Ultrastructural Alterations in Testis and Gastrodermis of Schistosoma mansoni Due to Treatment of Infected Mice with the New Rhodanine Derivative Ro-354
Mohammad I. Soliman
Ultrastructural changes in Schistosoma mansoni
isolated from mice up to 2 weeks after dosing with 50 mg kg1
body weight Ro-354 were investigated. The testicular follicles were completely
disorganized. Signs of degeneration of non-germinal cells, vacuolization
and swelling of spermatogonia, degeneration of spermatocytes and regression
of spermatids were also observed. All stages of spermiogenesis were regressed
and developing spermatozoa showed abnormal morphology. As to gastrodermis,
a dramatic decrease in the number of surface amplifications (microvilli)
was observed. Blebbing, formation of lipid granules, degeneration and
prominent flattening of the gastrodermis followed by a general erosion
of the tissue to the basal lamina.
Studies by Taha and Soliman (2007) have shown that Ro-354 (3-substituted-5-
(2-aryl-2-oxoethyl)-2,4-dioxo-1,3-thiazolidine), administered as a single
oral dose, has a high antischistosomal activity against Schistosoma
mansoni in experimentally infected animals. Diverse biological activities
such as anti-inflammatory, antiproteolytic, antibacterial, antiviral and
antihelminthic have been found to be associated with thiazolidinone derivatives
(Singh et al., 1981).
The use of new antischistosomal drugs is necessary nowadays because many
of the present drugs develop drug resistance (Cioli et al., 1995,
2004; Cioli, 1998; Doenhoff et al., 2000; WHO, 1999a, b; Liang
et al., 2001; Botros et al., 2005; Mattos et al.,
2007). Also reduced cure rates and the failure of treatment after praziquantel
(PZQ) treatment have been reported in patients (Fallon et al.,
1995; Ismail et al., 1996; Lawn et al., 2003; Magnussen,
Drug related testis and gastrodermis alterations of schistosomes and
other helminthes have received little attention (Bang and Hairston, 1946;
Khayyal, 1964; Molokhia and Smith, 1968; Stammers, 1975; Otubanjo, 1981;
Irie et al., 1989; Basch and Clemens, 1989; You et al.,
1992; Mohamed, 1999; Fawzi et al., 2001).
On the other hand, the schistosome digestive tract has been more or less
ignored in such investigatory efforts, although it long has been recognized,
because of its role in the physiology and development of the organism,
as an ideal system for study (Senft, 1969; Erasmus, 1977). There have
been a few reports on drug action relative to this system. For instance,
Yarinsky et al. (1970), Bogitsh (1975) and Clarkson and Erasmus
(1984) have shown that either in vitro or in vivo administration
of the drugs Astiban, Hycanthone, Lucanthone and Niridizole can precipitate
morphological changes in the gastrodermis of schistosomes similar to changes
resulting from starvation (Bogitsh, 1975).
In the present study, the effect of treatment with 3-substituted-5-(2-aryl-2-oxoethyl)-2,4-dioxo-1,3-thiazolidine
(Ro-354) on the ultrastructure of testis and gastrodermis of adult Schistosoma
mansoni worms has been examined using transmission electron microscopy.
MATERIALS AND METHODS
Experimental animals and infection: On March 2007, twenty Schistosoma
mansoni experimentally infected male mice were supplied by the Schistosome
Biological Supply Program (SBSP) at Thoedor Bilharz Research Institute,
Giza, Egypt. Thirty days post-infection, mice were divided into two groups
of 10 animals. The first group was considered as non-treated infected
control. Group 2 was treated intragastrically with 50 mg kg1
body weight of Ro-354 (3-substituted-5- (2-aryl-2-oxoethyl) -2,4- dioxo-
1,3-thiazolidine). Two weeks later, the worms were portally perfused from
untreated and drug treated mice by the perfusion technique described by
Smithers and Terry (1965).
Transmission electron microscopy: The male worms were carefully
cut into small pieces, dropped immediately into 3% phosphate buffered
glutaraldehyde (pH 7.3) for 2 h. Tissues were rinsed in 1% buffered osmium
tetroxide for 1-2 h at 4 °C, dehydrated in ascending grades of ethanol,
embedded in Spurr`s resin. Ultra-thin sections (60-90 nm) were mounted
on copper grids and stained with uranyl acetate and lead citrate. Stained
grids were then examined by JEOL 1200 EX2-transmission electron microscope
(60 kV) at the Central Lab., Faculty of Science, Ain Shams University.
Testicular tissues: Ultrastructural examination of the male reproductive
system of schistosomes, revealed that the testicular follicles of S.
mansoni are formed of germinal and non-germinal cells bounded by a
basal lamina enclosed by a thick coat of circularly arranged muscle fibers.
These cells are randomly arranged in the testicular follicle. The developing
germ cells appeared electron lucent while the non-germ cells were electron
dense. The spermatogonia, which are located peripherally near the basal
lamina, possessed oval or rounded nuclei with one large and spherical
nucleolus and patches of chromatin bodies; these cells were considerably
larger than the other germ cells (Fig. 1).
In treated testis, a significant alterations in testicular structures
was noticed everywhere. The muscle layer that surrounds the follicles,
increased in size and detached in some places. Also change in direction
of muscle fibers and deformation were noticed (Fig. 2).
The non germ cells were the most sensitive testicular component to the
drug. These cells exhibited different damage and some of them had completely
disintegrated. The cytoplasm became dark and the nucleus disappeared completely,
large intercellular spaces were noticed containing debris of degenerating
cells. Membranous bodies were also observed (Fig. 2,
The spermatogonia appeared swelling developing very large vacuoles that
squeezing nucleus to the cell side. Chromatin patches disappeared and
the nucleus became condensed. Lipid granules were observed in the cytoplasm
while mitochondria appeared as dark bodies with indistinct cristae (Fig.
Degenerative changes occurred in spermatocytes. A prolonged swelling
was observed in cytoplasm and nucleus. The swollen nuclei lost characteristic
chromatin patches. Mitochondria exhibited varying morphological changes:
some lost their cristae and appeared distended, whereas others contained
membrane whorls (Fig. 5). Degenerating spermatocytes
sometimes contained membrane whorls which lay directly below the cell
surface and around other cytoplasmic organelles (Fig. 5).
Rosette of early spermatids appeared pale with numerous mitochondria
accumulated at nuclear anterior end. Lipid granules were observed in the
cytoplasm. Nuclei became very pale with very few chromatin patches while
some nuclei degenerated completely (Fig. 6).
| Fig. 1:
Electron micrograph of a section of testis of S. mansoni
collected from control mice showing germinal cells (G), non-germinal
cells (NG), nucleus (N), nucleolus (NU), basal lamina (arrows) and
circular muscle (M). Bar = 2 μm
Degeneration and regression of late spermatids were also noted in testicular
follicles of treated worms. Cytoplasm develops vacuolization (Fig.
7) while other appears normal in the testicular follicle (Fig.
As to spermatozoa, some of them showed abnormal morphology and their
nuclei lost characteristic electron-lucent areas (Fig. 4,
The cytoplasm of parenchyma cells surround the vas deferens swollen.
Moreover, plasma membrane of these cells fragmented before the cell completely
degenerated. Nuclei swollen and became pale in colour lost their chromatin
patches (Fig. 9).
Gastrodermis: The first signs of anomaly appeared as blebbing
of small components from the apical surface of the gastrodermis into the
lumen and a dramatic decrease in the number of surface amplifications
(cytoplasmic extensions) became evident (Fig. 11) as
compared to control (Fig. 10). Moreover, an accumulation
of many small, Golgi-derived vesicles, a diminution in the Golgi apparatus
and the presence of larger, membrane-bound vacuoles, a number of which
contained remnants of rough
||Electron micrograph of a section of testis of S. mansoni
collected from mice treated with 50 mg kg?1 body
weight of Ro-354.
||Degeneration of non-germinal cells (NG), aggregation of lipid granules
(L). Note also disruption of follicular
wall (arrows). Bar = 500 nm
||Showing disorganized testicular tissue. Note swollen spermatogonia
(SG) developing pronounced vacuolization (V), non-germinal cell (NG)
degeneration leaving a large intercellular spaces (*), membranous
materials (arrows). Bar = 1 µm
||Showing abnormal swollen spermatogonia (SG) with large vacuoles
(V) that squeezing nucleus (N) sideward, cytoplasm full of lipid granules
(L) and fragmented endoplasmic reticulum (ER). Note also large intercellular
spaces (*) and regressed spermatozoa (SP). The circular muscles (M)
detached from the basal lamina in some places (arrows). Bar = 500
||Showing degenerated spermatocyte with abnormally enlarged nucleus
(N) and nucleolus (NU). Note whorled bodies (WB) in degenerated cytoplasm.
Mitochondria swollen and lost their cristae (MT). Bar = 1 µm
||Electron micrograph of a section of testis of S. mansoni
collected from mice treated with 50 mg kg?1 body weight of Ro-354.
||Showing rosette of early spermatids (ST1), with pale cytoplasm containing
numerous mitochondria, pale swollen nucleus (N) with nucleolus (NU)
and few chromatin patches. Note degenerated non-germinal cells (NG)
and late spermatids (ST2) some appeared normal and others degenerated.
Bar = 2 µm
||Showing regressed early spermatids (ST1) and late spermatids (ST2).
Inset: normal spermatid Bar = 2 m
||Showing spermatozoa (SP) with abnormal morphology and nucleus lost
characteristic electron-lucent area. Bar = 1 µm
||Degenerated parenchyma cells (P) surround vas deferens (VD). Note
degenerated cytoplasm and swelling nuclei. Bar = 2 µm
Section of gastrodermis of S. mansoni collected from
control mice showing syncytial gastrodermal cells with nucleus (N)
and numerous cytoplasmic extensions (CE). Bar = 2 µm
|Fig. 11 and 12:
||Electron micrographs of sections of gastrodermis of
S . mansoni collected from mice treated with 50 mg kg?1 body weight
||Showing disorganized gastrodermis containing large lipid droplets
(L) and blebbing (b) of small components from the apical surface (arrows).
Note extensively decreased cytoplasmic extensions (CE). Bar = 500nm
||Another region showing degenerated gastrodermis. Lysis of outer
membrane and the released cell content, lipid granules (L), degenerated
endoplasmic reticulum (ER). Note membrane bound vacuoles a number
of which contained remnants of ER (arrows). Bar = 500 nm inset: showing
flattening and erosion of gastrodermis. Bar = 2 µm
endoplasmic reticulum causing them to resemble autophagic vacuoles (Fig.
However, the most dramatic changes were twofold. Initially there was
a very prominent flattening of the gastrodermis, followed by a general
erosion of the tissue to the basal lamina and the gastrodermis was completely
degraded (Fig. 12).
Testicular tissues: Initially, variability and severity of Ro-354-induced
structural changes between different male worms and between different
regions of the same worm were seen. Similar observations have been made
for S. mansoni treated with praziquantel (Shaw and Erasmus, 1983)
and oxamniquine (Popiel and Erasmus, 1984). However, as we saw no evidence
that the extracellular space was formed as a result of cytolysis and cell
deletion, the drug-induced physiological and morphological changes which
gave rise to this bizarre condition is unknown.
Various alterations in testicular structure of S. mansoni have
been recorded in the present study as a result of treatment with Ro-354.
Such alterations included the disintegration of non-germinal cells, cellular
swelling, distortion and disorganization of germ cells, vacuolization
within the testis and regression of spermatocytes.
Non-germinal cells were completely disintegrated. Similar observations
have been reported in the testis of S. mansoni after treatment
with Astiban (Otubanjo, 1981) and Ro15-5458 (Mohamed, 1999). Moreover,
vacuolation of the non-germinal cells was reported in testis of S.
haematobium treated with Ro15-5458 (Fawzi et al., 2001). On
the other hand, non-germinal cells of S. mansoni showed increased
phagocytic activity in Astiban-induced testicular damage (Otubanjo, 1981).
In the present study, the germinal cells show swelling and contained
very large vacuoles. Nucleus squeezed by these vacuoles. Chromatin patches
disappeared and the nucleus became condensed. Cytoplasm developed lipid
granules while mitochondria abnormally increased and appeared as dark
bodies with indistinct cristae. Leitch and Probert (1984) similarly reported
abnormal increase of mitochondria as evident after treatment of S.
haematobium with amoscanate. Mohamed (1999) reported shrinkage of
germinal cells leads to separation and intercellular spaces after Ro15-5458
administration in case of S. mansoni. Similarly, Fawzi et al.
(2001) found that shrinkage and cell separation in S. haematobium.
On the other hand, Jiraungkoorskul et al. (2005) reported swelling
of the testicular tissue followed by degeneration leaving several hollow
spaces in Eurytrema pancreaticum after using PZQ and triclabendazole.
Vacuolation within the testis and regression of spermatocytes observed
in the present study are comparable to those recorded by Irie et al.
(1989) in their work on S. mansoni treated with PZQ and dextro-PZQ
and Mohamed (1999) in S. mansoni treated with Ro15-5458. The anticancer
drug procarbazine was found to be profoundly damaging to the primary and
secondary spermatocytes and spermatids which were replaced by amorphous
granular material (Basch and Clemens, 1989).
Degeneration and atrophy of the testis of S. japonicum was also
demonstrated by You et al. (1992) after artemether administration.
Otubanjo (1981) mentioned that testicular disorganization was prominent
initially in spermatozoa and spermatids but became more generalized with
Basch and Clemens (1989) reported disruption of meiotic process, spermatocytes
and spermatids were destroyed and replaces by amorphous granular material
in S. mansoni by procarbazine. The testicular cells were damaged
also due to the use of enaminone derivative against S. mansoni
(El-Shenawy et al., 2007). The swelling of parenchyma cells found
in the present study is similar to that reported by Xiao et al.
(2002) after treatment of S. mansoni with artemether.
Gastrodermis: Ultrastructural changes in the caecal cytoplasm
following Ro-354 treatment may interfere with digestion and, consequently,
have a determental effect on nutrient assimilation. The subsequent degeneration
of the gastrodermis may be attributed to further action of Ro-354 on the
luminal membrane of the gastrodermis. Howells et al. (1983) attributed
to suramin degenerative changes in the gastrodermis of Brugia pahangi
which are similar to those observed in S. mansoni in present investigation.
The drug may also labializes lysosomal and other membranes, since early
investigations (Williamson and MacAdam, 1965) indicate that lysosomal
membranes are altered by the drug to a point that acid hydrolases may
diffuse out and degrade cytoplasmic organelles, such as ribosomes. The
initial effects of Ro-354 on the gastrodermis of S. mansoni are
reminiscent of the early stages of starvation induced by various drugs
(Yarinsky et al., 1970; Bogitsh, 1975; Clarkson and Erasmus, 1984;
Popiel and Erasmus, 1984; Xiao et al., 2002). The increase in number
of gastrodermal vacuoles has been documented by Bogitsh (1975) and Clarkson
and Erasmus (1984) and is considered to be consistent with changes occurring
in the gastrodermis of S. mansoni when subjected to stress conditions,
such as starvation or drug treatment, either in vitro or in
vivo. Following this initial period, rapid degeneration of the gastrodermis
and, eventually, of the surrounding parenchyma occurs. It is not until
these last stages of degeneration that changes appear in the tegument,
indicating that the drug is most likely affecting the organism from within,
e.g., the cecum, while areas such as the tegument display changes reflecting
general systemic effects (Xiao et al., 2006; Taha, 2007).
Moreover, focal and extensive lysis, decrease in granular endoplasmic
reticulum, vacuolization and degeneration of mitochondria were observed
in gut epithelium of S. haematobium harbored in mice following
amoscanate (Lietch and Probert, 1984), artemether administration (Xiao
et al., 2002, 2006).
Last of all, it would seem that Ro-354 is capable of inducing a range
of ultrastructural abnormalities in both the testis and gastrodermal tissues.
However, the exact mechanism by which it brings about these changes must
await further information from both biochemical and physiological studies.
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