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Research Article
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A Study on the Effect of Thiotepa on Mice SpermatogenesisUsing Light and Electronic Microscope |
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D.M. Nejad,
J.S. Rad,
L. Roshankar,
M. Karimipor,
A.A. Ghanbari,
A. Aazami
and
M.R. Valilou
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ABSTRACT
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The present study was designed to explore the spermatogenic
effect of intraperitoneal administration of thiotepa in mice. It is known
the chemotherapy could affect testicular function and result in infertility.
There are few reports on the side effect of thiotepa on spermatogenesis
when are used as anticancer drug. Therefore, in the present study the
effect of thiotepa on spermatogenesis was evaluated by electron microscopy.
A total of 20 balb/c mice were used in this study. They were divided into
control and thiotepa treated groups. Thiotepa was administrated as 2.5
mg kg-1 for 5 days. The mice were sacrificed after 5 weeks
and testicular specimens were removed, fixed in boueins fixative and 2.5%
Glutaraldehide then prepared for light and electron microscopic study.
Light microscopy showed that the thickness of germinal epithelium was
decreased in thiotepa treated group and many seminiferous tubules have
lost germinal cells. Electron microscopy revealed that in experimental
group several intercellular spaces appeared between spermatogonial and
sertoli cells. The basement membrane became irregular and intercellular
vacuoles were present in sertoli and spermatogonial cells. Nuclear chromatin
was condensed and there were several vacuolated mitochondria. It was indicated
that thiotepa affected testicular germinal epithelium by both cytotoxic
effect and induction of apoptosis.
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INTRODUCTION
Todays, some of the malignancies such as Testicular tumors, acute Leukemia
Lymphomas can be cured by surgery, chemo or radiotherapy. For young male
patients, chemotherapy regimens that are toxic to testicular function
have made infertility an important problem. When the cancer is controlled,
quality of life, which often includes the ability to have a normal child,
becomes a major issue. Therefore, the long term toxicity following therapeutic
intervention and resulting quality of life becomes more important issue
for chemotherapy treatments. Testicular dysfunction is amongst the most
common long-term side effects of chemotherapy in men. Germinal epithelial
damage resulting in oligo or azoospermia has long been recognized as a
consequence of treatment with chemotherapeutic agents.
Spermatogenesis is influenced negatively by many factors such as radiation
therapy and cytotoxic drugs used for treatment of different cancers (Codrington
et al., 2007; Howell and Shalet, 2005; Howell and Shalet, 2001;
Liu et al., 2007; Meistrich et al., 2003). Recently, the
thiotepa is used for treatment of various malignancies including breast,
ovary, bladder cancers and Hodgkin and non-Hodgkin lymphoma (Bdul-Hai
et al., 2007; Biron et al., 2008; Burgues et al., 2007;
Tokuda et al., 2008). The previous studies on the side effect of
chemotherapeutic drugs such as procarbazine, cyclophosphamide and chlorambucil
showed long-term (up to years) azoospermia after treatment (Howell and
Shalet, 2005; Sieniawski et al., 2008). There are a few reports
on the side effect of thiotepa on the spermatogenesis when they are used
as anticancer drugs, in the literature (Khil`kevich and Kurilo, 1992).
Tumors are characterized by division, which is no longer controlled as
it is in normal tissue. Cancer cells no longer have the normal checks
and balances in place that control and limit cell division. The process
of cell division, whether normal or cancerous cell, is through the cell
cycle. The cell cycle goes from the resting phase, through active growing
phases and then to mitosis (division). The ability of chemotherapy to
kill cancer cells depends on its ability to halt cell division. So chemotherapy
is most effective at killing cells that are rapidly dividing. Unfortunately,
chemotherapeutic agents can not differentiate between the cancerous and
the normal cells. It has been shown that one of the mechanisms in cell
destruction following chemotherapy is apoptosis (Andriana et al.,
2004; Bakalska et al., 2004; Habermehl et al., 2006; Hou
et al., 2005; Sawhney et al., 2005). Some agents including
genes factors, Testicular ischemia, heat stress, exposure to irradiation
and toxic substances, could increase the rate of germ cell apoptosis in
testis (Andriana et al., 2004; Bakalska et al., 2004; Habermehl
et al., 2006; Hou et al., 2005; Liu et al., 2007;
Sakallioglu et al., 2007; Sawhney et al., 2005). Thiotepa
is a classical DNA directed alkylating agent currently used in high dose
therapy approaches to the treatment of both hematopoietic and solid tumors
(Bdul-Hai et al., 2007; Biron et al., 2008; Burgues et
al., 2007; Tokuda et al., 2008). Thiotepa induces S-phase cell
cycle arrest and cell death as a result of DNA cross-links or adduct S
formation. Several studies have shown the harmful impact of thiotepa on
the germinal epithelium of testis (Khil`kevich and Kurilo, 1992). These
studies have not provided enough information about ultra-structural changes
in germinal epithelium of testis and spermatogenesis. On other hand, the
results of electron microscope in evaluating morphological pattern of
nucleus, organelles, membrane and cell adhesion is more invaluable in
comparison to light microscope. In the meantime, the knowledge about the
ultra structural changes of germinal stem cells and spermatogenic cells
following the administration of thiotepa can help to understand the mechanisms
of action of thiotepa.
In the current study, the morphological pattern of nucleus, organelles,
membrane and cell junction of spermatogenic and somatic cells of mice
testis after treatment with thiotepa in evaluated.
MATERIALS AND METHODS
Animals: Twenty balb/c mice aging 6-8 weeks was used in this study.
The mice were divided into two groups, control and thiotepa treated. Thioteapa
was injected intraperitoneally for five days as 2.5 mg kg-1.
The dosage was selected as reported by previous studies. After 5 weeks
from beginning of treatment, all animals were sacrificed in both groups.
The testes were removed from the abdominal cavity and separated from the
epididymis with care by using a surgical blade and then testicular specimen
from right testis were fixed in boueins fixative for 48 h and then processed
for light microscopy. The paraffin sections stained with H and E and studied
with light microscope.
The specimens from the left testis were fixed in 2.5% Glutaraldehyde
(Pro. Sci. Tech. Au) for 12 h and washed with 0.2 M phosphate buffer and
post fixed with 2% osmium tetroxide (Taab, UK) for 2 h. After Dehydration,
Clearing and Infiltration they embedded in resin (Pro. Sci. Tech. Au)
and sectioned using ultramicrotome (Richert-Jung, Au). Semithin stained
with toluidine blue and studied with light microscope. The thin sections
(80 nm thickness) were stained with uranyl acetate and lead citrate and
were studied with Leo 906 transmission electron microscope (Leo, Germany).
RESULTS
Light microscopy: Light microscopy revealed that seminiferous
tubules have a thick germinal epithelium and their lumina contained numerous
mature sperms. Different stages of spermatogenesis (12 stages) could be
recognized based on morphological criteria. Interstitial spaces contained
connective tissue and lydig cells (Fig. 1a).
In the treated group, as compared to the control group, the spermatogenic
epitheliums were reduced in thickness and in some cases the tubules had
only sertoli cells and there were no mature sperms in any tubule. Therefore
recognition of different stages of spermatogenesis was impossible. There
were also several vacuoles between germinal cells in this group (Fig.
2a). Spermatogenic Index (SI) in control group was 3.01 ± 0.1
and in treated group it reduced to 0.32 ± 0.01 which is significant
(p = 0.001).
Electron microscopy: Electron microscopy revealed that in control
group, spermatogonia was rested on the basement membrane and had round
or oval nuclei and small mitochondria and less developed endoplasmic reticulum
(Fig. 1b). The sertoli cells could easily be distinguished
regarding their large nuclei with one or two deep indentation and large
nucleolus. Sertoli cells were rested on the basement membrane and the
junction between two adjacent sertoli cells was obvious.
This structure has important role in normal spermatogenesis by forming
Blood Testes Barrier (BTB) and is composed of three elements (Fig.
1b, c).
• |
Merged plasmalemma of adjacent sertoli cells in the center. |
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Dilated endoplasmic reticulum in both sides. |
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Bundles of filaments that are sandwiched between plasmalemma and
endoplasmic reticulum. |
Fig. 1: |
Light and electron micrographs from mouse testes in
control group |
a:
b:
c:
d:
e:
f:
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A cross section of seminiferous tubule showing germinal epithelium
with different cell type, H and E staining
Spermatogonium and sertoli cell on basement membrane. Junction between
two sertoli cells (blood testes barrier) (arrow head)
Blood testes barrier with higher magnification. Mp: merged plasma
membrane, Ac: actin filaments, Er: endoplasmic reticulum
Spermatids at different stages of spermatogenesis
Sperm head. Ectoplasmic specialization (arrow head)
Ectoplasmic specialization with higher magnification. Er: endoplasmic
reticulum, Ac: actin filaments, Mp: merged plasmalemma |
The spermatogenic cells, including round spermatids, could be seen at
various stages of spermatogenesis such as Golgi and cap stages (Fig.
1d).
In control group, spermatids were embedded in the sertoli cells and formed
Ectoplasmic Specialization (ES) which is an indication of final stages
of spermatogenesis. ES composed of three segments.
• |
Merged membrane of spermatid and sertoli cells. |
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Dilated endoplasmic reticulum in the sertoli cell. |
Fig. 2: |
Light and electron micrographs from mouse testes in
thiotepa treated groups |
a:
b:
c:
d:
e:
f: |
A cross section of seminiferous tubule. Note germinal cell depletion
Spermatogonia is separated from basement membrane. The basement membrane
is wavey. A contracted myoid cell is shown in the figure (M)
Spermatogonial cells are separated from sertoli cell and basement
membrane by large vacuoles Spermatogonium contain large intracytoplasmic
vacuoles (V)
Blood-Testes-Barrier is thinner and irregular in comparison to control
group
An apoptotic cell with crescent like chromatin condensation (arrow
head)
Vacuolated mitochondria and a deformed cilium (lower right corner)
in a spermatogonic cell and sperm |
• |
Bundles of actin filaments that are sandwiched between
sertoli plasmalemma and endoplasmic reticulum of sertoli cell (Fig.
1e, f). ES has important role in spermatogenesis especially in
converting of round spermatid to mature spermatozoa. |
Study of testis in the treated group showed that spermatogonia and sertoli
cells were separated from basal lamina and neighboring cells by large
spaces. There were also large vacuoles in the cytoplasm of sertoli and
spermatogonia cells. Basement membrane was morphologically irregular and
wavey. Myoid cells in the boundary tissue were thickened and condensed
(Fig. 2b, c).
BTB in this group showed a dramatic irregularity and appeared thinner
than in the control group (Fig. 2d).
In this group, a lot of apoptotic cells were observed, they were recognized
based on their crescent like chromatin condensation (Fig.
2e). Spermatogenic cells had also several damaged organelles such
as vacuolated mitochondria and abnormal axonem (Fig. 2f).
Due to destructive effect of thiotepa different spermatogenic cells and
stage of spermatogenesis was rarely observed.
DISCUSSION
In the present study, the effect of thiotepa, as an anticancer drug,
on germinal epithelium has been investigated. Present results showed that
the thickness of germinal epithelium decreased in the thiotepa treated
group. This is in agreement with the findings of other researchers that
chemotherapy could affect spermatogenesis (Codrington et al.,
2007; Howell and Shalet, 2005; Howell and Shalet, 2001; Khil`kevich and
Kurilo, 1992; Meistrich et al., 2003; Sieniawski et al.,
2008).
We also showed the presence of several spaces between the spermatogonial
and sertoli cells and between them and basal lamina. In this regard, it
has been shown that sertoli-germ cell adhesion enhances the survival of
germ cells and cadherins mediated adhesion between sertoli and germ cells
in vitro (Newton et al., 1993). Cadherins as an important
molecular system controls interaction between sertoli and germ cells and
promotes the survival of germ cells. It has thought that cadherin based
adhesion generates intracellular signaling cascades that control cell
survival, migration and maturation (Honecker et al., 2004). In
our TEM study both BM and myoid cells were altered structurally in experimental
group. BM in the treated group was irregular and had some swirls. Similar
findings were observed in irradiated rats (Sawada and Esaki, 2003) and
after efferent ligation (Richardson et al., 1998). The swirls may
be due to contraction of myoid cells or reduction of tubular diameter.
In this study the BM was continuous even in the absence of germ cells.
It means that germ cells are probably not involved in BM production. It
has been shown that during tubular injury, for survival and regeneration
of stem cells, the BM is produced (Sawada and Esaki, 2003). Testicular
BM is synthesized by both sertoli and myoid cells (Skinner et al.,
1985). In our TEM study it appeared that the thickness of BM is increased
in thiotepa treated group. Increasing of BM thickness similarly occurs
in: aging (Siu and Cheng, 2004), efferent ligation (Richardson et al.,
1998) and after irradiation (Sawada and Esaki, 2003). This alteration
either is due to increasing of production by sertoli or myoid cells or
reduction of proteolysis rate in ECM. In efferent ligation the gene expression
of laminin is changed in sertoli cells and their protein synthesis is
enhanced. The nucleolus of myoid cell in thiotepa treated groups were
appeared shorter and had a contractile form in comparison with control
group. Myoid cells contain abundant actin filaments which are distributed
in the cells in a species specific manner. The arrangement of actin filaments
in the cells changes during postnatal development and the distribution
of spermatogenesis such as cryptorchidism. Myoid cells also have myosin,
desmin and actinin (Maekawa et al., 1996). In other words they
are kind of smooth muscle cells (Virtanen et al., 1986). Several
substances such as prostoglandins, oxytocin, TGF-β have been suggested
to affect the contraction of these cells (Maekawa et al., 1996).
These finding suggest that probably after treatment with thiotepa, damaging
of epithelium stimulates secretion of some factors such as oxytocin or
prostaglandins and cause myoid cells contraction. Alteration in BTB could
affect normal spermatogenesis and may result in transient or long-term
spermatogenesis defect. Microtubular arrangement in the sperm flagella
(axonem) is important for energy production and sperm motility. Any changes,
in the arrangement of flagellar microtubules may affect motility and result
in subfertility. The changes, such as separation of spermatogonial cells
from sertoli cells and basal lamina could also be considered as preapoptotic
sign. Other finding such as nuclear condensation, chromatin margination
and mitochondrial vacuolization are additional evidence for appearance
of apoptotic cells. The cells with apoptotic feature were numerous in
experimental group than the control group. In support of this finding
there are some reports showing that anticancer drugs could induce apoptosis
in germinal epithelium (Andriana et al., 2004; Bakalska et
al., 2004; Habermehl et al., 2006; Hou et al., 2005;
Sawhney et al., 2005). It is also known that chemotherapy agent,
especially alkylating agents, tend to act on cells that have a high mitotic
activity such as spermatogonial cells (Codrington et al., 2007;
Howell and Shalet, 2001, 2005). On the other hand, apoptosis has a critical
role on the removal of damaged spermatogonial cells to prevent the formation
of abnormal sperms (Cai et al., 1997). It is also shown that spermatocytes
that fail to complete their mitotic division are removed by apoptosis
(Print and Loveland, 2000). It appears that thiotepa as an alkylating
agent induces apoptosis of spermatogenic cells.
CONCLUSION
Present results indicate that administration of thiotepa, as an anticancer
drug in mice, destroys testicular germinal epithelium by both cytotoxic
effect and induction of apoptosis.
ACKNOWLEDGMENT
The authors are thankful to Drug Applied Research Center, Tabriz University
of Medical Sciences, for their financial support of the project.
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