Chlorella Vulgaris alleviates Leadinduced testicular toxicity better than

Zingiber Officinale: An Ultrastructural

study
XXIV International Symposium on Morphological Sciences
Sep. 3, 2015

Introduction
References

Conclusion

Material and
Methods

Discussion

Results

Introduction

Introduction
The toxicity of lead is related to oxidative stress
through the creation of reactive oxygen species
(ROS).
The deterioration of reproductive ability in males
is the main manifestation of occupational
exposure to lead.

Introduction cont
Chlorella vulgaris is a green unicellular algae contains
metalion binding site affinity in the chloroplasts within
the cell wall that have sulfhydryl which chelates metals.
Zingiber officinale contains phytochemicals such as
shogaols and gingerols which recognized to have
antioxidant properties.

Aim of the current work

Aim of the current work

is to compare the chelating ability of Chlorella
vulgaris and the antioxidant ability of Zingiber
officinale in the alleviation of adverse
consequences of lead exposure.

Material and Methods

Material and Methods

Ethical Approval. by the Medical Research Ethics Committee of
Faculty of Medicine, King Abdulaziz University (Reference No
1167-13).
Sixty adult male Wistar rats were distributed into four groups.
Group 1 was considered control, group 2 received 200 mg/l
PbAc, group 3 received 50 mg/kg/rat of C. vulgaris extract and
200 mg/l PbAc, and group 4 received 100 mg/kg/rat of Z.
officinale and 200 mg/l PbAc for 90 days.

Material and Methods cont

Chlorella vulgaris Extract (CVE). CV Beijerinck (strain 072), a
strain of unicellular green algae was obtained from Medical
supplier (Kuala Lumpur, Malaysia).
Z. officinale root was obtained from the local market, cut into
tiny parts, and dried for 6 days. The dried root was ground in a
grinder and powder was obtained. Fifty grams of powder were
extracted in 250 ml of ethanol for 18 h in a Soxhlet extractor,
and then the extract was dried at low pressure and stored at
4C.

Material and Methods cont

Testicular Histology. with hematoxylin and eosin
(H&E) and examined using Olympus BX53
microscope equipped with a DP73 camera.

Material and Methods cont

Morphometry. At least 30 tubular profiles of each
animal. The mean seminiferous tubule diameter
(MSTD) was obtained by measuring across the
minor and major axes. The seminiferous
epithelium height was measured for the same
tubules. The measurements were taken at
different magnifications using Image-Pro Plus v6.0
(Media Cybernetics, Maryland, USA) and NIH
ImageJ (v1.50) (http://rsb.info.nih.gov/ij/).

Material and Methods cont

The Ultrastructural Picture. with a Philips 201 transmission
electron microscope at 6080 kV in the Transmission Electron
Microscope Unit (Philips Industries, Eindhoven, Netherlands).
Statistical Analysis. All data are presented as the mean
standard deviation (SD) of different parameters between
treated groups. Data evaluation was carried out using one-way
analysis of variance (ANOVA) followed by Tukeys post hoc test
using SPSS software v22.

Results
Histological Picture

Fig. (1A): Control. Fig. (1B): group 2 (Lead) showed spermatogenic cells with shrunken pyknotic nuclei
(arrow), hyaline material (H), and numerous vacuoles (V).
Fig. (1C): group 3 showed rebuilding of the spermatogenic layers.
Fig. (1D): group 4 showed restoration of the spermatogenic series
(H&E. Scale bar, 20 m).

Results cont
Morphometric Evaluation

Mean Seminiferous Tubular Diameter

m (Morphometry)

Germinal Epithelial Height m

(Morphometry)

Results cont
Semithin Picture

Fig. (2A): Control. Fig. (2B): group 2 shows depletion and separation of the spermatogenic cells with vacuoles
(V) and empty spaces between and inside the spermatogenic cells.
Fig. (2C): group 3 shows healthy spermatogenic series.
Fig. (2D): group 4 shows restoration of the spermatogenic cells.
(Toluidine Blue. Scale bar, 5 m).

Fig. (3A): Control. Fig. (3B): group 2 shows wide interstitial spaces with many interstitial cells of Leydig
(L), a lot of vacuolated cytoplasm (V). Note the large congested blood vessel (C).
Fig. (3C): group 3 showed almost healthy architecture.
Fig. (3D): group 4 shows restoration of interstitial cells of Leydig (L) with minimal vacuoles.
(Toluidine Blue. Scale bar, 5 m.)

Results cont
Ultrastructure Picture

Fig. (4A): group 2 shows a Sertoli cell (St),

basal lamina (BL), primary spermatocytes
(Sc), mitochondria (M), vacuoles (V), zone of
tight junctions (arrowhead) and smooth
endoplasmic reticulum (star). Fig. (4B):
Rounded spermatids (Rsd) with rounded or
oval nuclei that have disturbed chromatin;
peripherally arranged mitochondria with lost
cristae (M) and the characteristic acrosomal
cap (Ac) can be observed. Note the myelinlike figure (F) and numerous empty spaces
(V) all around. Fig. (4C): Cross-sections of
mid-pieces (MP) of the sperm, with
distortion of the central axoneme (Ax),
mitochondrial sheath (M), and fibrous sheath
(FS). Fig. (4D): Leydig cells near the basal
lamina (BL) with irregular euchromatic
nucleus (N) and peripheral heterochromatin.
Electron-dense bodies of variable sizes (D),
mitochondria (M), dilated sER (star), and
lipid droplets (L) are noticed.
Fig. 4A, 4B, and 4D, the scale bar is 2 m; for Fig. 4C, it is 500
nm.

Fig. (5A). group 3 that showed a Sertoli cell

(St) with a euchromatic-indented nucleus
and apparent nucleolus (N) and the
spermatogonia (Sg) resting on the thickened
basal lamina (BL) with an apparent myoid
cell (My) and collagen (C). Some
mitochondria are affected, while others are
healthy (M). Fig. (B): Rounded spermatids
(Rsd) with peripherally arranged unhealthy
mitochondria (M) and the characteristic
acrosomal cap (Ac) enwrapped by Golgi
saccules (G). Note that there are few empty
spaces (V) in the cytoplasm. Fig. (C): Crosssections in the mid-pieces (MP) of the
sperms that have a central axoneme (Ax)
surrounded by a mitochondrial sheath (M), a
fibrous sheath (FS), and a peripheral cell
membrane (P). Fig. (D): Leydig cells with
irregular euchromatic nucleus (N) and
peripheral heterochromatin. Electron-dense
bodies of variable sizes (D), mitochondria
(M), and lipid droplets (L) are present in the
cytoplasm. Fig. 5A, 5B, and 5D, scale bar is 2 m; for Fig.
5C is 500 nm.

Fig. (6A): An electron micrograph of a testis

from the group 4 shows a Sertoli cell (St)
with a euchromatic-indented nucleus
(arrow) resting on the thickened basal
lamina (BL) with an apparent myoid cell (My)
and collagen (C). Note the intact zone of
tight junctions (Z) and numerous unhealthy
mitochondria with lost cristae (M). Fig. (6B):
Rounded spermatids (Rsd) with the
characteristic acrosomal cap (Ac) and
peripherally arranged unhealthy
mitochondria (M) can be observed. Note
some empty spaces (V) all around. Fig. (6C):
Cross-sections of mid-pieces (MP) of the
sperms with minimal distortion of the
mitochondrial sheaths (M) and fibrous sheath
(FS). Note that the end-pieces (EP) have
central axoneme (Ax) surrounded by outer
cell membranes (P). Fig. (6D): Leydig cells
with irregular euchromatic nuclei (N) and
external heterochromatin. Variable-sized
electron-dense bodies (D), mitochondria (M),
and lymph (Ly) are observed in the
cytoplasm. Fig. 6A, 6B, and 6D, the scale bar is 2 m; for
Fig. 6C, it is 500 nm.

Discussion
C. vulgaris provides remarkable defensive mechanisms against
PbAc-induced injury; this consists of the elimination of heavy
metal through the digestive function, either affecting
absorption or excretion. When dietary C. vulgaris is given to
animals with heavy metal, this stimulates toxicant excretion in
the urine and feces.

Discussion cont
Previous research noted that the administration of C. vulgaris
extract (CVE) reduced the blood level of lead in PbAc-treated
groups, which is attributable to the chelating influence of the
sulfhydryl-containing elements in CVE.
As an additional factor, the mineral elements of CVE includes
iron, zinc, calcium, phosphorus, and magnesium may prevent
lead absorption from the alimentary tract, probably through
competition for shared absorptive receptors in the intestinal
mucosa.

Discussion cont
The defensive action of Z. officinale is due to its content of volatile oils,
which revealed immunomodulatory and anti-inflammatory outcomes that
can guard against testicular damage caused by the inflammatory process.
These volatile oils are able to suppress T-lymphocyte-dependent immune
reactions.
Furthermore, the anti-inflammatory action of Z. officinale is due to its
components as gingerol analogs (shogaols and paradols). Previous studies
have noted that these ingredients capable to inhibit prostaglandin and
leukotriene synthesis immediately.

Discussion cont
Results of presented experiments clarify the chelating effects
and the antioxidant effects of Chlorella vulgaris extract on the
ultrastructural changes of lead induced toxicity in rat testes.
The chelating effect of CVE gave better results as regards
diameter of seminiferous tubules and germinal epithelium
thickness, and the sperm structure.
While the antioxidant effects of Zingibar officinale extract was
less pronounced.

Conclusion
Chlorella vulgaris and Z. officinale can be
recommended as an adjunct therapeutic strategy
to combat lead-induced testicular damage.
C.vulgaris showed superior potentials through its
antioxidant ability and chelating properties of
heavy metals.

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