Chilling injury mechanism in the immature oocytes of zebrafish (Danio rerio)

Abstract

Immature zebrafish oocytes are sensitive to chilling, and their survival is markedly reduced by exposure to 0°C. In the present study, we investigated the involvement of cold-sensitive channels and lipid mediators in chilling injury in immature zebrafish oocytes. The oocytes were injected with inhibitors of a cold-sensitive channel (TRPA1), cytosolic phospholipase A₂α (cPLA₂α), cyclooxygenases (COXs), arachidonate 5-lipoxygenase (ALOX5), and lysophosphatidylcholine acyltransferase 2 (LPCAT2). The cells were then chilled at 0–12°C for 5–30 min, incubated at 25°C for 2 h, and stained with propidium iodide. Oocytes were damaged when exposed to temperatures below 12°C. When oocytes were chilled at 0°C for 15 min, the survival rate was very low (9%). However, when the oocytes were injected with a TRPA1-specific inhibitor, their survival markedly improved (70%). This strongly suggests that activation of the cold-sensitive TRPA1 channel triggers chilling injury in oocytes. When a cPLA₂α-specific inhibitor was injected, the survival of chilled oocytes markedly improved (60%). This strongly suggests that lipid mediators are involved in chilling injury in oocytes. When oocytes were injected with specific inhibitors of COXs, ALOX5, and LPCAT2, the survival of chilled oocytes significantly improved by 47%, 28%, and 43%, respectively. These results strongly suggest that eicosanoids and platelet-activating factor are involved in the chilling injury in oocytes. The results of this study may facilitate advancements in successful cryopreservation of fish oocytes.

Cryopreservation is widely used to preserve reproductive cells, including oocytes and embryos. This technique enables long-term storage of these cells and preserves their viability for future applications [1]. Successful cryopreservation of fish oocytes and embryos can enhance fish production, support genetic selection programs, and improve stock management [2].

Several studies have examined fish embryo cryopreservation. Among them, Khosla et al. successfully cryopreserved zebrafish embryos with laser gold nanowarming [3, 4]; to date, only this method has allowed for the reproducible cryopreservation of fish embryos. This study showed that a significant proportion of cryopreserved embryos developed into adult fish capable of breeding normally. However, cryopreserved embryos must be warmed individually using this method, which is not suitable for fish production in aquaculture or breeding stock.

Relative to their large size, fish embryos have low membrane permeability to water and cryoprotectants [5,6,7], which appears to be the most significant obstacle to cryopreservation. Consequently, dehydration and permeation by cryoprotectants require extended periods, making fish embryos more susceptible to damage by the toxicity of the cryoprotectant or the formation of intracellular ice. In contrast, immature fish oocytes have high permeability to water and cryoprotectants [8, 9]. Furthermore, we demonstrated that exogenous expression of water channels in immature fish oocytes increased permeability to water and cryoprotectants [10,11,12]. In contrast to fish embryos, fish oocytes are composed of a single compartment and do not have permeability barriers such as the multinucleated yolk syncytial layer found in fish embryos [13]. Although fish oocytes have not been successfully cryopreserved, they offer several key advantages.

To promote the cryopreservation of fish oocytes, all types of cell injury during cryopreservation must be overcome. Chilling injury is a major form of damage that occurs during cryopreservation, and fish oocytes are sensitive to chilling [14]. Several theories have been proposed to explain the mechanisms of chilling injury [15,16,17]. For example, the phase transition of membrane phospholipids may be related to chilling injury in mammalian spermatozoa [18,19,20], considering that the thermotropic lipid phase transition is associated with the lipid composition of the membranes [17, 21]. It has also been speculated that the high sensitivity of zebrafish embryos to chilling is related to the high lipid content of the yolk, because chilling sensitivity can be reduced by reducing the amount of yolk in the embryos [22]. However, there is no evidence that the phase transition of phospholipids in the plasma membrane causes chilling injury in immature fish oocytes.

Living organisms possess various sensory systems that enable them to respond to environmental changes. Transient receptor potential (TRP) channels are sensors that respond to environmental stimuli, including chemical and physical changes [23]. TRP channels are a family of nonselective cation channels that regulate the transmembrane flux of cations, including Ca²⁺. Some TRP channels are sensitive to warm and cold temperatures [24]. TRPA1 is activated at temperatures below 17°C in mammals and functions as a cold-sensitive receptor [25] with high permeability to cations, including Ca²⁺, and Ca²⁺ influx is involved in various biological processes [26]. The high permeability of TRPA1 to Ca²⁺ triggers the influx of extracellular Ca²⁺ and promotes Ca²⁺ release from intracellular stores, such as the endoplasmic reticulum, and the intracellular Ca²⁺ increase may enhance responses to other stimuli [27]. Therefore, cold-sensitive TRPA1 may trigger chilling injury in immature fish oocytes.

When TRPA1 is activated and intracellular Ca²⁺ increases in the cell, cytosolic phospholipase A₂α (cPLA₂α), a Ca²⁺-dependent enzyme, is activated and hydrolyzes phospholipids into arachidonic acid and lysophospholipids [28]. Considering that various bioactive lipid mediators, prostaglandins, leukotrienes, and platelet-activating factor are synthesized from these products, these lipid mediators may damage immature fish oocytes after chilling.

In this study, we investigated whether the activation of TRPA1 by cold triggers chilling injury in immature zebrafish oocytes. We further examined whether lipid mediators derived from arachidonic acid and lysophospholipids play important roles in chilling injury.

Materials and Methods

Collection of immature zebrafish oocytes

Approximately 50 mature zebrafish, purchased from a local fish dealer, were maintained in 60-l aquariums under 14-h light and 10-h dark periods at 26°C. They were fed twice a day with Tetramin dry flake fish food (Spectrum Brands Japan, Yokohama, Japan). To obtain immature oocytes at late stage III [29], actively spawning females were decapitated under anesthesia with 0.2 mg/ml tricaine (Sigma-Aldrich, St. Louis, MO, USA) in distilled water, 0–3 h before the end of the light period (just before the LH surge). The ovaries were recovered and placed in a 90% Leibovitz L-15 medium (Gibco, Waltham, MA, USA), pH 8.5, containing 100 μg/ml of gentamycin sulfate (90% LM) at 25°C based on the previous study [30], and follicles were manually separated using forceps and scissors. The size of the follicles was measured using a microscope with an ocular micrometer, and follicles with diameters of 0.60–0.69 mm were collected and used as immature oocytes at late stage III [29]. The collected oocytes were maintained in 90% LM. All the experiments were approved by the Animal Care and Use Committee of Kochi University (O-00083).

Chilling of the oocytes

To examine sensitivity to chilling, oocytes in 90% LM at 25°C were pipetted into 4 ml of the same medium precooled to 12, 10, 8, 5, or 0°C and treated for 5, 10, 15, 20, or 30 min. As controls, oocytes were maintained at 25°C for 30 min. Each treatment consisted of 10 oocytes and was repeated three times.

Injection of inhibitors into the oocytes

A 100-µl drop of 90% LM was prepared in a plastic Petri dish (90 mm × 10 mm, Thermo Scientific, Waltham, MA, USA), covered with liquid paraffin oil, and placed on the stage of an inverted microscope. An oocyte was placed in the drop and held on an inverted microscope using a holding pipette connected to a micromanipulator. Approximately 20 nl of the inhibitor solution was injected using an injection needle connected to another micromanipulator [10]. The experiment was conducted at room temperature (23–25°C).

The following inhibitors were injected into oocytes: 300 µM AP-18 (a TRPA1-specific inhibitor (Cayman Chemical, Ann Arbor, MI, USA)), 200 µM ruthenium red (a ryanodine receptor inhibitor (Cayman Chemical)), 5 µM pyrrophenone (a cPLA₂α-specific inhibitor (Cayman Chemical)), 10 µM indomethacin (a cyclooxygenase (COX)-specific inhibitor (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), 10 µM zileuton (an arachidonate 5-lipoxygenase (ALOX5)-specific inhibitor (ChemScene, Monmoth Junction, NJ, USA)), and 5 µM TSI-01 (a lysophosphatidylcholine acyltransferase 2 (LPCAT2)-specific inhibitor (ChemScene)); the concentrations of the inhibitors were 1–10 times higher than those of other papers [31,32,33,34,35,36] because inhibitor solutions were diluted in oocytes to 1/10–1/15. Inhibitors were dissolved in dimethyl sulfoxide (DMSO) and diluted with distilled water for injection. The final concentration of DMSO in the diluted inhibitor solutions was 0.01–0.1%.

After injection, oocytes were cultured in 90% LM at 25°C for 1 h, chilled, and then cultured in 90% LM at 25°C for 2 h. As a control, uninjected oocytes were cultured at 25°C for 1 h in 90% LM and cultured in the same medium at 25°C for 30 or 15 min. Each treatment consisted of 10 oocytes and was repeated three times.

Oocyte viability assessment

Oocytes were stained with 20 µg/ml of propidium iodide in 90% LM at 25°C for 10 min. The oocytes were examined using an inverted microscope equipped with a fluorescence device and a U-MWIG2 filter (excitation wavelength: 520–550 nm, emission wavelength: 580 nm; Olympus, Tokyo, Japan). Oocytes that emitted reddish-white light were considered dead, whereas those that did not emit light were considered alive.

Statistical analysis

Statistical differences between groups were analyzed using analysis of variance in GraphPad Instat software, v.7.04, with Tukey-Kramer multiple comparison tests, unless the groups did not have the same variance, as determined by Bartlett’s test, in which case Welch’s t-test was used. Statistical significance was set at P < 0.05.

Results

Chilling sensitivity of zebrafish oocytes

Figure 1 shows the sensitivity of immature zebrafish oocytes to chilling. At 12°C, the survival of oocytes chilled for 30 min remained high (83%) and was not significantly different from that of the control. At 10°C, however, survival significantly decreased after exposure for 5 min (59%) and continued decreasing as the exposure time increased (10% after 30 min). Similar results were obtained when oocytes were exposed to the solution at 0–8°C. These results indicate that immature zebrafish oocytes are sensitive to temperatures at or below 10°C, leading to cellular damage.

Fig. 1.

Survival of immature zebrafish oocytes after exposure to 0–12°C for 5–30 min. Control oocytes were incubated at 25°C for 30 min without any treatment. Each treatment consisted of 10 oocytes. Data are indicated as means ± SD from triplicate determinations. * Significantly different from control, as determined by ANOVA (P < 0.05).

Involvement of a cold-sensitive channel in the chilling injury of oocytes

We examined whether the cold-sensitive TRPA1 channel was involved in chilling injury in oocytes. Figure 2 shows the effect of AP-18, a specific TRPA1 inhibitor, on oocyte survival after chilling. The survival rate of oocytes exposed to 0°C for 15 min was low (9%). Injection of AP-18 markedly improved survival, and the survival rate (70%) was not significantly different from that of the control group (82%). This result strongly suggests that TRPA1 triggers chilling injury in immature zebrafish oocytes.

Fig. 2.

Survival of immature zebrafish oocytes injected with a specific TRPA1 inhibitor after being chilled. Immature zebrafish oocytes were injected with AP-18 (300 µM, ~20 nl) and incubated at 25°C for 1 h, chilled at 0°C for 15 min, and incubated at 25°C for 2 h. Control oocytes were incubated at 25°C for 15 min without injection. Each treatment consisted of 10 oocytes. Data are indicated as means ± SD from triplicate determinations. * Significantly different from control, as determined by ANOVA (P < 0.05).

Involvement of ryanodine receptors in the chilling injury of zebrafish oocytes

It has been shown that the activation of TRPA1 increases intracellular Ca²⁺ by transporting Ca²⁺ from the extracellular region and inducing Ca²⁺ release from the endoplasmic reticulum through ryanodine receptors [37]. Therefore, we examined whether ryanodine receptors are involved in chilling injury in immature zebrafish oocytes. Figure 3 shows the effect of ruthenium red, a ryanodine receptor inhibitor, on chilling injury in oocytes. Survival was originally low (10%) after chilling; however, injection of ruthenium red significantly improved survival (30%). This suggests that activated TRPA1 interacts with ryanodine receptors and amplifies the increase in intracellular Ca²⁺ by mobilizing Ca²⁺ from the endoplasmic reticulum.

Fig. 3.

Survival of immature zebrafish oocytes injected with a ryanodine receptor inhibitor after being chilled. Immature zebrafish oocytes were injected with ruthenium red (200 µM, ~20 nl) and incubated at 25°C for 1 h, chilled at 0°C for 15 min, and incubated at 25°C for 2 h. Control oocytes were incubated at 25°C for 15 min without injection. Each treatment consisted of 10 oocytes. Data are indicated as means ± SD from triplicate determinations. * Significantly different between two groups, as determined by Welch’s test (P < 0.05).

Involvement of lipid mediators in the chilling injury of oocytes

cPLA₂α is an intracellular Ca²⁺dependent enzyme. It hydrolyzes phospholipids into arachidonic acid and lysophospholipids; both products generate bioactive lipid mediators, eicosanoids, and platelet-activating factor [28]. Figure 4 shows the effect of pyrrophenone, a cPLA₂α-specific inhibitor, on chilling injury in oocytes. Most oocytes died when chilled, resulting in low survival (16%); however, survival markedly improved when pyrrophenone was injected (60%). This suggests that the lipid mediators produced from arachidonic acid and/or lysophospholipids play important roles in chilling injury in immature zebrafish oocytes.

Fig. 4.

Survival of immature zebrafish oocytes injected with a specific cPLA₂α inhibitor after being chilled. Immature zebrafish oocytes were injected with pyrrophenone (5 µM, ~20 nl) and incubated at 25°C for 1 h, chilled at 0°C for 15 min, and incubated at 25°C for 2 h. Control oocytes were incubated at 25°C for 15 min without injection. Each treatment consisted of 10 oocytes. Data are indicated as means ± SD from triplicate determinations. Bars with different superscripts differ significantly, as determined by ANOVA (P < 0.05).

Involvement of eicosanoids in chilling injury of oocytes

COXs are responsible for the synthesis of prostanoids from arachidonic acid. We examined the effect of indomethacin, a specific COX inhibitor, on chilling injury in oocytes (Fig. 5A). The survival of chilled oocytes markedly improved when indomethacin was injected (47%), suggesting that prostaglandins play an important role in chilling injury in immature zebrafish oocytes.

Fig. 5.

Survival of immature zebrafish oocytes injected with a specific inhibitor of cyclooxygenases (A) and arachidonate 5-lipoxygenase (B) after being chilled. Immature zebrafish oocytes were injected with indomethacin (10 µM, ~20 nl) or zileuton (10 μM, ~20 nl) and incubated at 25°C for 1 h, chilled at 0°C for 15 min, and incubated at 25°C for 2 h. Control oocytes were kept at 25°C for 15 min without injection. Each treatment consisted of 10 oocytes. Data are indicated as means ± SD from triplicate determinations. (A) Bars with different superscripts differ significantly, as determined by ANOVA (P < 0.05). (B) * Significantly different between two groups, as determined by Welch’s test (P < 0.05).

ALOX5 synthesizes leukotrienes from arachidonic acid. We examined the effects of zileuton, a specific ALOX5 inhibitor (Fig. 5B). The survival of chilled oocytes was significantly improved (28%) when zileuton was injected, suggesting that leukotrienes also play a role in chilling injury.

Involvement of platelet-activating factor in chilling injury in oocytes

LPCAT2 is responsible for the synthesis of platelet-activating factor from lysophospholipids. We examined the effect of TSI-01, a specific LPCAT2 inhibitor, on the chilling injury of oocytes (Fig. 6). The survival of chilled oocytes was significantly improved by injecting TSI-01 (43%), which suggests that platelet-activating factor play an important role in the chilling injury of immature zebrafish oocytes.

Fig. 6.

Survival of immature zebrafish oocytes injected with a specific inhibitor of lysophosphatidylcholine acyltransferase 2 after being chilled. Immature zebrafish oocytes were injected with TSI-01 (5 µM, ~20 nl) and incubated at 25°C for 1 h, chilled at 0°C for 15 min, and incubated at 25°C for 2 h. Control oocytes were incubated at 25°C for 15 min without injection. Each treatment consisted of 10 oocytes. Data are indicated as means ± SD from triplicate determinations. * Significantly different between two groups, as determined by Welch’s test (P < 0.05).

Discussion

In this study, we suggest that chilled oocytes are damaged by cold sensitivity through the activation of TRPA1 and that eicosanoids and platelet-activating factor play an important role in chilling injury. This is the first report to suggest that activation of cold-sensitive channels is a trigger for chilling injury.

When cells are damaged by chilling, various biological changes are observed, including changes in general cell metabolism [14], membrane rupture [38], membrane lipid phase transition [39], dehydration [40], loss of protein and enzyme functions [41], calcium imbalance [42], genome damage [43], and apoptosis [44]. Damage to the plasma membrane by chilling is considered one of the main mechanisms of chilling injury [45]. In amphibian oocytes, chilling reduces protein synthesis, followed by oocyte parthenogenetic activation [46]. Zebrafish embryos are highly sensitive to low temperatures. When zebrafish embryos were incubated at 18°C, the rate of survival and development was significantly low [47]. Zebrafish oocytes are also highly sensitive to low temperatures; Isayeva et al. showed that immature zebrafish oocytes at stage III are very sensitive to chilling, and their survival significantly decreased to less than 30% after only 15 min at 0°C [14]. Similar results were obtained in the present study; in immature zebrafish oocytes at late stage III, exposure to 0°C for 15 min markedly decreased survival (15%) (Fig. 1). However, the cause of this injury has not yet been elucidated.

In preliminary experiments, we examined the mRNA expression of TRPA1b and LPCAT2 in zebrafish ovaries by RT-PCR (data not shown). It has been shown that mRNA of cPLA₂α and COXs was expressed in zebrafish oocytes [48]. Additionally, the metabolite of ALOX5 was detected in zebrafish oocytes [49]. Therefore, TRPA1, cPLA₂α, COXs, ALOX5, and LPCAT may be expressed in zebrafish oocytes. Since it has been shown that oocytes express ryanodine receptors in medaka, another laboratory freshwater fish [50], zebrafish oocytes would also express ryanodine receptors. Therefore, the inhibitors used in the present study may affect oocyte survival through their inhibitory effects on channels and enzymes in oocytes.

It has been shown that there are two subtypes of TRPA1 in zebrafish, namely TRPA1a and TRPA1b, and only TRPA1b functions as a cold-sensitive channel [51]. In the present study, we showed that immature zebrafish oocytes are not injured at 12°C but become damaged at 10°C (Fig. 1) and that a specific TRPA1 inhibitor (AP-18) remarkably improved the survival of chilled oocytes (Fig. 2). Given that TRPA1b is activated at temperatures of 10.9°C or less [51], immature zebrafish oocytes appear to sense coldness through the activation of TRPA1b.

TRPA1 causes extracellular Ca²⁺ to enter cells after activation [27]. Chilling has been shown to induce an increase in intracellular Ca²⁺ in the oocytes of amphibians [46] and pigs [52]. Mattioli et al. investigated pig oocytes and suggested that ryanodine receptors, which release Ca²⁺ from the endoplasmic reticulum into the cytoplasm, are involved in the chilling-induced increase in intracellular Ca²⁺ levels [52]. Additionally, activated TRPA1 induces the release of Ca²⁺ from the sarcoplasmic reticulum through ryanodine receptors [37]. Therefore, we examined whether ryanodine receptors were involved in chilling injuries. The ryanodine receptor inhibitor significantly improved the survival of chilled oocytes; however, this effect was limited (Fig. 3). Thus, other Ca²⁺ channels may contribute to intracellular Ca²⁺ increase during chilling.

cPLA₂α is a Ca²⁺-dependent enzyme that produces arachidonic acid and lysophospholipids from membrane phospholipids and thus plays a key role in the synthesis of lipid mediators [53]. In the present study, survival of chilled oocytes markedly improved when a cPLA₂α-specific inhibitor was injected before chilling (Fig. 4). Therefore, the activation of cPLA₂α and the production of lipid mediators may be involved in chilling injury of immature zebrafish oocytes.

COXs and ALOX5 inhibitors significantly improved the survival of chilled oocytes (Fig. 5), and an LPCAT2 inhibitor also significantly improved their survival (Fig. 6). Hence, eicosanoids and platelet-activating factor are likely to be involved in chilling injury in immature zebrafish oocytes. Although eicosanoids and platelet-activating factor are signaling molecules that influence various cellular functions, their potential role in chilling injury has not been previously reported.

In the present study, we observed that many of the damaged oocytes chilled at 0°C for 15 min had translucent ooplasm (~50–80%), whereas those that survived had dark ooplasm (data not shown), suggesting that most egg yolks in damaged oocytes were degraded during culture for 2 h after chilling. This indicated that intracellular signaling by chilling caused a wide range of intracellular reactions, including the activation of various intracellular proteases. Further studies are required to clarify the mechanisms through which intracellular proteases are activated and damage fish oocytes during and/or after chilling.

In conclusion, activation of the cold-sensitive TRPA1 channel may trigger chilling injury in immature zebrafish oocytes, with lipid mediators potentially playing a key role in this process. The results of this study may facilitate advancements in successful cryopreservation of fish oocytes.

Conflict of interests

The authors declare no conflicts of interest.