Effects of manganese on maturation of porcine oocytes in vitro and their subsequent embryo development after parthenogenetic activation and somatic cell nuclear transfer.

This study was carried out to examine the effects of manganese (Mn) on the developmental competence of porcine oocytes during in vitro maturation (IVM) after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT). Upon treatment of porcine oocytes with different concentrations (0, 3, 6, and 12 ng/ml) of Mn during IVM, PA was performed to determine the optimum concentration. Following PA, the rate of blastocyst formation was higher significantly in treated porcine oocytes at 6 ng/ml of Mn than in other groups (P < 0.05). However, there was no substantial difference in the cleavage rate and total blastocyst cell numbers among all groups. SCNT was performed using the optimal concentration of Mn from PA, which showed an improved blastocyst formation rate in treated oocytes compared to that in control group (P < 0.05). However, the cleavage rate and total cell numbers per blastocyst were not different between the control and the Mn treated groups after SCNT. Additionally, oocyte nuclear maturation, intracellular glutathione (GSH), and reactive oxygen species (ROS) levels were assessed. There was no significant difference observed in nuclear maturation among all the groups. However, enhanced intracellular GSH levels while lower levels of ROS were seen in the Mn treated group compared to the control group (P < 0.05). Thus, these results indicate that Mn supplementation can improve the developmental competence of porcine PA and SCNT embryos by increasing GSH and decreasing ROS levels.

Synergistic effects of resveratrol and melatonin on in vitro maturation of porcine oocytes and subsequent embryo development.

Resveratrol and melatonin are known for their antioxidant properties and have various biological activities. The fact that they exhibit possible synergistic effects in phytomedicine researches suggests the use of a combination of these agents to promote porcine in vitro maturation (IVM) of oocytes. Therefore, we investigated the effects of resveratrol and/or melatonin on this process; cumulus-oocyte complexes underwent IVM culture with four different conditions (control, resveratrol, melatonin or their combination). Cumulus expansion, oocyte nuclear maturation and subsequent embryo development after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) were evaluated. In experiment 1, all treatment groups significantly increased the proportion of complete cumulus expansion (degree 4) compared to the control, showing no difference among the treatment groups (P = 0.30). In experiment 2, oocytes matured with resveratrol and the combination had significantly higher metaphase-II (MII) rates than the control and melatonin groups, showing the highest (P < 0.05) MII rates in the combination group. In experiment 3, all treatment groups significantly increased blastocyst formation rates and total blastocyst cell numbers after PA compared to the control, but especially the combination showed the highest (P < 0.05) total cell numbers. In experiment 4, we selected the combination as the optimal condition and used this IVM system prior to SCNT. The combination treatment showed a significant (P < 0.05) increase in blastocyst formation rate and total cell numbers after SCNT. In conclusion, our results suggest that the combination of resveratrol and melatonin supported a synergistic increase in oocyte nuclear maturation and total cell numbers of PA blastocysts and improved the development of SCNT embryos.

A potential role of knockout serum replacement as a porcine follicular fluid substitute for in vitro maturation: Lipid metabolism approach.

The use of supplements, such as porcine follicular fluid (pFF), fetal bovine serum and human serum albumin are widely used during in vitro maturation (IVM) in different species but these supplements contain undefined components that cause technical difficulties in standardization and influence the efficiency of IVM. Knockout serum replacement (KSR) is a synthetic protein source, without any undefined growth factors or differentiation-promoting factors. Therefore, it is feasible to use KSR as a defined component for avoiding effects of unknown molecules in an IVM system. In this study, the rates of oocyte maturation and blastocyst formation after parthenogenetic activation (PA), somatic cell nuclear transfer (SCNT) and in vitro fertilization (IVF) were significantly higher in the 5% KSR supplemented group than in the unsupplemented control group and more similar to those of the 10% pFF supplemented group. Moreover, the intensity of GDF9, BMP15, ROS, GSH, BODIPY-LD, BODIPY-FA, and BODIPY-ATP staining showed similar values between 5% KSR and 10% pFF, which have significant difference with control group. Most of the gene expression related to lipid metabolism with both supplements exhibited similar patterns. In conclusion, 5% KSR upregulated lipid metabolism and thereby provides an essential energy source to sustain and improve oocyte quality and subsequent embryo development after PA, SCNT, and IVF. These indications support the idea that KSR used as a defined serum supplement for oocyte IVM might be universally used in other species.

Melatonin influences the sonic hedgehog signaling pathway in porcine cumulus oocyte complexes.

Melatonin, which is synthesized in the pineal gland and peripheral reproductive organs, has antioxidant properties and regulates physiological processes. It is well known that melatonin affects in vitro maturation (IVM) of oocytes and embryonic development in many species. However, beneficial effects of melatonin on IVM have been explained mainly by indirect antioxidant effects and little information is available on the underlying mechanism by which melatonin directly acts on porcine cumulus oocyte complexes (COCs). Sonic hedgehog (Shh) signaling is important for follicle development, oocyte maturation, and embryo development, and there may be a relationship between melatonin and Shh signaling. To examine this, we designed three groups: (i) control, (ii) melatonin (10-9  mol/L), and (iii) melatonin with cyclopamine (2 µmol/L; Shh signaling inhibitor). The aim of this study was to investigate the effects of these agents on cumulus expansion, oocyte maturation, embryo development after parthenogenetic activation (PA), gene expression in cumulus cells, oocytes and blastocysts, and protein expression in COCs. Melatonin significantly increased the proportion of COCs exhibiting complete cumulus expansion (degree 4), PA blastocyst formation rates, and total cell numbers, which were inhibited by addition of cyclopamine. Simultaneously, the expression of cumulus expansion-related genes (Ptgs1, Ptgs2, and Has2) and Shh signaling-related genes (Shh, Pthc1, Smo, and Gli1) and proteins (Ptch1, Smo, and Gli1) in cumulus cells was upregulated in the melatonin-treated group, and these effects were also inhibited by cyclopamine. In conclusion, our results suggest that Shh signaling mediates effects of melatonin to improve porcine cumulus expansion and subsequent embryo development.