J. Anat. (1991), 178, pp. 1-9 1 With 8 figures Printed in Great Britain Evidence for a direct arteriovenous connection (A-V shunt) between the testicular artery and pampiniform plexus in the spermatic cord of the tree shrew (Tupaia glis) WORAWUT RERKAMNUAYCHOKE*, TAKAO NISHIDAt, MASAMICHI KUROHMARU AND YOSHIHIRO HAYASHI Department of Veterinary Anatomy, Faculty of Agriculture, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan (Accepted 20 March 1991) INTRODUCTION A high concentration of testosterone is essential for spermatogenesis in the testis (Steinberger, 1971; Steinberger, Root, Ficher & Smith, 1973) and for prolonging the life span of the epididymal sperm (Reeves, 1987). It has been demonstrated that in order to maintain high concentrations of testosterone intratesticularly, testosterone has to be transferred from the pampiniform plexus to the testicular artery. This has been shown in the rat (Free, Jaffe, Jain & Gomes, 1973; Free & Jaffe, 1975, 1978), ram (Jacks & Setchell, 1973; Ginther et al. 1974), rhesus monkey (Dierschke et al. 1975), cow (Amann & Ganjam, 1976) and man (Bayard, Boulard, Huc & Pontonnier, 1975). The structure of the vasculature in this area has been modified to some extent for this purpose. Although there is no doubt that a countercurrent system is present in this area, there is disagreement as to the way testosterone is transferred into the arterial blood. Some reports- have suggested that it is achieved through an arteriovenous connection (A-V shunt) in the cord (Godinho & Setchell, 1975; Noordhuizen-Stassen, Charbon, de Jong & Wensing, 1985). However, no morphological evidence for the existence of a shunt has been presented. Alternatively, the suggestion has been made that testosterone is transferred by diffusion (Free et al. 1973; Free, 1977). In this study, the first evidence for an A-V shunt between the testicular artery and the pampiniform plexus in the tree shrew spermatic cord is described by light and electron microscopy. The ultrastructural features and functional significance of the A-V shunts are discussed. MATERIALS AND METHODS Eight adult tree shrews (Tupaia glis) bought from the market in Thailand, weighing 120-170 g, were used in this study. Under ether and pentobarbital anaesthesia (5 mg/100 g body weight, i.p.), the thoracic aorta was cannulated just above the diaphragm. Ringer solution was perfused through the cannula followed by fixative, either 2 5 % glutaraldehyde in 0-1 M phosphate buffer for electron microscopy, or Bouin's fluid and/or 10% formalin for light microscopy. * To whom correspondence should be addressed. t Present address: Department of Animal Husbandry, College of Agriculture and Veterinary Medicine, Nihon University, Fujisawa 252, Japan. 2 W. RERKAMNUAYCHOKE AND OTHERS Light microscopy Two paraplast embedded samples were serially sectioned at 6-7 ,tm and stained with Masson's trichrome. Transmission electron microscopy Two samples were excised and cut into smaller blocks (1-2 mm thick), which were subsequently fixed in 2 5 % glutaraldehyde in 0 1 M phosphate buffer for 2 h. They were rinsed in the same phosphate buffer and postfixed in 1 % osmium tetroxide for 2 h. The specimens were then dehydrated in a graded series of ethanol and embedded in Epon 812 and Araldite 6005. Ultrathin sections were stained with uranyl acetate and lead citrate and observed with a JEM-1200EX transmission electron microscope at 80 kV. Scanning electron microscopy For the preparation of corrosion casts, methacrylic methyl ester monomer (Acry ester M., Mitsubishi Rayon Co. Ltd, Japan) was semipolymerised according to the method described by Murakami (1970, 1975). The semipolymerised ester was mixed with 25-30% hydroxypropyl methacrylate, 0-1 % Sudan III and 1-15% benzoyl peroxide (catalyst). Thereafter the mixture was supplemented with 1-1 5% N-N- dimethyl aniline (accelerator) just before injection. The resin was then perfused through the same cannula as described above. The resin-injected samples were immersed in warm water (60 °C) for 4 h, macerated in warm 2-5 % sodium hydroxide for 12-20 h, and then washed in running water followed by distilled water for 1-2 h. Finally, the casts were air-dried, mounted on stubs, and coated with platinum- palladium to be examined with a Hitachi S-4000 scanning electron microscope at 5-10 kV. RESULTS Testicular artery and pampiniform plexus The testicular artery arose from the abdominal aorta slightly below the origin of the renal artery. It was a small muscular type of artery consisting of 3-4 layers of smooth muscle cells in the tunica media. The testicular vein (pampiniform plexus), on the other hand, was composed of a single smooth muscle layer in the tunica media and shared a common tunica adventitia with the apposed artery. In the initial portion, the artery ran straight, accompanied by a single testicular vein (Fig. 1). At about one-third of the distance from the origin, the artery began to become convoluted. The degree of convolution gradually increased as the artery ran caudally to the testis and was prominent in the middle and distal portions of the cord. The vein in this portion also Fig. 1. LM of paraffin section showing testicular artery (A), vein (V) and capillary networks (*) in the proximal portion of the spermatic cord. The capillary networks are located on the surface of the spermatic cord. Masson's trichrome. x 160. Fig. 2. SEM ofcorrosion cast showing the course ofthe capillary network on the spermatic cord. The capillaries are long, tortuous and twist into U or S shapes (dotted line) and make several anastomoses with each other. Most of the capillaries show dichotomous branching (arrowheads). V, venule. x 670. Fig. 3. TEM of capillary network in the spermatic cord. The network is dominated by capillaries (C) and vesiculated cells. Arrowheads, vesiculated cell vesicles, Co, collagen; Nu, vesiculated cell nucleus. x 4320. Spermatic cord arteriovenous connection 3 I C ,}' F: es rX 7t C 4 .>... i.K.S-s.A.S.... 4. 4 W. RERKAMNUAYCHOKE AND OTHERS Fig. 4. SEM of the corrosion cast showing the arteriovenous connection (dotted lines) in the capillary network of the spermatic cord. The connection is formed between a small arteriole (A) derived from the testicular artery and a venule (V) from the testicular vein. x 340. Spermatic cord arteriovenous connection 5 divided into several branches, anastomosing with each other (pampiniform plexus) and covering the artery. Morphological architecture of A- V shunts Light microscopy and corrosion cast samples Five or six small ramifying arterioles arose directly from the testicular artery at regular intervals. In the initial portion, the tunica media of the arteriole consisted of two smooth muscle cell layers. The muscle layers became thinner as the vessel advanced peripherally. Thereafter, the arteriole gave rise to numerous capillaries. These capillaries were long, tortuous and twisted into U or S shapes in their course (Figs 2, 4). The capillary usually made a series of anastomoses with its counterpart derived from the same arteriole (Fig. 2). However, it occasionally anastomosed with the capillary derived from a different arteriole to make a connection between capillary networks of different arteriolar origins. Some capillary branches drained into a small venule which was directly connected with the testicular vein (pampiniform plexus). Thus a direct connection between the testicular artery and the pampiniform plexus was formed (Fig. 4). The capillary close to the venule had a less dilated lumen as compared with the remaining capillaries. Although most of the capillaries showed dichotomous branching (Fig. 2), trichotomous or tetramerous branching was also observed. The network was arranged in a localised mass and was situated on the surface of the spermatic cord (Figs 1, 4). Only a few portions of the network penetrated deep into the spermatic cord. In the initial portion of the cord, the capillary network was small and simple and A-V shunts were rare, compared with the middle and distal portions. Ultrastructure of the capillary network and its morphology The capillary network contained three kinds of cell, namely, endothelial cells, pericytes and vesiculated cells (Figs 3, 5). The name 'vesiculated cell' was adopted because of the presence of numerous cytoplasmic vesicles (Fig. 7). The endothelial cells with oval nuclei formed a continuous layer, whereas the pericyte layer was discontinuous. The pericytes, which contained microfilaments and possessed long processes within which were vesicles, were surrounded by the basal lamina of the endothelium and made direct contact with the epithelial cells (Fig. 5). The vesiculated cells which were large and irregular in shape consisted of cell bodies and cellular processes (Figs 3, 6). Because of their complicated cell processes, it was difficult to locate the boundaries of adjoining vesiculated cells. In the cell bodies, an abundance of mitochondria, rough endoplasmic reticulum, a well developed Golgi complex, large vesicles and numerous caveolae connecting with the plasma membrane were observed in the cytoplasm (Figs 6-8). The large vesicles varied in size and occupied almost the entire area of the cell body. Smaller vesicles were dispersed throughout the cell body and the cell processes (Figs 3, 7). The vesicles contained a fluid-like substance or lamellar structures, which seemed to be released (Fig. 7). The nucleus with heterochromatin showed invagination of the nuclear membrane. The heterochromatin of the vesiculated cell nucleus was less well developed than the euchromatin, differing from that of the endothelium and pericytic cells (Fig. 3). In the cell processes, a few mitochondria, small vesicles and numerous caveolae were recognisable. The elongated processes made contact with the adjoining cells and surrounded the capillary. In the areas where pericytes were absent, the cell processes were situated close to the capillary endothelium (Fig. 6). Although contact was 6 W. RERKAMNUAYCHOKE AND OTHERS Spermatic cord arteriovenous connection 7 observed between neighbouring vesiculated cells, no junctions were present between the vesiculated cells and the capillary endothelial cells.