Sources of the porcine testis innervation

ORIGINAL ARTICLE Sources of the porcine testis innervation W. Sienkiewicz Department of Animal Anatomy, University of Warmia and Mazury in Olsztyn, Faculty of Veterinary Medicine, Poland Keywords Autonomic—boar—ganglia—sensory—testis Correspondence Waldemar Sienkiewicz, 10-719 OlsztynKortowo, ul. Oczapowskiego 13, Bldg 105 J., Poland. Tel.: ++48 89 523 39 53; Fax: ++48 89 523 49 86; E-mail: [email protected] Accepted: August 1, 2010 Summary This study was carried out on three adult male pigs of the large White Polish breed weighing 110–130 kg each. The animals were anaesthetised and injected with retrograde tracer Fast Blue (FB) into right testis. Three weeks later, the pigs were deeply anaesthetised and perfused transcardially with fixative (4% paraformaldehyde in 0.1 m phosphate buffer pH 7.4). Collected ganglia were cut with freezing microtome into 12- lm-thick sections. The sections were examined under a fluorescent microscope (Zeiss). FB-positive neurones were found in pelvic ganglia (anterior pelvic ganglion) (15.4% of all FB+ neurones), prevertebral ganglia (caudal mesenteric, testicular, aortico-renal and renal ganglia) (59% of all FB+ neurones), sympathetic chain ganglia (last four lumbar and first three sacral ganglia) (18.1% of all FB+ neurones) and dorsal root ganglia (DRG) (first three lumbar and first three sacral ganglia) (7.4% of all FB+ neurones). The majority of FB-positive nerve cell bodies were observed in ipsilateral ganglia, but they were also found in contralateral ganglia (approximately 85% and 15% respectively). Thus, FB-positive neurones were located in the left prevertebral, sympathetic chain and DRG, but surprisingly, they were absent in left anterior pelvic ganglia. Introduction Retrograde tracing experiments dealing with the origin of nerve fibres that supply the mammalian testis are very scarce. Nevertheless, there is some limited evidence that the innervation of this organ originates from pelvic ganglia (PG-s), caudal mesenteric ganglion (CaMG), sympathetic chain ganglia (SCG-s) and dorsal root ganglia (DRG-s). It is known that in the pig nerve, fibres reach the gonad by the superior spermatic nerve running along testicular artery (Wrobel & Brandl, 1998). It was supposed that main sources of sympathetic innervation of rat testis are localised in the coeliac and aortic plexuses. The corresponding sympathetic preganglionic perikarya are localised in intermedio-lateral nucleus of the spinal cord in 10th and 11th thoracic spinal cord segments (Gerendai & Halasz, 1997). Parasympathetic nerve fibres originate from the vagus nerve (Gerendai & Halasz, 1997). The inferior spermatic nerve, which also supplies the testis, leads fibres from the caudal mesenteric and hypogastric ª 2010 Blackwell Verlag GmbH Æ Andrologia 42, 395–403 plexuses and runs along the vas deferens. Important sources of innervation for the testis are also neurones localised in the pelvic plexus, receiving preganglionic sympathetic fibres via the hypogastric nerve and parasympathetic through the pelvic nerve, which originates from the sacral plexus (Gerendai & Halasz, 1997). Studies performed with retrograde tracing allowed to precisely describe pathways and sources of the testis innervation in the rat. It was established that nerve fibres supplying rat testis reach the organ via spermatic nerves, and main sources of innervation are ganglia found in the pelvic plexus (pelvic accessory ganglia) and major pelvic ganglion [in the pig an analogue of this ganglion is anterior pelvic ganglion (APG)]. Neurones innervating the testis were disclosed also within lumbar SCG (from L1 to L5). Some smaller numbers of retrogradely labelled neurones were found in testicular and aortic ganglia (Rauchenwald et al., 1995). Studies performed on dogs revealed that the testis also receives sensory innervation from DRG (the last three thoracic and first four lumbar spinal cord 395 Origin of porcine testis innervation segments) (Tamura et al., 1996a,b). Using trans-synaptic viral tracers (pseudorabies virus) (Gerendai et al., 2000, 2001), preganglionic perikarya in the spinal cord and neurones in the brain innervating the epididymis and the testis in the rat were localised. These cells were found in sympathetic (spinal cord segments from Th10 to L1) and parasympathetic (spinal cord segments from L6 to S1) nuclei and also in the dorsal horn laminas II and IV of the spinal cord (Gerendai et al., 2000). Nerve cell bodies containing viral tracer were found in the brainstem, hypothalamus and telencephalon (Gerendai et al., 2000). As clearly seen from above-cited papers, there is lack of systematic studies dealing with localisation of nerve cell bodies involved in the male gonad innervation especially with regard to autonomic ganglia. It also should be stressed that all the literature describes the problem in the rat (Rauchenwald et al., 1995; Gerendai et al., 2000), dog (Tamura et al., 1996b) and rooster (Wakita et al., 1999), but there is no data regarding sources of the testis innervation in the pig; therefore, it was decided to study this topic. W. Sienkiewicz vertebral laminas, and spinal cords with spinal root ganglia were collected. The tissues were postfixed by immersion in the same fixative for 20–30 min, rinsed with phosphate buffer (PB) (pH 7.4) and transferred to and stored in 18% buffered sucrose solution (pH 7.4) until further processing. The ganglia were placed on specimen holder, covered with tissue freezing medium (Tissue-Tek OCT Compound) and left in cryostat chamber for several hours, until the specimen reached the temperature in the chamber ()24 C). Next, the tissues were cut into 12lm-thick cryostat sections, which were viewed under the fluorescent microscope equipped with a filter block for FB. To determine the relative number of FB-positive (FB+) neurones and to avoid double counting, the neurones were counted in every fifth section. Only neurones with a clearly visible nucleus were considered. Numerical data obtained from three animals were used to calculate the percentage of each subset of ganglia and particular ganglion in innervation of the organ studied. All data are expressed as mean ± SD. Results Material and methods This study was carried out on three sexually mature male pigs of the large White Polish breed weighing 110–130 kg each. The animals were housed and treated in accordance with the rules approved by the local Ethics Commission (confirming the principles of Laboratory Animal Care, NIH publication No. 86-23, revised in 1985). Animals were anaesthetised, 30 min before the main anaesthetic, pentobarbital (Vetbutal, Biowet, Poland; 25 mg kg)1 of b.w.) was given intravenously and all the pigs were pretreated with propionyl promazine (Combelen, Bayer, Germany; 0.4 mg kg)1 of b.w. i.m.). Then, the animals were injected with Fast Blue (FB) (5% suspension of the FB in distilled water) tracer into the right testis. Due to the size of the organ (around 200 g), tracer was given in approximately 30 injections, 2 ll of tracer each, to equally saturate the testis parenchyma. After a survival period of 3 weeks, the pigs were deeply anaesthetised (following the same procedure as described earlier). Then, the pigs were transcardially perfused with 0.5 l of preperfusion solution containing 0.9% sodium chloride (Chemia, Gliwice, Poland), 2.5% polyvinylpyrolidone (Sigma, Deisenhofen, Germany), 0.5% procaine hydrochloride (Polfa, Warsaw, Poland) and 20 000 IU of heparin (Heparinum; Polfa; added ex tempore), followed by 8–10 l of 4% ice-cold buffered paraformaldehyde (pH 7.4). After perfusion, the ganglia were macroscopically identified and collected by anatomical preparation. The collected ganglia (their morphology and localisation) are listed in Table 1. Then, the spinal canals were cut open by removal of the 396 After injections of the tracer to the right testis, FB+ neurones were found in APG but not in other pelvic ganglia. Numerous FB+ neurones (Fig. 1) were found to be evenly distributed throughout the ganglion. The average number of FB+ neurones was 1289 ± 152 (Table 2) within the ganglion, which constitutes 15.4 ± 0.3% (Fig. 2) of all FB+ neurones found. Tracer containing neuronal somata were localised only in the ipsilateral ganglion (Fig. 3). The traced perikarya were also found in prevertebral ganglia (PVG): CaMG (Fig. 4), testicular ganglion (TG) (Fig. 5), aortico-renal ganglion (ARG) (Fig. 6) and RG. Neurones found in PVG-s constituted 59 ± 2.3% (Fig. 7) of all FB+ cells. Among all the PVG-s, the main sources for testis innervation were CaMG-s. CaMG-s ‘testicular’ perikarya constituted 48 ± 2.5% of all FB+ neurones (Fig. 2). CaMG-located ‘testicular’ neurones constituted 81 ± 1.3% of FB-positive cells encountered within PVG-s. Within the left ganglia, mean number of FB+ cells was 726 ± 135, whereas in the right ganglia, 3300 ± 607 (Table 2) traced cells were encountered. In CaMG-s, numerous FB+ neurons were distributed unevenly throughout the ganglion. Some neurones were dispersed, but most of them were found to be located close to the lateral edge of the ganglion. These neurones formed a band of cells, extending from the cranial to the caudal pole of the ganglion, which may be considered as a ‘testicular centre’ (Figs 8 and 9). The cells were more numerous in the caudal part of the ganglion. In dorso-caudal part of the ganglion, another, much smaller number of the FB+ perikarya was found (Fig. 9). ª 2010 Blackwell Verlag GmbH Æ Andrologia 42, 395–403 W. Sienkiewicz Origin of porcine testis innervation Table 1 Description of the ganglia studied Subset of ganglia Name of the particular ganglia Abbreviation Morphological features Localisation Pelvic ganglia Pelvic accessory ganglia PAG Anterior pelvic ganglion APG Strip of small ganglia, containing tens to several neurones. Ganglia were numerous on cranial part of urethra, their number decreased caudally Relatively large ganglion (flat structure 1–1.5 mm thick, round or oval with diameter ranging from 3 to 5 mm Caudal mesenteric ganglion CaMG Elongated ganglion approximately 1 cm long, up to 4 mm wide Testicular ganglion TG Small, elongated ganglion, length – 2–3 mm Aortico-renal ganglion ARG Small round or oval ganglion, with diameter about 2 mm Renal ganglion RG Small round or oval ganglion, with diameter about 2 mm Coeliac-cranial mesenteric anglion complex CSMG Large complex of four ganglia- two cranial mesenteric and two coeliac ganglia. Dimensions of whole complex: 1.5–2 cm · 1.5–2 cm Paravertebral ganglia Sympathetic chain ganglia SCG-s Medium size ganglia elongated, sometimes of triangular shape, 4–5 mm long, in sacral and lumbar part of the sympathetic chain Sensory ganglia Dorsal root ganglia DRG-s Elongated ganglia, length 5–10 mm, width 3–4 mm Proximal ganglion of the vagus nerve PGn. X Small round ganglion 1–2 mm in diameter Distal ganglion of the vagus nerve DGn. X Medium size ganglion, oval with long and short diameters of 5 and 3 mm respectively Located on the dorso-lateral surface of the pelvic urethra extending from APG caudally, to the half of the length of bulbourethral gland Easy to localise macroscopically in triangular space between urethral end of the pelvic part of vas deferens and cranial part of the vesicular gland On the ventral surface of the aorta, on both sides of the caudal mesenteric artery In the prevertebral trunk, close to the outlet of the testicular artery from aorta on ventral surface of aorta In the prevertebral trunk, close to the exit of the renal artery from aorta on ventral surface of aorta In the prevertebral trunk, between ARG and CSMG on ventral surface of aorta Ganglia located on both sides of the coeliac and cranial mesenteric arteries, close to the outlet of arteries from aorta, on ventral surface of aorta On ventrolateral surface of the body of vertebras, close to the intervertebral foramen, the ganglia are connected by interganglionic branches forming sympathetic or paravertebral chain In the dorsal root of the spinal nerve, inside of the vertebral canal, close to the intervertebral foramen On the level of the pachymeninx, just beneath the entrance of the roots of the vagus into the opening in the dura mater In the vagosympathetic trunk, on the neck, 4–5 cm below the exit of the nerve trunk from skull cavity Prevertebral ganglia In TG, numerous retrogradely labelled neurones were found in both left and right ganglia. FB+ perikarya present in TG constituted 5.7 ± 0.3% of all FB+ cells (Fig. 2) and 9.9 ± 0.6% of FB+ neurones located in PVG-s. In the left ganglia, 91 ± 5 of FB+ cells were present; in the right ganglia, 396 ± 55 of FB+ perikarya were found (Table 2). The neurones observed were evenly dispersed within the ganglion (Fig. 5). In ARG-s, numerous retrogradely labelled neurones were found in both left (n = 43 ± 9) and right (n = 215 ± 18) ª 2010 Blackwell Verlag GmbH Æ Andrologia 42, 395–403 ganglia. (Table 2), which constituted 3.1 ± 0.1% of all FB+ neurones (Fig. 2). ARG-located FB-positive somata included 5.3 ± 0.4% of FB+ neurones located within PVG. FB+ neurones were evenly dispersed within the ganglion (Fig. 6). In RG-s, retrogradely labelled neurones were found in both left (n = 32 ± 4) and right (n = 151 ± 16) ganglia (Table 2), which constituted 2.2 ± 0.1% of all FB+ neurones (Fig. 2). RG-located FB-positive somata included 3.7 ± 0.2% of FB+ neurones located within PVG. FB+ neurones were evenly dispersed within the ganglion. 397 Origin of porcine testis innervation W. Sienkiewicz Fig. 1 Fast Blue-positive (FB+) neurones in the right anterior pelvic ganglion (APG). Table 2 Mean number ± SD of FB+ neurones in particular ganglia innervating the testis. For calculation, numerical data obtained from three animals were used Subset of ganglia PG-s PVG-s SChG-s DRG-s Ganglion No of FB+ neurones in right ganglion No of FB+ neurones in left ganglion No of FB+ neurones in both ganglia APG CaMG TG ARG RG SCG L3 SCG L4 SCG L5 SCG L6 SCG S1 SCG S2 SCG S3 DRG L1 DRG L2 DRG L3 DRG S1 DRG S2 DRG S3 1289 3300 396 215 151 120 169 233 279 269 171 47 96 127 125 131 53 25 0 726 91 43 32 27 30 39 39 42 32 11 9 11 7 13 10 7 1289 4026 487 258 183 148 200 271 318 311 202 58 105 138 132 143 62 32 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 152 607 55 18 16 23 32 23 15 13 18 10 17 37 25 14 12 8 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0 135 5 9 4 10 4 3 11 5 5 2 3 3 2 4 4 7 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 152 740 57 23 20 20 31 21 23 18 21 9 19 36 26 10 8 9 APG, anterior pelvic ganglion; ARG, aortico-renal ganglion; CaMG, caudal mesenteric ganglion; DRG, dorsal root ganglia; FB, Fast Blue; PVG, prevertebral ganglia; SCG, sympathetic chain ganglia; TG, testicular ganglion. Traced neurones were observed in both left and right PVG-s, but in the left ganglia only 18.0 ± 0.4% of the FB+ neurones were located, whereas in right PVG-s 82.0 ± 0.4% of these cells were found (Fig. 3). Sympathetic chain ganglia-s contained 18.1 ± 1.5% of all traced neurones (Fig. 7). FB+ perikarya were found in lumbar (L) (Fig. 10) and sacral (S) parts of the sympa398 thetic chain. The presence of labelled neurones was restricted to the last four lumbar (L3–L6) and first three sacral (S1–S3) ganglia (Table 2). The ganglia contained 0.7 ± 0.1% to 3.5 ± 0.5% of FB+ cells (Fig. 2). The smallest number of the traced neurones was observed in third sacral ganglia of the sympathetic chain, whereas the largest number of FB+ cells was found in fifth and sixth lumbar ganglia, and first sacral ganglia of the sympathetic chain. Left-sided ganglia contained 14.6 ± 1.1% of FBpositive neurones, whereas the opposite side ganglia contained 85.4 ± 1.1% of all FB+ cells counted in SCG-s (Fig. 3). Dorsal root ganglia-s contained 7.4 ± 0.7% of all FB+ neurones (Fig. 7). In all the animals studied, FB+ perikarya were found in lumbar and sacral (Fig. 11) sensory ganglia. The presence of the traced neurones was restricted to the first three lumbar (L1–L3) and first three sacral (S1–S3) ganglia. Sensory ganglia contained 0.4 ± 0.1% to 1.7 ± 0.2% of all FB+ counted (Fig. 2). The smallest number of traced neurones was observed in third sacral spinal ganglia (S3) (Table 2), while the highest number of FB+ cells was found in the third lumbar (L3) and first sacral spinal ganglia (S1) (Table 2). The contralateral spinal ganglia contained 9.2 ± 3.0% of DRG-s traced neurones, whereas the ipsilateral ganglia contained 90.8 ± 3.0% of these cells (Fig. 3). No FB+ neurones were found within the sensory ganglia of vagus nerve. Discussion Injection of the tracer to the testis has allowed to reveal that main sources of innervation of the porcine male gonad are PVG, containing about 60% of all retrogradely traced neurones. Contrary to these data, the main sources of nerves supplying the testis in the rat are pelvic ganglia, whereas only a few labelled cell profiles were present in the para-aortic plexus ganglia (Rauchenwald et al., 1995). In the rat, most of the traced neurones were found in the major pelvic (38%) and pelvic accessory ganglia (37%) (Rauchenwald et al., 1995). The boar APG (counterpart of the major pelvic ganglion in rats) contained only 15% of all traced neurones, and surprisingly, there were no FB+ cells in accessory pelvic ganglia. The majority of PVG-s-located traced perikarya were found in ipsilateral ganglia, whereas in contralateral ganglia only about 20% of the cells were harboured. This observation is in accordance with data regarding the rat (Rauchenwald et al., 1995). Similar localisation of the labelled perikarya to that described in the present paper was observed in studies dealing with the innervation of other genital organs in the pig, the vas deference (Kaleczyc et al., 1995), seminal vesicle (Kaleczyc, 1998) and bulbourethral gland (Klimczuk, 2004) in males, and ovary (Majewski & Heym, ª 2010 Blackwell Verlag GmbH Æ Andrologia 42, 395–403 W. Sienkiewicz Origin of porcine testis innervation 47.7 ± 2.5 Number of FB+ neurones in ganglion /total number of FB+ neurones (%) 50 40 30 20 15.4 ± 0.3 10 5.7 ± 0.3 3.1 ± 0.1 2.2 ± 0.1 1.8 ± 0.2 2.4 ± 0.4 3.3 ± 0.2 3.8 ± 0.5 3.7 ± 0.3 2.4 ± 0.1 0.7 ± 0.1 1.3 ± 0.2 1.7 ± 0.4 1.6 ± 0.5 1.7 ± 0.2 0.8 ± 0.1 0.4 ± 0.1 DRG S3 DRG S2 DRG S1 DRG L3 DRG L2 DRG L1 SCG S3 SCG S2 SCG S1 SCG L6 SCG L5 SCG L4 SCG L3 RG ARG TG CaMG APG 0 100.0 ± 0.0 100 90.8 ± 3.0 85.4 ± 1.1 82.0 ± 0.4 PG-s L PG-s R PVG-s L PVG-s R 75 SCG-s L SCG-s R DRG-s L 50 DRG-s R 25 14.6 ± 1.1 9.2 ± 3.0 DRG-s R DRG-s L SCG-s R SCG-s L PVG-s R PVG-s L 0.0 ± 0.0 PG-s R 0 18.0 ± 0.4 PG-s L ipsilateral-ganglia subset/total number of FB neurones in ganglia subset (%) Number of FB+ neurones in contra- and + Fig. 2 Percentages of Fast Blue-positive neurones in particular ganglia innervating the testis. (Percentage of the neurones was calculated as part of all neurons counted in particular animal. Numbers of neurones counted in both right and left ganglia were summed). Fig. 3 Percentages of Fast Blue-positive nerve cell bodies in particular left and right subsets of ganglia innervating the testis. For calculation, numerical data obtained from three animals were used. 1991), oviduct (Czaja, 2000; Czaja et al., 2001) and uterus (Wasowicz et al., 1998) in females. Considering traced neurones found in the PVG, the majority of them (over 80%) were localised in the CaMG-s, whereas TG-s contained only about 10% of the labelled cells. This information is somewhat surprising because the previous assessment regarding PVG revealed that in the rat the ª 2010 Blackwell Verlag GmbH Æ Andrologia 42, 395–403 testis receives sympathetic input particularly from the TG (Rauchenwald et al., 1995). CaMG is the ganglion that contributes to the innervation of different organs of the reproductive tract (Majewski & Heym, 1991; Kaleczyc et al., 1995; Kaleczyc, 1998; Wasowicz et al., 1998; Czaja et al. 2001, Klimczuk, 2004). The tracing studies dealing with the vas deferens, seminal vesicle (Kaleczyc et al., 399 Origin of porcine testis innervation Fig. 4 Fast Blue-positive (FB+) neurones in the dorso-caudal portion of ipsilateral caudal mesenteric ganglion (CaMG). W. Sienkiewicz Fig. 6 Fast Blue-positive (FB+) neurones in the right aortico-renal ganglion (ARG). Number of FB+ neurones in ganglia subset/total number of FB+ neurones (%) 75 APG-s 59.0 ± 2.3 PVG-s SCG-s 50 25 DRG-s 18.1 ± 1.5 15.4 ± 0.3 7.4 ± 0.7 0 APG-s PVG-s SCG-s DRG-s Fig. 7 Percentages of Fast Blue-positive nerve cell bodies in particular subset of ganglia innervating the testis. For calculation, numerical data obtained from three animals were used. Fig. 5 Fast Blue-positive (FB+) neurones in the right testicular ganglion. 1995; Kaleczyc, 1998) and bulbourethral gland (Klimczuk, 2004) showed the presence of FB+ neurones within this ganglion in male pigs. In female pigs, this ganglion is the source of the innervation of the ovary (Majewski & Heym, 1991), oviduct (Czaja, 2000; Czaja et al., 2001) and uterus (Wasowicz et al., 1998). In all studies aforementioned, FB+ neurons were localised in a discrete part of the ganglion, its lateral portion. The cells were mainly gathered within cranial and caudal poles of the ganglion. Very similar localisation of FB+ neurones was observed also in the present study. Such results obtained during studies dealing with the innervation of porcine genital organs suggest the existence of a ‘reproductive centre’ in CaMG of the pig. In available literature, there is no data on the involvement of SCG in the innervation of the testis except one 400 paper dealing with the innervation of the chicken testis (Wakita et al., 1999). In this study, sympathetic postganglionic neurones were located in the paravertebral ganglia Th3–L3 (10% of the total number of labelled neurones), most of them in ipsilateral ganglia. In the present study, traced ‘testicular’ neurones were found also in SCG-s. About 85% of the labelled cells were observed in ipsilateral ganglia, whereas only 15% in the contralateral ganglia. ‘Testicular’ perikarya located in SCG-s seem to be an important compartment of the organ innervation due to the number of these cells reaching up to 18% of the total number of the labelled neurones. These neurones were encountered in the last four lumbar (L3–L6) and first three sacral (S1–S3) paravertebral ganglia. This difference in the localisation of neurones supplying the testis in the chicken and pig can be explained by differences in the localisation of the target organ, which in birds is suspended in the body cavity, cranially to the kidney. In the ª 2010 Blackwell Verlag GmbH Æ Andrologia 42, 395–403 W. Sienkiewicz Origin of porcine testis innervation Fig. 9 Schematic drawing of the caudal mesenteric ganglion, with localisation of Fast Blue-positive perikarya within the ganglion. Abbreviations: V, ventral; D, dorsal; L, left; R, right; IMN, intermesenteric nerves; HGN, hypogastric nerves; CCN, caudal colic nerves; CAM, caudal mesenteric artery. Fig. 8 Microphotograph showing whole right CaMG with Fast Bluepositive neurones forming a band in lateral part of the ganglion. Abbreviations: CaMG, caudal mesenteric ganglion; CR, cranial; CA, caudal; LAT, lateral; ME, medial. rat exposed to tracing of the testis with pseudorabies virus, virus-infected perikarya were detected in the preganglionic neurones of the spinal cord (Th10–L1, L5–S1) and also in certain cell groups and areas of the brain stem, the hypothalamus and the telencephalon (Gerendai et al., 2000). In the brain stem, labelled neurones were also found in the nucleus of the solitary tract, which is the sensory nucleus of the vagus nerve. Contrary to these data, in the present study, primary sensory ‘testicular’ neurones were not observed in sensory ganglia of the vagus nerve. Taking under consideration all these observations, it should be concluded that the vagus nerve does not contribute to the innervation of the testis in the pig. It is also possible that due to the size of the animals and relatively short time of exposure to the tracer, FB+ did not reach sensory ganglia of the vagus, but to dispel this doubt, additional experiments involving longer experimental period should be performed. ª 2010 Blackwell Verlag GmbH Æ Andrologia 42, 395–403 Fig. 10 Fast Blue-positive (FB+) neurones in the right SCG, sixth lumbar ganglion. Abbreviations: SCG, sympathetic chain ganglion; L, lumbar. ‘Testicular’ neurones were also found in lumbar (L1– L3) and sacral (S1–S3) DRG-s. Similar results were obtained by Tamura et al. (1996b), who studied afferent innervation of the canine testis and epididymis. Tracing 401 Origin of porcine testis innervation Fig. 11 Fast Blue-positive (FB+) neurones in the right DRG, second sacral ganglion. Arrows indicate FB+ neurones. Abbreviations: DRG, dorsal root ganglion; S, sacral. All the bars = 50lm. of these organs resulted in labelling of cells located predominantly from Th10 to L4 (87%) and, to a lesser extent, in S1–S3 (13%) DRG-s. Wider extent of the traced neurones within DRG-s observed in experiments described by Tamura et al. (1996b) can be a result of the tracing of both organs: the testis and epididymis simultaneously. Cross-cut of the vas deferens prior to tracer injections diminished labelling in the S1–S3 DRG-s, but not at thoracolumbar levels (Tamura et al., 1996b). These observations indicate that primary afferent fibres supplying the canine testis and epididymis project to the DRG-s at the higher than L4 ganglion through the superior spermatic nerve. Some sensory nerve fibres project to the sacral DRG-s through the inferior spermatic nerve. In case of the pig, over 60% of primary sensory neurones supplying the testis are located in lumbar DRG-s, whereas <40% in sacral ganglia. Such localisation seems to be a result of similar routes of the sensory nerves reaching the testis in the pig. Findings described in papers dealing with the innervation of other genital organs of the pig (Kaleczyc, 1998; Wasowicz et al., 1998; Kaleczyc et al., 2002) confirm this assumption. As was already mentioned by Kaleczyc et al. (2002), similarities in the pattern of the distribution between primary sensory neurones projecting to the vas deferens in the pig (Kaleczyc et al., 2002) and to the testis and epididymis in the dog (Tamura et al., 1996a,b) suggest the existence of a relationship between the neurones in the lumbar and sacral ganglia and afferents that travel in the hypogastric and pelvic nerve respectively. Due to localisation of traced perikarya, it may be assumed that neurones located within SCG-s and PVG belong to the sympathetic component of autonomic 402 W. Sienkiewicz testicular supply. The APG is a mixture of sympathetic and parasympathetic nerve cell bodies (Kaleczyc et al., 2003), so probably it is a source of parasympathetic input for the testis, but real nature of these cells can be revealed by further immunohistochemical studies. Neurones located in DRG provide sensory input for the gonad. The present study allowed to disclose localisation and number of neurones supplying the porcine male gonad. All possible sources of localisation of afferent and efferent perikarya innervating the testis were precisely studied, so it is very unlikely that any neurones innervating the testis were undetected. Due to time of exposure of animals to the tracer and distance from the testis to sensory ganglia of the vagus, it is possible that these perikarya are involved in the gonad innervation, but they were not labelled. Such possibility was already discussed. As clearly seen, in available literature, there is very limited number of papers dealing with the origin of the testis innervation and even existing reports touch the problem very superficially. This comprehensive study describes localisation of autonomic and primary sensory neurones innervating the testis of the boar. The present results may be very useful for further studies, especially those dealing with immunohistochemical investigations of neurones supplying the testis and influence of the nervous system on the gonad function. Acknowledgement This research was supported by Grant nr P06K 003 27. References Czaja K (2000) Distribution of primary afferent neurons innervating the porcine oviduct and their immunohistochemical characterization. Cells Tissues Organs 166:275–282. Czaja K, Kaleczyc J, Pidsudko Z, Franke-Radowiecka A, Lakomy M (2001) Distribution of efferent neurones innervating the oviduct in the pig. Folia Morphol (Warsz) 60:243–248. 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