Chloroquine inhibits artificial oocyte activation induced by ethanol or Sr²⁺ but not by sperm in mice

Abstract

Calcium release from the endoplasmic reticulum via sperm-derived phospholipase C zeta is crucial for oocyte activation during fertilization. Chloroquine (CQ) inhibits the increase in cytoplasmic calcium. This study investigated the effects of CQ on fertilization and oocyte activation. Oocytes were collected from ICR mice, and in vitro fertilization and artificial oocyte activation with strontium ions (Sr²⁺) and ethanol were performed in the presence of 50 µM CQ. Pronuclear formation was assessed via Hoechst33242 nuclear staining, cortical granule release was evaluated using lens culinaris agglutinin-fluorescein isothiocyanate staining, and cytosolic calcium levels were measured using fluorescence microscopy with Cal-520 AM. In the presence of CQ, no pronuclei were formed even 8 h after Sr²⁺-induced oocyte activation. Furthermore, cortical granule release in CQ-treated oocytes was significantly suppressed, although not completely inhibited, and no increase in cytosolic calcium was detected. CQ also inhibited pronuclear formation during ethanol-induced oocyte activation. In in vitro fertilization, although the fertilization rate was decreased in the CQ-treated group, in which CQ treatment was continuously applied during insemination, pronuclear formation and cortical granule release were observed. The decrease in the fertilization rate was likely attributable to reduced sperm motility and decreased penetration of the zona pellucida. The findings indicate that the oocyte activation pathways triggered by ethanol/Sr²⁺ and sperm are distinguishable by CQ, and that CQ can be used as a selective inhibitor of oocyte activation induced by Sr²⁺ or ethanol treatment.

Ovulated mammalian oocytes arrested in metaphase II resume meiosis after sperm penetration. This process, called oocyte activation, occurs through a series of calcium oscillations induced by sperm-derived phospholipase C zeta (PLCζ) after fertilization [1]. These calcium oscillations are triggered by the release of calcium from the endoplasmic reticulum into the cytosol via inositol 1,4,5-trisphosphate (IP₃) receptors on the endoplasmic reticulum membrane [2, 3]. These oscillations drive activation-related events, including cortical granule exocytosis, completion of meiosis with the extrusion of the second polar body, and pronuclear formation. In addition to sperm PLCζ-induced oocyte activation, many artificial activation methods have been developed, including treatments with ethanol, electrical stimulation, and calcium ionophores [4, 5]. However, most artificial activation methods induce only transient increases in intracellular calcium levels in oocytes. In contrast, strontium ions (Sr²⁺) treatment induces repetitive increases in intracellular calcium levels similar to those observed during normal fertilization [6, 7]. Since Sr²⁺ treatment can mimic some of the oocyte activation-related events induced by sperm PLCζ, its characteristics have been considered similar to those of activation following normal fertilization [8]. In fact, Sr²⁺-induced oocyte activation is widely used in infertility treatments in humans, as well as in treatments after somatic cell nuclear transfer and during intracytoplasmic sperm injection in mice [9,10,11].

Sr²⁺ can release stored calcium through several pathways, including the activation of adenylyl cyclase 8 [12] and calcium-induced calcium release [8]. Additionally, calcium oscillations induced by Sr²⁺ require calcium release into the cytoplasm via IP₃ receptors, suggesting that the general activation mechanism is shared with sperm-induced activation [13]. Although Sr²⁺-induced oocyte activation has long been known, its mechanism of action is not fully understood.

Chloroquine (CQ) is an aminoquinoline compound synthesized in Germany in 1934 as an antimalarial drug, modeled after quinine extracted from the bark of cinchona trees [14]. However, CQ has multiple effects beyond malaria treatment, such as inducing changes in the intracellular localization of the Golgi apparatus and inhibiting the fusion of autophagosomes and lysosomes during autophagy [15]. Furthermore, CQ suppresses the increase of cytoplasmic calcium in somatic cells [16, 17]. Based on these findings, it is conceivable that CQ may affect oocyte activation. However, to date, no reports have been published on this matter. This study investigated the effects of CQ on fertilization and oocyte activation.

Materials and Methods

Animals

Female and male ICR mice, aged 8–12 weeks, were purchased from Shizuoka Laboratory Animal Center (SLC) Inc. (Hamamatsu, Japan). The mice were maintained in a specific pathogen free room (25°C, 50% relative humidity, and a 14/10-h light-dark cycle). The mice were provided ab libitum access to a standard pelleted diet and distilled water. All animal experiments were approved by the Animal Experimentation Committee (protocol number A4-10) at the University of Yamanashi, Japan, and were conducted in accordance with ethical guidelines.

Chemicals

The chemicals used in these experiments were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise indicated.

Collection of oocytes, oocyte activation, and in vitro fertilization (IVF) with CQ treatment

Mature oocytes were collected from the oviducts of 8–17 weeks-old female mice that were superovulated with 5 IU pregnant mare serum gonadotropin (ASKA Pharmaceutical, Tokyo, Japan), followed by 5 IU human chorionic gonadotropin (hCG) (ASKA Pharmaceutical) 48 h later. Cumulus–oocyte complexes (COCs) were collected from oviducts approximately 16 h after hCG injection. When the cumulus was removed, the COCs were placed in HEPES-buffered CZB medium and treated with 0.1% (w/v) bovine testicular hyaluronidase. After approximately 20 min, the COCs were washed twice and placed in a droplet of the CZB medium for culturing. For oocyte activation by Sr²⁺, cumulus-removed oocytes were cultured in Ca²⁺-free CZB medium containing 5 mM SrCl₂ (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) for 8 h at 37°C with 5% (v/v) CO₂. For oocyte activation with ethanol, cumulus-removed oocytes were cultured in CZB medium containing 5% (v/v) ethanol (Nacalai Tesque, Kyoto, Japan) for 5 min, and then cultured in CZB. For IVF, sperm collected from the caudal epididymis of 10–15 weeks-old mature ICR males were dispersed in human tubal fluid (HTF) medium and preincubated for 1 h at 37°C with 5% CO₂. For IVF, the collected COCs were incubated with sperm in HTF medium for 8 h. When CQ was used for the treatment, 50 µM were added to both the activation and insemination media [18, 19]. To observe pronuclei, activated or IVF oocytes were cultured in CZB medium containing 1 µg/ml Hoechst33342 for over 10 min and observed by fluorescence microscopy (BZ-X710 or 800, Keyence, Osaka, Japan).

Observation of cortical granule

Oocytes that were activated by Sr²⁺ treatment or IVF were cultured in CZB medium containing 5 µg/ml lens culinaris agglutinin-fluorescein isothiocyanate (LCA-FITC) (Vector Laboratories, Newark, CA, USA) for over 10 min and observed by fluorescence microscopy (BZ-X710 or 800, Keyence) at room temperature without the use of an incubator or environmental control. The patterns of cortical granule release were visually determined based on the presence (positive) or absence (negative) of LCA-FITC signals on the oocyte surface.

Analysis of calcium oscillation

Before activation with Sr²⁺ treatment, oocytes were preincubated in CZB medium containing 5 µM Cal-520 AM (AAT Bioquest, Pleasanton, CA, USA) for 1 h. After preincubation, oocytes were activated and observed by time-lapse imaging using fluorescence microscopy (BZ-X710 or 800, Keyence). Observations were conducted in a culture chamber at 5% CO₂, a top heater set at 45°C, and a bath heater at 41°C. Images were captured every 30 sec over a period of 3 h. The calcium signals were analyzed by measuring the average pixel values within the cytoplasmic regions using Python and the OpenCV library for image processing.

Analysis of sperm motility

After 1 h of incubation for capacitation, sperms were further incubated in HTF medium containing 50 µM CQ for 2 h. After incubation, sperm motility was measured using a sperm motility analysis system (SMAS; DITECT, Tokyo, Japan) at room temperature without the use of an incubator or environmental control. Analysis was performed using the software provided with the system.

Statistical analysis

All statistical analysis were performed using Fisher’s exact test in Google Colaboratory with Python.

Results

CQ inhibits pronuclear formation induced by artificial oocyte activation

To investigate the effect of CQ on oocyte activation, 50 µM CQ was added to oocytes during sperm insemination for IVF or during artificial activation with Sr²⁺ or ethanol. Pronuclear formation was observed using Hoechst staining after 8 h of Sr²⁺ treatment, 8 h of IVF, and 6 h of ethanol treatment (Fig. 1). Most fertilized embryos formed pronuclei (Supplementary Table 1, 2PN and 3PN columns). However, a limited number of embryos (Supplementary Table 1, 0PN columns) did not form pronuclei, although fluorescence resembling that of sperm was observed in the cytoplasm (Supplementary Fig. 1). However, it should be noted that in IVF, CQ treatment caused a significant decrease in the fertilization rate, including both 2PN and 3PN (Supplementary Table 1). In Sr²⁺-induced activation, pronuclei were not formed in any embryos in the CQ-treated group coincident with the activation treatment, except for two embryos that exhibited structures resembling unexpanded pronuclei (Supplementary Table 1). In contrast, in the group where CQ was added 2 h after the start of activation treatment, pronuclei were formed in all embryos, similar to the control group, as shown in ‘Late CQ’ in Fig. 1B and Supplementary Table 1. In the ethanol-treated group, no pronuclei formed in the embryos in the presence of CQ (Fig. 1C and Supplementary Table 1). These results indicate that CQ does not inhibit sperm-induced pronuclear formation, but that it completely inhibits pronuclear formation during artificial oocyte activation induced by Sr²⁺ or ethanol treatment when CQ is present in the medium from the initiation of activation.

Fig. 1.

DNA staining results using Hoechst after 8 h of IVF (ctrl: n = 60, CQ: n = 53) (A), after 8 h of activation with Sr²⁺ (ctrl: n = 46, CQ: n = 44, Late CQ: n = 49) (B), and after 6 h of activation with 5% (v/v) ethanol (EtOH) (ctrl: n = 47, CQ: n = 48) (C). ctrl, group without CQ addition; CQ, group with CQ added from the start of activation or insemination; Late CQ, group with CQ added 2 h after the start of activation; BF, bright-field image; Hoechst, fluorescent image. The images shown are representative examples. Scale bar = 25 µm. All experiments were performed three or more times.

CQ inhibits cortical reaction during Sr²⁺-induced oocyte activation

Since CQ treatment from the start of activation inhibited pronuclear formation in Sr²⁺-induced activation, whereas treatment starting 2 h after activation did not, it is assumed that CQ inhibits the early stages of the Sr²⁺-induced oocyte activation process. To investigate this possibility, cortical granule exocytosis using LCA-FITC staining was assessed 2 h after the start of activation with Sr²⁺ and 2 h post-insemination during IVF. In IVF, cortical granule release was observed in all fertilized embryos, including the CQ-treated group (Fig. 2A, Supplementary Table 2). However, in Sr²⁺-induced activation, the number of embryos exhibiting cortical granule release was significantly reduced in the CQ-treated group (Fig. 2B, Supplementary Table 2). Moreover, in embryos in which cortical granule release was observed, LCA-FITC signals appeared as small granule-like structures, which differed from the pattern observed in the control group (Fig. 2B). These findings demonstrate that CQ inhibits at an early stage prior to the cortical reaction during Sr²⁺-induced activation.

Fig. 2.

Cortical granule staining results using LCA-FITC at 2 h after IVF (ctrl: n = 70, CQ: n = 63) (A) or 2 h after activation with Sr²⁺ (ctrl: n = 47, CQ: n = 49) (B). In the CQ-treated group during Sr²⁺ activation, the right image shows an embryo without cortical granule exocytosis, whereas the left image shows an embryo with small, granule-like cortical granule exocytosis. BF, bright-field image; LCA-FITC, fluorescent image. The images are representative examples. Scale bar = 25 µm. All experiments were performed three or more times.

CQ inhibits calcium oscillations during Sr²⁺-induced oocyte activation

Since CQ was found to inhibit Sr²⁺-induced activation before the cortical reaction, we hypothesized that CQ might affect calcium oscillations, which occur prior to the cortical reaction and serve as the activation initiation mechanism. To test this, oocytes preincubated for 1 h with the cytosolic calcium fluorescent probe Cal-520 AM were activated with Sr²⁺, and changes in cytosolic calcium levels were monitored over a 3-h period. In the normal Sr²⁺ activation, all embryos exhibited increases in calcium signals (Figs. 3A, 3C). In contrast, in the CQ-treated group, no increase in calcium signal was observed in any embryo (Figs. 3B, 3C). These results indicate that CQ inhibits Sr²⁺-induced oocyte activation at a stage prior to the calcium oscillations that initiate activation.

Fig. 3.

Cytosolic Ca²⁺ fluorescence imaging results up to 3 h after the start of Sr²⁺-induced activation. (A) Control (ctrl) group; (B) CQ group: Changes in brightness values within the embryonic region. The horizontal axis represents elapsed time (min) from the start of observation, and the vertical axis represents brightness values within the embryo region. Graphs display imaging results from representative embryos. (C) Graph showing the proportion of embryos exhibiting an increase in brightness values in the ctrl and CQ groups, respectively. * Statistically significant difference (Fisher’s exact test), P < 0.05. Experiments were performed in triplicates. n = 27 for both experimental groups.

Decrease in fertilization rate due to CQ is caused by reduced sperm penetration through the zona pellucida

A significant decrease in the fertilization rate was observed with CQ treatment during insemination. To determine whether this decrease was due to the effects of CQ on the oocyte or sperm, IVF was conducted using oocytes from which the zona pellucida was removed using acidic Tyrode’s solution, and pronuclear formation were observed. In the CQ-treated group, removal of the zona pellucida led to a fertilization rate comparable to that of the control group, indicating the recovery of fertilization (Supplementary Table 3). Expanded pronuclei were observed in all the fertilized embryos (Fig. 4). Furthermore, polyspermy tended to increase with CQ treatment. These findings suggest that the decrease in the fertilization rate following CQ treatment is attributable to the reduced ability of sperm to penetrate the zona pellucida.

Fig. 4.

DNA staining results using Hoechst at 8 h after IVF using zona pellucida-free oocytes (ctrl: n = 32, CQ: n = 30). BF, bright-field image; Hoechst, fluorescent image. The images shown are representative examples. Scale bar = 25 µm. All experiments were performed in triplicates.

CQ reduces sperm straight-line motility

Since CQ has been suggested to inhibit sperm penetration through the zona pellucida, the effect of pre-incubation with CQ on sperm motility was investigated. After CQ treatment for 2 h, the proportion of sperm exhibiting A-grade straight-line velocity showed a significant decrease compared to the control group (Fig. 5A). Although not statistically significant, there was a tendency for reduced curvilinear velocity and average path velocity (Figs. 5B, 5C). Additionally, CQ treatment tended to increase the proportion of immotile (D-grade) sperm across all three velocity parameters (Fig. 5). A similar trend was observed with just 1-h CQ treatment (data not shown). These findings suggest that the reduced fertilization rate is likely due to decreased sperm motility, particularly a reduction in straight-line velocity.

Fig. 5.

Graphs of sperm straight-line velocity, curvilinear velocity, and average path velocity 2 h after CQ treatment following 1-h of preincubation. The horizontal axis (a–d) represents each velocity range (a: ≥ 25 µm/sec, b: 5–25 µm/sec, c: 0–5 µm/sec, d: 0 µm/sec). The vertical axis indicates the proportion of sperm within each velocity range. Data are expressed as mean ± standard error of the mean (SEM). * Statistically significant difference (Fisher’s exact test), P < 0.05. The experiments were performed in triplicates. n ≥ 300 for both experimental groups.

Discussion

This study demonstrated that CQ affects normal fertilization and oocyte activation induced specifically by Sr²⁺ and ethanol, but not by sperm, in mice.

In IVF, CQ treatment decreased the fertilization rate; however, this reduction was recovered upon the removal of the zona pellucida. Without the zona pellucida, fertilization occurs normally, followed by normal embryonic development [20]. This suggests that the decrease in fertilization rate in intact zona IVF was primarily due to reduced sperm penetration through the zona pellucida as a result of CQ treatment. Decreased sperm motility was consistently observed after the CQ treatment. Cortical granule exocytosis was observed in all fertilized embryos treated with CQ, and nearly all embryos formed fully expanded pronuclei. Although CQ treatment led to a decrease in the fertilization rate, primarily due to its effects on sperm, pronuclei formation in almost all fertilized embryos suggests that CQ does not significantly impact the sperm-induced oocyte activation pathway. However, the tendency for increased polyspermy observed with CQ treatment implied that CQ may play a role even in normal fertilization. Regarding the effects of CQ on sperm, decreased sperm quality due to autophagy inhibition by CQ has been reported in humans and rats [21,22,23], suggesting that the decreased sperm motility observed in this study might be due to a similar mechanism (Supplementary Fig. 2).

In Sr²⁺-induced oocyte activation, CQ treatment caused the disappearance of calcium oscillations, a significant reduction in cortical granule exocytosis, and the complete inhibition of normal pronuclear formation. However, when CQ treatment was initiated 2 h after the start of Sr²⁺ treatment, pronuclear formation was not suppressed. Additionally, in ethanol-induced activation, pronuclear formation was completely inhibited by CQ treatment. Thus, CQ completely inhibits Sr²⁺- and ethanol-induced oocyte activation, but does not inhibit normal sperm-induced oocyte activation.

Although the mechanism of CQ’s inhibition was not elucidated in the present study, it is unlikely that CQ directly interacts with Sr²⁺ and ethanol to inhibit their ability to activate the oocytes, as the concentration ratio of CQ to Sr²⁺ is, for example, 1:100. Presently, zinc and/or lysosomes were considered as possible targets of CQ for oocyte activation. CQ has been reported to act as a zinc ionophore to reduce the concentration of Zn²⁺ in oocytes [24]. Zn²⁺ is required for calcium oscillations during oocyte activation. However, the use of Zn²⁺ chelators, such as N,N,N,N-tetrakis (2-pyridinylmethyl)–1,2-ethylenediamine, indistinguishably disrupts the calcium oscillations initiated by both fertilization and SrCl₂ [25,26,27]. Lysosomes have been reported to function not only as organelles that degrade intracellular and extracellular components, but also as calcium store [28, 29]. Furthermore, CQ has been suggested to affect lysosomal function [15, 30]. Therefore, lysosome-derived signals may contribute to Sr²⁺-induced activation, and CQ may inhibit this process. Our data suggest the involvement of unidentified CQ target molecules in Sr²⁺- and ethanol-induced oocyte activation (Supplementary Fig. 2).

In IVF with CQ treatment, Hoechst fluorescence, presumed to be from sperm, was observed in the cytoplasm of CQ-treated oocytes; however, pronuclei were not formed. This suggest that CQ may affect oocyte activation, even when induced by sperm. The primary oocyte-activating factor from sperm, known as PLCζ [31,32,33,34], may not function properly, and while a backup activation pathway, such as the one with Sr²⁺, may induce activation, it appears that CQ inhibits this pathway as well, leading to failure in activation and pronuclear formation.

In Sr²⁺-induced activation, although several embryos in the CQ-treated group exhibited cortical granule exocytosis that differed in pattern from that of the control, no embryos formed pronuclei in the CQ-treated group. It is considered that slight increases in calcium, which were below the detection limit, occurred due to Sr²⁺ in the presence of CQ. This could partially cause cortical granule exocytosis but were insufficient for pronuclear formation.

In conclusion, this study demonstrates that the ethanol- or Sr²⁺- and sperm-triggered pathways leading to calcium oscillation required for oocyte activation are distinguishable in terms of their sensitivity to CQ. Furthermore, CQ can be used as a selective inhibitor of oocyte activation induced by ethanol or Sr²⁺.

Conflict of interests

The authors have nothing to declare.