Immune response following vasectomy in the rat: a study of

Transcription

1 J. Anat. (1992) 181, pp , with 11 figures Printed in Great Britain Immune response following vasectomy in the rat: a study of the stimulation of the regional lymph node 471 J. LEWIS AND S. W. McDONALD Department of Anatomy, University of Glasgow, UK (Accepted 5 October 1992) ABSTRACT Following vasectomy, sperm granulomas are generally believed to be important sites of access of spermatozoal antigens to the immune system. This study tests the validity of that assumption by grafting tissues from a sperm granuloma to an ectopic site (the scrotal skin) and studying the effect on the regional (inguinal) lymph node. Xiphoid cartilage provided the graft material in control animals. The experimental lymph nodes showed significant increases in weight and in the number of sectional profiles of cortical nodules indicating that they were stimulated by the presence of the granuloma tissue. To investigate the mechanism of lymph node stimulation further, a group of rats underwent unilateral vasectomy followed after 7 wk by ipsilateral orchidectomy. Three months after the initial operation the histological features of the regional (left renal) lymph node of the epididymis and granuloma were compared with corresponding nodes from rats 3 months following unilateral vasectomy only and following sham operation. The results indicate that continuous sperm production is required to sustain activity of the regional lymph nodes despite the continued presence of spermatozoa in the sperm granuloma. INTRODUCTION Following vasectomy serum antisperm antibody formation is well documented in man and laboratory animals (Samuel et al. 1975; Ansbacher et al. 1976; Kosuda & Bigazzi, 1979). Previous work has demonstrated that the regional lymph node of the testis and epididymis of vasectomised Albino Swiss rats show morphological changes consistent with this humoral response (McDonald & Scothorne, 1986, 1989; McDonald et al. 1991) but that in the rat, at least, whole spermatozoa rarely pass in local lymphatic vessels to the regional lymph node (McDonald & Scothorne, 1987); spermatozoal antigens must therefore reach the node in soluble or particulate form or be transferred by sensitised cells. The sperm granuloma, a chronic inflammatory lesion which forms at sites of leakage of spermatozoa from the vasectomised reproductive tract, is widely assumed to be an important site of access of spermatozoal antigens to the lymphatic vessels. Sperm granulomas invariably follow vasectomy in the rat and may form in up to 60% of vasectomised patients (Lee, 1986). This study investigates (1) the potential of sperm granulomas to stimulate an immune response by transplanting sperm granuloma tissue to an ectopic site and studying the effect on the local regional lymph nodes, and (2) the need for continued spermatozoal production in maintaining the immunological response of the lymph node by studying the effect on the node of excision of the testis from the vasectomised reproductive tract. MATERIALS AND METHODS The experiments were carried out on inbred Albino Swiss rats aged between 10 and 17 wk at the time of operation. All surgical procedures were performed with sterile precautions and under pentobarbitone sodium anaesthesia. Study 1: The effect of ectopic grafts of sperm granuloma tissue on the regional lymph node Vasectomy procedure for sperm granuloma donors The donor rats were vasectomised on the left side. The operation followed the procedure described in an Correspondence to Dr S. W. McDonald, Department of Anatomy, University of Glasgow, Glasgow G12 8QQ, UK.

2 472 J. Lewis and S. W. McDonald earlier paper (McDonald & Scothorne, 1986); the vasectomy site lay about 2 cm from the junction of the ductus deferens with the epididymis. L Collection of tissue for grafting Sperm granuloma tissue and, for control recipients, xiphoid cartilage, were obtained from the previously vasectomised animals. The rats were killed by intraperitoneal pentobarbitone sodium. The scrotum was shaved and, with sterile technique, the granuloma was excised and placed in sterile Hanks medium 199 on a dimple slide. A portion for grafting about 5 x 5 x 3 mm was prepared and placed on another dimple slide in sterile Hanks medium 199 until required. The xiphoid cartilage was then excised, using sterile techniques, and trimmed to provide a graft of similar size to that of sperm granuloma tissue. The cartilage for grafting was placed in sterile Hanks medium 199 until required. Grafting under scrotal skin 1 IL- it The scrotum of the host animals was shaved and swabbed with Hibitane in alcohol. A small transverse incision (approximately 4 mm long) was made in the skin of the left side and about 1 cm caudal to the root of the penis. Scissors were used to create a small pocket beneath the skin. This pocket extended up to the level of the penis and away from the midline. The graft was positioned as far up into the pocket as possible. The incision was closed with 5/0 silk sutures and protected with a plastic dressing. The operation site of each animal was inspected daily and only those animals showing good healing were included in the study. Two experimental rats developed severe scabbing at the operation site and were therefore excluded. Collection and processing of tissue -;; dc ai - Ten days after operation, 5 experimental and 5 control rats were killed. In each, the scrotum and abdomen were shaved. Using a 30G needle, 0.1 ml pontamine sky blue, a water soluble dye, was injected sub- 2..*t* s,>*j a ;41v~~' P.z Fig. 1. Surface of the left side of the scrotum showing the site of the graft of sperm granuloma tissue. The lower edge of the photograph is the most caudal part. The graft raises a noticeable lump (L). The incision site (arrow) is visible. Fig. 2. Histological section of the graft of sperm granuloma tissue. The epidermis (E) and dermis (D) cover the graft. The sperm granuloma consists of a layer of epithelioid macrophages (AM) surrounding a central mass of degenerating spermatozoa (S). Haematoxylin and eosin. x 100.

3 Immune response following vasectomy Figs 3-5. For legend see over ANA 181

4 474 J. Lewis and S. W. McDonald cutaneously at the graft site and the regional lymph node traced. The node was carefully excised and weighed and the graft and associated skin were cut from the scrotum. The nodes and graft sites were fixed in Bouin's fluid and processed for wax histology. Study 2: Effects of hemicastration on the granuloma Ten rats were vasectomised as before except that the operation was carried out close to the origin of the ductus deferens from the epididymis rather than about 2 cm away from the epididymis. This was done in the expectation that a sperm granuloma would form at the cauda epididymidis from which lymph consistently drains to the testicular lymph node (McDonald & Scothorne, 1988 a). Hemicastration The animals were left for 7 wk to allow a granuloma to form, and then the left testis of each rat was excised. Using microscissors the mesorchium between the testis and epididymis was divided except for the region of the hilum of the testis. Two 5/0 silk ligatures were tied round the testicular blood vessels as they entered the testis, and the vessels were transected between the ligatures. A single 5/0 silk ligature was tied round the fat at the lower pole of the testis, and the fat was cut between the ligature and the testis. The ligature occluded a prominent lymphatic vessel at this site. The testis was then excised, the epididymis returned to the scrotal cavity and the incisions closed as described earlier. Collection of tissue Seven weeks after hemicastration the rats were killed and, on opening the scrotal cavity, the position and appearance of the sperm granuloma was recorded. An injection of 0.05 ml 3 % pontamine sky blue dye was made through a 30G needle just beneath the surface of the left cauda epididymidis close the granuloma and the regional lymph node(s), usually the left renal node (Tilney, 1971), traced. The left renal node was excised, trimmed and weighed in those rats in which it received lymph directly from the epididymis. All nodes and epididymides were fixed in Bouin's fluid and processed for wax histology. Preparation of serial sections Serial sections of the inguinal and renal lymph nodes, of Studies 1 and 2 respectively, were cut at a section thickness of 5 gm and two 1 in 10 series prepared from each; one was stained with toluidine blue and eosin and the other with haematoxylin and eosin. Study I Quantitative analysis The weights of the left inguinal lymph nodes of experimental and control rats in Study 1 were compared by Student's t test. For each node, the number of cortical nodule profiles in every tenth section of the complete series of nodal sections was counted. Student's t test (2-tailed) was used to compare values obtained for experimental and control groups. Study 2 The quantitative data for this study consisted of the weights of the left renal lymph nodes and the number of profiles of cortical nodules in every tenth section of the nodes. Control values for this study comprised nodal weights and counts of sectional proffles of cortical nodules in every tenth section of the left renal nodes from vasectomised and sham-operated animals from a study previously presented by McDonald et al. (1991). The vasectomy technique was identical to the one used in the present study. For the sham operation the ligatures around the ductus deferens were only loosely tied and the ductus was not transected. The results were compared by one-way analysis of variance. RESULTS Study 1: Ectopic grafts of sperm granuloma tissue Following operation there was some bruising and swelling followed by light scabbing of the skin over the graft site. By the time of killing, however, all Fig. 3. Left inguinal node in situ following pontamine injection. Fig. 4. Histological structure of a left inguinal node draining a graft of sperm granuloma tissue. The cortex (C) and medullary cords (M) are well developed, indicating immunological activity. Haematoxylin and eosin. x 25. Fig. 5. Histological structure of a left inguinal node draining a control graft of xiphoid cartilage. The cortex (C) is less active than in experimental nodes and the medulla (M) is poorly developed. Haematoxylin and eosin. x 25.

5 Immune response following vasectomy ii..,t - * 4 i,,%." Fig. 6. Moderate-sized cortical nodule (outline), typical of those seen in the left inguinal node following grafting of sperm granuloma tissue beneath the scrotal skin; it shows light (L) and dark (D) poles. The capsule (arrows) and subcapsular sinus (asterisks) are visible. Toluidine blue and eosin. x 250. Fig. 7. Small cortical nodule (outlined), typical of those seen in the left inguinal node of control rats following grafting of cartilage beneath the scrotal skin. The capsule (arrows) and subcapsular sinus (asterisks) are shown. Haematoxylin and eosin. x 250. wounds had healed well. A lump measuring about 1 cm in diameter was present in the upper, outer aspect of the scrotum, and at the caudal end of this lay the scar at the site of incision (Fig. 1). These appearances were similar in experimental and control animals. Good healing of the operation site was important because inflammation or infection would have stimulated the node and obscured the effect of the graft. In histological sections, the grafts, both experimental and control, lay in the adipose tissue immediately deep to the dermis and were well vascularised; the overlying skin appeared healthy (Fig. 2). In the experimental grafts, the typical features of the sperm granuloma were evident: the central mass of degenerating spermatozoa with scattered leucocytes and surrounding macrophages and leucocyte-rich vascular connective tissue (Fig. 2); in some regions, however, the cellular wall of the granuloma appeared less organised. The chondrocytes of the cartilage grafts had died, leaving lacunar spaces in the cartilage matrix containing flattened pyknotic nuclei and cell debris. Lymph from the graft sites of all animals drained directly to the left inguinal node (as defined by Tilney, 1971) (Fig. 3); no other regional node was present. The left inguinal nodes of the experimental rats ranged in weight from to mg (mean mg, S.D mg) while those control animals ranged from to mg (mean mg, S.D mg). Student's t test showed a significant difference between the 2 groups (P < 0.02). The histological appearances of the experimental nodes were more reactive than the controls (Figs 4, 5). The development of medullary cords was greater in the experimental animals, in terms of thickness and plasma cell content. Larger germinal centre profiles were seen in the experimental nodes (Figs 6, 7) and there was some polarisation into light and dark poles; no germinal centre profiles, however, were well enough developed to show a corona of small lymphocytes. In both groups, the paracortex was similar in its development with relatively few lymphoblasts present. The number of profiles of cortical nodules counted in every tenth section of the inguinal nodes ranged 32-2

6 476 J. Lewis and S. W. McDonald from 104 to 285 (mean 161, S.D. 72) and 3 to 100 (mean 54, S.D. 43) for experimental and control rats respectively. Student's t test showed a significant difference (P < 0.05). Study 2: Hemicastration study After killing, the scrotum was opened and the contents examined. A granuloma was present on the surface of the left cauda epididymidis next to the vasectomy site in all cases (Fig. 8). The granuloma was smaller than those found 12 wk after vasectomy when the left testis was intact. The cauda epididymidis in the present study was devoid of sperm except for a small length of duct close to the granuloma. The corpus and caput of the epididymis were atrophic and mostly obscured by the surrounding fat. The contralateral testis and epididymis were normal in appearance. Histology showed the granuloma to consist of the typical central mass of spermatozoa surrounded by macrophages and a layer of vascular connective tissue rich in lymphocytes and plasma cells. The profiles of the epididymal duct adjacent to the granuloma were packed with sperm infiltrated with leucocytes. Many leucocytes were seen in the regions between duct profiles. The profiles of epididymal duct elsewhere in the cauda were smaller and devoid of spermatozoa as suggested by the gross appearance; this corresponded to observations made at the gross level. In 1 animal the epididymal lymphatics drained directly to the cisterna chyli and in another 2 the lymphatics passed the left iliac node first before continuing to the renal node; these animals were not used. Material was thus taken from a total of 7 animals whose epididymides drained directly to the left renal node. The weights of the left renal lymph nodes ranged from 4.30 to 8.25 mg (mean 6.29 mg, S.D mg; n = 7). The weights of the left renal lymph nodes from the previous study ranged from 4.72 to mg (mean mg, S.D mg, n = 8) in vasectomised rats and from 4.66 to 8.38 mg (mean 6.52 mg, S.D. 1.22, n = 9) in sham-operated controls. One-way analysis of variance (F) revealed a significant variance between the groups (F = 6.179, D.F. = 23, P < 0.01) and intergroup comparisons showed lymph nodes 8 I ' Fig. 8. Contents of the left side of the scrotum after left unilateral vasectomy followed by orchidectomy. The caput (Ca), corpus (Co) and cauda (Cd) of the epididymis and the ductus deferens (D) are shown. A sperm granuloma (G) is present at the cauda epididymidis. Ligatures are visible at the vasectomy site (arrows) and at the site of ligation of the testicular vessels (arrowhead). The caput and corpus epididymidis are atrophic. from vasectomised rats to be significantly heavier than either those from rats subjected to vasectomy and hemicastration or from sham-operated animals (P < 0.05). No differences were found in nodal weights between sham-operated rats and those with hemicastration and vasectomy. Histological examination of the renal nodes in the group of rats subjected to vasectomy followed by hemicastration showed lymphoid tissue which was better developed than in corresponding nodes from sham-operated rats but less reactive than in vasectomised animals (Figs 9-11). The cortex was poorly developed. Germinal centres were small and few in number. The medullary cords were more developed than those of sham-operated animals but not as well Fig. 9. Left renal lymph node of a rat having undergone vasectomy and hemicastration. The cortex (C) and medullary cords (M) are intermediate in development between those of vasectomised (Fig. 10) and sham-operated (Fig. 11) controls. The medullary cords show deposits of haemosiderin. Toluidine blue and eosin. x 100. Fig. 10. Left renal lymph node of a vasectomised rat. Two cortical nodules (arrows) are visible in the cortex. The medullary cords (M) are well developed. Toluidine blue and eosin. x 100. Fig. 11. Left renal lymph node of a sham-operated rat. The cortex (C) is quiescent and the medullary cords (M) are poorly developed and show deposits of haemosiderin. Toluidine blue and eosin. x 100.

7 Immune :s. response following 477 vasectomy 4''EN # fe,' *, w if; wv ow ro,'.,,.,...t Figs For legend see opposite.

8 478 J. Lewis and S. W. McDonald developed as in vasectomised animals. As in shamoperated rats, large amounts of haemosiderin were present in the medullary cords but appreciable numbers of plasma cells, indicative of immunological activity, were also present (Fig. 9). In the nodes collected from rats subjected to vasectomy and hemicastration, the total number of cortical nodule profiles observed in every tenth section ranged from 7 to 28 (mean 14, S.D. 8, n = 7) while the numbers previously determined for vasectomised and sham-operated rats were 5 to 337 (mean 108, S.D. 115, n = 8) and 0 to 12 (mean 4, S.D. 4, n = 9) respectively. One-way analysis ofvariance (F) revealed a significant variance between the groups (F = 6.078, D.F. = 23, P < 0.01) and showed that the cortical nodule content of lymph nodes from rats with vasectomy was significantly greater than in the other 2 groups (P < 0.05). No difference was found between the cortical nodule content of nodes collected from rats subjected to vasectomy with hemicastration and those from sham-operated animals. DISCUSSION Sperm granulomas, chronic inflammatory lesions at sites of spermatozoal extravasation, commonly form after vasectomy in man and laboratory animals (Bedford, 1976; Howards & Johnson, 1979; Lee, 1986). They may be responsible for postvasectomy pain and discomfort (Selikowitz & Schned, 1985) and are generally assumed to be important sites of access of spermatozoal autoantigens to the immune system allowing the production of antisperm antibodies following vasectomy. Clinical studies, probably because of difficulty in demonstrating the presence of small intraepididymal granulomas, have failed to establish a clear link between granuloma formation and humoral immunity to spermatozoa (Alexander & Schmidt, 1977; Crissey et al. 1980). In the rat, humoral immunity to spermatozoal antigens following vasectomy appears to be elicited by the regional testicular lymph nodes rather than by the spleen (McDonald & Scothorne, 1986, 1989; Al-Saffar, 1987). When grafted beneath the scrotal skin of adult rats, sperm granuloma tissue maintained its typical structure of deposits of spermatozoa surrounded by chronic inflammatory cells and provoked clear changes in the regional (inguinal) node. Compared with controls, the experimental nodes showed significant increases in weight and numbers of sectional profiles of cortical nodules. Some of the larger cortical nodules showed light and dark poles while medullary cords were thicker and more cellular than in controls. These features indicated that the experimental nodes were more immunologically active than the controls and confirmed that transplanted sperm granuloma tissue can stimulate an immune response in the regional lymph node. The experiment compared the morphology of the renal nodes of rats killed after (1) left unilateral vasectomy followed by hemicastration, (2) left unilateral vasectomy, and (3) left-sided sham operation. It suggests that a continuous supply of spermatozoa is necessary to maintain the nodal changes which follow vasectomy. The quantitative analyses showed no significant difference between the nodes from rats subjected to vasectomy with hemicastration and those from sham-operated controls. In contrast, those following vasectomy showed significant increases in weight and cortical nodule content compared with the other 2 groups. Histological examination confirmed that nodes from rats following vasectomy only were more reactive than in the other groups but also indicated that those following vasectomy with hemicastration were slightly more reactive than those following sham operation. Seven weeks after removing the ipsilateral testis from unilaterally vasectomised rats, sperm granulomas persisted. Histological examination showed that large numbers of spermatozoa were present in the centre; the granuloma, therefore, still had the potential to present antigen to the renal node. These sperm granulomas, however, were reduced in size compared to those seen following vasectomy only. The reduced size of the granuloma was associated with a marked reduction in the magnitude of the response of the renal node. The above observations suggest that the response of the node to spermatozoal antigens is dose-dependent. After vasectomy in the rat, spermatogenesis continues normally (McDonald & Scothorne, 1988b) and spermatozoa leak continuously in large quantities from the reproductive tract into the sperm granuloma. On castration, this large-scale spermatozoal antigen supply is removed. Once the epididymal duct has emptied, the only spermatozoal antigen remaining is in the central sperm mass of the granuloma. The observation that the granuloma was reduced in size compared with those at similar intervals following vasectomy without hemicastration indicates that phagocytosis of the extravasated spermatozoa in the granuloma had continued. The waning of the nodal response shows, however, that this level of antigen presentation was not sufficient to maintain the node in an active state. Despite the waning response, the nodes in this study were still slightly more active than

9 Immune response following vasectomy 479 those of sham-operated controls. Whether the granuloma presented sufficient antigen to maintain the nodes in this state of activity, or whether the nodes had not had sufficient time to return to their prevasectomy morphological state, is not known. Dose-dependency in the response to exposure to spermatozoa was suggested by Rumke & Titus (1970) who injected varying numbers of spermatozoa subcutaneously and found that at least 107 spermatozoa had to be injected before serum antisperm antibodies could be detected. Linnet & Hjort (1977) suggested that circulating antisperm antibodies tended to be found in men with higher prevasectomy sperm counts. In conclusion, this study shows that sperm granuloma tissue induces a response in the regional lymph node and indicates that a continued supply of large amounts of spermatozoal antigen to the granuloma is required to maintain the response in the node. ACKNOWLEDGEMENTS We are grateful to Mr N. K. Bennett and Miss M. Hughes for technical help. J. Lewis was supported by an award from the Wolfson Trust. REFERENCES ALEXANDER NJ, SCHMDT SS (1977) Incidence of antisperm antibody levels and granulomas in men. Fertility and Sterility 28, AL SAFFAR RA (1987) Morphological Studies of the Immune Response to Vasectomy. PhD thesis, University of Glasgow. ANSBACHER R, HODGE P, WILLIAMS A, MuMoFRD DM (1976) Vas ligation: humoral sperm antibodies. International Journal of Fertility 21, BEDFoRD JM (1976) Adaptations of the male reproductive tract and the fate of spermatozoa following vasectomy in the rabbit, rhesus monkey, hamster and rat. Biology of Reproduction 14, CRISSEY MM, SROUGI M, BIGAZZI P, MCDONALD J, GirrIS RF (1980) Correlation of sperm granulomas and sperm autoantibodies in inbred rats. Fertility and Sterility 33, HowARDS SS, JOHNSON AL (1979) Effects of vasectomy on intratubular hydrostatic pressure in the testis and epididymis. In Vasectomy: Immunologic and Pathophysiologic Effects in Animals and Man (ed. I. H. Lepow & R. Crozier), pp London: Academic Press. KOSUDA LL, BIGAZZI PE (1979) Influence of genetic factors on the production of autoantibodies to spermatozoa in vasectomized rats and mice of different inbred strains. In Vasectomy: Immunologic and Pathophysiologic Effects in Animals and Man (ed. I. H. Lepow & R. Crozier), pp London: Academic Press. LEE HY (1986) A 20-year experience with vasovasostomy. Journal of Urology 136, LINNET I, HJORT T (1977) Sperm agglutinins in seminal plasma and serum after vasectomy: correlation between immunological and clinical findings. Clinical and Experimental Immunology 30, McDoNALD SW, ScoTHORNE RJ (1986) On the response of the regional testicular lymph nodes after unilateral vasectomy in rats. Journal of Anatomy 144, McDoNALD SW, ScoTHORNE RJ (1987) On the mode of sperm autoantigen presentation to the regional lymph node of the testis after vasectomy in rats. Journal of Anatomy 153, McDoNALD SW, SCOTHORNE RJ (1988 a) The lymphatic drainage of the epididymis and of the ductus deferens of the rat, with reference to the immune response to vasectomy. Journal of Anatomy 158, McDoNALD SW, SCOTHORNE RJ (1988b) A quantitative study of the effects of vasectomy on spermatogenesis in rats. Journal of Anatomy 159, McDoNALD SW, SCOTHORNE RJ (1989) The response of the regional testicular lymph node six and nine months after vasectomy in rats. Journal of Anatomy 165, McDoNALD SW, AL SAFFAR RA, SCOTHoRNE RJ (1991) The response of the regional lymph node to epididymal sperm granulomas after vasectomy. Journal of Anatomy 176, RUJMKE P, TITus M (1970) Sperm agglutinin formation in male rats by subcutaneously injected syngeneic epididymal spermatozoa and by vasoligation or vasectomy. Journal of Reproduction and Fertility 21, SAMUEL T, KOLK AHJ, RUMKE P, VAN Lis JM (1975) Autoimmunity to sperm antigens in vasectomized men. Clinical and Experimental Immunology 21, SELIKOWITZ SM, SCHNED AR (1985) A late post-vasectomy syndrome. Journal of Urology 134, TILNEY NL (1971) Patterns of lymphatic drainage in the adult laboratory rat. Journal of Anatomy 109,