Altered actin cytoskeleton in ageing eggs of starfish affects fertilization process

Experimental Cell Research 381 (2019) 179–190

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Altered actin cytoskeleton in ageing eggs of starfish affects fertilization process

T

Nunzia Limatolaa,∗, Filip Vasilevb,1, Jong Tai Chunb, Luigia Santellaa a b

Department of Research Infrastructures for Marine Biological Resources, Italy Department of Biology and Evolution of Marine Organisms; Stazione Zoologica Anton Dohrn, Napoli, Italy

A R T I C LE I N FO

A B S T R A C T

Keywords: Ageing Oocyte quality Actin Starfish Polyspermy Calcium

Integrity of oocytes is of pivotal interest in the medical and zootechnical practice of in vitro fertilization. With time, oocytes undergo deterioration in quality, and ageing oocytes often exhibit compromised competence in fertilization and the subsequent embryonic development. With ageing oocytes and eggs of starfish (Astropecten aranciacus), we addressed the issue by examining changes of the subcellular structure and their performance at fertilization. Ageing eggs were simulated in two different experimental paradigms: i) oocytes were overmatured by 6 hours stimulation with 1-methyladenine (1-MA); ii) oocytes were removed from the gonad and maintained in seawater for 24 or 48 h before applying the hormonal stimulation (1-MA, 70 min). These eggs were compared with normally matured eggs (stimulated after isolation from the gonad with 1-MA for 70 min) with respect to the sperm-induced intracellular Ca2+ signaling and the structural changes of the egg surface. The cytoskeletal and ultrastructural differences in these eggs were assessed by confocal and transmission electron microscopy, respectively. In the two categories of ageing eggs, we have found remarkable structural modifications of the actin cytoskeleton and the cortical vesicles beneath the plasma membrane. At fertilization, these ageing eggs manifested an altered pattern of intracellular Ca2+ release, aberrant actin dynamics, and increased rate of polyspermy often despite full elevation of the fertilization envelope. Taken together, our results highlight the importance of spatio-temporal regulation of the actin cytoskeleton in the cortex of the eggs, and we postulate that the status of the actin cytoskeleton is one of the major determinants of the oocyte quality that ensures successful monospermic fertilization.

1. Introduction Optimal fertilization requires that oocytes should be fertilized at a specific meiotic stage, characteristic of the given species. For example, mammalian oocytes retain full competence for development only for several hours after ovulation during which their meiotic cycle is arrested at metaphase II [1]. If not fertilized within a certain stage or time of the maturation process, oocytes are destined to undergo time-dependent deterioration of their gamete quality [2,3]. The declining competence of the oocytes comes with alterations of the subcellular structures and improper interaction with the sperm at fertilization, which often leads to an aberrant embryonic development. At birth, a female body already possesses more than one million progenitor egg cells. Throughout life, the number of follicles decreases progressively, and only a few hundred will be released as mature eggs, whilst the

remaining cells will eventually degrade and die [4]. This gradual decrease in gamete quantity is accompanied by a decline in quality, which is associated with a progressive decrease in female fertility, higher rate of fetal pathologies, and increased risk to develop diseases in adulthood [5–7]. However, the physiological process of ‘oocyte ageing’ has remained largely unknown, and it is of fundamental importance to understand the cause of the deleterious cellular and molecular changes associated with this phenomenon. In this regard, starfish provide an invaluable experimental model system in which to analyze the molecular events taking place in the ageing oocyte. It has long been known that the optimal time frame for monospermic fertilization in starfish eggs is between the germinal vesicle breakdown (GVBD) and the extrusion of the first polar body [8–13]. For the sake of discussion, the oocytes in this interval will be referred to as “mature eggs” in this study. Fertilization outside this time

∗

Corresponding author. Department of Research Infrastructures for Marine Biological Resources, Stazione Zoologica Anton Dohrn, Villa Comunale 1, Napoli, 80121, Italy. E-mail address: [email protected] (N. Limatola). 1 Current affiliation: Centre de Recherche du Centre Hospitalier de l’Université de Montréal, (CRCHUM) Montréal, Canada. https://doi.org/10.1016/j.yexcr.2019.05.007 Received 30 November 2018; Received in revised form 3 May 2019; Accepted 4 May 2019 Available online 10 May 2019 0014-4827/ © 2019 Elsevier Inc. All rights reserved.

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polyspermy. These results underscore the importance of a spatio-temporal differentiation of the actin cytoskeleton structure of the egg necessary to ensure successful monospermic fertilization.

frame often displays polyspermy that inevitably leads to abnormal embryonic development. Indeed, when inseminated, the “immature” oocytes arrested at the GV-stage (prophase of the first meiotic division) are penetrated by multiple spermatozoa. During meiotic maturation, however, the cortex and cytoplasm of the oocytes undergo significant biochemical and morphological changes, enabling the eggs to show a normal Ca2+ response and monospermic incorporation. It has also been demonstrated that maturing oocytes of starfish become more sensitive to the Ca2+-mobilizing second messenger 1,4,5-inositol trisphosphate (InsP3) presumably owing to the extensive restructuring of the endoplasmic reticulum, the major intracellular Ca2+ store, which is concomitant with the significant reorganization of the actin cytoskeleton [14–16] in the cytoplasm and cortex of the maturing oocyte [17–19]. In line with the cortical restructuring, cortical granules are positioned just below the plasma membrane, thereby facilitating exocytosis of their contents into the perivitelline space at fertilization. The latter event leads to the separation of the vitelline coat and formation of the fertilization envelope (FE), which has been considered as the “mechanical block to polyspermy” [20–22]. However, it has been pointed out that starfish eggs treated with 1-MA for a prolonged period of time (i.e., overripe/ageing gametes) incorporate more than one spermatozoon despite the full elevation of the FE [11,23,24]. In the eggs of all animal species, interaction with the fertilizing spermatozoon gives rise to increases of intracellular Ca2+, which play a variety of roles toward successful egg activation [25,26]. Although the precise mechanism by which Ca2+ is increased has not been completely clarified, it is well known that the Ca2+ increase follows a precise spatio-temporal pattern characteristic of the species [27–29], which is necessary for the subsequent embryonic development [30–32]. In this regard, several studies have suggested that the reduced success rate of embryonic development after the fertilization of ageing oocytes could arise from abnormal calcium homeostasis [33] such as variations in the modality and frequency of the Ca2+ release at fertilization [34,35]. In echinoderm eggs, the cortical actin cytoskeleton plays a fundamental role in the modulation of the intracellular Ca2+ release, as judged by the experimental data obtained with various agents perturbing the structural organization of the actin filaments, i.e. actin drugs, the actin-severing/depolymerizing protein cofilin, anti-depactin antibody, heparin, and the sequester of PIP2 [36–40]. Accelerated actin polymerization at the time of Ca2+ increase after ionomycin treatment may also buffer the increase of Ca2+ [41]. The alteration of the actin cytoskeleton in the egg cortex with these agents commonly increased the rate of polyspermy and deregulated embryonic development. In particular, the latter consequence of ionomycin casts a warning sign to the use of this Ca2+ ionophore in the practice of artificial egg activation after intracellular sperm injection technique (ICSI) as a part of the protocol in advanced assisted reproductive technology (ART), which is now widely used to boost the successful implantation rate [42–45]. During this procedure, mature oocytes remain in the oviduct or incubation media for several hours before fertilization or activation, and the oocyte ‘ageing’ in this context of procedural handling could be one of the main causes of fertilization failure, increased embryo mortality and birth defects in most commonly used ART [46]. It has also been shown that the cleavage failure and the formation of multiple asters in equine zygotes following ICSI can be associated with alterations of Factin in the ageing oocytes [47]. In this study, we simulated the ageing process simply by leaving the oocytes in seawater for 24 or 48 hours before the addition of 1-MA, or by inducing overmaturation of freshly collected oocytes (1-MA, 6 h). After that, we investigated the effects of the ‘ageing’ on the cellular and molecular events of fertilization. To this end, we examined whether and how the changes of the actin cytoskeletal organization in the ageing eggs are linked to the rate of polyspermy and failed development, as well as to the alteration of the Ca2+ signaling patterns. We found that the ageing eggs at fertilization manifest an altered pattern of the Ca2+ release and deregulated actin dynamics, as well as an increased rate of

2. Materials and methods 2.1. Preparation of oocytes and reagents Starfish (Astropecten aranciacus) were collected in the Gulf of Gaeta and maintained in circulating cold seawater (16 °C). Female gonads were dissected from the central dorsal area near the arms and transferred to filter-sterilized seawater. Free individual oocytes were obtained by passing through gauze and rinsing in filtered seawater. After 30 min, only the fully grown immature oocytes containing the large nucleus (germinal vesicle, GV) were selected for the experiments. During the ‘ageing’ treatment, immature oocytes were incubated for 24 or 48 h in fresh filtered seawater at 18 °C, which was replaced every day. To determine the number of egg-incorporated spermatozoa, sperm were stained with Hoechst 33342 (Sigma–Aldrich), and the zygotes were observed through a CCD camera with the UV filter as described previously [41]. For fertilization, dry sperm were collected afresh and diluted in NSW. Following sperm count in hemocytometer, eggs were inseminated with the sperm density of 1 × 106 spermatozoa/ml. 2.2. Microinjection, confocal microscopy, and video imaging Microinjection was performed with an air pressure Transjector (Eppendorf) as described previously [37,38]. For Ca2+ imaging, Calcium Green 488 conjugated to 10 kD dextran (500 μM) was mixed with Rhodamine Red (35 μM) in the injection buffer (10 mM HEPES, 100 mM L-Asp, pH 7.0). To visualize F-actin in living cells, 50 μM AlexaFluor568–phalloidin (Molecular Probes) was microinjected into oocytes and eggs [38], and the cells were monitored with a TCS SP8 X confocal laser scanning microscope equipped with a White Light Laser and hybrid detectors (Leica Microsystem Srl). The images captured and saved in digital computer files were examined by using the LAS AF image analysis software (Leica Microsystem Srl) and MetaMorph (Universal Imaging Corporation). For Ca2+ imaging and to facilitate visualization of F-actin changes at the sites of multiple sperm entry throughout the entire cytoplasm of the oocytes and eggs, epifluorescence and bright field view images were preferred to confocal acquisitions and were recorded with a cooled CCD camera (MicroMax, Princeton Instruments, Inc.) mounted on a Zeiss Axiovert 200 microscope with a Plan-Neofluar 20x/0.50 objective with about 2 s time-resolution. The Ca2+ signals were quantified by MetaMorph at a given time point and normalized to the baseline fluorescence (F0) following the formula Frel = [F - F0]/F0, where F represents the average fluorescence level over the entire egg. Thus, Frel was defined in relative fluorescence units (RFU) for plotting Ca2+ trajectories in Fig. 6 A. To visualize the instantaneous increase of Ca2+ signals in subcellular regions, incremental changes of the Ca2+ rise were analyzed by applying the formula Finst = [(Ft - Ft-1)/Ft-1] and used for illustrations in Fig. 5. 2.3. Statistical analysis The average and variation of the data were reported as ‘mean ± SD (standard deviation)’ in all cases. The Student t-tests and the one-way ANOVA were performed by use of GraphPad Prism 3.0 (GraphPad Software, La Jolla, USA) and the P-values smaller than 0.05 (P < 0.05) were considered statistically significant. The results from One-way ANOVA were validated by post hoc tests. 2.4. Transmission electron microscopy (TEM) Starfish oocytes and eggs were fixed in filtered seawater containing 0.5% glutaraldehyde (pH 8.0) for 1 h at room temperature and post180

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Fig. 1. Monospermic fertilization in starfish takes place only within a short time frame during the meiotic maturation. A. aranciacus oocytes and eggs at various conditions were fertilized by Hoechst 33342-stained sperm, and the egg-incorporated spermatozoa and the fertilization envelope (FE) elevation were examined in the bright field view (upper panels) and in epifluorescence microscopy (lower panels), respectively. (A) Immature oocytes at the GV-stage. (B) Mature eggs (1-MA, 70 min). (C) Overripe eggs (1-MA, 6 h). (D) ‘Ageing oocyte’: a GV-stage oocyte kept 48 h in NSW. (E) Ageing oocytes matured by 1-MA (70 min) after the 48 h incubation. In A to E, white arrows indicate egg-incorporated spermatozoa. Note full elevation of the FE was observed only in mature (B) and overripe eggs (C). (F) Counts of the egg-incorporated spermatozoa. Histograms and error bars indicate mean and the standard deviations, respectively. The data were pooled from the eggs of three different animals. (G) A schematic diagram showing the experimental procedure and the stages of the oocytes and eggs being fertilized.

fixed with 1% osmium tetroxide for an additional 1 h. Specimens were dehydrated in increasing concentrations of ethanol and embedded in EPON 812. After being stained with UAR-EMS (Electron Microscope Science) for 30 min and 0.3% lead citrate for 30 s, the specimens were examined under a TEM (Zeiss LEO 912 AB).

3. Results 3.1. Incorporation of a single spermatozoon occurs only when the eggs are fertilized at the specific maturation stage To verify the correlation between polyspermy and the oocyte maturation stage, we examined the number of spermatozoa that were incorporated into the oocytes and eggs upon insemination. As expected, GV-stage oocytes, which do not elevate the FE, were invariably polyspermic at fertilization, and many spermatozoa (7.98 ± 1.88, n = 80) 181

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Fig. 2. The ultrastructure of the oocyte undergoes drastic reorganization during meiotic maturation and ageing. (A) The cortex of a GV-stage oocyte viewed by transmission electron microscopy (TEM). Visualization of cortical granules (CG) and microvilli (MV) embedded in the vitelline coat (VC) surrounded by the jelly coat (JC). (B) A mature egg from a fresh oocyte stimulated with 1-MA for 70 min. CG are seen positioned perpendicularly to the egg surface. (C) An overripe egg stimulated with 1-MA for 6 h. Unusually large vesicles were marked by asterisks (D) An ageing (24 h in NSW) oocyte arrested at GV-stage. (E) An oocyte matured with 1-MA for 70 min after 24 h incubation in NSW. Note the microvilli (MV) shortening due to hormone exposition (B) or ageing (C-D-E) and the accumulation of electron dense material (arrows) on the surface devoid of microvilli of an oocyte incubated in NSW for 24 h before and after hormonal treatment (D and E). Scale bar = 2 μm.

elevation of the FE at fertilization (Fig. 1 E, n = 60), suggesting that the property of the cortex has been markedly changed by the prolonged stay in seawater in such a way that the ability to form the FE at fertilization cannot be rendered by the hormone. Unlike the oocytes matured after 48 h stay in seawater, the overripe eggs (fresh oocytes stimulated with 1-MA for 6 h) did separate the FE at fertilization. To test the idea that the observed polyspermy in the overripe eggs may have been caused by slowed kinetics of the FE formation, which would not efficiently fend off supernumerary sperm, we assessed the speed of the FE formation by measuring the time required for the entire egg surface to be covered by the elevated FE. Our results showed that the time span between the onset of the FE elevation and its complete equidistance coverage of the egg surface was virtually unchanged for the 6 h overripe eggs (99.6 ± 20.5 s, n = 22), as compared with the mature eggs (107.6 ± 27.4 s, n = 26, P = 0.2644). Hence, the polyspermic fertilization displayed by the overripe eggs cannot be attributed to slow kinetics of the FE elevation, but may instead arise from another factor other than the failure of the intuitively conceived mechanical block to polyspermy rendered by the FE [21].

penetrated the oocyte surface and entered the cytoplasm (Fig. 1 A). In sharp contrast, the eggs matured for 70 min with 1-MA incorporated only a single spermatozoon in most cases (73 out of 80 eggs, 91.3%) under the same experimental conditions (Fig. 1 B). On the other hand, the oocytes exposed to 1-MA for 6 h (i.e. referred to as ‘overripe eggs’), were mostly polyspermic at fertilization (75 out of 80 eggs, 93.8%), as multiple spermatozoa entered despite the complete elevation of the FE (100% full-thickness elevation) (Fig. 1 C). While the ‘mature eggs’ fertilized in the right time frame (treated with 1-MA for 70 min) were mainly monospermic with the average egg-incorporated sperm count remaining at 1.25 ± 0.47 (n = 80), the overripe eggs exhibited a highly increased rate of polyspermy with the internalized sperm count reaching 4.1 ± 1.50 (n = 80), a difference statistically significant with respect to the mature eggs (P < 0.001) (Fig. 1 F). In line with the previous findings [11,23], the frequencies of polyspermy in the immature oocytes (100%, n = 80) and overripe eggs (93.8%, n = 80) were significantly higher (P < 0.001) than that of the mature eggs fertilized in the apt time interval (8.8%, n = 80). Likewise, the GVstage oocytes kept in seawater for 48 h (i.e. referred to as ‘ageing oocytes’) incorporated multiple spermatozoa at fertilization (4.20 ± 1.99, n = 60), but these oocytes sometimes failed to incorporate the sperm upon insemination (12 out of 60 oocytes, 20%). Even after stimulation with 1-MA for 70 min, these oocytes ‘ageing’ in seawater exhibited no signs of alleviation from the tendency of failed fertilization (11 out of 60 eggs, 18.3%) or polyspermy (egg-incorporated sperm count = 4.58 ± 2.73, n = 60). As expected, ageing GV-stage oocytes (after either 24 or 48 h) never manifested FE elevation (Fig. 1 D, n = 60). Interestingly, even after normal 70 min hormonal treatment, these polyspermic ageing eggs continued to fail to display

3.2. Alteration of the ultrastructural organization in the cortex of the ageing and overripe eggs The observation of multiple spermatozoa entering overripe eggs despite full elevation of the FE suggests that polyspermy may be determined by intracellular parameters, not the extracellular ones. In addition, the failure of FE elevation in ageing eggs at fertilization raised the possibility that the subplasmalemmal cortex of the oocytes may become aberrant during ageing and overmaturation. During meiotic 182

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to the pattern of the F-actin in the overripe eggs (Fig. 3 C,n = 25) which exhibited clear loss of their peculiar orientation in the subplasmalemmal zone. Hence, overripe eggs and the eggs matured from oocytes kept in NSW for 48 h commonly displayed increased rate of polyspermy (Fig. 1 F), and exhibited strikingly similar structure of subplasmalemmal F-actin (Fig. 3). These findings support the idea that the structural feature of the actin cytoskeleton may be closely linked to polyspermy. In addition to the structural differences, ageing and overripe eggs also displayed deregulated actin dynamics at fertilization. In normally matured eggs, the cortical actin filaments undergo orderly centripetal migration after fertilization [ [53], Supplemental Video 1], but such a characteristic movement of actin fibers was totally absent in the overripe eggs and in the eggs matured after oocyte ageing (Supplemental Video 2 and 3). Moreover, the intensely-stained thick actin bundles normally formed in the cytoplasm of the mature egg below the fertilization cone (Fig. 4 arrow, n = 9) were not clearly visible in the case of overripe eggs (Fig. 4 B, n = 10) and the eggs matured after ageing (Fig. 4 C, n = 6). Instead, the actin filaments polymerized near the internalized sperm head were rather faint, and the fertilization cones exhibited irregular shapes, which presumably prevented the spermatozoa from continuing their entry through the cytoplasm (Fig. 4 B, C; see also 1 E). This difficulty of the sperm to migrate towards the centre of the egg also seen in the GV-stage oocytes at fertilization [24] was evident in both overripe eggs (Fig. 4 B, n = 10) and in the eggs normally matured after 48 h stay of the oocytes in NSW (Fig. 4 C, n = 6). Taken together, our observation supports the idea that the structural and functional changes of the cortical actin filaments in the ageing and overripe eggs may contribute to the decaying quality of these cells as fertilization-competent eggs. Supplementary video related to this article can be found at https:// doi.org/10.1016/j.yexcr.2019.05.007

maturation, follicle cells adhering to the surface of the oocytes via cytoplasmic projections are progressively detached, and in consequence F-actin in the microvilli undergo accelerated structural reorganization [17,20,22,24,48,49]. In addition, the positioning of the vesicles in the cortex of the oocytes is expected to be influenced by the length of time individual oocytes have spent outside the ovary and after the stimulation by the maturation hormone. To address this question, we examined the ultrastructure of the cortices in these oocytes and eggs by TEM. The result showed intimate positioning of CG underneath the plasma membrane of the mature (Fig. 2 B) and overripe eggs (Fig. 2 C), while immature GV-stage oocytes lack such an orderly CG disposition (Fig. 2 A). In addition, with maturation, the profile of the microvilli layer became flat (Fig. 2 B, MV), reflecting the shortening of microvilli [22,24,50]. In some overripe eggs (1-MA, 6 h), unusually large vesicles were observed, which might represent several different vesicles being fused together during overmaturation (Fig. 2 C, asterisks). Nonetheless, when these overripe eggs were fertilized, cortical granules exocytosis still occurred in all cases, resulting in full elevation of the fertilization envelope (Supplemental Fig. 1). In 24 h ageing oocytes, some unidentified electron-dense material adhered to the surface lacking microvilli, probably representing remnants of the collapsed vitelline coat (VC) (Fig. 2 D). Even after 1-MA treatment, the CG neither moved toward plasma membrane nor re-oriented themselves perpendicular to it (Fig. 2 E). 3.3. The actin cytoskeleton of the egg cortex as a decisive factor controlling sperm penetration In starfish oocytes, the actin cytoskeleton undergoes finely regulated structural reorganization during meiotic resumption and fertilization [16,37,38]. The long acrosomal process of the sperm also contains F-actin [51,52]. At fertilization, the acrosomal filament of the spermatozoon appears to contact the cortical actin cytoskeleton of the egg. The establishment of this strong anchorage is an essential condition for the fertilizing spermatozoon to be incorporated into the egg [38]. In fact, when the structure of the actin cytoskeleton in starfish and sea urchin eggs is altered or its dynamic function is compromised by use of pharmacological agents, the sperm-incorporation process at fertilization is also heavily affected, often leading to polyspermy or failure of sperm entry [38,40,41]. Moreover, reorganization of F-actin in the maturing oocytes of starfish and sea urchin plays a key role in the translocation and reorientation of the CG beneath the plasma membrane (Fig. 2 B), which is thought to facilitate their exocytosis at fertilization [17,18]. However, their re-positioning, microvilli rearrangement and the restructuration of the cortex were all severely disabled in ageing oocytes and eggs (Fig. 2 D and E). Based on this, we examined the distribution of F-actin in the ageing oocytes and overripe eggs in order to correlate the F-actin structures with the differences in sperm receptivity and the tendency of polyspermy. As shown in the confocal microscopy images, the thick actin filaments randomly distributed throughout the inner cytoplasm of the GV-stage oocytes (Fig. 3 A, n = 15) almost completely disappeared in the mature eggs (Fig. 3 B, n = 28). Instead, F-actin in the mature eggs were more densely localized in the subplasmalemmal region, and oriented perpendicular to the egg surface (Fig. 3 B) [13,24]. This characteristically orderly arrangement of the subplasmalemmal actin filaments disappeared as the egg overmatured (Fig. 3 C). On the other hand, ageing of oocytes staled in NSW for 48 h caused profound changes in the structural organization of cortical actin filaments (Fig. 3 D, n = 12) as compared with the GVstage oocytes freshly taken from the gonad (Fig. 3 A). For instance, the individual actin bundles were much longer but sparser to the extent that actin filaments were hardly visible in certain areas of the cortex. Interestingly, when the ageing oocytes (48 h in NSW) were stimulated with 1-MA for 70 min, the pattern of the AlexaFluor-phalloidin staining in these eggs (Fig. 3 E, n = 17) was markedly different from that of the normally matured eggs (Fig. 3 B), but rather indistinguishably similar

3.4. Alteration of Ca2+ dynamics in the overripe and ageing eggs at fertilization To assess the physiological consequences of the cortical modification in ageing oocytes and overripe eggs, we examined their Ca2+ responses at fertilization. Being polyspermic [24], immature oocytes manifested multiple Ca2+ waves starting from several sperm interaction sites (3.02 ± 0.59, n = 50, Table 1). Likewise, the overripe eggs (1-MA, 6 h) exhibited multiple waves (2.96 ± 0.52, n = 26), reflecting the tendency of polyspermy (Fig. 5 B). These numbers were significantly higher than the average counts of the Ca2+ waves in the mature eggs of the same batches, which scored 1.5 ± 0.37 (n = 36, P < 0.001). As to the Ca2+ wave at fertilization, GV-stage oocytes showed a significantly slower wave propagation, as judged by the time required for the Ca2+ wave to traverse to the antipode (Table 1, traverse time). The peak amplitude of the Ca2+ wave was also significantly smaller in comparison with the mature eggs. On the other hand, the Ca2+ wave propagation in the overripe eggs (1-MA, 6 h) was not appreciably different from that in the mature eggs (1-MA, 70 min), as judged by the Ca2+ trajectories (Fig. 6 A), traverse time, and the peak amplitude (Table 1). Likewise, the latter aspects of Ca2+ wave propagation in the GV-stage oocytes at fertilization were virtually unchanged even after maintaining them in NSW for 24 h or 48 h (Table 2). However, when these oocytes were stimulated by 1-MA for 70 min, the duration of ‘ageing’ made a difference. Up to 24 h pre-incubation in NSW, all eggs exhibited virtually the same Ca2+ waves at fertilization (Fig. 5 C). When the incubation was prolonged to 48 h prior to the stimulation with 1-MA, only 17 out of 41 eggs exhibited a Ca2+ wave at fertilization (Fig. 5 D). Even when they did, the peak amplitude of the Ca2+ wave was significantly lower (Fig. 6 B) and the wave propagated faster (Table 2) in comparison with the freshly matured eggs or the eggs matured from oocytes kept in NSW for 24 h. Thus, it appears that GV stage-oocytes gradually lose their capability of responding to the maturation hormone to develop the characteristic features as competent 183

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Fig. 3. Alteration of the actin cytoskeleton in overripe eggs and in the eggs matured after 48 h incubation in NSW. Oocytes and eggs (A. aranciacus) were microinjected with phalloidin-AlexaFlour568 and F-actin changes were monitored by confocal microscopy. (A) A fresh GV-stage oocyte. (B) A mature egg (1-MA, 70 min) coming from a fresh oocyte. (C) An overripe egg (1-MA, 6 h). (D) A GV-stage oocyte maintained in NSW for 48 h. (E) An ‘ageing’ oocyte matured by 1-MA (70 min) after 48 h incubation in NSW. Scale bar = 50 μm.

other hand, prolonged time of “ageing” led to much longer latency, as was shown in ageing oocytes (48 h, 155.9 ± 92.2 s) and the eggs matured from oocytes pre-incubated in NSW for 48 h (227.6 ± 123.5 s) (Table 2). Taken as a whole, these data suggest that ageing reprograms the cortical properties of the oocytes and compromises their potentiality to undergo meiotic maturation to be competent for a normal fertilization.

eggs during the ‘ageing’ (24–48 h in NSW) and fail to display proper Ca2+ responses at fertilization. Interestingly, we found that the cortical flash (CF) was heavily influenced by the meiotic maturation stage of the oocytes and prolonged time of oocytes ageing (Fig. 5 and Table 3). Compared to sea urchin eggs, the CF in the fertilized eggs of starfish is generally modest in amplitude and lasts only few seconds, which makes it often difficult to detect. Indeed, when fertilized, only about 72% of fresh oocytes and mature eggs exhibited CF. Surprisingly, however, the CF was observed more frequently in the overripe eggs than in the normally matured eggs (Table 3). This is probably because the amplitude of the CF in the overripe (1-MA, 6 h) eggs was significantly higher (0.13 ± 0.03 RFU) than that of the mature eggs (0.06 ± 0.02 RFU, P < 0.001). Similarly, the amplitude of the CF in the fresh oocytes (0.13 ± 0.04 RFU) were twofold higher than that in the mature eggs (P < 0.001). Curiously, CF in the fresh oocytes at fertilization was peculiar in a sense that it took place after the onset of the Ca2+ wave in the majority of the cases (52%), while the CF in mature eggs (66.7%) and the ageing oocytes (24 h in NSW, 100%) predominantly preceded the onset of the Ca2+ wave (Table 3). As the oocytes undergo maturation and ageing, the responsiveness to sperm also changed considerably. When the latency of the Ca2+ response after insemination was examined, it took about 70–80 s for the fresh immature oocytes and mature eggs to show the Ca2+ responses to the fertilizing sperm in the given experimental conditions (Table 1). Conspicuously, the latency in overripe eggs (16.1 ± 2.1 s) and ageing oocytes (24 h in NSW, 11.2 ± 1.9 s) was much shorter than that of the fresh oocytes. On the

4. Discussion In this study, we examined the functional consequences of the structural changes of starfish eggs caused by overmaturation and by a condition simulating ageing. As aforementioned, the morphology of the oocyte drastically changes during the course of the meiotic progression, as evidenced by altered ultrastructure of the cortex and the actin cytoskeleton. According to the prevailing view, immature oocytes arrested at GV-stage are prone to polyspermy since they are unable to lift out the FE (Fig. 1 A); they lack an effective mechanical block to polyspermy [12,21,54,55]. However, the overripe eggs (1-MA, 6 h) fertilized after the extrusion of the second polar body did elevate the FE normally but manifested polyspermy (Fig. 1 C, Supplemental Fig. 1). On the other hand, ageing eggs matured following incubation of oocytes in NSW for 24–48 h were always unable to elevate the FE, but often managed to incorporate only one spermatozoon at fertilization (Fig. 5 C). This lack of correlation between FE elevation and monospermy raises skepticism about the physiological significance of the FE as a 184

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Fig. 4. Actin dynamics is compromised in the overripe eggs and in the ageing eggs matured after 48 h incubation in NSW. Epifluorescence images of F-actin (left panels) and the Hoechst 33342-stained spermatozoa incorporated into the egg (right panel, arrowheads). (A) A mature egg (1-MA, 70 min) after insemination (t = 11:56). (B) An overripe egg (1-MA, 6 h) (t = 12:48). (C) An ageing oocyte (48 h in NSW) matured by 1-MA (70 min) prior to insemination (t = 14:18). The moment immediately before sperm addition was taken as t = 0. Note the actin bundles near the internalized sperm head (left panels, white arrows). Scale bar = 50 μm. Fig. 5. Spatiotemporal patterns of intracellular Ca2+ increases in the overripe eggs and ageing oocytes at fertilization. A. aranciacus eggs were microinjected with Calcium dyes 10 min before fertilization, and the changes of the intracellular Ca2+ levels were monitored with the CCD camera. The pseudo-colored relative fluorescence images represent the sites of momentary Ca2+ increases at key time points. For the sake of comparison, the moment immediately before the detection of the first Ca2+ signal (CF) was set as t = 0. Numbers refer to ‘seconds’ after t = 0. (A) A mature egg (1-MA, 70 min). (B) An overripe (1-MA, 6 h) egg. (C) An ageing egg matured after incubation of immature oocytes in NSW for 24 h. (D) An ageing egg matured after incubation of immature oocytes in NSW for 48 h. The bright field view image of the same egg used in Ca2+ imaging was shown with or without the FE elevation on the right side of the pseudo-fluorescence images.

can now interact with the egg cortex and induce appropriate structural and biochemical changes in preparation for the physiological Ca2+ response at fertilization [57]. The contribution of the nucleus-borne signals in this process has been demonstrated in starfish eggs (A. pectinifera) that are partially matured only at the cortex without the occurrence of GVBD by the use of subthreshold dose of 1-MA. At fertilization, these cortically matured eggs displayed polyspermy despite the full elevation of FE and an increased sensitivity to the Ca2+-mobilizing second messenger InsP3 [58]. Thus, the formation of FE, which is a consequence of the cortical maturation, again does not play a decisive

mechanism to prevent the entry of additional spermatozoa in echinoderms. Our observations that the kinetics of the FE elevation in the overripe eggs were virtually the same as in the normally matured eggs and that the cortical granules exocytosis was not impaired by overmaturation suggest that the cause of the polyspermy in these eggs should be attributed to other physical parameters. Evidently, the cytoplasmic maturation in starfish eggs makes the cortical layer favor monospermic fertilization [56]. Once the GVDB is completed and the nuclear content is mixed with cytoplasm, substances previously trapped in the nucleus

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Fig. 6. The pattern of Ca2+ release at fertilization is severely compromised during ageing. (A) Ca2+ trajectories in mature (1-MA, 70 min) eggs (green curves) and overripe (1-MA, 6 h) eggs at fertilization (brown curves). Inset: the same curves on a different time scale to illustrate CF and the initial phase of the Ca2+ rise. Representative results from one of three independent experiments were presented here. (B) Comparison of the peak amplitude of the Ca2+ waves in the mature eggs prepared from the fresh oocytes, 24 h ageing oocytes, and 48 h ageing oocytes. Error bars represent standard deviation. *P < 0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

polyspermy in overripe eggs is also found in the electrical properties of the plasma membrane that are appreciably altered after overmaturation. The resting potential of the plasma membrane in a GV stage oocyte is about −70 mV, but it shifts to a stable level of −20 to −30 mV at the time of GVDB [64]. It has been reported that the membrane potential becomes more negative again in overripe eggs (−55 mV) as a result of the fluctuations in K+ permeability [64,65]. Moreover, the peak of the activation potential in the overripe eggs at fertilization (−4 mV) fails to reach a positive value, as opposed to the mature eggs fertilized in the optimal period (+12 mV). This compromised shift of the membrane potential at fertilization was postulated as the possible cause of multiple spermatozoa entry in overripe eggs based on the theory of the fast electric block to polyspermy [66,67]. Nonetheless, it has been rather controversial whether the abrupt shift of membrane potential at fertilization is the real cause of fast block to polyspermy or simply represents a rapid detectable change of the eggs concomitant with other physical alterations in the eggs [68–70]. Our demonstration of marked changes in the structure and functionality of microvilli and the subplasmalemmal actin filaments in the overripe eggs and ageing oocytes, which are either polyspermic or unfertilizable supports the idea that the status of the cortical actin

role in ensuring monospermic fertilization. In this context, it is noteworthy that starfish oocytes also undergo appreciable changes in their mechanical properties. In particular, during GVBD, the cellular rigidity is markedly reduced [59,60], and the stiffness of the cell remains low until the first polar body extrusion. Subsequently, an abrupt rise and fall in rigidity takes place during the formation of the second polar body [59,61]. The fluctuations in oocyte rigidity may be owing to the nucleoplasm-cytoplasm intermixing because there are no such variations in enucleated oocytes stimulated by 1-MA [62,63]. Interestingly, we noted that GV-stage oocytes ageing 24 and 48 h in NSW became progressively less resistant to microinjection and other manipulations. This simulated ‘ageing’ apparently interfered with the cortical maturation, as judged by the fact that the eggs matured after the 24–48 h pre-incubation of the oocytes in NSW mostly failed to elevate the FE at fertilization. In addition, only 25% of these eggs (5 out of 20) exhibited GVBD, a hallmark of meiotic resumption, while normally matured eggs showed GVBD in all cases (100%, n = 20). Thus, ‘ageing’ interfered with meiotic maturation, and it is also conceivable that ageing-induced morpho-functional changes compromised the responses of the ageing eggs to the fertilizing spermatozoa. One such change that might be accountable for the observed 186

100% (n = 50) 100% (n = 36) 100% (n = 26)

71.9 80.7 16.1 P<

± 31.9 ± 39.6 ± 2.1* 0.001

Latency of the Ca2+ response (after sperm addition) 3.02 ± 0.59* 1.5 ± 0.37 2.96 ± 0.52* P < 0.001

Number of Initial Ca2+ spots

187

n = 36/36 n = 9/9 n = 25/34

Mature eggs (1-MA, 70 min) Ageing oocytes (24 h in NSW) + 1-MA Ageing oocytes (48 h in NSW) + 1-MA One-way ANOVA

80.7 ± 39.6 50.6 ± 29.9 227.6 ± 123.5§ P < 0.01

71.9 ± 31.9 11.2 ± 1.9 155.9 ± 92.2** P < 0.01

Latency of the Ca2+ response (after sperm addition)

1.5 ± 0.37 1.3 ± 0.2 1.4 ± 0.3 n.s.

3.02 ± 0.59 3.8 ± 0.99 2.6 ± 0.89 n.s.

Number of Initial Ca2+ spots

0.13 0.06 0.13 P<

0.06 ± 0.02 RFU 0.07 ± 0.02 RFU 0.07 ± 0.03 RFU n.s.

0.13 ± 0.04 RFU 0.16 ± 0.02 RFU 0.08 ± 0.003 RFU n.s.

Amplitude of the CF

± 0.04 RFU ± 0.02 RFU ± 0.03 RFU 0.001

Amplitude of the CF ± 0.13 RFU* ± 0.11 RFU ± 0.08 RFU* 0.001

1.03 0.89 0.55 P<

± 0.11 RFU ± 0.10 RFU ± 0.10 RFU# 0.001

0.76 ± 0.13 RFU 0.84 ± 0.09 RFU 0.62 ± 0.16 RFU n.s.

Peak amplitude of the Ca2+ wave

0.76 1.03 1.15 P<

Peak amplitude of the Ca2+ wave

92.5 86.8 76.2 P<

± 8.7 s ± 16.8 s ± 9.8 s§ 0.01

127.2 ± 16.8 s 121.1 ± 28.2 s 89.1 ± 30.1 s n.s.

Traverse time

127.2 ± 16.8 s* 92.5 ± 8.7 s 88.6 ± 9.1 s P < 0.001

Traverse time

Note: Non-significant differences (n.s.). Values statistically significant in comparison with the control (fresh oocytes): *P < 0.01, ** P < 0.05; values statistically significant in comparison with the control (mature eggs): # P < 0.01, §P < 0.05 (post hoc analysis).

n = 50/50 n = 11/11 n = 17/41

Fresh oocytes Ageing oocytes (24 h in NSW) Ageing oocytes (48 h in NSW) One-way ANOVA

Presence of Ca2+ response

Table 2 Ca2+ responses at fertilization: comparison among fresh oocytes and ageing oocytes with or without maturation.

Note: Values statistically significant in comparison with the control (mature eggs): *P < 0.01, #P < 0.05 (post hoc analysis ).

Fresh oocytes Mature eggs (1-MA, 70 min) Overripe eggs (1-MA, 6 h) One-way ANOVA

Presence of Ca2+ response

Table 1 Ca2+ responses at fertilization: comparison among GV-stage oocytes, mature eggs, and overripe eggs.

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Table 3 Effects of oocyte maturation and ageing treatment on cortical flash.

Fresh oocytes Mature eggs (1-MA, 70 min) Overripe eggs (1-MA, 6 h) Ageing oocytes (24 h in NSW) Ageing oocytes (24 h in NSW) + 1-MA Ageing oocytes (48 h in NSW) Ageing oocytes (48 h in NSW) + 1-MA

No CF

CF before initial Ca2+ spot

CF simultaneous with initial Ca2+ spot

CF after initial Ca2+ spot

N

28% 27.8% 0% 0% 44.4% 82.9% 79.4%

14% 66.7% 88.5% 100% 55.6% 8.8% 8.8%

6% 5.5% 11.5% 0% 0% 0% 2.9%

52% 0% 0% 0% 0% 4.3% 8.8%

50 36 26 11 9 41 34

(11.5%) (Table 3). As the oocytes overmatured (1-MA, 6 h), the CF amplitude returned as high as in the fresh oocytes and its timing becomes uniform, i.e. always before the onset of the Ca2+ wave (Table 1). The tendency of firing high-amplitude CF displayed by fresh oocytes and overripe eggs might be related to the fact that the electric gradient across the plasma membrane at these stages is steeper than in the mature eggs bearing shorter microvilli [66,80]. In this regard it is important to note that while the structural arrangement of the subplasmalemmal actin meshwork in overripe egg is strikingly similar to that of the fresh oocytes (Fig. 3 A and C), the CF at insemination is altered or inhibited in the ageing oocytes (Fig. 5 C and D) in which microvilli morphology is heavily altered (Fig. 2 D). Thus, it would be of keen interest to resolve the causal relationship between the rearrangement of the core actin filaments of microvilli, which were suggested to act as a barrier to ionic fluxes across cell membranes [80,81] and the amplitude of the CF in future studies. Except for the mature eggs prepared from the fresh oocytes and fertilized during the period between GVBD and the extrusion of the first polar body, all the overripe eggs and the GV-stage oocytes both fresh and aged were prone to polyspermy (Fig. 1). However, the Ca2+ responses displayed by these ageing oocytes and overripe eggs were quite different from one another. Whereas overripe eggs and the moderately ageing oocytes (24 h) responded more promptly to the fertilizing sperm with Ca2+ increases (about 16 and 11 s, respectively), the Ca2+ responses in the oocytes and eggs with prolonged incubation in NSW (48 h) was much more delayed (about 156 s and 228 s, respectively) in comparison with the mature eggs (about 80 s) (Tables 1 and 2). The meaningful supernumerary sperm interactions are reflected by the number of initial Ca2+ spots in the oocytes and eggs, but it is not strictly correlated with the number of the egg-internalized sperm. For example, ageing eggs matured after 48 h pre-incubation in NSW displayed about 1.4 initial Ca2+ spots (Table 2) at fertilization, but the average number of egg-incorporated sperm actually mounted to about 4.3 (Fig. 1). It is also evident that the markedly high amplitude of the CF is no correlative indication of polyspermy. For instance, the oocytes ageing in NSW for 48 h or the ones matured after it were both highly polyspermic (Fig. 1) but displayed CF with the modest amplitude (0.08 and 0.07 RFU on the average) quite comparable to that of the mature eggs (0.06 RFU), which are mostly monospermic (Table 2). The results in our study underscore the importance of the structural integrity of the oocytes in ensuring successful fertilization and egg activation. The general decline of the ageing oocytes and eggs in terms of the capability of sperm interaction and Ca2+ responses calls for cautions in the biomedical and zoo-technological practices and researches. As the ultrastructure of the microvilli and cortical vesicles cytoskeletal elements are profoundly altered after a prolonged incubation in NSW, studies involving such treatment would require additional assessment of the data in the light of the fact that structures in the subplasmalemmal region is considerably altered and their functions are accordingly compromised.

cytoskeleton might play a decisive factor for a monospermic sperm-egg interaction and for guiding single sperm entry. The observation that the slow event of full elevation of the FE does not assure monospermic fertilization (Fig. 1) suggests that the major physiological role of FE may be to protect the embryo, but not to prevent polyspermy. Indeed, in the clam eggs where no thick FE is formed at fertilization, it has been demonstrated that the agent promoting actin depolymerization causes drastic increase of polyspermy [71]. Thus the intrinsic cytoskeletal nature of the eggs may be the decisive factor assuring monospermic fertilization. In this context, the cytological quality of the eggs, of which the actin cytoskeleton is considered as one of the key indices [24,38,40,72], bears great importance in successful fertilization and subsequent embryonic development. We found that one of the most evident morphological alterations in ageing and overripe eggs lies in extensive remodeling of the actin cytoskeleton within the cortex (Fig. 3). Although the precise molecular mechanism that leads to this structural reorganization is yet to be known, it has been suggested that dynamic changes of F-actin play crucial roles in controlling sperm interaction and its entry into the egg [73–75]. The different inclination to incorporate a single or multiple spermatozoa reflects variant actin dynamics at fertilization. In the eggs fertilized at the optimal period, actin filaments actively participate in the processes of sperm incorporation by implementing a specialized apparatus of actin polymerization at the sperm entry sites and by undergoing centripetal migration (Fig. 4). By contrast, ageing eggs and overripe eggs showed signs of deregulation in these processes (Fig. 4), as was the case with the GV-stage oocytes and ageing oocytes [24]. For the obvious reason that fertilization takes place at the surface of the eggs, a special attention has to be directed to microvilli, the outermost actin-filled structures. The oocytes and eggs are covered with a myriad of microvilli, which are thought to play important roles in establishing the sperm receptivity. While microvilli continuously change their length as a highly plastic structure [38], it has been reported that oocytes from aged woman have much reduced number of microvilli [76]. Here, we have shown that the profile of the microvilli is markedly changed on the surface of the starfish oocytes ‘ageing’ in NSW, as judged by the TEM images (Fig. 2). It is curious to know if such changes in microvilli are related to the increased rate of polyspermy in the ageing and overripe eggs. Artificial manipulation of actin cytoskeleton of eggs by use of chemical agents all increased the rate of polyspermy at fertilization [38–41,77]. To assess the roles played by microvilli in polyspermy and Ca2+ signaling (see below), it would be interesting to know whether or not microvilli are significantly altered by these actin drugs and agents inducing polyspermy. The actin cytoskeleton may modulate cellular Ca2+ homeostasis, regulating its influx and release from the intracellular stores in various ways [36,37,39,49,78,79]. In this study, we found that the pattern of the CF at fertilization undergoes conspicuous changes while the oocytes undergo meiotic maturation. When fresh oocytes are fertilized, a sizable CF is produced, but mostly with irregular timing, e.g. during the propagation of the Ca2+ wave (52%) (Table 3). As the oocytes became mature eggs (1-MA, 70 min), the amplitude of the CF at fertilization was reduced to a half (Table 1) but its occurrence was observed either before (88.5%) or simultaneously with the onset of the Ca2+ wave

Declarations of interest None. 188

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Funding

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