Can Ginger Protective Role Against Bisphenol A-Induction on NMRI Mice Ovarian?

Materials and Methods: adult female mice were divided into 4 groups [n=6]: control, BPA [240mg/kg/day], GE [500 mg/kg/day] and BPA+GE. Oral treatment of GE was performed during 30 days. After treatments, to histological evaluation, ovaries were removed and processed. Serum MDA levels were also determined by the spectrophotometric method. Data were analyzed with One Way ANOVA test and P<0.05 was considered as statistically significant.


Introduction
Since the last decades, there have been numerous concerns regarding the prevalence of reproductive abnormalities in people.
According to the previous studies, the incidence of reproductive abnormalities happens due to some environmental pollutants effecting on the endocrine system of mammals. They also believe these environmental pollutions disrupt the endocrine system [1]. These chemical constituents act like estrogens or they show anti-androgen activity. They employ this method to influence reproductive hormone-dependent activities. These pollutions can enter into the body in various ways which can be oral or respiratory [2].
BPA is one of the largest volumes of chemicals compound which has been producing. It will be produced more than six billion pounds annually [3]. Also, it is an external estrogen which pollutes both water and air. This chemical constituent is used in large scale in the industry. As a polycarbonate plastic monomer, BPA is a component of epoxy resin and polystyrene. It is also utilized in the production of various types of equipment and materials, such as dental filling material, disposable containers, the lining of cans, bottles of mineral water, children's bottles of milk, appliances, toys, and household materials. Moreover, it can be utilized in structures of some polycarbonate and polymer additives as a softening substance [4].
It has been reported that BPA causes depletion of the antioxidant system making stress oxidative. During the studies, this constituent causes shaping hydroxyl free radicals and increases of hydrogen peroxide in the cells [5]. Besides, when the cell is exposed to BPA, the production of free radicals from the electron transport chain in mitochondria is highly increased [6]. According to the studies, BPA causes deactivation and reduction of antioxidant enzymes, such as superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and it finally produces oxidative stress [7].
Also, this substance leads to apoptosis and imposes oxidative stress through increasing the concentration of intracellular calcium, production of reactive oxygen species, and activation of caspase.
Simply put these above-mentioned features make BPA as the main factor of damaged DNA [8].
BPA causes steroid inhibition, a decrease of progesterone, androstenedione, estradiol and disorder precursor in the synthesis of steroids, reducing the estrous cycle, and ovarian weight loss.
This constituent also leads to having ovarian cysts that have a large number, a lesser extent corpus luteum, and many more follicle atresia [9]. Other harmful and pernicious effects of BPA are inhibition of follicular development, reduction in the level of the epithelium, stroma, ovarian glands, and reduction of estrous cycle [10]. Conversely, ginger, a powerful antioxidant in eliminating the harmful effects of BPA on the ovary, is a molecule which is a molecule which has the capability of slowing down or preventing the oxidation of other molecules. Oxidation is a chemical reaction in which electrons are transferred from one material to the oxidizing agent. On one hand, the antioxidants cause a finishing chain of reactions through free intermediate radicals. On the other hand, the antioxidants make themselves oxidized to inhabit other oxidative reactions [11].
The plant phenolics are multi-functional. They, besides, to be used as regenerative and inert oxygen-free radicals, can cause oxidation-reduction [12]. Ginger can be used as a strong natural antioxidant due to having volatile and non-volatile antioxidant constituents in different parts especially in its rhizome [13]. Specific odour and its flavor are due to zingiberene mix, Shogoal, gingerols, and the volatile oils which constitute 3% of the weight of fresh ginger. Their antioxidant properties are related to zingiberene, shogaols, gingerols, and Paradol [14]. Because of these findings, this study was undertaken to determine the beneficial properties of ginger on the ovarian damages in mice.

Animals and Treatments
In the present study, the female mice [NMRI] were used. These animals were taken from Pasteur Institute in Tehran.

Tissue Preparation
After the treatment period [30 days], the mice were anaesthetized by diethyl ether. After that, in order to prove ovarian tissue, the ovaries were removed, and they were put in glasses containing freshly prepared Bruins fixative. For 24 hours, they were kept in laboratory temperature. After 24 hours, the ovaries processing, samples were blocked in paraffin blocks.

Morphometrical Study
After the tissue processing, blocking was performed by Isector method and IUR sections [17,18]. 5 and 20μm thick sections were provided by microtome and they were stained with Hematoxylin and Eosin [H&E].

Luteum [mm 3 ]
To examine the provided samples, morphometrical methods were used. To calculate the total ovarian volume by Cavalieri method [17], first 5 μm thick section were transferred on the work table.
Then randomly counting probe was superimposed on the image.
After that, the hit spots were counted with an image of the ovary.

The Volume of Oocyte, Follicular Cells and their Nuclei
To calculate the volume of oocyte and their nuclei the nucleator method were used. 12 sections from 20μm thick sections was randomly chosen and the selected follicles were studied using the  Germany] connected to a computer. The nuclei of follicular cells were sampled by an unbiased counting frame superimposed on the monitor. According to the counting method on special frame counter, those follicles are selected that their core is either inside of the frame counter or on the lines. And they must be acceptable.

The Number of Follicles
Those that are connected to forbidden lines are not acceptable and they're not counted. Then, the numerical density of follicles was calculated according to the following equation [20]:   After removing the samples from the water bath, they were quickly cooled and centrifuged for 10 minutes. The supernatant was carefully separated and its absorption was at 532 nm to compare a blank containing all the ingredients except the sample. MDA concentration by using the extinction coefficient was calculated and expressed in micro molar [23].

The Volume of Ovary, Cortex, Medulla and Corpus Luteum
The mean total volume of ovary, cortex, medulla and corpus luteum showed a highly significant reduction in BPA group compared to the others [p<0.01], while co-administration of BPA with GE increased the volume of ovary, cortex, medulla and corpus luteum to the control level (Table 1).

The Number of Follicles
The mean number of the primordial follicles, primary, preantral, and antral follicles in the GE group to control group a significant increase was observed when compared to the other groups [p<0/001]. Also, in the BPA + GE group, reduction of the number of follicles in comparison to BPA group was in the control group was significantly compensated [p <0/001] ( Table 2).

The Volume of Oocyte [μm 3 ]
The mean volume of oocyte in primordial, primary, preantral,

The Volume of Oocyte Nucleus [μm 3 ]
The mean volume of the nucleus of the follicular cell in primordial, primary, preantral and antral follicles showed a highly  Table 4).

The Thickness of Zona Pellucida [μm]
Comparing the mean thickness of zona pellucida in the preantral and antral follicles a significant reduction was found in the BPA group [p<0.001], while co-administration treatment of mice with BPA + GE compensated these reductions [p<0.001] to the level of the control group (Table 5).

MDA Serum Levels
Comparing  (Values are means ± SD. The means with the same code do not differ significantly (one-way ANOVA, Tukey test, p>0.05).

Discussion
This study revealed that BPA causes a reduction of the total volume of the ovary, cortex volume, medulla volume, and the corpus luteum volume. It also showed that the co_administration of BPA + GE can compensate for the destructive effects of BPA on the ovary.
In the other words, BPA can cause destruction of corpus luteum and the number in the ovary [24], also BPA can change the level of estradiol, testosterone and progesterone hormones. It would be seen that it changes the GnRH secretion; this could be a reason for reducing the amount of corpus luteum and thereby reducing the size of the ovarian cortex and the total volume. estrogens such as estradiol stimulate the growth of follicles and protect follicles from atresia. Thus, the disturbance of estradiol production by BPA can lead to follicle atresia and reduced ovarian volume [10].
Free radicals play physiological and pathological roles during follicular, oocyte maturation, fertilization the corpus luteum. High accumulation of free radicals can cause ovarian apoptosis. BPA increase lipid peroxidation and the production of free radicals in the ovary [25]. The most important results of them are denaturation of proteins and the membrane lipid peroxidation, as well as increased permeability of the membrane.
These factors can cause the loss of cells that is probably due to the reduction of ovarian volume. These factors can cause the loss of cells that is probably due to the reduction of ovarian volume.
GE accelerates the growth of ovarian follicles, increasing dens of connective tissue stroma, the rapid formation increasing secretion follicle and ultimately increasing the total volume of ovary, cortex and medulla [26]. Besides, the significant reduction of ovarian The analysis showed that BPA adversely affects the ovaries by increasing in production of reactive oxygen species by granulosa cells and inhibition of the growth of follicles [28] and finally reduces the number of them.
Also, BPA decreases oocyte volume and its nucleus by oxidative stress. BPA induce the apoptosis and inhibition of cell entry of the G2 phase of the cell cycle to M phase in the ovarian granulosa cells [29] leading to the inhibition of the growth and development of follicles.
ginger due to having saponins can increase LH and FSH hormones from the pituitary and improving the process of folliculogenesis [30]. GE causes an increase in the activity of superoxide dismutase, glutathione reductase and Glutathione peroxidase. GE inhibits lipid peroxidation and oxidative stress in the ovary [31]. This would be a reason to compensate for the destructive effects Bisphenol on the ovarian and a follicular reduction that is compensated for due to it.
The following study also illustrated that Bisphenol can cause decreasing of the thickness of zona pellucida in preantral follicles and antral follicles that zona pellucid has consisted of glycoprotein, and as it mentioned earlier, BPA can decrease the level of proteins [32]; Therefore, the reduction of the thickness of zona pellucida in follicles is something normal. On one hand, BPA results in a reduction in estradiol production, in a way that it can reduce the proliferation of granulosa cells and showed an increase in apoptosis [27]. Bisphenol induces apoptosis in granulosa cells thus, BPA by the induction of apoptosis in granulosa cells, decreasing estradiol and LH in both direct and indirect ways causes a decrease of the thickness of zona pellucida. GE by stimulating gonadotropin secretion of LH and FSH hormones can cause the production of estrogen and by boosting antioxidant systems can compensate for the reduction of the thickness of zona pellucida in follicles [30].
Besides, the measuring of lipid peroxidation showed that BPA would increase the production of MDA. When the cell exposed to BPA, the production of free radicals will increase in electron transport chain into the mitochondria [6]. According to the study, BPA causes deactivation and decrease of antioxidant enzymes such as superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase finally producing oxidative stress [31]. On the other side, GE due to having volatile and non-volatile antioxidant compounds especially in its different parts of the rhizome which can be used as a natural antioxidant, vitamin C and flavonoids and phenols contain in this extract can inhibit the lipid peroxidation.
By boosting the antioxidant system and improving the performance of ovarian cells, ginger can compensate for the reduction of the thickness of zona pellucida in follicles. This extract also inhabits protein denaturation and membrane lipid peroxidation [26] Thus, it prevents the loss of proteins and glycoproteins and eventually pellucida thickness would have remained.

Conclusion
Based on the results of this study GE can noticeably improve the adverse effects of BPA on ovarian tissue disorders in adult mice.
It can also be used because of the effective herbal antioxidants that have against toxicity oxidative stress caused by BPA on ovarian tissue.