NUCLEAR
ACCUMULATION OF PROTEIN p53 AND HISTOLOGICAL CHANGES IN THE RAT MODEL
OF UNILATERAL CRYPTORCHIDISM
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ATILLA ERTUGRUL, KAMIL CAM, TUFAN TARCAN, ATIF AKDAS, LEVENT TURKERI
Department of Urology, School of Medicine, Marmara University, Istanbul, Turkey
ABSTRACT
Introduction:
Cryptorchidism is accepted as a substantial risk factor for the subsequent
development of testis cancer. Since p53 is highly expressed in testicular
tumors, this study was undertaken to investigate the abnormal expression
of p53 protein and histological changes in a rat model of unilateral cryptorchidism.
Material and Methods: Prepubertal rats were
rendered mechanically rendered unilaterally cryptorchid on 15th day after
the birth. Subsequently, testes were harvested from experimental and sham
operated rats at two, four and six months for immunohistochemical and
histological studies. Contralateral testes in the experimental groups
served as the controls.
Results: There were no histological abnormalities
observed in the testes of both sham operated and control groups. However,
experimentally cryptorchid testes were smaller and accompanied by a prominent
change in color. A significant decrease in testicular volume in the cryptorchid
group was noted (p < 0.05). Seminiferous tubular atrophy, basement
membrane thickening, germ cell loss and spermatogenic arrest were progressively
more evident with time, being highest at the sixth month. Immunohistochemical
staining on paraffin sections demonstrated positive nuclear reaction for
p53 in 14 (93.3%) out of 15 cryptorchid testes. In contrast, sham operated
and control groups were completely devoid of any nuclear accumulation
of p53.
Conclusion: In conclusion, this study confirms
that intraabdominal cryptorchid testes are significantly impaired due
to abnormal localization. Immunohistochemical positivity for the p53 protein
in cryptorchid testes suggests a molecular alteration and may indicate
an association between cryptorchidism and testicular carcinogenesis.
Key words:
testis; cryptorchidism; protein p53; testicular neoplasms; rat
Braz J Urol, 28: 57-63, 2002
INTRODUCTION
Cryptorchidism
is a common congenital abnormality. The incidence of cryptorchidism in
term infants is approximately 3% and it decreases to 0.8% by one year
of age (1). Based on various clinical and experimental studies, cryptorchidism
is identified as a cause of testicular atrophy and subsequent development
of testicular neoplasm (2,3). It is known that 10% of testicular cancer
originate from the ipsilateral cryptorchid testis and the incidence of
carcinoma in situ in cryptorchid testes was reported as 1.7% (2). Despite
a large body of clinical and experimental evidence related to cryptorchidism
and its relation with testicular cancer, the mechanism of damage at molecular
level resulting in microscopic and macroscopic abnormalities has not been
explored in depth.
The
p53 gene is a tumor suppressor gene that exerts a negative regulatory
effect on cell growth (4). Different studies have demonstrated a significant
correlation between the functional inactivation of the p53 tumor suppressor
gene and tumor grade and stage. It is also found to be a predictor of
tumor behavior with a poor prognosis in tumors such as breast cancer,
colon cancer and other gastrointestinal malignancies (5,6). The wild-type
p53 gene product is undetectable by immunohistochemical methods due to
its short half-life and the low amount of p53 present (7). However, mutation
of p53 results in stabilization of the protein with a prolonged half-life
(8,9), and makes it detectable by immunohistochemistry (7). Abnormal p53
expression has been implicated as a valuable tumor marker for some neoplasms
including bladder cancer (10).
This
study was undertaken to define the status of nuclear accumulation of p53
indicating an aberrant protein and associated histological change in an
animal model.
MATERIAL AND METHODS
Animals
Fifteen-days-old
Wistar Albino Rats (20 - 30 gr.) were housed at the Laboratory Animal
Research Center at Marmara University. Experimental design and treatment
of these animals was approved by the Animal Care Committee of our institution.
Animals were caged under controlled lighting (12 hours light, 12 hours
dark) and temperature (24oC). Food and water were provided ad libitum.
The rat testes were divided into three groups: 1)- mechanically produced
left-sided cryptorchid testes (n = 15 testes); 2)- sham-operated testes
(n = 6 testes); and 3)- the right testes of the sham operated and cryptorchid
group were used as control (n = 15 + 6 = 21 testes). Rats were rendered
cryptorchid by obstructing left internal inguinal canal with a 4-0 vicryl
suture. Five rats from cryptorchid group and two rats from sham-operated
group were sacrificed after bilateral orchiectomy, in the second, fourth
and sixth month after first operation, respectively. After measurement
the three dimensions of each testis, the testicular volume was calculated
by using the “Ellipsoid formula”. Testes were then placed in
Bouin’s fixative solution for 6 hours at room temperature, embedded
in paraffin. Sections of 5-micrometer thickness were obtained using a
standard rotary microtome and fixed onto silanized slides for immunohistochemical
and histological studies.
Surgical Procedure
After
induction of anesthesia with an intraperitoneal injection of ketamine
100 mg/kg plus xylazine 1 mg/kg, rats were placed in a supine position
and a vertical midline incision of about 2 cm was made in the abdomen
just above the base of the penis. At this age, rat testes are still intraabdominal.
Through this approach intraabdominal, testes were identified on either
side. The left internal inguinal canal was closed with 4/0 vicryl suture
to prevent left testicular descend.
Sham-operated
rats (n = 6) were subject to a similar vertical midline incision of about
2 cm in the abdomen just above the base of the penis. Testes were identified
on either side. The left-sided testis and left internal inguinal ring
were touched with a cotton swab as in preparation for vicryl suture placement.
Abdomen was closed without further intervention.
The
effectiveness of the surgical procedure was evaluated by confirming the
intra-abdominal location of the experimental cryptorchid testis and by
verifying that the inguinal canal was closed.
p53 Immunohistochemistry
The
antibody used in this study was a mouse monoclonal antibody (pAb 1801)
against p53 mutant type. In preparation for immunohistochemical procedures,
the tissue was deparafined, rehydrated, and placed in a solution of hydrogen
peroxide for 10 min. An antigen retrieval method was used. Sections were
placed in 10 ml of citrate based retrieval solution, mixed with 30 ml
of water, and heated in a microwave for a total of 15 min at room temperature
at 100% power. Tissue slides were cooled for 20 min at room temperature,
rinsed with water and phosphate-buffered saline, and immersed into a blocking
serum (10% Rabbit non-immune serum, Zymed Laboratories Inc., South San
Francisco, CA) for 1 h. at room temperature. The antibody was then incubated
with the tissue sections overnight at 4oC. Tissue sections were incubated
for 30 min at room temperature with Biotinylated goat anti-mouse antibody
(Zymed Laboratories Inc, South San Francisco, CA). Color was developed
by diaminobenzidine tetrahydrochloride solution (Zymed Laboratories Inc,
South San Francisco, CA) and sections were counterstained with hematoxylin.
Immunohistochemical reaction was considered positive only in cases of
nuclear staining. P53 nuclear positivity was expressed in “%”
after counting a total of 400 cells from each slide by using the formula;
p53 (%) = 100 x (p53 positive cell number/400). Two investigators reviewed
all slides independently.
Differences
between the groups were tested for significance by “Dunn`s Multiple
Comparisons Test” and “Mann-Whitney Test”.
RESULTS
p53 Expression
Cryptorchidism
group - p53 expression: abnormal p53 nuclear accumulation was detected
by immunohistochemistry in 14 of 15 cryptorchid testes (93.3%) (Figure-1).
Ratio of nuclear p53 staining was 0.32 ± 0.28%, 0.41 ± 0.3%,
and 0.26 ± 0.06% at second, fourth, and sixth months, respectively
(Table-1).
Control
group - p53 expression: no abnormal nuclear accumulation was detected
in any one of the 21 testes.
Sham-operated
group -p53 expression: immunohistochemistry did not reveal any p53 expression
in sham-operated animals.
The
p53 nuclear staining of cryptorchid testes was considered statistically
significant. There was no statistical significance (p > 0.05) between
the nuclear p53 staining of the 2, 4 and 6-month cryptorchid testes (Table-1).
Effect
of Cryptorchidism on Testicular Volume
No
significant differences in testicular volume were noted between the left-sided
cryptorchid sub-groups of testes at month 2, 4 and 6 of operation (p >
0.05, Table-2). A significant decrease (p < 0.05) in testicular volume
(cm3 ± SD) was seen between the left-sided cryptorchid testes and
controls at each examination group. In fact, there was a significant increase
(p < 0.05) in the volume (cm3 ± SD) of testes of the control
group with time. Table-2 illustrates the comparison of testicular volume
changes between groups.
Effect
of Cryptorchidism on Testicular Histology
The histologic findings of the control and
sham-operated testes (n = 15, n = 12 respectively) were normal. In contrast
to sham-operated and control groups, undescended testis group was histologically
characterized by early maturation arrest, decrease in seminiferous tubule
diameter and thickening of basal membrane. These abnormal histological
changes were first observed at 2 months and were more prominent at 6 months.
The most distinctive histological abnormality seen at 6 months was the
“Sertoli cell only syndrome” in some areas of testicular tissue
(Figure-2).
DISCUSSION
Testis
cancer is one of the commonest cancers in young men with a substantial
increase in incidence in many white populations (11). Contemporarily,
cryptorchidism is the only well-established risk factor in etiology. The
risk of a cryptorchid testis developing cancer has been estimated up to
40 times higher than that of a scrotal testis (12).
The
objective of this study was to observe whether there was any correlation
between undescended testes and abnormal p53 protein expression and the
abnormal testicular histological findings caused by intraabdominal localization
of testes. In this animal model of cryptorchidism, testicular development
was arrested, and spermatogenesis was impaired dramatically as well. Both
macroscopic and microscopic changes in the left testis were readily apparent
at two months and became more obvious in the sixth month. Penson et al.
also recognized similar changes in testicular volume in their animal model
(13). It is interesting that we noted an increase in testicular volume
in contralateral testes of the cryptorchid group, particularly in the
sixth month in comparison to testes of the sham-operated rats. This may
indicate some sort of compensatory changes in opposite testes just like
compensatory renal hypertrophy. Microscopic findings of cryptorchid testes
were typical. There was prominent and progressive decrease in the diameters
of seminiferous tubules of cryptorchid testes when compared to the contralateral
and sham-operated testes. These changes were observed as early as in the
second month, and progressively became prominent in the fourth and sixth
months. The thickness of basal membrane was increased at fourth month,
and it was more evident by six months. In all cryptorchid testes spermatogenetic
arrest was observed. Mature spermatides could be seen in the second month
with a lesser degree of arrest. By the forth month spermatogenetic arrest
was at the stage of spermatocytes, and this was more obvious by the sixth
month. Subsequently, severe spermatogenetic arrest was seen in undescended
testes, which would correspond to “Sertoli cell only syndrome”.
However, there was no change in the number of the Leydig cells, which
constitutes the major part of interstitium. Penson et al also reported
no significant increase in the number of Leydig cells. In literature several
studies propose that the histological abnormalities observed in the cryptorchid
testes are due to a primary genetic alteration (14,15). They postulate
similar histological changes in the contralateral testes. Others propose
an autoimmune etiology as the causative factor in the development of these
histological changes in cryptorchid testes. However, in our study histological
changes were only observed in the cryptorchid testes. Contralateral testes
showed normal maturation, both macroscopically and microscopically. This
observation suggested that abnormal localization of the testes was the
principal factor for these histological alterations. Exposure to higher
temperatures due to abnormal position was accused as the cause of histological
and spermatogenetic defects in several studies (13, 16,17).
In
contrast to histological changes, which were extensively studied, any
alteration at the molecular level in the cryptorchid testes has not been
largely analyzed. Moreover, the association between cryptorchid testes
and testis cancer suggests a possible modification at the genetic level
in the cryptorchid testes, since a significantly higher incidence of testis
cancer has been recognized in cryptorchid testes (2,18-21). Increased
expression of p53 in germ cell tumors has been reported in several studies
(2,22,23). Bartkova et al. found nuclear p53 staining in 84% of the testis
cancers (20). The current study is valuable in terms of anticipating a
possible association between p53 expression and cryptorchidism. According
to our results, p53 expression was seen only in cryptorchid testes. Almost
all of the cryptorchid testes (14 cases out of 15) showed some degree
of positive staining for p53. Nuclear staining was observed between 0.25%
and 5% with the mean ratio of 1.53% of the nuclei examined in cryptorchid
testes. Although this was not statistically significant, p53 expression
was gradually increased with time being highest in the sixth month. This
observation suggests that there is also a molecular alteration beside
histological changes due to abnormal localization of the testes. Nuclear
accumulation of p53 in cryptorchid testes may cause a defect at the cell
cycle regulation in these cells, which in turn may result in initiation
of carcinogenesis. In a similar experimental model, Socher et al. rendered
male adult CD-1 mice unilaterally cryptorchid by a comparable surgical
method. They evaluated p53 expression by Western blot analysis (24). Altered
expression of p53 protein in the cryptorchid testis was seen beginning
on day 7. They also observed that the weights of the cryptorchid testes
decreased by approximately 40%. Histological evidence of germ cell loss
was also noted. They proposed that the p53 as an inducer of apoptotic
cell death has a significant role in temperature-mediated germ cell loss.
However, it would theoretically be also possible that the presence of
p53 expression in the cryptorchid testis might be due to the undescensus
itself, as the associated growth retardation, seminiferous atrophy, etc.
Similarly, there are several theories including endocrine dysfunction,
interference with blood supply, gonadal dysgenesis, etc. on neoplastic
process in cryptorchid testis leading to testis cancer (25). Nevertheless,
the results of the current study indicating p53 expression in cryptorchid
testis open new avenues in the research on cryptorchidism at molecular
level.
CONCLUSION
The results of this experimental study demonstrated both microscopic and macroscopic changes in cryptorchid testes. The documentation of nuclear p53 expression in cryptorchid testes suggests also an alteration at the genetic level. In this regard, observation of p53 expression in cryptorchid testis proposes a possible role of p53 in the development of testis cancer in cryptorchid testes. However, further experimental and clinical studies were required in reaching more definitive statements.
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____________________
Received: July 31, 2001
Accepted after revision: December 4, 2001
______________________
Correspondence address:
Dr. Levent Türkeri
Department of Urology
Marmara University School of Medicine
Tophanelioðlu Caddesi No: 13-15
81190 Altunizade-Istanbul, Turkey
Fax: + + (90) (216) 325-8579
E-mail: turkeri@marun.edu.tr
EDITORIAL COMMENT
Individuals born with an undescended testis have approximately a 40-fold increased incidence of testicular malignancy. The background of the increased risk for cancer is largely unknown. The present manuscript is, therefore, of great value since it suggests a genetic molecular pathogenesis, and quite convincingly so. This paper opens new avenues in the research around cryptorchidism. Subsequent research has to focus on the association between the abnormal expression of the p53 protein in undescended testes and the pathogenesis of cryptorchidism itself.
Dr.
Kelm Hjälmas
Section of Pediatric Urology
Göteborg University
Göteborg, Sweden