Morin improves Bisphenol-A-induced toxicity in the rat testicular mitochondria and sperms

Objective The present study aimed to examine the ameliorative effects of Morin (MRN) on Bisphenol-A (BPA)-induced oxidative stress in testicular mitochondria and sperm quality of rats. Methods BPA and MRN (25, 50, and 100 µM) were given to the spermatozoa and testicular tissue mitochondria. The sperm quality, mitochondrial viability, and MMP (mitochondrial membrane potential) were examined. Superoxide dismutase, CAT (catalase), malondialdehyde, and reactive oxygen species (ROS) levels of rat testicular mitochondria were measured. Results BPA raised mitochondrial oxidative stress biomarkers, whereas antioxidant acclivity and MMP were significantly lowered. BPA significantly lowered the normality, viability, and motility of the sperms. MRN dose-dependently lowered oxidative stress of the mitochondria, raised MMP, as well as improved the percentage of abnormality, motility, and viability of the sperms. Conclusions These data demonstrated that MRN dose-dependently attenuated BPA-induced mitochondrial damage and improved sperm quality by preventing oxidative stress.


INTRODUCTION
Bisphenol-A (BPA) is commonly used for producing clear and hard plastics for lining inside metal-based foods and beverage cans, sealants, toys, lenses, compact discs, etc… (Wazir & Mokbel, 2019;Vom Saal & Vandenberg, 2021). BPA through inhalation, ingestion, and dermal contact can penetrate into the human body. The exposure of individuals to BPA is associated with its environmental and food amounts (Ribeiro et al., 2017). BPA exists in breast milk, semen, plasma, and amniotic fluid (Suteau et al., 2020). BPA induces testicular damage and impairs the spermatogenesis of rats and other animals (Ullah et al., 2018;Anjum et al., 2011;Knez et al., 2014). BPA can impair mitochondrial function by lowering ATP, reducing mitochondrial mass, and altering the mitochondrial membrane integrity (Kaur et al., 2018). Mitochondrial dysfunction affects sperm generation and spermatozoa motility (Kamali Sangani et al., 2017). Additionally, BPA lowers antioxidant capacity and raises oxidative stress in testis (Chitra et al., 2003).
Current researches have focused on finding protective natural materials against male reproductive system dysfunctions. Recently, flavonoids have been extensively studied for ameliorating male reproductive damages (Farombi et al., 2012;Guvvala et al., 2017). Morin (MRN; a member of flavones) is plenty found in figs, sweet chestnuts, almonds, and other members of the Moraceae family. MRN was shown to have several beneficial impacts, including free radical scavenging, anti-tumorigenicity, anti-inflammatory, as well as DNA protection (Hussain et al., 2014;Meydanli et al., 2018). Recent studies have shown that MRN protects against sperm alterations and testicular oxidative stress induced by various chemicals in animals (Olayinka et al., 2018;Hussain et al., 2014).
The positive impacts of MRN against BSA-induced mitochondrial oxidative stress and impaired quality of rat sperms were the aim of the current study.

Study design (Figure 1)
The sperms and mitochondria were obtained from 15 male Wistar rats (180-200 g). The Ethics Committee of Animal Research approved current work (REC: 1398-016). The sperms were obtained from the epididymis of the rats and categorized into the below groups: Group 1. Treated with Ham's F-10 media only for 4 hours (Control) Group 2. Received BPA (0.8 μM) for 2 hours Groups 3-5: Received 25, 50, and 100 μM MRN, respectively, for 2 hours before BPA.
Group 6 (MRN): Received 100 μM MRN for 4 hours For each group 5×10 6 sperm/ ml were used for treatment with MRN or BPA. Because the unexposed sperms died after 4 hours, a total time of 4 hours was set for treatment with MRN or BPA. MRN (Sigma) or BPA (Sigma) were dissolved in 0.1% DMSO and then were diluted in Ham's F-10 media. BPA concentration was based on the MTT results (Table 1). The safety of DMSO was evaluated by MTT assay (Table 2).

Mitochondria isolation
The testicles of rats were dissected and minced in a buffer containing HEPES-KOH (5 mM), fat-free BSA (0.1%), EDTA (0.1 mM), EGTA (0.2 mM), and sucrose (250 mM), and then were homogenized. The mitochondrial fractions were centrifuged (at 4°C) 1 time for 10 minutes at 3,000·g and 3 times for 7 minutes at 10,000·g. The protein content was detected using the Bradford reagent (Invitrogen). An amount of 0.5 mg protein/ mL was exposed to BPA or MRN.

MTT assay
Spermatozoa (5×10 6 sperm/ml) or mitochondria were plated and exposed to MRN or BPA. MTT at a concentration of mg/ mL (Sigma, USA) was added to the treated subjects and incubated at 37°C for 1 hour. Then the media was   Values are expressed as mean ± SD (n=6). discarded, and DMSO (100 μL) was added to the wells, and the absorbance was read at 570 nm.

Sperm motility and morphology
In brief, 10 μL sperm suspension was placed in an analyzing semen chamber, and motility of at least 200 sperms was evaluated for each sample. Sperm motility was graded according to WHO guidelines to estimate the percentage of fast progressive (A), slow progressive (B), no progressive (C), and immotile sperm (D).
The sperm suspension was stained with 10%nigrosin and 1% eosin for morphological observations. For each group, 100 sperms with six replications were evaluated for abnormality estimation. The normal sperms had a hook-like shape acrosome with a straight neck and a free-end single tail. While the abnormal sperms had a smaller head, broken neck, branched tail, etc. The control and experimental samples were blindly analyzed with 3 co-workers.

Determining antioxidant levels, MDA content, and ROS formation
The mitochondria of each group were placed into a micro-tube, and DCFH-DA (10 μM; Sigma) plus Hank's buffered salt solution (100 μL) was added and incubated for 30 minutes. A spectrofluorometer (Ex: 490nm and Em: 570nm) was applied to detect ROS levels. Malondialdehyde (MDA), superoxide dismutase (SOD), and CAT (Catalase) levels were explored by an available commercial kit (Zell-Bio Company).

Statistical Analysis
Analysis of variance (one-way) in SPSS (version 21.0) followed by posthoc pairwise comparison was used in this study; p-values less than 0.05 were deemed significant.

MTT assay
The survival rate of spermatozoids and testicular mitochondria was decreased considerably following exposure to BPA (p<0.01). MRN raised the viability of the sperms and mitochondria in the BPA treatment (Figures 2 and 3). MRN at a concentration of 100 µM could reverse the viability of sperms and mitochondria near to the control (p<0.01).

Sperm quality
MRN slightly raised total sperm motility compared to controls. In the BPA treatment, the fast progressive percent (p<0.05) and total sperm motility (p<0.01) were significantly lowered, whereas the percentage of immotile spermatozoa was significantly raised (p<0.01). MRN at the concentrations of 50 and 100 µM could lower the percent of immotile sperm and raise total sperm motility after BPA treatment (Table 3 and Figure 3). The abnormality of sperms was significantly elevated in BPA-intoxicated sperms (p<0.01). Pretreatment with MRN could concentration-dependently attenuate the abnormality of the sperms in comparison to the BPA group.

Antioxidant levels, MDA content, and ROS formation
ROS and MDA levels were noticeably raised in the BPA group (p<0.01). MDA and ROS levels were lowered in the MRN-treated groups, compared with the control. MRN concentration-dependently lowered ROS generation in the BPA-exposed mitochondria (Figure 4). CAT and SOD activity were significantly lowered after treatment with BPA (p<0.01). In the MRN-treated mitochondria, the antioxidant activity was greater than the unexposed group. MRN concentration-dependently reversed the BPA-lowering antioxidant activity of the mitochondria (Figure 4).

MMP Assay
In the BPA group, MMP significantly lowered compared with the control (p<0.01). MRN dose-dependently could raise the MMP of BPA-treated mitochondria ( Figure 5). MRN at the concentration of 100 µM could reverse the MMP of BPA-exposed mitochondria near to the normal (p<0.01).

DISCUSSION
The present study evidenced that BPA significantly reduced motility, normal morphology, and survival of the spermatozoa. In parallel with our finding, BPA lowered the quality of the sperm in several human and animal studies (Knez et al., 2014;Kotwicka et al., 2016). We found that MRN concentration-dependently enhanced viability, normal morphology rate, and fast progressive movement of the  BPA-treated rat's sperms. The amelioration effect of MRN against BPA-impaired sperm quality was similar to the previous reports (Khaki et al., 2010;Merwid-Ląd et al., 2014). MRN considerably reversed the altered sperm parameters in the procarbazine (an alkylating agent)-induced sperm toxicity in rats (Olayinka et al., 2018). Arisha et al. (2019) have shown that silver nanoparticles reduce motility, viability, and concentration of sperms, disrupt the blood-testis barrier, and induce histological changes in rat testis. They have found that MRN can restore the reproductive alterations to their normal range. Hussein et al. (2019) reported that the administration of MRN with or without rutin led to a considerable decrease in the percentage of sperm abnormalities and alleviated the toxic effects of TiO 2 nanoparticles. MRN may improve sperm survival by preventing cell death signaling. MRN could significantly change the ratio of the pro-apoptotic and anti-apoptotic molecules and effectively prevent apoptosis induced by silver nanoparticles in the testicular cells . The MRN improved the motility of spermatozoa may be due to the beneficial impacts of MRN on the testicular mitochondrial (Ola et al., 2014).
The decreased sperm quality by the BPA was accompanied by enhancing oxidative stress. The BPA-raised ROS and MDA levels of the mitochondria were in line with other reports Kaur et al., 2018). The excessive generation of ROS attacks sperms and induces MDA production leading to lipid peroxidation Begum et al., 2018). MRN reversed MDA levels, ROS production, antioxidant biomarkers, and MMP in the BPA-exposed mitochondria. Therefore, MRN may protect spermatozoids via lowering ROS generation and lipid peroxidation. It has been documented that MRN protects mitochondrial from oxidative disorders in various pathological cases. MRN significantly enhanced antioxidant activity and lowered MDA level when co-administered with silver nanoparticles . MRN could preserve antioxidant defense systems against rat testicular damages induced by bicalutamide (Olayinka et al., 2018). MRN could reverse SOD, CAT, GSH, and MDA levels in rat testicular toxicity induced by dutasteride-tamsulosin. In another study, MRN ameliorated TiO2-Nanoparticles-induced testicular and prostatic toxicity by activating antioxidant systems and reducing apoptosis .
The BPA reducing MMP was similar to the previous studies (Wang et al., 2021;Mahdavinia et al., 2019;Barbonetti et al., 2016). MMP was positively correlated with total sperm numbers and progressive sperm motility (Zhang et al., 2016). The BPA reducing MMP was accompanied by enhancing mitochondrial oxidative stress, lowering spermatozoid motility, and attenuating sperm survival. BPA may cause mitochondrial damage by producing lipid peroxidation and impair spermatozoid function (del Hoyo et al., 2010). MRN concentration-dependently reversed the effects of BPA on MMP. Hence MRN may decrease ROS level by increasing MMP and consequently lowering germ cell loss. These results were in parallel to available documents that indicated the positive impacts of MRN on mitochondrial activity . Ola et al. (2014) have reported that MMP and sperm quality of infertile individuals enhance in MRN-exposed samples.

CONCLUSION
In summary, MRN could improve MMP and lowered mitochondrial oxidative stress. MRN could also effectively improve the normal morphology, survival, and motility of the rat sperms. MRN may ameliorate BPA-caused mitochondrial toxicity and rat sperm impairment by suppressing oxidative stress.