The extracellular reactive oxygen species levels in primary in vitro culture of human ovarian granulosa and cumulus cells

Inside the ovarian follicle, the oocyte microenvironment is formed by several layers of granulosa cells that differentiate into mural granulosa cells (GCs) and cumulus cells (CCs) during the final stages of folliculogenesis. The number and condition of cells surrounding the oocyte have been considered a biomarker for oocyte competence, embryo quality, and pregnancy outcome [1]. Bidirectional communication between follicular cells and oocyte is significant for successful maturation and the acquisition of developmental competence. The cumulus cells are located closer to the oocyte, and intercellular communication between CCs and the oocyte is conducted via cytoplasmic extensions of corona radiata cells passing through zona pellucida [2]. Granulosa cells not only provide oocytes with essential nutrients and maturation-related factors. The critical role of GCs and CCs is to protect oocyte from excessive oxidative stress damage through their antioxidant system during oocyte maturation [3, 4]

Follicular cells are sensitive to reactive oxygen species (ROS). Although ROS play an essential role as the signaling molecules in different cellular processes, their high levels induce various adverse effects, such as DNA damage, amino acids and polyunsaturated fatty acids oxidation, and enzyme deactivation co-factors oxidation. There are three major types of ROS: hydroxyl (OH^•), superoxide (O₂^•), and hydrogen peroxide (H₂O₂) [5]. ROS are produced due to electron leakage from the inner membrane of mitochondria during oxidative phosphorylation and ATP generation. Moreover, in steroidogenic tissues such as the ovary, cytochrome P450 enzymes represent an additional ROS source [6].

In the female reproductive tract, ROS may exert physiological and pathophysiological effects. Numerous studies have shown the presence of ROS in ovaries [7, 8, 9], fallopian tubes, and embryos [10]. Reactive oxygen species are also formed within the follicle during ovulation. Interestingly, inhibitors of acute inflammatory reactions that decrease ROS levels have been reported to suppress ovulation [11]. The fact that ovulation is accompanied by inflammation may suggest a role for ROS along this process. On the other hand, excessive ROS have been reported to play a critical role in GCs apoptosis [6, 12].

In the ovary, ROS are produced by inflammatory cells, such as neutrophils and macrophages, which are massively recruited to the ovarian tissues after the luteinizing hormone (LH) surge, and their depletion affects ovulation [13]. However, there is a lack of studies showing to which extend CCs and GCs can contribute to the development of oxidative stress within the ovarian follicle. The presented research was aimed to a) ascertain the presence of ROS in GCs, and CCs conditioned medium; b) quantify extracellular ROS; c) evaluate the changes in extracellular ROS concentration during the primary in vitro culture of GCs and CCs.

The obtained results revealed a downward trend in extracellular ROS levels during the seven days of in vitro primary culture of GCs (Fig. 1). There was a significant decrease in ROS concentration on the third day (D3, P < 0.05), which continued to drop during the further period of culture and resulted in the lowest level on the seventh day (D7) when compared to the previous time points (P < 0.001).

Figure 1

In contrast, we observed the opposite dynamics in the case of CCs culture (Fig. 2). The level of ROS in the conditioned medium grew significantly on the third day of culture (D3, P < 0.001). Although there was no significant difference in ROS between D3 and D7, the results demonstrated a slight increase in extracellular ROS accumulation during this period and a high level of significance (P < 0.001) when compared to the first day (D1).

Figure 2

In a healthy body, ROS and antioxidants remain in balance to ensure the proper functioning of physiological systems. When the balance is disturbed towards the excess of ROS, oxidative stress occurs. This condition has been reported to influence women’s entire reproductive lifespan and modulate menopause [5]. In the ovarian follicle, follicular fluid forms the oocyte’s environment before fertilization and may influence IVF outcome affecting fertilization and embryo cleavage [16]. This environment contains granulosa cells, leukocytes, and macrophages, all of which can produce ROS. Impaired metabolism of the oocyte may additionally contribute to ROS accumulation in follicular fluid [17].

Oocyte quality is a crucial determining factor in the outcome of IVF/ embryo transfer (ET).  8-hydroxy-2-deoxyguanosine is considered a reliable indicator of DNA damage initiated by oxidative stress. This compound has been used as an indicator of oxidative stress in various pathologies, such as renal carcinogenesis and diabetes mellitus. Higher levels of 8 hydroxy 2-deoxyguanosine correlated with lower fertilization rates and poor embryo quality [18]. Elevated concentrations of 8-hydroxy 2-deoxyguanosine have also been reported in GCs of women with endometriosis, which may impair oocyte quality [5].

In the present research, we observed a decrease in ROS concentration in GCs conditioned medium during the seven-day culture period, while CCs culture was characterized by an increasing extracellular ROS accumulation. We used a direct ROS detection method with 2′,7′-dichlorodihydrofluorescein diacetate solution, which is a widely known approach due to DCF-DA response to diverse and relevant oxidant species [19, 20, 21]. In general, ROS exert harmful effects on cells and organisms when generated in excess. However, the fine‐tuned maintenance of ROS is necessary for different signal transduction cascades. Reactive oxygen species represent the inevitable byproducts of metabolic processes and intracellular signaling pathways associated with oxidation‐reduction reactions in all aerobic organisms [22]. Although elevated ROS can reduce cell proliferation and induce cytotoxic effect, a growing body of evidence reveals that ROS act as fundamental signaling molecules. ROS can contribute to cell proliferation and differentiation either directly or indirectly via modulation of cell components’ redox status and by regulating the transcription factors related to proliferation and differentiation [23]. ROS are mainly considered as controlling signal transductions via the activation of mitogen-activated protein kinase (MAPK) to transcription factors, such as activator protein 1 (AP-1). Moreover, hydrogen peroxide stimulated MAPK-mediated cell proliferation, activation of MAPKs has been reported to coincide with superoxide anion formation, which in turn increased MAPK-mediated cell proliferation [24, 25]. A biphasic effect of ROS, especially superoxide and hydrogen peroxide, on cellular proliferation has been reported in previous studies, in which low concentrations (submicromolar concentrations) induced cell growth, but higher levels (usually more than 10–30 uM) induced apoptosis. The exact dosages of ROS resulting in cell growth or death vary significantly throughout available research and seem to be dependent on cell type [26]. Exogenous H₂O₂ at concentrations higher than 0.5 mM has been reported to rapidly induce cytotoxicity in human granulosa cell tumor line COV434, which possesses many characteristics of normal granulosa cells [27]. The generation of ROS caused by ionizing radiation or chemical toxicants has also been implicated in the toxicity of GCs [29]. But the mechanism of cytotoxicity induced by ROS is less known, as well as concentrations that could be beneficial for the proliferation of GCs [12]. Nakahara et al. studied three concentrations of H₂O₂ - 0.01, 0.1, and 1.0 mM – all of which induced apoptotic changes in cultured GCs [30]. The study of Shen et al. has also confirmed the pro-apoptotic effect of excessive H₂O₂ and revealed the involvement of FoxO1expression [28]. In the present study, we evaluated the level of ROS produced by cells without additional stimulation with exogenous ROS. Probably, due to this fact, we did not observe excessive apoptosis in cell culture even in the time points characterized by elevated ROS concentration in the extracellular medium. However, the changes in ROS levels during the in vitro culture may reflect the proliferation status and metabolic activity of GCs and CCs, as ROS could arise as a byproduct of different cellular processes [22].

On the other hand, the elevation of ROS level in CCs culture may be due to culture conditions, where the oxygen tension is much higher than in the female reproductive tract. A significant amount of early studies focused on characterizing the oxygen tension in human ovarian follicles. Despite this fact, knowledge of dissolved oxygen concentrations in follicular fluid remains elusive, and the reported results are highly variable. It has been accepted that levels of oxygen in follicular fluid decline through to the beginning of the preovulatory phase, and then pO₂ levels grow before the ovulation. Redding et al. calculated the model predictions suggesting that the mean oxygen concentrations in human follicular fluid during the late antral and preovulatory phases may range between 11 and 51 mmHg (∼1.5–6.7 vol%) [31]. Thus, it is possible that elevated atmospheric oxygen could negatively affect CCs by causing an elevation in the level of ROS during in vitro culture.

It has been shown that the variability of pO₂ levels in the follicular fluid may be attributed to the number of layers of mural GCs, which consume and impede oxygen diffusion to the follicular antrum. Clark et al.(2006), using the model of oxygen transport across the COC, demonstrated that CCs consume little oxygen, thus sparing and transmitting it to the oocyte [32]. Consequently, there is a possible difference between the CCs and GCs in terms of oxygen metabolism, which could explain the present research results. Interestingly, another mathematical model suggests that the oxygen levels within the antral follicle are dependent on follicle structure and size and that the mean level of dissolved oxygen in the follicular fluid may not correspond to that reaching the COC [33].

A recently discovered player in the oxygen balance maintaining is the intracellular synthesis of hemoglobin by both GCs and CCs.  Hemoglobin is known to possess an antioxidant function [34]. Its up-regulated expression over the periovulatory phase may assist with the protection of ovarian follicular cells from elevated ROS levels over this period. Cellular hemoglobin synthesis is dynamic, responding to the ovulatory luteinizing hormone (LH) surge [35]. In the present study, after the cell isolation, under the conditions of in vitro culture, the cells were not supplemented with any hormones, including LH. This lack of external hormonal stimulation, which is characteristic of in vivo environment, may at least in part explain the differences in ROS levels at the beginning of in vitro culture and during the following days. It is possible that CCs need systemic cues to launch their antioxidant defense system, including hemoglobin synthesis.

The present research revealed a steady decrease in extracellular ROS level during GCs primary culture. By contrast, ROS concentration in CCs conditioned medium increased gradually between the first and the seventh days of culture. The observed changes may reflect the proliferation status and metabolic activity of GCs and CCs during in vitro culture, as ROS could arise as a byproduct of different cellular processes. Additionally, the elevated ROS level at respective points of time could occur due to culture in atmospheric oxygen. The distinct function and localization within the ovarian follicle may explain the differences between GCs and CCs oxygen metabolism.