Chemosphere 268 (2021) 129282

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Endocrine disrupting effects of copper and cadmium in the oocytes of the Antarctic Emerald rockcod bernacchii

* Chiara Maria Motta a, Palma Simoniello b, , Mariana Di Lorenzo a, Vincenzo Migliaccio c, Raffaele Panzuto a, Emanuela Califano a, Gianfranco Santovito d a Department of Biology, University of Naples Federico II, Naples, Italy b Department of Science and Technology, University of Naples Parthenope, Naples, Italy c Department of Chemistry and Biology “Adolfo Zambelli”, University of Salerno, Fisciano, Italy d Department of Biology, University of Padova, Italy highlights

Risks related to metal contamination in Antarctica. Effects of waterborne Cd and Cu on fish oogenesis. Oocyte degeneration and changes in glycan composition. Cd downregulation of alpha and beta estradiol receptors. Cd and Cu reduce fecundity in T. bernacchii by impairing stock recruitment. article info abstract

Article history: Antarctica has long been considered a continent free from anthropic interference. Unfortunately, recent Received 5 October 2020 evidence indicate that metal contamination has gone so far and that its effects are still unknown. For this Received in revised form reason, in the present work, the potential endocrine disrupting effect of two highly polluting metals, 28 November 2020 copper and cadmium, were examined in the Antarctic teleost Trematomus bernacchii. After a 10 days Accepted 8 December 2020 waterborne exposure, ovarian metal uptake was determined by atomic absorption; in parallel, classical Available online 18 December 2020 histological approaches were adopted to determine the effects on oocyte morphology, carbohydrate Handling Editor: James Lazorchak composition and presence and localization of progesterone and estrogen receptors. Results show that both metals induce oocyte degeneration in about one third of the previtellogenic oocytes, no matter the Keywords: stage of development. In apparently healthy oocytes, changes in cytoplasm, cortical alveoli and/or Progesterone receptors chorion carbohydrates composition are observed. Cadmium but not copper also induces significant Estrogen receptors changes in the localization of progesterone and beta-estrogen receptors, a result that well correlates with Carbohydrate composition the observed increase in ovarian metals concentrations. In conclusion, the acute modifications detected PAS staining are suggestive of a significantly impaired fecundity and of a marked endocrine disrupting effects of Lectin staining copper and cadmium in this teleost . Oocyte degeneration © Heavy metals content 2020 Elsevier Ltd. All rights reserved. Ovarian morphology

1. Introduction Once taken up by diet, skin and/or gills (Komjarova and Bury, 2014), metals diffuse in the tissues interfering with conventional The marine environment is spoiled by a multitude of anthro- primary targets such as kidney and liver (Avallone et al., 2017; Koca pogenic pollutants (Di Lorenzo et al., 2020; Motta et al, 2018, 2019; et al., 2008) and with unconventional ones such as, for example, Zizza et al., 2018) including highly toxic metals (Mebane et al., muscles, retina or lateral line (Avallone et al., 2015a, 2015b; 2012; Motta et al., 2016; Rainbow, 2002). Hernandez et al., 2006). Toxic effects are exerted via multiple mechanisms but mainly via a marked prooxidant activity (Craig et al., 2007; Zheng et al., 2016) resulting in damage in protein and lipid metabolism and DNA integrity (Nawaz et al., 2005). * Corresponding author. E-mail address: [email protected] (P. Simoniello). Metal toxicity is also extended to the reproductive organs and https://doi.org/10.1016/j.chemosphere.2020.129282 0045-6535/© 2020 Elsevier Ltd. All rights reserved. C.M. Motta, P. Simoniello, M. Di Lorenzo et al. Chemosphere 268 (2021) 129282

Fig. 1. Cu (A) and Cd (B) accumulation in Trematomus bernacchii ovaries after 10 days exposure to CuCl2 (1.574 mM) or CdCl2 (0.890 mM). Values are media ±standard deviation (n ¼ 10). Significantly different from controls (**, p < 0.01; ***, p < 0.001). embryonic and larval development (Henson and Chedrese, 2004). alterations in carbohydrate composition (Eguchi et al., 2002; In particular, copper (Cu) and cadmium (Cd), are known to interfere Mehinto et al., 2014). In particular, three lectins were used to with fecundity, hatching and larval development in several aquatic highlight the presence and distribution of N-Acetyl-glucosamine species (Capriello et al., 2019; Driessnack et al., 2016). Reported (glcNAc), N-Acetyl-galactosamine (galNAc) and mannose (Man). effects are altered cadherin localization (Prozialeck et al., 2003), Presence and localization of progesterone and alpha and beta cytoskeleton organization (Perrin et al., 2017; Wang and estradiol receptors were also determined being sex hormones Templeton, 1996) and impaired sex hormone binding with re- master regulators of oogenesis also in teleosts (Miura et al., 2007). ceptors (Cao et al., 2019; Nesatyy et al., 2006). Moreover, significant Basic histopathological methods were applied: histology for interference with the expression of many genes, such as those morphology detection (Motta et al., 2005b), histochemistry for coding for ZPR1 zinc finger, metallothionein 1 and vitellogenin, are carbohydrate detection (PAS, Dahlqvist et al., 1965; lectin staining, reported (Craig et al., 2009; Hwang et al., 2000; Gonzalez et al., Motta et al., 2005a) and immunocytochemistry for receptor local- 2006). ization (Motta et al., 2020). Cu and Cd accumulate in the gonads of several teleosts (Karlsson-Norrgren and Runn, 1985; Zhang et al., 2016), including 2. Materials and methods the Antarctic ones, expected to live in an uncontaminated envi- ronment (Beltcheva et al., 2011; Illuminati et al., 2010). This 2.1. Experimental model apparent anomaly is partially explained by the natural occurrence of elevated Cu and Cd concentrations in coastal seawaters (70 ng/L Adult female specimens of Trematomus bernacchii were and 150 ng/L respectively; Westerlund and Ohoman, 1991) and collected in the proximity of Mario Zucchelli Station in Terra Nova surface sediments (from 0.05 to 0.49 mg/g dry mass; Terra Nova Bay, Bay, Antarctica (74420S, 16770E) and kept in aquaria supplied with Bargagli et al., 1996) caused by local volcanism and up-welling aerated seawater at approximately 1 C. After 5-day acclimati- phenomena (Kurtz and Bromwich, 1985). zation, were randomly divided in three 180 L aquaria: two Though the presence of metals in the gonads of Antarctic fish is groups of five specimens each were exposed to CuCl or CdCl at the demonstrated, their effects are far less understood. For this reason, 2 2 sub-lethal concentrations of 1.574 mM and 0.890 mM, respectively the aim of the present investigation was to carry out a comparative (Santovito et al., 2003); five unexposed animals were used as evaluation of the endocrine disrupting effects of Cu and Cd on the controls. Exposure occurred under static condition (Avallone et al., oocytes of the Nototheniid Trematomus bernacchii, the Antarctic 2015a; Motta et al., 2016): no metal was added during the experi- Emerald rockcod. T. bernacchii is a teleost widely distributed in mental period so to avoid overlapping the effects due to a daily many areas of Antarctica and for this reason it has often been used renewing. The limited exposure time and the reduced metabolism as a model organism for environmental studies, also in relation to of these fish at 1 C do not favour a significant reduction in the Global Change effects (Garofalo et al., 2019; Todgham and Mandic, concentration of metals, as shown by the analyses carried out on 2020). the water of the tanks before and after the experiment (Santovito To this purpose, adult females were exposed to waterborne et al., 2003). Despite selected exposure concentrations might copper chloride (CuCl ) or cadmium chloride (CdCl ) for 10 days, at 2 2 appear relatively high, they are still environmentally realistic a sub-lethal concentration of 1.574 mM and 0.890 mM, respectively considering the bioaccumulation of metals in the investigated site (Santovito et al., 2003). Ovarian metal uptake was then determined and in polluted areas. After 10 days, specimens were euthanized by atomic absorption (Santovito et al., 2012b). (tricaine methane sulfonate, MS-222; 0.2 g/L) and ovaries were The endocrine disrupting effects of the two metals were deter- quickly excised and stored. mined by using a histopathologic approach since it is reported that treatment and sample collection comply with Research the molecular events triggered by the endocrine disruptors end in regulations concerning activities and environmental protection in an interference with cell and tissue organization (van der Ven et al., Antarctica and with the Protocol on Environmental Protection to 2003). Changes in morphology were determined together with the Antarctic Treaty, Annex II, Art. 3. All the procedures were

2 C.M. Motta, P. Simoniello, M. Di Lorenzo et al. Chemosphere 268 (2021) 129282

Fig. 2. Effects of Cu and Cd on Trematomus bernacchii oocytes morphology. Controls. A) Perinucleolar oocytes (*); Balbiani’s body (Bb), multiple nucleoli (arrow) in nuclei (n). Alveolar oocyte with cortical alveoli (a). B) Early alveolar oocyte with alveoli (a) and multiple nucleoli (arrow). C) Mid alveolar oocyte with alveoli (a) containing nucleoids (arrow).

3 C.M. Motta, P. Simoniello, M. Di Lorenzo et al. Chemosphere 268 (2021) 129282 organized to minimize stress and the number of animals used. They were always completely unstained (data not shown). Label- ling was defined as positive or negative by two independent ob- 2.2. Quantification of metal concentrations servers (Motta et al., 2005a).

Ovarian tissues (2 g) were homogenized by Polytron in 4 vol/g of 2.5. Immunostaining tissue of 0.5 M sucrose, 20 mM TriseHCl buffer pH 8.6, supple- mented with 0.006 mM leupeptin, 0.5 mM PMSF (phenyl- Sections were dewaxed, rehydrated in a graded alcohol and methylsulfonyl fluoride) as anti-proteolytic agents, and 0.01% b- washed in PBS (pH 7.3). To unmask the antigens, they were mercaptoethanol as reducing agent. The homogenates were microwaved at 750 W for 15 min in citrate buffer (0.01 M, pH 6) and centrifuged at 48,000 g for 60 min at 4 C (Beckman model J2/21). washed in 0.1% bovine serum albumin in PBS followed by rinsing in Samples of the resulting supernatants were digested with 3% H2O2 for 20 min to block the endogenous peroxidases (Coscia AristaR nitric acid in Teflon vessels in a model MDS-2000 CEM et al., 2014). Incubation with the primary antibodies (progester- microwave under pressure, to eliminate the organic matrix. The one: Novocastra; mouse, monoclonal, A/B forms; estradiol: Abcam; digestion program consisted of four steps, each lasting 5 min, at rabbit polyclonal) was carried out after 1:150 dilution in PBS/BSA/ pressures of 137, 274, 411 and 548 kPa. The resulting extracts were Triton buffer, at 4 C for 24 h. Sections were repeatedly washed in made up to 2.5 mL with milliQ water and Cu and Cd contents were PBS and binding sites were revealed with a secondary peroxidase- determined by atomic absorption flame spectrophotometer (Per- conjugated antibody (1:400 in PBS, Sigma Aldrich, Italy) for kinElmer model 4000). The instruments for metal analysis were 90 min at room temperature followed by a tertiary anti-PAP anti- calibrated by standard addition methods and by reference to fresh body (1:100 in PBS) and developed with DAB tablets (Tammaro standard salt solution. Control blank solution on reagents and et al., 2007). Negative controls of the reaction were prepared by equipment revealed insignificant contamination of samples. Values omitting the primary antibody in the mixture; connective cells were expressed as ng of single metal/mg of total proteins assayed were used consistently as negative internal controls (Tammaro by the Folin phenol reagent method (Lowry et al., 1951) using et al., 2017). Labeling was considered positive or negative by bovine serum albumin as standard. three independent and experienced observers (Motta et al., 2020).

2.3. Statistical analysis of data 3. Results

Results were expressed as mean ± standard deviation (SD); 3.1. Cu and Cd ovarian content statistically significant differences among treatments were assessed using one-way ANOVA (PRIMER statistical software; Compared to the controls, ovaries in treated animals show a minimum accepted significance level p < 0.05). significant increase in both Cu and Cd content (p < 0.01 and p < 0.001, respectively). In particular, Cu concentration increases 1.6 2.4. Tissues sampling and staining times (Fig. 1A), whereas Cd concentration increases 14.8 times (Fig. 1B). For light microscopy, left ovaries (5 from control, 5 from Cd and 5 from Cu contaminated animals) were fixed in Bouin’s solution 3.2. Effects of Cu and Cd administration on oocyte morphology (8 h), dehydrated in graded ethanol and processed for paraffin embedding according to routine protocols (Bonucci, 1990). Serial The control ovary contains oocytes at different stages of growth sections (6 mm) were stained with haematoxylineeosin to show indicating that oogenesis is asynchronous with ovulatory waves. general morphology. Oocyte staging (perinucleolar, alveolar and Perinucleolar oocytes (Fig. 2A) show a dense cytoplasm, with early vitellogenic stages) were as in La Mesa et al. (2006). evident Balbiani’s body in the subcortical region. The nucleus is For PAS staining, sections were oxidized in 0.5% periodic acid large and contains multiple nucleoli located close to the nuclear solution for 10 min, rinsed in distilled water and stained with membrane. During the following alveolar phase (Fig. 2B), the Schiff’s reagent in the dark for 45 min. Reaction was stopped by cytoplasm progressively fills with small vesicles with translucent washing sections in 2.5% sodium bisulphite in 0.05 N HCl content. These alveoli gradually enlarge, and the amorphous con- (Simoniello et al., 2013). tent condenses, eventually forming a round and dense nucleoid Carbohydrate residues were identified by staining with FITC- (Fig. 2C). With the onset of vitellogenesis, the perinuclear cyto- conjugated lectins (Vector Laboratories Inc; 2 mg/ml). In partic- plasm fills with small yolk globules that progressively invade the ular, were used WGA (Triticum vulgaris agglutinin) specific for N- entire cytoplasm (Fig. 2D). At this stage the nucleus retains its Acetyl-glucosamine (glcNAc), DBA (Dolichos biflorus agglutinin) homogenous appearance and marginal nucleoli, the alveoli move specific for N-Acetyl-galactosamine (galNAc) and Concanavalin A, close to the oocyte membrane and the nucleoids decondense. Con A, specific for mannose (Man). Sections were covered with 1 ml After exposure to Cu (Fig. 2EeJ) or Cd (Fig. 2K-M), about one of lectin diluted in 19 ml of PBS and placed in a dark moist chamber third of the oocytes examined show marked alterations; contours at room temperature. After 15 min they were rinsed with PBS and appear indented (Fig. 2HeI, K-M) and/or the cytoplasm severely observed under a UV microscope (excitation maximum at 495 nm disorganized (Fig. 2J, K-M). and emission maximum at 515 nm). Negative controls were pre- The remaining two third of oocytes show an apparently normal pared by incubating slides with the lectin and the specific morphology (Fig. 2EeG; K, M) and have been analysed to test car- competing sugar or by omitting the lectin (Motta et al., 2005a). bohydrates composition and the presence and/or localization of sex

D) Perinuclear yolk globules (y), multiple nucleoli (arrow) in nucleus (n) of a late alveolar oocyte. Copper exposure. E) Perinucleolar oocytes; Balbiani’s body (Bb) and nuclei (n). F) Alveoli (a) and nucleus (n). G) Alveoli (a) with nucleoids (arrow). H) Perinucleolar and alveolar I) oocytes with indented contour (arrows) and disorganized cytoplasm (*). J) Detail: dense perinuclear cytoplasm containing large vesicles (*). Yolk (y), alveoli (a). Cadmium exposure. K) Perinucleolar oocyte with altered cytoplasm (*). Intact chromatin-nucleolar (small arrow) and vitellogenic (y) oocytes. Alveoli (a) with nucleoids (arrow). L) Inhomogeneous cytoplasm (*) and irregular contour (small arrow) in a perinucleolar oocyte. M) Alveolar oocytes; disorganized cytoplasm (*), intensely eosinophilic nuclei (n) and indented contours (arrows). Perinucleolar oocyte (small arrow). Haemalum-eosin staining. Bars: A: 100 mm; B: 10 mm; CeD: 5 mm.

4 C.M. Motta, P. Simoniello, M. Di Lorenzo et al. Chemosphere 268 (2021) 129282

Fig. 3. Effects of Cu and Cd on Trematomus bernacchii oocytes carbohydrate composition. PAS staining. Controls. A) Perinucleolar oocytes; stained chorion (arrow), pale cytoplasm and nuclei (n), unstained nucleoli (small arrow). BeC) Alveolar oocytes; stained chorion (c), nucleoids (arrows) in alveoli (a) and cytoplasm (*). Unstained nucleus (n) and nucleoli. D) Early vitellogenesis; stained yolk (y) and nucleoids (arrow) in alveoli (a). Copper exposure. E) Perinucleolar oocyte; stained chorion (arrow), pale cytoplasm (*) and unstained nucleus (n) and nucleoli (small arrow). F) Early alveolar oocyte; stained chorion (c), alveolar content (arrow) and cytoplasm (*). G) Detail. H) Early vitellogenic oocyte; stained yolk (y) and nucleoids (arrow) in alveoli (a). Cadmium exposure. I) Perinucleolar oocytes; stained chorion (arrow), unstained cytoplasm (*) and nucleus (n). J) Alveolar oocyte; stained chorion (c), nucleoids (arrow) in alveoli (a) and cytoplasm (*). K) Altered late alveolar oocyte; stained chorion (c) and nucleoids (arrow). Note the intensely stained fibro-granular cytoplasm (*) and the markedly indented contour (small arrow).

5 C.M. Motta, P. Simoniello, M. Di Lorenzo et al. Chemosphere 268 (2021) 129282

Fig. 4. Effects of Cu and Cd on Trematomus bernacchii oocytes carbohydrate composition. WGA staining for N-Acetyl-Glucosamine. Controls. A) Unlabeled perinucleolar oocytes (*) surrounded by fluorescent connectives (arrow). B) Labeled early alveoli (arrow) but not cytoplasm (*) in an early alveolar oocyte. C) Mid and late alveolar oocytes with labeled cytoplasm (*) and chorions (arrows) but unstained alveoli (a). D) Stained chorion (arrow) and yolk (y), unstained cytoplasm (*) and alveoli (a). Copper exposure. E) Unlabeled

6 C.M. Motta, P. Simoniello, M. Di Lorenzo et al. Chemosphere 268 (2021) 129282 hormone receptors. 3.4. Effects of Cu and Cd on steroid hormones receptors localization in oocytes

3.3. Effects of Cu and Cd on oocyte carbohydrates composition 3.4.1. Progesterone receptors In the control (Fig. 7AeC), progesterone receptor localizes in the 3.3.1. PAS staining cytoplasm of perinucleolar oocytes and, to a lesser extent, in their In control oocytes (Fig. 3AeD), PAS intensely stains the chorion nuclei (Fig. 7A). During the alveolar stage the cytoplasmic staining (Fig. 3AeC), the nucleoids in the cortical alveoli (Fig. 3BeD) and the reduces, probably due to the dispersion of the receptors in a yolk globules (Fig. 2D). The oocyte cytoplasm is almost unstained in growing volume (Fig. 7B); the nuclei remain moderately stained perinucleolar oocytes (Fig. 3A) and moderately stained in alveolar though in occasional oocytes, receptors temporarily concentrate in (Fig. 3BeC) and vitellogenic oocytes (Fig. 3D). The nuclei and the perinuclear cytoplasm (Fig. 7C). Later no receptors can be nucleoli are always unstained (Fig. 3A). detected, and late alveolar and early vitellogenic oocytes remain Comparable staining patterns are observed in oocytes exposed completely unstained (data not shown), as the negative controls to Cu (Fig. 3EeH) and Cd (Fig. 3IeJ) with intact morphology. prepared by omitting the primary antibody (Fig. 7D). e fi Conversely, in altered oocytes, either exposed to Cu or Cd, cyto- After exposure to Cu (Fig. 7E G) no signi cant differences are plasmic PAS staining is more intense and markedly fibro-granular noticed in receptor localization with respect to control oocytes. In fi in appearance (Fig. 3K). contrast, after exposure to Cd a signi cant increase in receptors occurs in the nuclei of perinucleolar oocytes and in the cortical cytoplasm of early and mid-alveolar oocytes (Fig. 7H). 3.3.2. Staining for N-Acetyl-glucosamine with fluorescent lectin WGA 3.4.2. Estrogen receptors In control ovaries (Fig. 4AeD), the glcNAc is present in the In control oocytes, alpha (Fig. 8A) and beta (Fig. 8BeC) estrogen alveoli of the early alveolar oocytes (Fig. 4B) and in the cytoplasm of receptors localize initially in the cytoplasm of perinucleolar oocytes mid and late alveolar oocytes (Fig. 4B). The sugar is also present in and, later, also in their nuclei (Fig. 8AeB). At the end of the alveolar the chorion (Fig. 4CeD) and in the yolk (Fig. 4D). stage, alpha (data not shown) and beta (Fig. 8C) receptors are After exposure to Cu (Fig. 4EeH) or Cd (Fig. 4I-L), the only sig- present in the oocyte nuclei and in follicular cells. nificant difference observed is the absence of glcNAc in the chorion. After exposure to Cu, the only significant variation observed In the alveolar oocytes with disorganized cytoplasm, labeling is occurs in the nuclei of the perinucleolar oocytes. With respect to irregularly distributed, often forming particularly dense areas the controls, they are richer in alpha (Fig. 8D) and poorer in beta (Fig. 4K-L). (Fig. 8F) receptors. No changes are observed in the localization of alpha (Fig. 8E) and beta (Fig. 8FeG) receptors in alveolar oocytes. In both cases, the receptors become scanty in the cytoplasm and 3.3.3. Staining for N-Acetyl-galactosamine with fluorescent lectin abundant in the nuclei (Fig. 8E, G). fi DBA After exposure to Cd, a signi cant decrease in alpha receptors is In control (Fig. 5AeC) and in Cu exposed oocytes (Fig. 5DeH), observed in the cytoplasm and nucleus of late perinucleolar oocytes the lectin stains exclusively the cytoplasm of the oocytes in the (Fig. 8H). In contrast, no differences are observed in earlier stages alveolar stage (Fig. 5AeC, 4E) and the yolk (Fig. 5FeH). Peri- when both nucleus and cytoplasm are intensely stained (Fig. 8I). In nucleolar oocytes (Fig. 5A, D) and the alveolar content (Fig. 5BeC, E- alveolar oocytes (Fig. 8J), the cytoplasm is initially rich in receptors G) are always unstained. The oocyte nuclei are also unstained (Fig. 8H) but later they concentrate in the nucleus (Fig. 8J). (Fig. 5AeB, D). As regard to beta receptors, a marked downregulation is After exposure to Cd (Fig. 5I-L), the lectin stains the nuclei and observed in all the oocytes, no matter the stage of differentiation the nucleoli of perinucleolar (Fig. 5IeJ) and early alveolar (Fig. 5J, L) (Fig. 8k-L). Nucleus and cytoplasm are consistently poorly stained oocytes and the cytoplasm of small perinucleolar oocytes indicating a substantial absence of receptors. (Fig. 5IeJ). Unlabeled cytoplasm is observed in all alveolar oocytes (Fig. 5I-L). 4. Discussion

In fish, waterborne metals enter primarily through the gills 3.3.4. Staining for mannose with fluorescent lectin Con A (Komjarova and Bury, 2014). Once inside, they are distributed in the In control (Fig. 6AeB) the lectin stains the cytoplasm of large different tissues, including the gonads, and are accumulated (Zysk, alveolar oocytes and, to a lesser extent, the cortical cytoplasm of 2020) at a rate depending on metal dynamics in distribution and early alveolar oocytes. The alveoli and the chorion are always elimination (Annabi et al., 2013). unstained. In the ovaries of the Antarctic teleost T. bernacchii, Cu accumu- After Cu exposure the oocytes (Fig. 6CeD) are completely un- lation results far less significant than that of Cd, probably because labeled, a significant fluorescence being present exclusively on the former is a nutritionally relevant metal whose presence in the connectives surrounding the follicle (Fig. 6 D). cell is finely tuned (Pena et al., 1999) while the latter has no role. After exposure to Cd (Fig. 6EeI), labeling is on the cytoplasm of Cells therefore lack specific homeostatic mechanisms actively the alveolar oocytes (Fig. 6EeF) and in the cortical alveoli in early controlling its internal concentration. The ion is partly neutralised (Fig. 6EeF) and mid alveolar (Fig. 6GeH) oocytes. In altered oocytes by binding to metallothioneins (Santovito et al., 2012b) or, even- (Fig. 6I) no alveolar labeling is observed. tually, as extreme remedy, by precipitation in the kidney or in the

perinucleolar oocytes (*). F) Labeled alveolar content (arrow) and cytoplasm (*) but not chorion (small arrow) in a mid-alveolar oocyte. G) Labeled yolk (y) among unlabeled alveoli (a), cytoplasm (*) and chorion (small arrow). H) Detail. Cadmium exposure. I) Labeled early alveoli (arrow) and cytoplasm in a mid-alveolar oocyte (**); unlabeled mid alveoli (a) and perinucleolar oocytes (*). J) Detail of labeled fibro-granular alveolar content (*). K) Altered alveolar oocyte with inhomogeneous cytoplasm (*); unstained alveoli (a), indented contour (small arrow). L) Detail of the fibro-granular cytoplasm (arrow) and unstained alveoli (a).

7 Fig. 5. Effects of Cu and Cd on Trematomus bernacchii oocytes carbohydrate composition. DBA staining for N-Acetyl-Galactosamine. Controls. A-B) Labeled cytoplasm (**) in alveolar oocytes; unstained nuclei (n), alveoli (a) and perinucleolar oocytes (*). C) Detail of labeled cytoplasm (*) and unlabeled alveoli (a). Copper exposure. D) Unlabeled oocyte cytoplasm (*) and nucleus (n). E) Stained yolk (y) among unstained cytoplasm (*) and alveoli (a). F) Labeled yolk (y), unlabeled alveoli (a) and alveolar content (arrow). G) Detail of an unlabeled nucleoid (arrow). H) Labeled yolk globules. Cadmium exposure. I) Perinucleolar oocytes with labeled nuclei (n) and nucleoli (arrows). Unlabeled alveoli (a) and cytoplasm (*)ina mid alveolar oocyte (a). J) Detail of figure I showing the labeled nucleoli (arrows), nucleoplasm (n) and cytoplasm of a small perinucleolar oocyte (**). Unlabeled cytoplasm (*) in the early alveolar oocyte. K) Detail of labeled nucleoli (arrow) in an alveolar oocyte (a). L) Unlabeled alveoli (a) and cytoplasm (*) in late alveolar oocytes. 8 C.M. Motta, P. Simoniello, M. Di Lorenzo et al. Chemosphere 268 (2021) 129282

Fig. 6. Effects of Cu and Cd on Trematomus bernacchii oocytes carbohydrate composition. Con A staining for mannose. Controls. A) Moderately labeled cytoplasm in late peri- nucleolar (*) and mid alveolar (a) oocyte; unstained nuclei (n). B) Labeled follicle cells (arrow) and cytoplasm of an alveolar oocyte (*); unlabeled early alveolar oocyte (**) and alveoli (a); Copper exposure. C and D) Unlabeled oocytes; cytoplasm (*), nuclei (n) and alveoli (a). Cadmium exposure. E) Intensely labeled early alveoli (arrow) and cytoplasm of a mid alveolar oocyte (a); unlabeled perinucleolar oocytes (*). F and inset) Details. G) Mid alveolar oocyte; labeled alveolar content (arrow) and alveolar margin (small arrow). Alveoli (a). I) Altered follicle; staining on cortical (*) but not perinuclear (**) cytoplasm and alveoli (a).

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Fig. 7. Effects of Cu and Cd on the localization of progesterone receptors in Trematomus bernacchii oocytes. Controls. A) Stained cytoplasm in perinucleolar (*) and early alveolar (a) oocytes. Perinuclear staining in a mid alveolar oocyte (arrow). B) Early alveolar oocyte with moderately stained cytoplasm (*) and nucleus (n); unstained alveoli (a). C) Intense perinuclear staining (arrow); moderately stained nucleus (n) and cytoplasm (*). Note the intensely stained cytoplasm in a perinucleolar oocyte (**). D) Unstained negative control. Copper exposure. E) Perinucleolar oocytes with stained cytoplasm (*) and nuclei (n). F) Stained cytoplasm (*) but not alveoli in an early alveolar oocyte. G) Unstained alveolar oocytes (a). Cadmium exposure. H) Stained cytoplasm (*) and nucleus (n) in a perinucleolar oocyte; notice the perinuclear (arrow) and cortical (**) staining in a mid alveolar oocyte. carcass (Szebedinszky et al., 2001) but these mechanisms are not homeostatic conditions. very efficient and Cd tends to accumulate in tissues (Jezierska and ROS have multiple effects on cell components and mechanisms, Witeska, 2006). including an interference on carbohydrate metabolism (Tolbert Despite the very different local concentrations, Cu and Cd et al., 1981). Cu and Cd in particular interfere at the post- induce essentially the same histopathological changes with oocytes translational level, directly or indirectly altering O- and/or N- appearing disorganized and deformed, in several cases clearly linked oligosaccharides composition (Almeida et al., 2002; Eguchi atretic. Comparable effects have been reported in other teleosts et al., 2002; Soengas et al., 1996). It is not surprising therefore (Chouchene et al., 2011; Szczerbik et al., 2006), in amphibians and that in T. bernacchii Cu and Cd have significantly altered oocytes reptiles (Lienesch et al., 2000; Simoniello et al., 2013). These al- composition in glcNAc, galNAc and mannose (Table 1). terations have been attributed to a single apical event: an increase The severe impact of two metals on glycan composition is in the production rate of reactive oxygen species (ROS) (Sanchez indicative of a reduced reproductive output. In fact, in fish, carbo- et al., 2005; Zhang et al., 2017). hydrates metabolism intensifies during oogenesis (Iwasaki et al., This event is all the more likely in organisms living at low 1992) as also evident in control T. bernacchii; here cytosolic and temperatures such as the Antarctic fish, due to the greater solubility chorionic positivity to PAS and lectins significantly increases during of gases and the particular blood condition, chronically deficient in the alveolar stage. Glycans are stored to provide the eggs with haemoglobin (Santovito et al., 2012a). In fact, Antarctic fish such as energy and reserve compounds necessary for the future, embryonic Trematomus bernacchii have evolved an efficient antioxidant synthesis of RNA, oligo and polysaccharides and glycoproteins defence system (Chatzidimitriou et al., 2020; Sattin et al., 2015; (Lahnsteiner, 2005). Glycoproteins also accumulate in the thick Tolomeo et al, 2016, 2019) that limits the risk of oxidative stress in chorion/vitelline envelope (Fausto et al., 2004) that will protect the

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Fig. 8. Effects of Cu and Cd on the localization of estradiol alpha and beta receptors in Trematomus bernacchii oocytes. Controls. A-B) Unstained nuclei and intensely stained cytoplasm in early perinucleolar oocytes (arrows). Stained cytoplasm (**) and nucleus (n) in late perinucleolar oocytes. Alveolar oocytes (a) with moderately stained cytoplasm (*) and intensely stained nuclei (n). C) Detail of stained nucleus (n) and cytoplasm (*) in an alveolar oocyte (a). Stained follicular epithelium (arrow). Copper exposure. D) Stained

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Table 1 Schematic representation of the presence (þ) or absence () of different carbohydrates in T. bernacchii oocytes after staining with FITC-conjugated lectins. Nucleus (nuc), nucleoulus (nucl), cytoplasm (cyt), cortical alveoli (alv), chorion (chor). glcNAc: N-Acetyl-Glucosamine stained with WGA; galNAc: N-Acetyl-Galactosamine stained with DBA; man: mannose stained with Con A.

perinucleolar early alveolar mid alveolar late alveolar glcNAc nuc nucl cyt nuc nucl cyt alv nuc nucl cyt alv nuc nucl cyt alv chor

cont ------þ -- þ -- - þ - þ Cu ------þ -- þ -- - þ -- Cd ------þ -- þ -- - þ --

galNAc nuc nucl cyt nuc nucl cyt alv nuc nucl Cyt alv nuc nucl cyt alv chor

cont -- --- þ -- - þ -- - þ -- Cu ------Cd þþ þþþ --þþ ------

man nuc nucl cyt nuc nucl cyt alv nuc nucl Cyt alv nuc nucl cyt alv chor

cont -- --- ± -- - þ -- - þ -- Cu ------Cd ------þ -- þþ-- þ --

developing embryo. too short to induce the expected effects. Investigations on receptors Rather surprisingly neither Cu nor Cd alter the cytosolic distri- mRNAs will probably clarify the point. Cu however does affect bution of glcNAc, a sugar involved in O-glyco-deglycosilation, a oogenesis as indicated by the increased number of degenerating function antagonistic to phosphorylation and responsible for oocytes, revealed by a significant increase in deformities important protein modification during the early oogenesis (Munkittrick and Dixon, 1989). The extensive disorganization of the (Lefebvre et al., 2004). In control T. bernacchii glcNAc becomes cytosol, in particular, suggests a direct interference of Cu on mi- abundant during the alveolar stage apparently reducing during crotubules assembly (Malea et al., 2013), a hypothesis also sup- vitellogenesis, probably because progressive dilution in an ported by the reduced cytosolic positivity to the lectin increasing cytoplasmic volume. No variation is observed in treated Concanavalin A, a marker for glycosylated tubulins (Hino et al., oocytes and this is unexpected since Cu and Cd interfere on tran- 2003). Alternatively, an indirect action can be postulated, with scription, either up- or down-regulating proteins involved in en- copper depressing the energy metabolism (Bundy et al., 2008) and, ergy metabolism, metal detoxification and protein protection consequently, reducing fish resources for reproduction, a stress (Casanova et al., 2013). signal conventionally activating mass atresia (Mendo et al., 2016; Another unexpected result, obtained after exposure to Cd, but Rideout et al., 2000). not to Cu, is the appearance of galNAc in nuclei and nucleoli of The disrupting effects of Cu and Cd are also evident at the level perinucleolar oocytes. Since a similar effect is not registered in of the chorion, a glycosylated envelope (Sugiyama et al., 1999) alveolar oocytes, a correlation with the intense rRNA synthesis and/ becoming depleted in glcNAc but not in mannose in late alveolar or with nucleolar fragmentation occurring at this stage can be oocytes. Data are too scanty to reach a conclusion, but a functional postulated (Prisco et al., 2004; Tammaro et al., 1998; Thiry and loss should be postulated. Poncin, 2005). A Cd disrupting effect is also suggested by the analysis of cortical In the same nuclei, an increase in progesterone receptors and a alveoli. As expected, being rich in poly sialo-glycoprotein (Asahina decrease in both alpha and beta estrogen receptors is observed. In et al., 2004), their content is always intensely stained by PAS, no teleosts, progesterone stimulates DNA duplication and initiates matter the stage of differentiation or the treatment. Lectins how- meiotic progression, controls maturation and ovulation (Miura ever demonstrate that Cd induces the appearance of mannose, in et al., 2007; Nagahama and Yamashita, 2008; Pinter and Thomas, mid and late alveoli (Table 1), those characterized by a progressive 1999). Estradiol controls ovogonial proliferation and modulates condensation of the nucleoid (Motta et al., 2005a). the anti-Müllerian hormone to cite only few actions (Fernandino The significance of such a change is not clear also because the et al., 2008; Miura et al., 2007). The observed changes indicate alveolar glycan pattern in T. bernacchii, the absence of galNAc in that Cd interferes on early oogenesis also behaving as an endocrine particular, is not consistent with previous report (Sarasquete et al., disruptor (Henson and Chedrese, 2004), confirming previous evi- 2002). At the moment this incongruence can be accounted only by dence in invertebrate and vertebrate species (Kitana and Callard, the extreme species-specific variability in oocyte glycan pattern 2008; Lienesch et al., 2000; Simoniello et al., 2010; Thompson (Accogli et al., 2014). and Bannigan, 2008). In conclusion, data collected indicate that Cu and Cd reduce In T. bernacchii, no alterations in sex steroids receptors are fecundity in T. bernacchii by increasing degeneration among pre- observed after Cu exposure, in clear contrast with the existing vitellogenic oocytes. Cd is the most toxic, accumulating at higher literature reporting that the ion interferes with sex steroid hor- concentrations in ovaries and significantly interfering with the mones secretion, increasing the expression of compensatory genes presence and localization of estradiol beta receptors. Both metals (Cao et al., 2019), A possible explanation for this apparent incon- alter carbohydrates composition of chorion and cytosol, Cd also gruence is that the dose was too low and/or the time of exposure altering the alveolar content.

perinucleolar oocytes. E) Alveolar oocyte (a) with stained nucleus (n) and cytoplasm (*). F) Perinucleolar oocytes with poorly stained nuclei (n) and stained cytoplasm (*). From mid (ma) to late (la) alveolar stage the nuclei (n) become moderately stained. G) Detail of a stained nucleus (n) and moderately stained cytoplasm (*) in an early alveolar oocyte. Cadmium exposure. H) Pale staining of nucleus (n) and cytoplasm (*) in two late perinucleolar oocytes. The alveolar oocytes (a) have a moderately stained cytoplasm (**). I) Group of intensely stained early perinucleolar oocytes. (J) Stained nucleus (n) and cytoplasm (*) in an early alveolar oocyte. K-L). Diffuse and pale staining of cytoplasm (*) and nucleus (n) in perinucleolar oocytes; unstained alveolar oocytes (**).

12 C.M. Motta, P. Simoniello, M. Di Lorenzo et al. Chemosphere 268 (2021) 129282

Considering that the Antarctic Ocean is already rich in metals aluminium and cadmium on hatching and swimming ability in developing fi e from natural sources and that the concentration of anthropic zebra sh. Chemosphere 222, 243 249. https://doi.org/10.1016/ j.chemosphere.2019.01.140. metals is growing very fast all over the world, a substantial decline Casanova, F.M., Honda, R.T., Ferreira-Nozawa, M.S., Aride, P.H.R., Nozawa, S.R., 2013. of fish stocks recruitment in Antarctica should be expected. Close Effects of copper and cadmium exposure on mRnA expression of catalase, monitoring would be appropriate to prevent the loss of this unique glutamine synthetase, cytochrome p450 and heat shock protein 70 in Tambaqui Fish (Colossoma Macropomum). Gene Expr. Genet. Genom. 6, 1. https://doi.org/ environment. 10.4137/GGG.S10716. Chatzidimitriou, E., Bisaccia, P., Corra, F., Bonato, M., Irato, P., Manuto, L., Toppo, S., Funding Bakiu, R., Santovito, G., 2020. Copper/zinc superoxide dismutase from the crocodile icefish Chionodraco hamatus: antioxidant defense at constant sub- zero temperature. Antioxidants 9, 325. https://doi.org/10.3390/antiox9040325. This work was supported by Research Funds from University of Chouchene, L., Banni, M., Kerkeni, A., Saïd, K., Messaoudi, I., 2011. Cadmium-induced Naples (Chiara Maria Motta) and from the Italian National Program ovarian pathophysiology is mediated by change in gene expression pattern of zinc transporters in zebrafish (Danio rerio). Chem. Biol. Interact. 193 (2), for Antarctic Research subprojects 2c.2.1 (Chiara Maria Motta) and 172e179. https://doi.org/10.1016/j.cbi.2011.06.010. 2c.3.1 (Gianfranco Santovito). Coscia, M.R., Simoniello, P., Giacomelli, S., Oreste, U., Motta, C.M., 2014. Investigation of immunoglobulins in skin of the Antarctic teleost Trematomus bernacchii. Fish Shellfish Immunol. 39, 206e214. https://doi.org/10.1016/j.fsi.2014.04.019. Author statement Craig, P.M., Wood, C.M., McClelland, G.B., 2007. Oxidative stress response and gene expression with acute copper exposure in zebrafish (Danio rerio). Am. J. Physiol. e In field experimentation and sampling (CMM, GS), metal anal- Regul. Integr. Comp. Physiol. 293 (5), R1882 R1892. https://doi.org/10.1152/ ajpregu.00383.2007. ysis, data collection and validation (GS), histological preparation, Craig, P.M., Hogstrand, C., Wood, C.M., McClelland, G.B., 2009. Gene expression data collection and validation (RP, EG, PS), immunocytochemistry, endpoints following chronic waterborne copper exposure in a genomic model fi e data collection, and validation (MDL, VM), Conceptualization, organism, the zebra sh, Danio rerio. Physiol. Genom. 40 (1), 23 33. https:// doi.org/10.1152/physiolgenomics.00089.2009. writing and reviewing (all the authors), editing supervision (PS) Dahlqvist, A., Olsson, I., Norden, Å., 1965. The periodate-Schiff reaction: specificity, Funding acquisition (CMM, GS) kinetics, and reaction products with pure substrates. J. Histochem. Cytochem. 13 (6), 423e430. https://doi.org/10.1177/13.6.423. Di Lorenzo, M., Barra, T., Rosati, L., Valiante, S., Capaldo, A., De Falco, M., Laforgia, V., Declaration of competing interest 2020. Adrenal gland response to endocrine disrupting chemicals in fishes, amphibians and reptiles: a comparative overview. Gen. Comp. Endocrinol. The authors declare that there is no conflict of interest that 113550 https://doi.org/10.1016/j.ygcen.2020.113550. Driessnack, M.K., Matthews, A.L., Raine, J.C., Niyogi, S., 2016. Interactive effects of could be perceived as prejudging the impartiality of the research chronic waterborne copper and cadmium exposure on tissue-specific metal reported. accumulation and reproduction in fathead minnow (Pimephales promelas). Comp. Biochem. Physiol. C Toxicol. 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