The mechanism of heat stress-induced injury in the immature oocytes of zebrafish (Danio rerio)

Abstract

Immature zebrafish oocytes are highly susceptible to high temperatures, making it difficult to warm cryopreserved oocytes rapidly. In the present study, we aimed to investigate whether thermosensitive channels, lipid mediators, and ferroptosis are involved in heat stress-induced injury in immature zebrafish oocytes. Oocytes were injected with inhibitors of a heat-sensitive channel (TRPV1) and multiple enzymes—cytosolic phospholipase A₂α (cPLA₂α), cyclooxygenases (COXs), arachidonate 5-lipoxygenase (ALOX5), and lysophosphatidylcholine acyltransferase 2 (LPCAT2). In addition, a ferroptosis-specific inhibitor was administered. The oocytes were then warmed at 45°C for 15 min, incubated at 25°C for 2 h, and then stained with propidium iodide. When the control oocytes were warmed at 45°C for 15 min, their survival was low (1%–8%). However, the survival of oocytes injected with the TRPV1-specific inhibitor markedly improved (40%), suggesting that TRPV1 activation triggers heat stress injury in oocytes. When a cPLA₂α-specific inhibitor was injected, survival of oocytes after warming significantly improved (30%), suggesting that lipid mediators or ferroptosis are involved in heat stress-induced injury in oocytes. In contrast, survival either slightly improved or did not improve when oocytes were injected with specific inhibitors of COXs, ALOX5, and LPCAT2 (16%, 8%, and 3%, respectively). Notably, the ferroptosis-specific inhibitor markedly improved oocyte survival (60%). These results may facilitate methodological advancements in fish oocyte cryopreservation. Additionally, they suggest that ferroptosis is involved in heat stress-induced injury in immature zebrafish oocytes, following TRPV1 activation and subsequent cPLA₂α activation.

Fish oocytes have not been successfully cryopreserved owing to several biological properties, including their size and sensitivity to low and high temperatures. During the vitrification of oocytes and embryos, the cooling and warming rates of the specimens are key factors for survival. For example, damage caused by intracellular ice formation can be prevented through rapid warming [1]. However, this is difficult to achieve, because heat stress-induced injury can damage fish oocytes and embryos during warming at high temperatures [2].

Khosla et al. successfully cryopreserved zebrafish embryos using laser gold nanowarming [3, 4], which is the only technique to date that has enabled reproducible cryopreservation of fish embryos. Nonetheless, cryopreserved embryos must be warmed individually using expensive equipment, which prevents practical application of the technique. In contrast, it is considerably simpler to plunge containers containing cryopreserved samples into hot water despite the risk of heat stress-induced injury.

Transient receptor potential (TRP) channels are sensitive to several chemical and physical stimuli [5]. Some TRP channels can sense warm and cold temperatures [6]. These thermo-sensor channels are present in the nerves of the skin and detect changes in the outside temperature, allowing cations, including Ca²⁺, to flow into the cells [7, 8]. In mammals, TRPA1 is activated at low temperatures [7], whereas TRPV1 and TRPV2 are activated at high temperatures [9]. In fish, a single TRPV1/2-like ortholog is expressed as a heat sensor [10, 11]. Our previous study findings strongly suggest that chilled oocytes are damaged by cold sensing through the activation of TRPA1 [12]. When TRPA1 senses low temperatures and is activated, the intracellular Ca²⁺ concentration increases in zebrafish oocytes. As a result, cytosolic phospholipase A₂α (cPLA₂α), one of the Ca²⁺-dependent enzymes, becomes activated and hydrolyzes phospholipids present in the membrane to arachidonic acid and lysophospholipids [13]. Considering that various bioactive lipid mediators, such as prostaglandins, leukotrienes, and platelet-activating factors, are synthesized from these products, lipid mediators may damage immature zebrafish oocytes after chilling [12]. Therefore, it is possible that the detection of high temperatures by heat-sensitive TRPV1 is the trigger for heat stress-induced injury and that eicosanoids and platelet-activating factors play an important role in the injury.

Recently, it has been suggested that a diverse range of polyunsaturated fatty acids, including arachidonic acid, participate in cell death through the ferroptosis pathway [14,15,16]. Specifically, lipid peroxidation of fatty acid chains by enzymes such as arachidonate lipoxygenase promotes the accumulation of lipid reactive oxygen species [15], serving as the ultimate cause of ferroptosis and cell death [16].

In this study, we aimed to investigate whether TRPV1 activation triggers heat stress-induced injury in immature zebrafish oocytes. We also examined whether cPLA₂α and lipid mediators derived from arachidonic acid and lysophospholipids play an important role in preventing heat stress-induced injury. Finally, we examined whether arachidonic acid-induced ferroptosis is involved in the injury. Figure 1 outlines our hypotheses regarding heat stress-induced injury in oocytes.

Fig. 1.

Potential mechanism of heat stress-induced injury in fish oocytes. When TRPV1 senses high temperatures, it gets activated, facilitating transmembrane Ca²⁺ entry and thus increasing intracellular Ca²⁺ levels. As a result, cytosolic phospholipase A₂α (cPLA₂α), a calcium-dependent enzyme, hydrolyzes phospholipids to arachidonic acid and lysophospholipids. Cyclooxygenases (COXs) are responsible for the synthesis of prostanoids (prostaglandins and thromboxanes) from arachidonic acid, and arachidonate 5-lipoxygenase (ALOX5) converts arachidonic acid to leukotrienes. Lysophosphatidylcholine acyltransferase 2 (LPCAT2) is involved in the biosynthesis of platelet-activating factor (PAF) biosynthesis from lysophospholipids. The increased levels of arachidonic acid and subsequent lipid peroxidation act as a trigger for ferroptosis.

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 Tetramin dry flake fish food (Spectrum Brands Japan, Yokohama, Japan) twice a day. To obtain late stage III oocytes (immature oocytes), actively spawning female fish 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 90% (v/v) Leibovitz L-15 medium (Gibco, Waltham, MA, USA), pH 8.5, containing 100 μg/ml gentamycin sulfate (90% LM) at 25°C [12] based on a previous study [17], and follicles were manually separated using a pair of forceps and a pair of 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 [18]. The collected oocytes were maintained in 90% LM. All experiments were approved by the Animal Care and Use Committee of Kochi University (O-00083).

High temperature treatment

To examine the sensitivity of immature zebrafish oocytes to high temperatures, the collected immature zebrafish oocytes were placed in 90% LM solution preheated at 45°C for 0, 5, 10, 15, 20, or 30 min. The oocytes placed at 45°C for 0 min were incubated at 25°C for 30 min, without heat treatment. They were then transferred to the same medium at 25°C and incubated for 2 h.

Injection of the inhibitors into the oocytes and high temperature treatment

A 100-µl drop of 90% LM was placed 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 (IX-71, Olympus Corporation, Tokyo, Japan). An oocyte was placed in the drop and held with a holding pipette connected to a micromanipulator under the inverted microscope. Approximately 20 nl of an inhibitor solution was injected using an injection needle connected to another micromanipulator under the inverted microscope [19]. The experiment was conducted at room temperature (23°C–25°C).

The following inhibitors were injected into the oocytes: 100 µM BCTC (a TRPV1-specific inhibitor; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), 5 μM pyrrophenone (a cPLA₂α-specific inhibitor; Cayman Chemical, Ann Arbor, MI, USA), 50 µM indomethacin (a cyclooxygenase (COX)-specific inhibitor; FUJIFILM Wako Pure Chemical Corporation), 100 µM zileuton (an arachidonate 5-lipoxygenase (ALOX5)-specific inhibitor; ChemScene, Monmouth Junction, NJ, USA), 10 μM TSI-01 (a lysophosphatidylcholine acyltransferase 2 (LPCAT2)-specific inhibitor; ChemScene), and 100 μM liproxstatin-1 (a ferroptosis-specific inhibitor; FUJIFILM Wako Pure Chemical Corporation). The concentrations of the inhibitors were ~10 times higher than those used in previous studies [20,21,22,23,24,25] because the inhibitor solutions were further diluted within the oocytes to 1/10–1/15 of their original concentration [12]. The inhibitors were dissolved in dimethyl sulfoxide (DMSO) and diluted with distilled water for injection. The final DMSO concentration in the diluted inhibitor solutions was 0.01%–0.1% (v/v).

After being injected with the inhibitors, the oocytes were incubated in 90% LM at 25°C for 1 h, treated at a high temperature of 45°C for 15 min, and then incubated in 90% LM at 25°C for 2 h. As a control, oocytes not injected with any inhibitor were first incubated at 25°C for 1 h in 90% LM, then in the same medium at 25°C for 15 min, and further incubated in the same medium at 25°C for 2 h. Each treatment consisted of 10 oocytes and was repeated 3–6 times.

Oocyte viability assessment

Oocytes were stained with 20 µg/ml 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 Corporation). Oocytes that emitted reddish-white light were considered dead, whereas those that did not emit light were considered alive.

Statistical analysis

Statistical differences among groups were analyzed using the analysis of variance in GraphPad Instat software, v.7.04, with Tukey–Kramer multiple test as a post hoc test after confirming that the groups had the same variance using Bartlett’s test. Results with P-values less than 0.05 were considered significant.

Results

Heat sensitivity of the oocytes

As shown in Fig. 2, the survival of immature zebrafish oocytes significantly decreased when exposed to 45°C for 5–30 min. The survival of immature zebrafish oocytes decreased significantly after exposure for 5 min and markedly decreased after exposure for 15 min or more, indicating that immature zebrafish oocytes are highly susceptible to 45°C.

Fig. 2.

Survival of immature zebrafish oocytes after exposure to 45°C for 5–30 min. Oocytes placed at 45°C for 0 min were incubated at 25°C for 30 min without heat treatment. Each treatment consisted of 10 oocytes. Data are indicated as mean ± SD from 3–5 replicates. Bars with different superscripts indicate significant differences (P < 0.05).

Involvement of a heat-sensitive channel in heat stress-induced injury

Figure 3A shows the effect of BCTC, a TRPV1-specific inhibitor, on heat stress-induced injury in oocytes. When oocytes were exposed to 45°C for 15 min, most of them did not survive (1%); however, the injection of BCTC into oocytes markedly improved their survival (40%). This finding suggests that TRPV1 activation triggers heat stress-induced injury in oocytes.

Fig. 3.

Survival of immature zebrafish oocytes injected with a specific inhibitor for TRPV1 (A) and cPLA₂α (B) after being warmed. Immature zebrafish oocytes were injected with BCTC (100 µM, ~20 nl) or pyrrophenone (5 µM, ~20 nl) and incubated at 25°C for 1 h, warmed at 45°C for 15 min, and incubated at 25°C for 2 h. Control oocytes were placed at 25°C for 15 min without any injection. Each treatment consisted of 10 oocytes. Data are indicated as mean ± SD from 3–4 replicates. Bars with different superscripts indicate significant differences (P < 0.05).

Involvement of cPLA₂α in heat stress-induced injury

Figure 3B shows the effect of pyrrophenone, a cPLA₂α-specific inhibitor, on heat stress-induced injury in oocytes. The survival of control oocytes was low (3%) when they were warmed at 45°C for 15 min, but their survival markedly improved following pyrrophenone injection (30%). This finding suggests that cPLA₂α and the lipid mediators, which are produced from arachidonic acid and/or lysophospholipids, play an important role in heat stress-induced injury.

Involvement of eicosanoid production in heat stress-induced injury

Figure 4A shows the effect of indomethacin, a COX-specific inhibitor, on heat stress-induced injury in oocytes. The survival of oocytes warmed at 45°C for 15 min improved slightly but significantly following indomethacin injection (16%), suggesting that prostanoids play a role in heat stress-induced injury.

Fig. 4.

Survival of immature zebrafish oocytes injected with a specific inhibitor for cyclooxygenases (A) and arachidonate 5-lipoxygenase (B) after being warmed. Immature zebrafish oocytes were injected with indomethacin (50 µM, ~20 nl) or zileuton (100 μM, ~20 nl) and incubated at 25°C for 1 h, warmed at 45°C for 15 min, and incubated at 25°C for 2 h. Control oocytes were placed at 25°C for 15 min without any injection. Each treatment consisted of 10 oocytes. Data are indicated as mean ± SD from 3–6 replicates. Bars with different superscripts indicate significant differences (P < 0.05).

Conversely, the survival of oocytes injected with zileuton, an ALOX5-specific inhibitor, did not significantly differ from that of the control (8%) (Fig. 4B), suggesting that leukotrienes do not play a significant role in heat stress-induced injury.

Involvement of platelet-activating factor production in heat stress-induced injury

Figure 5 shows the effect of TSI-01, an LPCAT2-specific inhibitor, on heat stress-induced injury in oocytes. When the oocytes were injected with TSI-01, their survival after warming at 45°C for 15 min (3%) was as low as that of the control (7%). This finding suggests that platelet-activating factors do not play a significant role in heat stress-induced injury.

Fig. 5.

Survival of immature zebrafish oocytes injected with a specific inhibitor for lysophosphatidylcholine acyltransferase 2 after being warmed. Immature zebrafish oocytes were injected with TSI-01 (10 µM, ~20 nl) and incubated at 25°C for 1 h, warmed at 45°C for 15 min, and incubated at 25°C for 2 h. Control oocytes were placed at 25°C for 15 min without any injection. Each treatment consisted of 10 oocytes. Data are indicated as mean ± SD from triplicate determinations. Bars with different superscripts indicate significant differences (P < 0.05).

Involvement of ferroptosis in heat stress-induced injury

Figure 6 shows the effect of liproxstatin-1, a ferroptosis-specific inhibitor, on heat stress-induced injury in oocytes. The survival of oocytes warmed at 45°C for 15 min markedly improved following liproxstatin-1 injection (60%). This result suggests that heat stress-induced injury in oocytes is caused by ferroptosis.

Fig. 6.

Survival of immature zebrafish oocytes injected with a specific inhibitor of ferroptosis after being warmed. Immature zebrafish oocytes were injected with liproxstatin-1 (100 µM, ~20 nl), incubated at 25°C for 1 h, warmed at 45°C for 15 min, and incubated at 25°C for 2 h. Control oocytes were placed at 25°C for 15 min without any injection. Each treatment consisted of 10 oocytes. Data are indicated as mean ± SD from 4–6 replicates. Bars with different superscripts indicate significant differences (P < 0.05).

Discussion

The findings of this study suggest that warmed oocytes are damaged by the activation of the heat-sensitive channel TRPV1. To our knowledge, this is the first study to suggest that the activation of thermosensitive channels triggers heat stress-induced injury in fish oocytes. This mechanism is similar to the chilling injury mechanism observed in immature zebrafish oocytes in our previous study [12]. Our findings also suggest that ferroptosis may be involved in the injury.

In a preliminary experiment, we examined the mRNA expression of TRPV1 and LPCAT2 in zebrafish ovaries using RT-PCR and detected their expression (Supplementary Fig. 1). Furthermore, the mRNAs of cPLA2α and COXs are expressed in zebrafish oocytes [26], and the metabolite of ALOX5 has been detected in zebrafish oocytes [27]. Therefore, TRPV1, cPLA2α, COXs, ALOX5, and LPCAT2 may be expressed in zebrafish oocytes, and inhibitors of the channel TRPV1 and the enzymes in oocytes may affect oocyte survival.

TRPV1 and TRPV2 are involved in heat sensing in mammals. TRPV1 is activated at 40°C–45°C, but TRPV2 is activated by higher temperatures (> 52°C) [28]. In contrast, zebrafish expresses a single TRPV1/2-like ortholog that may be derived from an evolutionary precursor of TRPV1 and TRPV2 [10, 11]. The threshold for the activation of TRPV1 in zebrafish (25°C) is considerably lower than that in mammals [11]. Considering the high warming temperature required for cryopreserved fish oocytes, we treated immature zebrafish oocytes at a very high temperature (45°C) in this study. Notably, when oocytes were injected with a specific TRPV1 inhibitor, their survival at 45°C for 15 min was markedly improved (40%) (Fig. 3A). Therefore, even at 45°C, the activation of TRPV1 accounts for a significant percentage of heat stress-induced injury in immature zebrafish oocytes.

cPLA2α is a Ca²⁺-dependent enzyme responsible for the production of arachidonic acid and lysophospholipids from membrane phospholipids. It plays a key role in the synthesis of lipid mediators, contributing significantly to various physiological processes [29] and chilling injury in immature zebrafish oocytes [12]. In the present study, as expected, the cPLA₂α-specific inhibitor (Fig. 3B) and COX-specific inhibitor significantly improved the survival of warmed oocytes (Fig. 4A); however, the effect of the COX-inhibitor was limited (16%). In contrast, injection of specific inhibitors of ALOX5 and LPCAT2 did not improve oocyte survival (8% and 3%, respectively) (Figs. 4B and 5). These results suggest that the production of prostaglandins plays a role in heat stress-induced injury at 45°C in immature zebrafish oocytes, whereas other eicosanoids and platelet-activating factors have little or no effect. These results differ from those observed in oocytes with chilling injury, in which both prostaglandins and platelet-activating factors contribute to cell damage [12]. One possibility is that the concentration of the inhibitors is not suitable for examining their effects on the survival of warmed oocytes. The injection of indomethacin and zileuton significantly decreased the survival of oocytes without warming (Figs. 4A and B), and the concentration of the inhibitors was considerably higher than that used in our previous study [12]. As the viability of the injected oocytes decreased before warming, further studies are needed to clarify this aspect. Another possibility is that arachidonic acid and lysophospholipids are metabolized, injuring oocytes via other pathways. As shown in Fig. 6, the ferroptosis-specific inhibitor markedly improved oocyte survival after warming, suggesting that heat stress-induced injury is principally caused by ferroptosis. Ferroptosis is a type of cell death caused by lipid peroxidation and requires iron [30]. In the mouse liver, TRPV1 plays a role in triggering cPLA₂ activation and increasing the levels of its metabolite arachidonic acid, thus promoting lipid peroxidation and ferroptosis [31]. Similar mechanisms may be involved in heat stress-induced injury in immature zebrafish oocytes. Further investigation is needed to clarify why the arachidonic acid metabolic pathway differs between low and high temperatures in zebrafish oocytes.

In the present study, higher survival (60%) after warming was observed in oocytes injected with the ferroptosis inhibitor than in oocytes injected with the TRPV1-specific inhibitor (40%). As we examined survival at a single concentration of inhibitors, this discrepancy could have been caused by the concentration of the inhibitors.

In conclusion, the results of our study facilitate methodological advancements in fish oocyte cryopreservation. On the basis of our results, we hypothesize that the activation of TRPV1 triggers heat stress (45°C)-induced injury in immature zebrafish oocytes and that ferroptosis is involved in this injury. However, further experiments are required to test these hypotheses.

Conflict of interests

The authors declare no conflicts of interest associated with this manuscript.