Potassium bromate disrupts mitochondrial distribution within murine oocytes during in vitro maturation.

PURPOSE: As disturbed mitochondrial distribution is thought to be a cause of the aging of oocytes, it was investigated whether oxidizing agents exert harmful effects on nuclear maturation and mitochondrial cluster formation in murine oocytes and whether antioxidants could rescue such harmful effects in vitro.
METHODS: Oocytes were obtained from female Institute of Cancer Research mice 48 h after an intraperitoneal injection of 7.5 IU pregnant mare serum gonadotropin. The oocytes were cultured with potassium bromate, an oxidizing agent, in the presence or absence of the antioxidant, resveratrol. After 12 h, the nuclear phases and mitochondrial distribution were observed.
RESULTS: Significantly decreased rates of metaphase II (MII) oocytes were observed with 750 μM and 1000 μM of potassium bromate, while a significant increase in abnormal mitochondrial clusters was induced at 500 μM, 750 μM, and 1,000 μM. The addition of 10 μM or 20 μM resveratrol improved both MII maturity and the cluster formation rates in the presence of potassium bromate.
CONCLUSIONS: The addition of potassium bromate reduced MII maturity rates and induced abnormal mitochondrial cluster formation. This effect was alleviated by the antioxidant, resveratrol. The in vitro model used herein is useful for investigating the functions of antioxidants in the aging of oocytes.

INTRODUCTION

In vitro maturation (IVM) is an assisted reproductive technique to obtain mature mammalian oocytes involving a culture of immature oocytes that were harvested at the germinal vesicle (GV) stage from ovarian follicles. In vitro maturation technology has led to the mass production of livestock because it allows mature oocytes to be obtained in large quantities from immature oocytes.

For human‐assisted reproductive technology, the first pregnancy and delivery with IVM was reported in 1991.1 Using an IVM technique to induce mature oocytes with gonadotropin helps to avoid the risk of some of the harmful effects of ovarian hyperstimulation syndrome.2, 3 In vitro maturation is also useful in conserving the fertile ovaries of patients who require chemotherapy or radiotherapy.4 However, the embryonic development rates using IVM remain quite low in both animals and humans.5, 6, 7, 8, 9, 10, 11, 12 This is thought to be one of the causes of insufficient cytosolic maturation in IVM.13 Intracellular glutathione concentrations,14, 15 as well as the distribution patterns of free calcium ions16 and cortical granules,17 are factors that are reportedly related to cytoplasmic maturation. Notably, it has been observed in recent years that a decline in mitochondrial function is related to poor oocyte quality.18

Aged murine oocytes often show an abnormal mitochondrial distribution.19 It has been reported that abnormalities in the mitochondrial distribution of aged oocytes are associated with decreased mitochondrial function. Oxidative stress is considered to be a primary cause of this mitochondrial abnormality. Therefore, it was investigated whether the disruption of oocyte nuclear maturation and abnormal mitochondrial distribution could be induced artificially by using compounds with an oxidizing effect to create an aged oocyte model. Potassium bromate, which is used as a food additive, reportedly acts as an oxidizing agent to increase the amount of 8‐hydroxydeoxyguanosine, a marker for DNA oxidative damage.20

In this study, the effects of potassium bromate as an oxidizing agent on oocyte nuclear maturation and mitochondrial distribution patterns were investigated. It was revealed that high doses of potassium bromate decreased the percentage of oocytes at the metaphase II (MII) stage and increased abnormal mitochondrial distribution. Furthermore, as the efficacy of resveratrol as an antioxidant has been reported,21, 22 it was investigated whether resveratrol could improve the oxidizing actions that had been elicited by potassium bromate.

MATERIALS AND METHODS

Collection of immature oocytes

Pregnant mare serum gonadotropin (7.5 IU; Teikokuzohki, Tokyo, Japan) was administrated intraperitoneally to 3 week old female Institute of Cancer Research mice (Japan SLC, Shizuoka, Japan). After 48 h, their ovaries were collected after euthanasia by cervical spine fracture dislocation. The follicles were punctured by needle in Leibovitz's L‐15 culture medium (Invitrogen, Carlsbad, CA, USA), containing 5% fetal bovine serum (FBS) (Invitrogen). Oocytes with several layers of cumulus cells were used for the experiments.

In vitro maturation and mitochondrial distribution

Experiments 1 and 2

In Experiment 1, the oocytes were cultured in maturation medium (Waymouth MB 752/1, containing 5% FBS and 0.23 mM pyruvic acid) with 0, 10, 100, 500, 750, or 1000 μM potassium bromate (Nacalai Tesque, Kyoto, Japan). In Experiment 2, the oocytes were cultured in maturation medium with 0, 10, or 20 μM resveratrol (Wako Pure Chemical Industries, Osaka, Japan) in the presence of 500 μM potassium bromate.

Culture method

The culture was maintained by using the hanging‐drop method. This single‐oocyte culture system provides high through‐put and excludes the paracrine effects that are elicited by the presence of other oocytes.23 Ninety‐six‐well plates were used for the culture of the oocytes in 10 μL of culture medium and 10 μL of mineral oil was used to cover each well. The culture was performed with the plate upside down at 37°C in 5% CO2 and 95% air.

Assessment of nuclear maturation and mitochondrial distribution

Twelve hours later, the cumulus cells were removed and the number of oocytes that released the first polar bodies was assessed under the microscope to calculate the MII maturation rate. For the observation of the mitochondrial distribution patterns, 250 nM of MitoTracker® Red CMXRos (Invitrogen) was added to the medium and the oocytes were incubated for 30 min. The oocytes then were washed with Leibovitz's L‐15‐containing culture medium with 5% FBS and mounted on glass slides that were covered with cover glass. The observations were made by using a confocal laser microscope (LSM‐710; Carl Zeiss, Jena, Germany). The mitochondrial distribution patterns were determined by the presence or absence of cluster formations (Figure 1). The mitochondria that aggregated to a diameter of ≥5 μM were judged as a cluster formation.

Figure 1

Fluorescent images of mitochondrial distribution and transmission images of metaphase II‐stage murine oocytes during in vitro maturation. The mitochondrial distribution patterns were determined by the presence or absence of cluster formation (stained with MitoTracker® Red CMXRos; Invitrogen, Carlsbad, CA, USA). A, Without potassium bromate treatment, no cluster formation was observed. B, Potassium bromate treatment resulted in cluster formations. Scale bar, 50 μm

Statistical analysis

For the obtained MII maturation rate and mitochondrial cluster formation rate, a significant difference test between a control and the chemically treated group was conducted by the chi‐square test. P‐values < 0.05 were considered to be statistically significant.

Ethical considerations

All the experimental procedures conformed to the “Regulations for Animal Experiments and Related Activities at Tohoku University,” were reviewed by the Institutional Laboratory Animal Care and Use Committee of Tohoku University, and finally approved (2016 AgA‐048) by the President of the University.

RESULTS

Experiment 1

On adding 0, 10, 100, 500, 750, or 1000 μM of potassium bromate to the maturation medium, the observed rates of MII maturation were 95.6% (43/45), 87.9% (29/33), 86.1% (31/36), 86.3% (88/102), 65.3% (32/49), and 18.8% (24/128), respectively. The maturation rates were significantly decreased by the addition of 750 and 1000 μM potassium bromate, with <18.8% of the oocytes maturing in the latter condition (Figure 2).

Figure 2

Effects of potassium bromate on the metaphase II (MII) oocyte maturation rate. The values represent the mean ± standard error of the mean. *P < .05 and ** P < .01

The mitochondria clustering rates were 7.0% (3/43), 13.8% (4/29), 6.5% (2/31), 59.1% (52/88), 59.4% (19/32), and 70.8% (17/24), respectively. Although clustering within the MII oocytes was observed in all conditions, it was especially evident with the addition of >500 μM potassium bromate (almost 60% of the oocytes exhibited clustering), indicating a remarkable increase, compared with the medium to which <100 μM had been added (Figures 3 and 4).

Figure 3

Effects of potassium bromate on the mitochondrial cluster formation rate. The values represent the mean ± standard error of the mean. **P < .01

Figure 4

Changes in the intracellular distribution of mitochondria on the addition of varying concentrations of potassium bromate, which were (A) 0 μM, (B) 500 μM, (C) 750 μM, and (D) 1000 μM. Scale bar, 50 μm

Experiment 2

Zero, 10, or 20 μM of resveratrol was added to the culture medium in order to examine the antioxidant effects in the presence of potassium bromate. As 500 μM potassium bromate did not affect the MII rate, but increased the cluster formation rate (as shown in Experiment 1), this concentration was chosen for this experiment. The MII maturation rates at 0, 10, and 20 μM were 86.3% (88/102), 83.3% (85/102), and 74.5% (79/106), respectively. The addition of 20 μM resveratrol significantly decreased the MII rate. The mitochondrial cluster formation rates were 59.1% (52/88), 11.8% (10/85), and 12.7% (10/79), respectively. Both 10 μM and 20 μM of resveratrol significantly decreased the cluster formation rate (Table 1).

Table 1

Effects of resveratrol on metaphase II (MII) maturation and mitochondrial cluster formation rates in the presence of potassium bromate

Concentration of resveratrol (μM)	MII maturation rate (%)	Cluster formation rate (%)
0	86.3 (88/102)	59.1 (52/88)
10	83.3 (85/102)	11.8** (10/85)
20	74.5* (79/106)	12.7** (10/79)

*P < .05 and **P < .01 indicate a significant difference between the control and the resveratrol‐treated group.

DISCUSSION

For patients with ovulatory disorders, such as polycystic ovary syndrome, the IVM technique is important in making effective use of rare oocytes, although further improvements are required. Immediately after starting IVM, the mitochondria aggregate just below the cell membrane and around the GV, then distribute throughout the cytoplasm at the MII stage. It is important that the mitochondria are properly distributed within the oocytes.24, 25 In addition, it has been concluded that the appropriate distribution of the mitochondria is necessary for proper cytoplasmic maturation of the oocytes.19 In this way, it is suggested that the distribution of the mitochondria can be an index of oocyte quality.

Potassium bromate, which is used as a food additive, reportedly acts as an oxidizing agent to increase the amount of 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG), a marker for DNA oxidative damage,20 and the increase in the levels of 8‐OHdG in the granulosa cells during the ovulatory process has been shown to lead to a lowering of the fertilization rate and a decrease in the good‐quality embryo rate in in vitro fertilization programs. As a marker of oxidative stress in the granulosa cells during the ovulatory process, 8‐OHdG is one of the factors that adversely affects not only fertilization, but the growth of embryos.26 Therefore, this study's result suggests that potassium bromate causes oxidative stress and reduces oocyte quality, although it needs to be addressed whether this drug increases reactive oxygen stress in the oocytes and/or granulosa cells in future studies.

In the present study, the addition of potassium bromate resulted in the inhibition of mitochondrial spreading throughout the cytoplasm and abnormal mitochondrial aggregation. It has been reported that the prominent aggregation of mitochondria is observed in aged oocytes and it is considered as one of thephenotypes.19, 27 Therefore, this study's model seems to be useful for partially mimicking the abnormal mitochondrial aggregation that is seen in aged oocytes.

Also examined was whether potassium bromate‐induced mitochondrial aggregation could be rescued by the addition of the antioxidant, resveratrol, which has been suggested to be useful in IVM and embryo culture systems. Several studies have reported improvements with resveratrol: the fertilization rate was increased by a reduction of active oxygen,28 the pregnancy rate was increased by improved mitochondrial function,21 and fertility and embryo developmental capacities were improved by an increased number of active mitochondria, leading to better‐quality oocytes.29 Collectively, these reports suggest that resveratrol improves mitochondrial function. This study also showed the utility of resveratrol as an antioxidant, as the appearance of mitochondrial aggregation within the oocyte cytoplasm was successfully avoided, even in the presence of the oxidizing agent. Resveratrol has been reported to have an antioxidant effect against cumulus cells.22 In this study, the culture occurred in the cumulus–oocyte complex, instead of the denuded oocyte. Therefore, cytoplasmic maturation of the cumulus cells could be promoted by a reduction in oxidative stress of the cumulus cells. However, it is still necessary to examine whether the addition of this antioxidant is useful for enhancing fertilization rates and early embryonic development under similar conditions. The authors believe that the current observations yield insight for the potential improvement of the IVM system by reducing oxidization of the immature oocytes in order to enhance mitochondrial function.

It was concluded that there was success in constructing an in vitro experimental system that can control the promotion and suppression of mitochondrial cluster formation in MII oocytes by using potassium bromate and resveratrol. This experimental model could help to evaluate oocyte cytoplasm maturation. It also would be useful to compare and analyze the effects of other antioxidants for oocyte maturation.

DISCLOSURES

Conflict of interest: The authors declare no conflict of interest. Human and Animal Rights: All the experimental procedures conformed to the “Regulations for Animal Experiments and Related Activities at Tohoku University” and were reviewed by the Institutional Laboratory Animal Care and Use Committee of Tohoku University, finally being approved (2016 AgA‐048) by the President of the University. This article does not contain any study with human participants that was performed by any of the authors.