The current study revealed that CP treatment caused a decrease in the total number of Sertoli, Leydig, and spermatogonia cells and total testicular volume, as well as structural deterioration in the testis and epididymis. It also caused a significant increase in apoptosis ,oxidative stress, and DNA damage indexes. Due to these effects of CP, there have been impairments in testosterone secretion and spermatogenesis. However, GA significantly improved the side effects induced by CP, by reducing testicular structural deformities, apoptosis, DNA damage, oxidative stress, and improving testosterone secretion.
Chemotherapeutic agents are considered to be among the high-risk factors of infertility (Nna et al. 2020). Typically, infertility occurs after chemotherapy and radiotherapy. CP, an antineoplastic agent, is widely used to treat tumors. It causes cytotoxic effects in proliferating cells by damaging the binding of DNA base pairs. Excessive cell proliferation in spermatogenesis makes the testis more sensitive to these agents (Mercantepe et al. 2018). Therefore, protecting the testicles from CP-induced toxicity has become an important issue in chemotherapy with CP (Afsar et al. 2017). Previous studies have reported that CP-induced testicular toxicity can be prohibited with treatment of antioxidants such as melatonin, vitamin C, and resveratrol (Reddy et al. 2016; Almeer and Abdel Moneim 2018; El-shafaei et al. 2018). Additionally reported that GA has protective effects against CP-induced nephrotoxicity, ototoxicity and genotoxicity of the bone marrow (Akomolafe et al. 2014; Shruthi et al. 2014; Kilic et al. 2019). However, in our literature review, no study was found on the effects of GA on testicular and epididymal damage induced by CP.
It has been reported that CP increases ROS production, lipid peroxidation, and denaturation of structural proteins, additionally decreases the activity of the antioxidant system and promotes cell apoptosis by activating p53 (Dasari and Tchounwou 2014; Kohsaka et al. 2020). Oxidative stress is accepted as the result of the disruption of the balance between ROS production and antioxidant enzyme levels (Ekinci Akdemir et al. 2019). Oxidative stress causes testicular damage, testicular apoptosis, DNA damage, and male reproductive dysfunction, which can result in infertility (Shati 2019). Previous studies have reported that CP treatment caused the increased in level of lipid peroxidation and the decreased in level of SOD, CAT and GSH (Reddy et al. 2016; Azarbarz et al. 2020). Similarly, Afsar et al. (2017) reported that CP increased the level of oxidative stress parameters while decreased the activity of antioxidant enzymes. In line with these studies, we observed that CP increased MDA level and decreased CAT level in testicular tissue. Decreased antioxidant enzymes cause the testis to remain vulnerable to the toxic effect of CP. However, the finding of our study indicated that GA can protect the testis against oxidative damage by improving the increased MDA and decreased CAT activity in rats treated with CP. The beneficial effects of GA are mainly related to its antioxidant properties as it acts as a radical scavenger. Previous studies have reported that in doxorubicin (DOX) -induced testicular injury, GA significantly improves increased MDA and decreased antioxidant enzymes (Olusoji et al. 2016). In addition, Oyagbemi et al. (2016) reported that GA significantly improved MDA, nitrite, and H2O2 levels and decreased SOD, GSH cyclophosphamide-induced testicular and epididymal damage.
Apoptotic proteins classified according to their role in apoptosis are classified as pro-apoptotic and anti-apoptotic proteins (Abotaleb et al., 2018). Bax protein, a proapoptotic protein, increases under oxidative stress conditions and subsequently release the cytochrome C impairing the mitochondrial membrane permeability (Fouad et al. 2017). Cytochrome C binds to apoptotic protease-activating factor 1 (Apaf-1) and activates it. Activated Apaf-1 forms the apoptosome. Apoptosis is maintained by caspases formed in response to cell death stimuli. The apoptotic protease-activating factor 1 (Apaf-1) controls caspase activation. As a result, activation of caspase-3, which plays a key role in the execution of apoptosis, is stimulated (Riedl et al. 2005). Since the activation of caspase-3 is an irreversible stage that induces apoptosis, it is frequently used in studies to evaluate apoptosis (Mercantepe et al. 2018; Nna et al. 2020). Proteins in the Bcl-2 families control cell death and survival by regulating the mitochondrial apoptotic pathway. Bcl-2, an anti-apoptotic protein, maintains mitochondrial membrane integrity (Fouad et al. 2017). Previous studies have reported that CP causes upregulation of caspase-3 and downregulation of Bcl-2 (Nna et al., 2020; Kohsaka et al. 2020). Similarly, Aly and Eid (2020) have reported that CP treatment increased the activity of caspase-3 and bax while decreased the activity of bcl-2 in testis. They also showed that resveratrol decreased activity of caspase-3 and bax but increased activity of bcl-2. In another study, Eren et al. (2020) reported that CP increased the caspase-3 immunoreactivity in the germinal epithelium and Leydig cells. However, they also revealed that amifostine and melatonin decreased the caspase-3 immunoreactivity in the germinal epithelium and Leydig cells. The present study revealed that CP treatment significantly increased the expressions of caspase-3 and Bax, which are important markers of the apoptotic index, while significantly decreased Bcl-2 expression in the testis. However, GA treatment significantly decreased an increase in expression of caspase-3 and Bax and increased a decrease in expression of Bcl-2. Thus, these findings of our study proved that GA could protected against CP-induced testic toxicity by regulating expressions of caspase-3, Bax and Bcl-2.
Morphometric examination of the testis is very important to determine male reproductive injury by CP-induced. Based on histological semi-quantitative evaluation, Jahan et al. (2018) reported that CP decreased sperm production, seminiferous tubule diameter, number of spermatogonia, spermatocytes and spermatids compared to control group. They also showed that rutin co-treatment could ameliorated a decreases in sperm production, cell number and tubule diameter. Similarly, Azarbarz et al. (2020) revealed that CP treatment significantly decreased the seminiferous tubules diameter, germinal epithelium thickness, the number of spermatogonia, spermatocyte, and Sertoli cells. In another study, semiquantitative data of Afsar et al. (2017) revealed that CP caused a reducing of the number of Leydig cells inducing the degradation in Leydig cells. They also showed that Acacia hydaspica extract ameliorated the degradation in Leydig cells. The quantitative findings of our study revealed that CP decreased the total number of spermatogonia, Sertoli, and Leydig cells in the morphometric examination performed by unbiased stereological methods. This decrease in testicular cells may be a result of apoptosis induced by increased ROS production caused by CP (Köroğlu et al. 2019). Also, quantitative data of our study obtained using stereological methods demonstrated that total volume of testis, total volume of seminiferous tubule and germinal epithelial height significantly decreased in animals that received CP compare to control groups. We estimate that the decrease in these parameters of our study may have been due to the decrease in the total number of testicular cells caused by CP. These changes in testicular morphometric parameters confirmed that CP treatment causes toxicity of testicular tissue. However, GA treatment inhibited the total volume of testis, total volume of seminiferous tubule and germinal epithelial height.
According to histopathological examinations of previous studies, Köroğlu et al. (2019) reported that CP caused the atrophy in seminiferous tubules, an decrease in spermatogenic cell lines and an increase in number of abnormal spermatozoa. They also showed that Apocynin ameliorate the these pathological changes due to CP. Shati (2019) reported that CP increased abnormal sperm number, decreased the sperm count and motility. Besides, they demonstrated that Resveratrol aleviated these structural distortions. Azarbarz et al. (2020) revealed that CP caused a significantly decrease in Johnsen’s testicular score. In accordance with these studies, The histopathological findings of our study proved that CP treatment caused the atrophy in the seminiferous tubules, a decreased sperm cells in tublulus lumen, and a decrease in the number of germinal epithelial layers, and a significant decrease in the Johnsen testicular score. Additionally, CP caused the reduced mature sperm count in epididymis. However, GA significantly improved these structural deteriorations caused by CP in testis. It is estimated that the decrease in sperm count in the epididymis may be related to the deterioration in spermatogenesis, as a consequence of the decrease in the number of spermatogenic cells caused by CP. In our study, the decreases in the total number of testicular cells, the germinal epithelium height, the Johnsen score, and the increase in the number of caspase-3 and Bax immunopositive cells confirm each other.
The previous studies reported that CP causes DNA damage by causing cross-linking of DNA double strands of proliferating cells especially in the testis (Mohammadnejad et al., 2012). 8-OHdG, an important biomarker of DNA damage and oxidative stress, is widely used in experimental studies to determine DNA damage (Köroğlu et al. 2019). Mitochondrial and nuclear DNA damage caused by ROS can be detected with 8-OHdG (Mercantepe et al. 2018).
Köroğlu et al. (2019) found that CP treatment induced a higher levels of 8-OHdG compared to conrtol group in testis of rats. But, they also showed that Apocynin alleviated the DNA damage reducing the levels of 8-OHdG. In another study, Eren et al. (2020) reported that the 8-OHdG was increased in testis of CP-received rats in the germinal epithelium and Leydig cells. However, they also revealed that amifostine and melatonin decreased the levels of 8-OHdG in the germinal epithelium and Leydig cells. In our study, CP significantly increased 8-OHdG expression, which is a marker of DNA damage and oxidative injury. These findings of our study are consistent with previous studies (Amin et al. 2012; Mohammadnejad et al. 2012). However, GA treatment significantly reduced 8-OHdG expression in testis of CP-applied rats.
Testosterone, which plays a prominent role in the initiation and maintenance of spermatogenesis, is secreted by Leydig cells. Previous studies demonstrated that CP decreased the testosteron but, rutin increased the decrease in testosteron caused by CP (Jahan et al. 2018). Similarly, Salem et al. (2012) reported that CP treatment caused the decrease in the testosteron level and Selenium and Lycopene attenuated the decrease in testosteron levels induced by CP in the Wistar rat model. In addition, Salah Azab et al. (2019) found that CP treatment sgnificantly decreased the testosteron levels but taurine sgnificantly increased the testosteron levels in cisplatin plus taurine group compared to cisplatin group. In our study, serum testosterone levels significantly decreased in rats applied CP. According to findings of our study, it is estimated that the decrease in serum testosterone level caused by CP application, may be related to the decrease in Leydig cell number as a result of increased apoptosis in Leydig cells. Because testosterone secretion is directly related to Leydig cell function and number (Gholami Jourabi et al. 2021). Decreased testosterone level caused by may cause disruption of the spermatogenesis process and thus may cause male infertility.