time intervals which could demonstrate the adaptive changes of autonomic neurons after different experimental conditions, e.g. testosterone application or physical exercise, are not yet available in the applied literature.

The analysis of the effects of training and of testosterone application on the number of fluorescing fibers and tissue catecholamines indicates that the stimula-

96

tory influences of these two conditions seem to be comparable. This corresponds to earlier biochemical investigations which indicate an activation of the catecholamine synthesizing enzymes in the sympathetic neurons after strenous swimming [8], immobilization [33] or testosterone application [10]. The catabolic enzymes. catechol-O-methyl-transferase (COMT) and monoamine oxidase (MAO). are in- hibited under the same conditions [10,33,43].

One-week testosterone application or physical exercise provokes an increase in the number of fluorescing fibers as well as tissue catecholamines. These results correspond to those describing the reaction of acute testosterone application [22,48]. Our da ta - -a f te r a 3-week treadmill training or testosterone application-- are related to those results describing sympathetic activity influenced by chronic treat- ment with testosterone [19]. In our experiments a significant decrease in the number of fluorescing fibers is not only visible in relation to the first week but also to group C. Following 6 weeks of treadmill training or testosterone application, there is again a change in the number of fluorescing fibers and catecholamine concentration. This illustrates signs of adaptation to the stimuli of training and of testosterone. The adaptive system is assumed to develop degenerative and then regenerative patterns, since ultrastructural observations give evidence of degenerations such as axop- lasmalysis, membrane fractures and retractions of the cytoplasm of Schwann's cells at the 3rd week, and evidence of regeneration such as growth cones at the 6th week. This regeneration reveals itself fluorescence microscopically in the shape of branch- ing of the adrenergic nerve fibers [1].

A perpetual change in the number of varicosities and arborizations in the terminal regions of the sympathetic postganglionic neurons can be presumed [28,29]. These observations confirm our ultrastructural findings under control conditions with a constant amount of axons and axon varicosities. The change of the quotient of axons/varicosities in groups T and TP indicates nervous activity. The axonal varicosities are no static structures but may undergo relevant changes even in the fully developed adult nervous system [17]. The complex structural and functional changes which may take place in the varicosities under experimental conditions constitute the basis of nervous plasticity [2]. The increase of varicosities and signs of degeneration are highly sensitive indicators of neuronal activity. Although the fluorescing intensity--after 3 weeks of treadmill training or testosterone application - -does not differ, the number of degenerated axons is significantly higher in group TP. This indicates a more intensive stimulation of the sympathetic nerve fibers caused by testosterone. Bearing in mind that the quotient of axons/varicosities does not differ statistically between the two experimental groups (3rd week) we must assume that testosterone induces an activation of the neuronal metabolism as a result of a trophic factor [9,50].

Special types of hypertrophied endoplasmic reticulum with concentric lamellar bodies occur in activated sympathetic neurons [24,36,37]. These lamellar bodies may demonstrate their involvement in an increased activity of protein synthesis [11]. The frequent occurrence of nematosomes (or thread-like bodies [20]) following immobili- zation stress is related to chronic neuronal hyperactivity [24]. In the neuronal postganglionic profiles the nematosomes--as described for the first t ime--are

97

regarded as the morphological expression of the cell and hence as the response to the experimental conditions (testosterone). In former investigations these structures were interpreted as results of degenerative processes [42,45]. According to Heym and Addicks [24] it is more likely that the nematosomes provide morphological evidence for a differentiated sympathetic neuronal adaptation to long-term activa- tion.

The analysis of the effect of the training period as well as of testosterone application can be interpreted as follows: the adaptive reactions of the sympathetic nerve fibers under both experimental conditions are comparable. They cannot be regarded as a monodirectional system since there are signs of adaptation in several directions, viz. activation by an increased number of fluorescing fibers and of tissue catecholamines (first week), contra-regulatorv reactions reflected by a decrease in fluorescing fibers and tissue catecholamines and an increase in degenerated axons (3rd week) and regeneration established by an increase in fluorescing fibers, tissue catecholamines and a decrease in degenerated axons (6th week).

According to these reaction patterns there are also signs of an increase and decrease in heart weights after physical training. These differences in weight depend on the physical exercise as well as on its frequency of repetition [26,27]. After an early, temporary increase (5%, 2 exercises/2 days) the heart weight of the mice (NMRI) drops below the control level (4%, 7 exercises/9 days), returns to the control level (20 exercises/4 weeks), and finally the heart weight increased by up to 7% of its original weight (20 exercises/6 weeks) [26,27].

The differences in heart weight correspond to our findings concerning the number of fluorescing fibers, biochemical results as well as the quotient of axon calibers/varicosities. The increase in heart weight can be explained by an increase in tissue catecholamines and the noradrenaline turnover in the heart which induces cardiac hypertrophy [40]. It can be stated that physical exercise provokes sym- pathetic activity and a moderate cardiac hypertrophy. This is confirmed by other studies [3,7].

The combination of both st imuli-- t readmil l training and tes tosterone--af ter 1, 3 and 6 weeks provides strong evidence for an adapted overstimulated system of sympathetic nerve fibers. The overstimulated system is recognizable by an initial decrease in fluorescing fibers and in tissue catecholamines after one week. This reduction is probably caused by an increased number of degenerated axons. Although the number of fluorescing fibers and of tissue catecholamines decreases eventually up to the 3rd week, a point of stabilization with regard to the degener- ated axons is detectable. The quantity of these axons decrease and the number of varicosities increase. After the 6th week of testosterone and training a remarkable increase in fluorescing fibers and of tissue catecholamines is observed which exceeds the level of groups T and TP.

These observations underline the strong effects of testosterone and training. They are well in accordance with the assumption that the reaction of the terminal nerve fibers depends on the quality of the stimuli, e.g. physical training, testosterone application, and testosterone and training.

98

Acknowledgements

The authors wish to thank Frau W. Brunner and Herrn Chr. Hoffmann for their skilful technical assistance. This work has been kindly supported by Fisher Scien- tific Co., Munich, F.R.G.

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