Meretrix meretrix individuals (shell length, 46.52 ± 0.28 mm; shell width, 23.51 ± 0.22 mm; shell height, 37.73 ± 0.21 mm; weight, 27.25 ± 0.22 g) were purchased in July 2020 from Lingkun aquafarm, Wenzhou, Zhejiang, China. The seawater of the aquafarm where the clams were raised had a Cd²⁺ concentration of 0.54 μg L^-1, which is within the Chinese standard for first-class seawater quality. The clams were randomly assigned to five groups and each group was exposed to a different Cd²⁺ concentration (0, 1.5, 3, 6, or 12 mg L⁻¹). These concentrations of Cd²⁺ correspond to 1/10, 1/5, 1/2.5, and 1/1.25 of the LC₅₀ (15.01 mg L⁻¹) previously determined from a 4-day Cd²⁺ exposure (Xia et al., 2016). Each group consisted of 120 clams that were kept in three separate aquariums, with 40 clams per aquarium. The clams were exposed to the Cd²⁺ solutions without food for 5 days at 22 ± 1°C and pH 8.0. A 5-day exposure was used instead of a 4-day exposure to maximize the effect of the Cd exposure, and at the same time without killing the clams, since prolonged exposure to 12 mg L^-1 Cd²⁺ has been found to result in inactivity and even death for the clams. The aquaculture water in each aquarium was replaced daily with fresh water (prepared from double distilled water and artificial seawater salt) containing the same Cd²⁺ concentration. At the end of the exposure period, all clams were dissected, and the ovaries were quickly removed from the female clams and processed according to the following experiments. All animal experimental procedures used complied with the Wenzhou University Animal Care and Use Committee’s (WU-ACUC) guidelines, and this experiment was approved by the WU-ACUC.

To determine the Cd²⁺ content of the ovary, two female clams were randomly selected from each aquarium (sample size = 6/treatment) to harvest their ovaries. The concentration of Cd²⁺ in the ovary was measured following the experimental methods of Lin et al. (2017).

Two clams were randomly taken from each aquarium (sample size = 6/treatment) and their ovaries were extracted and then homogenized in 9 vol of 0.9% ice-cold physiological saline using a high throughput tissue grinder (SCIENTZ-48, China) kept on ice. The homogenate was then centrifuged at 12000 × g and 4°C for 5 min and the supernatant was retained, yielding an ovary extract. The levels of MDA, PCO, DPC, ROS and T-AOC in the ovary extract were then measured as described below.

The morphological changes of the ovary after Cd²⁺ treatment were observed by Hematoxylin-eosin (H&E) staining. One clam was randomly taken from each aquarium (sample size = 3/treatment) and its ovary was extracted and immobilized in 4% (v/v) paraformaldehyde for 24 h, followed by dehydration in different concentrations of ethanol (75%, 85%, 90%, 95% and 100%). The dehydrated samples were embedded in melted paraffin and sectioned with a microtome (YD-315, China) to yield 7-μm thick slices. The slices were then deparaffinized, dehydrated, stained with hematoxylin and eosin, and finally sealed with neutral balsam and observed with a light microscope (Olympus BX51, Japan).

Total RNA was extracted from the ovaries extracted from two clams randomly taken from each aquarium (sample size = 6/treatment). The RNA was extracted using a UNIQ-10 column Trizol Total RNA Extraction Kit (Shanghai Sangon, China) according to the manufacturer’s protocol. The quality of the RNA was evaluated by agarose gel electrophoresis, while its content was determined by UV spectrophotometry. The extracted RNA was reverse transcribed into cDNA using PrimeScript RT Reagent Kit (TaKaRa) according to the manufacturer’s instructions, and 1 μg of RNA was used as a template in a 20 μL reaction. RT-PCR was performed on a Roche LC480 PCR instrument (Switzerland) using a TB Green PCR kit (TaKaRa, Dalian, Liaoning, China) with β-actin as the housekeeping gene. The qPCR primers for p53, MT, Bax, Bcl-2, caspase-3 and β-actin genes are listed in Table 1 . The PCR conditions were as follows: an initial step at 95°C for 5 min followed by 40 cycles of 15 s at 95°C, 15 s at 58°C, and 20 s at 72°C. The relative expression level was calculated by using the 2 ^{- Δ Δ Ct} method (Livak and Schmittgen, 2001; Huang et al., 2020).

Statistical analysis of the data was performed with the SPSS Statistical Package (Version 23.0, Chicago, USA) and Origin Statistical Package (Version 9.2, Northampton, USA). All data were expressed as means ± standard errors (mean ± S.E.). Significance differences between the control group and treatment groups were determined using a one-way ANOVA. In addition, correlation analysis was performed with Pearson Correlation Coefficient using SPSS 23.

The Cd²⁺ level for the control group was 0.338 ± 0.009 mg kg⁻¹ wet tissue whereas the Cd²⁺ levels for the groups treated with 1.5, 3, 6, 12 mg L⁻¹ Cd²⁺ were 1.696 ± 0.010, 2.443 ± 0.012, 3.350 ± 0.012, and 4.637 ± 0.061 kg⁻¹ wet tissue, respectively. All Cd²⁺-treated groups exhibited a significant (P<0.05) increase in accumulated Cd²⁺ in the ovary compared with the control, with the group treated with 12 mg L⁻¹ Cd²⁺ displaying the maximum Cd²⁺ level. The accumulated Cd²⁺ level in the ovary of the clams clearly showed a dose-response relationship with respect to the concentration of Cd²⁺ in the water that the clams were exposed to.

The effect of Cd²⁺ on the tissue structure of M. meretrix ovary was observed by light microscopy. In the control group, the gonadal structure was complete, the ovarian cells had a normal shape and were tightly arranged, and the number of follicular epithelial cells was higher compared with those of the Cd²⁺-treated groups ( Figure 2A ). In the Cd²⁺-treated groups, the number of follicular cells gradually decreased with increasing Cd²⁺ concentrations ( Figures 2B, C, E, G ) and ovarian cells had a loose and more irregular arrangement (yellow arrows in Figures 2B, C, E, G ) compared with the control group. Cadmium treatment caused the loss of gonadal tissue structure (rectangles in Figures 2B, C, E, G ), with some ovarian cells being deformed in the group treated with 12 mg L^-1 Cd²⁺ ( Figures 2G–I ). In addition, chromatin condensation, a typical apoptotic feature, was observed in the nucleus of ovarian cells of the higher Cd²⁺ treated groups ( Figures 2D, F, H, I , red arrows). The size of the nucleus in the ovarian cells of the group treated with 12 mg L^-1 Cd²⁺ was clearly smaller compared with the ovarian cells of 3 and 6 mg L^-1 Cd²⁺-treated groups ( Figures 2D, F, H, I ).

The clams treated with Cd²⁺ showed a significantly higher level of MT mRNA than the control clams, but the initial increase was followed by a substantial decrease at the highest Cd²⁺ concentration ( Figure 6A , white bars). The maximum level was found in the group treated with 3 mg L⁻¹ Cd²⁺, and it was significantly (P < 0.05) different from the control group and the groups treated with 1.5 and 12 mg L⁻¹ Cd²⁺. The level of p53 mRNA increased with increasing Cd²⁺ concentrations ( Figure 6A , black bars), but a significant increase over the control group was only detected for the groups treated with Cd²⁺ higher than 1.5 mg L⁻¹. Cadmium, therefore, appeared to induce the expression of p53 and MT in the ovary of M. meretrix, with a more obvious concentration effect seen in the case of p53 expression.

The level of Bax mRNA increased in a concentration-dependent manner after Cd²⁺ treatment ( Figure 6B white bars), with the groups treated with 3, 6 and 12 mg L^-1 Cd²⁺ all exhibited significantly (P < 0.05) higher levels than the control group, while there was no significant (P > 0.05) difference between the control group and the group treated with the lowest Cd²⁺ concentration. As for the level of Bcl-2 mRNA, it increased for the group treated with 1.5 mg L⁻¹ Cd²⁺ but then decreased with increasing Cd²⁺ concentrations relative to the control group ( Figure 6B black bars), and the difference in Bcl-2 mRNA level between the control and each of the Cd²⁺-treated group was significant (P<0.05). The level of Caspase-3 mRNA increased with increasing Cd²⁺ concentrations and all Cd²⁺-treated groups exhibited significantly (P < 0.05) higher levels than the control group ( Figure 6C ). This indicated an obvious degree of apoptosis in ovarian cells linked to the concentration effect of Cd²⁺. Overall, the results did suggest that Cd²⁺ could activate the p53-mediated apoptosis pathway in ovarian cells, although the changes in these apoptosis-associated markers did not seem to have a consistent response to Cd²⁺ concentration.

The p53 mRNA level of the ovary was positively correlated with ROS, MDA, PCO, DPC and MT, Bax mRNA levels and negatively correlated with T-AOC and Bcl-2 mRNA levels ( Figure 7 ). This indicated that oxidative damage and p53 apoptosis pathway were mutually stimulating in the ovarian cells of Cd²⁺ treated M. meretrix, such that when the p53 apoptosis pathway was activated, the function of the intracellular antioxidant system may be inhibited.

Aquatic environment is the final acceptor of various cadmium pollutions and cadmium can remain and become enriched in aquatic organisms in tissues such as the gonads, gills, foot and visceral mass for a long time through the food chain (Revathi et al., 2011; Nunez-Nogueira et al., 2012; Chen et al., 2020). If cadmium is accumulated in the gonads, it can result in serious reproductive toxicity (Fleege et al., 2003). Long-term exposure to cadmium can inhibit ovulation and affect the development of the embryo and larva of fish (Szczerbik et al., 2006; Wu et al., 2012). Therefore, it is of great significance to study the effect of cadmium on the ovary to better understand the relationship between aquatic ecological pollution and aquatic organisms. In this study, Cd²⁺ was found to accumulate in M. meretrix ovary in a concentration-dependent manner. This finding is consistent with that of Chen et al. (2019) who found that exposure to Cd²⁺ led to a significant accumulation of Cd²⁺ in the ovary of E. sinensis in a concentration-dependent manner. Xu et al. (2016) also found the accumulation of cadmium in the ovary of the crab Sinopotamon henanense is time-dependent. Cd²⁺ toxicity has been comprehensively explored in many in vitro and in vivo studies, and various molecular and cellular mechanisms have been proposed to explain the toxicity of this metal. Among these mechanisms, oxidative stress is considered to play an important role (Matovic et al., 2015; Ren et al., 2019; Chen et al., 2020). The enrichment of cadmium to a certain level within an aquatic animal will lead to the production of a large amount of ROS (Gao et al., 2021; Wang et al., 2021). The increased extent of lipid peroxidation we observed in M. meretrix ovary manifested itself as increased ROS, MDA, DPC and PCO levels and decreased T-AOC and MT mRNA levels ( Figure 1 & Figure 6A ). Exposure to cadmium, therefore, led to the production of an excessive amount of ROS which subsequently induced lipid peroxidation in the ovary. The excess ROS can also cause the cross-linking of proteins, nucleic acids and other macromolecules, making them unable to carry out their normal functions in the ovarian cells, thereby adding further stress to the survival of the ovarian cells. The appearance of MDA, the final product of lipid peroxidation, could lead to the denaturation of the cell membrane (Gao et al., 2021). PCO is an irreversible protein modification that occurs under the action of oxidation, and such change in protein structure will result in the loss of the protein function (Fedorova et al., 2014). Other well-known forms of cellular damage induced by toxic agents are protein crosslinking and DNA fragmentation, commonly measured by the DPC assay (Jena, 2012; Cheng et al., 2021). In the case of M. meretrix ovary, DPC level was found to increase significantly in the group exposed to the lowest Cd²⁺ concentration (1.5 mg L⁻¹), however, the levels of MDA and PCO in this group were not significantly different from those of the control group ( Figures 1A–C ). This suggested that within the M. meretrix ovarian cells, DNA might be more sensitive to cadmium compared with lipids and proteins.

Excessive production of ROS can likely trigger an antioxidant defense response, which can subsequently work to purge the system of these free radicals. Experimentally, the purging of free radicals is measured in terms of T-AOC. The rising level of T-AOC in M. meretrix ovary following Cd²⁺ exposure further demonstrated the ability of Cd²⁺ to promote the production of ROS in the cells. The rise in T-AOC was accompanied by a rising level of MT mRNA. Both T-AOC level and MT expression were stimulated by Cd²⁺, but their increases appeared to drop at the higher ends of Cd²⁺ concentration ( Figures 1D , 6A ), suggesting that the antioxidant capacity of the ovary might have been overwhelmed or might have reached a maximum level. Similar findings have been reported for E. sinensis following exposure to Cd²⁺, where the activities of antioxidant enzymes such as SOD, CAT and GPx were found to increase at lower Cd²⁺ concentrations but decreased at higher Cd²⁺ concentrations (Chen et al., 2019). This suggested that there could be a threshold to which the antioxidant capacity of the cells can deal with the rise in oxidative stress induced by an external agent, and the likelihood of the cells surviving the onslaught of oxidative stress is pretty much dependent on the upper threshold of the anti-oxidative response intrinsic to that organism.

The accumulation of free radicals caused by cadmium can inhibit the development of the ovary through affecting the normal morphology of the membrane system and cells (Xu et al., 2016). The accumulation of Cd²⁺ in M. meretrix ovary was also manifested as a severely damaged cellular structure revealed by microscopic examination ( Figure 2 ). The normal survival of ovarian cells depends on the transport of substances from other tissues to these cells. Cadmium may enter the ovarian cells through the routes of material transport and accumulate in the cells, gradually increasing the survival pressure for the ovarian cells. Some ovarian cells were able to withstand the oxidative damage caused by cadmium and became deformed, and the distance between different ovarian cells became larger ( Figure 2 ). Revathi et al. (2011) found that in cadmium-treated Macrobrachium rosenbergii, the ovary has both immature oocytes and hypertrophied primary oocytes with more vacuoles. When the fish Oreochromis niloticus was exposed to Cd²⁺ for 30 days, a seemingly increase in the number of primary follicles and secondary follicles was noticed in all Cd²⁺-exposed female fish relative to the control, and after Cd²⁺ exposure for 60 days, deformed follicles were observed in the ovaries (Luo et al., 2015). Cadmium-treated rats have been shown to exhibit irregular morphology, vacuolated primary follicles and reduced luteal volume (da Costa et al., 2021). The number of primordial follicles and primary follicles in these rats was significantly lower than that in the control rats. Cadmium-induced destruction of ovarian tissue appears to be definite, universal and irreversible.

Patel et al. (2021) found that cadmium has a great toxic effect on the reproductive system, and this may consequently have a negative impact on the fertilization process, embryonic development and embryo survival rate. Cadmium can cause thinning of the ovarian cortical area, damage or loss of oocytes as well as reducing the growth and maturation of the follicles or turning them into vacuoles, thereby reducing the ovulation and fertilization ability in mammals (Thompson & Bannigan, 2008; Ikeh-Tawari et al., 2013). Wu et al. (2013) observed a 60% decrease in the mating rate for female zebrafish after exposure to 8.9–35.6 µM Cd²⁺ for 72 h. Likewise, cadmium exposure was found to result in a lower number of normal ovarian cells ( Figures 2D–I ), suggesting that cadmium may reduce ovulation in M. meretrix. The quality of the ovarian cells determines the success rate of fertilization and the normal development of the embryo. In addition, cadmium-induced DNA damage may affect the integrity of the genetic material carried by the ovarian cells, leaving a potential threat to embryonic development. Our previous study has found that cadmium exposure can disrupt the membranous structure of M. meretrix testis and significantly reduce sperm survival (Gao et al., 2021). In addition, the levels of Cd enrichment, ROS, MDA, DPC and PCO in the testis are also lower than those found in the ovary, although the number of normal sperm is significantly reduced in M. meretrix individuals exposed to increasing Cd²⁺ concentrations as in the case of the ovary observed in this study ( Figures 1 – 4 ). This indicated that the ovary might have suffered a more serious degree of oxidative damage than the testis, consistent with Luo et al. (2015) who found that female tilapia is more sensitive to Cd than male tilapia. However, the number of ovarian cells in M. meretrix was much lower than that of sperm, and since the nutrients needed for embryonic development are mainly stored in the ovary, this could mean that the detrimental effects of reduced ovarian cell count and structural damage on M. meretrix reproduction may be greater than the adverse effects of reduced sperm count. In M. meretrix, fertilization of the ova takes place outside the body. This means that cadmium not only can affect the formation and survival of the sperms and eggs by entering the body tissues of M. meretrix, but it can also interfere with the fertilization process by directly affecting the sperms and eggs that were successfully released into seawater. Therefore, the damage to ovarian cells caused by cadmium could directly affect the fecundity and seedling rate of M. meretrix. Our result provided support for the notion that Cd might pose a significant threat to the reproduction of clams through its damaging impact on ovarian cells. Apart from the biological impact that cadmium can inflict on aquatic animals, cadmium exposure can also adversely affect the economics of shellfish aquaculture (Chen et al., 2020) by decreasing the reproductive potential of the animals, and hence their production output. However, further research is required to examine the effect of Cd on the reproduction potential of M. meretrix cultured in cadmium-polluted water.

Apoptosis, a genetically controlled self-destructive process under stress, plays a crucial role in maintaining tissue homeostasis by ridding the system of abnormal or damaging cells (Elmore, 2007; Xu and Yang, 2018). A large number of studies in recent years have shown that cadmium can induce apoptosis in many tissues and cells, which include rat osteoblasts (Zheng et al., 2020), duck ovaries (Cao et al., 2016), carp neutrophils (Sun et al., 2020), crab hepatopancreas (Cheng et al., 2021) and ovary (Chen et al., 2019), clam gill (Wang et al., 2021) and hepatopancreas (Xia et al., 2016), demonstrating that apoptosis is one of the important consequences of cadmium toxicity. The study of cadmium-induced apoptosis has, therefore, become a hot topic to unravel the toxicity of cadmium at the molecular level. According to the AO/EB and flow cytometry data, the number of apoptotic M. meretrix ovarian cells increased with an increase in Cd²⁺ exposure concentration, with the number of apoptotic cells accounting for more than half of the total cells number at the highest Cd²⁺ concentration used ( Figures 3 & 4 ). This suggested the existence of a concentration-dependent relationship between the extent of apoptosis and the accumulation of cadmium, consistent with our previous study (Gao et al., 2021), where we showed that increasing the Cd²⁺ concentration to which M. meretrix is exposed can lead to a significant increase in the number of apoptotic sperm cells. Apoptosis induced by heavy metals is a very complex process, involving a series of pro-apoptotic genes and proteins, including the Src-family tyrosinase kinase, Bax, Fas and p53, and anti-apoptotic genes and related proteins such as Bcl-2 and Myc (Kurochkin et al., 2011; Jena, 2012). We have previously shown that in M. meretrix testis, Cd²⁺-induced apoptosis can up-regulate the levels of ROS and caspase-3, -9 activities, while decreasing the levels of Δψm and CCO activity, activating caspase signaling and triggering apoptosis, with p53 playing a vital role in the Cd²⁺-induced apoptosis (Gao et al., 2021). Furthermore, Cd²⁺ was also found to induce apoptosis in clam gill via the mitochondrial caspase-dependent pathway (Wang et al., 2021). In vertebrates, many genes or proteins, such as the caspase family, p53, Bax and Bcl-2, are considered to be key factors in both pathways (Huang et al., 2017; Sun et al., 2020). Caspase activation plays a central role in the execution of apoptosis. Caspase-8 and Caspase-9 are key proteins of the extrinsic (Huang et al., 2017; Sun et al., 2020) and intrinsic (Ren et al., 2019; Cheng et al., 2021) pathways of apoptosis, respectively. However, apoptosis ultimately relies on caspase-3 to regulate endonucleases to promote DNA cleavage (Kumar, 2007). The activation of caspase-3, -8 and -9 in M. meretrix ovary demonstrated that both the intrinsic and extrinsic pathways were involved in the apoptosis ( Figure 5 ). However, caspase-8 activity in the ovary appeared to be insensitive to Cd²⁺ concentration ( Figure 5B ). This suggested that the extrinsic pathway of apoptosis in the ovarian cells may have reached saturation. Instead, caspase-9 activity appeared to correlate with Cd²⁺ concentration ( Figure 5C ). It could also suggest that within a certain Cd²⁺ concentration range (1.5–12 mg L⁻¹), the extrinsic pathway of apoptosis was not significantly correlated with Cd²⁺ concentration, while the intrinsic pathway of apoptosis was positively correlated with Cd²⁺ concentration. Thus, under the Cd²⁺ concentrations used in this study, the intrinsic apoptotic pathway seemed to be the main route by which M. meretrix ovarian cells would undergo apoptosis.

Among the intrinsic apoptosis pathways, the mitochondrial pathway is the most classic apoptosis pathway. The Bcl-2 family controls the permeability of the mitochondrial membrane and is the main regulator of the mitochondrial apoptosis pathway, while p53 plays a key role in the regulation of the Bcl-2 family of proteins (Ostrakhovitch et al., 2016; Yamada and Yoshida, 2019). The expression of these apoptosis-related genes (caspase-3, p53, Bcl-2 and Bax) was found to be regulated by Cd²⁺ ( Figure 6 ). For example, after Cd²⁺ treatment, the level of Bcl-2 expression was decreased under higher Cd²⁺ concentrations (3-12 mg L⁻¹) while the levels of caspase-3, Bax and p53 expressions increased in a concentration-dependent manner. These results implied that elevation of ROS level could directly lead to DNA damage, and at the same time, it may also activate the mitochondrial-related apoptosis signaling pathway, such as by activating the expression of p53, triggering the expression of Bax. The activation of caspase-3 may be due to the direct effect of Cd²⁺ on Bax protein, which can subsequently induce mitochondrial membrane permeability with the consequent release of cytochrome c into the cytoplasm. Furthermore, the data also implied that Cd may deplete the level of mitochondrial Bcl-2 protein, and increase both the levels of caspase-3 activity and mRNA in M. meretrix ovary, probably partly because of an increase in the amount of ROS. The positive correlations observed between p53 and each of the oxidative damage indexes and apoptosis-related factors ( Figure 7 ) indicated the important role of p53 in mediating Cd²⁺-induced apoptosis in M. meretrix ovarian cells.