Integrity Of Zona Pellucida Research Articles

SIRT1 overexpression by melatonin and resveratrol combined treatment attenuates premature ovarian failure through activation of SIRT1/FOXO3a/BCL2 pathway

AimTo evaluate the synergistic effect of combined treatment with melatonin (MEL) and resveratrol (RES) in cisplatin (CIS)-induced premature ovarian failure (POF) model in rats and to elucidate the molecular mechanism of this therapeutic effect. Material & methodsFemale Sprague Dawley rats were divided into 7 experimental groups as follows; CONT (Control), CIS, MEL, RES, POF + MEL, POF + RES, and POF + MEL + RES. H&E staining was performed to evaluate follicular cell vacuolization/degeneration, vascular congestion/hemorrhage, and inflammation, by using an ordinal scale from 0 to 4 to grade the severity of observed changes (0 = normal, 1 = mild, 2 = moderate, 3 = severe, 4 = very severe). Zona pellucida integrity and connective tissue amount in the ovarian tissue were detected using PAS & Masson Trichrome staining. The immunofluorescence method was used to determine the immune localizations of pH2Ax, SIRT1, FOXO3a, and BCL2. The connective tissue amounts and immunoreactivity staining intensities were measured using ImageJ. The gene expression of SIRT1, FOXO3a, and BCL2 was determined using RT-PCR. Serum estrogen hormone levels were measured by ELISA. Statistically, Bonferroni correction was performed, and p < 0.002 were considered significant. ResultsA significant difference was observed in the POF group compared to the CONT group in all parameters except tertiary follicle count and hemorrhage. The decrease in the number of atretic follicles in the POF + MEL + RES group was found significant compared to both POF + MEL and POF + RES groups. The expression of pH2Ax, SIRT1, FOXO3a, and BCL2 at the protein level and SIRT1 and BCL2 at the mRNA level were significant in the POF + MEL + RES group compared to the POF group. Between the single and combination treatment groups, the difference in protein level was found in pH2Ax, SIRT1, FOXO3a, and BCL2 expression. The POF + MEL + RES group exhibited significantly higher SIRT1 mRNA expression compared to the groups receiving single treatments. ConclusionThe present study provides evidence that MEL and RES have synergistic effects in preventing the decrease in follicle reserve and increase in DNA break (pH2Ax) and follicle atresia in POF ovaries. This therapeutic effect is mediated by SIRT1 overexpression and activation of the SIRT1/FOXO3a/BCL2 pathway.

Parental genetic material and oxygen concentration affect hatch dynamics of mouse embryo in vitro

BackgroundHatching is crucial for mammalian embryo implantation, since difficulties during this process can lead to implantation failure, ectopic pregnancy and consequent infertility. Despite years of intensive researches, how internal and external factors affecting embryo hatch are still largely unclear.MethodsThe effects of parental genetic material and oxygen concentration on hatch process were examined. Fertilized and parthenogenetic mouse preimplantation embryos were cultured in vitro under 5 and 20% oxygen for 120 h. Zona pellucida drilling by Peizo micromanipulation were performed to resemble the breach by sperm penetration.ResultsFirstly, parthenogenetic embryos had similarly high blastocyst developmental efficiency as fertilized embryos, but significantly higher hatch ratio than fertilized embryos in both O2 concentrations. 5% O2 reduced the hatch rate of fertilized embryos from 58.2 to 23.8%, but increased that of parthenogenetic embryos from 81.2 to 90.8% significantly. Analogously, 5% O2 decreased the ratio of Oct4-positive cells in fertilized blastocysts, whereas increased that in parthenogenetic blastocysts. Additionally, 5% O2 increased the total embryonic cell number in both fertilized and parthegenetic embryos, when compared to 20% O2, and the total cell number of fertilized embryos was also higher than that of parthegenetic embryos, despite O2 concentration. Real-time PCR revealed that the expression of key genes involving in MAPK pathway and superoxide dismutase family might contribute to preimplantation development and consequent blastocyst hatch in vitro. Finally, we showed that fertilized and parthenogenetic embryos have diverse hatch dynamics in vitro, although the zona pellucida integrity is not the main reason for their mechanistic differences.ConclusionBoth parental genetic material and O2 concentration, as the representative of intrinsic and extrinsic factors respectively, have significant impacts on mouse preimplantation development and subsequent hatch dynamics, probably by regulating the gene expression involving in MAPK pathway and superoxide dismutase family to control embryonic cell proliferation and allocation of ICM cells.

Characterization of immature sheep oocytes and zona pellucida morphology using scanning electron microscope (SEM)

The aim of the present work was to study the differences among different categories of immature sheep oocytes. Oocytes were divided into three categories: excellent, good and fair oocytes. Results showed that cumulus cells were close to each other and zona pellucida in all the three oocytes categories. All oocytes categories with cumulus cells exhibited an irregular shape, mucous material inter-granular cells, low number of small droplets and minutes tapes on the cell surface. Good oocytes category was characterized by the presence of a belt surrounding the oocytes. Moreover, there are large numbers of small holes on the ZP surface in all categories. The good oocytes also contained a large number of big holes. In contrast, fair and excellent oocytes have low number of large holes in the ZP surface. In conclusion, there are several differences between oocytes categories in the morphology of oocytes and ZP characteristics. This study recommends using excellent and good oocytes in the in vitro embryo production for quality of the outer surface and integrity of zona pellucida than fair oocytes.

Variations in the gene expression of zona pellucida proteins, zpb and zpc, in female European eel (Anguilla anguilla) during induced sexual maturation

Vertebrate eggs are surrounded by an extracellular glycoprotein coat termed zona pellucida (ZP). Integrity of ZP is critical for a correct embryo development. Two zona pellucida protein genes (zpb and zpc) from European eel were characterized, specific qPCR assays developed and their expression in immature males and females carried out.An experimental group of silver-stage eel females was maintained at 18°C and hormonally induced to sexual maturation by weekly injections of carp pituitary extract during 12weeks. Changes in zpb and zpc expression during sexual maturation were studied in liver and ovary by qPCR. In liver, no changes were recorded during hormonal treatment, while in ovary expression of both genes decreased during sexual development.These results are a first step in the characterization of ZP in European eel and in the understanding of the mechanism underlying egg envelope formation.

Sheep embryo cryopreservation by vitrification and conventional freezing

The survival of ovine embryos (morulae and blastocysts) either frozen by a conventional method or vitrified was investigated in culture. In Experiment I, embryos were vitrified using a solution containing 25% propylene glycol and 25% glycerol. A group of embryos (simulated control) was processed without freezing to evaluate the toxicity of the vitrification solution. In Experiment II, embryos were exposed to a solution of PBS containing 10% glycerol and 0.25 M sucrose placed horizontally in a programmable freezer. Automatic seeding was applied at −7°C in 2 positions on straws and cooled at −0.3°C/min to −25°C and then stored in liquid nitrogen. In vitro development rates of vitrified embryos were 12% (morulae) and 19% (blastocysts). Simulated embryos showed a higher rate of survival than embryos cryopreserved by vitrification (67 and 63%, morulae and blastocysts respectively). In conventional cooling, the blastocysts showed the highest viability percentage (67%) of all the experimental groups but these values decreased significantly in morulae (31%). Differences in temperature between straws placed in distinct positions in the freezing chamber and thermic deviation were observed when automatic seeding was applied. Embryo viability differed from 51 to 75% according the relative position of the embryos within the chamber. Survival was higher when automatic seeding was applied on the meniscus of the embryo column versus the central point of this column (65 vs 21%). The damage of both cryopreservation methods on zona pellucida integrity (27 and 35% in vitrified and conventionally frozen embryos, respectively) had no effect on the in vitro survival.