Introduction
The incidence of infertility has increased in recent
years. Among all infertile couples, the proportion of infertility caused by
male factors accounts for about 50% and affects one man in 20 in the general
population (Agarwal et al. 2020). One
of the proposed mechanisms for idiopathic infertility is oxidative stress (OS)
and reactive oxygen species (ROS). Evidence now suggests ROS-mediated damage to
sperm is a significant contributing pathology in 30–80% of cases (Agarwal et al. 2006; Tremellen 2008).
Alzheimer's disease (Vergallo
et al. 2018), cancer (Ma-On et al. 2017), heart failure (Li et al. 2016) and obesity (Dursun et al. 2016) are just a few of the
disorders that have been connected to OS's ubiquitous effect in humans.
Moreover, roughly 35% of infertile men have OS, making it a common biomarker in
their semen. Male reproductive diseases such as varicocele, inflammation and prostate
cancer have all been linked to increased levels of seminal ROS (Lanzafame et al. 2009; Kurfurstova et al. 2016; Nowicka-Bauer and Nixon
2020).
Thioacetamide (TAA)
In place of hydrogen sulfide, thioacetimidic acid/acetothioamide (CH3CSNH2) is
a sulfur-containing chemical frequently utilized in healthcare and
manufacturing (Al-Attar 2011). TAA is often used to induce liver injury
(hepatic necrosis/apoptosis) (Wang et al.
2019), hepatic fibrosis (Makled et al.
2019) and cirrhosis (Keshk et al.
2019). It also affects the cardiovascular, urinary, and nervous systems (Amirtharaj
et al. 2017; Khiat et al. 2019). Although TAA is often used
to induce liver damage, it causes damage to the testis (Kang et al. 2006; Celik et al. 2016; Karabulut et al.
2020). Antioxidant enzyme activities have been reported to be decreased in the
testes of thioacetamide-induced cirrhotic rats (Abul et al. 2002). Therefore, antioxidants may inhibit the production of
ROS, allowing for the healing of damaged cells (Lin et al. 2015).
The treatment of
infertility has evolved through time to incorporate a wide range of methods,
from the use of synthetic medications to that of natural goods and supplements (Oyewopo
et al. 2021). Numerous plant species
contain the flavonoid and antioxidant luteolin (3′, 4′, 5,
7-tetrahydroxyflavone, LUT). Broccoli, pepper, thyme, and celery are just a few
of the many fruits and vegetables that naturally contain glycosylated LUT (Al‐Megrin et al. 2020). Moreover,
LUT may powerfully scavenge free radicals, which helps reduce oxidative damage
to the biosystem (Ijaz et al. 2022).
Epidemiological studies have shown that the high consumption of LUT-containing
foods can reduce the risks of chronic diseases (Kerimoğlu et al. 2016). Antioxidant and
anti-inflammatory effects (Wang et al.
2020), anticancer (Xu et al. 2019)
and neuroprotective (Theoharides et al.
2016), as well as its ability to inhibit the production of free radicals, are
the basis for LUT's pharmacological. In addition, new evidence suggests that
Luteolin inhibits systemic and neuroinflammatory reactions in 2019 Coronavirus
disease (COVID-19) (Kempuraj et al.
2021).
Therefore, in the
current study, we established an animal model of
testicular toxicity induced by Thioacetamide in male rats. Thioacetamide-induced
spermatotoxicity and testicular histopathology were also evaluated, with
Luteolin supplementation at high doses. This study was designed to provide a
reference for further revealing the hazrad of OS and decreasing the damage with
Luteolin supplementation via histopathological,
morphometrical and biochemical evalaution.
Materials and Methods
Drugs and chemicals
Thioacetamide was used as an
inducer of testis damage nad Luteolin (LUT) as
an antioxidant substance in the experiment. They were obtained from Sigma Chemical Co., MO, USA.
Experimental animals
Thirty adult
male Wistar albino rats weighing 180 – 250 g were obtained from King Fahd
animal house in Jeddah, Saudi Arabia. The animals were housed in plastic cages
(10 animals for
each) at a temperature of 25°C and 50–70% humidity, with 12 h light/ dark
cycles and were fed a freely standard rodent diet and water.
Ethical approval statement
All the animals received human care
according to the standard guidelines. Ethical
approval for the study was obtained from the Research Ethics Committee,
which is in force at the University of Jeddah. The rats were treated in accordance with the
Laboratory Animal Treatment Agreement of the Kingdom of Saudi Arabia and the ethical
regulations were followed in accordance with the national and institutional
guidelines with the protocol published by the National Institutes of Health.
Experimental design
The rats used
in the experiment were randomly divided into three groups (ten animals each)
and placed in cages as follow: Negative control group (G1)
rats were given 1 mL/kg body weight (BW) normal saline solution (0.9% NaCl)
i.p. three times a week at 24 h intervals for four weeks, in addition to 3
mL/kg body BW of normal saline solution orally once a day. Positive
control group (G2) rats were injected i.p. with TAA at the dose of (200
mg/kg BW) 3 times weekly at 24 h intervals for four weeks to induce testicular toxicity (Celik et al. 2016). TAA +
LUT group (G3) rats received Luteolin at a daily dose of (50 mg/kg BW orally) for four weeks (Kalbolandi et al. 2019) concurrently
with i.p. injection of TAA in the same dose as G2.
Testicular weight measurements
At the end of experiment, the testes
was weighed to collect data for statistical analysis.
Sample collection
Twenty-four hours after the last
treatment, all animals were were ethically anesthetized by i.p. injection of 40 mg/kg thiopental ether. Testis was excised immediately. Dissected right testes were processed for microscopic
examination and stained with different histological and immuno-histochemical
stains for mapping Sertoli Cells with an anti-vimentin antibody. In
addition, the dissected left testis was prepared for tissue homogenate for biochemical assays. Also, blood
samples were collected for sexual hormones assays.
Biochemical studies
Determination of sexual hormones
Blood was collected from the retro-orbital veins in which 3 cm of blood into a plain tube was taken and
centrifuged at 3000 rpm at 4°C for 5 min to get the serum. The separated sera were used for the
estimation of serum levels of testosterone hormone (TH), luteinizing hormone (LH) and
follicle-stimulating hormone (FSH). TH serum levels were identified by
by enzyme-linked immunosorbent assay (ELISA) according to the instructions of
the manufacturer (The BioVendor Mouse /Rat Testosterone). LH and FSH levels in the serum
were calculated using ELISA kits per the manufacturer's instructions (Cat.: MBS764675 and MBS2021901, MyBioSource, San Diego,
California, United States)
ELISA kits for LH
and FSH, respectively.
Determination of the testicular oxidative stress status and antioxidant
enzymes
The left testis was homogenized from each rat in
ice-cold (10% w/v) phosphate-buffered saline (50 mM K2HPO4, pH 7.4).
The supernatant was separated by centrifugation at 1000 g for 20 min at 4oC.
Antioxidant enzymes such reduced glutathione (GSH), superoxide dismutase (SOD)
and catalase (CAT) (Beutler 1963; McCord and
Fridovich 1969; Aebi 1984), as well as malondialdehyde (MDA) were
measured in the supernatant to determine the level of oxidative stress present (Ohkawa et al. 1979). Analyses were made
according to the manufacturer's instructions (Cat.:
MBS738685, MBS724319, MBS036924 and MBS701908, MyBioSource, San Diego,
California, United States) ELISA kits for MDA, GSH, SOD and
CAT,
respectively. Colorimetric analysis was performed on a homogenate of
testes to determine their concentration. Spectrophotometry was used to analyze
the concentrations of MDA, GSH, SOD and CAT, which were determined with the
help of standard curves. MDA activity was expressed as nmol\mL, GSH as ng\mL,
SOD as U/mL and GAT as Mu\L.
Histological and
immunohistochemical studies
Histological studies for light
microscopy: A 4% buffered formalin solution was used to preserve the right testes for
14 days after they were removed. Tissues were fixed, then dehydrated in a
series of graded alcohols, and finally cleaned with xylene. Paraffin was then
used to impregnate the samples. Histopathological and immunohistochemical
analysis was performed on transverse sections cut with a rotary microtome at a
thickness of six micrometers. Hematoxylin and eosin (H & E) stain was used
for routine histological examination, whereas Masson trichrome stain was used
to identify collagen fibers in histopathological sections (Suvarna et al. 2018).
Immunohistochemical study: Deparaffinized,
positively charged slide sections (6 µm) embedded in paraffin were soaked in
PBS and pretreated with 3% hydrogen peroxide in distilled water to inactivate
endogenous peroxidase activity. Antibodies were diluted with 10% normal horse
serum (NHS) in phosphate-buffered saline (PBS) (1: 200) before being applied to
the sections and incubated overnight with a polyclonal Rabbit anti-goat
antibody to Vimentin from Santa Cruz Biotechnology in Dallas, Texas, USA.
Finally, the sections were incubated with biotinylated immunoglobulin (1:300,
purchased from Boster Biotechnology Co., Ltd., CA, USA) diluted in NHS at 2%
for 30 min at room temperature after being washed three times in PBS. After
three washes in PBS, the sections were incubated at room temperature for 30 min
with peroxidase-conjugated streptavidin diluted in PBS. Finally, the sections
were counterstained with Mayers hematoxylin, dried in a sequence of ethanol and
xylene, mounted and finally dyed using a diaminobenzidine (DAB) kit (purchased
from Boster Bio-Technology Co., Ltd., CA, USA). The positively stained area was
brown. A specific primary antibody was switched out for PBS saline as a negative
control.
Morphometric analysis
To conduct the morphometric analysis, a
plugin for the image analyzer Olympus Image J, NIH, 1.41b, America Inc.,
Melville, NY, USA was used on tissue samples from all rats in each group (n = 10).
In order to get accurate results, we measured five separate slides from each
rat in each group. On average, we looked at five distinct fields every slide. Briefly, the average diameter of the testicular seminiferous
tubules (H & E X20) was calculated and those tubules were selected which
were rounded on the transverse cut. Then, their diameters in two perpendicular
directions were measured to determine the average diameter of the seminiferous
tubules (Narayana et al. 2006). In
the case of oblique sections, only the minor axis was considered during the
measurement process (Stumpp et al.
2004). The average thickness of the germinal epithelium was also measured
(using H&E X20). The percentage of collagen fibers in Masson
trichrome-stained sections at a magnification of X20 and the percentage of
vimentin-positive cells were then calculated as the means (X20).
Statistical Analysis
Data were analyzed using SPSS for Windows (version 26;
IBM Corp., Armica, NY, USA). Statistics were summarized by standard deviation
of means. One-way analysis of variance followed by a post hoc LSD test was used
to assess differences among the groups. P = 0.05 was considered
significant. Information was presented graphically using GraphPad® Prism 9
(2022), Statistical Package.
Results
Testicular weight measurements
Concerning right and left testis
weight, there were highly significant (P < 0.0001 and P < 0.0001; respectively) increases in
negative control G1 as compared to positive control treated G2. Also,
significant (P < 0.01, P < 0.0001
and P < 0.001;
respectively) increase in body weights (249.0 ± 25.07) , right
(1.50 ± 0.17) and left (1.50 ± 0.17) testis weights were detected in G3 (TAA + LUT) as compared to G2 (TAA). Interestingly,
there was a non-significant (P = 0.39, P = 0.21, P = 0.17;
respectively) difference in G3 (TAA + LUT) regarding rat body weights, right
testis weight and left testis weight as
compared to a negative control group (G1) (Table 1 and Fig. 1).
Evaluation of sexual hormones
Table 2 and Fig. 2 show different groups' mean ± SD of TH, LH, and FSH
levels. Group II (positive control group) showed a highly significant decrease (P < 0.0001, P <
0.0001 and P <
0.0001; respectively) in serum TH (118.6 ± 10.43), LH (0.75 ± 0.12), and FSH
(0.95 ± 0.32) compared Table 1: Testis weight of all experimental groups
|
Groups |
Negative control group (G1) |
Positive control group (G2) |
TAA + LUT group (G3) |
P value |
|
n |
10 |
10 |
10 |
|
|
Right testis weight (g) |
1.59 ± 0.05 |
1.22 ± 0.05 a (****) |
1.50 ± 0.17 a (ns) b (****) |
< 0.0001 < 0.0001 |
|
Left testis weight (g) |
1.60 ± 0.07 |
a (****) |
a (ns) b (***) |
< 0.0001 0.1714 0.0001 |
Data were presented
as mean and standard deviation (Mean ± SD), n= Number of rats /group, a: compared to Negative control group (G1), b: compared
to Positive control group (G2), ns = non-significant,
** P <
0.01, *** P < 0.001, **** P < 0.0001.
TAA: Thioacetamide. LUT: Luteolin
Table 2: Comparison between all experimental groups according to the levels of Serum
testosterone (TS, ng\dL), Serum luteinizing hormone
(LH, IU\L), and Serum follicle-stimulating hormone (FSH, IU\L)
|
Groups |
Negative control group (G1) |
Positive control group (G2) |
TAA + LUT group (G3) |
P value |
|
n |
10 |
10 |
10 |
|
|
292.4 ± 29.74 |
118.6 ± 10.43 a (****) |
270.6 ± 41.38 a (ns) b (****) |
< 0.0001 0.30 < 0.0001 |
|
|
4.34 ± 0.66 |
0.75 ± 0.12 a (****) |
3.96 ± 1.85 a (ns) b (****) |
< 0.0001 0.84 < 0.0001 |
|
|
4.80 ± 1.01 |
0.95 ± 0.32 a (****) |
4.72 ± 0.85 a (ns) b (****) |
< 0.0001 0.99 < 0.0001 |
Data were presented
as mean and standard deviation (Mean ± SD), n= Number of rats /group, a:
compared to Negative control group (G1), b: compared to Positive control
group (G2), ns= non-significant, **** P < 0.0001. TAA: Thioacetamide. LUT: Luteolin
Table 3: Comparison between all experimental groups according to the levels of
oxidative stress enzyme malondialdehyde (MDA, nmol\mL) and the antioxidant enzymes such as reduced glutathione (GSH,
ng\mL), superoxide dismutase (SOD, µ\mL),
and catalase (CAT, Mu\L) in testis tissue homogenate
|
Groups |
Negative control group (G1) |
Positive control group (G2) |
TAA + LUT group (G3) |
P value |
|
n |
10 |
10 |
10 |
|
|
a (****) |
a (*) b (****) |
< 0.0001 0.01 < 0.0001 |
||
|
Reduced glutathione (GSH, ng\mL) |
16.12 ± 2.32 |
2.18 ± 0.67 a (****) |
16.12 ± 2.29 a (ns) b (****) |
< 0.0001 > 0.99 < 0.0001 |
|
Superoxide dismutase (SOD, µ\mL) |
170.40 ± 9.34 |
79.60 ± 18.96 a (****) |
163.2 ± 12.53 a (ns) b (****) |
< 0.0001 0.60 < 0.0001 |
|
a (****) |
a (ns) b (****) |
< 0.0001 0.29 < 0.0001 |
Data were presented
as mean and standard deviation (Mean ± SD), n= Number of rats /group, a:
compared to Negative control group (G1), b: compared to Positive control
group (G2), ns = non-significant, **** P < 0.0001. TAA: Thioacetamide. LUT: Luteolin
with
the negative control group (292.4 ± 29.74, 4.34 ± 0.66, 4.80 ± 1.01; respectively) and TAA + LUT treated group (270.6 ±
41.38, 3.96 ± 1.85, 4.72 ± 0.85; respectively). Whereas G3 (TAA + LUT treated
rats) showed a highly significant (P <
0.0001, P <
0.0001 and P <
0.0001; respectively) increased serum TH, LH, and FSH versus the G2 (positive control
group). Meanwhile, G3 showed a non-significant difference in serum TH, LH, and
FSH levels (P = 0.30, 0.84, 0.99, respectively) compared with the
negative control group (G1).
Evaluation of the testicular
oxidative stress status and antioxidant enzymes
MDA and antioxidants markers GSH, SOD
and CAT, were expressed as mean ± SD in all experimental groups (Table 3 and Fig. 3). The mean value
of the MDA level demonstrated a significant increase (1.45
± 0.09, P < 0.0001). In
contrast, a significant reduction (P < 0.0001) in the levels of antioxidant markers such as GSH (2.18 ± 0.67), SOD (79.60
± 18.96) and CAT (55.40 ± 11.52) were seen in rats of G2 versus G1 and
TAA + LUT group (G3). There were no significant changes (P > 0.99, P = 0.60, P = 0.29;
respectively) detected in the levels of GSH, SOD and CAT in TAA + LUT group
(G3) (16.12 ± 2.29, 163.2 ± 12.53, 117.6 ± 1.95; respectively) as compared to
the negative control group (16.12 ± 2.32, 170.40 ± 9.34, 112.2 ± 4.96;
respectively). While there was a significant increase in the MDA level (0.56 ±
0.20, P = 0.01) in G3 compared to the MDA level (0.37 ± 0.10) in G1.
%20391-404_files/image002.png)
Fig.
1: Chart showing: (A)
Right testis weight (g), and (B)
Left testis weight (g) among all experimental groups after the administration
of Thioacetamide (TAA) and Luteolin (LUT). Data were presented as mean and
standard deviation (Mean ± SD), ns = non-significant, ** P < 0.01, *** P < 0.001, **** P < 0. 0001
%20391-404_files/image004.png)
Fig. 2: Chart showing: (A) Serum testosterone (TS,
ng\dL), (B) Serum
luteinizing hormone (LH, IU\L), and (C)
Serum follicle-stimulating hormone (FSH, IU\L) among all experimental groups
after the administration of Thioacetamide (TAA) and Luteolin (LUT). Data were
presented as mean and standard deviation (Mean ± SD), ns= non-significant, ****
P < 0. 0001
Histological results
H & E results: Examination of H& E stained sections from negative control group (G1) testis showed normal histological
architecture. Clusters of interstitial Leydig cells with acidophilic cytoplasm
and vesicular nuclei were seen in the interstitial spaces between the firmly
packed seminiferous tubules in the testicular tissue. Each seminiferous tubule
had a distinct basement membrane and was lined by myoid cells with flat nuclei.
Semineferous tubules bordered with Sertoli cells and stratified germinal
epithelium. The germinal epithelium is formed of spermatogonia, primary
spermatocytes, early and late spermatids and sperms. Spermatogonia appeared as
small rounded cells with rounded nuclei present in the basal part of the
tubules. Primary spermatocytes were larger in size than spermatogonia with
large rounded nuclei. Early spermatids appeared as small rounded cells with
paler nuclei. Sperms were demonstrated by their long tails in the lumen of
tubules. The Sertoli cells established a pyramidal shape between the
spermatogenic cells and a smooth basement membrane. They had large pale
vesicular nuclei and prominent nucleoli. Even spermatozoa could be seen in the
seminiferous tubules lumen (Fig. 4).
%20391-404_files/image006.png)
Fig. 3: Chart showing: (A) Malondialdehyde (MDA,
nmol\ml) and the antioxidant enzymes such as (B) Reduced glutathione (GSH, ng\mL), (C) Superoxide dismutase (SOD, µ\mL), and (D) Catalase (CAT, MU\L) in testis tissue homogenate among
all experimental groups after the administration of Thioacetamide (TAA) and
Luteolin (LUT). Data were presented as mean and standard deviation (Mean ± SD),
ns = non-significant, * P < 0.
05, **** P < 0. 0001
In the positive control group (G2) (Fig. 5), impairment
of normal architecture of testicular tissue with wide interstitial spaces
between tubules and arrested spermatocytes in a different stage in the
division was seen. Sloughing of the basal lamina of many seminiferous tubules
from lamina propria, loss of stratal
arrangement of germinal epithelium with wide intercellular separation,
occluded lumen with vacuolated eosinophilic substance and wide
interstitial space filled with eosinophilic material. The Sertoli cell
showed pale nuclei, distorted and destructed
cytoplasmic extension, few elongated spermatids and round spermatids, and few
flagella of mature sperms in lumina.
Wide interstitial spaces had Leydig cells, dilated congested blood
vessels between seminiferous tubules and eosinophilic material. Germinal
epithelium seemed thinner, spermatogenic cells appeared deformed and their
nuclei were apaptotoic, and there were large gaps between spermatogenic cells. The absence of sperms inside the lumen of seminiferous
tubules was also noticed.
However, TAA + LUT treated group (G3) showed
normal seminiferous tubules surrounded by a basement membrane lined by Sertoli
cells and stratified germinal epithelium. Spermatogonia rested on the basal
lamina and was closely related with little intercellular spacing. Spermatids in
various stages of spermiogenesis were present as rounded spermatids, and
elongated spermatids embedded in cytoplasmic extensions of Sertoli cells were
seen. Sperms were observed in the seminiferous tubules lumen. Leydig cells were
noticed in the normal interstitial tissue (Fig. 6).
Masson trichrome stained sections: Collagen fibers in
the
%20391-404_files/image008.png)
Fig. 4: Testicular sections of adult male rats in the
negative control group (G1) showing:
(A)
Normal testicular architecture of seminiferous tubules (St).
The regular arrangement of closely packed seminiferous tubules (St). Multiple
seminiferous tubules (St) with regular outlines, lined by stratified germinal
epithelium (GE) and lumen (L) filled with sperm flagella. Clusters of
Interstitial cells of Leydig (curved arrow) with acidophilic cytoplasm and
vesicular nuclei around blood vessels (v) in the interstitial spaces (I)
in-between tubules. (H&E stain X 100, scale bar 100 µm)
(B)
Each seminiferous tubule (St) is lined by Sertoli cells
(S) and stratified germinal epithelium (GE). Each seminiferous tubule is
surrounded by a well-defined basement membrane and flattened myoid cells (blue
arrow) with flattened nuclei. GE is formed of spermatogonia (G), primary
spermatocytes (Ps), early (Es) and late spermatids (Ls), and lumen (L) filled
with sperm flagella. (H&E stain X 20, scale bar 50 µm)
(C)
Spermatogonium (G) small, rounded cells with rounded
nuclei resting on the basement membrane, primary spermatocyte (Ps) larger
rounded cells with large, rounded nuclei, early spermatids (Rs) small, rounded
cells with paler nuclei, and many elongated spermatids (Ls) are detected.
Sertoli cell (S) appears with a vesicular nucleus and a well-formed cytoplasmic
extension (arrowhead). (H&E stain X 40, scale bar 50 µm)
%20391-404_files/image010.png)
Fig. 5: Testicular sections of adult male rats in the
positive control group (G2) show:
(A) loss
of normal architecture of testicular tissue with wide
interstitial spaces (I) between tubules and arrested spermatocytes in a
different stage in the division are seen. Many other tubules (St) are
depleted of most of the spermatogenic cells. Note,
occluded lumen (L) with remnants of spermatogenic cells(n). (H&E stain
X 100, scale bar 100 µm)
(B)
another section shows the sloughing of
the basal lamina of many seminiferous tubules (St) from lamina propria (*) and wide interstitial (I) spaces filled with
eosinophilic material (E) and dilated congested blood vessel (V). The
absence of sperms inside the lumen of seminiferous tubules (St) is sometimes
noticed and other lumens fill with remanent of
spermatogenic cells (n). (H&E stain X 100, scale bar 100 µm)
(C) Severely
affected tubules (St) with extensive loss of their germinal epithelium. Note,
occluded lumen with remnants of spermatogenic cells(n). Distorted
tubules with deeply stained pyknotic nuclei of germinal epithelium (↑)
and a few elongated spermatids (bifid arrow) are also noticed. (H&E stain X
20, scale bar 50 µm)
(D) Sloughing
of the basal lamina of many seminiferous tubules (St) from lamina propria,
loss of stratal arrangement of germinal epithelium with wide intercellular
separation (*), occluded lumen with remanent of spermatogenic cells (n).
Distorted tubules with deeply stained pyknotic nuclei of germinal epithelium
(↑) and a few elongated spermatids (bifid arrow) are
seen. Wide interstitial spaces (I) filled with eosinophilic material (E) are
also noticed. (H&E stain X 20, scale bar 50 µm)
(E) higher
magnification of shows wide intercellular separation. Sloughing of the basal
lamina of many seminiferous tubules (St) from lamina propria (*) and wide
interstitial space (I). Some Sertoli cells (S) appear with pale nuclei and
destructed cytoplasmic extension. Note, occluded lumen (L) with vacuolated
eosinophilic substance (n). Sloughing of the basal lamina of many seminiferous
tubules (St) from lamina propria (*) and wide interstitial space (I). (H&E
stain X 40, scale bar 50 µm)
higher magnification shows part of a seminiferous tubule
(St) with distorted germ cells, spermatogonia (↑) have oval dark stained
nuclei and Sertoli cell (S) with a pale nucleus and
destructed cytoplasmic extensions with wide interstitial spaces (I) between
tubules. Note, a seminiferous tubule (St) with extensive loss of germinal
epithelium (GE). (H&E stain X 40, scale bar 50 µm)
%20391-404_files/image012.png)
Fig. 6: Testicular sections of adult male rats in TAA+LUT
treated group (G3) showing:
(A)
Normal testicular architecture of seminiferous tubules
(St). The regular arrangement of closely packed seminiferous tubules (St).
Multiple seminiferous tubules (St) with regular outlines, lined by stratified
germinal epithelium (GE) and lumen (L) filled with sperm flagella. Clusters of
Interstitial cells of Leydig (curved arrow) with acidophilic cytoplasm and
vesicular nuclei around blood vessels (v) in the interstitium (I) in-between
tubules. (H&E stain X
100, scale bar 100 µm)
(B)
Each seminiferous tubule (St) is lined by Sertoli cells
(S) and stratified germinal epithelium (GE). Each seminiferous tubule is
surrounded by a well-defined basement membrane and flattened myoid cells (blue
arrow) with flattened nuclei. GE is formed of spermatogonia (G), primary
spermatocytes (Ps), early (Es) and late spermatids (Ls), and lumen (L) filled
with sperm flagella. (H&E stain X 20, scale bar 50 µm)
(C)
Spermatogonium (G) small, rounded cells with rounded
nuclei resting on the basement membrane, primary spermatocyte (Ps) larger
rounded cells with large, rounded nuclei, early spermatids (Rs) small, rounded
cells with paler nuclei, and many elongated spermatids (Ls) are detected.
Sertoli cell (S) appears with a vesicular nucleus and a well-formed cytoplasmic
extension (arrowhead). (H&E stain X 40, scale bar 50 µm)
%20391-404_files/image014.png)
Fig. 7: Testicular sections of adult
male rats in
(A)
Negative control group (G1) shows a normal
distribution of collagen fibers (↑) in
capsule (C), and in the basal lamina of seminiferous tubules (St).
(B)
Positive control group (G2) shows
a marked increase of collagen fibers (↑) in capsule (C), basal lamina of
seminiferous tubules (white arrows) and in the wall of blood vessels (V).
(C) TAA+LUT treated group (G3) shows the decreased distribution of collagen
fibers (↑) in capsule (C), basal lamina of seminiferous tubules (St) and
in the wall of blood vessels (V). Masson trichrome stain (X 100, scale bar 100
µm)
tunica albuginea, basal lamina of the
seminiferous tubules, and vessel walls were uniformly distributed in rat testes
stained with Masson's trichrome of negative control (Fig. 7A). Increased collagen fibers were found in the testicular
capsules, vessel walls and seminiferous tubule basal lamina of the positive
control group (Fig. 7B).
Collagen fiber distribution was nearly normal in the TAA+LUT treated group
compared to G1 (Fig. 7C).
Immunohistochemical reaction results
Midportion and apices of Sertoli cells
were immunostained positively for vimentin in G1 as well as their walls with
adjoining germ cells and spermatozoa (Fig. 8A_B).
Weak vimentin immunoreaction, in the
form of streaks, was observed in G2 at Sertoli-spermatocyte contact points. In
addition, the presence of large vacuoles in the %20391-404_files/image016.png)
Fig. 8: Testicular sections of adult male rats in
(A & B) The negative control group (G1) shows the appearance
of Sertoli (S) vimentin filaments (immunohistochemical staining), Sertoli cell
(S) has perinuclear positivity (dot arrow) and in
cytoplasmic extensions (↑).
(C & D) The positive control group (G2) shows a marked
decrease in the number of Sertoli cells (S) with distorted perinuclear positive
reaction (dot arrow) and fragmentation of cytoplasmic extensions (↑)
irregular basal lamina (curved arrows).
(E & F)
TAA+LUT treated group (G3) shows an
apparent increase of positively stained Sertoli cells (S), perinuclear
positivity (dot arrow), continuously extended positive cytoplasmic extensions
(↑), and few seminiferous tubules (St) appear with fragmented cytoplasmic
reaction (*). Vimentin immunohistochemistry (X 100, 200, scale bar 100, 50 µm)
place of degraded
spermatocytes was observed in several seminiferous tubules, as evidenced by
vimentin immunostaining in those sections. At the same time, mild
immunoreactivity was seen in the interstitial cells. G2 showed that the number of Sertoli cells presenting positive vimentin
reaction was reduced with disturbed apical cytoplasmic extensions and
marked irregularity of the seminiferous tubule basement membrane (Fig. 8C–D).
In TAA and LUT
treated group, vimentin filaments' positive reaction within Sertoli cells was
concentrated around the nucleus (perinuclear) in the basal part of the cell,
then radiating apically toward the apical part of the cell. In addition, a
regular basement membrane of the seminiferous tubule was detected. Moreover,
the number of Sertoli cells presenting positive vimentin reaction was
apparently increased and normally distributed vimentin filaments with
cytoplasmic extensions. (Fig. 8E–F).
Morphometrical results
There was a significant difference in
the TAA+LUT group in the mean diameter of the
seminiferous tubules, the mean germinal epithelial
thickness, mean area (%) of collagen fibers content
in Masson trichrome stained sections and mean area (%) of vimentin-positive
cells (Table 4; Fig. 9).
In the Masson trichrome staining of tissue sections, the positive control
group had a significantly higher mean area percent of collagen fibers content
(20.55 ± 1.75) than the control (8.72 ± 0.98) and TAA+LUT (10.00 ± 0.3) groups
(P < 0.0001). While the
TAA+LUT group showed a slight increase relative to the negative control group.
There was no statistically significant difference (P = 0.065) between
the two groups. (Table 4, Fig. 9C).
Comparing the area percent of vimentin-positive staining between the
control (15.38 ± 0.48) and TAA+LUT (15.71 ± 0.69) groups, we find that the
positive control group shows a statistically significant (P < 0.0001) reduction in area percent (11.57
± 0.24) compared to both control and TAA+LUT groups. Additionally, the TAA+LUT
group did not significantly change from the negative control group (P =
0.4017) (Table 4, Fig. 9D).
Discussion
Chemicals and diseases can alter
physiological and biochemical activities that impact a man's reproduction
ability. Classical qualitative chemical analysis frequently uses. Thioacetamide
as an in situ source for sulfide ions; nevertheless, it is harmful to the liver
in experimental animal models of exposure (Czechowska et al. 2015; Lebda et al.
2018). The literature contains only a small number of papers
Table 4: Morphometric analysis in all experimental groups according to the mean
diameter of seminiferous tubules (mm), mean thickness of germinal epithelium (µm), mean area % of collagen fibers
content in Masson trichrome stained sections (%), and Mean area % of vimentin-positive
cells (%)
|
Groups |
Negative
control group (G1) |
Positive
control group (G2) |
TAA
+ LUT group (G3) |
P
value |
|
n |
10 |
10 |
10 |
|
|
414.7 ± 54.50 |
163.7 ± 43.16 a
(****) |
454.6 ± 27.90 a
(ns) b
(****) |
< 0.0001 0.1398 < 0.0001 |
|
|
Mean
thickness of germinal epithelium (µm) |
74.55 ±
3.10 |
46.98 ±
4.77 a
(****) |
72.29 ±
4.20 a
(ns) b
(****) |
< 0.0001 0.53 < 0.0001 |
|
Mean
area % of collagen fibres content in Masson trichrome stained sections (%) |
8.72 ±
0.98 |
20.55 ±
1.75 a
(****) |
10.00 ±
0.30 a
(ns) b
(****) |
< 0.0001 0.0650 < 0.0001 |
|
Mean
area % of vimentin-positive cells (%) |
15.38 ± 0.48 |
11.57 ± 0.24 a
(****) |
15.71 ± 0.69 a
(ns) b
(****) |
< 0.0001 0.4017 < 0.0001 |
Data were presented as mean and standard deviation (Mean ± SD), n = Number
of rats /group, a: compared to Negative control group (G1), b: compared to Positive control
group (G2), ns = non-significant, **** P < 0.0001. TAA: Thioacetamide. LUT: Luteolin
%20391-404_files/image018.png)
Fig. 9: Chart showing: (A) the mean diameter of seminiferous
tubules (mm), (B) mean thickness of
germinal epithelium (µm), (C) mean area % of collagen fibers
content in Masson trichrome stained sections (%) and (D) Mean area % of vimentin-positive cells (%). Data were presented
as mean and standard deviation (Mean ± SD), ns = non-significant, **** P < 0. 0001
mentioning TAA concerning its effects on
the testes. Due to the current buzz surrounding the potential health benefits
of herbal remedies and nutritional supplements, this research is particularly
timely. The antioxidant, anti-inflammatory, anti-carcinogenic, neuroprotective,
and antineoplastic effects of LUT are well-documented (Manju et al. 2005).
In the current study,
there were statistically significant decreases in right testis weight, and left
testis weight in G2 versus G1. These findings corroborate previous studies that
TAA exposure significantly altered animals, notably reducing the testicular
weight of male rats (Nourozi and Shariati 2020). Testicular weight was also
significantly different between TAA-treated and control rats, which may be
attributable to the atrophy of the germinal epithelium observed in prior
studies following TAA treatment (Celik et
al. 2016; Lebda et al. 2018;
Nourozi and Shariati 2020). In addition, Explained that the reduced weight of
the testes due to decreases in androgen levels (Elmallah
et al. 2017). In addition, we
discovered that weight loss was lessened in the TAA + LUT group compared to G2,
indicating a beneficial impact for LUT.
Spermatogenesis is
known to be regulated in great portion by gonadotropins and testosterone (Johnston
et al. 2004; An et al. 2020; Behairy et al.
2020). Testosterone and FSH work together to produce sperm in men. There is evidence
that both FSH and testosterone stimulate each stage of spermatogenesis (Oduwole
et al. 2018). LH stimulates testosterone production and secretion by Leydig cells, while
FSH acts directly on the seminiferous tubules (MacLachlan et al. 2002; Spaliviero et
al. 2004). In the present study, the serum testosterone level was
significantly decreased in G2 (testicular toxicity rat model induced by TAA). A significant decrease in LH
and FSH concentration was also noted. Studies have shown that LH, which is
generated by the pituitary, stimulates TH production in the male testes.
Testosterone levels change dramatically during the life cycle of males (An et al. 2020; Kamińska et al. 2020; Albasher et al. 2021). Found that high OS reduced
levels of essential enzymatic and non-enzymatic antioxidants in Leydig cells,
leading to a decrease in TH release, which may explain this phenomenon (Cao et al. 2004). The reduction in
testosterone and LH levels, according to is attributed to the failure of LH
binding sites in the body's Leydig cells (El-Sayed and El-Neweshy 2010). There may have been a decrease in the total quantity of spermatids and
testosterone, as indicated by Cormier and his coworkers. They also noted that
testis weight is directly related to testis function (Cormier
et al. 2018). Moreover, the
current study showed a significant increase in testosterone, LH, and FSH
concentration in TAA+LUT treated rats. These results agree with those of in
rats pretreated with LUT in lead-induced testicular toxicity. The observed
increase in plasma levels suggests that these effects are due to the
antioxidant effect of LUT and its metal chelating activity; LUT binds with lead
and decreases its toxic effect (Al-Megrin et al. 2020).
There has been speculation that oxidative damage is responsible for the
observed testicular damage after TAA exposure. Oxidative stress results when
there is a discrepancy between the generation and clearance of ROS and free
radicals, TAA-induced lipid peroxidation, and the activity of enzymes that
prevent oxidative damage is reduced (Celik et
al. 2016). TAA also generated many ROS, which could interfere with the
antioxidant defense system. The overproduction of ROS was harmful to several
parts of the cell (Türkmen et al.
2022). When ROS peroxidizes fatty acids, indicators of lipid peroxidation, such
as MDA, are formed, leading to permanent cell damage and a decrease in the
efficacy of the antioxidant defense system, including SOD and CAT activity and
GSH levels. Because of their large concentration of polyunsaturated membrane
lipids cells equipped with an antioxidant mechanism, the testes are the
principal target organs for OS (Sedha et
al. 2015). Our data demonstrated that TAA exposure promoted lipid
peroxidation as measured by a rise in MDA levels and weakened the antioxidant
defense system as measured by a decrease in CAT, SOD activity, and GSH. Prior
research has demonstrated that TAA causes serious oxidative damage to liver
tissue, but there has been considerably less investigation into the effects of
TAA on testicles. These research are in agreement with our findings (Czechowska
et al. 2015; Celik et al. 2016; Karabulut et al. 2020). Our results show that LUT
protects rat testes homogenate from TAA-induced oxidative stress by bolstering
the rats' antioxidant defenses. Similarly, Showed that the antioxidant Luteolin
significantly mitigated the harmful effects of electromagnetic fields (Yahyazadeh and Altunkaynak 2019).
Histological analysis of TAA-treated rats revealed progressive
degeneration of the germinal epithelium lining most of the seminiferous
tubules. Other researchers have found similar results, describing the
TAA-induced cell death as a "washed out" appearance (Celik et al. 2016). The present study found
that the testicular tissue architecture in the positive control group showed
many forms of abnormalities as vacuolations. Some seminiferous tubules were
sloughed from lamina propria, had an irregular outline and were filled with
remnants of germinal epithelium. Interstitial tissue was wide, containing
eosinophilic exudate, and dilated congested blood vessels. Such an observation
is a hallmark of testicular toxicants such as Lead (Albasher et al. 2021). Other seminiferous tubules
with discarded germinal epithelium from the basal lamina showed darkly stained,
apoptotic nuclei of spermatogenic cells. Also, distorted tubules with wide and
empty lumina were detected. These were in agreement with Johnson, who explained
the destruction of testicular tissue and subsequent infertility by Sertoli cell
fragmentation, which provides support and nutrition for the spermatic cells, so
its destruction results in the loss of spermatic cells (Johnson
2014). We also found that the lack of intimate interaction of spermatogenic
cells may impair proper spermatogenesis, as seen in the high power results.
This was characteristic of seminiferous tubule degeneration as evidenced by
degenerating spermatids and a few germinal epithelial cells in tubular lumens. Reported
that the close relationship between germ cells and consequently the
intercellular bridges was an important landmark of normal spermatogenesis (Lara
et al. 2018). The use of LUT reduced
the severity of the damage, which is in line with previous findings about the
potential benefit as a preventative measure (Yahyazadeh and Altunkaynak 2019; Al-Megrin
et al. 2020).
These histological findings were supported by the current histomorphometric data, which demonstrated a considerable
decrease in the diameter and thickness of the germinal epithelium lining the
seminiferous tubules in G2 compared to G1. In contrast, we observed that the
TAA + LUT group had significantly less damage to the testes. But the
significant observation that LUT can minimise the impact of TAA exposure is
truly remarkable.
During
spermatogenesis, Sertoli cells, which play a crucial role in the formation of
germ cells within functional testes, display a range of morphologies (Ahmed et al. 2016). The current H&E
histopathological findings shows that Sertoli cells were fully mature in the
control group (G1) and the TAA + LUT treated group (G3), spermatogenic cells
had close relations to each other and Sertoli cytoplasmic extensions, but that
these relations had been lost in the G2 group. Sertoli cells make contact with
growing germ cells by stretching their cytoplasm. The same was observed in the
findings by (Mohammed and Sabry 2020). While in the
TAA group, Sertoli cells appeared with destructed cytoplasmic extension, few
elongated spermatids, round spermatids and few flagella of mature sperms in
lumina. Also, there was an apparent decrease in the Sertoli cells. It has been an
archetypal indicator of direct toxicant action on Sertoli cells (Johnson 2014).
Vimentin
immunoreactivity was detected in the nucleus and apical area of Sertoli cells,
between spermatogonia and primary spermatocytes and in the intercellular spaces
between these cell types in G1. Similarly, Reported that in the normal testis,
the Sertoli vimentin filaments are either arranged around the nucleus or extend
to the apical region of Sertoli cells (Lydka et al. 2011). In the positive
control group, this immunoreactivity staining diminished. Because vimentin
filaments collapsed away from the cell membrane of Sertoli cells, this decrease
in vimentin expression harmed testicular tissue. Possible outcomes include
Sertoli cell detachment and subsequent apoptosis of detached spermatogenic
cells due to lack of nourishment and support ( Johnson 2014; Alam et al. 2010). Vimentin immunohistochemistry
results in Sertoli cells were stronger and apparently increased in the TAA+LUT
treated rats compared to the positive control group (testicular toxicity
model), suggesting the development of a cytoskeleton that promotes higher
mechanical force and the creation of a lengthy sperm nucleus.
These results also
imply that intermediate filaments form in a different pattern during late
spermatogenesis when they are positioned close to the nucleus of germ cells and
that their distribution is highly cyclical during the early stages of the
process. Vimentin filaments likewise maintain Sertoli cells' connections with
the seminiferous epithelium surrounding spermatogenic cells. Therefore, they
are crucial for the completion of spermatogenesis and for gap junction intercellular
communication (Show et al. 2003; Kopecky
et al. 2005; ElGhamrawy et al. 2014). A considerable drop in the
mean area percentage of vimentin-positive cells in the G2 phase was seen in the
present morphometric experiments, corroborating the present histology and
immunohistochemical findings. Furthermore, their G3 (TAA + LUT) scores improved
significantly.
Conclusion
Especially LUT is effective in reducing
TAA testicular toxicity by ameliorating histopathological changes and restoring
the normal testicular tissue architecture to a great extent, balancing the
oxidative tissue state, and increasing gonadotropins and testosterone hormones.
Thus, LUT could afford a feasible and useful food-based approach for improving
male fertility. Moreover, the Clinical application and therapeutic efficacy of
LUT need further investigations and more research to guide its optimal use.
Conflicts of Interest
The authors declare no conflict of interest.
Ethical Approval
All the animals received human care according to the
standard guidelines. Ethical approval for the study was obtained from the
Research Ethics Committee, which is in force at the University of Jeddah. The
rats were treated in accordance with the Laboratory Animal Treatment Agreement
of the Kingdom of Saudi Arabia and the ethical regulations were followed in
accordance with the national and institutional guidelines with the protocol
published by the National Institutes of Health.
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