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Morphological and biochemical studies on the innervation of the testis of Salamandra salamandra (L.) (Amphibia, Urodela)
Journal of the Autonomic Nervous System, 8 (1983) 65-78 Elsevier Biomedical Press
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Morphological and biochemical studies on the innervation of the testis of Salamandra salamandra (L.) (Amphibia, Urodela) Jochen Schindelmeiser l, Martin Bergmann 1, Heidrun Straub 2 and H a r t m u t Greven 2 I Anatomical and 2 Zoological Institutes, University of Mfinster, Vesaliusweg 2 - 4 and Hufferstr. 1, D-4400 Manster (F. R. G.) (Received November 18th, 1982) (Accepted December 28th, 1982)
Key words: i n n e r v a t i o n - t e s t i s - fluorescence m i c r o s c o p y - electron microscopy - - high-performance liquid chromatography - - noradrenergic nerves
Abstract
The innervation of the male gonad of Salamandra salamandra was studied by fluorescence microscopy using the glyoxylic acid method, acetylcholinesterase histochemistry, electron microscopy using glutaraldehyde/osmium tetroxide and chromate/dichromate fixation, and high-performance liquid chromatography (HPLC) with electrochemical detection of biogenic amines in homogenates of the testicular tissue. Noradrenaline has been found to be the prevailing neurotransmitter in the testicular nerves; dopamine could be identified only in minor concentration. The relatively scarce noradrenergic innervation is mainly restricted to the connective tissue septa between the immature and the mature part of the testis and between the mature part and the glandular tissue. Most of the fluorescent fibers have a close connection to blood vessels. After chromate/dichromate fixation the nerve profiles contain in most cases small vesicles with electron-dense reaction products, indicating the presence of catecholamines. Varicosities are to be found near the capillaries, in the vicinity of vascular (in the wall of arterioles) and non-vascular (near the testicular surface) smooth muscle cells; no relationships were found between nerve fibers and glandular (steroid hormone-secreting) or germinal cells. Cholinergic fibers could not be identified, non-adrenergic/non-cholinergic fibers were present only in very small numbers.
0165-1838/83/0000-0000/$03.00 © 1983 Elsevier Science Publishers
66 Introduction A number of studies on the structure and function of the autonomic nervous system in the male gonad of vertebrates have revealed a non-uniform pattern regarding the different effector cells and their relations to nerve fibers. In the testis of birds [3,20] and reptiles [30] extended plexuses of adrenergic fibers have been demonstrated by histochemical and electron microscopical techniques to make close contact with Leydig cells. Gresik [17] noted the presence of nerve terminals with clear or dense vesicles near Leydig cells in the testis of the teleost Orvzias latipes. In the mammalian testis, however, such contacts appear to be less numerous; here the adrenergic fibers seem to have mainly vasomotor functions [2,4,5,14,24]. Information concerning the innervation of the male gonad in Amphibia is rather inadequate and limited to studies of anurans [32]. In the testis of Xenopus laevis, Rana temporaria and Bufo bufo, noradrenaline has been detected, whereas neither adrenaline nor dopamine nor non-adrenergic fibers could be demonstrated. A weak adrenergic innervation was confined to blood vessels; gonadal ducts and the non-vascular intratesticular smooth muscle cells present in Xenopus laevis [31] were not innervated [32]. The present investigation of the testis of the European fire salamander, Salamandra salamandra, was undertaken in the course of our studies of the general organization of the male gonad in this species (see refs. 6 and 7). We also wished to extend our knowledge of the innervation pattern of male gonads to Amphibia other than the Anura.
Materials and Methods Eight adult fire salamanders, Salamandra salamandra, were examined during this investigation. The animals were obtained from a commercial dealer in the autumn and kept at 16°C in a terrarium until December-February, the period during which the work described was done. For fluorescence and enzyme histochemistry as well as for the H P L C of biogenic monoamines, the testes were quickly removed under diethyl ether anesthesia and frozen in iso-pentane cooled by liquid nitrogen. For light and electron microscopy the testes were removed and fixed appropriately [6,7].
Fluorescence histochemistry For the demonstration of tissue monoamines, specimens were processed following the procedures of de la Torre [13,14]. They were photographed with a Leitz orthoplan microscope equipped for fluorescence studies. The primary filter was BG 12 (3 mm), the secondary filter a Leitz K490.
A cetylcholinesterase histochemistry For the histochemical localization of acetylcholinesterase, the direct colouring thiocholine method [21] was employed following the modification by E1-Badawi and Schenk [15]. Tetraisopropyl-pyrophosphoramide (iso-OMPA, Sigma) was used to
67 inhibit pseudocholinesterases [27] in a concentration of 10 ~mol/l, both in the preincubation and the final incubation medium. The sections were incubated for 150 min at 37°C in the dark, counterstained with hematoxylin and embedded in DePeX.
Electron microscopy For electron microscopy small pieces of the whole testes were fixed in 2.5% glutaraldehyde buffered with 0.1 mol/1 cacodylate buffer, pH 7.2 (20 h), washed in cacodylate buffer (0.1 mol/1, pH 7.2), postfixed in 2% osmium tetroxide in the same buffer (1 h), dehydrated and embedded in Epon 812. Sections were cut with a LKB Ultramicrotome, stained in a saturated aqueous solution of uranyl acetate followed by lead citrate and examined in a Philips 300 electron microscope. For the demonstration of biogenic amines the fixation method of Tranzer and Richards [29], as modified by Campbell et al. [11], was used. Small pieces of the tissue were fixed in a solution containing 1% glutaraldehyde, 0.4% formaldehyde (generated from paraformaldehyde) in 0.1 mol/1 sodium chromate/potassium dichromate buffer, pH 7.2, for 15 min at 4°C. The samples were then transferred to a solution of 0.2 mol/1 sodium chromate/potassium dichromate, pH 6.2, for 18-20 h at 4°C, followed by post-fixation in 2% OsO 4 in a 0.1 mol/1 sodium chromate/potassium dichromate buffer, pH 7.2, for 1 h at 4°C. The sections were prepared as for normal electron microscopy (see above) with and without staining. Catecholamine and serotonin assay procedure (HPLC with electrochemical detection) The testes were weighed and each homogenized in toto, following the method of Sasa and Blank [26], in 10/~1 of 0.1 mol/1 EDTA, sodium salt, 50/~1 of epinine (1 m g / m l , used as an internal standard) and 100 /~1 of 25 mmol/1 HC1. The homogenate was saturated with sodium chloride (approximately 1 g) and extracted with 2.2 ml of butanol by stirring for 60 min, 1.1 ml of the butanol layer was transferred to a 5-ml vial containing 2.5 ml of heptane and 200/~1 of 10 mmol/1 HC1 followed by the extraction of the amines into the aqueous phase by shaking for 10 min. Finally, 20/~1 of this aqueous solution were injected into the valve (Rheodyne, 20/~1 loop), using a microsyringe with a stainless-steel needle. A stainless-steel column (i.d. 2.1 mm, length 500 mm, DuPont) was packed with Zipax SCX (DuPont). An acetate-citrate buffer, pH 5.2 [22], was used for the elution. A flow rate, adjusted to about 0.6 ml/min, was produced by a Milton Roy high-pressure pump with pulse dumper (working pressure 30-50 bar). For the electrochemical detection an amplifier [22] completed by addition of a Metrohm 3-electrode detector (with a glassy carbon working electrode, potential set to + 600 mV) was used. All procedures were performed at room temperature. For the quantitative estimation of the amounts of biogenic amines the schedule proposed by Refshauge et al. [25] was followed. Results
In Urodela each testicular lobe shows a well marked cephalo-caudally orientated zonation. In a given zone, seminiferous units (lobules) contain germinal cysts in an
68 identical developmental stage, i.e. along the length of the testis successive zones of spermatogonia, spermatocytes, spermatids and mature spermatozoa can be recognized. Due to the season and the composition of the testicular lobes which varies with the seasons (for details see refs. 19, 23), the males of Salamandra salamandra investigated were able to mate successfully.
Fluorescence histochemistry On the basis of this methodology the aminergic innervation of the male gonads of Salamandra salamandra can be regarded as being rather weak. It is restricted to the connective tissue, which surrounds each lobule, separates bulks of lobules containing the same stages of spermatogenesis, represents in modified form the main constituent of the glandular tissue in the most caudal part of the testis, and underlies the monolayered epithelium ensheathing the whole testis. There are no connections between the fluorescent fibers and the germinal cells or any other constituents of the lobules (follicle or Sertoli cells) or to the seminal efferent ductules. Aminergic yellow-green fluorescent fibers mostly follow the main blood vessels that reach the testis via the hilus region and then branch in the main connective tissue septa that are localized between the immature (containing spermatogonia, spermatocytes and spermatids) and mature (containing mature spermatozoa) parts of the testis and between the mature part and the glandular tissue. Finally, most of the nerves follow the smaller vessels deeper into the testis. Most of the fluorescent fibers are found near the walls of arterioles or in the vicinity of capillaries, but some do run independently, having no association with vessels. A very few can be observed to have close contact with groups of cells in the zone of spermatids. These cells have a yellow-brownish autofluorescence (Fig. lb), and possibly are steroid hormone-secreting cells. Our studies showed that the greatest density of aminergic nerve plexuses is found in the zone between the part of the testis containing mature spermatozoa and the developing glandular tissue (Fig. ld-e). In the latter, a great number of delicate varicose single fibers or nerve plexuses could be observed between the autofluorescent glandular cells. Regularly, but less often, nerves are to be found in the zone between the immature and mature parts of the testis (Fig. lb). In the small connective tissue septa between the lobules in the cephalad (containing primordial germ cells and primary spermatogonia) and central (containing secondary spermatogonia and primary spermatocytes) parts of the immature zone (Fig. la), between the most central lobules in the mature part of the testis (Fig. lc), in the most caudal part of the glandular tissue (Fig. If) and in the testicular wall, nerve fibers are to be found only very rarely.
A cetylcholinesterase histochemistry There was no clear evidence for a cholinergic innervation of the testis; only a weak acetylcholinesterase-positive reaction of the germinal cells and of some cells in the lobule boundary could be observed (cf. ref. 27).
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8
8 C: C
Fig. 1. Fluorescent histochemistry, a: fluorescent adrenergic fibers (arrow) running along the connective tissue septa, mostly near blood vessels (v); zone of secondary spermatogonia (sg). ×250. b: scarce fluorescent varicose fibers (arrow) and groups of autofluorescent cells (ac); transition between the immature and mature zone of the testis. × 250. c: zone of mature spermatozoa (arrow; weakly autofluorescing); fluorescent nerve fibers are missing. × 100. d: transition between the mature zone containing spermatozoa (sz) and the glandular tissue (below); ac, autofluorescing glandular cells; few adrenergic fibers (arrow). × 100. e: glandular tissue with groups of autofluorescing cells (ac) and fluorescent varicose fibers (arrow). × 250. f: most caudal part of the glandular tissue near the testis wall (tw). x 100.
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Electron microscopy In the connective tissue septa between the immature and mature parts (for definition see above) of the male gonad, unmyelinated preterminal axon bundles are to be found; some show a close contact to bundles of collagenous fibers, others are localized near the nucleated part of fibrocytes (minimal distance nucleus-axon about 150 nm; Fig. 2a). The diameter of the axons is between 100 and 750 nm; neurotubules, neurofilaments, mitochondria, and after glutaraldehyde/osmium tetroxide fixation, some small agranular electron-lucent synaptic vesicles (diameter 30 nm) can be visualized (Fig. 2a). Most of the terminal nerve profiles in this region are to be found near to capillaries (Fig. 3a) as dilated naked axons (diameter 300-700 nm) without a basal lamina. After chromate/dichromate fixation these terminals show a few agranular vesicles (diameter 35-55 mm), but mostly vesicles with an electron-dense reaction product (diameter 30-50 and 60-120 nm); the latter contain a dense-core with a light halo (Fig. 3a, e). According to the specificity of the c h r o m a t e / d i c h r o m a t e reaction, these vesicles are to be regarded as containing catecholamines. As already stated, fluorescence microscopy revealed that the density of innervation in the region between the caudal part of the mature zone of the testis and the cranial part of the glandular tissue is much higher than in the immature part of the testis. Myelinated nerves are lacking; larger axon bundles surrounded by Schwann cells are to be found regularly in the connective tissue septa, sometimes without any specific orientation to the blood vessels (Fig. 2b, d). In the glandular tissue adjacent to the zone containing mature spermatozoa, the highest number of nerve profiles can be observed. They contain neurotubules, neurofilaments and mitochondria (Fig. 2d). After glutaraldehyde/osmium tetroxide fixation granular and agranular synaptic vesicles are to be seen in intercalated transmitter segments (Fig. 2d). Some varicosities approach smooth muscle cells in the walls of arterioles (Fig. 4a), non-vascular smooth muscle cells in the connective tissue septa near to the testicular surface (Fig. 4b) and the lobule boundary. The neuron-to-tissue gap is about 500 nm; in all cases, the varicosities also are separated from adjacent cells by a small layer of collagenous fibers. A contact of nervous fibers with glandular cells, germinal cells or efferent ductules could not be identified. Most of the terminal naked axons, their diameters ranging between 300 and 900 nm, show a close connection to capillaries (Fig. 3c-e). After c h r o m a t e / d i c h r o m a t e fixation, vesicles are to be found therein, which apparently contain catecholamines. A few varicosities were observed that contain mostly small agranular (diameter 30-70 nm, sometimes elongated) and a few dense-core vesicles without a distinct reaction to the chromate/dichromate fixation. These axons do not seem to contain biogenic amines and might be classified as non-adrenergic/non-cholinergic [11].
Catecholamine and serotonin assay by high-performance liquid chromatography (HPLC) with electrochemical detection Noradrenaline could be identified in all cases as the main testicular neurotransmitter of the catecholamine plus serotonin group. Dopamine was present in a lower
71
Fig. 2. Electron microscopy, a: lobule boundary, zone of primary spermatocytes; bundle of axons (a) surrounded by collagenous fibers (co), near the nucleated part of a fibrocyte (fc). × 32,000. b: transition between the zone of mature spermatozoa and the glandular tissue; axon bundles (a) in the connective tissue between groups of glandular cells (gc). x 7700. c: glandular tissue; axon bundles (a) near a capillary (lu, lumen). × 6700. d: glandular tissue; axon bundles (a) without any relation to blood vessels; collagenous fibers (co); note the electron-dense core in some of the vesicles (arrow). x 23,400. a - d glutaraldeh y d e / o s m i u m tetroxide fixation.
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Fig. 3. a: i m m a t u r e zone of the testis; n a k e d adrenergic axon (arrow) near the e n d o t h e l i u m (e) of a capillary, x 33,000. b: adrenergic axons (arrow) without relation to blood vessels in the c o n n e c t i v e tissue septa of the i m m a t u r e testis, x 5200. c: n o n - c h o l i n e r g i c / n o n - a d r e n e r g i c axons near a capillary; g l a n d u l a r tissue, x 31,400. d: adrenergic axons near the e n d o t h e l i u m (e) of a capillary; g l a n d u l a r tissue, x 15,600. e: transition between the m a t u r e testis and the g l a n d u l a r tissue. A d r e n e r g i c axons near capillary endot h e l i u m (e), p a r t l y s u r r o u n d e d by collagenous fibers (co). X 18,800. f: adrenergic axons s u r r o u n d e d partly by collagenous fibers (co); g l a n d u l a r tissue, x 18,300. a - f c h r o m a t e / d i c h r o m a t e fixation.
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Fig. 4. a: transition between the mature testis and the glandular tissue; axon bundles (a) near smooth muscle cells (sin) in the wall of an arteriole (lu, lumen) and glandular cells (gc).x 3300. b: glandular tissue; axon bundles (a) near non-vascular smooth muscle cells ( s m ) . x l 4 , 3 0 0 . a and b: g l u t a r a l d e h y d e / o s m i u m tetroxide fixation.
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Fig. 5. High-performance liquid chromatography with electrochemical detection, a: homogenate of one testis, b: control, f, front; na, noradrenaline; a, adrenaline; da, dopamine; se, serotonin; epi, epinine (standard).
concentration (about 5-10% compared to that of noradrenaline); adrenaline and serotonin could be found only in very small amounts (Fig. 5). The presence of noradrenaline and dopamine in the testis and their relative amounts compared with the fresh weight of the organ (about 50 mg) was nearly constant. The testicular noradrenaline concentration was normally in the range of 25-30 n g / g fresh weight; in one case, however, a concentration higher by a factor of more than 10 was obtained with the same ratio between noradrenaline and dopamine.
75 Discussion In a comparative investigation of the type of catecholamines in the brain of several vertebrates, a clear predominance of adrenaline was demonstrated in amphibia, whereas in other species noradrenaline appears to be the principal transmitter substance [8]. In general, this pattern of distribution could be confirmed also for peripheral tissues of anurans [1,8]. However, some exceptions, mainly in the urodeles, have been found. With respect to the innervation of the male urogenital tract in anurans, the investigations of Unsicker et al. [32] confirm the generally accepted view that adrenaline is the predominant transmitter in Amphibia. Adrenaline was detected by thin-layer chromatography in the bladder, ureter and kidneyadrenal of Xenopus laevis, Rana temporaria and Bufo bufo, but some noradrenaline was present in the ureter of Xenopus laevis and in all kidney-adrenal preparations. In the testes of the species mentioned, however, only noradrenaline could be demonstrated after tissue homogenization. Nevertheless, on the basis of pharmacological experiments and differentiated formaldehyde treatment of testis sections, Unsicker et al. [32] postulated the presence of adrenaline too, at least in very small amounts. They suggested that the testicular noradrenaline might come chiefly from small, intensely fluorescent (SIF) noradrenaline-containing cells comparable to those in the epididymis and the vas deferens of the tortoise Testudo hermanni [28]. On the basis of assays of peripheral organs of some urodeles, Brodie et al. [8] reported that noradrenaline was predominant in the heart, stomach and small intestine (see also ref. 1). The HPLC analyses of the Salamandra testis reported here also provided clear evidence for the presence of considerable amounts of noradrenaline; furthermore, small amounts of dopamine could be identified, whereas adrenaline or serotonin were not present in measurable quantities. The low concentration of dopamine, the ratio being approximately constant relative to noradrenaline, may be explained by its role as a direct precursor in the biosynthesis of noradrenaline (cf. ref. 10). The predominance of noradrenaline in some peripheral tissues of urodeles is also confirmed by our studies of the innervation of the uterus of Salamandra salamandra. Moreover, in the brain of this species, noradrenaline is present, even in a concentration twice that of adrenaline, but distinctly lower than that of serotonin [18]. The absolute concentration of noradrenaline in the testis of Salamandra salamandra is relatively low (about one-third) compared to that of the uterus [18]. The finding that noradrenaline had a 10 times higher concentration than expected in one case, cannot be explained at present. Perhaps noradrenaline storing (SIF) cells, like those described by Sj6strand [28] in Testudo hermanni, had been obtained in different amounts during the preparations of the testes. Fluorescence microscopy points to a relatively weak and non-uniform innervation of the male gonad in Salamandra salamandra. Compared to the uterus of the same species, where large plexuses have been observed [18], nerve fibers in the testis are to be found exclusively in the connective tissue septa near the blood vessels. Only a few fibers were seen to run independently of arterioles or capillaries, but a contact with glandular or germinal cells could never be observed. In this respect, the urodele as
76
well as the anuran testis [32] resembles the mammalian testis, where adrenergic fibers obviously have a vasomotor function [2,4,5,12,14,24]. The limitation of nerve fibers to the zones between the immature and mature parts, with lobules of the latter containing spermatids or spermatozoa, and between the mature part and the glandular tissue of the testis in Salamandra salamandra is due to the fact that the main septa of connective tissue contain the principal blood vessels which extend from the testicular hilus between these compartments. The nerve fibers are orientated along these vessels and branch to other parts of the male gonad with smaller septa and capillaries. In the testicular zones mentioned, spermatids and mature spermatozoa develop and in the more caudal region lobule boundary cells differentiate into steroid hormone-secreting cells [6]. The influence of the nervous system on these events may be limited only to the regulation of the blood supply. We failed to demonstrate cholinergic fibers in the testis of Salamandra salamandra. Also in the literature there are no hints given of a cholinergic innervation of the amphibian testis, whereas a few fibers identified as cholinergic innervate the mammalian seminal efferent pathways (for references see ref. 32). Our ultrastructural observations confirm the light microscopic and biochemical findings. The chromate/dichromate fixation [11, 29] permits the identification of nearly all of the nerve profiles in the salamander testis as being catecholaminergic. The nature and function of the very scarce non-adrenergic/non-cholinergic axons (for definition see ref. 11) is unknown. As has been demonstrated already by fluorescence microscopy, the main parts of nerve endings show a close affinity to blood vessels. Axon profiles may be found near smooth muscle cells in the walls of arterioles, and also near the capillary endothelium (cf. ref. 16). Direct neuro-muscular or neuro-endothelial contacts, however, could not be observed. Axons approach, but never make contact with the non-vascular smooth muscle cells in connective tissue septa near the testicular surface [7,31,32]. In any case, the nerves and the myolemma are separated by bundles of collagenous fibers. Thus, a possible influence on muscle cells may occur, but solely by circulating catecholamines as is assumed in general for lower vertebrates [9].
Acknowledgements
We are indebted to Prof. Dr. D. Kuhlmann, Zoological Institute of the University of M/)nster, who provided the facilities for HPLC, to B. Kerkhoff for technical assistance, and to Miss E. McLure for reading the English manuscript.
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77 2 Baumgarten, H.G. and Holstein, A.F., Catecholaminhaltige Nervenfasern im Hoden des Menschen, Z. Zellforsch., 79 (1967) 389-395. 3 Baumgarten, H.G. and Holstein, A.F., Adrenerge Innervation im Hoden und Nebenhoden vom Schwan (Cygnus olor), Z. Zellforsch., 91 (1968) 402-410. 4 Baumgarten, H.G. and Holstein, A.F., Noradrenerge Nervenfasern im Hoden von Mammaliern und anderen Vertebraten, J. neurovisc. Rel., Suppl. 10 (1971) 563-572. 5 Baumgarten, H.G., Falck, B., Holstein, A.F., Owman, Ch. and Owman, T., Adrenergic innervation of the human testis, epididymis, ductus deferens and prostate: a fluorescence microscopic and fluorimetric study, Z. Zellforsch., 90 (1968) 81-95~ 6 Bergmann, M., Schindelmeiser, J. and Greven, H., The zone of mature spermatozoa in the testis of Salamandra salamandra (L.) (Amphibia, Urodela), Z. mikrosk.-anat. Forsch., 96 (1982) 221-234. 7 Bergmann, M., Greven, H. and Schindelmeiser, J., The fine structure of the testis wall in Salamandra salamandra (L.) (Amphibia, Urodela), Arch. histol, japon., 45 (1982) 317-324. 8 Brodie, B.B., Bogdanski, D.F. and Bonomi, L., Formation, storage and metabolism of serotonin (5-hydroxytryptamin) and catecholamines in lower vertebrates. In D. Richter (Ed.), Comparative Neurochemistry, Pergamon Press, Oxford, 1964, pp. 367-377. 9 Burnstock, G., Evolution of the autonomic innervation of visceral and cardiovascular system in vertebrates, Pharmacol. Rev., 21 (1969) 247-324. 10 Burnstock, G. and Costa, M., Adrenergic neurons. Their organization, function and development in the peripheral nervous system, Chapman and Hall, London, 1975. 11 Campbell, G., Huller, C.J. and Rogers, D.C., Fine structural and cytochemical study of the innervation of smooth muscle in an amphibian (Bufo marinus) lung before and after denervation, Cell Tiss. Res., 194 (1978) 419-432. 12 Dayan, A.D., Variation between species in the innervation of intratesticular blood vessels, Experientia, 26 (1970) 1359-1360. 13 De la Torre, J.C. Standardization of the sucrose-potassium phosphate-glyoxylic acid histofluorescence method for tissue monoamines, Neurosci. Lett., 17 (1980) 339-340. 14 De la Torre, J.C., An improved approach to histofluorescence using the SPG method for tissue monoamines, J. neurosci. Meth., 3 (1980) 1-5. 15 El-Badawi, A. and Schenk, E.A., Histochemical methods for separate, consecutive and simultaneous demonstration of acetylcholinesterase and norepinephrine in cryostat sections, J. Histochem. Cytochem., 15 (1967) 580-588. 16 Gabella, G., Structure of the Autonomic Nervous System, Chapman and Hall, London, 1976. 17 Gresik, E.W., Fine structural evidence for the presence of nerve terminals in the testis of the teleost, Oryzias latipes, Gen. Comp. Endocrinol., 21 (1973) 210-213. 18 Greven, H., Schindelmeiser, J. and Straub, H., On the innervation of the uterus of Salamandra salamandra (L.) (Amphibia, Urodela), Cell Tiss. Res., in press. 19 Joly, J., Les cycles sexuels de Salamandra (L.) I. Cycle sexuel des males, Ann Sci. Nat. Zool., 264 (1971) 451-504. 20 Haase, E., Histochemische und elektronenmikroskopische Untersuchungen 0bet die Innervation des Hodens vom Bergfink (Fringilla montifringilla), Verh. Dtsch. Zool. Ges., 66 (1973) 106-110. 21 Karnovsky, M.J. and Roots, L., A 'direct-coloring' thiocholine technique for cholinesterases. J. Histochem. Cytochem., 12 (1964) 219-221. 22 Keller, R., Oke, A., Mefford, I. and Adams, R.N., Liquid chromatographic analysis of catecholamines. Routine assay for regional brain mapping, Life Sci., 19 (1976) 995-1004. 23 Lofts, B., Reproduction. In B. Lofts (Ed.), Physiology of the Amphibia, Vol. II, Academic Press, New York, London, 1974, pp. 107-218. 24 Norberg, K.-A., Risley, P.L. and Ungerstedt, U., Adrenergic innervation of the male reproductive ducts in some mammals. I. The distribution of adrenergic nerves, Z. Zellforsch., 76 (1967) 278-286. 25 Refshauge, C., Kissinger, P.T., Dreiling, R., Blank, L., Freeman, R. and Adams, R.N., New high performance liquid chromatographic analysis of brain catecholamines, Life Sci., 14 (1974) 311-322. 26 Sasa, S. and Blank, L., Determination of serotonin and dopamine in mouse brain tissue by high performance liquid chromatography with electrochemical detection, Analyt. Chem., 49 (1977) 354-359. 27 Silver, A., The Biology of Cholinesterases, North-Holland, Oxford, Amsterdam, 1974.
78 28 SjOstrand, N.O., Noradrenaline-containing cells in the epididymis and vas deferens of the tortoise Testudo hermanni, Acta zool. (Stockh.), 50 (1969) 271 275. 29 Tranzer, J.-P. and Richards, J.G., Ultrastructural cytochemistry of biogenic amines in nervous tissue: methodological improvements, J. Histochem. Cytochem., 24 (1976) 1178-1193. 30 Unsicker, K., Innervation of the testicular interstitial tissue in reptiles, Z. Zellforsch., 146 (1973) 123-138. 31 Unsicker, K., Fine structure of the male genital tract and kidney in the amphibia Xenopus laevis Daudin, Rana temporaria L., and Bufo bufo L. under normal and experimental conditions. I. Testicular interstitial tissue and seminal efferent ducts, Cell Tiss. Res., 158 (1975) 215-240. 32 Unsicker, K., Axelsson, S., Owman, Ch. and Svensson, K.-G., lnnervation of the male genital tract and kidney in the amphibia, Xenopus laevis Daudin, Rana ternporaria L., and Bufo bufo L., Cell Tiss. Res.. 160 (1975) 453-484.
65
Morphological and biochemical studies on the innervation of the testis of Salamandra salamandra (L.) (Amphibia, Urodela) Jochen Schindelmeiser l, Martin Bergmann 1, Heidrun Straub 2 and H a r t m u t Greven 2 I Anatomical and 2 Zoological Institutes, University of Mfinster, Vesaliusweg 2 - 4 and Hufferstr. 1, D-4400 Manster (F. R. G.) (Received November 18th, 1982) (Accepted December 28th, 1982)
Key words: i n n e r v a t i o n - t e s t i s - fluorescence m i c r o s c o p y - electron microscopy - - high-performance liquid chromatography - - noradrenergic nerves
Abstract
The innervation of the male gonad of Salamandra salamandra was studied by fluorescence microscopy using the glyoxylic acid method, acetylcholinesterase histochemistry, electron microscopy using glutaraldehyde/osmium tetroxide and chromate/dichromate fixation, and high-performance liquid chromatography (HPLC) with electrochemical detection of biogenic amines in homogenates of the testicular tissue. Noradrenaline has been found to be the prevailing neurotransmitter in the testicular nerves; dopamine could be identified only in minor concentration. The relatively scarce noradrenergic innervation is mainly restricted to the connective tissue septa between the immature and the mature part of the testis and between the mature part and the glandular tissue. Most of the fluorescent fibers have a close connection to blood vessels. After chromate/dichromate fixation the nerve profiles contain in most cases small vesicles with electron-dense reaction products, indicating the presence of catecholamines. Varicosities are to be found near the capillaries, in the vicinity of vascular (in the wall of arterioles) and non-vascular (near the testicular surface) smooth muscle cells; no relationships were found between nerve fibers and glandular (steroid hormone-secreting) or germinal cells. Cholinergic fibers could not be identified, non-adrenergic/non-cholinergic fibers were present only in very small numbers.
0165-1838/83/0000-0000/$03.00 © 1983 Elsevier Science Publishers
66 Introduction A number of studies on the structure and function of the autonomic nervous system in the male gonad of vertebrates have revealed a non-uniform pattern regarding the different effector cells and their relations to nerve fibers. In the testis of birds [3,20] and reptiles [30] extended plexuses of adrenergic fibers have been demonstrated by histochemical and electron microscopical techniques to make close contact with Leydig cells. Gresik [17] noted the presence of nerve terminals with clear or dense vesicles near Leydig cells in the testis of the teleost Orvzias latipes. In the mammalian testis, however, such contacts appear to be less numerous; here the adrenergic fibers seem to have mainly vasomotor functions [2,4,5,14,24]. Information concerning the innervation of the male gonad in Amphibia is rather inadequate and limited to studies of anurans [32]. In the testis of Xenopus laevis, Rana temporaria and Bufo bufo, noradrenaline has been detected, whereas neither adrenaline nor dopamine nor non-adrenergic fibers could be demonstrated. A weak adrenergic innervation was confined to blood vessels; gonadal ducts and the non-vascular intratesticular smooth muscle cells present in Xenopus laevis [31] were not innervated [32]. The present investigation of the testis of the European fire salamander, Salamandra salamandra, was undertaken in the course of our studies of the general organization of the male gonad in this species (see refs. 6 and 7). We also wished to extend our knowledge of the innervation pattern of male gonads to Amphibia other than the Anura.
Materials and Methods Eight adult fire salamanders, Salamandra salamandra, were examined during this investigation. The animals were obtained from a commercial dealer in the autumn and kept at 16°C in a terrarium until December-February, the period during which the work described was done. For fluorescence and enzyme histochemistry as well as for the H P L C of biogenic monoamines, the testes were quickly removed under diethyl ether anesthesia and frozen in iso-pentane cooled by liquid nitrogen. For light and electron microscopy the testes were removed and fixed appropriately [6,7].
Fluorescence histochemistry For the demonstration of tissue monoamines, specimens were processed following the procedures of de la Torre [13,14]. They were photographed with a Leitz orthoplan microscope equipped for fluorescence studies. The primary filter was BG 12 (3 mm), the secondary filter a Leitz K490.
A cetylcholinesterase histochemistry For the histochemical localization of acetylcholinesterase, the direct colouring thiocholine method [21] was employed following the modification by E1-Badawi and Schenk [15]. Tetraisopropyl-pyrophosphoramide (iso-OMPA, Sigma) was used to
67 inhibit pseudocholinesterases [27] in a concentration of 10 ~mol/l, both in the preincubation and the final incubation medium. The sections were incubated for 150 min at 37°C in the dark, counterstained with hematoxylin and embedded in DePeX.
Electron microscopy For electron microscopy small pieces of the whole testes were fixed in 2.5% glutaraldehyde buffered with 0.1 mol/1 cacodylate buffer, pH 7.2 (20 h), washed in cacodylate buffer (0.1 mol/1, pH 7.2), postfixed in 2% osmium tetroxide in the same buffer (1 h), dehydrated and embedded in Epon 812. Sections were cut with a LKB Ultramicrotome, stained in a saturated aqueous solution of uranyl acetate followed by lead citrate and examined in a Philips 300 electron microscope. For the demonstration of biogenic amines the fixation method of Tranzer and Richards [29], as modified by Campbell et al. [11], was used. Small pieces of the tissue were fixed in a solution containing 1% glutaraldehyde, 0.4% formaldehyde (generated from paraformaldehyde) in 0.1 mol/1 sodium chromate/potassium dichromate buffer, pH 7.2, for 15 min at 4°C. The samples were then transferred to a solution of 0.2 mol/1 sodium chromate/potassium dichromate, pH 6.2, for 18-20 h at 4°C, followed by post-fixation in 2% OsO 4 in a 0.1 mol/1 sodium chromate/potassium dichromate buffer, pH 7.2, for 1 h at 4°C. The sections were prepared as for normal electron microscopy (see above) with and without staining. Catecholamine and serotonin assay procedure (HPLC with electrochemical detection) The testes were weighed and each homogenized in toto, following the method of Sasa and Blank [26], in 10/~1 of 0.1 mol/1 EDTA, sodium salt, 50/~1 of epinine (1 m g / m l , used as an internal standard) and 100 /~1 of 25 mmol/1 HC1. The homogenate was saturated with sodium chloride (approximately 1 g) and extracted with 2.2 ml of butanol by stirring for 60 min, 1.1 ml of the butanol layer was transferred to a 5-ml vial containing 2.5 ml of heptane and 200/~1 of 10 mmol/1 HC1 followed by the extraction of the amines into the aqueous phase by shaking for 10 min. Finally, 20/~1 of this aqueous solution were injected into the valve (Rheodyne, 20/~1 loop), using a microsyringe with a stainless-steel needle. A stainless-steel column (i.d. 2.1 mm, length 500 mm, DuPont) was packed with Zipax SCX (DuPont). An acetate-citrate buffer, pH 5.2 [22], was used for the elution. A flow rate, adjusted to about 0.6 ml/min, was produced by a Milton Roy high-pressure pump with pulse dumper (working pressure 30-50 bar). For the electrochemical detection an amplifier [22] completed by addition of a Metrohm 3-electrode detector (with a glassy carbon working electrode, potential set to + 600 mV) was used. All procedures were performed at room temperature. For the quantitative estimation of the amounts of biogenic amines the schedule proposed by Refshauge et al. [25] was followed. Results
In Urodela each testicular lobe shows a well marked cephalo-caudally orientated zonation. In a given zone, seminiferous units (lobules) contain germinal cysts in an
68 identical developmental stage, i.e. along the length of the testis successive zones of spermatogonia, spermatocytes, spermatids and mature spermatozoa can be recognized. Due to the season and the composition of the testicular lobes which varies with the seasons (for details see refs. 19, 23), the males of Salamandra salamandra investigated were able to mate successfully.
Fluorescence histochemistry On the basis of this methodology the aminergic innervation of the male gonads of Salamandra salamandra can be regarded as being rather weak. It is restricted to the connective tissue, which surrounds each lobule, separates bulks of lobules containing the same stages of spermatogenesis, represents in modified form the main constituent of the glandular tissue in the most caudal part of the testis, and underlies the monolayered epithelium ensheathing the whole testis. There are no connections between the fluorescent fibers and the germinal cells or any other constituents of the lobules (follicle or Sertoli cells) or to the seminal efferent ductules. Aminergic yellow-green fluorescent fibers mostly follow the main blood vessels that reach the testis via the hilus region and then branch in the main connective tissue septa that are localized between the immature (containing spermatogonia, spermatocytes and spermatids) and mature (containing mature spermatozoa) parts of the testis and between the mature part and the glandular tissue. Finally, most of the nerves follow the smaller vessels deeper into the testis. Most of the fluorescent fibers are found near the walls of arterioles or in the vicinity of capillaries, but some do run independently, having no association with vessels. A very few can be observed to have close contact with groups of cells in the zone of spermatids. These cells have a yellow-brownish autofluorescence (Fig. lb), and possibly are steroid hormone-secreting cells. Our studies showed that the greatest density of aminergic nerve plexuses is found in the zone between the part of the testis containing mature spermatozoa and the developing glandular tissue (Fig. ld-e). In the latter, a great number of delicate varicose single fibers or nerve plexuses could be observed between the autofluorescent glandular cells. Regularly, but less often, nerves are to be found in the zone between the immature and mature parts of the testis (Fig. lb). In the small connective tissue septa between the lobules in the cephalad (containing primordial germ cells and primary spermatogonia) and central (containing secondary spermatogonia and primary spermatocytes) parts of the immature zone (Fig. la), between the most central lobules in the mature part of the testis (Fig. lc), in the most caudal part of the glandular tissue (Fig. If) and in the testicular wall, nerve fibers are to be found only very rarely.
A cetylcholinesterase histochemistry There was no clear evidence for a cholinergic innervation of the testis; only a weak acetylcholinesterase-positive reaction of the germinal cells and of some cells in the lobule boundary could be observed (cf. ref. 27).
69
8
8 C: C
Fig. 1. Fluorescent histochemistry, a: fluorescent adrenergic fibers (arrow) running along the connective tissue septa, mostly near blood vessels (v); zone of secondary spermatogonia (sg). ×250. b: scarce fluorescent varicose fibers (arrow) and groups of autofluorescent cells (ac); transition between the immature and mature zone of the testis. × 250. c: zone of mature spermatozoa (arrow; weakly autofluorescing); fluorescent nerve fibers are missing. × 100. d: transition between the mature zone containing spermatozoa (sz) and the glandular tissue (below); ac, autofluorescing glandular cells; few adrenergic fibers (arrow). × 100. e: glandular tissue with groups of autofluorescing cells (ac) and fluorescent varicose fibers (arrow). × 250. f: most caudal part of the glandular tissue near the testis wall (tw). x 100.
70
Electron microscopy In the connective tissue septa between the immature and mature parts (for definition see above) of the male gonad, unmyelinated preterminal axon bundles are to be found; some show a close contact to bundles of collagenous fibers, others are localized near the nucleated part of fibrocytes (minimal distance nucleus-axon about 150 nm; Fig. 2a). The diameter of the axons is between 100 and 750 nm; neurotubules, neurofilaments, mitochondria, and after glutaraldehyde/osmium tetroxide fixation, some small agranular electron-lucent synaptic vesicles (diameter 30 nm) can be visualized (Fig. 2a). Most of the terminal nerve profiles in this region are to be found near to capillaries (Fig. 3a) as dilated naked axons (diameter 300-700 nm) without a basal lamina. After chromate/dichromate fixation these terminals show a few agranular vesicles (diameter 35-55 mm), but mostly vesicles with an electron-dense reaction product (diameter 30-50 and 60-120 nm); the latter contain a dense-core with a light halo (Fig. 3a, e). According to the specificity of the c h r o m a t e / d i c h r o m a t e reaction, these vesicles are to be regarded as containing catecholamines. As already stated, fluorescence microscopy revealed that the density of innervation in the region between the caudal part of the mature zone of the testis and the cranial part of the glandular tissue is much higher than in the immature part of the testis. Myelinated nerves are lacking; larger axon bundles surrounded by Schwann cells are to be found regularly in the connective tissue septa, sometimes without any specific orientation to the blood vessels (Fig. 2b, d). In the glandular tissue adjacent to the zone containing mature spermatozoa, the highest number of nerve profiles can be observed. They contain neurotubules, neurofilaments and mitochondria (Fig. 2d). After glutaraldehyde/osmium tetroxide fixation granular and agranular synaptic vesicles are to be seen in intercalated transmitter segments (Fig. 2d). Some varicosities approach smooth muscle cells in the walls of arterioles (Fig. 4a), non-vascular smooth muscle cells in the connective tissue septa near to the testicular surface (Fig. 4b) and the lobule boundary. The neuron-to-tissue gap is about 500 nm; in all cases, the varicosities also are separated from adjacent cells by a small layer of collagenous fibers. A contact of nervous fibers with glandular cells, germinal cells or efferent ductules could not be identified. Most of the terminal naked axons, their diameters ranging between 300 and 900 nm, show a close connection to capillaries (Fig. 3c-e). After c h r o m a t e / d i c h r o m a t e fixation, vesicles are to be found therein, which apparently contain catecholamines. A few varicosities were observed that contain mostly small agranular (diameter 30-70 nm, sometimes elongated) and a few dense-core vesicles without a distinct reaction to the chromate/dichromate fixation. These axons do not seem to contain biogenic amines and might be classified as non-adrenergic/non-cholinergic [11].
Catecholamine and serotonin assay by high-performance liquid chromatography (HPLC) with electrochemical detection Noradrenaline could be identified in all cases as the main testicular neurotransmitter of the catecholamine plus serotonin group. Dopamine was present in a lower
71
Fig. 2. Electron microscopy, a: lobule boundary, zone of primary spermatocytes; bundle of axons (a) surrounded by collagenous fibers (co), near the nucleated part of a fibrocyte (fc). × 32,000. b: transition between the zone of mature spermatozoa and the glandular tissue; axon bundles (a) in the connective tissue between groups of glandular cells (gc). x 7700. c: glandular tissue; axon bundles (a) near a capillary (lu, lumen). × 6700. d: glandular tissue; axon bundles (a) without any relation to blood vessels; collagenous fibers (co); note the electron-dense core in some of the vesicles (arrow). x 23,400. a - d glutaraldeh y d e / o s m i u m tetroxide fixation.
72
Fig. 3. a: i m m a t u r e zone of the testis; n a k e d adrenergic axon (arrow) near the e n d o t h e l i u m (e) of a capillary, x 33,000. b: adrenergic axons (arrow) without relation to blood vessels in the c o n n e c t i v e tissue septa of the i m m a t u r e testis, x 5200. c: n o n - c h o l i n e r g i c / n o n - a d r e n e r g i c axons near a capillary; g l a n d u l a r tissue, x 31,400. d: adrenergic axons near the e n d o t h e l i u m (e) of a capillary; g l a n d u l a r tissue, x 15,600. e: transition between the m a t u r e testis and the g l a n d u l a r tissue. A d r e n e r g i c axons near capillary endot h e l i u m (e), p a r t l y s u r r o u n d e d by collagenous fibers (co). X 18,800. f: adrenergic axons s u r r o u n d e d partly by collagenous fibers (co); g l a n d u l a r tissue, x 18,300. a - f c h r o m a t e / d i c h r o m a t e fixation.
73
Fig. 4. a: transition between the mature testis and the glandular tissue; axon bundles (a) near smooth muscle cells (sin) in the wall of an arteriole (lu, lumen) and glandular cells (gc).x 3300. b: glandular tissue; axon bundles (a) near non-vascular smooth muscle cells ( s m ) . x l 4 , 3 0 0 . a and b: g l u t a r a l d e h y d e / o s m i u m tetroxide fixation.
74
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concentration (about 5-10% compared to that of noradrenaline); adrenaline and serotonin could be found only in very small amounts (Fig. 5). The presence of noradrenaline and dopamine in the testis and their relative amounts compared with the fresh weight of the organ (about 50 mg) was nearly constant. The testicular noradrenaline concentration was normally in the range of 25-30 n g / g fresh weight; in one case, however, a concentration higher by a factor of more than 10 was obtained with the same ratio between noradrenaline and dopamine.
75 Discussion In a comparative investigation of the type of catecholamines in the brain of several vertebrates, a clear predominance of adrenaline was demonstrated in amphibia, whereas in other species noradrenaline appears to be the principal transmitter substance [8]. In general, this pattern of distribution could be confirmed also for peripheral tissues of anurans [1,8]. However, some exceptions, mainly in the urodeles, have been found. With respect to the innervation of the male urogenital tract in anurans, the investigations of Unsicker et al. [32] confirm the generally accepted view that adrenaline is the predominant transmitter in Amphibia. Adrenaline was detected by thin-layer chromatography in the bladder, ureter and kidneyadrenal of Xenopus laevis, Rana temporaria and Bufo bufo, but some noradrenaline was present in the ureter of Xenopus laevis and in all kidney-adrenal preparations. In the testes of the species mentioned, however, only noradrenaline could be demonstrated after tissue homogenization. Nevertheless, on the basis of pharmacological experiments and differentiated formaldehyde treatment of testis sections, Unsicker et al. [32] postulated the presence of adrenaline too, at least in very small amounts. They suggested that the testicular noradrenaline might come chiefly from small, intensely fluorescent (SIF) noradrenaline-containing cells comparable to those in the epididymis and the vas deferens of the tortoise Testudo hermanni [28]. On the basis of assays of peripheral organs of some urodeles, Brodie et al. [8] reported that noradrenaline was predominant in the heart, stomach and small intestine (see also ref. 1). The HPLC analyses of the Salamandra testis reported here also provided clear evidence for the presence of considerable amounts of noradrenaline; furthermore, small amounts of dopamine could be identified, whereas adrenaline or serotonin were not present in measurable quantities. The low concentration of dopamine, the ratio being approximately constant relative to noradrenaline, may be explained by its role as a direct precursor in the biosynthesis of noradrenaline (cf. ref. 10). The predominance of noradrenaline in some peripheral tissues of urodeles is also confirmed by our studies of the innervation of the uterus of Salamandra salamandra. Moreover, in the brain of this species, noradrenaline is present, even in a concentration twice that of adrenaline, but distinctly lower than that of serotonin [18]. The absolute concentration of noradrenaline in the testis of Salamandra salamandra is relatively low (about one-third) compared to that of the uterus [18]. The finding that noradrenaline had a 10 times higher concentration than expected in one case, cannot be explained at present. Perhaps noradrenaline storing (SIF) cells, like those described by Sj6strand [28] in Testudo hermanni, had been obtained in different amounts during the preparations of the testes. Fluorescence microscopy points to a relatively weak and non-uniform innervation of the male gonad in Salamandra salamandra. Compared to the uterus of the same species, where large plexuses have been observed [18], nerve fibers in the testis are to be found exclusively in the connective tissue septa near the blood vessels. Only a few fibers were seen to run independently of arterioles or capillaries, but a contact with glandular or germinal cells could never be observed. In this respect, the urodele as
76
well as the anuran testis [32] resembles the mammalian testis, where adrenergic fibers obviously have a vasomotor function [2,4,5,12,14,24]. The limitation of nerve fibers to the zones between the immature and mature parts, with lobules of the latter containing spermatids or spermatozoa, and between the mature part and the glandular tissue of the testis in Salamandra salamandra is due to the fact that the main septa of connective tissue contain the principal blood vessels which extend from the testicular hilus between these compartments. The nerve fibers are orientated along these vessels and branch to other parts of the male gonad with smaller septa and capillaries. In the testicular zones mentioned, spermatids and mature spermatozoa develop and in the more caudal region lobule boundary cells differentiate into steroid hormone-secreting cells [6]. The influence of the nervous system on these events may be limited only to the regulation of the blood supply. We failed to demonstrate cholinergic fibers in the testis of Salamandra salamandra. Also in the literature there are no hints given of a cholinergic innervation of the amphibian testis, whereas a few fibers identified as cholinergic innervate the mammalian seminal efferent pathways (for references see ref. 32). Our ultrastructural observations confirm the light microscopic and biochemical findings. The chromate/dichromate fixation [11, 29] permits the identification of nearly all of the nerve profiles in the salamander testis as being catecholaminergic. The nature and function of the very scarce non-adrenergic/non-cholinergic axons (for definition see ref. 11) is unknown. As has been demonstrated already by fluorescence microscopy, the main parts of nerve endings show a close affinity to blood vessels. Axon profiles may be found near smooth muscle cells in the walls of arterioles, and also near the capillary endothelium (cf. ref. 16). Direct neuro-muscular or neuro-endothelial contacts, however, could not be observed. Axons approach, but never make contact with the non-vascular smooth muscle cells in connective tissue septa near the testicular surface [7,31,32]. In any case, the nerves and the myolemma are separated by bundles of collagenous fibers. Thus, a possible influence on muscle cells may occur, but solely by circulating catecholamines as is assumed in general for lower vertebrates [9].
Acknowledgements
We are indebted to Prof. Dr. D. Kuhlmann, Zoological Institute of the University of M/)nster, who provided the facilities for HPLC, to B. Kerkhoff for technical assistance, and to Miss E. McLure for reading the English manuscript.
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