Mitochondria are well known to regulate the mammalian embryo development. Recent studies showed that the mitochondrial dynamics responses are mainly generated through mitochondrial membrane potential (MMP) and cellular ATP production, which is dependent on mitochondrial reactive oxygen species (ROS). However, these mechanisms are unclear on development process of preimplantation porcine embryos. The aim of this study was to evaluate that difference of the mitochondrial dynamics-derived various functions on the embryo development according to lipid composition of zygote. First, zygote was classify two groups (high lipid, grade 1: G1 and low lipid, grade 2: G2) by lipid composition of cytoplasm. And, we performed the in vitro culture (IVC) using zygote of divided groups. The nuclei numbers and developmental rates of blastocysts were lower in G2 than those of G1 embryos. Next, we investigated the intracellular ROS and mitochondrial derived superoxide production in porcine embryos by using DCF-DA and Mito-SOX staining. As expected, both intracellular ROS and mitochondrial derived superoxide were significantly increased (p<0.05) in the preimplantation stage embryos of G2 group compared with G1 group. In addition, to observe difference of the mitochondrial functions, we investigated the mitochondrial membrane potential (MMP, ΔΨ) and contents of ATP in the preimplantation stage embryos by using JC-1 kit and ATP determination kit. These functions of mitochondria were dramatically reduced in cleavage stage embryos or blastocysts of G2 group. Finally, to verify the difference of the mitochondrial dynamics-derived various functions, we investigated the expressions of mitochondrial fission (Drp1, pDrp1-616) and fusion (Mfn1, Mfn2) factors by Western blotting analysis. Interestingly, the protein levels of pDrp1-616 in embryos of G1 group were continuously increased until blastocyst stage. Whereas, the expression patterns of Mfn1/2 in embryos of G2 group were significantly reduced during IVC progression. The expression patterns of mitochondria dynamic between the two groups were shown opposite. These results demonstrated that the lipid contents of zygote were related the positive-correlation with mitochondrial dynamics-derived functions in porcine embryos. Moreover, we suggest that lipid of zygote is play a important role on mitochondrial functions and dynamics during preimplantation embryos development in pigs.

Autocrine or paracrine mediators released by the early embryo are implicated in the support of embryonic development. Their mechanisms and optimal embryo density in the medium, however, are uncertain. This study was conducted to establish the optimal embryo density and culture medium volume in mouse parthenogenetic embryo culture. In experiment 1, culture of parthenogenetirally activated oocytes at a concentration of 2~4 embryos/ uL significantly improved development to the blastoryst stage (72%≤) compared with culture at the lower (0.2~1 embryos㎕, 0~37.5%) and the higher (5~6 embryos㎕, 30~53%) concentration for 120 h when the oocytes were cultured in a 5㎕ drop under mineral oil In experiment 2, the embryos cultured at a concentration of 2~4 embryos㎕ in a 10㎕ drop (81.1%) showed significantly higher blastocyst rates than those in a 5㎕ drop (68.5%). This study optimizes in vitro culture condition by modifying embryo density and the volume of culture medium It may give appropriate level of autocrine and/or paracrine factors to enhance viability and subsequent normal development of mouse parthenogenetic embryos in vitro.

당근종자의 발아율의 낮음과 발아지정의 문제점을 해결하기 위해서 Danver 126품종을 공시하여 여러가지 prime처리와 density separation 방법을 이용한 결과 다음과 같은 효과를 얻었다. 1. 여러가지 prime처리중 SMP처리(수분함량 90%, 6일간처리)에서 배의 생장이 아주 양호하였고 발아속도 및 발아율이 가장 좋았으며 종자에 피해도 없었다. 2. 무기염처리(0.2M KNO3 ＋0.1M K2 HPO4 )에 서는 배의 생장은 좋았으나 염에 의해 종자가 피해를 입었다. 3. SMP처리(수분함량 90%, 6일간)된 종자를 density separation한 결과 종자의 비중이 낮을수록 배의 생장이 컸으며 발아율 및 발아속도가 빨랐다.