Effects of nTiO₂ on Oocytes of the Marine Bivalve Tegillarca granosa: Implications for Fertilization Success

Abstract

The increasing environmental release of nano-titanium dioxide (nTiO₂) due to its widespread industrial application raises concerns about its potential effects on aquatic ecosystems, particularly marine organisms. Fertilization, a critical reproductive process for broadcast-spawning bivalves, is highly sensitive to environmental pollutants. In the present investigation, we explored the effects of nTiO₂ at environmentally relevant concentrations on oocyte quality and the fertilization process in the economically important marine bivalve Tegillarca granosa. nTiO₂ exposure significantly reduced fertilization success and sperm–egg fusion efficiency, while markedly increasing polyspermy incidence. Mechanistically, nTiO₂ triggered oxidative stress in oocytes, elevating ROS and MDA levels and causing structural damage to the oocyte membrane. Moreover, nTiO₂ exposure disrupted cellular energy metabolism by inhibiting PK and PFK activities, depleting ATP content, and reducing MMP. Additionally, nTiO₂ exposure impaired Ca²⁺ homeostasis by suppressing Ca²⁺-ATPase activity, which reduced intracellular Ca²⁺ levels. These cellular disruptions collectively compromised the cortical reaction by inhibiting cortical granule exocytosis and microfilament migration. Our findings suggest that nTiO₂-induced oxidative stress, coupled with an imbalance in energy and Ca²⁺ homeostasis, impairs the cortical reaction and fertilization capacity in T. granosa. This study provides valuable insights into the mechanistic pathway underlying the reproductive toxicity of nTiO₂ in marine invertebrates, offering a basis for evaluating the ecological risks associated with the presence of nanomaterials in marine environments.

1. Introduction

The rapid advancement of nanotechnology has resulted in the widespread use of various nanoparticles (NPs) in a wide range of consumer products [1]. Among these, nano-titanium dioxide (nTiO₂, nano-TiO₂, or TiO₂ NPs) is one of the most extensively used artificial nanomaterials [2]. Due to its nanoscale properties, nTiO₂ exhibits superior photocatalytic activity, as well as enhanced chemical corrosion resistance and thermal stability compared to conventional TiO₂ (particle size > 500 nm) [3]. These properties render it ideal for various commercial sectors, including electronics, coatings, personal care products, food, and pharmaceuticals [4]. Projections indicate that by 2025, the global annual output of nTiO₂ will amount to 2.5 million tons [5]. However, as the scale of its production and usage continues to expand, nTiO₂ is expected to be released into the natural environment, potentially posing risks to organisms and ecosystems.

The ocean serves as an ultimate reservoir for NPs, which enter the marine ecosystems via atmospheric inputs, wastewater effluents, and terrestrial runoff, thereby intensifying pollution risks to marine organisms [6]. Research has reported high nTiO₂ concentrations in several marine regions. Specifically, the highest nTiO₂ concentration in the waters of San Francisco Bay (United States) was recorded at 103 μg/L [7]. Along the French Mediterranean coast, nTiO₂ concentrations were reported as high as 900 μg/L [8]. Notably, it was reported that nTiO₂ concentrations in Laizhou Bay sediments were up to 122.66 mg/kg [9]. Moreover, numerous studies have documented a range of adverse effects following exposure to nTiO₂. For instance, nTiO₂ elevates oxidative stress and suppresses digestive enzyme activity in Mytilus coruscus [10,11]. In the marine scallop Chlamys farreri, dietary exposure to nTiO₂ leads to significant accumulation in digestive tissues, provoking oxidative stress and mitochondrial ultrastructural damage [12]. Previous studies have found that nTiO₂ could impair gamete, fertilization, and embryo development in marine invertebrates. For example, nTiO₂ exposure reduces the sperm velocity and fertilization in the sea urchin Heliocidaris erythrogramma [13]. Furthermore, nTiO₂ has been shown to exacerbate total ammonia nitrogen-induced developmental neurotoxicity in Danio rerio embryos [14]. Therefore, elevated levels of nTiO₂ in marine sediments pose a significant ecological threat to benthic organisms.

Broadcast spawning is a widely recognized and common reproductive mode among many marine invertebrates [15]. During the reproductive season, they release both male and female gametes directly into seawater, where fertilization occurs via the interaction and fusion of sperm and eggs [16]. Consequently, successful fertilization in these organisms is largely dependent on gamete quality (e.g., egg morphology, size, and sperm motility) and ambient seawater conditions (e.g., temperature, pH, and pollutants) [17,18,19,20]. Accumulating evidence indicates that nanomaterials can impair gamete function and embryonic development. Nanomaterials exhibit a range of toxic effects on the gametes of aquatic organisms, such as Danio rerio, Oryzias latipes, and Daphnia magna [21,22,23]. Moreover, studies have shown that exposure to nTiO₂ reduces sperm motility in Crassostrea gigas [24]. Further research confirms that nTiO₂ induces DNA damage in the sperm [25]. In Paracentrotus lividus, nTiO₂ triggers oxidative stress and developmental abnormalities in fertilized eggs [26]. Moreover, exposure to nNiO significantly suppresses egg production and hatching rates in Centropages ponticus [27]. While early research has explored the effects of nTiO₂ on fertilization in aquatic species, most have primarily focused on its toxicity to sperm. Thus, a significant knowledge gap remains regarding nTiO₂ toxicity on oocytes.

Fertilization is crucial in the life cycle of marine organisms, as it influences population recruitment [28]. This process encompasses a sequence of intricate events, including sperm activation, the acrosome reaction, sperm–egg fusion, and egg activation [29]. In most marine invertebrates, fertilization generally involves the fusion of one sperm with a single oocyte, resulting in zygote formation. However, polyspermy occurs when multiple sperm enter a single egg [30], typically resulting in abnormal cell division and embryonic developmental defects and embryonic mortality [31]. A key block to polyspermy is the cortical reaction [32], characterized by exocytosis of cortical granules and a process triggered by intracellular Ca²⁺ oscillations [33]. Cortical granule exocytosis is an energy-intensive process that requires functional mitochondria and sufficient ATP [34]. Consequently, disruption of the cortical reaction can lead to fertilization failure or increased polyspermy, severely compromising embryonic development [35].

The blood clam (Tegillarca granosa) is a widely distributed organism of broadcast-spawning benthic bivalves. It is an economically important aquaculture species in coastal regions of East and Southeast Asia [36] and plays a vital role in coastal intertidal ecosystems [37]. Due to its limited mobility, T. granosa is particularly vulnerable to environmental pollutants [38]. Therefore, it is extensively employed as a model organism to investigate a wide range of anthropogenic stressors in marine ecotoxicology, including the toxicity of heavy metals, the ecological risks of pesticides, the effects of emerging contaminants like microplastics, and the impacts of non-traditional pressures, including anthropogenic noise [39,40,41,42,43].

Although the reproductive toxicity of nTiO₂ in aquatic organisms is being increasingly recognized, mechanistic studies specifically investigating its effects on gamete function and the fertilization process remain limited, particularly in marine bivalves. To fill this critical knowledge gap, the present study examines the toxicological mechanisms of nTiO₂ on T. granosa oocytes and fertilization. Specifically, we examined the impacts of nTiO₂ exposure on oocyte ultrastructure, oxidative stress, and the cortical reaction, with a focus on the related processes of energy metabolism, calcium homeostasis, cortical granule exocytosis, and microfilament dynamics. The research aims to elucidate the cellular mechanisms by which nTiO₂ impacts fertilization in marine bivalves, providing a theoretical foundation for assessing its reproductive toxicity and ecological risks.

2. Materials and Methods

2.1. Reagents and Preparation of nTiO₂ Solutions

nTiO₂ (≥99.8%) and non-nanoscale titanium dioxide (TiO₂ bulk) were obtained from Shanghai Kermel Chemical Reagent Co., Ltd. (Shanghai, China). Before the experiment, particle morphology and size were analyzed using a transmission electron microscope (TEM, FEI Talos F200X G2, Hillsboro, OR, USA, Figure S1A). The crystalline structure of the particles was characterized by X-ray diffraction (XRD, Ultima IV model, Rigaku, Tokyo, Japan, Figure S1B). The surface area of the particles was measured with Micromeritics ASAP 2460 (Table S1). The stock solution (1 g/L) was prepared in ultrapure water and stored at 4 °C in the dark. For each experiment, working solutions were freshly prepared in filtered (0.22 μm) seawater. The stock was sonicated for 30 min using a probe sonicator (VCX 750 model, Sonics & Materials, Inc., Newtown, CT, USA) to ensure dispersion and minimize aggregation. Based on actual environmental investigation, the exposure concentrations were selected as 0 μg/L (control group), 10 μg/L, and 100 μg/L for nTiO₂ and 100 μg/L for bulk TiO₂.

2.2. T. granosa Acquisition and Acclimation

Sexually mature blood clams were collected and manually excavated from intertidal flats in Yueqing Bay, China (28°9′11″ N, 121°5′28″ E), during low tide. After collection, they were placed in insulated containers with site seawater and transported to the laboratory within 2 h under cool, aerated conditions. The blood clams were brought to the lab and acclimated in 1000 L tanks with 800 L of sand-filtered seawater. During acclimation, seawater was maintained at a temperature, pH, salinity, and dissolved oxygen of 27.24 ± 0.87 °C, 8.12 ± 0.11, 19.47 ± 0.87‰, and 7.98 ± 0.31 mg/L, respectively. Clams were fed with Platymonas subcordiformis once a day, and the seawater was replaced to maintain water quality after feeding for 2 h.

2.3. Gamete Collection and nTiO₂ Exposure

Prior to the experiment, approximately 200 clams were transferred to the laboratory. Spawning was induced using air exposure overnight at 20 °C, followed by thermal shock in 25.0 ± 0.3 °C seawater, according to the established methods [17,44]. Individual clams were placed in separate beakers with 500 mL of filtered seawater and continuously aerated between 8:00 and 9:00. After about 30 min, clams were removed, and gametes were collected separately. Gametes were assessed microscopically. Sperm with rapid motility and less than 5% non-motile sperm were used. Oocytes were selected based on morphological integrity, spherical shape, and uniform size (59.00 ± 2.35 μm).

The obtained oocytes were randomly divided into 12 portions (3 biological replicates per treatment) and exposed for 2 h in 500 mL of the respective treatment media (Control: normal filtered seawater; Bulk TiO₂: 100 μg/L TiO₂; nTiO₂ group: 10 μg/L and 100 μg/L nTiO₂). After 2 h of exposure, oocytes were collected on a sieve with a 30 μm mesh and washed with clean seawater three times.

2.4. Fertilization and Polyspermy Assay

After 2 h of exposure, oocytes were transferred to 500 mL of clean seawater. Untreated sperm were introduced to each beaker at a ratio of 100 sperm to 1 oocyte. Following a 0.5 h fertilization, the fertilized oocytes were collected, filtered, and subsequently fixed in 4% paraformaldehyde (PFA). The fixed oocytes were then subjected to three washes, each lasting 10 min with 0.01 M phosphate-buffered saline (PBS, P1003, Solarbio, Beijing, China) to remove excess fixative. For nuclear staining, they were incubated with Hoechst 33258 (C1018, Beyotime Biotechnology, Shanghai, China) in the dark for 40 min. After washing, samples were mounted in an anti-fluorescence quenching agent (P0123, Beyotime). At least 100 oocytes per replicate were analyzed under a fluorescence microscope. Six biological replicates were used for this assay (each replicate consisted of an independent fertilization trial), with at least 100 oocytes scored per replicate. Fertilization success was scored by the presence of a male pronucleus. Polyspermy was identified by the presence of two or more male pronuclei within a single oocyte.

2.5. Gamete Fusion Efficiency

Sperm motility was quantitatively evaluated using computer-assisted sperm analysis (CASA) with a plugin for Image J. To assess sperm motility, 100 μL of untreated sperm suspension was transferred to a concave slide and covered with a coverslip for observation. The curvilinear velocity (VCL) of at least 100 sperm tracks from six males was recorded. Spermegg fusion probability was calculated based on the Vogel fertilization kinetics model formula [45]:

$$\begin{array}{c}{\displaystyle \frac{\mathsf{\beta }}{{\mathsf{\beta }}_0}}={\displaystyle \frac{\ln (1 - \mathit{p}) \times {\mathit{E}}_0}{({\mathit{e}}^{{\mathsf{\beta }}_0{\mathit{E}}_0{\mathit{t}}_{\mathit{c}}}-1) \times { \mathit{S}}_0}}\end{array}$$ (1)

where p denotes the fertilization rate, S₀ and E₀ represent sperm and oocyte concentration (cells/μL), β is the fertilization constant, β₀ stands for the probability of collision, and t_c is the gamete contact time (0.5 h). E₀/S₀ was fixed at 0.01. The fertilization rate used in the model calculation was derived from the results of Section 2.4.

2.6. Oocyte Ultrastructure Observation

The sample preparation for electron microscopy followed well-established protocols for bivalve oocytes [46,47,48]. Oocytes intended for scanning electron microscopy (SEM) were fixed in 2.5% glutaraldehyde (GA, P1126, Solarbio) in 0.1 M phosphate-buffered saline (PBS, pH 7.4, P1003, Solarbio) for 12 h, followed by post-fixation in 1% osmium tetroxide. After washing, the oocytes underwent dehydration through a graded series of ethanol. Subsequently, they were immersed for 30 min in a 1:1 mixture of alcohol and isoamyl acetate, followed by overnight immersion in pure isoamyl acetate. Samples were critical-point dried using a Hitachi HCP-2 (Hitachi, Tokyo, Japan), then coated with gold-palladium using a Hitachi E-1010 sputter coater. The samples were observed with a Hitachi SU-8010 SEM at an accelerating voltage of 20 kV. For transmission electron microscopy (TEM), oocytes were fixed following the same procedure as SEM and then embedded in Spurr’s resin after dehydration. Ultrathin sections were prepared using a Leica EM UC7 ultramicrotome, stained with uranyl acetate (5 min) and alkaline lead citrate (10 min), and analyzed under a Hitachi H-7650 TEM operating at 80 kV. Three oocytes from both the control group and the high-concentration (100 μg/L) treatment group were examined.

2.7. Oxidative Stress Biomarkers

Intracellular reactive oxygen species (ROS) levels were quantified using a commercial assay kit (S0033S, Beyotime). After 30 min incubation with DCFH-DA at 25 °C, oocytes were washed and visualized under a fluorescence microscope (excitation/emission: 488/525 nm). Mean fluorescence intensity was calculated using Image-J with the following formula: Mean = IntDen/Area, where IntDen denotes the integrated fluorescence density and Area refers to the total fluorescence area. For this assay, there were six biological replicates with at least 100 oocytes assessed per replicate. Malondialdehyde (MDA) levels were measured using the thiobarbituric acid reactive substance (TBARS) assay (A003-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) to evaluate lipid peroxidation. A 0.1 mL sample was combined with reagents, incubated at 95 °C for 40 min, and absorbance was measured at 532 nm in a 96-well plate. Protein concentration was determined using a BCA protein assay kit (P0010, Beyotime). For this assay, six biological replicates were used. Each replicate involved a pooled sample of approximately 1 × 10⁷ oocytes for biochemical extraction.

2.8. Oocyte Energy Supply

Pyruvate kinase (PK) activity was measured using a commercial kit (BC0545, Solarbio). Absorbance readings were recorded at 340 nm at 20 s (A1) and after 2 min incubation at 25 °C (A2). Phosphofructokinase (PFK) activity was similarly determined using a kit (BC0535, Solarbio) with a 10 min incubation, and the results were expressed as nmol per mg protein. ATP content was measured using a commercial kit (A095-1, Nanjing Jiancheng) by evaluating absorbance at 636 nm.

Mitochondrial membrane potential (MMP) was assessed using the JC-1 probe (C2003S, Beyotime). Oocytes were stained with JC-1 in the dark for 20 min, then washed and resuspended in anti-fade medium (P0128M, Beyotime). Fluorescence was visualized under a microscope (excitation/emission: 490/530 nm), and the mean fluorescence intensity was quantified by Image J. The mean intensity was calculated using the formula: Mean = IntDen/Area, where IntDen represents the integrated fluorescence density and Area represents the total fluorescent area.

For PK, PFK, and ATP assays, six biological replicates were analyzed. Each replicate consisted of a pooled sample of approximately 1 × 10⁷ oocytes. For MMP, six biological replicates were used, with at least 100 oocytes imaged and analyzed per replicate.

2.9. Oocyte Calcium Homeostasis

Intracellular Ca²⁺ levels were detected using the Fluo-4 AM fluorescent probe (S1060, Beyotime). For this assay, six biological replicates were used, with at least 100 oocytes assessed per replicate. Oocytes were incubated with Fluo-4 AM for 0.5 h at 25 °C, then washed and imaged using fluorescence microscopy (excitation/emission: 488/525 nm). Mean fluorescence intensity was quantified with Image J, where the formula for determining the mean is Mean = IntDen/Area, with IntDen representing the total integrated of Ca²⁺ in the oocytes and Area referring to the total fluorescent area of the Ca²⁺ signal within the oocytes.

Ca²⁺-ATPase activity was assessed using an ultramicro assay kit (A070-4, Nanjing Jiancheng) based on ATP hydrolysis. For this assay, six biological replicates were used, and each replicate involved a pooled sample of approximately 1 × 10⁷ oocytes for enzyme activity measurement. Absorbance was measured at 636 nm, and the enzyme activity was expressed as μmol of inorganic phosphorus released per mg of protein per hour (U/mg prot).

2.10. Cortical Granule Exocytosis and Microfilament Migration

Cortical granule exocytosis was assessed via fluorescent lectin staining. Oocytes were fixed, stained with FITC-conjugated peanut agglutinin (PNA-FITC, L7381, Sigma, St. Louis, MO, USA) for 40 min, and co-stained with the lipophilic dye DiI (C1036, Beyotime) to visualize the plasma membrane. Samples were visualized via a confocal microscope (BH-2, Olympus, Tokyo, Japan). Mean fluorescence intensity of PNA-FITC per oocyte, representing residual CG content, was quantified using ImageJ 1.53 (National Institutes of Health, Bethesda, MD, USA).

Microfilament distribution was visualized using Phalloidin-FITC (C1033, Beyotime). Oocytes were permeabilized with Triton X-100 (0.1%), stained with Phalloidin-FITC for 60 min, then observed under a fluorescence microscope (BH-2, Olympus). To quantify peripheral cytoskeletal recruitment, the outer layer–whole-cell fluorescence ratio (OWR) was computed as OWR = OF/TF, where OF represents the fluorescence intensity of the outer layer ring of the oocyte, and TF refers to the total fluorescence intensity of the oocyte. For these assays, six biological replicates were analyzed, with at least 50 oocytes imaged per replicate.

2.11. Statistical Analysis

Normality and homogeneity of variance were assessed using the Shapiro–Wilk and Levene tests, respectively. For data that did not conform to parametric assumptions, transformations were applied (arcsine square root transformation for percentages). Overall statistical significance was evaluated using one-way analysis of variance (ANOVA), with post hoc comparisons conducted with Tukey’s HSD test. All analyses were conducted using OriginPro 2021 (OriginLab Corp., Northampton, MA, USA). Statistical significance was defined as p < 0.05. Data were presented as mean ± SD.

3. Results

3.1. nTiO₂ Exposure Impairs Fertilization Success and Induces Polyspermy

nTiO₂ exposure significantly compromised the fertilization success of blood clam oocytes in a concentration-dependent manner (Figure 1A). Following 2 h exposure, the fertilization rate decreased significantly by 7.07% and 12.70% in the nTiO₂ treatment group (10 μg/L and 100 μg/L). In contrast, bulk TiO₂ exposure at 100 μg/L had no marked effect. Concurrently, compared to the control, nTiO₂ with 100 μg/L induced a pronounced polyspermy phenotype, with the polyspermy rate increasing 5.10 times (Figure 1B). Neither the lower nTiO₂ concentrations (10 μg/L) nor bulk TiO₂ exposure significantly altered the polyspermy rate.

Figure 1.

nTiO₂ exposure impacts fertilization and polyspermy rate in blood clam. (A) Fertilization success. (B) Polyspermy rates. Data are presented as mean ± SD (n = 6). Statistical significance among means is denoted by different superscript letters (a,b,c), where p < 0.05.

3.2. nTiO₂ Reduces Gamete Fusion Probability

Analysis based on the Vogel fertilization kinetics model revealed that the impairment in fertilization was underpinned by a significantly reduced probability of successful gamete fusion. The calculated gamete fusion probability decreased by 43.42% and 47.72% for oocytes exposed to nTiO₂ groups (10 μg/L and 100 μg/L), respectively (Figure 2). This effect was specific to the nanoscale form, as bulk TiO₂ exposure did not significantly affect fusion probability.

Figure 2.

nTiO₂ exposure impacts the gamete fusion of blood clam. Data are presented as mean ± SD (n = 6). Statistical significance among means is denoted by different superscript letters (a,b,c), where p < 0.05.

3.3. nTiO₂ Disrupts Oocyte Plasma Membrane Ultrastructure

Ultrastructure analysis via SEM and TEM confirmed significant nanoscale-specific damage to the oocyte surface. Oocytes exposed to 100 μg/L nTiO₂ exhibited severe disruption to the microvillar layer, characterized by membrane lysis, detachment, and a loss of structural connectivity (Figure 3B,D,F,H). This was in stark contrast to the intact, densely packed microvilli observed in control oocytes (Figure 3A,C,E,G). No internalization of nTiO₂ aggregates was observed.

Figure 3.

nTiO₂ exposure alters oocyte ultrastructure. Control (A,C,E,G) and nTiO₂ exposure groups (100 μg/L, (B,D,F,H)) are shown. Damaged or disconnected microvilli are indicated by arrows.

3.4. nTiO₂ Triggers Oxidative Stress in Oocytes

The mechanism underlying membrane damage was linked to the induction of oxidative stress. nTiO₂ exposure caused a significant and concentration-dependent increase in key oxidative stress biomarkers. After 2 h exposure to nTiO₂, ROS levels in oocytes rose by 21.59% and 43.83% in the nTiO₂ group (10 μg/L and 100 μg/L), respectively (Figure 4A and Figure S2). This redox imbalance led to lipid peroxidation, similarly evidenced by 76.14% and 157.53% increases in MDA content at the same concentration (Figure 4B). Bulk TiO₂ exposure did not elicit a significant oxidative stress response in oocytes.

Figure 4.

nTiO₂ exposure induces oxidative stress in blood clam oocytes. (A,B) show the relative ROS level and MDA content. Data are presented as mean ± SD (n = 6). Statistical significance among means is denoted by different superscript letters (a,b,c), where p < 0.05.

3.5. nTiO₂ Disrupts Cellular Energy Supply

nTiO₂ exposure significantly inhibited key glycolytic enzymes. PK and PFK activities were reduced by 58.88% and 58.47% in nTiO₂ treatment (10 μg/L and 100 μg/L), respectively (Figure 5A,B). This led to a concomitant depletion of cellular ATP, with ATP content decreasing by 23.46% and 51.64% in the nTiO₂ treatment groups (Figure 5C). Furthermore, exposure to high-concentration nTiO₂ (100 μg/L) induced mitochondrial dysfunction, as indicated by the 20.38% decrease in mitochondrial membrane potential (Figure 5D and Figure S3). Bulk did not alter these metabolic parameters.

Figure 5.

nTiO₂ exposure disrupts energy metabolism in blood clam oocytes. (A) PK activity. (B) PFK activity. (C) ATP content. (D) MMP in oocytes. Data are presented as mean ± SD (n = 6). Statistical significance among means is denoted by different superscript letters (a,b,c), where p < 0.05.

3.6. nTiO₂ Dysregulates Intracellular Ca²⁺ Homeostasis

Calcium signaling, essential for triggering the cortical reaction, was also disrupted. Intracellular Ca²⁺ levels in fertilized oocytes were significantly reduced by 23.07% following exposure to 100 μg/L nTiO₂ (Figure 6A and Figure S4). This depletion was mechanistically linked to the inhibition of Ca²⁺-ATPase activity, which was suppressed by 35.23% and 65.03% in the nTiO₂ treatment groups (10 μg/L and 100 μg/L), respectively (Figure 6B). Bulk TiO₂ did not affect Ca²⁺ levels or Ca²⁺-ATPase activity.

Figure 6.

nTiO₂ exposure affects Ca²⁺ homeostasis in blood clam oocytes. (A) Ca²⁺ content. (B) Ca²⁺-ATPase activity. Data are presented as mean ± SD (n = 6). Statistical significance among means is denoted by different superscript letters (a,b,c), where p < 0.05.

3.7. nTiO₂ Inhibits the Cortical Reaction by Impeding Granule Exocytosis and Microfilament Dynamics

The cortical reaction, a critical block to polyspermy, was profoundly inhibited by nTiO₂ exposure. Fluorescence quantification of cortical granules impaired exocytosis. Oocytes exposed to nTiO₂ retained significantly more cortical granules, with relative fluorescence intensity increasing by 38.57% and 109% in nTiO₂ treatments (10 μg/L and 100 μg/L), respectively (Figure 7A and Figure S5). Additionally, the migration of actin microfilaments to the cell cortex was significantly inhibited, with migration rates reduced by 30.94% and 41.68% in nTiO₂ groups (10 μg/L and 100 μg/L, Figure 7B and Figure S6). Bulk TiO₂ did not affect these processes.

Figure 7.

nTiO₂ exposure impairs cortical granule exocytosis and actin filament translocation of fertilized blood clam oocytes. (A) Cortical granule exocytosis. (B) Microfilament migration. Data are presented as mean ± SD (n = 6). Statistical significance among means is denoted by different superscript letters (a,b,c), where p < 0.05.

4. Discussion

The increasing environmental release of engineered nanoparticles and their potential ecotoxicological effects, particularly on marine organisms, has attracted growing attention in recent years [49,50,51,52]. Although the general toxicity of nTiO₂ to aquatic biota has been widely documented, its specific mechanisms of action on reproductive processes in marine invertebrates, particularly broadcast-spawning species like T. granosa, remain poorly understood. The present study provides a comprehensive framework demonstrating that exposure to environmentally relevant concentrations of nTiO₂ significantly impairs oocyte quality and fertilization success. Our results reveal that nTiO₂ toxicity operates through a sequential and interlinked pathway in which oxidative stress-induced membrane damage, disruption of cellular energy metabolism, and Ca²⁺ homeostasis collectively impair the cortical reaction, thereby increasing polyspermy. Importantly, this study further indicates that the observed reproductive toxicity is closely associated with the nanoscale properties of nTiO₂ rather than its intrinsic chemical composition.

The integrity of the oocyte plasma membrane is fundamental to successful gamete interaction, including sperm–oocyte recognition, binding, and fusion [53,54]. Ultrastructural observations revealed pronounced nanoscale-specific damage to oocyte membranes following nTiO₂ exposure, particularly manifested as microvillar disintegration and membrane irregularities [55,56]. Given that microvilli are enriched in membrane receptors and adhesion molecules involved in species-specific sperm–oocyte interaction, their structural disruption is likely to directly impair gamete fusion competence [57,58]. The strong association between membrane damage and oxidative stress was supported by the dose-dependent increases in ROS and MDA, altered membrane fluidity, permeability, and membrane protein functionality [59]. These alterations provide a mechanistic explanation for the observed decline in sperm–oocyte fusion efficiency, as quantitatively supported by the Vogel model analyses.

Beyond ultrastructural damage, nTiO₂ exposure profoundly compromised the biochemical integrity of the oocyte membrane. Phospholipids containing unsaturated fatty acids are essential for maintaining membrane fluidity, a critical determinant of membrane fusion events during fertilization [60,61]. However, these lipid components are particularly vulnerable to nanoparticle-induced oxidative stress [62,63]. Comparable mechanisms have been reported in other aquatic species. For instance, nCuO exposure in Paracentrotus lividus resulted in elevated ROS production, lipid peroxidation, mitochondrial dysfunction, and impaired sperm quality [64], while nZnO exposure in D. rerio oocytes induced oxidative DNA damage and activated autophagic and apoptotic pathways [65]. Consistent with these findings, our study demonstrated that nTiO₂ significantly elevated ROS levels and promoted lipid peroxidation in T. granosa oocytes. When lipid peroxidation exceeds a critical threshold, membrane fluidity is markedly reduced, thereby directly interfering with sperm–oocyte fusion [66]. Notably, nTiO₂ particles may penetrate the nuclear envelope and induce DNA damage, further compromising the cellular conditions required for successful fertilization [48]. Collectively, these findings suggest that excessive ROS generation induced by nTiO₂ leads to lipid peroxidation, reduced membrane fluidity, impaired gamete fusion, and ultimately decreased fertilization success in blood clam oocytes.

Successful fertilization not only requires gamete fusion but also the rapid and effective prevention of polyspermy, a process predominantly mediated by the cortical reaction in broadcast-spawning organisms [67,68]. This reaction constitutes a long-lasting block to polyspermy and relies critically on regulated intracellular Ca²⁺ signaling, actin cytoskeleton dynamics, and sufficient ATP availability [35]. In the present study, nTiO₂ exposure significantly reduced intracellular Ca²⁺ levels in fertilized oocytes. Given that a transient intracellular Ca²⁺ wave is the primary trigger for cortical granule exocytosis, Ca²⁺ homeostasis disruption is expected to severely impair cortical reaction initiation [35,69,70]. Furthermore, Ca²⁺-ATPase is crucial to regulate both Ca²⁺ wave generation and intracellular Ca²⁺ balance [71,72]. The observed inhibition of Ca²⁺-ATPase activity following nTiO₂ exposure likely contributes directly to the attenuation of intracellular Ca²⁺ signaling in fertilized oocytes.

In parallel, nTiO₂ exposure substantially disrupted cellular energy metabolism in fertilized T. granosa oocytes. The significant suppression of PK and PFK activities indicates impaired glycolytic ATP production [73]. Additionally, the marked reduction in MMP reflects mitochondrial dysfunction, further limiting ATP synthesis [74]. Elevated ROS levels induced by nTiO₂ exacerbate mitochondrial damage by compromising membrane integrity and electron transport efficiency [75,76]. As the cortical reaction is a highly energy-dependent process, the combined inhibition of glycolysis and mitochondrial ATP production severely restricts the energy supply required for cytoskeletal remodeling and vesicular transport during oocyte activation [35].

The disruption of energy and Ca²⁺ homeostasis ultimately converges on the inhibition of cortical granule exocytosis and microfilament migration. Previous studies have demonstrated that actin microfilaments are essential for the transport and fusion of cortical granules with the oocyte membrane [69]. In our study, exposure to high concentrations of nTiO₂ (100 μg/L) markedly inhibited cortical granule release and prevented the proper migration of microfilaments to the oocyte cortex. Cortical granule exocytosis is initiated by intracellular Ca²⁺ elevation and finalized through ATP-dependent vesicle transport along the actin cytoskeleton [68,77,78,79]. Moreover, actin polymerization itself is tightly regulated by both Ca²⁺ availability and cellular ATP levels [80,81]. Therefore, nTiO₂-induced depletion of intracellular Ca²⁺ and ATP likely disrupts actin assembly and dynamics, thereby impeding cortical granule trafficking and exocytosis. Consequently, insufficient cortical reaction execution fails to establish an effective block to polyspermy, explaining the elevated polyspermy observed following nTiO₂ exposure.

5. Conclusions

In conclusion, our study promotes understanding of the reproductive toxicity of nTiO₂ by elucidating a coherent mechanistic pathway that involves oxidative membrane damage, bioenergetic failure, and Ca²⁺ signaling dysregulation. These combined effects incapacitate the oocyte’s ability to complete fertilization, ultimately leading to reduced fertilization success and increased polyspermy. By identifying the cortical reaction as a critical vulnerability, our findings offer new insights into how nanoparticles can specifically compromise reproductive success in marine invertebrates. This work underscores the need to consider subcellular and functional endpoints in gametes when evaluating the ecological risks associated with nanoparticle pollution, particularly in species of economic importance. Future studies employing high-throughput molecular approaches, such as transcriptomic or proteomic analyses, could further refine our understanding of the upstream molecular events that initiate these cellular disruptions.

Abbreviations

The following abbreviations are used in this manuscript:

ATP	Adenosine triphosphate
MDA	Malondialdehyde
MMP	Mitochondrial membrane potential
NPs	Nanoparticles
PFK	Phosphofructokinase
PK	Pyruvate kinase
ROS	Reactive oxygen species
SEM	Scanning electron microscopy
TEM	Transmission electron microscopy

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/toxics14020132/s1, Figure S1: TEM micrograph and XRD pattern of nTiO₂ in present study; Table S1: Properties of nTiO₂ in the present study; Figure S2: Representative micrograph of ROS in T. granosa fertilized oocytes after nTiO₂ exposure.; Figure S3: Representative micrograph of MMP fluorescent in T. granosa oocytes after nTiO₂ exposure; Figure S4: Representative micrograph of Ca²⁺ fluorescent in fertilized oocytes of T. granosa after nTiO₂ exposure; Figure S5: Representative micrograph of nTiO₂ on cortical granule exocytosis in fertilized oocytes of T. granosa; Figure S6: Representative micrograph of nTiO₂ on microfilament migration in fertilized oocytes of T. granosa.

Author Contributions

J.Q.: Writing—original draft, Methodology, Investigation, Formal analysis. Y.C.: Writing—original draft, Methodology, Investigation. Y.Z.: Visualization, Software, Investigation. Y.Y.: Software, Formal analysis. S.Z.: Resources, Project administration. X.Z.: Visualization, Software. Y.H.: Writing—review and editing, Methodology, Investigation, Formal analysis; G.L.: Funding acquisition, Formal analysis. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Ethical review and approval were waived for this study due to REASON (Tegillarca granosa is a commercially farmed invertebrate species not subject to formal ethics committee approval under our institutional policies.). All procedures were in accordance with relevant animal welfare guidelines.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors.

Conflicts of Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Funding Statement

This research was supported by the National Natural Science Foundation of China (Grant No. 42307357) and Zhejiang Key Laboratory of Coastal Biological Germplasm Resources Conservation and Utilization Open Fund (J2024008), funded by Yu Han.