MOLECULAR REPRODUCTION AND DEVELOPMENT 75:1127–1135 (2008) Anti-Apoptotic Effect of Melatonin on Preimplantation Development of Porcine Parthenogenetic Embryos JIHO CHOI, 1 SEON-MI PARK, 1 EUGINE LEE, 1 JI-HYE KIM, 1 YEON-IK JEONG, 1 JONG-YUN LEE, 1 SUN-WOO PARK, 1 HUEN-SUK KIM, 1 MOHAMMAD SHAMIM HOSSEIN, 1 YEON-WOO JEONG, 1 SUE KIM, 1 SANG-HWAN HYUN, 1,2 AND WOO-SUK HWANG 1 * 1 SooAm Biotech Research Foundation, Bangbae3-dong, Seocho-gu, Seoul, Korea 2 Laboratory of Veterinary Biotechnology, College of Veterinary Medicine, Chungbuk National University, Cheongju, Korea ABSTRACT In the present study, we investi- gated the effect of melatonin on the preimplantation development of porcine parthenogenetic and somatic cell nuclear transfer (SCNT) embryos. Parthenogenetic embryos were cultured in mNCSU-23 supplement- ed with various concentrations of melatonin for 7 days. The results revealed that 100 pM was the optimal concentration,whichresultedinsignificantlyincreased cleavage and blastocyst formation rates. Additionally, 100 pM melatonin provided the highest increase in total cell number of blastocysts. Therefore, the subsequent experiments were performed with 100 pM melatonin. ROS level in 2–8 cell stage embryos in the presence or absence of melatonin was evaluated. Embryos cultured with melatonin showed significantly decreased ROS. Blastocysts cultured with melatonin for 7 days were analyzed by the TUNEL assay. It was observed that melatonin not only increased (P < 0.05) the total cell number but also decreased (P < 0.05) the rate of apoptotic nuclei. Blastocysts cultured with melatonin were assessed for the expression of apoptosis-related genes Bcl-xl and Bax, and of pluripotency marker gene Oct-4 by real-time quanti- tative PCR. Analysis of data showed that the expres- sion of Bcl-xl was higher (1.7-fold) compared to the control while the expression of Bax was significantly decreased relative to the control (0.7-fold) (P < 0.05). Moreover, the expression of Oct-4 was 1.7-fold higher than the control. These results indicated that melatonin had beneficial effects on the development of porcine parthenogenetic embryos. Based on the findings of parthenogenetic embryos, we investigat- ed the effect of melatonin on the development of porcine SCNT embryos. The results also demonstrated increased cleavage and blastocyst formation rates, and the total cell numbers in blastocysts were significantly higher when the embryos were cultured with melatonin. Therefore, these data suggested that melatonin may have important implications for improving porcine preimplantation SCNT embryo development. Mol. Reprod. Dev. 75: 1127–1135, 2008. ß 2008 Wiley-Liss, Inc. Key Words: parthenogenetic development; somatic cell nuclear transfer; melatonin; reactive oxygen species; apoptosis INTRODUCTION Parthenogenesis is a useful tool to investigate the biochemicalmechanismsinthefertilizationprocessand thecomparativerolesofpaternalandmaternalgenomes in early embryo development (Kim et al., 1996; Cheng etal.,2007).Furthermore,parthenogeneticactivationis closely related to cloning research because artificial activation of oocytes is a crucial step in somatic cell nucleartransfer(SCNT)bywhichonly1–2%ofembryos reconstructed are able to develop to term (Colman, 2000). Apoptosis, in response to inappropriate culture con- ditions and stress, is a common physiological process in invitroembryodevelopment(Nanassyetal.,2007).Hao et al. (2003) suggested that apoptosis might contribute to the progressive loss of embryos during the in vitro production procedure. Therefore, in the present study, we mainly focused on sub-optimal culture conditions that lead to programmed cell death via ROS-mediated oxidative stress (Pierce et al., 1991; Yang et al., 1998; Trinei et al., 2002). The level of ROS production by embryos is partic- ularly important during in vitro culture at various stages of development. During in vitro culture, embryos areexposedtorelativelyhighoxidativestresscompared to the environment in vivo, thus the production of ROS within embryos is increased. Accordingly, in vitro ß 2008 WILEY-LISS, INC. Grant sponsor: SooAm Biotech Research Foundation, Republic of Korea. *Correspondence to: Dr. Woo-Suk Hwang, DVM, PhD, SooAm Biotech Research Foundation, Sooambuilding 1027-4, Bangbae3-dong, Seocho-gu, Seoul, 137-851, Korea. E-mail: hwangws@sooam.org Received 3 September 2007; Accepted 29 October 2007 Published online 6 March 2008 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/mrd.20861