Anti-Oxidative and Anti-Apoptotic Effects of Apigenin on Number of Viable and Apoptotic Blastomeres, Zona Pellucida Thickness and Hatching Rate of Mouse Embryos

Embryonic development in culture medium may be affected by several stressors such as high oxygen concentration and high level of reactive oxygen species (ROS) (1). It is important that embryos in culture media to be protected from oxidative stress. For this purpose, antioxidants are valuable candidates (2).

Apigenin is a plant-derived compound belonging to the flavonoids category presented in various fruits and vegetables, such as parsley, onion, celery, chamomile and orange (3). Apigenin has different biological activities such as anti-oxidative, anti-inflammatory, anti-cancer and anti-tumorigenic properties (4, 5). Apigenin protects DNA from oxidative stress by binding to nucleic acids. Moreover, apigenin prevents cell apoptosis by suppressing ROS compounds (6) and decreasing the expression of caspase-3, caspase-9 and TNF-α (7).

By studying the embryo morphology, prediction of embryo fate is largely possible. The most morphological indicators to select the best embryos for transferring are zona pellucida (ZP) thickness and blastomere quantity (8). ZP thickness is a reliable indicator of in vitro fertilization (IVF) success rate which can be applied as a criterion for embryo selection. Actually, ZP thickness is inversely correlated with embryo viability and hatching rate (9). Moreover, cleavage rate and development to blastocyst are applied as two quality parameters of mammal embryos (8).

Although the beneficial effects of apigenin on different cells and tissues have been investigated (10, 11), there is no report yet concerning the effect of apigenin on growth and quality of embryos. So, in this study, we evaluated for the first time the impact of apigenin on some morphological indicators of pre-implantation mouse embryos including ZP thickness, viable and apoptotic blastomere quantity and hatching rate. To evaluate the anti-oxidant and anti-apoptotic effects of apigenin, we used H₂O₂ and actinomycin D in the culture medium to create ROS and apoptosis.

In this experimental study, female C57BL/6 mice (6-8 weeks) were kept under controlled temperature (25 ± 2°C) and light (12 hours light/12 hours dark), with free access to food and water. All animal protocols were approved by the Research Council of Semnan University of Medical Sciences (Semnan, Iran).

During development of embryos in vitro, various harmful factors can affect embryo quality and fertilization, although it seems that anti-oxidants can reduce the amount of damage (2, 18). In this study, for the first time, we evaluated the effect of apigenin on some morphological indicators of pre-implantation mouse embryos including ZP thickness, number of viable and apoptotic blastomeres and hatching rate. Moreover, to evaluate the anti-oxidant and anti-apoptotic effects of apigenin, we used H₂O₂ and actinomycin D in the culture medium to induce oxidative stress and apoptosis. Overall, the results showed that 10 µM apigenin, by protecting the embryos against H₂O₂ and actinomycin D, was able to enhance the quality and development of embryos and reduce apoptosis in the blastomeres.

Anti-oxidant capacity of apigenin has been shown in different cell types. So that Zhang et al. (19) reported that apigenin has neuro-protective effect on rats after contusive spinal cord injury. Zhao et al. (20) reported that apigenin has neuro-protective, anti-amyloidogenic and neuro-trophic effects on an Alzheimer disease mouse model. Liu et al. (21) reported that apigenin expresses Oct-4, Sox2, and c-Myc in dental pulp cells which helps maintain the dental pulp cells in an undifferentiated stage. However, no report concerns the effect of apigenin on growth and quality of embryos.

In the present study, to evaluate the anti-oxidant effect of apigenin, H₂O₂ was used, which similar to ROS easily penetrates from the cell membrane, causing damage and apoptosis (22). Sharma et al. (6) reported that apigenin attaches to nucleic acid bases and decreases oxidative DNA damage in epithelial cells of prostate. Lagoa et al. (23) showed that flavonoids including apigenin inhibit H₂O₂ production by increasing mitochondrial activity. The purpose of exposing embryos to H₂O₂ was to exacerbate the conditions of ROS in the culture medium (24) and evaluate the anti-oxidant effect of apigenin on protection of the embryos. The results of present study showed that apigenin by reducing the effects of H₂O₂ could protect the embryos and improve embryonic development. These results were in agreement with the other related study (25).

Moreover, to evaluate the anti-apoptotic effect of apigenin, actinomycin D was used as an inducer of apoptosis on different cell types by connecting to guanine-cytosine base pairs and inhibiting DNA transcription (26). Niknafs et al. (27) showed that melatonin improved development of the early mouse embryos exposed to actinomycin D. Abdelrazik et al. (13) reported that l-carnitine reduces apoptosis rate in blastomeres of mouse embryos exposed to actinomycin D. The results of present study showed that apigenin could protect the embryos exposed to actinomycin D and decrease the rate of apoptosis. These results were in agreement with other related studies (13, 27, 28).

Embryo quality is evaluated with morphological parameters. Viable and apoptotic blastomeres quantity, ZP thickness and ability to hatch of blastocyst are some of the most important morphological parameters of embryo (29, 30). Various studies have reported that reducing number of blastomeres could decrease chance of survival of embryos (31, 32). While the number of blastomeres increase, ZP thickness is decreased; in contrast the probability of blastocyst hatching and successful implantation are increased (15, 33).

Regarding the anti-oxidant and anti-apoptotic properties of apigenin, protective effect of this agent on improvement embryo growth is probably due to reduction of the ROS level (34), maintaining the mitochondrial activity (11, 35) and upregulating the gene expression of anti-oxidant enzymes like glutathione peroxidase (25). Glutathione peroxidase is an enzymatic anti-oxidant expressing in many cells and tissues during embryo formation and protecting the embryo against oxidative stress (18, 36). Since the glutathione peroxidase removes H₂O₂ , apoptosis in embryonic cells reduces (18). Han et al. (37) reported that apigenin reduces oxidative stress and neuronal apoptosis in early brain injury following subarachnoid hemorrhage.

ZP thickness is a marker to select the best frozen-thawed embryos for transfer (38), because thin ZP increases the probability of hatching rate and implantation. ZP thickness depends on inherent features of embryos to generate the lytic factors needed for ZP thinning (9, 39). There are many ways to thin or remove ZP such as partial zona dissection, using proteolytic enzymes, laser and Tyrode’s solution (2). But those methods are invasive regarding that adding anti-oxidant into the embryo culture medium is probably less invasive and may cause thinning ZP thickness (15, 40). The present study showed apigenin could decrease ZP thickness of blastocysts. These results are in agreement with the results of Khanmohammadi et al. (15) indicating that l-carnitine, as an antioxidant, has the ability to reduce ZP thickness.

Despite obtaining these results, there are some limitations in this study. More research is required to clarify the molecular mechanisms underlying apigenin function on development and qualifying embryos. In addition, the number of samples was low. Hence, more samples would be needed in different conditions and with different doses of apigenin.

The results of this study suggest that apigenin with anti- oxidant and anti-apoptotic properties may protect the embryos against H₂O₂ and actinomycin D. Apigenin can probably increase the number of viable blastomeres and decrease the number of apoptotic blastomeres, which may cause expanding blastocysts, thinning ZP thickness and increasing the rate of hatching in mouse embryos.