Effect of eicosapentaenoic acid on bovine cumulus-oocyte complex in vitro.

The aim of the present study was to investigate the effects of eicosapentaenoic acid (EPA) supplementation during in vitro maturation (IVM) of bovine oocytes. The concentrations tested in all experiments were 1 nM, 1 µM, and 1 mM EPA. The effect of EPA was evaluated on cumulus-oocyte complexes (COC) by oocyte maturation (cumulus expansion area and oocyte nuclear maturation), genotoxicity [single cell gel electrophoresis (SCGE)], and cytotoxicity (apoptosis, viability, and MTT assays) end points. The maturation parameters were affected by exposure of COC to different EPA concentrations in the IVM medium. Cumulus expansion area increased in the presence of 1 nM EPA (P < 0.05) whereas addition of 1 nM EPA (P < 0.05) decreased cumulus expansion after 24 h of IVM. Moreover, the maturation rate significantly decreased when 1 mM of EPA was assayed (P < 0.001). EPA at 1 nM induced genotoxic and cytotoxic effects on bovine cumulus cells (CC) and primary DNA lesions (P < 0.001). A significant increase in the frequency of apoptotic (P < 0.01) and necrotic (P < 0.001) cells was observed after 24 h of treatment with 1 nM, 1 µM, and 1 mM EPA. Mitochondrial activity was altered with 1 mM EPA (P < 0.001). We inferred that optimal oocyte quality was partially dependent on the presence of adequate EPA concentrations; EPA could be beneficial to improve oocyte quality in the maturation process, because low concentration tested (1 nM EPA) improved cumulus expansion.

The presence of acylated ghrelin during in vitro maturation of bovine oocytes induces cumulus cell DNA damage and apoptosis, and impairs early embryo development.

The aim of this study was to investigate the effects of acylated ghrelin supplementation during in vitro maturation (IVM) of bovine oocytes. IVM medium was supplemented with 20, 40 or 60 pM acylated ghrelin concentrations. Cumulus expansion area and oocyte nuclear maturation were studied as maturation parameters. Cumulus-oocyte complexes (COC) were assessed with the comet, apoptosis and viability assays. The in vitro effects of acylated ghrelin on embryo developmental capacity and embryo quality were also evaluated. Results demonstrated that acylated ghrelin did not affect oocyte nuclear maturation and cumulus expansion area. However, it induced cumulus cell (CC) death, apoptosis and DNA damage. The damage increased as a function of the concentration employed. Additionally, the percentages of blastocyst yield, hatching and embryo quality decreased with all acylated ghrelin concentrations tested. Our study highlights the importance of acylated ghrelin in bovine reproduction, suggesting that this metabolic hormone could function as a signal that prevents the progress to reproductive processes.

Reproductive hormones influence zinc homeostasis in the bovine cumulus-oocyte complex: Impact on intracellular zinc concentration and transporters gene expression.

Zinc (Zn) is a vital trace element for the body and its bioavailability influences numerous reproductive events. However, the mechanisms that regulate Zn homeostasis in the cumulus-oocyte complex (COC) are yet to be elucidated. The aim of this study was to investigate the role of estradiol 17-beta (E2), FSH and LH in Zn homeostasis regulation in bovine COC matured in vitro and Zn transporters gene expression. For this purpose, intracellular Zn levels in oocytes and cumulus cells (CC) were assessed using a Zn-specific fluorescent indicator. In addition, gene expression and sequencing of six Zn transporters (Slc39a6, Slc39a8, Slc39a14, Slc30a3, Slc30a7 and Slc30a9) were assessed. Our results demonstrated that the simultaneous presence of E2, FSH, and LH during oocyte maturation altered intracellular zinc levels and transporters expression in both oocytes and CC. Transporter's gene expression was different in oocytes and CC, possibly due to cell-specific changes in Zn levels during maturation. The interaction effects of Zn with hormonal treatments influenced the results. This study emphasizes that Slc39a6 is highly sensitive to hormone induction. Overall, the hormonal modulation of Zn homeostasis in the COC was evidenced. Also, a preponderant role of FSH as a modulator of Zn intracellular levels and transporter gene expression is suggested.

Effects of EPA on bovine oocytes matured in vitro with antioxidants: Impact on the lipid content of oocytes and early embryo development.

The eicosapentaenoic acid (EPA) is an n-3 polyunsaturated fatty acid (PUFA) present in the lipid composition of bovine oocytes. Little is known about the importance of EPA in bovine oocyte maturation and embryo development in vitro. Although previous work suggest that n-3 PUFAs may inhibit oocyte maturation, the available data are inconsistent. In this study, we evaluated the effect of EPA (1, 10, 100 nM) during in vitro maturation (IVM) of bovine oocytes, alone and in combination with vitamin E (VE) or cysteamine (CYS). EPA treatment in IVM decreased oocyte lipid content and affected lipid droplets pattern (P < 0.05). EPA 100 nM reduced oocytes maturation rate (P < 0.05), without affecting cumulus expansion. At the concentrations tested, EPA did not modify embryo development. However, the addition of antioxidants during IVM reduced the levels of reactive oxygen species in the culture system by increasing intracellular glutathione content (P < 0.05). Besides, the combination of EPA with VE or CYS reduced the percentages of MI oocytes after 24 h of IVM (P < 0.05). EPA reduced oocyte lipid content without any detrimental for embryo development.

High copper concentrations produce genotoxicity and cytotoxicity in bovine cumulus cells.

The aim of this study was to investigate the cytotoxic and genotoxic effects of high copper (Cu) concentrations on bovine cumulus cells (CCs) cultured in vitro. We evaluated the effect of 0, 120, 240, and 360 µg/dL Cu added to in vitro maturation (IVM) medium on CC viability assessed by the trypan blue (TB)-fluorescein diacetate (FDA) and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assays, apoptosis, and DNA damage. Differences in cell viability assessed by TB-FDA were not significant among CC treated with 0, 120, 240, and 360 µg/dL Cu. However, mitochondrial activity assessed by MTT was lower in CC cultured with 120, 240, and 360 µg/dL Cu as compared with the control (p < 0.01). Percentages of apoptotic cells were higher when CCs were treated with 120, 240, and 360 µg/dL Cu (p < 0.05) due to higher frequencies of late apoptotic cells (p < 0.05). The frequency of live cells diminished in a dose-dependent manner when Cu was added to the culture medium. Whereas genetic damage index (GDI) increased significantly in CC cultured in the presence of 240 and 360 µg/dL Cu (p Ë‚ 0.05), DNA damage increased at all Cu concentrations tested (p Ë‚ 0.05). These results indicate that Cu induces cytotoxic and genotoxic effects in bovine CC.