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Effect
of testicular capsulotomy on fertility of rats
Da-Nian
QIN, Mary A. Lung1
Department
of Physiology, Shantou University School of Medicine, Shantou 515031,
China
1Department of Physiology, University of
Hong Kong, Hong Kong, China
Asian J Androl 2001 Mar; 3: 21-25
Keywords:
testis;
testicular capsulotomy; fertility
Abstract
Aim: To demonstrate the effect of capsulotomy on the fertility of male rats. Methods: Testicular capsulotomy was carried out in immature (21 days) and adult (60 days) male rats. The fertility of them was assessed by cohabitation with proestrus females overnight and 20 days later, the females were examined for impregnation. Morphological changes at the site of the capsulotomy were observed under light microscope. Results: In rats capsulotomized at Day 60, the fertility was gradually depressed and all the rats completely lost their fertility 2 months post-operation. At that time, a partial regeneration of the capsule at the site of capsulotomy was observed. Immature rats capsulotomized at Day 21 were found to possess normal fertility at maturity. The capsulotomy site was almost completely recovered 60 days post-operation. Conclusion: In male rats, testicular capsulotomy at the age of Day 60 will damage fertility. However, when capsulotomy is performed at Day 21, fertility is preserve.
1 Introduction
In
the rat testicular albuginea, smooth muscle cell is not identifiable before
the age of 24 days, during which only myocytes are present at various
stages of differentiation. These myocytes reach morphological maturity
at Day 30. Full differentiation of smooth muscle cells in tunica albuginea
is completed just prior to sexual maturity[1]. It is important
to note that the development of smooth muscle cells in the tunica coinside
well with the evolution of spermatogenesis and seminiferous tubular secretion.
In rats sperm first appear in the seminiferous tubules at about 50 days
of age[2,3] and seminiferous fluid secretion begins just after
puberty, with a sharp increase at approximately 30 days and reaching adult
rate at about 40 days[4-6].
It
was shown in our previous study that a progressive degeneration of seminiferous
tubules was induced by testicular capsulotomy in adult male rats with
a progressive reduction in the testosterone concentration in the testicular
venous blood and a gradual increase in the
LH and FSH levels in the peripheral blood from approximately 30 days post-capsulotomy[7,8].
2 Materials and methods
2.1
Animals and testicular capsulotomy
Immature
(21 days of age) and mature (60 days) male Sprague-Dawley rats were obtained
from the Laboratory Animal Unit, University of Hong Kong and each divided
into sham-operated control and capsulotomized groups at random. Both groups
were further divided into 6 subgroups with 8 rats each. Surgical intervention
was performed in the
Minimal Disease Operation Theatre of the University. The animals were
anaesthetized intraperitoneally with sodium pentobarbitone (Sigma, USA)
at a priming dose of 60 mg/kg and a maintenance dose of 10 mg/kg.h. Testicular
capsulotomy was carried out as previously described[1]. Briefly,
with the aid of a
dissecting microscope (Wild M60, Switzerland), the two outer layers of
the capsule, i.e., tunica vaginalis and tunica albuginea, were carefully
incised starting half-way down the rostral half of the testis along the
two lateral borders to the middle of the caudal half of the testis.
2.2
Postoperative care
After
the operation, rats were immediately kept in a section of the animal quarters
reserved for postoperative recovery care. The postoperative cage was large
enough to accommodate them in a completely outstretched position. They
were kept warm by
a lamp placed well out of their reach.
Rats were frequently examined during the first 3-5 days post-operation,
to see whether there was any abnormality in their activity, and food and
water intake.
The
positions of the capsulotomized testes and spermatic cords were checked
regularly to see whether the testes were located at the normal position
and the spermatid
cords not distorted.
2.3
Fertility and sexual activity observation
For
rats sham-operated or capsulotomized at Day 60, fertility tests were perfomred
at 10-day intervals for a period of 60 days after the operation. For rats
sham-operated or capsulotomized at Day 21, fertility test was performed
at 10-day intervals from Day 40 to Day 90 post-operation.
Female
Sprague-Dawley rats, weighing 280-320 g, with a regular 4-day estrous
cycle as documented by vaginal smear examination were used for the fertility
test. Each male were cohabited with 2 proestroous females overnight and
successful mating was confirmed by the presence of copulatory plugs. Twenty
days after mating, the
females were killed and the numbers of pregnant females recorded.
The fertility of males was assessed either by the percentage of
males able to impregnate at
least one female or by the percentage of pregnant females.
The
sexual activity was assessed by placing each male alone in a Plaxiglas
arena for 10-min adaptation
period, after which a receptive female (ovariectomized and primed with
subcutaneous injection of 10 g estradiol benzoate 3 days prior to testing
and 0.5mg progesterone in the morning of the test) was introduced, and
observed for 20 min. The number of mounts and the number of mounts with
intromissions were recorded.
2.4
Histological examination
Rats
were killed and testes were incised, fixed in Helly's fluid[9]
overnight, cut into two parts and placed again in the Helly's solution
for another 32 hours. Paraffin section were cut in ribbon at a thickness
of 4 m and stained with
Weigert's stain. Morphological changes at the site of the capsulotomy
were observed under light microscope.
2.5
Statistical analysis
3 Results
3.1
General condition
During
the observaton period, The testes were located at the normal position
and there was no torsion of spermatid cords. Hence, the surgical intervention
did not disturb the testes within the scrota. Moreover, no abnormality
in eating and drinking behavior was observed in the postoperation period,
indicating that testicular capsulotomy did not induce apparent ill effect
on the general health of the animals.
3.2
Sexual activity
None
of the sexual activity parameters was significantly different between
the sham-operated controls and the capsulotomized rats.
3.3
Fertility-adult males
The fertility of the males, sham-operated or capsulotomized at Day 60, is summarized in Figure 1. The fertility of the sham-operated animals remained relatively steady throughout the post-operative period of 60 days. On the other hand, The capsulotomized males gradually lost their fertility with the elapse of time. During the first 20 days after capsulotomy, no deleterious effect was observed on their fertility. However, it was significantly inhibited on day 30: only 6/8 males were fertile, and the female pregnant rate was 62% (P<0.05, compared with the controls). The numbers of both fertile males and pregnant females were further reduced at day 40 to 3/8 and 31%, respectively. By day 50 post-operation, 2/8 males were fertile and the pregnant rate of females was 18%. At 60 days post-operation, males completely lost their fertility and none of the females was pregnant.
Figure
1. Effect of sham-operation and testicular capsulotomy at Day 60 on fertility
of male rats. (A) SM (%), male rats successfully impregnate at least one
female (n=8). (B) SF (%), pregnant females (n=16). bP<0.05,
compared with corresponding sham-operated control, 2 test.
3.4
Fertility-immature males
The
fertility of male rats sham-operated or capsulotomized at Day 21 is
summarized in Figure 2. The fertility
of the sham-operated controls gradually increased to a steady level by
day 40 post-operation and this remained unchanged up to the
end of the observation period of 90 days. Similar changes were observed
in the capsulotomized group, i.e., there was no significant decline in
the percentages of either fertile male or pregnant females throughout
the observation period of
90 days post-operation. The males preserve their normal fertility when
matured.
Figure
2. Effect of
sham-operation and testicular capsulotomy at Day 21 on fertility of male
rats. (A) SM (%), male rats successfully impregnated at least one female
(n=8). (B) SF (%), pregnant females (n=16). 2 test.
3.5
Changes in capsule at site of capsulotomy
In
rats capsulotomized at Day 21, a partial regeneration of the capsule at the
site of capsulotomy was observed 10 days post-operation (Figure
3A) and almost complete recovery by the 60th day (Figure
3B).
For rats capsulotomized at Day 60, an insignificant regeneration of the capsule was observed by 10 days post-operation (Figure 4A) and only partial regeneration by 60 days post-operation. (Figure 4B).
Figure
3. Light micrographs of testis at site of capsuloltomy performed at Day
21. (A) At 10 days post-operation. Seminiferous tubules were well enclosed
by tunica vasculosa (big arrow) with a partially regenerated connective
tissue layer (small arrow). (B) At 60 days post-operation. Seminiferous
tubules were well enclosed by tunica vasculosa (big arrow) with an almost
completely regenerated connective tissue layer (small arrow). Weigert's
stain 120.
Figure 4. Light micrographs of testis at site of capsuloltomy performed at Day 60. (A) At 10 days post-operation. Seminiferous tubules were well enclosed by tunica vasculosa (big arrow). (B) At 60 days post-operation. Seminiferous tubules were well enclosed by tunica vasculosa (big arrow) with a partially regenerated connective tissue layer (small arrow). Weigert's stain 120.
4 Discussion
The
sexual activity of capsulotomized rats was not statistically different
from that of sham-operated controls, suggesting that capsulotomy did not
influence the sexual activity of male rats.
In capsulotomized adult male rats, there was a progressive loss of fertility
from
30 days post-operation. This might be related to a disturbance in
either
spermatogenesis or sperm transport. The duration of spermatogenesis in
the rat is about
50 days[10]. The transport of sperm along the epididymis and
vas deferens takes about 10 days[11]. Consequently, the time
for a type A spermatogonium to
yield sperm in the ejaculate is about 60 days.
With this in mind, if the effect of an experimental treatment is
evident in the first two weeks, it may be the result of an epididymal
action. If the effect becomes evident two weeks after treatment, it is
most likely due to a testicular effect. We indicated that in adult rats,
testicular capsulotomy had no significant influence on male fertility
in the first 20 days post-operation, but the fertility was progressively
depressed from 30
days onwards. Therefore, testicular capsulotomy impairs male fertility
probably by affecting the testicular function rather than epididymal spermatozoa.
In
Day 21 male rats, spermaogenesis and seminiferous tubular function have
not been well developed.
It is unlikely that testicular capsulotomy at this stage will disturb
testicular function that will be fully developed at least 30 days later.
Preservation of fertility of the immature rats seen in the present study
is in accordance with the conservation of normal seminiferous tubular
morphology reported by us previously[7,8].
If
the capsule is incised at Day 21, it is capable of restoration as it is
not yet fully developed and still at the stage of development and differentiation.
The complete restoration of the structural integrity of the capsule may
explain the fact that in rats capsulotomized at Day 21, the testicular
function and fertility will be normal when they are grown up. Although
certain degree of capsular regeneration occurred in rats capsulotomized
at Day 60, full functional integrity of the capsule could not be restored,
thus leading to seminiferous tubular degeneration and infertility.
Acknowledgements
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Correspondence
to: Dr
Da-Nian QIN, Shantou University School of Medicine, Shantou 515031, China.
Tel: +86-754-856 6776
Fax: +86-754-855 7562
e-mail: lqchen@mailserv.stu.edu.cn
Received
2000-12-22 Accepted 2001-02-20
