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Histologic
changes in the mouse testis after bilateral vasectomy
Shio
Kumar Singh, Sumana Chakravarty
Department
of Zoology, Banaras Hindu University, Varanasi 221 005, India
Asian J Androl 2000 Jun; 2: 115-120
Keywords:
vasectomy;
testis; mouse; contraception
Abstract
Aim: To study the effect of vasectomy on histological appearance of the testis. Methods: Parkes strain mice were used as the animal model; they were bilaterally vasectomized (Vx) or sham-operated (So) and killed at intervals of 4, 6, 9, and 12 months after the operation. Testes were excised from 5 Vx and 5 So mice at each interval and processed for histological examination. Results: Testes of So mice showed normal histological features. By contrast, marked alterations were observed in the seminiferous tubules in testes of Vx mice, except in those killed 4 months after the operation. The seminiferous epithelium in the tubules was only 2-3 layers thick and showed much depletion of germ cells; in severe cases, the epithelium consisted of only a thin layer of Sertoli cells, spermatogonia and a few spermatocytes. Exfoliation of germ cells, occurrence of multinucleated giant cells and vacuolated appearance of the epithelium were of common features in the tubules. Furthermore, lumen of the retetestis in Vx mice was greatly dilated and showed accumulation of spermatozoa with immature germ cells; in mice vasectomized for 6-12 months, several macrophages ingesting spermatozoa were often observed in the lumen of the rete testis. Spermatic granuloma was also sometimes noticed in corpus or in cauda regions of the epididymis in mice vasectomized for 6-12 months. Conclusion: We suggest that consequences of vasectomy should be thoroughly understood in order to make this method rather more popular as a reversible method of male contraception.1 Introduction
Vasectomy is a simple, effective and one of the most widely used methods of male contraception. The operation causes interruption in the luminal continuity of the vas deferens and, thus, interferes with transport of spermatozoa from the testis and epididymis into the ejaculatory duct. However, there is no unanimity in opinion among authors on the effect of vasectomy on the testis. On one hand, several authors have reported marked alterations in the histoarchitecture of the testis after vasectomy (rat : Flickinger et al[1]; Sarrat et al[2]; guinea pig: Alexander[3]; Tung and Alexander[4]; monkey: Lohiya et al[5]). On the other hand, there are authors who did not find alterations in the testis after the operation (rat : Flickinger[6]; Heller and Rothchild[7]; McDonald and Scothorne[8]; rabbit: Paufler and Foote[9]; monkey: Alexander[10]; Chapman et al[11]; Hadley and Dym[12]). However, there has been an increasing demand in recent years for reversal of vasectomy, so the findings of the effect of vasectomy on the testis are important because lesions induced in the organ by the operation have potential influence on fertility after reversal of the operation. Therefore, there is a need to examine the effect of vasectomy on the testis. In the present study, we have investigated the effect of bilateral vasectomy on the testis of the Parkes strain mouse, which we have been using as the animal model in our laboratory[13].2 Materials and methods
Forty
adult (age: 12-14 weeks) male mice belonging to the Parkes (P) strain,
weighing 30-40 g, were used in the investigations.
The animals were housed under standard laboratory conditions and
maintained on pelleted food (Lipton India Ltd) and water ad libitum.
After recording initial body weights, they were divided into 4
groups of 10 each and operated upon and killed at intervals of 4
(Group I), 6 (Group II), 9 (Group III) and 12 (Group IV) months.
Within each group, 5 animals were bilaterally vasectomized, and
5 were sham-operated
as controls.
The
operations were performed under sodium pentobarbital (60 mg/kg body weight, ip)
anesthesia via a lower mid-abdominal incision.
The vas deferens on each side was exposed without causing injury
to the adherent blood vessels and a double ligature, about 1 cm apart,
was applied using silk thread.
The portion
of the vas between the two ligatures was removed and the ligated ends
were replaced in the abdominal cavity; the incision was then closed in
two layers. The scrotal sacs were checked on alternate days for two weeks
and then at weekly intervals to ascertain that the animals did not become
cryptorchid after the operation.
For sham operation, the same procedure was applied except that
the vas deferens was neither ligated nor divided.
All operations were carried out under sterile conditions as described
by Heller and Rothchild[7].
After
recording final body weights, animals were sacrificed by dislocation of
the cervical vertebrae, and the testes and epididymes were dissected out
and weighed.
The testes were then fixed for histological studies in freshly
prepared Bouin's fluid, dehydrated in graded ethanol series, cleared in
benzene and embedded in
paraffin wax.
Tissues were sectioned longitudinally at 6 m and serial sections
were obtained, which were stained with periodic acid-Schiff (PAS) and
counterstained with Harris haematoxylin.
3
Results
All
animals maintained a healthy appearance throughout the period of study
and no significant differences were found between the initial and final
body weights of the vasectomized animals and controls.
However, a significant reduction was noted in the weight of the
testis in vasectomized mice as compared with controls.
The epididymal weight, on the other hand, showed a significant
increase in vasectomized mice in comparison to controls (Table 1).
Table
1.
Effect of vasectomy on body weight and weight of the testis and
epididymis in the laboratory mouse. So: Sham-operated; Vx: Vasectomized.
aValues are meanSEM (n=5) and refer to the weight
of the single
organ. Significantly different from controls: bP<0.05;
cP<0.02; dP<0.01; eP<0.001.
|
Group
and duration |
Animals |
Body
weight (g) |
Sex
organs weighta |
||
|
Initial |
Final |
Testis |
Epididymis |
||
|
I
Four months |
So |
33.601.03 |
40.601.08 |
276.646.02 |
132.251.51 |
|
Vs |
34.401.44 |
40.802.35 |
236.6514.54b |
219.7127.48c |
|
|
II
Six months |
So |
34.800.86 |
42.000.32 |
219.706.89 |
103.313.57 |
|
Vx |
33.000.32 |
42.801.32 |
176.4510.37d |
136.084.56e |
|
|
III
Nine months |
So |
34.000.71 |
42.200.66 |
225.262.57 |
106.742.43 |
|
Vx |
33.800.58 |
38.601.08 |
181.7612.27d |
197.3623.45e |
|
|
IV
Twelve months |
So |
34.400.51 |
44.400.51 |
224.791.28 |
101.262.67 |
|
Vx |
34.400.51 |
45.000.84 |
168.069.79e |
177.506.97e |
|
The
testes of controls (Figure 1)
exhibited normal histological features; the seminiferous tubules showed
spermatogenic activity with successive stages of transformation of spermatogonia
into spermatozoa.
By contrast, marked histologic changes were observed in the semini-ferous
tubules in testes of vasectomized mice, except
those killed 4 months after the operation.
However, individual differences were noticed in response of the
testis to the operation, and some animals showed more alterations in the
seminiferous tubules than others.
Four months after vasectomy, almost normal histological features
were observed in the seminiferous tubules in the testis, except that small
separations were sometimes noticed between cells in the seminiferous epithelium.
However, at six months post-vasectomy (Figure
2), marked regressive changes were noticed in the seminiferous tubules;
the seminiferous epithelium was only 2-3 layers thick and showed much
depletion of germ cells and was lined by Sertoli cells, spermatogonia, spermatocytes
and few spermatids; the lumen of the tubules was wide and devoid of spermatozoa.
Nine months after vasectomy, regressive changes in the seminiferous
tubules were further pronounced and the seminiferous epithelium presented
a vacuolated
appearance.
By 12 months of vasectomy, maximal regressive changes were observed
in the seminiferous tubules in the testis (Figures
3-5).
The seminiferous
epithelium was disorganized and showed much depletion of germ cells.
In severe cases, the tubules were lined with only a thin layer
of Sertoli cells, spermatogonia and a few spermatocytes.
The nuclei of the Sertoli cells were sometimes dislocated from
the basal portion towards the luminal portion.
The changes in the tubules included exfoliation of germ cells,
occurrence of giant cells, and presence of vacuoles in the epithelium.
Giant cells containing 2-9 round nuclei of early spermatids were
seen in the lumen of the seminiferous tubules; occasionally, giant cells
containing flattened nuclei of late spermatids were also
seen (Figure 4).
Sometimes, macrophages ingesting spermatozoa were also noticed
in the lumen of the seminiferous tubules in testes of mice vasectomized for
nine and twelve months (Figure 5).
The appearance of the Leydig cells in vasectomized mice at each
interval was considered normal.
Figure
1.
Testis of a sham-operated mouse.
Note the normal appearance of the organ.
160.
Figure 2.
Testis of a mouse six months after vasectomy.
Note marked depletion of
germ cells in the seminiferous tubules; the germinal epithelium is lined
by mainly Sertoli cells, spermatogonia, spermatocytes and few spermatids.
The lumen of the tubules is wide.
160.
Figure 3.
Testis of a mouse twelve months after vasectomy.
Note marked regressive changes in the seminiferous tubules; the
tubules are showing
intraepithelial
vacuoles
and
exfoliation of germ cells. 200.
Figure 4.
Portion of seminiferous tubule from the testis of a mouse twelve
months after vasectomy to show occurrence of giant cells.
Giant cells (arrow head) with flattened nuclei of late spermatids
and those (arrows) with round nuclei of early spermatids are seen in the
lumen of the tubule.
510.
Figure 5.
Testis of a mouse twelve months after vasectomy to show maximal
regressive changes in the seminiferous tubules.
Lumen of a tubule (centre) shows macrophages containing sperm debris
and PAS-positive material.
250.
In
controls, the lumen of the rete testis was narrow and for the
most part empty or
contained
only
a
few spermatozoa and immature germ cells (Figure
6). By contrast, in general, in vasectomized animals the lumen of
the rete testis was greatly dilated and showed accumulation of spermatozoa,
with immature germ cells.
However, in mice vasectomized for 6-12 months, several macrophages
ingesting spermatozoa were often encountered in the lumen of the rete
testis (Figures 7 and
8).
In vasectomized mice, there was always distension of epididymis
and so an increase in the size of the organ than in controls.
However, in 7 (in 3 mice at each interval
of six and nine months and in 1 at twelve months) of the 20 vasectomized
mice, spermatic granuloma was observed either in the corpus or in cauda
regions of the epididymis; histologically, such granuloma consisted of
spermatozoa, PAS-positive material and macrophages containing sperm debris
and PAS-positive material.
Figure
6.
Region of rete testis from a sham-operated mouse.
Note that the rete (R) is lined by cuboidal epithelium and the
lumen is almost empty or contains only a few spermatozoa
and immature germ cells.
Portions of normal seminiferous tubules are also seen.
200.
Figure 7.
Rete testis of a mouse twelve months after vasectomy.
Note marked dilation of the rete and the lumen contains exfoliated
germ cells, sperm debris and macrophages (arrows). 200.
Figure 8.
A portion of Figure 7
enlarged to show macrophages containing
sperm debris.
510.
4 Discussion
In
the present study in P mouse, vasectomy for 4 months did not cause appreciable
changes in the seminiferous tubules, but by 6, 9, and 12 months of the
operation, marked alterations were noticed in the tubules in the testis.
Regressive changes in the seminiferous tubules have also been observed
following vasectomy in several species including rat[1,2],
guinea pig[3,4], rabbit[14],
dog[2] and monkey[5].
In general, the regressive changes observed in
the seminiferous tubules in testes of vasectomized mice in the present
study included occurrence of multinucleated giant cells, exfoliation of
germ cells, appearance of intraepithelial vacuoles, and depletion of germ
cells in the epithelium.
Similar changes have also been noticed in mouse testis after treatment
with several antispermatogenic agents[15]
or after ligation of the efferent duct[16,17].
The occurrence of giant cells in the testis is considered to be
an expression of germ cell degeneration.
The present results showed that giant cells were formed by round nuclei
of early spermatids or sometimes by flattened nuclei of late spermatids. It
has been suggested that giant cells are formed as a result of fusion of
spermatids due to alterations in the intercellular bridges[17].
Furthermore, as in guinea
pig[3], dog[18] and musk shrew[19], individual
differences in response of the testis to vasectomy were also noticed in
P mouse.
Spermatic granuloma as observed in the present study is also reported
in several other species after vasectomy[20].
In
the present study, macrophages
in vasectomized mice ingesting spermatozoa were often observed
in the lumen of the rete testis and sometimes in the lumen of the seminiferous
tubules.
This suggests that macrophages may play a role in the elimination
of degenerated spermatozoa from the testis after the operation.
Ingestion of spermatozoa by macrophages has also been noticed in
the efferent ducts of
monkey[10] and in the epididymis of mouse[21] after
vasectomy; these authors have postulated that macrophages play a role
in the removal of degenerated spermatozoa from the tract after the operation.
Vasectomy
is shown to cause regressive changes in the seminiferous tubules in testes
of several mammalian species[13].
However, it is still not clear as to how the
operation induces such effects, though several explanations have been
put forth.
According to Oslund[22], vasectomy does not cause regressive
changes in the testes of experimental animals viz. rat and guinea pig
and the changes noticed in some cases are mainly due to the side effects
such as post-operative cryptorchidism.
However, in vasectomized mice in the present study, testes were
found in scrotal position as confirmed by regular palpation and also during
autopsy. Thus,
vasectomy-induced changes in the testes of P mouse do not seem to be caused
by cryptorchidism.
Furthermore, Heller and Rothchild[7] reported that in
rat vasectomy performed under
sterile surgical conditions has no adverse effect on the
testis, while the same performed under nonsterile conditions has the opposite
effect.
On the other hand, Plaut[23] has shown that bilateral
vasectomy with ligation
and resection of the deferential blood vessels causes a significant decrease
in the weight of the left testis only; he attributed the observed atrophic
changes in the left testis to circulatory disturbances.
Thus, Heller and Rothchild[7] and Plaut[23]
suggested that vasectomy has no deleterious effect on the testis
and that the testicular alterations seen after vasectomy are caused by
procedural artefacts such as infection and circulatory disturbances.
In the present study, operation was performed under sterile conditions
with adequate care to avoid any injury to the adherent blood vessels.
Therefore, it seems reasonable to assume that the regressive changes
noticed in the seminiferous tubules in testes of vasectomized mice in
the present study are not caused by procedural artefacts, but are the
results of the operation per se.
It has also been suggested that
blockade of the vas deferens induces an increase in the hydrostatic pressure
in the testis and epididymis, and this, in turn, causes alterations in
the testis[24-26].
Spermatic
granulomas have been observed after vasectomy in several species including
P mouse in the present study. It is believed that formation of spermatic
granuloma after vasectomy has a protective effect on the testis as this
causes relief in the pressure build-up in the tract[27,28].
In the
present study, however, regressive changes were noticed in the seminiferous
tubules in testes of all the 15 mice vasectomized for 6 to 12 months,
though spermatic granulomas were seen only in 7 of the 15 vasectomized
mice. Thus, in P mouse formation of spermatic granuloma could not prevent
regressive changes from occurring in the seminiferous tubules in testes
of vasectomized individuals, and this also
suggests that perhaps hydrostatic pressure may not be the causative
factor in inducing testicular alterations in P mouse.
Testicular changes noticed after vasectomy have also been described
as a consequence of immunological response[3,4].
In P mouse, however, it is not clear from the present study as
to whether the alterations induced in the seminiferous tubules after vasectomy
are caused by immunological response, but such a possibility can not be ruled
out altogether.
It is relevant to mention here that phagocytosis of spermatozoa
by macrophages was seen in the rete testis and sometimes in the lumen
of the seminiferous tubules in testes of the vasectomized mice in the
present study, and such a phenomenon is believed to offer an important
mechanism for processing sperm to elicit an immunological response after
vasectomy[29].
However, further studies would be needed to ascertain the involvement
of immunological mechanism(s) in causing regressive changes in the testis
of P mouse after vasectomy.
In
conclusion, we suggest that consequences of vasectomy should be thoroughly
understood, so this method becomes rather more popular as a reversible
method of contraception after successful vasovasostomy.
5
Acknowledgements
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Correspondence
to Dr.
Shio Kumar Singh, Department of Zoology, Banaras Hindu University, Varanasi
221 005, India.
Fax : +91-542-317 074
Received
1999-12-24 Accepted 2000-03-13
