Histological analysis of the effects of thymoquinone on testicular damage in pentylenetetrazole-induced temporal lobe epilepsy model

Fatma Bedia Karakaya1 | Melis Yavuz2 | Serap Sirvanci1

1Department of Histology and Embryology, School of Medicine, Marmara University, Istanbul, Turkey
2Department of Medical Pharmacology, School of Medicine, Marmara University, Istanbul, Turkey

Correspondence
Fatma Bedia Karakaya, Department of Histology and Embryology, School of Medicine, Bezm-i Âlem University, Istanbul, Turkey.
Email: [email protected]
Present address
Fatma Bedia Karakaya, Department of Histology and Embryology, School of Medicine, Bezm-i Âlem University, Istanbul, Turkey Melis Yavuz, Department of Pharmacology, School of Pharmacy, Acibadem Mehmet Ali Aydinlar University, Istanbul, Turkey
Funding information
Marmara Üniversitesi, Grant/Award Number: SAG-C-YLP-110618-0309

Abstract

In this study, it was aimed to investigate possible ameliorating effects of thymo- quinone on testicular damage in an epilepsy model. Adult male Wistar rats were divided into 4 groups. The animals in sham-operated groups were given saline or thymoquinone (s.c.); and the animals in pentylenetetrazole (PTZ) group were applied PTZ. The animals in PTZ+thymoquinone group were given thymoquinone (i.p) for 6 days after applying PTZ. Hematoxylin-eosin, periodic acid–Schiff and TUNEL stain- ing and PCNA, StAR, inhibin β-B immunohistochemistry and ZO-1 immunofluores- cence methods were applied. Staining intensity and cell numbers were determined. Degeneration of seminiferous tubules was observed in PTZ group. Most of the tu- bules showed normal morphology in the PTZ+thymoquinone group. Apoptotic cell index was found to be increased and proliferative index decreased in PTZ group. Thymoquinone administration decreased apoptotic index and increased proliferation index. In PTZ group, ZO-1, StAR and inhibin β-B immunohistochemical staining inten- sity was observed to be decreased and after thymoquinone application, ZO-1 was increased. StAR and inhibin β-B-positive cell numbers were decreased in PTZ group and increased in the PTZ +thymoquinone group. In this study, it was observed that PTZ-induced epileptic seizures caused testicular damage in the rat and thymoqui- none ameliorated these effects.

K E Y WO R D S
kindling, pentylenetetrazole, temporal lobe epilepsy, testis, thymoquinone

1 | INTRODUC TION

Epilepsy is a neurological disease, affecting approximately 50 mil- lion people, with a prevalance of 1% in the world (Shokri et al., 2015; Zupec-Kania & Spellman, 2008). Temporal lobe epilepsy is the most common form of this disease (Engel, 2001).
Reproductive and sexual function disorders are frequent in male and female patients with epilepsy (Luef & Madersbacher, 2015; Webber et al., 1986). 90% of males with epilepsy were reported to have abnormal sperm count, morphology and motility (Montouris & Morris, 2005). Both epilepsy and antiepileptic drugs play a role in the pathophysiology of reproductive disorders (Herzog, 2008; Rattya et al., 2001). Studies have shown alterations in endocrine system and hormonal levels as a result of epileptiform activity (Hauser & Beghi, 2008; Veliskova & Desantis, 2013). Abnormal serum testos- terone and gonadotropin concentrations, abnormal LH response to GnRH stimulation and increased serum prolactin levels were reported to be related to epileptic discharges (Montouris & Morris, 2005).
Nigella sativa (N. sativa), belonging to Ranunculaceae family, is a plant frequently used in modern and alternative medicine (Bamosa et al., 2015). Thymoquinone (C10H12O2, 2-isopropyl-5-methyl 1, 4-benzoquinone) is the most important bioactive compound found in the volatile oil of N. sativa. It was reported that thymoquinone has antioxidant, antihyperlipidemic, antidiabetic, antiinflammatory, gastroprotective, hepatoprotective, antihypercholesterolemic, anti- carcinogenic and analgesic effects (Güllü & Gülcan, 2013).
To our knowledge, there is no study about the effects of pentylenetetrazole-induced temporal lobe epilepsy model on testis morphology, and the ameliorating effect of thymoquinone on testic- ular damage. Other studies focused on the effects of antiepileptic drugs, rather than the effects of epilepsy itself. The present study aimed to investigate ameliorating effect of thymoquinone on testic- ular damage in pentylenetetrazole-induced temporal lobe epilepsy model by morphological and immunohistochemical methods.

2 | MATERIAL S AND METHODS

Animals were obtained from Marmara University, The Experimental Animal Implementation and Research Center. All experiments were done according to the national guidelines on animal experimentation and were approved by the Marmara University Local Ethical Committee for Experimental Animals (43.2018.mar). Four-month-old male Wistar albino rats weighing 250–300 g were used in the present study. The animals were housed with free access to water and food in a 12-hr light/dark cycle and humidity controlled room (21 ± 2°C and 65%–70% humidity). Seizure stages were evaluated Racine’s 5 stage scale (Luttjohann et al., 2009).

Groups were as follows:
1. Sham-operated (saline) group (n = 6): This group of animals were injected DMSO (i.p.) 6 consecutive days after saline injection.
2. Sham-operated (thymoquinone) group (n = 6): Animals were first injected saline, and then 10 mg/kg (Fouad & Jresat, 2015) thymo- quinone 6 consecutive days.
3. Pentylenetetrazole (PTZ) group (n = 6): This group of animals were injected PTZ (35 mg/kg, s.c.) (Tekgul et al., 2020) every other day until they had 5 times grade 5 seizures, and then they were in- jected DMSO (i.p.)
4. Pentylenetetrazole+thymoquinone (PTZ+thymoquinone) group (n = 6): This group of animals were injected PTZ (35 mg/kg, s.c.) every other day until they had 5 times grade 5 seizures, and then, they were injected 10 mg/kg thymoquinone (i.p.) 6 consecutive days.

2.1 | Perfusion fixation
After deep anaesthesia by ketamine (100 mg/kg) and xylazine hydro- chloride (10 mg/kg), animals were perfused with a fixative solution con- taining 4% paraformaldehyde. After decapitation, testes tissues were obtained and incubated in the same fixative solution overnight at 4°C.

2.2 | Morphological examination
Tissues were washed in tap water, dehydrated by increasing series of ethanol, cleared in xylene, incubated in liquid paraffin and embedded in paraffin. Four-micron-thick sections were stained with haematoxy- lin and eosin, and PAS reaction was applied. Micrographs were taken with Olympus DP72 CCD camera attached Olympus BX51 micro- scope at 40× magnification. A total of 100 seminiferous tubules were examined from each section. Tubular damage was scored according to Johnsen’s scoring method (Gozen et al., 2013; Johnsen, 1970).

2.3 | TUNEL method
TUNEL method was applied according to manufacturer’s instruction (ApopTag Plus, In Situ Apoptosis Detection Kit, S7101, Millipore). A total of 20 seminiferous tubule, each containing at least 3 TUNEL- positive cells, were examined at 40× magnification. Apoptotic index was found by dividing the number of seminiferous tubules contain- ing 3 or more TUNEL-positive cells to the number of total seminifer- ous tubules (Koroglu et al., 2019).

2.4 | Immunohistochemistry
Paraffin sections were incubated in 37°C, and then xylene was ap- plied for deparaffinisation. After incubating in 96% ethanol and 3% hydrogen peroxide, citrate buffer was applied for antigen retrieval. Sections were incubated in blocking solution and anti-PCNA (Abcam, ab15497, 1:100), anti-ZO-1 (Thermo Fisher Scientific, 61–7300, 1:100), anti-StAR (Abcam, ab2031931, 1:100) and anti-inhibin β-B (Santa Cruz, sc376971, 1:100) primary antibodies. For anti-PCNA, anti-StAR and anti-inhibin β-B antibodies, antipolyvalent secondary antibody (Scytek SHP125), streptavidin peroxidase, DAB chromogen (Thermo Fisher Scientific, 34002) and Mayer’s haematoxylin were applied and sections were coverslipped with entellan. For anti-ZO1 antibody, Dylight 488 conjugated goat anti-rabbit secondary antibody (Thermo Pierce 35502, 1:100) was used and sections were coverslipped with DAPI containing mounting medium (Vectashield, ZE0618). Negative control staining was achieved by omitting the primary antibodies.
For PCNA immunohistochemistry, 20 seminiferous tubules were pho- tographed in each section. Proliferation index was calculated by dividing the number of PCNA-positive cells in each seminiferous tubule to the total number of cells (Takagi et al., 2001). For ZO-1 immunohistochemis- try, 20 seminiferous tubule were examined at ×40 magnification and im- munoreactivity was scored as none (0), very mild (+), mild (++), moderate (+++) and intense (++++). For StAR and inhibin β-B immunohistochem- istry, cell density was calculated by dividing the number of cells in the interstitial area to the area of the interstitial region. Immunoreactivities for StAR and inhibin β-B were scored as none (0), very mild (+), mild (++), moderate (+++) and intense (++++) (van Diest et al., 1997).

2.5 | Statistical analysis
Statistical analysis was performed by using GraphPad Prism 7 pro- gram. Data were analysed by using one-way ANOVA and post hoc Bonferroni tests. p < 0.05 was accepted as statistical significance level.
FI G U R E 1 Normal morphology of seminiferous tubules in sham-operated saline (a) and thymoquinone (b) groups. (c) Left: Atrophic tubule (asterisk) in PTZ group. Right: Detachment of the tubule (arrow) and vacuoles between spermatogenic cells (asterisk). (d) Normal morphology of seminiferous tubules in PTZ+thymoquinone group. (e) Normal morphology of basement membrane in sham-operated saline group and thymoquinone (f) group. (g) Disorganisation of basement membrane in PTZ group (arrow). (h) Normal morphology of basement membrane in PTZ+thymoquinone group. (a-d) Haematoxylin and eosin staining, (e-h) PAS reaction. Bars: 100 µm

3 | RESULTS

3.1 | Morphological examination
Haematoxylin and eosin staining demonstrated normal morphology in sham-operated groups. Atrophic seminiferous tubules and cell debris in the lumen of the tubules were observed in PTZ group. Also, detach- ment of spermatogenic cells and vacuolisation in interstitial area and in germinal epithelium were observed. In some tubules, spermatogenic cell number was decreased. PTZ+thymoquinone group showed similar morphology with the sham-operated groups (Figure 1).
PAS reaction showed normal morphology of basement membrane and seminiferous tubule borders in sham-operated groups. Most of the tubular borders and basement membrane were disorganised in PTZ group. PTZ+thymoquinone group showed normal morphology of the basement membrane and tubular borders (Figure 1).
Johnsen scoring was significantly decreased in PTZ group com- pared with the sham-operated groups; and thymoquinone adminis- tration increased the score (p < 0.001) (Figure 2).

3.2 | TUNEL staining
TUNEL-positive cells were observed in dark brown colour. Apoptotic index was significantly increased in PTZ group com- pared with the sham-operated groups (p < 0.01), and decreased in PTZ+thymoquinone group compared with the PTZ group (p < 0.05). (Figure 3).

3.3 | PCNA immunohistochemistry
Proliferative cell index was significantly decreased in PTZ group compared with the sham-operated groups (p < 0.001). Index wasFI G U R E 2 Statistical analysis of Johnsen scoring.
***: Compared with the sham-operated groups; +++: compared with the PTZ group. ***, +++: p < 0.001
significantly increased in PTZ+thymoquinone group compared with the PTZ group (p < 0.001) (Figure 4). Negative control staining showed no immunoreactivity.

3.4 | ZO-1 immunohistochemistry
ZO-1 immunoreactivity was observed in the basolateral area of Sertoli cells and spermatogenic cells in the basal area in the sham-operated groups. Immunoreactivity was decreased and irregular in the PTZ group. Although some of the tubules in the PTZ+thymoquinone group showed irregular immunoreactivity, most of the tubules demonstrated similar normal staining as sham-operated groups. Immunoreactivity was decreased in the PTZ group compared with the sham-operated groups (p < 0.001) and increased in the PTZ+thymoquinone group compared with the PTZ group (p < 0.001) (Figure 5). Negative control staining showed no immunoreactivity.

3.5 | StAR immunohistochemistry
StAR immunoreactivity was observed in the interstitial area of sham- operated groups. Immunoreactive Leydig cell density was signifi- cantly decreased in the PTZ group compared with the sham-operated
FI G U R E 3 TUNEL staining in sham-operated saline (a) and
thymoquinone (b) groups, and PTZ (c) and PTZ+thymoquinone (d) groups. Arrows: TUNEL-positive cells. Bars: 100 µm. (e) Statistical analysis of apoptotic index.
**: Compared with the sham-operated groups; +: compared with the PTZ group. +: p < 0.05; **: p < 0.01
FI G U R E 4 PCNA
immunohistochemistry in sham-operated
saline (a) and thymoquinone (b) groups,
and PTZ (c) and PTZ+thymoquinone (d)
groups. Arrows: PCNA-positive cells.
Bars: 100 µm. (e) Statistical analysis of proliferation index. ***: Compared with the sham-operated groups; +++:
compared with the PTZ group. ***, +++: p < 0.001
groups (p < 0.05), and increased in PTZ+thymoquinone group com- pared with the PTZ group (p < 0.05). Staining intensity was sig- nificantly decreased both in PTZ and PTZ+thmoquinone groups, compared with the sham-operated groups (p < 0.001) (Figure 6). Negative control staining showed no immunoreactivity.

3.6 | Inhibin β-B immunohistochemistry
Inhibin β-B immunoreactivity was observed in the interstitial area of sham- operated groups. Inhibin β-B cell density was decreased in PTZ group com- pared with the sham-operated groups (p < 0.001). There was no difference in the staining intensity between PTZ (Figure 4g) and PTZ+thmoquinone groups; however, inhibin β-B-positive cell density was increased in the PTZ+thymoquinone group compared with the PTZ group (p < 0.05). Staining intensity was decreased both in PTZ and PTZ+thymoquinone groups, compared with the sham-operated groups (p < 0.01) (Figure 7). Negative control staining showed no immunoreactivity.

4 | DISCUSSION

To our knowledge, there is no study showing testicular dam- age caused by epileptic seizures and ameliorating effect of thymoquinone in the literature. In a previous study, an antiepilep- tic agent, valproic acid, was shown to cause desquamation of germ cells, vacuolisation in germinal epithelium and decreased sperm con- centration (Iamsaard et al., 2017). In another study, a neurodegen- erative disease, Huntington disease, was demonstrated to result in decreased spermatocyte end spermatid and irregularities in germinal epithelium (Van Raamsdonk et al., 2007). It was reported that the disease affected hypothalamic–hypophyseal–gonadal axis. Similarly, we observed deterioration in spermatogenic series in seminiferous tubules, vacuolisation in germinal epithelium and interstitial area, detachment of germinal epithelium from basal lamina, decreased number of spermatogenic cells, abnormally positioned germ cells and degenerated and atrophic tubules. PTZ-induced seizures ruin hypothalamic regulation of hypophyseal secretions and affect go- nadal function and reproduction (Herzog, 2008). Besides, decrease in gonadotropins and testosterone cause germ cell degeneration in the testes (Kiess & Gallaher, 1998). In the present study, PTZ- induced seizures might have affected hypothalamic-hypophyseal gonadal axis, causing testicular damage.
Gonadotropin deficiency is related to germ cell apoptosis (Sinha Hikim & Swerdloff, 1999). Deterioration of the balance between cell proliferation and apoptosis ruins spermatogenesis. Disruption of spermatogenesis is related to male reproduction disorders (Sakkas et al., 2003). Epileptic seizures disrupt steroidogenesis and cause
FI G U R E 5 ZO-1 immunohistochemistry in sham-operated saline (a) and thymoquinone (b) groups, and PTZ (c) and PTZ+thymoquinone (d) groups. Arrows: Decreased ZO-1 immunoreactivity in PTZ group. Bars: 20 µm. (e) Statistical analysis of ZO- 1 staining intensity. ***: Compared with the sham-operated groups; +++: compared with the PTZ group. ***, +++: p < 0.001
oxidative stress, inducing apoptosis. In a TLE model, induced by lithium-pilocarpine, apoptotic cell number increased in epileptic group (Shokri et al., 2015). Similarly, we observed increase in apop- totic cells in PTZ group. Thymoquinone administration decreased apoptotic cell index.
PCNA is a marker of cell proliferation. Decrease in PCNA expres- sion in testis germ cells is an indication of disrupted spermatogenesis and decrease in proliferative activity (Erboga et al., 2016). To our knowledge, there is no study in the literature investigating PCNA expression in testicular damage caused by epileptic seizures. Other studies showed decrease in proliferative cells after arsenic (Uygur et al., 2016), streptozotosin (Kaplanoglu et al., 2013) and cadmium (Koyuturk et al., 2006) administration. In line with these studies, we observed decreased number of proliferative cells after PTZ-induced epileptic seizures and increased number after thymoquinone administration.
Primary proteins forming blood–testis barrier are occludin; clau- din 3, 5, 11; ZO-1, 2, 3; and junctional adhesion molecule (JAM) A and B (Gow et al., 1999). Disruption of blood-testis barrier causes de- creased fertility (Xu et al., 2009). To our knowledge, there is no study in the literature showing the effect of epileptic seizures on blood– testis barrier. Other studies showed decreased expression of ZO-1 after exposure to low-dose radiation (Son et al., 2015), flutamide (Chojnacka et al., 2016), fluorochloridone (Liu et al., 2018) and bi- sphenol A and diethylhexylphthalate (Salian et al., 2009; Sobarzo et al., 2009). In the present study, it was observed that ZO-1 immu- noreactivity was decreased and even absent in some areas in the seminiferous tubules, and the structure of blood-testis barrier was disrupted after PTZ-induced seizures. Thymoquinone administrated group demonstrated similar structure of the barrier to the sham- operated control groups.
TLE causes a decrease in serum concentration of testosterone by affecting sex steroid hormones. Hormonal changes resulting in alter- ations of androgen synthesis and gonadotropin levels might be a cause of sexual function disorders in epileptic patients (Szupera, 2007). Testes contain StAR protein involved in steroidogenesis. StAR pro- tein is involved in the transfer of cholesterol from outer to inner mi- tochondrial membrane. Decrease in the regulation of StAR protein results in decrease of steroidogenesis (Leers-Sucheta et al., 1999; Temel et al., 1997). There is no study in the literature about StAR protein in epileptic models. Other sudies have shown decrease in the expression of StAR as a result of exposure to diethylhexylphthal- ate (Chen et al., 2015), heat stress (Li et al., 2015), diethylstilbestrol (Guyot et al., 2004) and triclosan (Kumar et al., 2009). Similarly, in our study, PTZ-induced epileptic seizures decreased the number of StAR immunoreactive cells and the staining intensity in Leydig cells.
FI G U R E 6 StAR
immunohistochemistry in sham-operated
saline (a) and thymoquinone (b) groups,
and PTZ (c) and PTZ+thymoquinone (d)
groups. Arrows: StAR-positive cells. Bars:
50 µm. (e, f) Statistical analysis of StAR (+)
cell density and StAR staining intensity.
*, ***: Compared with the sham-operated groups; +: compared with the PTZ group.
*, +: p < 0.05; ***: p < 0.001
We observed that thymoquinone administration reversed these ef- fects. We suggest that decrease in the expression of StAR protein might result in decreased levels of testosterone and in reproductive disorders.
Inhibin B is a hormone secreted from testes and regulates FSH secretion. Leydig cells produce inhibin in response to LH secretion (O'Connor & De Kretser, 2004). In a previous study, inhibin B levels were found to be decreased after cisplatin-induced testicular dam- age (Kilarkaje et al., 2013). Leydig cell tumour cells also show posi- tive inhibin immunoreactivity (Taniyama et al., 2001). Azoospermic patients were shown to express inhibin β-B immunoreactivity in the interstitial cells (Dong et al., 2008). Similarly, we detected inhibin im- munoreactivity in the Leydig cell cytoplasm. Staining intensity and immunoreactive cell number in PTZ group were less than that of the sham-operated groups. We concluded that this reduction might af- fect spermatogenesis and reproductive function.
Testis torsion, undescended testis, varicocele, diabetes, infections, hyperthyroidism and epilepsy are some of the disorders causing oxidative stress in testis tissue (Draper & Hadley, 1990; Erboga et al., 2016; Shokri et al., 2015). Thymoquinone is an anti- oxidant and has effects on cell cycle and proliferation, angiogenesis, apoptosis, migration, inflammation, oxidative stress, invasion and metastasis (Güzelsoy et al., 2018). The effect of thymoquinone on testis tissue in animal models of epilepsy has not been investigated before. In a previous study, thymoquinone was shown to prevent apoptosis in testis tissue after methotrexate exposure (Sheikhbahaei et al., 2016). Other studies have shown the ameliorating effect of thymoquinone on testicular damage after morphine (Salahshoor et al., 2018), cadmium (Fouad & Jresat, 2015; Sayed et al., 2014) and lead (Mabrouk, 2018) exposure. It was reported that ischaemia- reperfusion caused decreased PCNA expression and increased apoptotic cells; and thymoquinone reversed these effects (Erboga et al., 2016). In obese rats, it was shown that spermatogenic and Leydig cell number was decreased and spermatozoa showed ab- normal morphology; and thymoquinone administration ameliorated these effects (Tufek et al., 2015). In line with the above studies, the present study showed that thymoquinone increased Johnsen score, decreased the number of TUNEL-positive cells and increased the number of PCNA-positive cells. Moreover, thymoquinone adminis- tration reversed decreased ZO-1, StAR and inhibin β-B immunore- activities in epileptic group to control levels. We thought that these findings are the result of positive effect of thymoquinone on Leydig cells, reproductive organs and sex hormones, and its antioxidant effect.
In conclusion, PTZ administration resulted in severe morphological damage in testis tissue. The reduction in the expressions of
FI G U R E 7 Inhibin β-B immunohistochemistry in sham-operated saline (a) and thymoquinone (b) groups, and PTZ (c) and PTZ+thymoquinone (d) groups. Arrows: Inhibin β-B-positive cells. Bars: 50 µm. (e, f) Statistical analysis of inhibin β-B cell density and inhibin β-B staining intensity. **, ***: Compared with the sham-operated groups; +: compared with the PTZ group. +: p < 0.05; **: p < 0.01; ***, +++: p < 0.001
ZO-1, StAR and inhibin β-B suggests that hypothalamic–hypophyseal– gonadal axis is affected from epileptic seizures. Abnormality in this axis affects spermatogenesis, resulting in dysfunction in male reproductive disorders. Reduction in the ZO-1 expression points to damage in the blood–testis barrier, and this condition is related to infertility. The ameliorating effect of thymoquinone on seizure- induced testicular damage suggests that thymoquinone is a prom- ising agent in male epilepsy patients, whose reproductive system is affected. Besides, the present study is a basis for future experimen- tal studies and will contribute to therapeutic approaches about epi- lepsy related infertility.

ACKNOWLEDG EMENTS
This study was supported by Marmara University Research Fund (SAG-C-YLP-110618-0309).

DATA AVAIL ABILIT Y STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.

ORCID
Fatma Bedia Karakaya https://orcid.org/0000-0001-6054-0752

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