The porcine zygotic genome activation occurs along with global epigenetic remolding at the 4-cell stage. The histone acetylation, regulating DNA transcription, replication and so on, requires adequate acetyl-CoA. Acetyl-CoA produced by translocated pyruvate dehydrogenase in the nucleus of mammalian cells has been reported, which is commonly considered locating in the mitochondria. To find out whether the nuclear pyruvate dehydrogenase regulating the histone acetylation by controlling generation of acetyl-CoA, a multiple sgRNAs-CRISPR/Cas9 targeting strategy was employed to generate a pyruvate dehydrogenase E1 alpha1 (Pdha1) knockout (KO) parthenogenetic embryo model. Results showed that the targeting efficiency of Pdha1 reached more than 90%. Hence, this model was used in the subsequent experiments. Furthermore, a translocation of Pdha1 during zygotic genome activation was found by immunofluorescent staining and was significantly inhibited by Pdha1 KO. Meanwhile, the 8-cell stage embryo rate significantly decreased after 72 h (24.19% vs 12.53%, control vs Pdha1 KO), indicating a 4-cell arrest. In addition, the nuclear histone acetylation level significantly decreased when Pdha1 was KO. To determine whether the zygotic genome transcription was affected, the qPCR was performed and showed that the mRNA level of Eif1A, Acly, Sqle and Pdha1 all dropped significantly in the Pdha1 KO group compared to the control. In conclusion, the translocated Pdha1 generates acetyl-CoA for histone acetylation inside the nucleus of porcine embryos, which promotes the zygotic genome activation of porcine embryos.

본 연구는 호르몬과 Na-pyruvate의 첨가가 개 미성숙난자의 체외성숙에 미치는 영향을 검토하였다. 개의 발정주기와 관계없이 암캐의 난소에서 회수한 난자를 NCSU-37 배양액에서 72시간 체외 배양하였다. 호르몬의 영향을 검토하기 위하여 배양액 중에 다양한 호르몬을 24{sim}72시간 첨가하여 성숙율을 검사하였으며, Na-pyruvate 첨가 농도의 영향을 검토하였다. E2 단독 첨가구에서는 제2감수분열 중기(MII) 단계까지 성숙이 관찰되었으나(5.7%), 타 처리구에서는 MII기까지의 성숙이 이루어지지 않았다(P<0.05), E2를 6~24시간만 처리하였을 때는 MII기로 성숙되는 난자가 없었으나 72시간 처리하였을 때는 6.0%로 유의적으로 높게 나타났다(P<0.05). Na-pyruvate의 농도를 0.25 mM 첨가한 구보다 2.5 mM 첨가한 구에서 제1감수분열 중기(MI) 단계까지의 성숙율이 증가하는 경향을 보였으나, MII 단계까지의 성숙율은 차이가 없었다. 본 실험의 결과는 개 난자의 체외성숙시 E2가 첨가된 배양액에서 72시간 배양시에 성숙율을 증진시킬 수 있으나, Na-pyruvate의 농도는 개 체외배양에 있어 큰 영향을 미치지 않는 것을 나타낸다.

Oxidative stress is one of the major causes of failure in in vitro storage of boar semen. Reactive oxygen species (ROS) are known to be important mediators of such stress. The present study examined the effects of pyruvate and taurine on sperm motility and expression of BAD, Cytochrome c, Caspase-3 and Cox-2 protein in in vitro storage of boar semen, and tested the effect of semen treated with antioxidant with or without hydrogen peroxide on the development of IVM/IVF porcine embryos. Semen samples were transported to the laboratory at 17℃ within 2 hr after collection and were treated with different concentration of pyruvate (1~10mM) and taurine (25~100mM) with or without 250uM H2O2 respectively. The supplementation of pyruvate and taurine increased sperm motility in boar semen during in vitro incubation at 37℃. Expression of apoptosis protein (BAD, cytochrome c, caspase-3 and cox-2) were reduced in the group of boar semen treated with pyruvate and taurine when compared to the other groups. The developmental rates of IVM/IVF porcine embryos fertilized by semen treated with pyruvate and taurine were significantly increased when compared to control (P<0.005). These results indicate that supplementation of pyruvate and taurine as antioxidants in boar semen extender can improve the semen quality and increase in vitro development of porcine IVM/IVF embryos when boar semen treated with antioxidants was used for in vitro fertilization.

당 대사에 관여하는 Pyruvate dehydrogenase phosphatase (PDP)는 해당과정에서의 대사 산물인 pyruvate를 acetyl CoA로 만들어 구연산 회로로 진입시켜주는 효소인 pyruvate dehydrogenase complex (PDC)의 활성을 조절하는 중요한 효소이다. PDP의 catalytic subunit는 PDC의 dihydrolipoamide acetyltransferase(E2), PDP regulatory subunit (PDPr), 그리고 칼슘 결합 도메인 등으로 구성되어 있는 것으로 추측되어지고 있다. 본 연구에서는 PDP 단백질을 분리정제하고 결정화 하고자 하였다. PDP는 catalytic subunit(PDPc, Mr 52,600 Da)과, regulatory subunit (PDPr 95,600 Da)으로 구성되어 있으며 칼슘 존재하에 PDPc는 dihydrolipoamide acetyltransferase(E2) component와 결합하여 기질인 인산 E1 component의 탈 인산화율을 증가시킨다. PDPc는 intrinsic 칼슘 결합부위를 가지며 두 번째 칼슘 부위는 E2 존재 하에 형성된다. 이러한 특이한 상호반응을 이용한 GSH-Sepharose-GST-L2 matrix를 이용하여 약 1000 U/㎎의 specific activity를 갖는 순수 PDPc를 약 80%의 yeild로 얻어 결정화에 사용하였다.

We have studied the technology to extend the storage period of ‘Seolhyang’ strawberries using modified atmosphere package (MAP) and ethyl pyruvate (EP) treatment for domestic distribution and export. The selected ripe strawberries harvested on December 28, 2016 at the Sancheong farmhouse were transported to the laboratory for 2 h and tested. After a day’s precooling at 4℃, the strawberries were divided into seven experimental groups. These groups were control, active MAP using low density polyethylene (LDPE), active MAP using polyamide (PA), active MAP using PA with EP treatment, passive MAP using LDPE, passive MAP using PA and passive MAP using PA with EP treatment. Quality analysis was carried out every 4 days during the storage period of 16 days. During the storage period of 16 days, MAP decreased from 3.5% to less than 1.1% in weight loss ratio compared with control, and decreased from 36% to less than 7% in fungal incidence. In the case of fungi in the EP treatment group, hyphae did not grow on the outside of the strawberry but grew to the inside. This tendency was similar to that in the low oxygen and high carbon dioxide environment of the MAP, the mycelium of the fungus did not grow outside of the strawberry. Fungi are the biggest problem in the distribution and export of strawberries, and these results suggest that MAP alone could inhibit mold and increase shelf life.