Direct injection of CRISPR/Cas9 into zygotes enables the production of genetically modified nonhuman primates (NHPs) essential for modeling specific human diseases, such as Usher syndrome, and for developing novel therapeutic strategies. Usher syndrome is a rare genetic disease that causes loss of hearing, retinal degeneration, and problems with balance, and is attributed to a mutation in MYO7A, a gene that encodes an uncommon myosin motor protein expressed in the inner ear and retinal photoreceptors. To produce an Usher syndrome type 1B (USH1B) rhesus macaque model, we disrupted the MYO7A gene in developing zygotes. Identification of appropriately edited MYO7A embryos for knockout embryo transfer requires sequence analysis of material recovered from a trophectoderm (TE) cell biopsy. However, the TE biopsy procedure is labor intensive and could adversely impact embryo development. Recent studies have reported using cell-free DNA (cfDNA) from embryo culture media to detect aneuploid embryos in human in vitro fertilization (IVF) clinics. The cfDNA is released from the embryo during cell division or cell death, suggesting that cfDNA may be a viable resource for sequence analysis. Moreover, cfDNA collection is not invasive to the embryo and does not require special tools or expertise. We hypothesized that selection of appropriate edited embryos could be performed by analyzing cfDNA for MYO7A editing in embryo culture medium, and that this method would be advantageous for the subsequent generation of genetically modified NHPs. The purpose of this experiment is to determine whether cfDNA can be used to identify the target gene mutation of CRISPR/Cas9 injected embryos. In this study, we were able to obtain and utilize cfDNA to confirm the mutagenesis of MYO7A, but the method will require further optimization to obtain better accuracy before it can replace the TE biopsy approach.

도시의 역사와 건설 환경의 대 발전은 언제나 다음과 같은 딜레마에 직면해 있다. 온전한 역사적 보호인가 아니면 도시재생인가? 어반 아큐펀쳐는 도시의 고유한 분위기 유지와 도시의 전반적인 발전, 이 두 가지를 모두 충족할 수 있는 또 하나의 도시발전 전략이다. 어반 아큐펀쳐는 엄격한 정의와 보편적인 표준 패턴이 없이도 내포된 내용이 풍부하고 개방적인 도시 발전 전략으로서 상당히 다양한 해석과 이해를 가지고 있는데, 한국의 도시재생과 관련된 본 연구에는 마누엘 데 모랄레스의 개념이 큰 계기를 마련해주었다. 한국과 유럽, 중국의 다양한 문헌 자료 조사 연구를 통해, 문화와 환경의 관점에서 거리 조각을 도시 촉매로 삼아 지역의 발전 잠재력을 촉진하고 도시재생과의 관계를 탐구하는 연구는 많지 않다는 것을 발견했기 때문이다. 그리하여 도시재생과 어반 아큐펀쳐에 대한 연구 및 그와 관련된 사례는 여전히 건축 및 구역의 개조, 공공 인프라의 업그레이드, 공공 공간 및 도로 교통의 최적화라는 것을 알 수 있었다. 문화적 측면에서도 도시재생과 어반 아큐펀쳐에 대한 연구 및 사례는 비교적 거시적일 뿐만 아니라 주로 도시설계에 관한 주제가 주를 이루고 있다. 따라서 본 연구에서는 어반 아큐펀쳐와 거리에 우뚝자리 잡고 있는 조각을 결합하고 이 조각을 통해 거리의 관건이 되는 혈점을 선택하여 소규모로 개입하거나 마이크로 임플랜테이션할 것을 주장하였다. 본 연구의 목적은 이를 바탕으로 도시재생의 큰 틀에서 조각의 아큐펀쳐과 조각의 마이크로 임플랜테이션이라는 개념을 정련하는 것이다. 따라서 이를 계기로 도시재생의 새로운 방향을 제시하고 향후 도시재생의 실천에도 참고할 수 있는 사료를 제공하고자 하였다. 이에 본 연구는 어반 아큐펀쳐와 도시 내 조각품의 사례를 각각 분석하고 마이크로 임플랜테이션이라는 개념 아래에서 양자의 비교를 통한 결합 가능성을 모색함으로써 도시재생에 있어서 도시공학이나 도시디자인의 범주 아래에서 공공조형의 역할과 기능을 통합할 수 있다는 사실을 확인하고 제시했다는 점에서 의의가 있을 것이다.

Interferon tau (IFNT) regulation, an anti-luteolytic factor produced by conceptuses of the ruminant ungulates, is essential for the maintenance of early pregnancy, but a definitive mechanism for its temporal transcription has not been elucidated. We and others have observed the T-box protein eomesodermin (EOMES) exhibited high mRNA expression in the ovine embryonic trophectoderm; thus, both caudal-relatedhomeobox-2 (CDX2) and EOMES coexist during the early stages of conceptus development. Objective of this study was to examine the effect of EOMES on ovine IFNT gene transcription when evaluated with CDX2, ETS2 and AP1 transcription factors implicated in the control of cell differentiation in the trophectoderm. In this study, quantitatively via reverse transcription-polymerase chain reaction (RT-PCR) analysis between ovine trophoblast cells was initially performed, finding that transcription factors CDX2 and ‘EOMES transcription factor mRNAs’ were specific to trophectoderm cells. These mRNAs were also found in days 15, 17, and 21 ovine conceptuses. Furthermore, human choriocarcinoma JEG3 cells (trophoblast cell line) were cotransfected with an ovine IFNT (-654bp)-luciferase reporter (-654-oIFNT-Luc) construct and several transcription factor expression plasmids. Cotransfection of the reporter construct with CDX2, ETS2 and AP1 increased transcription of -654-oIFNT-Luc by about 11-fold compared with transfection of the construct alone. When cells were initially transfected with EOMES followed by transfection with CDX2, ETS2 and/or AP1, the expression of -654-oIFNT-Luc was decreased. Also, EOMES factor inhibited the stimulatory activity of CDX2 alone. These results suggest that when conceptuses attach to the uterine epithelium, ovine IFNT gene transcription is down-regulated by an increase of EOMES factor expression in the attached ovine trophoblast cells.

Polo-like kinase 1 (Plk1) has multiple roles in somatic cell and mammalian oocyte division. In mice, Plk1 distributes to the centromeres from prophase to anaphase and compose spindle apparatus in mitosis stages. Somatic cell nuclear transfer (SCNT) has diverse advantages. However, low cloning efficiency of SCNT procedure causes difficulty to application. The causes of this low efficiency are still unclear. However, they are attributed to the cumulative results of several biological and technical factors. In this study, Plk1, a biological factor, was investigated. B6D2F1 mice (7 weeks old) were superovulated with 10 IU of pregnant mare’s serum gonadotropin and 9 U of human chorionic gonadotropin (HCG) 48 hr later. The oocytes were collected 14 hr after HCG injection and cultured on potassium simplex optimized medium. The BI2536, Plk1-specific inhibitor, was used to understand the influence of Plk1. Also, the embryos were assessed by immunofluorescence. All BI2536-treated embryos failed to the first mitotic division. It showed Plk1 has a critical role in the first mitotic division of the mouse embryo. Moreover, there were significant differences between the control and SCNT embryos in the patterns of Plk1. All SCNT embryos which failed 2-cell development presented incorrect positioning and low expression of Plk1. On the other hand, the control embryos which failed to 2-cell division showed only low expression of Plk1. Taken together, this results demonstrate that Plk1 is critical for successful mitotic division of mouse embryos. Also, correct localization of Plk1 has crucial effect in the development of murine SCNT embryos.

One of the major hallmarks of uterine diseases is disruption of ovarian steroid hormone control of uterine cell proliferation and differentiation. Estrogen (E2) stimulates proliferation of uterine epithelial cells while progesterone (P4) is inhibitory to E2-mediated proliferation of the epithelium. Mitogen inducible gene 6 (Mig-6) is an important mediator of P4 signaling to inhibit E2 signaling in the uterus. Uterine-specific knockout of Mig-6 caused endometrial P4 resistance and infertility. Levels of ErbB2 (also known as HER2) and phospho-ERK1/2 are significantly higher in Mig-6 knockout mice as well as infertile women with endometriosis. To determine the interplay between Mig-6 and the Erbb2 signaling pathway in the uterus, we generated mice with Mig-6 and Erbb2 conditionally ablated in progesterone receptor-positive cells (Pgrcre/+ Mig-6f/f Erbb2f/f; Mig-6d/d Erbb2d/d). Mig-6d/d mice were infertile whereas control and Mig-6d/d Erbb2d/d mice exhibited normal fecundity. The uterine horns of Mig-6d/d mice had no implantation sites, whereas control and Mig-6d/d Erbb2d/d mice had averaged implantation sites. Additionally, aberrant increment of epithelial proliferation in uterus of Mig-6d/d mice did not show in Mig-6d/d Erbb2d/d mice uterus at pre-implantation stage. Microarray analysis revealed that almost altered genes in Mig-6d/d mice were recovered their expression levels in Mig-6d/d Erbb2d/d mice. The altered pathways such as cell-cycle control, DNA replication, and modification processes by Mig-6 ablation were rescued in Mig-6d/d Erbb2d/d mice. The infertility seen in Mig-6d/d mice is recovered in Mig-6d/d Erbb2d/d mice. These results suggest that Mig-6 mediates a critical P4 function to inhibit E2 signaling by inhibiting ErbB2 signaling. As MIG-6 is a mediator of P4 signaling, the activity of which can suppress unopposed-E2 signaling, our studies provide a potential new drug target for the intervention of female infertility.

Embryo transfer is one of the important process of assisted reproductive technology (ART) and that is associated with uterus endometrial receptivity. Recently, mouse endometrial stimulation by artificial injury had shown the favorable effect on conception. In this experiment, we used uterus stimulation method that injury the endometrium to increase implantation rate for spontaneous Diabetes Mellitus (sDM) rat. Rats are divided into several groups involved a control group. We performed the surgical method to Experimental group bilaterally or unilaterally After that, we investigated morphological change and calculated implanted embryos respective sides of the uterus. The number of implanted embryo in the experimental group was significantly higher and there were lots of morphological changes including glands and endometrial cells that support implantation. Our results showed that rat uterus endometrial injury in ART help enhancing implantation rate.

Interferon tau (IFNT), produced by the mononuclear trophectoderm, signals the process of maternal recognition of pregnancy in ruminants, but its expression in vivo is not well characterized. Objectives of this study were to determine IFNT gene isoforms expressed in the bovine uterus, and to identify differences in promoter sequences of IFNT genes that differ in their expression. Through the RNA-seq analysis of bovine conceptuses on days 17, 20 and 22 (day 0 = day of estrus), the expression of only two IFNT transcripts, IFNT1 and IFNTc1, were found, which were indeed classified into the IFNT gene clade. IFNT mRNAs were highest on day 17, and then decreased on days 20, and 22, which were also supported by the results of quantitative RT-PCR. Bovine ear-derived fibroblast (EF) cells were then cotransfected with luciferase reporter constructs carrying 5‘-upstream (positions -1000 to +51) regions of IFNT1 or IFNTc1 and various transcription factor expression plasmids. CDX2, either alone or with other Ap-1, ETS2 and/or CREBBP transcription factors, was found to increase luciferase activity approximately 10 and 18 fold more than twice of those cotransfected with bIFNT1, c1-Luc construct. Furthermore, The degree of transcriptional activation by a combination of the AP1, ETS2, CREBBP and/or CDX2 expression vectors was similar to that of CDX2 along plasmid. However, expression patterns of these Luc activity differented. Whereas bIFNTc1-Luc showed lowest antivity had than bIFNT1-Luc reports. Although, lowest antivity had of the bIFNTc1 –Luc report, cotransfected with the bIFNTc1-Luc construct and AP1(JUN) or/and ETS2 expression plasmid, Luc activity was enhanced approximately 2 and 4-fold more than the bIFNT1-Luc. Furthermore, along CDX2 expression factor had high effect on activity of bIFNT1-Luc reporter than the c1 gene in EF cells. These results suggest that two forms of IFNT genes are expressed in utero and their transcriptional regulations differ.

ZnO with wurtzite structure has a wide band gap of 3.37 eV. Because ZnO has a direct band gap and a large exciton binding energy, it has higher optical efficiency and thermal stability than the GaN material of blue light emitting devices. To fabricate ZnO devices with optical and thermal advantages, n-type and p-type doping are needed. Many research groups have devoted themselves to fabricating stable p-type ZnO. In this study, As+ ion was implanted using an ion implanter to fabricate p-type ZnO. After the ion implant, rapid thermal annealing (RTA) was conducted to activate the arsenic dopants. First, the structural and optical properties of the ZnO thin films were investigated for as-grown, as-implanted, and annealed ZnO using FE-SEM, XRD, and PL, respectively. Then, the structural, optical, and electrical properties of the ZnO thin films, depending on the As ion dose variation and the RTA temperatures, were analyzed using the same methods. In our experiment, p-type ZnO thin films with a hole concentration of 1.263 × 1018 cm−3 were obtained when the dose of 5 × 1014 As ions/cm2 was implanted and the RTA was conducted at 850 oC for 1 min.

목 적: 후방안내렌즈삽입술 후 이중경로방식(double-pass technique)으로 시력의 질을 평가하였다. 방 법: 중등도 및 고도 근시안(평균 등가구면굴절력: -9.26±1.67 D) 30명(57 안, 평균연령: 26.87± 7.26세)을 대상으로 후방안내렌즈 삽입 전과 삽입 1개월 후 OQAS(Optical Quality Analysis System) 장비 를 이용하여 MTF(modulation transfer function), Strehl ratio, OSI(objective scattering index) 및 객관 적 시력 OVs(OQAS values)를 측정하였다. 수술 전과 수술 1개월 후 모든 측정값을 비교하여 시력의 질을 평가하였으며, 중등도 근시안과 고도 근시군의 수술 후 시력의 질을 비교하였다. 결 과: 후방안내렌즈 삽입 1개월 후 MTF cutoff 값, Strehl ratio, OSI, OV100%, OV20% 및 OV9% 는 각 각 31.23±8.85 cycles/degree, 0.18±0.05, 0.76±0.37, 1.04±0.30, 0.73±0.24, 0.44±0.14로, MTF cutoff 값, Strehl ratio, OSI는 수술 전과 유의한 차이가 없었고(p=0.552, p=0.579, p=0.364), 객관적 시 력 OV100%, OV20%, OV9%도 후방안내렌즈 삽입 전과 유의한 차이를 보이지 않았다(p=0.561, p=0.671, p=0.522). 등가구면 굴절력 –6.00 D를 기준으로 나눈 두 그룹 사이에서 수술 1개월 후 MTF cutoff, Strehl ratio, OSI, OV100%, OV20% 및 OV9% 는 모두 유의한 차이를 보이지 않았다(p>0.05). 결 론: 후방안내렌즈 삽입 1개월 후 시력의 질을 나타내는 측정값은 수술 전 안경으로 교정한 시력의 질 과 비교하여 차이가 없었으며, 고도근시안에서도 중등도 근시안과 차이를 보이지 않아 굴절력에 관계없이 좋 은 시력의 질을 제공하는 것으로 평가된다.

Three-dimensional (3D) culture system is useful technique for study of in vivo environment and it was used various experiments. This study was investigated to establish of embryo co-culture system and changes of PAs activity in 3D cultured endometrial cells of pigs. In results, growth of stromal cells into gel matrix were detected only with endometrial and myometrial cells. The most rapid growth of stromal cells were confirmed in 2.5x105cells/ml and gel matrix containing 15% FBS. Expression of urokinase-PA (uPA) after treatment of hCG (0.5, 1.0, 1.5 and 2.0 IU/ml) were higher than without hCG, but, there are not significant difference among the treatment. On the other hand, expression of uPA after treatment of IL-1β (0.1, 1, 10 and 100 ng/ml) were higher than without IL-1β, but, there are not significant difference. Expression of uPA after treatment of estrogen (0.2, 2, 20 and 200 ng/ml) were not difference, but PA activity was significantly decreased (p＜0.05). Blastocyst was producing in PZM-3 medium containing FBS and endometrial cells were grown in PZM-3 medium. When embryos development with cultured endometrial cells, cleavage rates were not significant difference and blastocyst were not produced in co-culture with stromal cells and 3D culture system. 3D culture system had similar activity to in vivo tissue and these features are very useful for study of in vivo physiology. Nevertheless 3D culture system was not proper in embryo co-culture system. Therefore, we suggest that 3D culture system with embryo co-culture need continuous research.

For successful embryo implantation, the communication of the maternal endometrium with the conceptus trophec-toderm is required essentially. In pigs, conceptuses undergo morphological change in length to enlarge the physical contact area with the maternal endometrium and secrete estrogen to induce the maternal recognition of pregnancy during the peri-implantation period. Conceptus-derived estrogen prevents luteolysis by conversion in direction of PGF2α secretion from the uterine vasculature to the uterine lumen as well as it affects on expression of the uterine endo-metrial genes. In addition to estrogen, conceptuses release various signaling molecules, including cytokines, growth factors, and proteases, and, in response to these signaling molecules, the maternal uterine endometrium also syn-thesizes many signaling molecules, including hormones, cytokines, growth factors, lipid molecules, and utilizes ions such as calcium ion by calcium regulatory molecules. These reciprocal interactions of the conceptus trophectoderm with the maternal uterine endometrium make development and successful implantation of embryos possible. Thus, signaling molecules at the maternal-conceptus interface may play an important role in the implantation process. This review summarized syntheses and functions of signaling molecules at the maternal-conceptus interface to further understand mechanisms of the embryo implantation process in pigs.

목적: 백내장환자를 대상으로 비구면 단초점 인공수정체 삽입 후 시력과 시기능 변화를 분석하였다. 방법: 단안 또는 양안에 HOYA PC-60AD 비구면 단초점 인공수정체를 삽입한 백내장 환자 25명의 30안을 대상으로 수술 전, 수술 후 1일, 1주일, 1개월 및 3개월 후에 원거리 시력과 객관적인 시기능을 평가하고, 수술 3개월 후 인공수정체 삽입안의 시기능을 40대 이상의 정상안 18명(36안)과 비교하였다. 객관적인 시기능은 원거리 시력과 OQAS 장비를 이용하여 4 mm 동공크기에서 측정된 객관적 산란지수(objective scatter index, OSI), 변조전달함수(modulation transfer function, MTF) 및 Strehl ratio를 측정하여 평가하였다. 통계분석은 SPSS ver. 18.0로 대응표본 T 검정과 선형 회귀분석을 이용하여 비교하였고 유의수준은 p

Successful pregnancy requires well-coordinated interactions between the maternal uterus and the developing embryo in pigs. In pigs, implantation begins around Day 12 of pregnancy. During this period, conceptus undergoes a dramatic morphological change and secretes various factors such as estrogens, interleukin-1 beta (IL1B), and interferons. Estrogens produced by conceptuses act as the signal for maternal recognition of pregnancy, and the mechanism of estrogen action is explained by the endocrine and exocrine theory. The uterine endometrium becomes receptive to the conceptus by changing cell adhesion molecules, polarizing epithelial cells and increasing secretory activity. Some changes of uterine activity are affected by the ovarian hormone, progesterone, but the presence of conceptus in the uterus also induces changes of endometrial functions, including most importantly maternal recognition of pregnancy. Many factors, such as hormones, cytokines, enzymes, extracellular matrix proteins, and transport proteins are reported to be present at the maternal-fetal interface and function in the establishment of pregnancy in pigs. However, understanding of the cellular and molecular events occurring in the endometrium is not complete. In recent studies we made some progress on understanding of expression and function of genes involved in maternal-fetal interaction for the establishment and maintenance of pregnancy in the uterine endometrium in pigs. Firstly, we found that lysophosphatidic acid (LPA) was present at the maternal-and fetal interface at the time of implantation and LPA receptor 3 was uniquely expressed in the endometrium during early pregnancy. Secondly, we observed that salivary lipocalin (SAL1), a lipid-binding protein, was uniquely expressed in the uterine endometrium at the time of embryo implantation, and its expression was regulated by IL1B. Furthermore, expression of IL1B receptors are regulated by estrogen and IL1B, and IL1B functions in expression of genes related to prostaglandin synthesis and transport. Thirdly, we found that calcium regulatory molecules TRPV6 and S100G were dynamically regulated in the uterine endometrium during pregnancy, suggesting that regulation of calcium ion concentration may important for the embryo implantation and the maintenance of pregnancy. Finally, we observed that an MHC class II molecule, SLA-DQ, is expressed in the uterine endometrium at the time of conceptus implantation and its expression is essential for successful pregnancy, indicating that appropriate maternal-fetal immune interaction is required for the maintenance of pregnancy. Further analysis of these molecules will provide insights into the cellular and molecular basis of maternal-and fetal interaction during pregnancy in pigs.

The objective of this study was to determine the mRNA expression patterns of several putative imprinted genes in in vivo and in vitro fertilized, parthenogenetic, and cloned porcine preimplantation embryos. Both maternally (Dlk1, IGF2, Peg1/Mest and Ndn) and paternally (IGF2r, H19 and Xist) imprinted genes were selected. We have used reverse transcription polymerase chain reaction (RT-PCR) to investigate gene expression patterns in the porcine embryos. IGF2 transcripts were detected in the most of embryos. In nuclear transfer (NT), Peg1/MEST transcripts showed fluctuating pattern. Dlk1 was only expressed partially from the morula and blastocyst stage of NT embryos. Ndn gene expression was started somewhat early for in vivo embryos. However, the expressions of maternally imprinted genes were similar in all types of blastocysts (NT, in vivo and in vitro fertilized, and parthenogenetic embryos). The IGF2R gene expression level was somewhat irregular and varied among samples. However, for the majority samples of all types of embryos, IGF2R expression was diminished after one- to two-cell stages and reappeared at the morulae or blastocyst stage embryos. H19 gene was only expressed early in parthenogenetic and in vivo embryos. For NT embryos, H19 was only expressed in blastocysts. Xist expression was detected in all blastocysts with the earliest being in vivo 8-cell stage embryos and the last one being NT blastocysts. These putative imprinted genes appeared to have stage specific expression patterns with a fluctuating pattern for some genes (Peg/Mest, IGF2r, H19). These results suggest that stage specific presence of imprinted genes can affect the embryo implantation and fetal development.

Pregnancy is a unique event in which a fetus develops in the uterus despite being genetically and immunologically different from the mother, and the underlying mechanisms remain poorly understood. To analyze the differential gene expression profiles in nonpregnant and 7 days post coitus (dpc) pregnant uterus of mice, we performed a global proteomic study by 2‐D gel electrophoresis (2‐DE) and MALDI‐TOF‐MS. The uterine proteins were separated using 2‐DE. Approximately 1,000 spots were detected on staining with Coomassie brilliant blue. An image analysis using Melanie III (Swiss Institute for Bioinformatics) was performed to detect variations in protein spots between pregnant and nonpregnant uterus. Twenty‐one spots were identified as differentially expressed proteins, of which 10 were up‐regulated proteins such as alpha‐fetoprotein, chloride intracellular channel 1, transgelin, heat‐shock protein beta‐1, and carbonic anhydrase II, while 11 were down‐regulated proteins such as X‐box binding protein, glutathione S‐transferase omega 1, olfactory receptor Olfr204, and metalloproteinase‐disintegrin domain containing protein TECADAM. Most of the identified proteins appeared to be related with catabolism, cell growth, metabolism, regulation, cell protection, protein repair, or protection. Our results uncovered key proteins of mouse uterus involved in pregnancy.

In this study, we aimed to determine whether the evaluated markers of cell death could be found at particular developmental stages of normal porcine in vitro fertilization (IVF) embryos. We investigated the characteristics of spontaneous and induced apoptosis during preimplantation development stages of porcine IVF embryos. In experiment 1, to induce apoptosis of porcine IVF embryos, porcine IVF embryos at 22h post insemination were treated at different concentration of actinomycin D (0, 5, 50 and 500 ng/ml in NCSU medium). Treated embryos were incubated at in 5% , 5% for 8h, and then washed to NCSU medium and incubated until blastocyst (BL) stage. We examined cleavage rate at 2days and BL development rate at 7days after in vitro culture. A significantly lower rate of cleavage was found in the 500 ng/ml group compared to others (500 ng/ml vs. 0, 5, 50 ng/ml; 27.8 % vs. 50.0%, 41.2%, 35.9%), and BL formation rate in 500 ng/ml was lower than that of others (500 ng/ml vs. 0, 5, 50 ng/ml; 8.0% vs. 12.6%, 11.2%, 12.6%). In experiment 2, to evaluate apoptotic cells, we conducted TUNEL assay based on morphological assessment of nuclei and on detection of specific DNA degradation under fluorescence microscope. This result showed that apoptosis is a normal event during preimplantation development in control group (0 ng/ml actinomycin D). A high number of BL derived control group contained at least one apoptotic cell. Actinomycin D treated BLs responded to the presence of apoptotic inductor by significant decrease in the average number of blastomeres and increase in the incidence of apoptotic cell death. In 500 ng/ml group, the incidence of apoptosis increased at 4-cell stage and later. This result suggested that apoptosis is a process of normal embryonic development and actinomycin D is useful tool for the apoptosis study of porcine preimplantation embryos.