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The citrus flavonoid hesperetin has a variety of pharmacological actions, including antioxidant, antiinflammatory, and anticancer activities. This study investigated whether hesperetin prevents aging of oocytes in vitro in which it determined the maturation of nuclear and cytoplasm and the developmental capacity of embryo by modulating the reactive oxygen species (ROS) level. Porcine oocytes were matured in vitro for 44 hr (control) and for an additional 24 hr in the presence of 0, 1, 10, 100, and 250 μM hesperetin (aging, H-1, H-10, H-100, and H-250, respectively). Although there was no difference in the rate of maturation among all the groups, both the control and H-100 groups significantly increased in the rate of cleavage and blastocyst formation compared to the aging group. The H-100 group significantly decreased ROS activity and increases the level of glutathione (GSH) and expression of the antioxidant genes (PRDX5, NFE2L, SOD1, and SOD2) compared with the aging group. The H-100 groups prevented aberrant spindle organization and chromosomal misalignment, blocked the decrease in the level of phosphorylated-p44/42 mitogen-activated protein kinase and increased the messenger RNA expression of cytoplasmic maturation factor genes (GDF9, CCNB1, BMP15, and MOS). Subsequently, both the control and H-100 groups significantly increased the total cell number and decreased the apoptosis cells at the blastocyst stage compared with aging group. The results indicate that hesperetin improves the quality of porcine oocytes by protecting them against oxidative stress during aging in vitro.

Culture media modifications, including the addition of various factors, are important for the in vitro production of oocytes and embryos. In this study, we investigated the effects of lysophosphatidic acid (LPA) on porcine embryo development. Porcine parthenogenetic embryos were cultured with 0, 0.1, 1, and 10 μM LPA for 7 days, or cultured in basic medium until Day 4 and then treated with LPA from Days 4 to 7. No difference in the in vitro development of embryos cultured with LPA for 7 days was observed. Conversely, rates of blastocyst and over-expanded blastocyst formation were higher in the 0.1 and 1 µM LPA-treated versus the other groups of embryos treated from Days 4 to 7. Moreover, formation of early blastocysts occurred earlier and embryo size was larger in LPA-treated compared to control embryos. Expression of Connexin 43 and gap junction and cell adhesion-related genes (GJC1 and CDH1, respectively) was also higher in LPA-treated compared to control embryos. Despite no difference in the blastocyst total cell number between groups, the apoptotic index was lower in the LPA-treated group than in the control group; indeed, BCL2L1 (B-cell lymphoma 2-like protein 1) expression increased while BAK (Bcl-2 homologous antagonist killer) decreased in the LPA-treated group. Thus, addition of LPA to the medium from Days 4 to 7 of culture improves blastocyst formation and aids the development of preimplantation embryos.

SUMMARY Growth factors synthesized by ovarian somatic cells affect cumulus cell expansion and oocyte maturation in vitro. Fibroblast growth factor 10 (FGF10), for example, is a known regulator of mammalian cumulus-oocyte complex maturation. In this study, we investigated the effects of 0, 5, 10, 50, and 100 ng/mL FGF10 (5F, 10F, 50F, and 100F, respectively) on in vitro cumulus cell expansion, oocyte maturation, and embryo development. The percentage of fully expanded cumulus cells at the oocyte's metaphase-II (MII) stage was significantly higher in the 10F-treated group than in the control. Transcript abundance of the cumulus cell expansion-related gene encoding hyaluronian synthase 2 (HAS2) in cumulus cells at oocyte germinal vesicle breakdown (GVBD) was significantly higher in the 10F- and 50F-treated groups compared to untreated controls, whereas the mRNA abundance of the protease cathepsin B (CTSB) at the oocyte MII stage was remarkably decreased in the 10F-treated group. The percentage of oocytes with normal spindles was greater in the 10F- and 50F-treated group at GVBD than in the other groups; the 5F-, 10F-, and 100F-treated groups were higher than the control; and the 50F-treated group was highest at MII. The abundance of GDF9 and BMP15 transcript at GVBD and BMP15 and CCNB1 transcripts at MII increased in the 10F-treated group. Cleavage rate, blastocyst formation rate, and total cell number were significantly higher in the 5F- to 50F-treated groups. These results demonstrate that FGF10 markedly improves cumulus cell expansion, oocyte maturation, and subsequent embryo development. Mol. Reprod. Dev. 84: 67–75, 2017. © 2016 Wiley Periodicals, Inc.