Immunological and Morphological Effects of Vasectomy in the Rabbit NANCY J. ALEXANDER AND KENNETH S . K. TUNG Reproductive Physiology, Oregon Regional Primate Research Center, 505 N.W. 185th Avenue, Beaverton, Oregon 97005 and Department of Pathology, University of New Mexico School of Medicine, Albuquerque, New Mexico 87131 ABSTRACT Half of the rabbits developed antisperm antibodies (measured by either indirect immunofluorescence or sperm immobilization tests) after either a unilateral or bilateral vasectomy. The raised antibody levels, particularly six months or longer after vasectomy, often accompanied patchy orchitis. Seminiferous tubules from such animals exhibited sloughed, multinucleated, and immature germinal cells which were engulfed by phagocytic cells. Mononuclear infiltrates were occasionally present. The basal lamina infolded and thickened by means of supernumerary layers and appeared to be endocytosed by cells of the seminiferous tubules. Four months after vasectomy, numerous phagocytic cells were seen to migrate through the intact epithelium of zone 1 in the caput epididymidis, and were particularly prevalent in animals that exhibited testicular damage. These macrophages may serve to present sperm antigens to lymphocytes. The increasing popularity of vasectomy as a method of permanent birth control has spurred investigations into the safety of the procedure in men as well as experimental animal models. With ligation of the vas deferens, the millions of spermatozoa produced are imprisoned within the epididymis and vas deferens, resulting in complex physiological and immunologicaI effects. Sperm antigens from these sperm may reach the lymphoid system and elicit antisperm antibodies. Such antibodies have been described in men (Ansbacher, '71; Alexander et al., '74), rhesus monkeys (Alexander et al., '74), guinea pigs (Alexander, '73a), rats (Rumke and Titus, '70), and other species. Our work in rhesus monkeys shows that after vasectomy, the number of macrophages in the epididymis increases markedly, particularly in the efferent ducts and caput epididymidis. These phagocytic cells engulf and degrade spermatozoa, removing them from the epididymis. Additionally, deposits of the third component of complement, C3, were found surrounding the efferent ducts of an animal vasectomized for three years (Alexander, '72). Spermatozoa are similarly phagocytosed in men after ligation of the vas ANAT. REC., 188: 339-350. deferens (Phadke, '64), although it is not known whether immune complex deposition is present. An immune response to spermspecific antigens may lead to allergic orchitis, an autoimmune disease of the testis. In fact, a recent paper on vasectomy in rabbits (Bigazzi et al., '76a) describes testicular changes related to an autoimmune response to vasectomy. The present study with rabbits indicates that increased phagocytosis of sperm and sperm remnants in the caput epididymis and patchy degeneration of seminiferous tubules coincides with the development of antisperm antibodies and describes effects on the testis and the epididymis after vasectomy in rabbits. METHODS Surgery Bilateral or unilateral vasectomy was performed on adult New Zealand white rabbits which were anesthetized with Fluothane. All surgeries were performed with sterile procedures. A midline abdominal Received Oct. 18, '76. Accepted Nov. 19, '76. 1 Publication No. 896 from the Oregon Regional Primate Research Center, supported by NIH Grants HD05969 and RR00163. The excellent technical assistance of Jeanne Hren and Barbara Mixon are gratefully appreciated, a s well as the expertise of the core facilities personnel at the Primate Center. 339 340 NANCY J. ALEXANDER AND KENNETH S. K. TUNG results were considered borderline positive; a higher ratio was considered very positive. Indirect immunofluorescence Smears were made from thrice-washed (10 minutes a t 2,000 g), freshly ejaculated or epididymal rabbit spermatozoa diluted to 60 X lo6 spermlml. The smears were air-dried, methanol-fixed for 30 minutes, and frozen a t - 70°C. The slides were incubated for 30 minutes with sera taken both pre- and postvasectomy, washed, and incubated for 45 minutes at room temperature with fluorescein-conjugated goat anti-rabbit IgG and IgM (Cappel, Cochranville, Pennsylvania); they were washed again, mounted in buffered glycerol, and studied under a Zeiss epi-fluorescence microscope. Fig. 1 Normal rabbit testis and epididymis partially dissected to show the zones of the epididymis (14). de, ductuli efferentes; v, vas deferens; p, proximal, and d, distal, represent areas of the testis where tissue samples were taken for electron microscopy. incision was made in the suprapubic area, both spermatic cords were located with blunt dissection, and the vas deferens was cut and doubly ligated with 4-0 chromic suture. Some animals were vasectomized with no ligatures by removing a 1-cm piece of vas deferens and replacing the vas within the body. After surgery, all animals were examined to ensure that the testes could freely enter the scrotum. Serum Serum from normal and experimental rabbits (prevasectomy and a t sacrifice) was collected from the ear by venipuncture, heated to 56°C for half a n hour to inactivate complement, and stored at - 70 C until used. O Sperm immobilization test The procedure of Isojima et al. ('68) was used. The number of motile sperm exposed to normal serum and complement was divided by the number of motile sperm exposed to the serum being tested and complement. When this ratio was 3-12, the Electron microscopy Two samples of testes were taken, one adjacent to area of the rete testes, and one distal (fig. 1). Additionally, samples of rete testis, ductuli efferentes, caput (zones 1, 2, 3), corpus (zone 4), cauda (zones 5, 6) (fig. l ) , and occasionally vas deferens were taken from 35 mature rabbits at sacrifice which had (N = number of rabbits): (1) Bilateral vasectomy with ligatures (N = 12; tissue taken 2 weeks and 1,2, 4 [ N = 211, 6.8 [N = 2 ] , 9 , 10, 12, and 18 months after vasectomy). (2) Bilateral vasectomy without ligatures (N = 9; tissue taken a t 3 days, 10 days, and 1, 2, 4 [ N = 21, 6 [N = 21, and 8 months). (3) Left unilateral vasectomy with ligatures (N = 8; tissue taken at 2 weeks and 1, 2, 4, 6, 8, 10, and 12 months). (4) Right unilateral vasectomy with ligatures (N = 2; sacrificed at 4 months and 8 months). ( 5 ) No treatment (N = 4; normal adult males). The tissues were fixed for two hours in paraformaldehyde-glutaraldehyde (Alexander, '72), and postfixed in Karnovsky's osmium fixative (Karnovsky, '71), dehydrated in alcohol, and embedded in Araldite. Sections 1-pm thick were stained with 1% toluidine blue with 1% borax. Thin sections were stained with alcoholic uranyl acetate and lead citrate and viewed with a Philips 300 electron microscope. 341 EFFECTS OF VASECTOMY IN THERABBIT TABLE 1 Morphological a n d serological f i n d i n g s in 31 rabbits vasectomized either bilaterally or u n i laterally f o r periods ranging f r o m 3 d a y s t o 18 m o n t h s . Since no d r f e r e n c e was s e e n b e t w e e n a n i m a l s vasectomzzed w i t h a n d t h o s e vasectomized w i t h o u t ligation of t h e v a s deferens, t h e y w e r e grouped together. S e v e n a n i m a l s had a left unilateral v a s e c t o m y , a n d t w o a n i m a l s had a r i g h t u n i l a t e r a l v a s e c t o m y ; since no difference w a s n o t e d , t h e s e a n i m a l s are reported as o n e g r o u p . De refers t o d u c t u l i e f f e r e n t e s a n d z o n e 1 i s part of t h e c a p u t e p i d i d y m i d i s Bilateral vasectomy No. of rabbits Testicular pathology 3 days-2 weeks 1-2 m o n t h s 3 4 4 4 months 7 6 1 0 0 2 2 0 Time after vasectomy 8-12 m o n t h s 18 m o n t h s Macrophages present De or Zone 1 Presence of antibodies Unilateral vasectomy Operated side Time after vasectomy 2 weeks 1-2 ..~ m o ~~.~ nths ~ 4 4months 8-12 months Nonoperated side No. of rabbits Testicular pathology Macrophages present DelZone 1 Testicular pathology 1 2 3 4 0 0 0 3 0 0 1 4 0 0 0 1 Macrophages present DelZone 1 0 0 3 4 Presence of antibodies 0 1 0 4 Spermatogenesis continued after vasectoAcid phosphatase my, as evidenced by the presence of all Tissue was fixed in 4.6 % glutaraldehyde stages of the spermatic cycle as well as in cacodylate buffer for two hours and mature spermatozoa in the vas deferens. washed in buffer overnight. Forty-micron However, rabbits vasectomized for six sections were cut on a freezing microtome months or longer often exhibited focal deand incubated in Gomori’s substrate. Sec- generation of the seminiferous tubules. tions for light microscopy were rinsed in Sloughing of immature as well as abnoracetate buffer, washed in acetic acid, am- mal germinal cells (fig. 2) was evident in monium sulfide, and water and mounted the tubules of 43% of the rabbits vasectoin glycerine jelly. mized for eight or more months (table 1). RESULTS Macrophages were observed engulfing clumped and disrupted spermatozoa (fig. 3 ) . Morphological findi?lgs Numerous multinucleate and other abnorBilateral vasectomy mal spermatids with irregular acrosomal Vasectomy caused visible morphological formation, vacuoles, and discontinuities of changes in the epididymis and testis. The membranes were abundant in the lumen caudal epididymis (particularly zone 6 - of the testis and epididymis (fig. 2). In adsee fig. 1) and proximal vas deferens were dition, the Sertoli cells of these testes conmarkedly distended three to four times be- tained unusually large amounts of lipid yond normal. The adjacent zone 5 sus- droplets. The basal lamina underwent a tained less distention. Granulomas occurred general thickening because of development in some of the rabbits vasectomized eight of supernumerary layers (fig. 6). Rather months or longer. Spermatozoa apparently than smoothly surrounding the seminifercontinued to migrate through the epididy- ous tubules, the basal lamina because inmal lumen and were retained, particularly folded and developed spikes (figs. 2, 9). in zone 6 and the vas deferens. Rarely, discrete granular deposits that reIn the animals vasectomized for periods sembled immune complexes appeared withof six months or longer, the testes frequent- in the thickened basement membrane (fig. ly became flaccid and reduced in size. 9). The cells of the seminiferous tubules 342 NANCY J . ALEXANDER AND KENNETH S. K. TUNG appeared to endocytose onion-skin whorls of basal lamina (fig. 8). Whether this is infolding or endocytosis would require further study. The Sertoli cell cytoplasm contained numerous phagocytic vacuoles adjacent to these whorls. Occasionally, lymphocytes and macrophages were seen within the tubules (figs. 2, 3 ) and in several cases, abundant mononuclear cells infiltrated between seminiferous tubules. The cuboidal epithelium of the rete testis was studded with microvilli and remained unchanged after vasectomy. The surrounding connective tissue area exhibited an increase in histiocytes or macrophages. A greatly increased number of macrophages engulfing and degrading spermatozoa appeared within the lumen of the rete eight months after vasectomy. The ciliated ductuli efferentes exhibited few changes until eight months or longer, when - as in the rete - macrophages became abundant within the lumen. In zone 1 of the caput epididymis, presumptive macrophages increased in numbers from two weeks after vasectomy through the end of the experiment (figs. 4, 7). The epididymides of animals that exhibited testicular damage especially revealed numerous phagocytic cells interspersed within the epithelium of zone 1 of the epididymis as well as in the surrounding interstitium and adjacent blood vessels (figs. 4, 5). The presence of these cells in the connective tissue, at all levels of the epididymal epithelium as well as in the epididymal lumen suggests an active migration of macrophages in and out of the epididymis. Electron-dense phagocytic granules in the macrophages may represent the end products of ingested spermatozoa and spermatids (figs. 4, 5, 7). Both lymphocytes and macrophages invaded the epithelium of zone 1 (table 1). The last segment of the caudal epididymis (zone 6) became packed with spermatozoa after vasectomy but, unlike zone 1, macrophages did not invade the cauda. This region increased to almost the size of the testis itself. However, distended and flattened epithelial cells appeared active, with abundant rough endoplasmic reticulum, a large Golgi apparatus, and smooth endoplasmic reticulum. Sperm ingestion by any of the epithelial cells of the epididymis was not evident. One normal animal and one vasectomized bilaterally ten months earlier showed equal amounts of acid phosphatase staining. In both cases, zones 1 through 6 and the vas deferens exhibited some staining. Furthermore, there was no evidence of sperm breakdown and release of lytic enzymes in the epididymal lumen. This observation supports that of Flickinger ('75a) and indicates that the epididyma1 epithelium is not actively involved in sperm phagocytosis after vasectomy. Unilateral vasectomy Numerous macrophages infiltrated zone 1 of the epididymis in all seven animals that were unilaterally vasectomized for four months or more; they were found on the contralateral as well as on the operated side. Seminiferous tubule degeneration was found on the operated side of three of the seven animals, and one had tubular degeneration on both sides. Since one side (either right or left) was intact, changes could not be attributed to manipulation or to semicryptorchidism. Two animals were given a right rather than a left unilateral vasectomy in order to establish the relevance of surgically manipulating one side or the other. No difference was found. Serologicalfindings Vasectomy resulted in a gradual increase in antibody levels (table 1) measured either by sperm immobilization or indirect immunofluorescence. When serum samples taken before vasectomy from 19 of the experimental animals were tested by immunofluorescence, one was found to be positive. A few others had low levels of antisperm fluorescence on some test runs. Fluorescent levels were similar when either epididymal or ejaculated spermatozoa were used, evidence that the antibodies revealed by this test were sperm-specific rather than against sperm-coating antigens from the seminal plasma. After six months, half the rabbits had antibodies as measured by the two methods. The animals with prevasectomy antibodies developed higher levels after vasectomy. Anti-acrosomal antibodies were the most common type (figs. 10, 11) but occasionally post-acrosomal (fig. 1I), equatorial, or tail fluorescence was observed. Although both IgM and IgG conjugated antisera were used, most of the antibodies were of the IgG class. The development of E F F E C T S OF VASECTOMY IN T H E R A B B I T 343 Fig. 2 Part of a left seminiferous tubule eight months after a left unilateral vasectomy. Spermatogenesis is impeded and the basement membrane is thickened and reveals the formation of spikes (arrows; see also fig. 7). The lumen contains sloughed multinucleate spermatids and phagocytic cells. X 700. Fig. 3 The lumen of a seminiferous tubule from the same rabbit as figure 5 contains this macrophage which has engulfed spermatozoa and a spermatid. x 3,100. Fig. 4 Zone 1 of a rabbit’s right epididymis 12 months after a left unilateral vasectomy. The number of phagocytic cells (arrows) resembling macrophages within the epididymal epithelium increases after vasectomy, even on the contralateral side. X 375. Fig. 5 Zone 1 six months after a bilateral vasectomy without ligatures. An accumulation of dense phagocytic cells (arrows) is found i n the connective tissue surrounding a n epididymal tubule (upper right). x 375. 344 NANCY J . ALEXANDER AND K E N N E T H S . K. TUNG Fig. 6 The right testis of a rabbit eight months after a left unilateral vasectomy. Even the contralateral side of unilaterally vasectomized animals reveals infolding and layering of the basal lamina as well as increased accumulations of residual bodies within the Sertoli cells. X 9,300. Fig. 7 Epididymal zone 1 of a rabbit six months after bilateral vasectomy, revealing presumptive phagocytic cells and a lymphocyte within the apparently intact epithelium. X 7,800. EFFECTS O F VASECTOMY IN T H E R A B B I T 345 Fig. 8 Part of a seminiferous tubule 12 months after bilateral vasectomy. Whorls of basal lamina appear pinched off within the cells. X 6,200. Fig. 9 The right testis of a rabbit eight months after a right unilateral vasectomy. This micrograph depicts a spike of layered basal lamina containing a rare granular deposit resembling a n immune complex (arrows) and adjacent to a possible lymphocyte. X 9,000. 346 NANCY J . ALEXANDER AND KENNETH S. K. TUNG Fig. 10 Indirect immunofluorescent localization of antisperm antibodies on rabbit spermatozoa. The most common pattern is acrosomal. Serum from a rabbit bilaterally vasectomized six months earlier. X 500. Fig. 1 1 Indirect immunofluorescence. Antibodies to the acrosomal and postacrosomal region in a rabbit six months after bilateral vasectomy. X 500. sperm immobilizing antibodies did not correlate with antibodies revealed by immunofluorescence; some animals had only one type and others had both. However, elevated levels of antisperm antibodies were found in all animals with testicular damage. Neither antibody type nor time of development correlated with the presence or absence of visible granulomas, distended epididymides, or flaccid testes. One group of 12 rabbits was vasoligated at the time of bilateral vasectomy; the vasa in another group of nine were cut but not ligated. In the second group, spermatozoa were not impeded from draining into the peritoneum. Presumably, those animals with no ligatures would have a greater antigen input and a higher antisperm antibody response, but sperm immobilization and indirect immunofluorescence revealed little difference between the two groups, nor did they differ in testicular damage or macrophage migration into the epididymis. Examination of the cut vas end at the time of sacrifice revealed no sperm granuloma formation or tissue reaction of any kind. The epididymides from animals of both groups distended in a manner similar to the vasoligated group, that is, zone 6 became particularly enlarged. Perhaps cutting the vas terminated peristalis; except during ejaculation, peristalis may be a more important factor than intraductal pressure in moving sperm through the vas. DISCUSSION This study confirms that both unilateral and bilateral vasectomy in rabbits can cause a proliferation of acrosomal antibodies as revealed by indirect immunofluorescence or complement dependent immobilizing antibodies. Degenerative changes in the testis were always associated with at least one of the two antibody types tested. Rabbits with raised antibody levels often develop patchy orchitis, sloughing of the germinal epithelium, and a thickening of the basal lamina. After vasectomy in the rabbit, macrophage involvement and humoral immunity were both factors. Different species respond variously to vasectomy. For example, the testes of vasectomized dogs seem to suffer some early morphological damage which may or may not be reparable (Kothari and Mishra, '73; Grewel and Sachan, '68; Vare and Bansal, '73; Derrick et al., '74). Rats respond to vasectomy uniquely; most develop sperm granulomas at the site of ligature (Flickinger, '72a; Alexander, '73). Bedford ('76) postulated that granulomas develop because the rat vas deferens is not extensi- EFFECTS OF VASECTOMY IN THERABBIT ble. The rabbit, on the other hand, has a vas that is very expansible and granulomas do not readily develop in this species. The cauda epididymis can increase greatly in size (MacMillan et al., '68; Swanson and Hafs, '69). Our study supports the observation that lesions of the rabbit epididymis do not usually occur until 8 to 12 months after vasectomy (Flickinger, '75a; Bedford, '76). Flickinger ('75a) showed as does our study, that the rabbit epididymal epithelium remains relatively unchanged morphologically. The rabbit epididymis, particularly zone 6 and the vas, undergoes marked distention after vasectomy. Bedford ('76) postulates that phagocytes are first attracted to and enter through focal lesions of the caudal epididymal epithelium and later migrate within the duct to ingest sperm. Our study demonstrates that phagocytic cells invade the intact epithelium of zone 1 of the caput epididymis of the rabbit (figs. 2, 3) and probably migrate both in and out. Macrophage-like cells increased both within the epididymal epithelium and in the surrounding interstitial area after vasectomy. Animals with testicular damage always had numerous macrophages in this region. Luminal macrophages with identifiable sperm remnants, although occasionally present in normal rabbits, were more common in animals vasectomized for longer periods (over 8 months). These findings suggest active macrophage phagocytosis of spermatozoa in the caput epididymis of the rabbit may be an important mechanism for sperm removal. That Flickinger ('75a) did not observe lumenal macrophages was probably due to the shorter duration of his study. Macrophages were found in the epididymides of most animals studied four months after vasectomy, before seminiferous tubule damage was observed. It seems likely that macrophages migrate back and fourth between the connective tissue and the epididymal tubules, thus allowing presentation of sperm antigen to antigen-specific lymphocytes (both T and B). In animals vasectomized for longer periods (8 months or more), macrophages were sometimes seen in the seminiferous tubules. It is doubtful that this macrophage invasion occurred via migration through the epididymal tubules to the seminiferous tubules, which would have been 347 against the normal gradient of flow. Macrophage accumulation in seminiferous tubules was more prevalent in tubules which exhibited degeneration, including a thickened basal lamina. The development of antisperm antibodies after vasectomy as well as the role of these antibodies in sperm degradation are not well defined. Granulomas from epididymal lesions are probably not the cause of antibody development in rabbits, since antisperm antibodies are detectable before granulomas are found; furthermore, no difference in antibody level or type was observed between rabbits with greatly distended epididymides and those with granulomas and less distended epididymides. We included both right and left unilateral vasectomies in our study to ensure that differences were not the result of the side of vasectomy. Bilateral vasectomies were done both with and without ligation in order to check morphological and immunological differences. Rumke and Titus ('70), for example, found that a higher percentage of rats develop sperm-agglutinin titers after vasectomy without ligatures. Brannen and Coffey ('74), on the other hand, found that rats with ligated vasa developed high antibody levels, whereas those with no ligation after vasectomy developed a cell-mediated immunity. Our study revealed no differences in antibody level dependent on side or type of surgery. Antibody development in rabbits parallels that seen in men: after vasectomy not all subjects developed antibodies and antibody elaboration was relatively slow (Ansbacher, '71; Alexander et al., '74). Only one of four of the rabbits in the 6-month group had antibodies, whereas 10 of the 11 animals vasectomized for eight months or more had some type of antibody level, and five had sperm immobilizing antibodies. Swanson and Hafs ('69) reported no antibody levels to spermatozoa in rabbits ten weeks following unilateral vasectomy. Rhesus monkeys, on the other hand, rapidly develop antisperm antibodies after vasectomy; most show antibodies by two weeks, after which there is a decline (Alexander, '75). Acrosomal antibodies were the most common type in the rabbit, but other patterns -namely postacrosomal, equatorial, and tail -found in human sera (Hansen and Hjort, '71; Tung, '75), are reported 348 NANCY J. ALEXANDER AND KENNETH S. K. TUNG here for the first time. The increase in lipofuscin within the Sertoli cells of rabbits also mirrors the effects of vasectomy in other species, i.e., rhesus monkeys (Alexander, unpublished) and men (Davis and Lubell, '75). Testicular damage in the rabbit is not a direct immediate result of vasectomy. No damage occurs (Flickinger, '75b; Jones, '73; Glover, '69; Paufler and Foote, '69; Moore and Quick, '24) until approximately eight months after vasectomy. The considerable distention of the rabbit epididymis suggested to Flickinger ('75b) that the testicular damage was due to a state of semicryptorchidism. Another explanation involves the immune system; our study confirms that of Bigazzi and his associates C75a,b) as well as Alexander's work in the guinea pig ('73). Bigazzi et al. ('76a,b) clearly demonstrated that vasectomized rabbits develop antisperm antibodies and consequent immune complexes. In their studies, circulating antibodies to testicular antigens -measured by tanned cell hemagglutination, complement fixation, and indirect immunofluorescence -were found in 58 % of the bilaterally vasectomized rabbits. Four of the rabbits in their study had immune complexes in the testis with deposition of IgG and C3 in the basement membrane of the seminiferous tubules, presumably antigen-antibody complexes with sperm antigens. Ultrastructurally, these deposits appeared as electron-dense accumulations. Such deposits were dissociated and eluted from homogenates and the recovered immunoglobulin reacted with sperm acrosomes. Our study did not reveal obvious immune complexes in the basal lamina around the seminiferous tubules even at regions of extensive layering. The layering effect seemed to be independent of antibody levels. Perhaps the dearth of immune complexes is due to sampling. We did not collect tissue for immunofluorescence studies and location of such complexes with electron microscopy is most easily accomplished by observing material adjacent to that studied with immunofluorescence. Perhaps the time span of the study made observation of immune complexes unlikely. Only five animals in our study were vasectomized for over ten months; since immune complexes only occur in a fraction of the animals with antisperm antibodies, it is reasonable to assume that such animals were not in our sample. The other features described by Bigazzi and associates ('76a) -spikes and layered basal lamina - were observed in the present study; these may indicate T-cell-mediated injury or transitory immune complex deposition and resorption. The finding of sperm-immune complexes in the basal lamina surrounding seminiferous tubules strongly suggests that the bloodtestis barrier must be penetrable in some animals as a result of vasectomy. The rete testis is considered to be a weaker barrier than the seminiferous tubules (Waites and Setchell, '69; Tung et al., '71; Koskimies and Kormano, '73), because of the tight junctions of the Sertoli cells plus the surrounding myoid cells (Dym and Fawcett, '70). In the present study, macrophages were revealed, particularly in the caput epididymis and in the rete. They appeared to invade the intact epithelium of zone 1 rather than the simple cuboidal rete testis epithelium. Furthermore, since some macrophages are present in normal animals, the macrophage movement was probably due to a greatly increased rate of a normal process. It is likely that several events are occurring either simultaneously or in a time sequence that cannot presently be separated, including (1) increased macrophage migration into the head of the epididymis, (2) occasional lymphocyte infiltration into the seminiferous tubules, (3) increased stimulation of B-cells, resulting in antibody formation, and (4) degenerative changes in the seminiferous tubules. The complex histopathology of allergic orchitis includes at least three changes : (1) mononuclear invasion of the seminiferous tubules, (2) degenerative changes in the germinal epithelium, and (3) neutroPhil-rich lesions in the efferent ducts (Tung and Alexander, '77). All these changes were found in some rabbits in this study. Initiation of such reactions can involve interaction between antibody andlor lymphocytes and sperm antigen. In no case did we observe damage in the absence of antibodies; however, the presence of antibodies did not automatically indicate testicular damage. A time sequence was noted in the development of antisperm antibodies measured by either sperm immobilization or indirect immunofluorescence: high levels EFFECTS OF VASECTOMY IN THERABBIT 349 of antisperm antibodies were more fre- Bigazzi, P. E., L. L. Kosuda, K. C. Hsu and G. A. Andres 1976a Immune complex orchitis in quent after six months; similarly, seminifvasectomized rabbits. J. Exp. Med., 143: 382erous tubule degeneration was not ob404. served until six months and was then gen- Bigazzi, P. E., L. L. Kosuda, L. L. Harnick, R. C . Brown and N. R. Rose 1976b Antibodies to erally found in the same animals that had testicular antigens i n vasectomized rabbits. Clin. high antisperm antibodies. However, tesImmunol. Immunopathol., 5: 182-194. ticular damage and antibody development Brannen, G. E., and D. S. Coffey 1974 Immunocould well have been parallel events. logic implications of vasectomy: 11. Serum-mediated immunity. Fertil. Steril., 25: 515-520. Studies on antibody localization in the seminiferous tubules prior to testicular Davis, J. E., and I. Lube11 1975 Advances i n understanding the effects of vasectomy. Mt. Sinai damage would shed more light on this subJ. Med. N.Y., 42: 391-397. ject. Lymphocytes were found both as in- Derrick, F. C., W. L. Glover, 2. Kanjuparamban, C. B. Jacobson, M. McDougall, K. McCowin, filtrates and as individual cells either adH. D. Mercer and L. D. Rollins 1974 Histojacent to the seminiferous tubules or zone logic changes in the seminiferous tubules after 1. Small numbers of lymphocytes have vasectomy. Fertil. Steril., 25: 649-658. been found in normal epididymides of other Dym, M., and D. W. Fawcett 1970 The bloodtestis barrier i n the rat and the physiological species (Dym and Romrell, '75; Alexander, compartmentation of the seminiferous epithe'73b), but infiltrates are representative of lium. Biol. Reprod., 3: 308326. an abnormal condition. T-lymphocytes are Dym, M., and L. J. Romrell 1975 Intraepitheprobably the most antigen-reactive cells lial lymphocytes in the male reproductive tract of rats and rhesus monkeys. J. Reprod. Fertil., in this system and have several functions, 42: 1-7. including acting as killer cells directly, as Flickinger, C. J. 1972a Alterations in the fine helper cells in conjunction with B cells for structure of the rat epididymis after vasectomy. antibody production, or as mediators of a Anat. Rec., 173: 2 7 7 3 0 0 . 1972b Ultrastructure of the rat testis delayed hypersensitivity reaction. In order after vasectomy. Anat. Rec., 174: 477494. to more fully elucidate the role of T-cells 1975a Fine structure of the rabbit epiin vasectomy, adoptive transfer studies didymis and vas deferens after vasectomy. Biol. would be necessary. The occasional infilReprod., 13: 50-60. 1975b Fine structure of the rabbit testis trates of mononuclear cells could either be after vasectomy. Biol. Reprod., 13: 61-67. caused by a delayed hypersensitivity reT. D. 1969 Some aspects of function i n sponse, by the presence of humoral anti- Glover, the epididymis. Experimental occlusion of the body (Oldstone and Dixon, '70), or by the epididymis in the rabbit. Int. J. 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