Effects of glucuronic acid and N-acetyl-D-glucosamine supplementation on the perivitelline space during the IVM of pig oocytes.

The objective of this study was to minimise polyspermic penetration by increasing the perivitelline space (PVS) thickness through supplementation of the hyaluronic acid components glucuronic acid and N-acetyl-d-glucosamine (GlcNAc). Oocytes (n=4690) were supplemented during the first 24h and/or the remainder of maturation (final 16-18h) with 0.01mM glucuronic acid and 0.01mM GlcNAc and then evaluated for PVS thickness, hyaluronic acid, glutathione and glutathione peroxidase concentrations. Fertilised oocytes were evaluated for polyspermic penetration and embryo development. The PVS thickness and amount of hyaluronic acid was significantly (P<0.05) greater in oocytes supplemented with 0.01mM glucuronic acid and 0.01mM GlcNAc during the second part or all of maturation compared with the other treatments. In addition, polyspermic penetration was significantly (P<0.05) less in oocytes supplemented with 0.01mM glucuronic acid and 0.01mM GlcNAc during the second part or all of maturation compared with the other treatments. Supplementing 0.01mM glucuronic acid and GlcNAc during maturation significantly (P<0.05) increased the percentage of cleaved embryos by 48h after IVF and blastocysts formed by 144h after IVF compared those not supplemented. These results indicate that supplementing PVS components during maturation decreases polyspermic penetration by increasing PVS thickness.

Deleterious effects of nicotine on the ultrastructure of oocytes: role of gamma-tocotrienol.

BACKGROUND: Previous studies have shown that nicotine enhances oxidative DNA damage and leads to increased lipid peroxidation, which affects embryo development. The present study investigated the effect of daily supplementation of gamma-tocotrienol on oocytes of nicotine-treated mice. MATERIAL/METHODS: Immature female mice (18-25 g) were divided into three groups. For 30 days, group A (control group) received saline (0.2 ml/day s.c.), group B nicotine (5 mg/kg/day s.c. in saline), and group C nicotine with gamma-tocotrienol (60 mg/kg/day p.o.). The animals were superovulated following these schedules. RESULTS: Scanning electron microscopy (SEM) showed that the nicotine-treated oocytes appeared nonspherical with rough surface and the zona pellucida (zp) was torn and became irregular. Supplementation with gamma-tocotrienol in the nicotine-treated mice retained the spherical shape of the oocytes with intact zp; however, the surfaces of the oocytes remained irregular and rough. Transmission electron microscopy (TEM) following chronic nicotine treatment showed loosening of the boundary and tearing of the zp. The perivitelline space was also widened. The cytoplasm of the oocytes retained abundant rough endoplasmic reticulum (rER) with numerous vesicles. Mitochondria were highly dense, with no cristae. The administration of gamma-tocotrienol partially reduced the detrimental effects of nicotine by retaining the smooth boundary of the zp with the tight perivitelline space. There was less rER with no visible vesicle and a lower amount of dense mitochondrial matrix. CONCLUSIONS: This study documented that chronic nicotine treatment adversely affects the ultrastructure of oocytes, while gamma-tocotrienol treatment at least minimizes the nicotine-induced damage to oocytes.

Development of in vitro embryo production systems for red deer (Cervus elaphus). Part 2. The timing of in vitro nuclear oocyte maturation.

The time course of in vitro red deer nuclear oocyte maturation was determined. Ovaries were obtained at slaughter and oocytes were aspirated from follicles greater than 2mm in diameter. Oocytes with compact cumulus cells were matured in 50 microl microdrops (10 per drop) under mineral oil containing TCM 199 supplemented with 0.33 mM pyruvate, 10 microg LH and FSH, 1 microg oestradiol and 10% foetal bovine serum. Oocytes were matured at 39 degrees C and 5% CO(2) in air. At 3h intervals (0-27 h) oocytes were removed from incubation, cumulus expansion scored and removed, and fixed oocytes in ethanol:acetic acid (3:1) for 48 h. Oocytes were stained with lacmoid (1%) and nuclear maturation assessed. Oocytes were arrested in the germinal vesicle (GV) stage at aspiration and up to 6h of incubation. The nuclear membrane began to disperse after 6h and by 10.6+/-0.6h of incubation 75% of the oocytes exhibited germinal vesicle breakdown (GVBD). The mean time for 50% of the oocytes to reach metaphase one (MI) and metaphase two (MII) was 11.7+/-0.4 and 24.8+/-0.9h, respectively. Cumulus oophorus were tightly compacted at aspiration and did not begin expansion until 12h of culture. Full expansion was complete by 18 h of culture. Corona radiata cells did not begin expansion until 15 h and were fully expanded by 24h. Results indicate that in vitro red deer oocyte maturation follows a similar time course of nuclear maturation as reported for bovine and ovine oocytes.