Cecilia Furnus

The importance of having high glutathione (GSH) level after bovine in vitro maturation on embryo development: Effect of β-mercaptoethanol, cysteine and cystine

Supplementation of IVM medium with cysteamine, beta-mercaptoethanol, cysteine and cystine induced bovine oocyte glutathione (GSH) synthesis, but only the effect of cysteamine on the developmental competence of these oocytes was tested. During IVM of sheep oocytes, cysteamine but not beta-mercaptoethanol increased embryo development. However, it is not known how long the high intracellular oocyte GSH levels obtained after IVM with thiol compounds, can be maintained. Thus, the present study was carried out to evaluate the effects of supplementing maturation medium with 100 microM beta-mercaptoethanol, 0.6 mM cysteine and 0.6 mM cystine on 1) intracellular GSH level after IVM, 2) after IVF, 3) in 6 to 8-cell embryos and 4) on embryo development. In oocytes after IVM and in presumptive zygotes after IVF, intracellular GSH levels were significantly higher in the treated groups (P < 0.05). While, GSH content in 6 to 8-cell embryos was similar among treatment groups (P > 0.05). Differences in cleavage rates and the percentage of embryos that developed to morula and blastocyst stages were significantly higher (P < 0.05) for treated oocytes than for those matured in the control medium. We conclude from the results that the high intracellular GSH levels after induction of GSH synthesis in bovine IVM by thiol compounds remain during IVF and are still present at the beginning of IVC, improving developmental rates. Moreover, the results indicate that this metabolic pathway is an important component of the cytoplasmic maturation process that affects the subsequent steps of in vitro embryo production.

The hyaluronic acid receptor (CD44) is expressed in bovine oocytes and early stage embryos.

Hyaluronic acid (HA) is a high molecular weight polysaccharide found in the extracellular matrix of most animal tissues, that exerts a profound influence on cell behavior. HA is one of the most abundant glycosaminoglycans (GAGs) in the uterine, oviductal and follicular fluids in mouse, pig, human and cattle. CD44, the principal cell membrane receptor for HA, is expressed from the 1- to 8-cell stage in human embryos, during post-implantation mouse embryogenesis and on the surface of differentiated embryonic stem cells. In the present study, we have analyzed by immunofluorescence, whether CD44 is present in bovine oocytes, fertilized oocytes and early stage embryos. Bovine cumulus-oocyte complexes (COCs) were aspirated from follicles (2-5mm) and were selected for IVM and incubated for 24h. Oocytes showing an expanded cumulus (generally 90-95%) were used for IVF. Fertilized oocytes were separated for immunofluorescence assay after 16h of sperm incubation in order to fix the eggs at the pronuclear stage. The embryos were cultured for 8 days and the different stages of development for immunofluorescence assay were separated every 24h of culture. The CD44 receptor was detected at every observation time examined. Fluorescence-tagged HA for the internalization assay was prepared by mixing fluorescein amine, Isomer I and 1mg of HA from umbilical cord. Fluorescence-tagged HA was internalized in 2-, 4-, 8- and 16-cell-stage embryos, morulae and blastocysts. CD44 is expressed on the surface and in the cytoplasm of bovine oocytes and embryos in different stages of development.

Effect of increasing zinc sulphate concentration during in vitro maturation of bovine oocytes.

The objective was to investigate the effects of supplementary zinc (Zn) during in vitro maturation (IVM) of bovine oocytes. The DNA damage in cumulus cells was low with supplemental Zn concentrations of 1.1 and 1.5 μg/mL in the IVM medium (mean ± SEM index of DNA damage was 67.52 ± 9.32, 68.52 ± 13.34, 33.80 ± 4.89, and 34.65 ± 7.92 for supplementation with 0, 0.7, 1.1, and 1.5 μg/mL Zn, respectively; P < 0.01). Total glutathione concentrations did not differ following Zn supplementation of 1.1 and 1.5 μg/mL (3.7 ± 0.4 vs. 4.0 ± 0.5 pmol, respectively, in oocytes; and in cumulus cells, 0.5 ± 0.04 nmol/10(6) cells, combined for both treatments), but were greater (P < 0.01) than supplementation with 0.7 μg/mL (1.8 ± 0.5 pmol in oocytes and 0.2 ± 0.02 nmol/10(6) cumulus cells). Cleavage rate increased (P < 0.05) when Zn was added to the IVM medium at any concentration (67.16 ± 1.17, 73.15 ± 1.15, 74.05 ± 1.23, and 72.76 ± 0.74 for 0, 0.7, 1.1, and 1.5 μg/mL Zn). For these concentrations, subsequent embryo development to the blastocyst stage was 17.83 ± 2.15, 21.95 ± 0.95, 27.65 ± 1.61, and 30.33 ± 2.78%, highest (P < 0.01) in oocytes matured with 1.5 μg/mL Zn. There was an increase (P < 0.05) in mean cell number per blastocyst obtained from oocytes matured with 1.1 and 1.5 μg/mL Zn relative to 0 Zn (IVM alone) and 0.7 μg/mL Zn. In conclusion, Zn during oocytes maturation significantly affected intracellular GSH content and DNA integrity of cumulus cells, and improved preimplantational embryo development. We inferred that optimal embryo development to the blastocyst stage was partially dependent on the presence of adequate Zn concentrations.

Effects of copper sulphate concentrations during in vitro maturation of bovine oocytes

THE OBJECTIVES WERE TO EVALUATE: 1) copper (Cu) concentrations in plasma and follicular fluid (FF) from cattle ovaries; 2) the effects of supplemental Cu during in vitro maturation (IVM) on DNA damage of cumulus cells and glutathione (GSH) content in oocytes and cumulus cells; and 3) supplementary Cu during IVM on subsequent embryo development. Copper concentrations in heifer plasma (116 ± 27.1 μg/dL Cu) were similar (P > 0.05) to concentrations in FF from large (90 ± 20.4 μg/dL Cu) and small (82 ± 22.1 μg/dL Cu) ovarian follicles in these heifers. The DNA damage in cumulus cells decreased with supplemental Cu concentrations of 4 and 6 μg/mL (P < 0.01) in the IVM medium (mean ± SEM index of DNA damage was: 200.0 ± 27.6, 127.6 ± 6.0, 46.4 ± 4.8, and 51.1 ± 6.0 for supplementation with 0, 2, 4, and 6 μg/mL Cu respectively). Total GSH concentrations increased following supplementation with 4 μg/mL Cu (4.7 ± 0.4 pmol in oocytes and 0.4 ± 0.04 nmol/10(6) cumulus cells) and 6 μg/mL Cu (5.0 ± 0.5 pmol in oocytes and 0.5 ± 0.05 nmol/10(6) cumulus cells, P < 0.01) compared with the other classes. Cleavage rates were similar (P ≥ 0.05) when Cu was added to the IVM medium at any concentration (65.1 ± 2.0, 66.6 ± 1.6, 72.0 ± 2.1, and 70.7 ± 2.1 for Cu concentrations of 0, 2, 4, and 6 μg/mL). Percentages of matured oocytes that developed to the blastocyst stage were 18.7 ± 0.6, 26.4 ± 0.03, and 29.0 ± 1.7% for 0, 2, and 4 μg/mL Cu, and was highest (33.2 ± 1.6 %) in oocytes matured with 6 μg/mL Cu (P > 0.01). There was an increase (P > 0.05) in mean cell number per blastocyst obtained from oocytes matured with 4 and 6 μg/mL Cu relative to 0 Cu (IVM alone) and 2 μg/mL Cu. In conclusion, Cu concentrations in the FF and plasma of heifers were similar. Adding copper during oocyte maturation significantly increased both intracellular GSH content and DNA integrity of cumulus cells. Since embryo development was responsive to copper supplementation, we inferred that optimal embryo development to the blastocyst stage was partially dependent on the presence of adequate Cu concentrations during IVM.

Analysis of apoptosis and DNA damage in bovine cumulus cells after exposure in vitro to different zinc concentrations.

The purpose of this study was to investigate the effect of Zn (zinc) concentration on CCs (cumulus cells) during in vitro maturation. For this purpose, DNA integrity of CCs by addition of different Zn concentrations [0 (control); 0.7 μg/ml (Zn1); 1.1 μg/ml (Zn2) and 1.5 μg/ml (Zn3)] to the culture medium was evaluated by comet assay. In addition, early apoptosis was analysed by annexin staining assay. CCs treated with Zn showed a significant decrease in the DNA damage in a dose‐dependent manner. Comet assay analysed for TM (tail moment) was significantly higher in cells cultured without Zn (control, P<0.01) with respect to cells treated with Zn (control: 5.24±16.05; Zn1: 1.13±5.31; Zn2: 0.10±0.36; Zn3: 0.017±0.06). All treatments were statistically different from the control (P=0.014 for Zn1; P<0.01 for Zn2 and Zn3). The frequency of apoptotic cells was higher in the control group (control: 0.142±0.07; Zn1: 0.109±0.0328; Zn2:0.102±0.013; Zn3: 0.0577±0.019). Statistical differences were found between control and Zn1 (P=0.0308), control and Zn2 (P=0.0077), control and Zn3 (P<0.0001), Zn1 and Zn3 (P<0.001) and Zn2 and Zn3 (P=0.0004). No differences were found between Zn1 and Zn2. In conclusion, low Zn concentrations increase DNA damage and apoptosis in CCs cultured in vitro. However, adequate Zn concentrations ‘protect’ the integrity of DNA molecule and diminish the percentage of apoptotic CC.

The induction of reciprocal translocations in mouse germ cells by chemicals and ionizing radiations II. Combined effects of mitomycin C or thio-tepa with two doses of γ-rays

The combined effects of mitomycin C (MMC) and thio-tepa (TT) with gamma-ray doses of 5 and 9 Gy on mouse stem cells were studied using the spermatocyte test. Both chemicals induced very low yields of translocations after single treatments. In combined treatments with a dose of 5 Gy, a subadditive effect of MMC and an additive effect of TT were found. Combined with a dose of 9 Gy the compounds potentiated the effect of radiations. Up to now, most of the chemicals tested have shown additive effects when combined with doses of the ascending part of the dose-response curve and potentiating effects when combined with doses of its descending part. This has been considered additional confirmation of the concept that depletion of any kind of spermatogonia is sufficient to modify the genetic response of stem cells. However, the subadditive and additive responses found could be considered evidence that common biological mechanisms can modulate the response to combined treatments of chemicals and ionizing radiations.

Influence of manganese on apoptosis and glutathione content of cumulus cells during in vitro maturation in bovine oocytes.

We have investigated the effect of different Mn concentrations on (1) DNA integrity of cumulus cells by olive tail moment (OTM); (2) cumulus cells apoptosis by Annexin V staining assay; (3) intracellular total glutathione (GSH‐GSSG) content; and (4) oocyte nuclear maturation and embryo cleavage after in vitro fertilisation (IVF). For this purpose, 0 (control), 2 (Mn1), 5 (Mn2) and 6 ng/mL (Mn3) Mn concentrations were added to IVM medium. Comet assay analysed by OTM was significantly higher in cumulus cells arising from COCs matured without Mn (control, P < 0.01) respect to cumulus cells obtained from COCs matured with Mn (control: 5.18 ± 2.3; Mn1: 2.93 ± 2.2; Mn2: 2.63 ± 2.4; Mn3: 2.92 ± 2.4). The frequency of apoptotic cells was higher in the control group (control: 6.63 ± 0.59; Mn1: 5.05 ± 0.5; Mn2: 4.61 ± 0.49; Mn3: 3.33 ± 0.42). Intracellular concentration of GSH‐GSSG increased in oocytes and cumulus cells matured in the presence of Mn (P < 0.01). There were no differences in percentages of nuclear maturation when Mn was added to IVM medium at any concentration, but at 6 ng/mL Mn a higher cleavage rate was observed respect to the control group (P < 0.05). In conclusion, deficiency in Mn concentration during in vitro maturation increased the damage in the DNA molecule and the frequency of apoptotic cumulus cells. However, the addition of an adequate Mn concentration (6 ng/mL Mn) to IVM medium improved the health of cumulus‐oocyte complexes and produced more cleaved embryos 48 h after IVF.

Influence of vascular endothelial growth factor and Cysteamine on in vitro bovine oocyte maturation and subsequent embryo development.

The objective of this study was to investigate the effect of VEGF and Cysteamine during in vitro maturation (IVM) of bovine oocytes on GSH content and developmental competence. For this purpose, experiments were designed to evaluate the effect of 0, 100, 300, and 500 ng/mL VEGF in IVM medium on: GSH content in oocytes and cumulus cells (Exp. 1) and subsequent embryo development (Exp. 2). Also, influence of adding 500 ng/mL VEGF and 100 μM Cysteamine to IVM medium on GSH content in oocytes and cumulus cells (Exp. 3) and oocyte developmental capacity (Exp. 4) were evaluated. Oocytes were matured in: a) Control; b) VEGF 0–3 h; c) Cysteamine 4–24 h; d) VEGF 0–3 h + Cysteamine 4–24 h; and e) VEGF + Cysteamine 24 h. The results showed that: i) VEGF did not alter GSH content in oocytes and cumulus cells; (ii) supplementation of 300 and 500 ng/mL VEGF increased blastocyst yield; (iii) the presence of VEGF + Cysteamine simultaneously during 24 h improved GSH content but not embryo development; and (iv) the presence of VEGF during the first 3 h + Cysteamine from 4 to 24 h increased GSH concentrations and subsequent embryo development. In conclusion, the addition of VEGF and Cysteamine in two sequential steps to maturation medium result in an improvement of cytoplasmic maturation, with a positive impact on oocyte developmental capacity by increasing the efficiency of in vitro blastocyst production. However, the effect was detrimental when both VEGF and Cysteamine were present during 24 of IVM.

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.

Analyses of apoptosis and DNA damage in bovine cumulus cells after in vitro maturation with different copper concentrations: consequences on early embryo development

The aim of this study was to investigate the influence of copper (Cu) during in vitro maturation (IVM) on apoptosis and DNA integrity of cumulus cells (CC); and oocyte viability. Also, the role of CC in the transport of Cu during IVM was evaluated on oocyte developmental capacity. Damage of DNA was higher in CC matured without Cu (0 µg/dl Cu, P < 0.01) with respect to cells treated with Cu for cumulus-oocyte complexes (COCs) exposed to 0, 20, 40, or 60 µg/dl Cu). The percentage of apoptotic cells was higher in CC matured without Cu than in CC matured with Cu. Cumulus expansion and viability of CC did not show differences in COC treated with 0, 20, 40, or 60 µg/dl Cu during IVM. After in vitro fertilization (IVF), cleavage rates were higher in COC and DO + CC (denuded oocytes + CC) with or without Cu than in DO. Independently of CC presence (COC, DO + CC or DO) the blastocyst rates were higher when 60 µg/dl Cu was added to IVM medium compared to medium alone. These results indicate that Cu supplementation to IVM medium: (i) decreased DNA damage and apoptosis in CC; (ii) did not modify oocyte viability and cumulus expansion; and (iii) improved subsequent embryo development up to blastocyst stage regardless of CC presence during IVM.