The objective of this study was to examine the effects of high concentrations of glucose on porcine parthenotes developing in vitro. Addition of 55 mM glucose to the culture medium of embryos at the four-cell-stage significantly inhibited blastocyst formation, resulting in fewer cells in blastocyst-stage embryos and increased levels of apoptosis and autophagy compared to control. Quantitative reverse transcriptase (RT) PCR analysis revealed that the expression of pro-apoptotic genes (Caspase 3, Bax and Bak) and autophagy genes (Atg6 and Atg8/Lc3) were increased significantly by the addition of 55 mM glucose to the culture medium compared to control. MitoTracker Green fluorescence revealed a decrease in the overall mitochondrial mass compared to control. However, the addition of 55 mM glucose had no effect on mRNA expression of the nuclear DNA-encoded mitochondrial-related genes, cytochrome oxidase (Cox) 5a, Cox5b and Cox6b1. These results suggest that hyperglycemia reduced the mitochondrial content of porcine embryos developing in vitro and that this may hinder embryonic development to the blastocyst stage and embryo quality by increasing apoptosis and autophagy in these embryos.

It is well established that mitochondrial genome is strictly maternally inherited in mammalian, despite the fact that paternal mitochondria enter into oocyte during fertilization. To date, although some mechanisms have been extrapolated to interpret the elimination of paternal mitochondria, the exact mechanism still is unclear. Recent studies suggest that autophagy process and the ubiquitin-mediated degradation pathway may be involved in elimination of paternal mitochondria. However, the dynamic profiles of autophagy and ubiquitination associated with paternal mitochondria degradation have not been determined in mouse model. Through immunostaining with specific antibody LC3 and Ubiquitin and confocal microscopy, we investigated the dynamic profiles of LC3 and Ubiquitin signals in mouse embryos during preimplantation development. In addition, embryos were stained with MitoTracker Red for tracking the degradation process of paternal mitochondria. Our results showed that paternal mitochondria gradually degraded during postfertilization development, and sporadic paternal mitochondria were found at least in 16 cell embryos. LC3 and Ubiquitin signals appeared in the midpiece of sperm at 3 h postfertilization, and they were strictly colocalizated with paternal mitochondria from zygote to 2 cell embryo. Nevertheless, the colocalization became loose at 4 cell embryos, and gradually disappeared beyond 4 cell embryos. Our results confirmed that autophagy process and the ubiquitin-mediated degradation pathway may take part in the postfertilization remove of paternal mitochondria.

Superovulation, or ovarian stimulation is a commonly used ART for treatment of human infertility/subfertility. Recent studies suggest that superovulation unaffects methylated imprints acquisition in mouse oocytes during oogenesis, whereas disrupts DNA methylation maintenance in embryos during preimplantation development. However, the mechanisms of defects in methylation maintanence caused by superovulation remain largely unclear. We hypothesized that superovulation may disrupt the expression of DNA methyltransferases (Dnmts), the enzymes which catalyze DNA methylation acquisition and maintenance. The mice were subjected to superovulate with low (6 IU) and high (10 IU) dosage hormone. We examined the global DNA methylation levels in zygotes and DNA methylation of repeated sequences (IAP and Line 1) in blastocyst stage embryos. In addition, we investigated the expression of Dnmts (Dnmt3a, Dnmt3b, Dnmt3l and Dnmt1o) in ovulated oocytes and zygotes. Through staining with antibody 5mC and Di-H3K9 coupled with confocal microscopy, we found that global methylation profiles in zygotes derived from females after low or high dosage hormone treatment were not affected when compared to control counterpart. Moreover, methylation at IAP in blastocysts also was unaffected by superovulation, irrespective of hormone dosage. In contrast, methylation level at Line 1 decreased when the females were administered by high dosage hormone. Furthermore, expression of de novo DNA methyltransferase Dnmt3a, Dnmt3b, Dnmt3L, as well as maintenance Dnmt1o in MII oocytes and zygotes was not disrupted by superovulation. Given superovulation adversely affected methylation maintenance in blastocysts during preimplantation development but with normal expression of Dnmts in oocytes and zygotes, it is indicated that defects of embryonic methylation didn’t originate from abnormal expression of Dnmts.