Ameliorative Effect of Crocin on Sperm Parameters and In Vitro Fertilization in Mice under Oxidative Stress Induced by Paraquat

Paraquat (PQ) (N, N'-dimethyl-4,4'-bipyridinium dichloride) is a pyridine compound that contains an ammonia sodium moiety, which activates oxidation. Oxidation begins with the methylation of chloromethane. PQ is a nonselective contact herbicide that is used to control annual weeds (1).

In general, the fertility rate of men exposed to toxins in the workplace is significantly lower (2). PQ is a highly effective, fast-acting, and nonselective herbicide widely used throughout the world. Human exposure by either respiratory or systemic routes leads to the accumulation of PQ in the lungs, resulting in pulmonary oedema, bronchial and alveolar destruction, and ultimately fibrosis with a high mortality, which is in part caused by the lack of a specific antidote to PQ (3). Chronic exposure to PQ is associated with liver damage, kidney failure, and Parkinsonian lesions, in addition to fibrosis (4). Upon entering the cells, PQ undergoes cyclic single-electron reduction/ oxidation through its quaternary ammonium nitrogen atoms and bipyridyl ring, and produces reactive oxygen species (ROS) and PQ radicals. Redox cycling is believed to play an important role in initiating lung damage and fibrosis by paraquat. The mechanism by which oxidative signals from PQ interact with the pathways that underlie the lung fibrogenic response is poorly understood (5). PQ reduces the cellular oxidation cycle with its tangible presence in this process, which results in the production of ROS (6). In a rat model, free radicals have been shown to accumulate in the testicular torsion, causing inflammation and damage to the tissues by membrane lipid peroxidation (7). Because of the scant amount of cytoplasmic antioxidant enzymes, the sperm cannot repair the damage caused by oxidative stress. Studies have shown that antioxidants such as vitamin C have extensive ameliorative effects on sex hormonal status and can protect sperm from ROS-induced abnormalities. These compounds also inhibit ROS produced by leukocytes, improve the quality of semen, and prevent DNA fragmentation and premature sperm production (8). Accordingly, antioxidants such as Fumaria parviflora on testicular injury induced by torsion/ detorsion in adult rats have confirmed reduced DNA damage and apoptosis in sperms, as well as increased implantation and pregnancy (9).

Carotenoids, by acting as biological antioxidants, protect cells and tissues from the damaging effects of free radicals and singlet oxygen, and play a significant role in human health. Crocus sativus L., commonly known as saffron, is a stemless herb of the Iridaceae family. The major bioactive compounds in saffron are crocin, safranal, and picrocrocin. Crocin, glycosyl esters of crocetin, are unusual water-soluble carotenoids, and are responsible for the characteristic colour of saffron (10). Numerous studies have shown that crocin (Cr) can produce a variety of pharmacological effects, such as protection against cardiovascular diseases (11), inhibition of tumour cell proliferation (12), and neuroprotection (13). Saffron has a role in sexual enhancement (14). It also has been reported that Cr inhibits lipid peroxidation in the kidneys (15). The antioxidant and radical scavenging activities of Cr have also been reported in several in vitro models (16). In the current study, we aimed to evaluate the protective capacity of Cr on quality and fertilization potential of mice sperm against the toxicity caused by paraquat.

We purchased PQ with formulation of SL20%, (Exir Co., Iran). Cr was purchased from Sigma-Aldrich (USA) in the form of a powder.

In this experimental study, 28 adult mice (20 to 25 g) were randomly divided into four equal groups and allowed to adjust to their surroundings for one week before the start date of the experiments. All the ethical issues were carried out based on guidelines of the Ethics Committee of Urmia University, Faculty of Veterinary Medicine (ethics number: ECVU-173-2018). The mice were kept on a 12-hour light/12-hour dark schedule with free access to adequate water and food. The animals were fed with pellets and wheat, and tap water was used as drinking water. The treatment period lasted 35 days.

The mice were assigned to the following experimental groups: control group, which received intraperitoneal (IP) injections of normal saline (0.1 ml/day); PQ group received PQ (5 mg/ kg/day, IP) (17); experimental (PQ+Cr) group received Cr (200 mg/kg/day, IP) two hours before PQ (5 mg/ kg/day, IP) (18); and positive control (Cr) group received Cr (200 mg/kg/day, IP).

At the end of the treatment all mice were weighed one hour before the beginning of the sampling. They were anesthetized by ketamine (40 mg/kg) and xylazine (5 mg/ kg), and were euthanized by dislocation of their neck vertebrae.

In the present study, we evaluated the experimental groups in two sections: spermatogenesis and early embryonic growth. The results of the first part of the experiment showed that PQ could significantly decrease sperm quality; thereby, in the second part, the PQ group had decreased percentages of fertilization, two-cell embryos, four-cell embryos, blastocysts, and hatched embryos. This group also had increased percentages of whole arrested embryos, and types I, II, and III arrested embryos. In this study, PQ could significantly reduce the number of sperm, the average percentages of sperm motility, and sperm viability, and could significantly increase the percentages of immature sperm and those with damaged DNA, which significantly differed from the control and Cr groups. The results of the two sections of the experiments showed that Cr in the experimental group significantly improved the damage induced by paraquat. In most of the studied parameters, we observed a significant difference between the PQ+Cr and PQ groups.

In previous studies, it has been reported that oxidative stress in animals can cause infertility by affecting the genital organs (25). In a study conducted on men, it has been shown that psychological stress can reduce both motility and sperm quality, and lead to infertility (26). Genital damage is one of the known side effects of PQ poisoning, which has a toxic effect on reproductive systems of both sexes, and it can disrupt the process of spermatogenesis and oogenesis (27). The effects of low doses of PQ on the spermatozoids of Sprague-Dawley rats were studied. The researchers noted that the mean number of sperm decreased on the seventh and fourteenth days after the injection, and abnormal sperm significantly increased. Sperm mortality also increased with higher doses. In this study, it was found that PQ has genotoxic and cytotoxic effects on male germinal cells (28).

The first indication of an increase in ROS is the loss of sperm motility (29). The production of free radicals in mitochondria damage the DNA of mitochondria, and it can also damage the mitochondrial region of the middle part of the spermatozoid (30). Mitochondrial damage of the middle part of the sperm leads to a progressive decrease in sperm motility in terms of decreasing the numbers of motile sperm and decreasing the vehemence of motility (31).

Antioxidants in semen are categorized within the endogen antioxidant group. Several studies have shown that antioxidants do not reduce sperm motility; however, they increase Sperm capability (32). Laboratory studies have also confirmed the role of antioxidants in reducing ROS production and improving the evolutionary ability of the foetus (33). Results of a study revealed that administration of citrus flavonoid extract significantly increased the total antioxidant capacity (TAC) and superoxide dismutase (SOD) levels, and sperm percentage, viability, and motility, and decreased MDA levels. This suggested that citrus, as an antioxidant, may be promising for enhancement of healthy sperm parameters (34).

In the present study, the number of fertilized oocytes in the PQ group significantly differed from the control and Cr groups, which indicated that PQ had a negative effect on fertility. This might be due to an increase of free radicals in the testicular tissue and semen, and ultimately damage the membrane of the gametes; it can reduce the percentage of IVF. However, Cr with PQ compensates for this failure.

Cr appears to have an antioxidant activity when tested in vitro. This activity is linked to half of its sugar content (35). In one study, the antioxidant activity of Cr was evaluated and showed that administration of Cr reduced the level of MDA and increased ferric reduction antioxidant power (FRAP) following stress from renal ischemic reperfusion (36). In one study, the results of IVF showed an increase in fertilization, two-cell embryos, and blastocysts in the group that received Cr and cyclophosphamide (CP) in comparison with the CP-only group. It was also shown that in mice that received Cr along with CP the overall numbers of the arresting embryos was decreased in comparison with the CP-alone group. In this study, administration of Cr along with CP reduced MDA malondialdehyde and sperm with damaged DNA in comparison with the CP group (19).

In the present study, the average number of blastocytes and embryos at the hatching stage in the PQ group significantly differed from the control group. In both cases, the combined use of PQ with Cr ameliorated this effect. This finding showed the antioxidant effect of crocin, while the use of Cr alone did not show any negative effect on any of the above parameters. The PQ group had a significantly different number of arrested embryos compared to the control group, and this effect might be due to the destructive effect of PQ on the membrane and genome of the embryos, whereas Cr has a recovery effect. PQ also caused a negative effect on the percentage of the type I arrested embryos, and Cr ameliorated the percentage of the type III arrested embryos, which was due to its antioxidant properties.

The onset of fragmentation was also caused by the effect of PQ on the fertilized oocytes, which might be due to the effects of PQ on genetic and intracellular factors. This decrease was also observed in the four-cell zygotes, while Cr showed a better protective effect on two-cell zygotes. The effect of PQ on the cellular stage was such that Cr could not compensate for it at this stage. The two factors that protect the sperm DNA against oxidation are the density of the DNA nucleus and presence of antioxidant agents in semen plasma (37). In a study on diabetic men, it was found that the mechanisms that led to the damage of sperm DNA with increasing ROS, which could be a factor in the glycosylation of the final products of advanced glycation end products (38). In addition, the increase of oxidative stress and high fragmentation of DNA continuously occurs with apoptosis (39).

According to the findings of this study, we concluded that crocin, as an antioxidant, protected the male genital organs against the impacts of oxidative stress induced by PQ and significantly ameliorated both sperm quality and IVF outcomes in paraquat-treated mice. However, this study should be performed at the serological and molecular levels because there is not an adequate knowledge about the effects of PQ poisoning on in vivo embryo development.