Journal of Embryo Transfer 2015; 30(1): 23-31
Published online March 29, 2015
Copyright © The Korean Society of Animal Reproduction and Biotechnology.
Yong-Hee Lee, Jin-Woo Kim, Sung-Kyu Chae, Jae-Hyun Ahn, Geon-Yeop Do, and Deog-Bon Koo†
Department of Biotechnology, College of Engineering, Daegu University, Gyeongsan 712-714, Republic of Korea
Correspondence to: Correspondence :
Catalpol, an iridoid glucoside, isolated from the root of
Keywords: catalpol, ROS, antioxidant, apoptosis, pig
Catalpol, a major chemical constituent of
Apoptotic cell death in preimplantation mammalian embryos has been well described. Apoptosis in response to improper culture conditions and stress is a common physiological process that occurs in embryo development
The aim of the present study, we demonstrated the effects of catalpol on developmental competence of preimplantation porcine embryos cultured under oxidative stress conditions. Furthermore, the expression levels of ROS and the apoptotic index in blastocyst stage embryos derived from catalpol treatment were measured under oxidative stress conditions.
All chemicals used in this study were purchased from Sigma Chemical Co. (St Louis, MO, USA) unless otherwise indicated.
Porcine ovaries were collected from at a local abattoir and transported to the laboratory at 30~35°C in 0.9% saline supplement with 75 mg/ml potassium penicillin G. Immature cumulus- oocyte complexes (COCs) were aspirated from follicles between 3 and 6 mm through an 18-gauge needle into a disposable 10-ml syringe (
In this study used medium, designated as modified IVF of porcine oocytes was performed as described by
For all experiments, the groups of 25~30 embryos were cultured in 50-μl drops of PZM-3 medium with 3 mg/ml BSA at 38.5°C and under 5% CO2 in air. After 2 days of culture, cleaved embryos were further cultured in a 50-μl drop of PZM-3 medium supplemented with 3 mg/ml BSA at 38.5 °C and under 5% CO2 in air for 4 days. To modulate oxidative stress, fertilized embryos were treated with oxidative inducer H2O2 (200 μM; Lee
The level of H2O2 in each embryo was measured using the difluorodihydrofluorescein diacetate method (H2DCFDA; Invitrogen, Molecular Probes, Willow, USA) described previously (
The number of apoptotic nuclei in the blastocysts was detected using an
All experiments were repeated at least three times. All percentage data were subjected to arcsine transformation. All percentage data and datasets obtained in the present study are presented as the mean ± standard deviation (S.D.). Rates of preimplantation embryos development, ROS levels, cell numbers and apoptotic proportions of blastocysts were analyzed by ANOVA and Student’s
There are no previous reports about the concentration and effects of catalpol to treatment in porcine culture experiments. In this study, we investigated the optimal catalpol concentration of culture medium in preimplantation development of porcine embryos. After completion of IVF, porcine embryos were cultured in IVC medium supplemented with 100, 200 and 400 μM catalpol for 6 days at 38.5°C under 5% CO2 in air. As shown in Table 1, developmental competence of blastocysts formation significantly not increased between control groups and groups treated group with 100 μM catalpol. However, developmental competence of blastocysts formation in the presence of 200 and 400 μM catalpol was significantly decreased when compared with the untreated control group (
We next investigated the intracellular levels of ROS and the apoptotic index in catalpol treated blastocysts. Intracellular levels of ROS were significantly reduced in 100 μM catalpol treated group compared with other groups (
Comparison of antioxidant and apoptotic characteristics in porcine blastocysts derived from catalpol treatment groups. Fluorescence microscopy imaging of intracellular ROS expression (A) and level of hydrogen peroxide (B) in blastocysts. Epifluorescent images of porcine blastocysts derived from various concentration of catalpol undergoing apoptosis
Presumptive zygotes were cultured in the presence or absence of catalpol and/or H2O2 for 6 days at 38.5°C and under 5% CO2 in air. As shown in Table 2, the rates of cleavage and blastocyst formation under the in duced H2O2 oxidative stress were significantly improved in the presence of catalpol group when compared with absence of catalpol group (
As shown in Fig. 2A and 2B, the intracellular levels of ROS in blastocysts under the induced H2O2 oxidative stress that developed from embryos cultured in the presence of catalpol was lower than that of blastocysts from embryos cultured in the absence of catalpol. Moreover, the number of apoptotic nuclei was significantly lower in blastocyst stage embryos derived from the catalpol treated group when compared with the group not treated with catalpol (Fig. 2C, 2E;
Comparison of antioxidant and anti-apoptotic characteristics in porcine blastocysts derived from H2O2 and/or catalpol treatment groups. Fluorescence microscopy imaging of intracellular ROS expression (A) and level of hydrogen peroxide (B) in blastocysts. Epifluorescent images of porcine blastocysts stage embryos cultured with H2O2 and/or catalpol under going apoptos is
In the present study, we demonstrated that the catalpol significantly increased the embryo development and embryo quality such as increasing blastocyst cell number, reduction of ROS and apoptosis in the pig embryos under oxidative stress culture condition
Oxidative stress can damage oocytes and cause mitochondrial alterations, embryo block, adenosine triphosphatase depletion and apoptosis (
Catalpol dependently attenuated H2O2-induced ROS production (
Previous studies have shown that apoptosis plays an important role in embryonic development (
In this study, these results suggest that decreased developmental competence caused by H2O2 induced oxidative stress, resulting in an increased number of apoptotic nuclei and decreased cell number in blastocysts. In contrast, the addition of catalpol under H2O2 induced oxidative stress improved developmental competence to the blastocyst stage, reduced the number of apoptotic nuclei an decreased the cell number in porcine IVP embryos by preventing oxidative stress. Therefore, we suggest that catalpol may be improved the development and quality of porcine embryos by preventing oxidative stress.