Protective effect of resveratrol against cadmium-induced toxicity on ovine oocyte in vitro maturation and fertilization.

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.

Selection of Immature Cat Oocytes with Brilliant Cresyl Blue Stain Improves In Vitro Embryo Production during Non-Breeding Season.

In domestic cats, the maturation, fertilization, and development potential in vitro decreases during the non-breeding season. This study aims at evaluating the efficacy of Brilliant Cresyl Blue (BCB) staining in selecting developmentally competent oocytes to be used in in vitro embryo production (IVEP) programs in order to overcome the season variability in blastocyst yield. Cumulus-oocytes complexes (COCs) collected from antral follicles of domestic cat ovaries during the anestrus phase (July to November) were selected by BCB staining and classified as BCB+ (colored cytoplasm) and BCB- (colorless cytoplasm). COCs not exposed to BCB staining were used as control. Before and after in vitro maturation mitochondrial activity and reactive oxygen species (ROS) were measured. Following in vitro fertilization, blastocyst rate, hatching rate, and blastocyst cell numbers were recorded. The results show that BCB staining did not alter the mitochondrial function and ROS production in cat oocytes. BCB+ oocytes presented a higher (p < 0.05) blastocyst rate, hatching rate, and blastocyst cell number than BCB- and control oocytes. In conclusion, BCB staining does not affect the bioenergetic/oxidative status of the oocyte while being a useful tool for selecting good quality oocytes to increase IVEP in domestic cats during non-breeding season.

Effect of vitrification of in vitro matured prepubertal goat oocytes on embryo development after parthenogenic activation and intracytoplasmic sperm injection.

This work studies the effect of vitrification of in vitro matured (IVM) prepubertal goat oocytes on: 1) oocyte damage assessed by reactive oxygen species (ROS) level and apoptosis and 2) embryo development after Intracytoplasmic sperm injection (ICSI) and Parthenogenic Activation (PA). Oocytes were IVM in supplemented TCM-199 for 22-24 h. Control group oocytes matured during 24 h were directly used for the analysis after IVM. Vitrified/warmed IVM-oocytes were vitrified after 22 h of IVM in 15% ethylene glycol (EG), 15% dimethyl sulfoxide (Me2SO) and 0.5 M sucrose and after subjected to warming procedure. Oocyte ROS level was measured by staining denuded IVM-oocytes with 10 µM 2'7' dichlorodihydrofluorescein diacetate. Apoptosis was analyzed by Annexin V (AV) Apoptosis Detection kit and Propidium iodide (PI) signal and oocytes were classified as: Live (AV- PI-), early apoptotic (AV+ PI-), dead non-apoptotic (AV- PI+) and necrotic (AV+ PI+). Developmental competence of vitrified/warmed oocytes was assessed by PA (5 min in 5 µM Ionomycin plus 4 h in 2 mM 6-Dimethylaminopurine), and by ICSI fertilization. Presumptive zygotes were in vitro cultured for 8 days in commercial media BO-IVC. Vitrified/warmed oocytes showed higher ROS levels (P < 0.0001), lower live oocytes (44 vs. 66%; P: 0.0025) and higher dead non-apoptotic oocytes (33 vs. 13% P: 0.023) compared to control. No differences were found on normal zygote formation (2 PN) (32 vs. 25%) or blastocyst development (0 vs. 4%) after ICSI fertilization. However, after PA, significant differences were found in cleavage rate (59 vs.78%; P < 0.0343) and blastocyst formation (1 vs. 25%; P < 0.0001). In conclusion, vitrification reduced oocyte competence by increasing dead oocytes and ROS levels.

Resveratrol treatment during maturation enhances developmental competence of oocytes after prolonged ovary storage at 4 °C in the domestic cat model.

Resveratrol (Resv; 3,4,5-trihydroxy-trans-stilbene) is a phytoalexin with antioxidant activity that modulates redox homeostasis in oocytes and improves in vitro embryo production. Cold storage of cat ovaries for a period longer than 24 h alters oxidative status of oocytes after in vitro maturation and reduces their developmental competence. The aim of this study was to evaluate the effect of resveratrol supplementation to the maturation medium on embryo development of oocytes after storage of domestic cat ovaries at 4 °C for 24 h or 48 h. Cumulus-oocyte complexes (COCs) were recovered from ovaries of domestic queens and cultured in maturation medium supplemented with (+) or without (-) 5 µM resveratrol for 24 h. COCs collected from fresh ovaries were matured in vitro (IVM) in standard conditions as control. After IVM, oocytes were in vitro fertilized (IVF) and presumptive zygotes cultured for 7 days. Oocyte nuclear maturation, reactive oxygen species (ROS) and glutathione (GSH) levels as well as cleavage, blastocyst formation and blastocyst cell number were determined. There were no differences in the maturation rates of oocytes between the control and stored groups, irrespective of resveratrol supplementation. Resveratrol treatment during IVM significantly increased the level of GSH and reduced the level of ROS of oocytes recovered from ovaries stored for 48 h as compared to the non-treated group (48 h-). The rate of blastocyst formation from oocytes recovered from ovaries after 48 h storage that underwent IVM with resveratrol was higher (P < 0.05) than that of oocytes matured without resveratrol and similar to that of control oocytes. Resveratrol treatment increased (P < 0.05) cell number in blastocysts from 24 h + and 48 h + groups as compared to their respective counterparts. In conclusion, our results demonstrated that resveratrol supplementation during IVM can reverse the adverse effect of oxidative stress on oocytes, and enhances embryo development after ovary storage at 4 °C for 48 h. These results may provide a basis for improving culture conditions and extend the possibility of storage of cat ovaries for more than 24 h thus ensuring successful in vitro embryo production.

Resveratrol supplementation during in vitro maturation improves embryo development of prepubertal goat oocytes selected by brilliant cresyl blue staining.

This study aimed to investigate the effect of resveratrol supplementation in maturation medium on the developmental ability and bioenergetic\oxidative status of prepubertal goat oocytes selected by brilliant cresyl blue (BCB). Oocytes collected from slaughterhouse-derived ovaries were selected by 13 µM BCB staining and classified as grown BCB+ and growing BCB- oocytes. All oocytes were matured in vitro in our conventional maturation medium and supplemented with 1 µM (BCB+R and BCB-R) and without (Control groups: BCB+C and BCB-C) resveratrol. After 24 h, IVM-oocytes were fertilized with fresh semen and presumptive zygotes were in vitro cultured for 8 days. Oocytes were assessed for blastocyst development and quality, mitochondrial activity and distribution, and levels of GSH, ROS, and ATP. BCB+R (28.3%) oocytes matured with resveratrol presented significantly higher blastocyst development than BCB+C (13.0%) and BCB- groups (BCB-R: 8.3% and BCB-C: 4.7%). Resveratrol improved blastocyst development of BCB-R oocytes at the same rate as BCB+C oocytes. No differences were observed in blastocyst quality among groups. GSH levels were significantly higher in resveratrol groups (BCB+R: 36554.6; BCB-R: 34946.7 pixels/oocyte) than in control groups (BCB+C: 27624.0; BCB-C: 27655.4 pixels/oocyte). No differences were found in mitochondrial activity, ROS level, and ATP content among the groups. Resveratrol-treated oocytes had a higher proportion of clustered active mitochondria in both BCB groups (BCB+R: 73.07%; BCB-R: 79.16%) than control groups (BCB+C: 19.35%; BCB-C: 40%). In conclusion, resveratrol increased blastocyst production from oocytes of prepubertal goats, particularly in better quality oocytes (BCB+).

Effects of melatonin on oocyte developmental competence and the role of melatonin receptor 1 in juvenile goats.

Melatonin enhances in vitro embryo development in several species by improving the oocyte developmental competence during in vitro maturation (IVM). Melatonin has a wide range of actions, from scavenging reactive oxygen species (ROS) to regulating gene expression, and it can also act by way of melatonin receptors. The aim of this study was to determine the mechanism of action of melatonin during the IVM of juvenile goat oocytes and the role of the membrane receptors. Melatonin receptor 1 was immunolocalized in cumulus cells and oocytes before and after 24 hr of IVM. The effect of melatonin on oocyte developmental competence was tested in three experimental IVM groups: (a) control, (b) 10-7  M melatonin, and (c) 10-7  M melatonin +10-7  M luzindole (an inhibitor of both melatonin receptors). After IVM oocytes were assessed for ROS levels, mitochondrial activity, adenosine 5'-triphosphate (ATP) concentration and relative gene expression (ACTB, SLC1A1, SOD1, GPx1, BAX, DNMT1, GCLC and GDF9). IVM-oocytes were in vitro fertilized and cultured under conventional conditions. Blastocyst rate and quality (differential cell count) were assessed at 8 days post-fertilization. Melatonin decreased ROS levels, increased mitochondrial activity and ATP content and increased blastocyst quality compared to control group (55.8 vs. 30.4 inner cell mass ICM, p < 0.05). There was no effect on the relative gene expression due to treatment with melatonin. In conclusion, we have showed that melatonin improves oocyte developmental competence in juvenile goats by reducing ROS levels and improving mitochondrial activity.

Structure of preantral follicles, oxidative status and developmental competence of in vitro matured oocytes after ovary storage at 4 °C in the domestic cat model.

BACKGROUND: Storage conditions during transportation of explanted ovaries are a critical step in setting up fertility preservation protocols in both animal and human fields. Here, we evaluated the effects of ovary storage at 4 °C on the preservation of preantral follicles and oocytes retrieved from antral follicles using the domestic cat as model. METHODS: Ovaries were harvested from fifty-five healthy domestic queens during ovariectomy and stored at 4 °C for 0 (control), 24, 48, 72 and 96 h. In Experiment 1, the effects of the storage period at 4 °C on the morphology, cytoskeleton (α/ß tubulin) and DNA integrity (phosphorylation of histone H2AX) of preantral follicles were investigated. In Experiment 2, oocytes recovered from antral follicles were matured and fertilized in vitro to evaluate their meiotic and developmental competence. Reactive oxygen species (ROS), glutathione (GSH) and lipid peroxidation were measured in matured oocytes. RESULTS: The results showed that: a) storage up to 24 h did not affect the morphology and the DNA integrity of preantral follicles; b) extended storage times caused progressive morphological abnormalities, disassembling of microtubules and DNA damage; c) storage up to 48 h did not influence in vitro meiotic maturation of oocytes nor cleavage after in vitro fertilization. However, only oocytes stored within the ovary for 24 h produced blastocysts in a percentage similar to control oocytes; d) GSH levels of in vitro matured oocytes did not change at any time during ovary storage; a progressive increase in ROS levels was detected from 48 h associated with elevated lipid peroxidation at 72 and 96 h of storage. CONCLUSIONS: Storage of cat ovaries for up to 24 h caused minimal alteration of preantral follicles and oocytes. The extension of the storage period beyond 24 h progressively impaired the structure of follicles, and modified the oxidative status of in vitro matured oocytes and their developmental competence after in vitro fertilization. This information may help when setting up programs for fertility conservation, especially for wild feline species which die in geographic areas located far away from ARTs centers.