Elsevier

Reproductive Toxicology

Volume 98, December 2020, Pages 278-285
Reproductive Toxicology

Cell-free mitochondrial DNA increases granulosa cell apoptosis and reduces aged oocyte blastocyst development in the mouse

https://doi.org/10.1016/j.reprotox.2020.10.012Get rights and content

Highlights

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    Cf-mtDNA can reduce ATP content in mouse ovarian granulosa cells cultured in vitro.

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    Cf-mtDNA can induce apoptosis of mouse ovarian granulosa cells cultured in vitro.

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    Granulosa cell apoptosis induced by cf-mtDNA is related to TLR9/NF-кB p65 or MAPK p38 signal pathway.

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    Cf-mtDNA decreases blastocyst development in aged mouse oocytes matured in vitro.

Abstract

Cell-free mitochondrial DNA (cf-mtDNA) released into the extracellular environment can cause cellular inflammatory responses and damage. Here, we investigated the effects of cf-mtDNA on mouse ovarian granulosa cell function and on the developmental competence of oocytes matured in vitro. Granulosa cells in the cf-mtDNA treatment group had a lower ATP content (P < 0.05), a higher apoptotic cell percentage (P < 0.01), and higher mRNA and protein levels of apoptosis-related factors than the control group (P < 0.01). TLR9, NF-кB p65 and MAPK p38 expression levels in granulosa cells were significantly increased in the cf-mtDNA treatment group (P < 0.05). The blastocyst formation rate of aged mice oocytes matured in vitro decreased significantly (P < 0.05) when cf-mtDNA was added to the media, compared with the control. However, the oocytes from young mice were not affected. Our results suggest that cf-mtDNA may impair granulosa cell function and induce granulosa cell apoptosis, subsequently decreasing blastocyst development in aged oocytes. This role of cf-mtDNA may be associated with the binding to TLR9 and the activation of NF-кB p65 and MAPK p38 signaling pathways.

Introduction

Cell-free mitochondrial DNA (cf-mtDNA) was found for the first time in the serum and plasma by Zhong et al. in 2000 [1]. Later, cf-mtDNA was also detected in other body fluids, such as the semen, urine, follicular fluid, embryo culture media, and cerebrospinal fluid [[2], [3], [4], [5], [6]]. It is thought to have great application value in the diagnosis of cancer and other diseases [3,[7], [8], [9]] because of its unique characteristics compared with nuclear DNA (nDNA), including the short length, the simple molecular structure, and the multiple copy number. For example, in spent embryo culture media, cf-mtDNA levels are higher than those of cf-nDNA and the cf-mtDNA/cf-nDNA ratio may be a novel, non-invasive biomarker of blastocyst potential [[9], [10], [11], [12]].

Our previous study confirmed the presence of cf-mtDNA in human follicular fluid, and quantitative analysis showed that the relative content of cf-mtDNA in follicular fluid was positively correlated with patient age [4]. Moreover, the relative cf-mtDNA content in follicular fluid was significantly lower in the group with blastocyst development than in the non-blastocyst group, suggesting that the relative content of cf-mtDNA in follicular fluid may be closely related to oocyte developmental competence.

Recent studies have shown that mtDNA can cause inflammation as a damage-associated molecular pattern (DAMP) molecule when it is released out of the cells [13,14]. Similarly to bacterial DNA, mtDNA is a covalently closed circular double-stranded molecule that lacks histone protection and contains a large number of unmethylated CpG sequences. Under normal circumstances, mtDNA exists in mitochondria, isolated from the immune system and is not recognized. However, when mtDNA is released due to mitochondrial damage or an increase in the membrane permeability, an immune response may be induced. For example, during acute trauma, a large amount of mtDNA is released into the blood. MtDNA binds to the toll-like receptor (TLR) to activate the classical NF-кB p65 and MAPK p38 pathways, and then induces the release of inflammatory factors, such as TNF-α, IL-1, IL-6, among others, and causes acute lung injury [15]. Cf-mtDNA has also been reported to cause inflammation of cardiomyocytes and result in myocarditis and cardiomyopathy [16].

It is worth noting that in addition to traditional immune cells, TLRs are widely expressed on the surface or inside of non-immune cells in the vagina, fallopian tubes, uterus and the ovaries of various mammals. Human ovarian epithelium and granulosa cells express various TLR mRNAs and proteins, including TLR 2, 4, 5, 6, 7, 9, and 10 [17,18]. In addition, it is believed that there is a close relationship between the oocyte and the surrounding granulosa cells. Granulosa cells facilitate oocyte development and maturation by providing small nutrient molecules, such as cyclic nucleotides, glucose metabolites, and amino acids [19,20]. We were interested in whether the increase in follicular fluid cf-mtDNA content can cause damage to granulosa cells and, subsequently, to oocytes developmental competence during follicular development.

Therefore, in the present study, we investigated the effect of purified mtDNA on mouse ovarian granulosa cells and on the developmental competence of mouse oocytes matured in vitro.

Section snippets

Ethics statement

The study was approved by the Ethics Committee of Tongji Medical College of Huazhong University of Science and Technology. All experiments were conducted in accordance with the guidelines approved by the Huazhong University of Science and Technology (IORG No: IORG0003571).

Animals

C57BL/6 J mice were obtained from the Animal Center of Tongji Medical College of Huazhong University of Science and Technology. They were kept in a room a controlled temperature and humidity and 12 h/12 h light-dark cycles.

Statistical analysis

All data are described as the mean ± standard deviation (Mean ± SD). Statistical analyses were performed using the Mann-Whitney test or one-way ANOVA using the statistical software SPSS 12.0. P < 0.05 was considered statistically significant.

In vitro granulosa cells culture and identification

As shown in Fig. 1, the mouse ovarian granulosa cells appeared to have undergone significant proliferation after 48 h of in vitro culture. Most of the cells in the cultures were granulosa cells, which were characterized by a positive FSHR staining (Fig. 1). Non-specific staining was not detected (Fig. 1).

Effect of cell-free mitochondrial DNA on granulosa cell viability in vitro

The results showed an obvious decrease in granulosa cells viability incubated when a 10-3 ng/μl (P<0.0001), 10-2 ng/μl (P = 0.002), 1 ng/μl (P = 0.001), and a 5 ng/μl (P = 0.017) cf-mtDNA

Discussion

In the present study, we found that cf-mtDNA induced apoptosis of mouse ovarian granulosa cells, which was related to the binding of cf-mtDNA to TLR9 receptors and the subsequent activation of the NF-κB p65 and MAPK p38 pathways. Interestingly, cf-mtDNA supplemented into IVM media did not affect the developmental competence of young mice oocytes matured in vitro, however, it significantly reduced blastocyst development in those oocytes from aged mice.

Our results showed that the addition of 10-3

Conclusions

In conclusion, cf-mtDNA can induce mouse ovarian granulosa cell apoptosis, which may be associated with the TLR9- NF-κB p65/MAPK p38 pathway. Moreover, cf-mtDNA can lead to a decrease in oocyte developmental competence in aged oocytes in vitro. This study provides a new perspective to understand poor oocyte quality in women of an advanced age.

Author contribution statement

Y.L. carried out experimental work, conducted the statistical analysis and wrote the manuscript. Q.S. carried out experimental work and prepared the pictures and revised the manuscript. H.L. took part in experimental work and discussed the results. W.X. took part in experiment design and helped with data analysis. L.Z. designed experiments, interpreted the data and revised the manuscript. All authors read and approved the manuscript.

Funding

This work was supported by National Key Research and Development Program of China (grant numbers 2017YFC1002001) and Youth Program of National Natural Science Foundation of China (grant numbers No. 81000274).

Declaration of Competing Interest

None declared.

Acknowledgement

We would like to thank Editage (www.editage.com) for English language editing.

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