The combination of rolipram and cilostamide improved the developmental competence of cloned porcine embryos

Abstract

In vitro maturation of porcine oocytes is characterized by asynchronous cytoplasmic and nuclear maturation, leading to less competent oocytes supporting embryo development. The purpose of this study was to evaluate the combined effect of rolipram and cilostamide as cyclic Adenine monophosphate (cAMP) modulators to find the maximum cAMP levels that temporarily arrest meiosis. We determined the optimal time to maintain functional gap junction communication during pre-in vitro maturation to be four hours. Oocyte competence was evaluated by the level of glutathione, reactive oxygen species, meiotic progression, and gene expression. We evaluated embryonic developmental competence after parthenogenetic activation and somatic cell nuclear transfer. The combined treatment group showed significantly higher glutathione and lower reactive oxygen species levels and a higher maturation rate than the control and single treatment groups. Cleavage and blastocyst formation rates in parthenogenetic activation and somatic cell nuclear transfer embryos were higher in two-phase in vitro maturation than in the other groups. The relative levels of BMP15and GDF9 expression were increased in two-phase in vitro maturation. Somatic cell nuclear transfer blastocysts from two-phase in vitro maturation oocytes showed a lower level of expression of apoptotic genes than the control, indicating better pre-implantation developmental competence. The combination of rolipram and cilostamide resulted in optimal synchrony of cytoplasmic and nuclear maturation in porcine in vitro matured oocytes and there by enhanced the developmental competence of pre-implantation embryos.

Figure 1

Images of the open status of functional GJCin porcine oocytes after pre-IVM rolipram–cilostamide treatment. Open functional GJC, almost all layers of cumulus cells are stained with lucifer yellow dye after intraoocyte microinjection (a,a′); partially open functional GJC, only one or two layers of cumulus cells are stained (b,b′); and closed functional GJC (c,c′), in bright light and UV, respectively.

Figure 2

The optimal time for pre-IVM incubation of porcine oocytes with rolipram–cilostamide treatments. Percentage of oocytes with open, partially open, and closed GJC after 2 (A), 4 (B), 6 (C), and 8 (D) h of pre-IVM incubation. A significant difference (P < 0.05) was observed between the control and rolipram–cilostamide-treated groups for open, partially open, and closed GJC at 2 and 4 h but not 6 or 8 h. The percentage of oocytes with open and partially open GJC was less than 10%. As seen in (A,B), after 4 h of pre-IVM incubation, rolipram–cilostamide treatment significantly affected functional GJC status, and this period was determined to be the optimal time for the two-phase IVM of porcine oocytes using rolipram and cilostamide.

Figure 3

Schematic diagram of the experimental design for two-phase IVM systems using the PDE inhibitors rolipram and cilostamide, with embryonic development after parthenogenesis and SCNT.

Figure 4

Epifluorescence photomicrographic images of IVM porcine oocytes. (A) Oocytes were stained with (a,b) Cell Tracker Blue and (a′,b′) 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) to detect intracellular GSH and ROS levels of control (a,a′) and rolipram–cilostamide-treated oocytes (b,b′). (B) Effects of treatments on intracellular levels of IVM porcine oocytes (GSH and ROS levels in each group), bars with *indicate significant differences (P < 0.05). To measure the GSH and ROS levels, at least 40 oocytes were used for each experiment, which was replicated 8 times.

Figure 5

The relative level of gene expression on quantitative analysis of mRNA transcripts by real-time PCR in control, rolipram, cilostamide, and rolipram–cilostamide-treated porcine oocytes during IVM. At least 80 oocytes were obtained per sample and 8 biological replications were performed. Relative expression of BMP15 (A) and GDF9 (B) to determine the level of cytoplasmic maturation were statistically significantly different among groups. (C) Relative level of CX43 gene expression for level of functional GJC is significantly different between cilostamide and rolipram–cilostamide-treated groups versus the control and rolipram-treated groups (P < 0.05). Values in columns with different superscript letters are significantly different. Control samples were set as arbitrary units, and the target genes were expressed as a fold-change of the corresponding control relative to the house keeping gene (β-actin).

Figure 6

Effect of combined two-phase IVM with rolipram and cilostamide on the embryonic development of transgenic cloned porcine embryos. Representative images of blastocysts developed from oocytes matured in control (A) and rolipram–cilostamide (B) during IVM. At least 68 blastocysts were stained from each group. (C) Inner cell mass to TE ratio in differential staining. The inner cell mass to TE ratio in blastocysts from two-phase IVM differed significantly from that from the control (0.399 vs 0.269, respectively, P < 0.05). The nuclei of ICM and TE cells were stained with Hoechst (blue) and PI (red) dye, respectively. The data are from at least three independent experiments, and the values represent the mean ± SEM.

Figure 7

Relative quantitative analysis of mRNA transcript expression by real-time PCR in control and two-phase IVM porcine oocyte blastocysts. At least 5 blastocysts were obtained per sample and 8 biological replications were performed. Control samples were set as arbitrary units, and the target genes were expressed as the fold-change of the corresponding control relative to the house keeping gene (β-actin). *Indicates a significant difference at P < 0.05.