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Immunological and morphological effects of vasectomy in the rabbit.

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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.
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Flickinger, C. J. 1972a Alterations in the fine
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structure of the rat epididymis after vasectomy.
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350
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Note added in proof: Immunofluorescent study of a rabbit that had been vasectomized four years earlier revealed circulating antisperm antibodies as
well as immune complex disposition -particularly
IgM -surrounding
the seminiferous tubules and parts of the epididymis. Heavy staining of
the luminal contents was observed in the epididymis.
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