Background Heavy metal cadmium (Cd) is a widespread environmental contaminant with a potential toxicity that might negatively affect female reproduction and fertility. It has been reported that Cd exposure impaired the quality of oocytes and led to a defective maturation and fertilization, through oxidative stress induction. Resveratrol (Res) is a natural polyphenol with strong antioxidant properties that exhibited protective role in preventing oocyte redox homeostasis disruption and quality decline. Here, we explored whether the addition of Res to in vitro maturation (IVM) medium might act as a protection against Cd-induced toxicity on ovine oocyte maturation and fertilization. Firstly, we evaluated the effect of supplementing IVM medium with two different Res concentrations (1 and 2 μmol/L) on nuclear maturation and fertilization of oocytes matured under CdCl2 (2 μmol/L) exposure. Therefore, the concentration of 1 μmol/L Res was selected to analyse the effects of this compound on intracellular ROS levels, mitochondrial (mt) distribution and activity, chromatin configuration, cytoskeleton morphology, cortical granules (CGs) distribution and mRNA expression of genes associated with cellular response to oxidative stress (i.e. SIRT1, SOD 1, GPX1, GSR, CAT) in Cd-exposed in vitro matured oocytes. Results We found that 1 μmol/L Res restored the reduced oocyte meiotic competence induced by Cd exposure as well as, Res sustained oocyte ability to be normally fertilized and decreased polyspermic fertilization at both tested concentrations. Moreover, we demonstrated that 1 μmol/L Res mitigated Cd-induced alterations of oocyte cytoplasmic maturation by reducing reactive oxygen species (ROS) accumulation, preventing mt dysfunction, maintaining the correct meiotic spindle and cortical F-actin assembly and the normal cortical granule distribution as well as up-regulating SIRT1, SOD1 and GPX1 genes. Conclusions Taken together, our findings highlighted the beneficial influence exerted by Res in preventing Cd-induced disturbance of nuclear and cytoplasmic maturation and subsequent fertilization in ovine oocytes. Res treatment may help to establish defence strategies counteracting Cd-induced toxicity on the female gamete.

Background: Heavy metal cadmium (Cd) is a widespread environmental contaminant with a potential toxicity that might negatively affect female reproduction and fertility. It has been reported that Cd exposure impaired the quality of oocytes and led to a defective maturation and fertilization, through oxidative stress induction. Resveratrol (Res) is a natural polyphenol with strong antioxidant properties that exhibited protective role in preventing oocyte redox homeostasis disruption and quality decline. Here, we explored whether the addition of Res to in vitro maturation (IVM) medium might act as a protection against Cd-induced toxicity on ovine oocyte maturation and fertilization. Firstly, we evaluated the effect of supplementing IVM medium with two different Res concentrations (1 and 2 μmol/L) on nuclear maturation and fertilization of oocytes matured under CdCl2 (2 μmol/L) exposure. Therefore, the concentration of 1 μmol/L Res was selected to analyse intracellular ROS levels, mitochondrial (mt) distribution and activity, chromatin configuration, cytoskeleton morphology, cortical granules (CGs) distribution and mRNA expression of genes associated with cellular response to oxidative stress (i.e. SIRT1, SOD 1, GPX1, GSR, CAT ) in Cd-exposed in vitro matured oocytes. Results: We found that 1μmol/L Res was effective to recover the reduced oocyte meiotic competence induced by Cd exposure, while treatment with Res, at both tested concentrations, sustained oocyte ability to be normally fertilized and decreased polyspermic fertilization. Moreover, we demonstrated that 1 μmol/L Res mitigated Cd-induced alterations of oocyte cytoplasmic maturation by reducing reactive oxygen species (ROS) accumulation, preventing mt dysfunction, maintaining the correct meiotic spindle and cortical F-actin assembly and the normal cortical granule distribution as well as up-regulating SIRT1 , SOD1 and GPX1 genes. Conclusions: Taken together, our findings highlighted the beneficial influence exerted by Res in preventing Cd-induced disturbance of nuclear and cytoplasmic maturation and subsequent fertilization in ovine oocytes. Res treatment may help to establish defence strategies counteracting Cd-induced toxicity on the female gamete.

Cadmium (Cd) is one of the most toxic environmental and industrial heavy metals, with adverse effect on female reproduction. Earlier invitro studies have demonstrated that exposure to Cd during IVM impairs oocyte fertilization through the induction of oxidative stress (Martino et al. 2017 Reprod. Toxicol. 69, 132-145). The aim of this study was to evaluate the effect of supplementing IVM medium with resveratrol (Res), a phytoalexin with antioxidant activity, on IVF of ovine oocytes matured under Cd exposure and to evaluate whether this effect could be mediated by alterations of cortical granules (CGs) distribution and/or intracellular levels of reactive oxygen species (ROS). Cumulus-oocyte complexes collected from slaughterhouse-derived ovaries of prepubertal ewes were invitro matured with 0μM CdCl2 (control), 2μM CdCl2 (Cd group), or 2μM CdCl2 + 1μM Res (Cd-Res). Groups of MII oocytes were invitro fertilized with ram semen (Bogliolo et al. 2011 Reprod. Fertil. Dev. 23, 809-817). After 16h, oocytes were fixed with absolute ethanol, stained with Hoechst 33342, and classified as follows: normally fertilized (two pronuclei), polyspermic (more than two pronuclei), and abnormally fertilized (asynchronous pronucleus formation). Levels of ROS and distribution of CG in MII oocytes (n=25 each group) were evaluated by confocal laser scanning microscopy after staining with 2’,7’dichlorodihydrofluorescein diacetate and Alexa Fluor 488-conjugated lectin peanut agglutinin, respectively (Jiao et al. 2013 PLoS One 8, 58018). Data of fertilization and CGs distribution were analysed by Chi-squared test and ROS levels (mean±standard error) by Kruskal-Wallis test with STATA\IC 11.0 (Stata Corp.). Oocytes matured in presence of Res had higher normal fertilization and lower polyspermy rates than those matured without Res (Table 1). Levels of ROS in oocytes were lower in the Cd-Res group than in the Cd group (74.4±1.3 vs. 122.4±1.5 pixel/oocyte; P<0.05), and similar to those of the control (71.1±2.3 pixel/oocyte). The ratio of oocytes of Cd-Res group with CGs normally distributed in the peripheral region was higher than that of Cd group (91.3% vs. 65.2%; P<0.05) and comparable to that of control oocytes (95.8%). Collectively, our results showed that resveratrol protects oocytes from Cd-induced alteration of normal fertilization by preventing oxidative stress and improving the major mechanism responsible for blockage of polyspermic fertilization. Table 1.Effect of resveratrol on IVF rate of oocytes matured under cadmium exposure Group No. of total oocytes No. of MII oocytes No. of fertilized oocytes (%) Normal Polyspermy Abnormal Control 136 105 (77.2) 59 (56.2)a 23 (21.9)a 6 (5.7) Cd 146 106 (72.6) 39 (36.8)b 38 (35.8)b 7 (6.6) Cd-Res 114 88 (77.2) 47 (53.4)a 16 (18.2)a 7 (7.9) a,bValues in the same column with different superscripts differ significantly (P<0.05). This project was supported by FDS 2016 (CUP J86C18000780005).