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The effect of long-term castration on the histochemically demonstrable catecholamines in the hypogastric ganglion of the rat
Journal o f the Autonomic Nervous System I (1979) 139--147 © Elsevier/North-Holland Biomedical Press
139
THE EFFECT OF LONG-TERM CASTRATION ON THE HISTOCHEMICALLY DEMONSTRABLE CATECHOLAMINES IN THE HYPOGASTRIC GANGLION OF THE RAT
MATTI P A R T A N E N and ANTTI H E R V O N E N
Department of Biomedical Sciences. Universityof Tampere, Box 607. SF-33101 Tampere I 0 (Finland) (Received June 9th, 1979) (Accepted August 24th, 1979)
Key words: adrenergic n e u r o n s - - hypogastric ganglion -- castration -- testosterone
ABSTRACT
The effect of long-term castr~ltion on the hypogastric ganglion of the rat was studied using the formaldehyde-induced fluorescence (FIF) method. After castration the fluorescence intensity was lower and the size of the adrenergic neurons was smallel than in normal or in testosterone-treated castrated rats. The fluorescence profile of the ganglia of castrated rats differed from the profiles of control or testosterone-treated castrated rats. Vacuolated neurons were seen ill the hypogastric ganglion of controls but not in the ganglia of castrated an:imals. After long-term castr~:tion the size of the ventral prostate was drastically reduced. The density o ~ adrenergic nerves was similar in castrated, normal and testosterone-treated castrated rats. It is concluded that long-term castration has an effect on adrenergic neurons by decreasing the FIF and by producing other morphological changes. The effect can be reversed by testosterone treatment.
INTRODUCTION
The "short" adrenergic neurons innervating the genit~d organs in the female and in the male rat or rabbit differ from the ordinary "long" adrenergic neurons [13]. In female rat and rabbit these neurons react to estrogen treatment by increasing the amouut of noradrenaline (NA) in nerve terminals [22] and by ultrastructural changes [6]. Furthermore the formaldehydeinduced fluorescence (FIF) increases in the perikarya of the short adrenergic neurons in the paracervical ganglion of the female rat dtadng the second week of pregnancy [8]. In the castrated male rat tastt;st~rone-treatment
140
induces an increase in the total amount of NA but the concentration of NA decreases in genital organs [23,25]. The corresponding neurons in the hypogastric ganglion react to testosterone treatment by increasing the FIF, the cell size and the number of vacuolated neurons [14,16] during the normal development, suggesting the presence of peripheral neuroendocr/ne interaction also in male animals. This study was made to follow the effect of longterm castration on the adrenergic neurons in the hypogastric ganglion and on the terminal network of adrenergic fibers innervating the ventral prostate and to determine the effect of testosterone treatment after castration. MATERIAL AND METHODS
Material. Twenty-four male rats were castrated prepubertally at the age of 4 weeks and were killed at the age of 8 and 12 months, 12 rats in each age grovp. Control rats were from the same litters. In each age group 4 castrated rats were injected with testosterone enanthate/propionate (Primoteston Depot) 10 mg/kg twice a week for one and two weeks before sacrifice. Formaldehyde.induced fluorescence. The standardized formaldehydeinduced fluorescence method [5] was used. The specimens were taken under visual control and frozen immediately in liquid nitrogen. Later they were freeze-dried for 5 days at --40°C under a vacuum of 10 -4 tort in the presence of phosphorous pentoxide as a water trap. The freeze-dried tissue pieces wer~ exposed at 80°C to vaporous paraformaldehyde previously equilibrated to 60% relative humidity for one hour. The specimens were embedded in paraffin under vacuum and sectioned serially at 10 pm. The diameters of the fluorescent neurons were measured from photographs with magnification of 40)<. Only neurons in which the nucleus was visible were measured -- in two directions at right-angles to each other. Means, S.D., the number of observations and the values of P from the Student's t-test are given in the text. Microspectrofluorimetry. The formaldehyde-induced fluorescence (FIF) was measured using Leitz MPV-2 microspectrophotometer equipped with band interference filter VERIL S-60 and S-20 type photomultiplier. The filters BG 3, KP 425 and TK 455 for excitation light and K 460 for emission light were used..4, stabilized HBO 100 mercury lamp (Osram) served as light source. The band interference filter on the emission side was fixed at 480-490 nm for maximal emission of the FIF of catecholamine. A Leitz NPL 25X objective was used in measurements. The measuring spot was 10 ~um. After identification of the ganglion the measuring spot was moved manually from one fluorescent neuron to the other; the automatic control unit performed one measurement/sec. Four cross-sections from each ganglion were measured. The numerical data, means, S.D., the number of observations and the values of P are expressed in the text. The distribution of the recordings of the fluorescence intensities of the neurons were divided into classes (17)
141
according to the calibration of the instrament. The classes are expressed graphically as columns each representing the percentage of the recordings in the given intensity classes (Fig. 3). The graphic presentation is referred to as the fluorescence profile. The effect of fading of fluorescence was eliminated by excitation field-iris diaphragm and standardized measuring procedure. RESULTS
Four different cell types were seen in the hypogastric ganglion of the normal adult rat under fluorescence microscopy: (1) fluore~en't (adrenergic) neurons; (2) nonfluorescent neurons considered -~s nonadrenergic; (3) vacuolated neurons; and (4) small intensely fluorescent (SIF) cells. Only the specific catecholamine fluorescence of the neurons was recorded microspectrofluorimetrically. In normal 8- and 12-month-old rats and in testosterone-treated castrated rats the distribution of the specific cytoplasmic formaldehyde-induced fluorescence (later referred as FIF) was homogeneous ~md the intensity varied from neuron to neuron (Fig. l a ) only in the weakly fluorescent neurons. The nonspecific granular yellow fluorescence of the pigment was seen (Fig. la). The a m o u n t of pigment increased with ageing. In castrated rats in both age groups the specific catecholamine fluorescence in most of the neurons was n o t strong enough to be differentia~d from the nonspecific fluorescence due to pigment (Fig. l b ) , but in testos~rone.treated castrated rats the specific FIF was as intense as in the controls and covered the pigment fluorescence (Fig. la). In the castrated rats the peripheral parts of the cytoplasm provided a nonfluorescent halo while the perinuclear region emitted fluorescence due to catecholamine and pigment or only to pigment (Fig. l b ) . Around the neurons, probably in the satellite ceLs, typical pigment fluorescence was seen (Fig. l b ) . Cell size was smaller in castrated rats than in normal or testosterone-treated castrated animals. The values of the mean cell size were in normal, castrated and testosterone-treated (two weeks) animals of 12 months, 60 + 13 urn, 49 ± 11 um and 57 + 14 urn respectively. The differences were significant between castrated and control animals (P<: 0.001) and between castrated and testostel'one-treated castrated rats (P .v. 0.001). The number of observations varied between 200 and 300. No vacuolated neurons were seen in castrated rats, while they occurred regularly in normal animals. M ic rospec tro fi'u oro m e try
The mean values of the relative intensity of the FIF in normal, castrated and testosterone-treated castrated animals (one and two weeks) were 11.5 ± 3.0, 9.9 -+ 9.4, 11.8 -+ 3.5 and 11.7 -+ 3.4 respectively. Student's t-test showed that the differences between castrated and normal animals (P < 0.05) and between castrated and testosterone-treated for one and two weeks
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144
% C~ltr~ton 30 L~ 1(] 2 4 6 S ~0121416
2 4 6 8 10121416
% Cimtratk~ and testosterone1~). Castration arwttestostorone2 30 20 10
2 4 6 8 10121416
3 Fig. 3. The fluorescent profiles of castrated, normal and testosterone-treated (for one and two weeks) castrated rats. The fluorescent neurons are divided into 17 classes according to the instrument. Each column represents the percentage of the fluorescent neurons in each intensity class. In castrated rats the profile differs from the others by shifting to the left.
castrated animals were significant (P < 0.025; P < 0.05 respectively). The number of observations varied between 200 and 400 in each animal. The fluorescence ])rofile reflected changes between the intensity classes of the adrenergic he,irons. In castrated rats the profile moved to the left, indicating a decrease in fluorescence intensity (Fig. 3). In normal aged and in testosterone-treated castrated animals the profiles were almost identical (Fig. 3). The patterns of thq~:fluorescence profiles were similar in 8. and 12-month-old rats. After long-I;erm castration the size of the ventral prostate was reduced. After testoste:~one treatment for two weeks the size became normal again in castrated rats. The amount and the size of alveoli decreased after castration (Fig. 2b) and ;vere normal after the testosterone treatment of two weeks in castrated rats ( Fig. 2a). Because of the shrinkage of the stroma, evaluation of the density of ~;heterminal network was difficult, but it seemed to be similar in normal, castrated and testosterone-treated castrated rats (Fig. 2a and b). By visual estimation under fluorescence microscopy no changes in the intensity of FIF in axons could be observed. DISCUSSION In the present study long-term castration was found to induce a chm~ge in the fluorescence intensity of the adrenergic neurons and in the cytoplasmic distribution of the formaldehyde-induced fluorescence (FIF). Also the fluorescence profile differs from the controls. The recovery after one week's testosterone treatment was complete.
145
The short adrenergic neurons are k n o w n to react to estrogen treatment in female rat and rabbit [12,13]. In the castrated male rat the total amount of noraclrenaline (later referred to as N A ) increases [10,23,25] but the concentration in genital glands decreases [23] after t~stosterone treatment. Only after neonatal castration does the amount of N A decrease significantly [2]. In this present study the size of the ventral prostate decreased dramatically after castration, but the density of innervation was the same in castrated, normal and testosterone-treated castrated animals. By visual estimation the intensity of FIF in axons was the same in castrated, normal and testosteronetreated animals. This observation, however, m a y be unreliable since Jonson [7] and Schipper et ",d.[21] have noted that even highly significantchanges in the fluorescent intensity to the axcns in the irism a y remain hidden to visual quantitation. Our efforts to regmter the FIF of the axons were prevented by the changed patterns of the fluorescent fibers in the c~trated animals. Furthermore, there was always a marked backgrovnd fluorescence due to the shrinkage of the stroma and the pigrn~nt fluorescence in epithelial cells of alveoles in the castrated animals. W h e n testosterone is given to castrated rats, the amount of fluorescent fibres increases with th~ regeneration of the alveoli.Thus a considerable part of the terminal axons must have lost their N A content in the degenerated gland, which effect is reversible.This reflects a marked capability to regulate the amou;it of N A in terminal axons and the N A turnover in adrenergic neurons. The reduction in ~;hesize of the ventral prostate is in accordance with previous knowledge [3] The perikarya of the short adrenergic neurons in the paracer~qcal ganglion has been noted to react during the second week of pregnancy in the female rat by increasing the FIF [8]. In the hypogastric ganglion, the :o~esponding ganglion in males, the concentration of N A in the adrenergic neurons decreases during normal development up to the age of 1 ~ weeks, and increases in testosterone-treated rats between 2 and 6 weeks h~ the rat [14]. Also the cell size of the fluorescent neurons increases more rapidly, and the number of vacuolated neurons also increases after testosterc,ne treatment [14,16] when compared to normally developing neurons. The vacuolated neurons (VN) are a normal cell type in pelvic autonomic ganglia bo~h in the male [16] and in the female [9] adult rat and the quantity of V N is under the hormonal con~ol of sex steroids [11,16--20,24]. In this study it was found that the intensity of FIF and the size of the neurons decrease alld no vacuolated neurons are seen in the hypogastric ganglion in prepubertally castrated old rats. In normal 8--12-month-old ra~ the distribution of the FIF in the adrenergic neuron is homogeneous but in castrated rats there is a nonfluorescent halo in the periphery of the cytoplasm of the neuron. The fluorescent material surrounding the neuron is thought to be due to pigment in satellite cells found with electronmicroscopy (unpublished data). In ~.estosterone-treated castrated rats the distribution of the cytoplasmic fluorescence is identical to that of the controls. Thus the short adrenergic neurons are sensitive to the androgenic hormone.substitution. By the auto-
146
radiographic method it has been shown that tritiated testosterone incorporates into the neurons of the hypogastric ganglion (unpublished data). The neurons can be classified and grouped in classes according to the intensity of the cytoplasmic FIF recorded microspectrofluorimetrically [1]. This method is useful in following minor changes in neuronal catecholamine content. This method has been used to follow changes in peripheral autonomic ganglia during development [15]. Another variation of this method has been used in the central nervous system [4]. In hypogastric genglion in normally developing rats the profile shifts to the left, indicating an increase in weakly fluorescent neurons, but in testosterone-treated rats the profile shifts to the right up to the age of 6 weeks [15]. After long-term castration the profile moves to the left, indicating an increase in the percentage of weakly fluorescent neurons. The profiles are almost identical in normally aged animals and in testosterone.treated castrated animals. The short adrenergic neurons in the hypoga~tric ganglion in the male rat seem to be under the control of androgenic hormone [14--16]. The nature of this control might be a direct effect upon the neurons or an indirect effect of a trophic nature via their target organs. REFERENCES 1 Alho, H. and Hervonen, A., Mierospectrofluorimetric quantitation of cathecholamine fluorescence in rat sympathetic ganglia. I. Estimation of the fluorescence profiles, Histochem, J., (1979) in press. 2 Broberg, A., Nubell, G., Owman, Ch., Rosengren, E. and SjSberg, N.-O., ConseqiJence of neonatal androgenization and castration for future levels of norepinephrine transmitter in uterus and vas deferens of the rat, Neuroendocrinology, 15 (1974) 308-312. 3 Burns, R.K., Role of hormone~ in the differentiation of sex. In W.G. Yound led.), Sex and Internal Secretion, Williams and Wilkins, Baltimore, 1961, pp. 76--158. 4 Einarsson, P., Hallman, H. and Jonsson, G., Quantitative microfluorimetry of formaldehyde induced fluorescence of dopamine in the caudate nucleus, Med. Biol., 53 (1975) 15--24. 5 Er~/nkS, O., The practical histochemical demonstration of catecholamines by formaldehyde-induced fluorescence, J. roy. micr. Soc., 87 (1967) 259--276. 6 Hervonen, A. and Kanerva, L., Effect cf ~7-~-estradiol on the axoplasmic organellcs of the adrenergic axons of the rabbit ,ay~metrium, as revealed aftt~r KMnO4-fixation, Z. ZeUforsch., 144 (1973) 219--229. 7 Jonsson, G., Microfluorimetric studi~;~ ~.,~'~the l'ormaldehyde-induced fluorescence of noradrenaline in adrenergic nerves of rat iris, j. Histochem. Cytochem., 17 (1969) 714--723. 8 Kanerva, L., Lietz~n, R. and Ter~iv~iinen, H., Catecholamines and cholinesterases ill the paracervical (Frankenh/iuser) ganglion of normal and pregnant rats, Acta physiol. scand., 86 (1972) 271--277. 9 Kanerva, L. and Ter//v~/inen, H., Electron Microscopy of the paracervical (Frankenh~iuser) ganglion of the adult rat, Z. Zellforsch., 129 (1972) 161--177. 10 Kvist, U. and SjSstrand, N.-O., A further contribution to the question of trophic and hormonal influences on the noradrenaline content of the male reproductive tract: effect of combined androgen and estrogen treatment of prepubertally castrated rats, Acta physiol, scand., 98 (1976) 339--346.
147 11 Lehman, H.J. und Stange, H.H., ~)ber das Verkommen vakuolen-haltiger Ganglienzellen im Ganglion cervicale uteri tr~/chtiger und nichttr~ichtiger Ratten, Z. Zellforsch., 38 (~953) 230--236. 12 Owman, Ch. and Falck, B., Functional Approach to the Histochemistry of Catecholamines in peripheral Sympathetic Nerves. In M. Santini (Ed), Golgi Centennial Symposium Proceedings, Raven Press, New York, 1975, pp. 415--436. 13 Owman, Ch., S.i~berg, N.-O. and Sj~istrand, N.O., Short adrenergic neurons, a peripheral neuroedocrine mechanism. In M. Fujiwara and C. Tanaka (Eds.), Amine Fluorescence Histochemistry, Igaku Shoin, Tokyo, 1974, pp. 47--66. 14 Partanen, M. and Hervonen, A., The formaldehyde-induced fluorescence of the developing hypogastric (main pelvic) ganglion of the rat. Short adrenergic neurons and the effect of testosterone, Histochemistry, 62 (1979) 249--258. 15 Partanen, M., Hervonen, A. and Alho, H., Microspectrofluorimetric estimation of the formaldehyde-induced fluorescence of the developing main pelvic ganglion of the rat, Histochem. J., (1979) in press. 16 Partanen, M., Hervonen, A., Vaalasti, A., Kanerva, L. and Hervonen, H., Vacuolated neurons in the hypogastric ganglion of the rat, Cell and Tissue Res., (1979) in pre.~s. 17 Pawlikowski, M., Das Ganglion prostaticum und das Ganglion cervicale superius normaler und gonadopriver Ratten, Endokr. pol., 10 (1959) 449--458. 18 Pawlikowski, M., The occurrence of vacuoles in the nerve cells of autonomic ganglia as a sign of neurosecretion, Polish reed. Sci. Hist. Bull., 4 (1961) 110--112. 19 Pawlikowski, M.0 Studies on peripheral neurosecretion. I. Morphological and topographic features of neurosecretion in mammalian autonomic ganglions, Endokr. pol. 13 (1962) 153--170. 20 Pawlikowski, M., The effect of gonadal and gonadotrophic hormones on the prostatic ganglion and the superior cervical ganglion in male rats, Acta reed. pol. 3, 2 (1962) 171--183. 21 Schipper, I., Tilders F.J.H. and Proem, I.S., Micrc fluorimetric scanning of sympathetic nerve fibers. An improved method to quantitative formaldehyde induced fluorescence of biogenic amines, J. Histochem. Cytochem., 2 (1978) 1057--1066. 22 Sj~berg, N.-O., Increase in transmitter conten.~ of adrenergic nerves in the reproductive tract of female rabbits after oestrogen treatment, Acta endocr, (KrJh.), 57 (1968) 45--513. 23 Sj~strand, N.-O. and Swedin, G., Influence of age, growth, castrath" i and testosterone treatment on the noradrenaline levels of the ductus deferens and the auxiliary male reproductive glands of the rat, Acta physiol, scand., 98 (1976) 323--338. 24 Stange, H.H. and Drescher, I., Tierexperimentelle Untersuchungen am Frankenh~userschen Ganglion zum Problem der peripheren Neurosekretion, Arch. Gyn~/k., 184 (1954) 530--542. 25 Wakade, A.R. and Kirpekar, S.M., "Trophic" influence on the sympathetic nerves of the vas deferens and seminal vesicle of the guinea-pig, J. Pharmacol. exp. Ther., 186 (1973) 528--536.
139
THE EFFECT OF LONG-TERM CASTRATION ON THE HISTOCHEMICALLY DEMONSTRABLE CATECHOLAMINES IN THE HYPOGASTRIC GANGLION OF THE RAT
MATTI P A R T A N E N and ANTTI H E R V O N E N
Department of Biomedical Sciences. Universityof Tampere, Box 607. SF-33101 Tampere I 0 (Finland) (Received June 9th, 1979) (Accepted August 24th, 1979)
Key words: adrenergic n e u r o n s - - hypogastric ganglion -- castration -- testosterone
ABSTRACT
The effect of long-term castr~ltion on the hypogastric ganglion of the rat was studied using the formaldehyde-induced fluorescence (FIF) method. After castration the fluorescence intensity was lower and the size of the adrenergic neurons was smallel than in normal or in testosterone-treated castrated rats. The fluorescence profile of the ganglia of castrated rats differed from the profiles of control or testosterone-treated castrated rats. Vacuolated neurons were seen ill the hypogastric ganglion of controls but not in the ganglia of castrated an:imals. After long-term castr~:tion the size of the ventral prostate was drastically reduced. The density o ~ adrenergic nerves was similar in castrated, normal and testosterone-treated castrated rats. It is concluded that long-term castration has an effect on adrenergic neurons by decreasing the FIF and by producing other morphological changes. The effect can be reversed by testosterone treatment.
INTRODUCTION
The "short" adrenergic neurons innervating the genit~d organs in the female and in the male rat or rabbit differ from the ordinary "long" adrenergic neurons [13]. In female rat and rabbit these neurons react to estrogen treatment by increasing the amouut of noradrenaline (NA) in nerve terminals [22] and by ultrastructural changes [6]. Furthermore the formaldehydeinduced fluorescence (FIF) increases in the perikarya of the short adrenergic neurons in the paracervical ganglion of the female rat dtadng the second week of pregnancy [8]. In the castrated male rat tastt;st~rone-treatment
140
induces an increase in the total amount of NA but the concentration of NA decreases in genital organs [23,25]. The corresponding neurons in the hypogastric ganglion react to testosterone treatment by increasing the FIF, the cell size and the number of vacuolated neurons [14,16] during the normal development, suggesting the presence of peripheral neuroendocr/ne interaction also in male animals. This study was made to follow the effect of longterm castration on the adrenergic neurons in the hypogastric ganglion and on the terminal network of adrenergic fibers innervating the ventral prostate and to determine the effect of testosterone treatment after castration. MATERIAL AND METHODS
Material. Twenty-four male rats were castrated prepubertally at the age of 4 weeks and were killed at the age of 8 and 12 months, 12 rats in each age grovp. Control rats were from the same litters. In each age group 4 castrated rats were injected with testosterone enanthate/propionate (Primoteston Depot) 10 mg/kg twice a week for one and two weeks before sacrifice. Formaldehyde.induced fluorescence. The standardized formaldehydeinduced fluorescence method [5] was used. The specimens were taken under visual control and frozen immediately in liquid nitrogen. Later they were freeze-dried for 5 days at --40°C under a vacuum of 10 -4 tort in the presence of phosphorous pentoxide as a water trap. The freeze-dried tissue pieces wer~ exposed at 80°C to vaporous paraformaldehyde previously equilibrated to 60% relative humidity for one hour. The specimens were embedded in paraffin under vacuum and sectioned serially at 10 pm. The diameters of the fluorescent neurons were measured from photographs with magnification of 40)<. Only neurons in which the nucleus was visible were measured -- in two directions at right-angles to each other. Means, S.D., the number of observations and the values of P from the Student's t-test are given in the text. Microspectrofluorimetry. The formaldehyde-induced fluorescence (FIF) was measured using Leitz MPV-2 microspectrophotometer equipped with band interference filter VERIL S-60 and S-20 type photomultiplier. The filters BG 3, KP 425 and TK 455 for excitation light and K 460 for emission light were used..4, stabilized HBO 100 mercury lamp (Osram) served as light source. The band interference filter on the emission side was fixed at 480-490 nm for maximal emission of the FIF of catecholamine. A Leitz NPL 25X objective was used in measurements. The measuring spot was 10 ~um. After identification of the ganglion the measuring spot was moved manually from one fluorescent neuron to the other; the automatic control unit performed one measurement/sec. Four cross-sections from each ganglion were measured. The numerical data, means, S.D., the number of observations and the values of P are expressed in the text. The distribution of the recordings of the fluorescence intensities of the neurons were divided into classes (17)
141
according to the calibration of the instrament. The classes are expressed graphically as columns each representing the percentage of the recordings in the given intensity classes (Fig. 3). The graphic presentation is referred to as the fluorescence profile. The effect of fading of fluorescence was eliminated by excitation field-iris diaphragm and standardized measuring procedure. RESULTS
Four different cell types were seen in the hypogastric ganglion of the normal adult rat under fluorescence microscopy: (1) fluore~en't (adrenergic) neurons; (2) nonfluorescent neurons considered -~s nonadrenergic; (3) vacuolated neurons; and (4) small intensely fluorescent (SIF) cells. Only the specific catecholamine fluorescence of the neurons was recorded microspectrofluorimetrically. In normal 8- and 12-month-old rats and in testosterone-treated castrated rats the distribution of the specific cytoplasmic formaldehyde-induced fluorescence (later referred as FIF) was homogeneous ~md the intensity varied from neuron to neuron (Fig. l a ) only in the weakly fluorescent neurons. The nonspecific granular yellow fluorescence of the pigment was seen (Fig. la). The a m o u n t of pigment increased with ageing. In castrated rats in both age groups the specific catecholamine fluorescence in most of the neurons was n o t strong enough to be differentia~d from the nonspecific fluorescence due to pigment (Fig. l b ) , but in testos~rone.treated castrated rats the specific FIF was as intense as in the controls and covered the pigment fluorescence (Fig. la). In the castrated rats the peripheral parts of the cytoplasm provided a nonfluorescent halo while the perinuclear region emitted fluorescence due to catecholamine and pigment or only to pigment (Fig. l b ) . Around the neurons, probably in the satellite ceLs, typical pigment fluorescence was seen (Fig. l b ) . Cell size was smaller in castrated rats than in normal or testosterone-treated castrated animals. The values of the mean cell size were in normal, castrated and testosterone-treated (two weeks) animals of 12 months, 60 + 13 urn, 49 ± 11 um and 57 + 14 urn respectively. The differences were significant between castrated and control animals (P<: 0.001) and between castrated and testostel'one-treated castrated rats (P .v. 0.001). The number of observations varied between 200 and 300. No vacuolated neurons were seen in castrated rats, while they occurred regularly in normal animals. M ic rospec tro fi'u oro m e try
The mean values of the relative intensity of the FIF in normal, castrated and testosterone-treated castrated animals (one and two weeks) were 11.5 ± 3.0, 9.9 -+ 9.4, 11.8 -+ 3.5 and 11.7 -+ 3.4 respectively. Student's t-test showed that the differences between castrated and normal animals (P < 0.05) and between castrated and testosterone-treated for one and two weeks
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]"i~'. '2. T h e ~,~,ntr~d p r ~ s t a t e or" t h e t e S t ~ s t e z ' o n e - l r ~ ' a t e d e a s l r a t t ' d r;ut (~') :kllcl tl'n' ~'m, tr~l~'tl rill (11) ;It |}|~' ilL~l' ()t' ].~ l'll()tlth~. A3;.~)IIS i|l'l' lll;}.l']~t'(.l w i t h ;irr¢>whe[icls ,~rkd ;,h~',~]i ,,,,itl~ .\ A h ' n ~ s t f i l e w h o l e p r o s t a l e ~>I" the, c a s t Y a t e d rat is seen in h . ~ I ~ ' tllq' l>,i~.'l~l~i't~land tlu~r~,,, c.',,n¢',, in lh,, ~'astr,lt~,d r ; i t . . ~ 1 ~ i ~ n i l i ~ ' a l i o n : I 0 0 .
144
% C~ltr~ton 30 L~ 1(] 2 4 6 S ~0121416
2 4 6 8 10121416
% Cimtratk~ and testosterone1~). Castration arwttestostorone2 30 20 10
2 4 6 8 10121416
3 Fig. 3. The fluorescent profiles of castrated, normal and testosterone-treated (for one and two weeks) castrated rats. The fluorescent neurons are divided into 17 classes according to the instrument. Each column represents the percentage of the fluorescent neurons in each intensity class. In castrated rats the profile differs from the others by shifting to the left.
castrated animals were significant (P < 0.025; P < 0.05 respectively). The number of observations varied between 200 and 400 in each animal. The fluorescence ])rofile reflected changes between the intensity classes of the adrenergic he,irons. In castrated rats the profile moved to the left, indicating a decrease in fluorescence intensity (Fig. 3). In normal aged and in testosterone-treated castrated animals the profiles were almost identical (Fig. 3). The patterns of thq~:fluorescence profiles were similar in 8. and 12-month-old rats. After long-I;erm castration the size of the ventral prostate was reduced. After testoste:~one treatment for two weeks the size became normal again in castrated rats. The amount and the size of alveoli decreased after castration (Fig. 2b) and ;vere normal after the testosterone treatment of two weeks in castrated rats ( Fig. 2a). Because of the shrinkage of the stroma, evaluation of the density of ~;heterminal network was difficult, but it seemed to be similar in normal, castrated and testosterone-treated castrated rats (Fig. 2a and b). By visual estimation under fluorescence microscopy no changes in the intensity of FIF in axons could be observed. DISCUSSION In the present study long-term castration was found to induce a chm~ge in the fluorescence intensity of the adrenergic neurons and in the cytoplasmic distribution of the formaldehyde-induced fluorescence (FIF). Also the fluorescence profile differs from the controls. The recovery after one week's testosterone treatment was complete.
145
The short adrenergic neurons are k n o w n to react to estrogen treatment in female rat and rabbit [12,13]. In the castrated male rat the total amount of noraclrenaline (later referred to as N A ) increases [10,23,25] but the concentration in genital glands decreases [23] after t~stosterone treatment. Only after neonatal castration does the amount of N A decrease significantly [2]. In this present study the size of the ventral prostate decreased dramatically after castration, but the density of innervation was the same in castrated, normal and testosterone-treated castrated animals. By visual estimation the intensity of FIF in axons was the same in castrated, normal and testosteronetreated animals. This observation, however, m a y be unreliable since Jonson [7] and Schipper et ",d.[21] have noted that even highly significantchanges in the fluorescent intensity to the axcns in the irism a y remain hidden to visual quantitation. Our efforts to regmter the FIF of the axons were prevented by the changed patterns of the fluorescent fibers in the c~trated animals. Furthermore, there was always a marked backgrovnd fluorescence due to the shrinkage of the stroma and the pigrn~nt fluorescence in epithelial cells of alveoles in the castrated animals. W h e n testosterone is given to castrated rats, the amount of fluorescent fibres increases with th~ regeneration of the alveoli.Thus a considerable part of the terminal axons must have lost their N A content in the degenerated gland, which effect is reversible.This reflects a marked capability to regulate the amou;it of N A in terminal axons and the N A turnover in adrenergic neurons. The reduction in ~;hesize of the ventral prostate is in accordance with previous knowledge [3] The perikarya of the short adrenergic neurons in the paracer~qcal ganglion has been noted to react during the second week of pregnancy in the female rat by increasing the FIF [8]. In the hypogastric ganglion, the :o~esponding ganglion in males, the concentration of N A in the adrenergic neurons decreases during normal development up to the age of 1 ~ weeks, and increases in testosterone-treated rats between 2 and 6 weeks h~ the rat [14]. Also the cell size of the fluorescent neurons increases more rapidly, and the number of vacuolated neurons also increases after testosterc,ne treatment [14,16] when compared to normally developing neurons. The vacuolated neurons (VN) are a normal cell type in pelvic autonomic ganglia bo~h in the male [16] and in the female [9] adult rat and the quantity of V N is under the hormonal con~ol of sex steroids [11,16--20,24]. In this study it was found that the intensity of FIF and the size of the neurons decrease alld no vacuolated neurons are seen in the hypogastric ganglion in prepubertally castrated old rats. In normal 8--12-month-old ra~ the distribution of the FIF in the adrenergic neuron is homogeneous but in castrated rats there is a nonfluorescent halo in the periphery of the cytoplasm of the neuron. The fluorescent material surrounding the neuron is thought to be due to pigment in satellite cells found with electronmicroscopy (unpublished data). In ~.estosterone-treated castrated rats the distribution of the cytoplasmic fluorescence is identical to that of the controls. Thus the short adrenergic neurons are sensitive to the androgenic hormone.substitution. By the auto-
146
radiographic method it has been shown that tritiated testosterone incorporates into the neurons of the hypogastric ganglion (unpublished data). The neurons can be classified and grouped in classes according to the intensity of the cytoplasmic FIF recorded microspectrofluorimetrically [1]. This method is useful in following minor changes in neuronal catecholamine content. This method has been used to follow changes in peripheral autonomic ganglia during development [15]. Another variation of this method has been used in the central nervous system [4]. In hypogastric genglion in normally developing rats the profile shifts to the left, indicating an increase in weakly fluorescent neurons, but in testosterone-treated rats the profile shifts to the right up to the age of 6 weeks [15]. After long-term castration the profile moves to the left, indicating an increase in the percentage of weakly fluorescent neurons. The profiles are almost identical in normally aged animals and in testosterone.treated castrated animals. The short adrenergic neurons in the hypoga~tric ganglion in the male rat seem to be under the control of androgenic hormone [14--16]. The nature of this control might be a direct effect upon the neurons or an indirect effect of a trophic nature via their target organs. REFERENCES 1 Alho, H. and Hervonen, A., Mierospectrofluorimetric quantitation of cathecholamine fluorescence in rat sympathetic ganglia. I. Estimation of the fluorescence profiles, Histochem, J., (1979) in press. 2 Broberg, A., Nubell, G., Owman, Ch., Rosengren, E. and SjSberg, N.-O., ConseqiJence of neonatal androgenization and castration for future levels of norepinephrine transmitter in uterus and vas deferens of the rat, Neuroendocrinology, 15 (1974) 308-312. 3 Burns, R.K., Role of hormone~ in the differentiation of sex. In W.G. Yound led.), Sex and Internal Secretion, Williams and Wilkins, Baltimore, 1961, pp. 76--158. 4 Einarsson, P., Hallman, H. and Jonsson, G., Quantitative microfluorimetry of formaldehyde induced fluorescence of dopamine in the caudate nucleus, Med. Biol., 53 (1975) 15--24. 5 Er~/nkS, O., The practical histochemical demonstration of catecholamines by formaldehyde-induced fluorescence, J. roy. micr. Soc., 87 (1967) 259--276. 6 Hervonen, A. and Kanerva, L., Effect cf ~7-~-estradiol on the axoplasmic organellcs of the adrenergic axons of the rabbit ,ay~metrium, as revealed aftt~r KMnO4-fixation, Z. ZeUforsch., 144 (1973) 219--229. 7 Jonsson, G., Microfluorimetric studi~;~ ~.,~'~the l'ormaldehyde-induced fluorescence of noradrenaline in adrenergic nerves of rat iris, j. Histochem. Cytochem., 17 (1969) 714--723. 8 Kanerva, L., Lietz~n, R. and Ter~iv~iinen, H., Catecholamines and cholinesterases ill the paracervical (Frankenh/iuser) ganglion of normal and pregnant rats, Acta physiol. scand., 86 (1972) 271--277. 9 Kanerva, L. and Ter//v~/inen, H., Electron Microscopy of the paracervical (Frankenh~iuser) ganglion of the adult rat, Z. Zellforsch., 129 (1972) 161--177. 10 Kvist, U. and SjSstrand, N.-O., A further contribution to the question of trophic and hormonal influences on the noradrenaline content of the male reproductive tract: effect of combined androgen and estrogen treatment of prepubertally castrated rats, Acta physiol, scand., 98 (1976) 339--346.
147 11 Lehman, H.J. und Stange, H.H., ~)ber das Verkommen vakuolen-haltiger Ganglienzellen im Ganglion cervicale uteri tr~/chtiger und nichttr~ichtiger Ratten, Z. Zellforsch., 38 (~953) 230--236. 12 Owman, Ch. and Falck, B., Functional Approach to the Histochemistry of Catecholamines in peripheral Sympathetic Nerves. In M. Santini (Ed), Golgi Centennial Symposium Proceedings, Raven Press, New York, 1975, pp. 415--436. 13 Owman, Ch., S.i~berg, N.-O. and Sj~istrand, N.O., Short adrenergic neurons, a peripheral neuroedocrine mechanism. In M. Fujiwara and C. Tanaka (Eds.), Amine Fluorescence Histochemistry, Igaku Shoin, Tokyo, 1974, pp. 47--66. 14 Partanen, M. and Hervonen, A., The formaldehyde-induced fluorescence of the developing hypogastric (main pelvic) ganglion of the rat. Short adrenergic neurons and the effect of testosterone, Histochemistry, 62 (1979) 249--258. 15 Partanen, M., Hervonen, A. and Alho, H., Microspectrofluorimetric estimation of the formaldehyde-induced fluorescence of the developing main pelvic ganglion of the rat, Histochem. J., (1979) in press. 16 Partanen, M., Hervonen, A., Vaalasti, A., Kanerva, L. and Hervonen, H., Vacuolated neurons in the hypogastric ganglion of the rat, Cell and Tissue Res., (1979) in pre.~s. 17 Pawlikowski, M., Das Ganglion prostaticum und das Ganglion cervicale superius normaler und gonadopriver Ratten, Endokr. pol., 10 (1959) 449--458. 18 Pawlikowski, M., The occurrence of vacuoles in the nerve cells of autonomic ganglia as a sign of neurosecretion, Polish reed. Sci. Hist. Bull., 4 (1961) 110--112. 19 Pawlikowski, M.0 Studies on peripheral neurosecretion. I. Morphological and topographic features of neurosecretion in mammalian autonomic ganglions, Endokr. pol. 13 (1962) 153--170. 20 Pawlikowski, M., The effect of gonadal and gonadotrophic hormones on the prostatic ganglion and the superior cervical ganglion in male rats, Acta reed. pol. 3, 2 (1962) 171--183. 21 Schipper, I., Tilders F.J.H. and Proem, I.S., Micrc fluorimetric scanning of sympathetic nerve fibers. An improved method to quantitative formaldehyde induced fluorescence of biogenic amines, J. Histochem. Cytochem., 2 (1978) 1057--1066. 22 Sj~berg, N.-O., Increase in transmitter conten.~ of adrenergic nerves in the reproductive tract of female rabbits after oestrogen treatment, Acta endocr, (KrJh.), 57 (1968) 45--513. 23 Sj~strand, N.-O. and Swedin, G., Influence of age, growth, castrath" i and testosterone treatment on the noradrenaline levels of the ductus deferens and the auxiliary male reproductive glands of the rat, Acta physiol, scand., 98 (1976) 323--338. 24 Stange, H.H. and Drescher, I., Tierexperimentelle Untersuchungen am Frankenh~userschen Ganglion zum Problem der peripheren Neurosekretion, Arch. Gyn~/k., 184 (1954) 530--542. 25 Wakade, A.R. and Kirpekar, S.M., "Trophic" influence on the sympathetic nerves of the vas deferens and seminal vesicle of the guinea-pig, J. Pharmacol. exp. Ther., 186 (1973) 528--536.