Cell Tissue Res (1984) 237:31-38
a n d T'msue Reseaw.h 9 Springer-Verlag 1984
Vascular morphology of the bovine spermatic cord and testis I. Light- and scanning electron-microscopic studies on the testicular artery and pampiniform plexus* Herbert Hees 1, Rudolf Leiser 2, Toni Kohler 2, and Karl-Heinz Wrobel 1 1 Institut fiir Anatomic der Universitfit Regensburg, Bundesrepublik Deutschland; 2 Institut ffir Tieranatomie der Univcrsitfit Bern, Schweiz
Summary. The highly coiled testicular artery within the bovine spermatic cord has a constant luminal diameter but a continuously decreasing mural thickness. The pampiniform plexus is composed of three interconnected venous networks differing in mesh sizes and calibres. The large veins of the first network display pouches and permanent constrictions, which may serve as throttle devices. The constitutents of the third network are venules or venous capillaries with diameters between 10 and 20 ~tm; they favor a periarterial position or even occupy the media-adventitia border of the testicular artery. All plexus veins are devoid of valves. The existence of true arteriovenous anastomoses between smaller branches of the testicular artery and plexus veins was established by serial sections. The vascular morphology of the spermatic cord is discussed with special attention to a postulated venous-arterial steroid transfer in this region. Key words: Testis Blood vessels - Androgen, transfer Light and scanning electron microscopy - Corrosion casts Bovine
Physiological studies in various species including man and bull have proven that the androgen concentration in the testicular artery is significantly higher than in the systemic circulation. The reason for this is an androgen transfer from the venous into the arterial compartment within the area of the pampiniform plexus (Amann and Ganjam 1976; Free 1977; Damber et al. 1979). An analogous exchange has also been confirmed in female animals; Krzymowski et al. (1982) reported a transfer of steroids from the porcine ovarian vein into the ovarian artery. The morphological basis for such an androgen exchange in the area of the spermatic cord is poorly understood. Previous anatomical studies on the bovine testicular artery and veins have discussed problems of blood pressure and temperature regulation (Hofmann 1960) in the male gonad Send offprint requests to: Dr. Dr. H. Hees, Institut ffir Anatomic, Universitfitsstral3e 31, D-8400 Regensburg, Federal Republic of Germany * Supported by the Deutsche Forschungsgemeinschaft and the Stiftung zur F6rderung der wissenschaftlichen Forschung an der Universit~t Bern
but lack the details necessary to evaluate an androgen transfer. Therefore the present study was undertaken to establish a detailed description of the vascular architecture within the bovine spermatic cord and to discuss how the topographic situation is able to favor exchange processes between testicular veins and the artery.
Materials and methods Testes of 15 adult bulls were obtained 5-10 min after death. Light-microscopic studies. A bulb-headed cannula was inserted into the straight proximal portion of the testicular artery of five testes. Rinsing procedures and perfusion with Bouin's solution or formol were performed as described elsewhere (Wrobel et al. 1978) by use of a gas-driven perfusion pump (Scheubeck and Wrobel 1983). Ligature of the spermatic cord vessels at the level of the epididymal head had no influence on the perfusion of the pampiniform plexus due to the existence of arteriovenous anastomoses. Paraplast-embedded material was sectioned (thickness 5 ~tm) and stained according to the methods of Masson-Goldner, van Gieson, Crossmon and Pasini, and also with resorcinfuchsin. In addition, larger segments of the spermatic cord were cut in thick serial sections (approximately 100 ~tm) and stained with iron-hematoxylin. For transmission electron-microscopic studies five testes were treated as described by Wrobel et al. (1982). Scanning electron-microscopic studies. Five testes were perfused with Ringer's solution (at 38 ~ C, containing 1 ml/1 Liquemine and 0.5% Procaine) and stored in ice water for 3 h to prevent vasoconstriction and premature polymerisation of the injection mass. Following a second perfusion with Ringer's solution (at 25 ~ C, mixture as mentioned above) methyl-methacrylate was infused according to the following scheme: Testis 1. Injection via the testicular artery proximal to the pampiniform plexus. Testes 2, 3. Injection via the straight portion of the testicular artery running parallel to the epididymal body. Testes 4, 5. Retrograde injection via one of the testicular veins proximal to the pampiniform plexus.
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Fig. 1. Cross section showing general vascular topography of the spermatic cord. The large coiled vessel is the testicular artery, which is embedded in the spongy network of the venous pampiniform plexus. Note that the surface areas of the artery are not covered by veins. • 4 Fig. 2. Venous corrosion cast of spermatic cord (above) and cranial pole of testis, seen from the epididymal side. (Injection method as described for testes 2 and 3). Many parallel-running superficial testicular veins fuse to form the pampiniform plexus, in which the arterial course is represented by empty hollows. Arrows point to epididymal vein. x 4.5 Fig. 3. Cast of large plexus veins of the first network, which run parallel and anastomose frequently. Together they form a venous coat around the testicular artery (represented by the central empty space). Where the artery reaches the surface of the spermatic cord, the coat is absent. Bar = 200 gm F o r injection, low viscosity methyl-methacrylate-mixtures (Kohler and Leiser 1983; Leiser and Kohler 1983) were used: A) Batson No. 17 corrosion c o m p o u n d (Polysciencess, Inc. Warrington, PA, USA). 25 ml m o n o m e r base solution, 4 ~ C; 7.5 ml catalyst, room temperature; 0.5 ml promotor, 4 ~ C. B) 12 ml Sevriton (available from any dental supplier), 4 ~ C. Following injection, testes were stored at room temperature for 30 min, then transferred into a water bath of 80 ~ C for 4 h to enable polymerisation. Tissue corrosion was performed by alternating submersion in 40% K O H and distilled water (60 ~ C) for a few days. The liquids were changed once a day. To achieve smooth fractures, the finished corrosion casts were immersed in a warm 20% gelatine solution, which was slowly hardened in liquid nitrogen. Small pieces of selected areas were removed and cleaned from the gela-
tine using an additional corrosion treatment, distilled water and 10% Extran (Merck). The vascular casts were air-dried, m o u n t e d on scanning electron-microscopic specimen holders using a fast-drying conductive adhesive (Leit-C nach G6cke, Neubauer-Chemikalien, D-4000 Mfinster), dried in an exsiccator, sputtercoated with gold (30 nm), and examined in a Philips 500 scanning electron microscope. Results
General vascular arrangement in the spermatic cord In the spermatic cord the testicular artery is highly coiled (pars convoluta). The windings increase in n u m b e r from proximal (abdominal side) to distal (testicular side) and are arranged in all planes of space but avoid forming larger retrograde loops (Figs. 1, 2). As a whole the pars convoluta is cone-shaped with a length of 10-15 cm and a maximal
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Fig. 4. Cast of testicular artery with a small branch. Note constriction at the base of the small vessel and its typical, sharp angular bend. Indentations on the surface of arterial casts correspond to perikarya of endothelium protruding into the arterial lumen (see inset). Bar = t00 p_m; inset • 3500 Fig. 5. Arterial termination of an arteriovenous anastomosis (evidenced by serial sections). Note basal constriction and angular course of the small vessel within the media of the testicular artery. Pasini. x 300 Fig. 6. Nearly complete arteriovenous anastomosis connecting testicular artery (above) with a large vein of the first network. Adjacent serial sections showed the termination of this anastomosis. Crossmon. x 300
diameter o f 5-6 cm, The base o f this cone covers the cranial pole o f the testis in a cap-like fashion. The overall length of the convoluted p o r t i o n of the testicular artery a m o u n t s to 3.40-4.55 m ( H o f m a n n 1960). The testicular artery is o f the muscular type with a well-developed wavy m e m b r a n a elastica interna, at certain sites split into two or three lamellae. The media is practically devoid o f elastic material. In the adventitia, otherwise n o t sharply d e m a r c a t e d against the perivascular connective tissue, elastic fibres are again discernible but do not form a distinct m e m b r a n a elastica externa. The elastic adventitial network is more p r o n o u n c e d in the p r o x i m a l region o f the pars convoluta than in the distal one. The luminal diameter o f the testicular artery measures constantly 1.6-2 ram, whereas the thickness o f the arterial wall (intima and media) decreases regularly in orthograde
direction: from a p p r o x i m a t e l y 300 lam in the proximal portion, to 2 0 0 - 2 5 0 g m in the middle p o r t i o n and to 100-120 I-tm in the distal p o r t i o n o f the pars convoluta. This corresponds to 45-50 layers o f s m o o t h muscle cells in the proximal portion, 35~40 layers in the middle p o r t i o n and 20-30 layers in the distal p o r t i o n o f the artery. Occasionally, in limited areas of the arterial wall the musculature of the media is highly reduced a n d partially replaced by periarterial connective tissue. These locations, where the thickness o f the arterial wall m a y measure n o t more than 10 pm, are prefereably found on the inner side o f sharp bendings but are also situated in straight running portions of the artery. Sinusoidal venous networks ( p a m p i n i f o r m plexus) lie between the convolutions of the testicular artery. They fill the available space in such a way that the central arterial
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Fig. 7. Venous cast showing constituents of the first and the intermediate network of the pampiniform plexus. Note varicose pouches of the large veins. Arrows indicate connections between the two networks. Arrowheads point to connecting veins with the third periarterial venous network (C), partially perceptible in the background. Small branches (SA) of the testicular artery are also seen. Bar = 100 gm Fig. 8. Cord-like arrangement of constituents of the intermediate venous network from the surface area of the pampiniform plexus. In the centre of the cord runs a small, slightly bent artery (SA). Bar = 100 gm
windings are completely surrounded by the densely lying veins. Only the superficial convolutions of the artery are not covered by veins on their convex sides (Figs. 1-3). The pampiniform plexus, which receives the venous blood from the testis, gives origin to several large venous trunks in the proximal area of the spermatic cord. These trunks are the concomitant veins of the straight proximal portion of the testicular artery. All plexus veins are devoid of valves.
Branches of the testicular artery The pars convoluta of the testicular artery gives rise to the epididymal artery and to smaller arteries which ramify further and finally supply the capillary network of the spermatic cord. At their origin the smaller arteries possess structural peculiarities which may be interpreted as throttle de-
vices: (i) permanent constrictions of the arterial lumen, and (ii) sharp angular bending (Figs. 4, 5, 14). Continuous serial thick sections reveal the existence of arteriovenous anastomoses between the smaller branches of the testicular artery and the large plexus veins (Figs. 5, 6, 14). Perfusion experiments, however, suggest that these anastomoses are of functional importance only for that portion of the vascular bed that is situated close to the epididymal head. A ligature distal to this area resulted in a complete perfusion of all vessels of the spermatic cord, and the perfusion solution left the proximal venous trunks without having circulated through the testis. Ligature of the spermatic cord proximal to the level of the epididymal head, on the other hand, prevented a proper perfusion of the spermatic cord vessels. In this case all arteries were filled and dilated considerably but no fluid left the veins. Contrary to the light-microscopic results and the perfusion experiments we have not been
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Fig. 9. Cast of the third periarterial venous network consisting of venules and venous capillaries. Note characteristic constrictions in the course of the venules. Connecting veins (arrowhead)run from the periarterial to the first venous network (top). Bar = 100 gm Fig. 10. Detailed view of the third venous network. On the left a capillary with wavy course emptying into a venule (bottom). In the center venules with many asymmetric constrictions. Bar = 10 p,m Fig. 11. Elements of the third venous network in periarterial and intramural position. Connecting veins to the larger networks are indicated by arrowheads. Crossmon. x 300 Fig. 12. Casts of two veins of the first network exhibit deep, permanent constrictions serving as throttle devices (compare with Fig. 15). Impressions of the endothelium are very prominent. Bar = 100 I~m
able so far to identify unequivocally the existence o f arteriovenous anastomoses in our SEM-specimens. The corrosion casts o f the testicular artery and its branches up to the capillary bed show in the S E M longitudinally oriented furrows between which the p r o m i n e n t perik a r y a o f the endothelial cells are e m b e d d e d like spindleshaped cavities (Figs. 4, 8).
General organization of the pampiniform plexus The p a m p i n i f o r m plexus is c o m p o s e d o f three interconnected venous networks differing in mesh sizes and calibers : 1) The first network is formed by large veins running mostly parallel to each other, surrounding the testicular
artery (Fig. 3), and m a y be identified in corrosion casts by the n a k e d eye. The thickness of the venous walls measures 20-30 gm. F o u r to six layers o f smooth muscle cells can be identified within the media. The expansion o f the connective tissue spaces between the plexus veins in this network averages 120 g m in the p r o x i m a l p o r t i o n o f the spermatic cord b u t is reduced to a p p r o x i m a t e l y 50 gm in the distal portion. The luminal diameter, however, remains relatively unchanged, i.e., there are m o r e plexus veins in a given cross section in the distal portion. 2) The intermediate network is well developed (Fig. 7). Its constituent veins are in continuation with the first network as well as with the venules and venous capillaries forming the third network. Occasionally, at certain sites
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Fig. 13. Large plexus vein with pouches (*) and constrictions (arrows), devices that may influence velocity of blood flow (compare wi~h Fig. 15). Impressions of endothelial cells and their perikarya are very obvious. Bar = t00 pm on the surface of the pampiniform plexus the intermediate network shows a cord-like arrangement, often surrounding a small, centrally located artery (Fig. 8). The veins of the intermediate network exhibit diameters between 40 and 70 l.tm. Histologically, the vessels can be classified as small veins or venutes. 3) The third network consisting of venules and venous capillaries with diameters between 10 and 20 ~tm (Figs. 7, 9, 10, 14) either connects veins of the intermediate network or empties directly into the larger veins of the first network. The third network occupies a periarterial or intramural position and favors the border region between media and adventitia of the testicular artery but may also enter the media (intramural vessels). The main constituents of this third network run parallel to each other and may be found in either longitudinal, diagonal or transverse position when compared to the course of the artery. Typically, the distribution of the vessels of the third network in cross sections of the artery is not uniform. In most cases only one sector of the arterial wall is equipped with or covered by a b u n d a n t venules and venous capillaries, whereas the remaining circumference is virtually or actually free from these vessels. Structural specializations o f the pampiniJorm plexus |"T
Fig. 14, Schematic drawing of the threevenous networks forming the pampiniform plexus and their mutual connections. A small arterial branch of the testicular artery, a direct arteriovenous anastomosis and a portion of the testicular artery are also depicted. M, L media and lumen of testicular artery; SA small artery branching off from testicular artery; A VA arteriovenous anastomosis; V1, V2 veins of the first (V 0 and the intermediate (V2) venous network; VL, C venules and venous capillaries of the third periarterial network; CO connecting veins between the periarterial network and the two larger venous networks; IVA intervenous network anastomosis; IMV intramural veuule of the periarterial network; P. CS pouches and constrictions in the course of veins
The large thin-walled veins of the first network display pouches and bulbous ectasiae (dilations) which resemble varices or aneurysmata (Figs. 7, 13). These vessels are devoid of true valves but exhibit constantly characteristic constrictions of their lumina. The pointed projections of the venous wall responsible for these constrictions are mainly composed of smooth musculature (Fig. 15). This light-m~croscopic result is in accordance with the SEM-findings: The corrosion casts of the large venous network exhibit circular indentations of various depths which seem to divide the injection mass into subsequent segments. Constrictions of the lumen are not only a characteristic of the longer straight portions of the first venous network but are also
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Fig. 15. Vascular arrangement in vicinity of testicular artery (upper right). Large plexus veins of the first network (V1) exhibit pouches and luminal constrictions. In the connective tissue veins of the two smaller networks are seen. EA one of the epididymal arteries. MassonGoldner, x 60 Fig. 16. Distribution of elastic fibers in the media of testicular artery (M) in the interstitial connective tissue and in the walls of veins of the plexus pampiniformis (V1). Resorcinfuchsin, x 140 found in shorter intervenous anastomoses (Figs. 12, 13). The intermediate-sized veins of the second network are devoid of luminal constrictions; therefore the surface area of corrosion casts is generally smooth with a shallow circular pattern (Figs. 7-9). The venules of the third (periarterial) network are characterized by a strikingly flexed or spiral course caused by deep unilateral constrictions of their lumina (Figs. 9, 10). Responsible for these constrictions seen on the SEM-casts are again asymmetric accumulations of smooth muscle cells within the walls of the venules. The smaller venous capillaries have practically no constrictions, but occasionally shallow pouches (Fig. 10) and run generally a wavy course. On the corrosion casts, especially of the larger plexus veins, superficial impressions of perikarya and even cell borders of endothelial cells can be observed. The impressions of the perikarya have round or oval shape and display dimples corresponding to microvilli (Fig. 13). The interstitial connective tissue of the pampiniform plexus area is especially rich in elastic fiber networks with intermingled bundles of unmyelinated nerves. Compared with the findings in the intervascular area the elastic components of the vessel wall are less well developed (Fig. 16).
Discussion
The remarkable vascular topography in the mammalian spermatic cord has stimulated functional considerations by many investigators. Generally accepted is the importance of this region for a heat exchange between the relatively cool venous and the arterial blood. Lately, discussion has centered on another possible biological function of the complicated arteriovenous interactions: venous-arterial steroid transfer with the purpose to supply the gonad with higher testosterone concentrations. For some species there exists convincing physiological evidence that such a transfer actually occurs. In man, testosterone concentration in the blood of the testicular vein is 500 ng/ml, of the testicular artery 17.3 ng/ml, and the systemic arteries 5.7ng/ml (Bayard et al. 1975). In the bovine, the corresponding data are 142 ng/ml, 6.8 ng/ml, and 4.1 ng/ml, respectively (Amann and Ganjam 1976). From the studies of Amann and Gan-
jam (1976) and Free (1977) it may be concluded that the venous-arterial steroid transfer occurs by diffusion. In the present investigation we have focussed on a detailed account of the vascular arrangement in the bovine spermatic cord in order to evaluate various diffusion parameters from a morphological point of view, i.e., expansion of exchange area as well as thickness and construction of diffusion barrier. With the exception of short distances, where the testicular artery touches the surface of the spermarie cord, all other portions of the highly coiled artery are invested by vessels of the first and second venous network. Since the length of the arterial pars convoluta may amount to 4.5 m (Hofmann 1960) and the arterial diameter measures between 1.8 and 2.6 mm, the total exchange area between testicular artery and venous plexus vessels is very large. Concerning the diffusion barrier between arterial and venous lumina the following morphological features can influence speed and amount of venous-arterial transfer: 1) The thickness of the arterial wall (intima and media) decreases from 300 gm in the proximal portion (abdominal side) to 100-120 gm in the distal portion (testicular side) of the spermatic cord, and the thickness of the venous walls never surmounts 30 gin. The expansion of the connective tissue compartment between the vessels is likewise reduced from the abdominal to the testicular extremity of the spermarie cord. 2) The third venous network occupies a periarterial and often an intramural (media, adventitia) position; hence, the venous-arterial diffusion barrier is considerably reduced at these sites. The constituents of the third venous network must not be confused with nutritive capillaries, which are common in the walls of larger arteries but are characteristically lacking in the testicular artery. Generally, only one sector of a cross section of the testicular artery is equipped with venules of the third network, whereas the remaining circumference is free from small vessels. The above-mentioned arrangements, which can directly influence diffusion processes, are supplemented by other morphological peculiarities of the spermatic cord vasculature influencing the rate of velocity of the blood within the plexus veins. The well-developed venous networks of the pampiniform plexus are found not only in immediate
38 proximity o f the artery but occupy practically most o f the spermatic cord. This augmentation o f the total cross-sectional area in the venous system o f the spermatic cord causes r e t a r d a t i o n o f b l o o d flow and consequently an increase in contact time between b l o o d and vascular walls. The delay o f the venous blood in the networks o f the pampiniform plexus is furthermore prolonged by the lack o f valves and the existence o f permanent constrictions in the large plexus veins, which serve as throttle devices. Owing to the thin venous walls and on behalf o f the delayed blood flow it is concluded that the intervasal connective tissue c o m p a r t m e n t is practically soaked with androgens, so that a p e r m a n e n t diffusion gradient to the arterial b l o o d is maintained. M o r p h o l o g i c a l and physiological d a t a give evidence for a venous-arterial androgen transfer in the area of the spermatic cord. However, in view o f other possibilities to maintain and regulate a higher testicular androgen level (androgen distribution by testicular lymph, leakage of androgens from intratesticular venules, high androgen content o f testicular interstitial fluid, androgen binding by special testicular proteins), it is more than doubtful that the additional androgen input via testicular artery plays an i m p o r t a n t role in g o n a d a l a n d r o g e n supply. Perhaps, the real target organ for the augmented androgen level in the blood o f the testicular artery is represented by the epididymis. In this respect, it must be kept in mind that the epididymal arteries branch off from the testicular artery in the spermatic cord. References Amann RP, Ganjam VK (1976) Steroid production by the bovine testis and steroid transfer across the pampiniform plexus. Biol Reprod 15 : 695-703
Bayard F, Boular PY, Huc A, Pontonnier F (1975) Arterio-venous transfer of testosterone in the spermatic cord of man. J Clin Endocrinol Metabol 40:345-346 Damber JE, Tomic R, Bergmann B, Bergh A (1979) Increased concentration of testosterone in testis artery blood as compared to peripheral venous blood in man. lnt J Androl 2:315-318 Free MJ (1977) Blood supply to the testis and its role in local exchange and transport of hormones. In: Johnson AD, Gomes WR (eds) The testis, Academic Press, New York San Francisco London, vol. IV, pp 39-90 Hofmann R (1960) Die Geffil3architektur des Bullenhodens, zugleich ein Versuch ihrer funktionellen Deutung. Zbl Vet Med 7 : 59-93 Kohler T, Leiser R (1983) Blood vessels of the bovine chorioidea. Acta Anat 116: 55-61 Krzymowski T, Kotwica J, Stefancyk S, Czarnocki J, Debek J (1982) A subovarian exchange mechanism for the countercurrent transfer of ovarian steroid hormones in the pig. J Reprod Fertil 65:457 465 Leiser R, Kohler T (1983) The blood vessels of the cat girdle placenta, observations on corrosion casts, scanning electron microscopical and histological studies, I : Maternal vasculature. Anat Embryol 167 : 85-93 Scheubeck M, Wrobel KH (1983) Eine einfache transportable Apparatur zur Durchfiihrung yon Perfusionsfixierungen. Mikroskopie (in press) Wrobel KH, Sinowatz F, Kugler P (1978) Zur funktionellen Morphologie des Rete testis, der Tubuli recti und der Terminalsegmente der Tubuli seminiferi des geschlechtsreifen Rindes. Zbl Vet Med C Anat Histol Embryol 7 : 320-335 Wrobel KH, Sinowatz F, Mademann R (1982) The fine structure of the terminal segment of the bovine seminiferous tubule. Cell Tissue Res 225 : 29-44
Accepted March 1, 1984