Ubiquitin-proteasome system modulates zygotic genome activation in early mouse embryos and influences full-term development.

Maternal RNA/protein degradation and zygotic genome activation (ZGA), occurring during maternal-to-zygotic transition (MZT), are the first essential events for the development of pre-implantation embryos. Previously, we have shown the importance of the ubiquitin-proteasome system (UPS) for initiation of minor ZGA at the 1-cell stage of mouse embryos. However, little is known about the mechanism of involvement of the UPS-degraded maternal proteins in ZGA. In this study, we investigated the effect of inhibiting maternal protein degradation by the reversible proteasome inhibitor, MG132, on post-implantation development and ZGA regulation during early cleavage stages. Our study revealed that zygotic transcription by RNA polymerase II (Pol II) at the 1-cell stage was delayed and the full-term development was affected by transient proteasome inhibition during 1 to 9 h post-insemination (hpi). Furthermore, we found that the transient inhibition of proteasome activity at the 2-cell stage delayed the onset of transcription of some major ZGA genes. These results support the model hypothesizing the requirement of sequential degradation of maternal proteins by UPS for the proper onset of ZGA and normal progression of MZT in early mouse embryos.

Peroxiredoxin as a functional endogenous antioxidant enzyme in pronuclei of mouse zygotes.

Antioxidant mechanisms to adequately moderate levels of endogenous reactive oxygen species (ROS) are important for oocytes and embryos to obtain and maintain developmental competence, respectively. Immediately after fertilization, ROS levels in zygotes are elevated but the antioxidant mechanisms during the maternal-to-zygotic transition (MZT) are not well understood. First, we identified peroxiredoxin 1 (PRDX1) and PRDX2 by proteomics analysis as two of the most abundant endogenous antioxidant enzymes eliminating hydrogen peroxide (H2O2). We here report the cellular localization of hyperoxidized PRDX and its involvement in the antioxidant mechanisms of freshly fertilized oocytes. Treatment of zygotes at the pronuclear stage with H2O2 enhanced pronuclear localization of hyperoxidized PRDX in zygotes and concurrently impaired the generation of 5-hydroxymethylcytosine (5hmC) on the male genome, which is an epigenetic reprogramming event that occurs at the pronuclear stage. Thus, our results suggest that endogenous PRDX is involved in antioxidant mechanisms and epigenetic reprogramming during MZT.

Perturbation of maternal PIASy abundance disrupts zygotic genome activation and embryonic development via SUMOylation pathway.

During the maternal-to-zygotic transition (MZT), mRNAs and proteins stored in oocytes are degraded and zygotic genes are activated. We have previously shown that the ubiquitin-proteasome system (UPS)-mediated degradation of maternal proteins plays a role in the onset of zygotic transcription. However, it is still unclear which maternal proteins should be degraded for zygotic genome activation and ensuring subsequent embryonic development. In this study, we screen for these maternal factors that are degraded via the UPS. We thus identified a maternal protein PIASy (protein inhibitor of activated STATy), which is an E3 SUMO ligase. The overexpression of PIASy in fertilized embryos causes developmental arrest at the two-cell stage due to severe abnormal chromosome segregation and impaired zygotic transcription. We find that this developmental role of PIASy is related to its SUMOylation activity. Moreover, PIASy overexpression leads to increased trimethylation of histone H3 lysine 9 (H3K9me3) in two-cell nuclei and enhanced translocation of H3K9me3 methyltransferase to the pronucleus. Hence, PIASy is a maternal factor that is degraded after fertilization and may be important for the proper induction of zygotic genome activation and embryonic development.

Anesthetic and cardiorespiratory effects of single-bolus intravenous alfaxalone with or without intramuscular xylazine-premedication in calves.

The anesthetic and cardiorespiratory effects of xylazine-alfaxalone combination were evaluated in calves. Six calves (age: 6-9 months old; weight: 114-310 kg) were anesthetized with intravenous alfaxalone 15 min after administration of intramuscular saline (0.5 ml/100 kg) or xylazine (0.1 mg/kg; 0.5 ml/100 kg of a 2% xylazine solution). Anesthesia induction was smooth and orotracheal intubation was achieved in all calves. The calves anesthetized with xylazine-alfaxalone required a smaller induction dose of alfaxalone (1.23 ± 0.17 mg/kg, P=0.010) and accepted endotracheal intubation for a significantly longer period (16.8 ± 7.2 min, P=0.022) than the calves anesthetized with alfaxalone alone (2.28 ± 0.65 mg/kg 7.3 ± 1.6 min). At 5 min after induction, tachycardia (heart rate: 166 ± 47 beats/min of heart rate), hypertension (mean arterial blood pressure: 147 ± 81 mmHg) and hypoxemia (partial pressure of arterial blood oxygen [PaO2]: 43 ± 10 mmHg) were observed in the calves anesthetized with alfaxalone alone, whereas hypoxemia (PaO2: 47 ± 7 mmHg) and mild hypercapnia (partial pressure of arterial blood carbon dioxide: 54 ± 5 mmHg) were observed in the calves anesthetized with xylazine-alfaxalone. Premedication with xylazine provided a sparing effect on the induction dose of alfaxalone and a prolongation of anesthetic effect. Oxygen supplementation should be considered to prevent hypoxemia during anesthesia.