Cellular and exosome mediated molecular defense mechanism in bovine granulosa cells exposed to oxidative stress

Abstract

Various environmental insults including diseases, heat and oxidative stress could lead to abnormal growth, functions and apoptosis in granulosa cells during ovarian follicle growth and oocyte maturation. Despite the fact that cells exposed to oxidative stress are responding transcriptionally, the potential release of transcripts associated with oxidative stress response into extracellular space through exosomes is not yet determined. Therefore, here we aimed to investigate the effect of oxidative stress in bovine granulosa cells in vitro on the cellular and exosome mediated defense mechanisms. Bovine granulosa cells were aspirated from ovarian follicles and cultured in DMEM/F-12 Ham culture medium supplemented with 10% exosome-depleted fetal bovine serum. In the first experiment sub-confluent cells were treated with 5 μM H2O2 for 40 min to induce oxidative stress. Thereafter, cells were subjected to ROS and mitochondrial staining, cell proliferation and cell cycle assays. Furthermore, gene and protein expression analysis were performed in H2O2-challenged versus control group 24 hr post-treatment using qRT-PCR and immune blotting or immunocytochemistry assay, respectively. Moreover, exosomes were isolated from spent media using ultracentrifugation procedure, and subsequently used for RNA isolation and qRT-PCR. In the second experiment, exosomes released by granulosa cells under oxidative stress (StressExo) or those released by granulosa cells without oxidative stress (NormalExo) were co-incubated with bovine granulosa cells in vitro to proof the potential horizontal transfer of defense molecules from exosomes to granulosa cells and investigate any phenotype changes. Exposure of bovine granulosa cells to H2O2 induced the accumulation of ROS, reduced mitochondrial activity, increased expression of Nrf2 and its downstream antioxidant genes (both mRNA and protein), altered the cell cycle transitions and induced cellular apoptosis. Granulosa cells exposed to oxidative stress released exosomes enriched with mRNA of Nrf2 and candidate antioxidants. Subsequent co-incubation of StressExo with cultured granulosa cells could alter the relative abundance of cellular oxidative stress response molecules including Nrf2 and antioxidants CAT, PRDX1 and TXN1. The present study provide evidences that granulosa cells exposed to oxidative stress conditions react to stress by activating cascades of cellular antioxidant molecules which can also be released into extracellular environment through exosomes.

Oxygen tension modulates extracellular vesicles and its miRNA contents in bovine embryo culture medium

“…Finally, it is known that embryos release EVs during the early embryonic development (Saadeldin et al, ) but there is still controversy regarding the function of embryo secreted vesicles and its consequences on further development (Mellisho et al, ). The release of EVs by the embryos and the content of these vesicles, especially miRNAs, undergo several changes, which may be caused by the culture conditions directly on the embryo or influenced by the cells present in the microenvironment, in alignment with previous reports that oxidative stress can alter the composition of granulosa cells from the EVs (Saeed‐zidane et al, ).…”

Sexual dimorphic expression and release of transcription factors in bovine embryos exposed to oxidative stress

“…Accordingly, our results indicated that the higher number and bigger size EV (Figure c) were found in spent media of male embryos cultured under high oxygen level. Similar results were obtained in our previous study in granulosa cells exposed to OS (Saeed‐Zidane et al, ). In contrast, the female embryos exhibited a lower number and bigger size EVs upon exposure to OS.…”

“…As shown in Figure , EVs from male embryos at higher oxygen level contained higher level of NFE2L2 and SOD1 transcripts compared to those cultured under low oxygen level, which might be beneficial of development of embryos (Pavani et al, ). The role of EVs is not only in cell–cell communication (Saeed‐Zidane et al, ) but also in the removal of undesirable cellular molecules (Takahashi et al, ). Contrary to cellular expression, the NOTCH1 was increased in EVs derived from male embryos exposed to OS compared with female counterparts, which may indicate the selectivity of cargo molecules to be exported by EVs (Bhome et al, ; Hinger et al, ) to facilitate cell to cell communication or maintain of cellular homeostasis.…”

“…On the basis of their size, EVs can be subdivided into apoptotic bodies (1,000–5,000 nm), microvesicles (100–1,000 nm), and exosomes (30–200 nm; Lawson et al, ; Sun et al, ; de la Torre Gomez, Goreham, Bech Serra, Nann & Kussmann, ). They carry a cargo of bioactive molecules such as DNA, RNAs, proteins to be transferred into the extracellular environment to maintain cellular homeostasis (Takahashi et al, ) and/or transfer genetic material to neighbor or distant recipient cells (Saeed‐Zidane et al, ). In addition, its cargo content varies depending on the cell origin (de la Torre Gomez et al, ), physiological status (Hung et al, ; Navakanitworakul et al, ), inflammation (Jolly, Thompson, & Winchester, ) and stress status (Beninson et al, ; Bewicke‐Copley et al, ) of the donor cells.…”

A New Biochemical Approach to Detect Oxidative Stress in Infertile Women Undergoing Assisted Reproductive Technology Procedures

“…Supporting these data, other authors [30] demonstrated that increased 8OHdG concentrations in the FF of women undergoing IVF are associated with oocyte degeneration, suggesting that oxidative stress in the follicular compartment has deleterious effects on the oocyte. Our data are in agreement with a report showing lower vitamin E concentrations in the FF of women with infertility related to endometriosis [31] but are in contrast with another study reporting both increased 8OHdG and an higher vitamin E concentration in the FF of women with endometriosis. These data probably indicate an up-regulation of the follicular antioxidant system in an attempt to neutralize, but not successfully, reactive species and to prevent oxidative damage to oocytes.…”