Porcine Preimplantation Embryo Development Research Articles

46 ANTI-APOPTOTIC EFFECT OF INSULIN-LIKE GROWTH FACTOR-1 AND ITS RECEPTOR ON DEVELOPMENT OF PORCINE PRE-IMPLANTATION EMBRYOS PRODUCED BY IN VITRO FERTILIZATION AND SOMATIC CELL NUCLEAR TRANSFER

Insulin-like growth factor (IGF)-I is a receptor-mediated autocrine and/or paracrine growth and/or survival factor for mammalian embryo development. It is known to promote the growth and development of mouse pre-implantation embryos. The present study was designed to investigate the effects of IGF-I (50 ng/mL), anti-IGF-I receptor (IGR-IR) antibody (0.05 �g/mL), and their combination on porcine pre-implantation embryo development. Furthermore, the mechanism underlying the embryotropic effects of IGF-I was evaluated by monitoring the incidence of apoptosis and expression of apoptosis-related genes. In both IVF and SCNT embryos, culturing with IGF-I increased the rate of blastocyst formation and this embryotropic effect was neutralized by culturing with IGF-I and anti-IGF-I receptor antibody. Significant effects on the development of blastocysts (P < 0.05) were found in IVF (16.9, 22.6, 9.3, and 13.5% for control, IGF-I, anti-IGF-IR antibody, and their combination, respectively) and SCNT (13.2, 21.0, 5.4, and 15.7%) embryos. Culturing IVF and SCNT embryos with IGF-I significantly increased the total number of cells in IVF blastocysts (58.3, 72.4, 41.1, and 55.2; P < 0.05), and SCNT blastocysts (49.2, 60.1, 35.2, and 43.1; P < 0.05), and it decreased the number of apoptotic nuclei in IVF blastocysts (3.9, 2.8, 5.5, and 3.9; P < 0.05) and SCNT blastocysts (4.6, 3.0, 6.1, and 4.9; P < 0.05). These effects of IGF-I were also neutralized by culturing with IGF-I along with anti-IGF-IR antibody. Expression of the anti-apoptotic Bcl-2 gene was increased, whereas expression of the pro-apoptotic Bax gene was decreased in both IVF and SCNT embryos cultured with IGF-I. In both IVF and SCNT embryos, anti-IGF-IR antibody along with IGF-I neutralized the effect of IGF-I on expression of Bcl-2 and Bax genes. In conclusion, the present study demonstrated that IGF-I through its specific receptors improved the developmental competence of IVF and SCNT embryos by decreasing the incidence of apoptosis and regulating apoptosis-related genes in porcine pre-implantation embryos. This study was supported by grants from the Korean MOST (Top Scientist Fellowship) and MAF (Biogreen 21 #20050301-034-443-026-01-00).

Embryotropic effect of glycosaminoglycans and receptors in development of porcine pre-implantation embryos

The present study investigated the expression of receptors for glycosaminoglycans (GAGs) and the effect of GAGs supplementation on development of porcine IVF embryos. Total RNA was prepared from oocytes, 2-, 4- and 8-cell embryos, morulae and blastocysts. The expression of hyaluronic acid receptor (CD44) and heparin (HP) interacting protein (HIP) was determined using RT-PCR and Southern blot analysis. The CD44 and HIP mRNA were detected from in vitro matured oocytes and all stages of pre-implantation embryos. The IVF embryos were cultured in modified NCSU-23 medium supplemented with various concentrations (0, 0.1, 0.5 or 1.0 mg/mL) of hyaluronic acid (HA) or heparin. Supplementing with 0.5 mg/mL HA significantly increased total cell number compared to other experimental groups, due to increase in trophectoderm cells. Supplementing with 1.0 mg/mL, HP significantly increased blastocyst formation rate compared to the control group. Supplementing media, in which IVF embryos were cultured with 0.5 mg/mL HA + 1.0 mg/mL HP, significantly increased blastocyst formation rate compared to the control group. In conclusion, the present study demonstrated the expression of HA and HP receptors and the embryotrophic effect of HA or HP on porcine IVF embryos.

The role of gonadotropin-releasing hormone (GnRH) and its receptor in development of porcine preimplantation embryos derived from in vitro fertilization

This study was performed to investigate the expression of embryo-derived gonadotropin-releasing hormone (GnRH) and its receptor, and to determine the role of GnRH in porcine preimplantation embryos. In Experiment 1, porcine blastocysts derived from in vitro fertilization (IVF) and cultured in North Carolina State University (NCSU)-23 medium were subjected to reverse transcription polymerase chain reaction (RT-PCR) amplification with specific primers for GnRH and its receptor. The results showed that GnRH and its receptor were expressed in porcine IVF blastocysts. In order to investigate the role of GnRH in embryo development, porcine IVF embryos were cultured in NCSU-23 supplemented with different concentrations (0, 0.1, 1, or 10 μM) of a GnRH agonist (leuprolide, Experiment 2) or GnRH antagonist (antide, Experiment 3). Supplementing the culture medium with 0.1 or 1 μM leuprolide increased the rate of blastocyst formation (28.5 or 27.6% versus 20.2%) and mean total cell number (129 versus 104) compared to the control group. In contrast, antide significantly decreased the rate of blastocyst formation [12.6% (0.1 μM), 10.2% (1.0 μM), or 8.9% (10.0 μM) versus 22.8% (control)] and total cell number [69 (1 μM) or 68 (10 μM) versus 104 (control)]. In Experiment 4, porcine IVF embryos were cultured in NCSU-23 medium containing 1 μM antide plus 1 μM leuprolide. The embryotrophic effect of GnRH agonist was reversed by co-supplementing with GnRH antagonist. In conclusion, the present study demonstrated that supplementing a culture medium with GnRH agonist can improve blastocyst formation and the quality of porcine IVF embryos, and that this action was mediated through GnRH receptors.

Serial analysis of gene expression (SAGE) during porcine embryo development.

Functional genomics provides a powerful means for delving into the molecular mechanisms involved in pre-implantation development of porcine embryos. High rates of embryonic mortality (30%), following either natural mating or artificial insemination, emphasise the need to improve the efficiency of reproduction in the pig. The poor success rate of live offspring from in vitro-manipulated pig embryos also hampers efforts to generate transgenic animals for biotechnology applications. Previous analysis of differential gene expression has demonstrated stage-specific gene expression for in vivo-derived embryos and altered gene expression for in vitro-derived embryos. However, the methods used to date examine relatively few genes simultaneously and, thus, provide an incomplete glimpse of the physiological role of these genes during embryogenesis. The present review will focus on two aspects of applying functional genomics research strategies for analysing the expression of genes during elongation of pig embryos between gestational day (D) 11 and D12. First, we compare and contrast current methodologies that are being used for gene discovery and expression analysis during pig embryo development. Second, we establish a paradigm for applying serial analysis of gene expression as a functional genomics tool to obtain preliminary information essential for discovering the physiological mechanisms by which distinct embryonic phenotypes are derived.

Effect of Replacement of Pyruvate/Lactate in Culture Medium with Glucose on Preimplantation Development of Porcine Embryos In Vitro

The effect of glucose supplementation at different times in in vitro culture on the developmental competence of in vitro produced (IVP) porcine embryos was examined. In Experiment 1, when IVP embryos were cultured in modified NCSU-37 supplemented with pyruvate and lactate (IVC-pyr/lac) for 0 h, 24 h, 48 h, 72 h, 96 h, or 118 h and subsequently in modified NCSU-37 supplemented with glucose (IVC-glu) until Day 6 (Day 0=day of in vitro fertilization), the rates of blastocyst formation were significantly higher in embryos cultured in IVC-pyr/lac for 24 or 48 h (24.4% and 23.0%, respectively) than in embryos cultured in IVC-pyr/lac for the whole culture period (14.5%). However, there were no significant differences between embryos obtained after the energy source replacement and embryos cultured in IVC-glu for the whole culture period on the rates (15.2%-24.4%, and 16.8% respectively). Replacement of pyruvate/lactate with glucose at 58 h of culture in Experiment 2 significantly enhanced the rate (31.3%) compared to those after replacement at 48 h, 53 h and 63 h of culture (20.6%, 20.8%, and 21.1%, respectively). In conclusion, replacement of pyruvate/lactate with glucose as the energy substrate was optimal at 58 h of culture for the development of porcine embryos to the blastocyst stage.

Serial analysis of gene expression (SAGE) during porcine embryo development

Functional genomics provides a powerful means for delving into the molecular mechanisms involved in pre-implantation development of porcine embryos. High rates of embryonic mortality (30%), following either natural mating or artificial insemination, emphasise the need to improve the efficiency of reproduction in the pig. The poor success rate of live offspring from in vitro-manipulated pig embryos also hampers efforts to generate transgenic animals for biotechnology applications. Previous analysis of differential gene expression has demonstrated stage-specific gene expression for in vivo-derived embryos and altered gene expression for in vitro-derived embryos. However, the methods used to date examine relatively few genes simultaneously and, thus, provide an incomplete glimpse of the physiological role of these genes during embryogenesis. The present review will focus on two aspects of applying functional genomics research strategies for analysing the expression of genes during elongation of pig embryos between gestational day (D) 11 and D12. First, we compare and contrast current methodologies that are being used for gene discovery and expression analysis during pig embryo development. Second, we establish a paradigm for applying serial analysis of gene expression as a functional genomics tool to obtain preliminary information essential for discovering the physiological mechanisms by which distinct embryonic phenotypes are derived.

48EFFECT OF INSULIN-LIKE GROWTH FACTOR-1 SUPPLEMENT TO NCSU-23 MEDIUM ON PREIMPLANTATION DEVELOPMENT OF PORCINE EMBRYOS DERIVED FROM IN VITRO FERTILIZATION AND SOMATIC CELL NUCLEAR TRANSFER

The developmental potential of in vitro production of embryos is affected by various factors, including the culture system, oocyte quality, the presence of serum, and embryo paracrine and autocrine growth factors. Insulin-like growth factor is a good stimulator of oocyte maturation and embryo development. The present study investigated the effect of insulin-like growth factor-I (IGF-I) supplement on the preimplantation development of porcine embryos derived from in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT). Developmental competence was evaluated by monitoring the numbers of 2-cell embryos and blastocysts at Days 2 and 7, respectively. The number of total cells and inner cell mass (ICM) cells in blastocysts were counted after differential staining at Day 7. All data were analyzed by ANOVA using a Generalized Linear Model (SAS). In Experiment 1, a total of 2,462 in vitro-matured oocytes (527, 458, 498, 481 and 498, respectively) were inseminated with frozen-thawed boar semen and subsequently cultured in North Carolina State University (NCSU)-23 medium supplemented with various concentrations of IGF-1 (0, 1, 10, 50 and 100ngmL−1). As a result, significant model effects on the development to the 2-cell stage (P=0.033) and to the blastocyst stage (P=0.0067) were found, and more blastocysts (16.9, 16.6, 17.5, 21.8 and 14.7 %, respectively) were obtained in medium supplemented with 50ngmL−1 of IGF-I. Moreover, increase in the total cell number (56.5, 53.2, 74.0, 76.4 and 58.4) and ICM (6.6, 5.8, 9.3, 9.4 and 6.1) cells was observed in IVF embryos cultured in NCSU-23 medium supplemented with 50ngmL−1 IGF-1. In Experiment 2, porcine cloned embryos were produced by our standard protocol using fetal fibroblasts as donor cells (Hyun SH et al., 2003 Theriogenology 59, 1641–1649) and cultured in NCSU-23 supplemented with the same concentration of IGF-1 as Experiment 1. As a result, a total of 501 reconstructed oocytes (99, 98, 102, 99 and 96, respectively) were cultured and significant model effects on the development to the 2-cell stage (P=0.0179) were found. More blastocysts (10.5, 11.2, 11.8, 20.8 and 10.1%) were produced when embryos were cultured in NCSU-23 medium supplemented with 50ngmL−1, even though no statistical significance was found (P=0.1182). Increases in the total cell number (42.7, 46.0, 45.9, 51.1 and 38.2) and ICM cells (3.8, 3.8, 5.6, 6.6 and 4.8, respectively) were observed in cloned embryos cultured in NCSU-23 medium supplemented with 50ngmL−1 of IGF-I. In conclusion, the present study demonstrated that IGF-1 at the concentration of 50ngmL−1 improves the development of preimplantion embryos derived from IVF and SCNT. This study was supported by the Advanced Backbone IT Technology Development (IMT 2000-C1-1).

Apoptosis and in vitro development of preimplantation porcine embryos derived in vitro or by nuclear transfer.

Apoptosis occurs during preimplantation development in both in vivo- and in vitro-produced embryos, and it may contribute to embryonic loss. The present study investigated the development of porcine nuclear transfer (NT) embryos reconstructed by using fetal fibroblasts as compared to embryos produced by in vitro fertilization (IVF). The onset and the frequency of apoptosis in NT and IVF embryos were examined via morphological and nuclear changes and TUNEL assay. The NT blastocysts had a similar number of nuclei as compared to IVF blastocysts and appeared to be morphologically similar. Relative to IVF embryos, the NT embryos had a lower cleavage rate (42.7% vs. 71.0%) and a lower developmental rate (11.1% vs. 28.6%) to the blastocyst stage. The earliest positive TUNEL signals were detected in the NT embryos on Day 5 of culture. The percentage of cells undergoing apoptosis in the NT embryos was higher than that of the IVF embryos and increased with time in vitro. Some of the abnormal morphological changes observed during early development related to apoptosis. Cytoplasmic fragmentation, developmental arrest, and nuclear condensation were typical characteristics of embryos undergoing apoptosis. Some mechanisms of the apoptotic pathway were triggered by changes in the NT embryos. The developmental rates of NT embryos might be improved by identifying specific apoptotic pathways and then intervening in these pathways to improve development.

Development and viability of in vitro derived porcine blastocysts cultured in NCSU23 and G1.2/G2.2 sequential medium

Porcine embryo development in vitro is relatively inefficient compared to other domestic species. Currently, a single culture medium (NCSU23) is the standard for porcine in vitro systems. However, the G1.2/G2.2 sequential culture system has been beneficial for embryo development in other species. The objective of this study was to compare porcine preimplantation embryo development in vitro and subsequent blastocyst viability and metabolic activity using NCSU23 and G1.2/G2.2 culture media. Oocytes were matured in defined TCM199 base medium for 45 to 47 h and fertilized in mTBM for 4 h. Embryos were cultured in either NCSU23 for 146 h or G1.2 medium for 72 h followed by culture in G2.2 medium for an additional 74 h. Blastocyst substrate use was measured using a modification of the hanging drop technique. Culture in NCSU23 resulted in a higher percentage (P < 0.05) of embryo cleavage (74.0%) and blastocyst development (14.6%) than culture in G1.2/G2.2 (67.8% and 7.8%, respectively). Both NCSU23 and G1.2/G2.2 produced blastocysts with similar mean cell numbers (51.5 ± 4.3 and 47.1 ± 4.3, respectively), similar glucose use (10.81 ± 1.39 and 10.12 ± 1.72 pmol/embryo/3h, respectively) and pyruvate use (1.08 ± 0.056 and 0.88 ± 0.048 pmol/embryo/3h, respectively). These data indicate that a sequential culture system can support porcine embryo development in vitro without compromising embryo viability. However, the G1.2/G2.2 system was not as effective as NCSU23 in supporting blastocyst development. Sequential media should be formulated specifically for porcine embryos to improve embryonic cleavage and blastocyst development.