cAMP Modulators before In Vitro Maturation Decrease DNA Damage and Boost Developmental Potential of Sheep Oocytes

Abstract

To date, the underlying mechanisms by which cAMP modulators act during in vitro maturation to improve oocyte developmental competence are poorly understood. Here, we sought to fill this knowledge gap by evaluating the use of phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) and adenylyl cyclase activator forskolin during a culture period of 2 h before in vitro maturation (pre-IVM) on the nuclear and cytoplasmic maturation features in essential organelles, cumulus cells activity, and in vitro developmental potential of sheep oocytes. Results showed that pre-IVM treatment significantly decreased (p < 0.05) the DNA damage of mature oocytes (pre-IVM = 2.08% ± 3.51% vs. control = 20.58% ± 3.51%) and increased (p ≤ 0.05) expanded blastocyst rates compared to the control (from the total of oocytes: pre-IVM = 23.89% ± 1.47% vs. control = 18.22% ± 1.47%, and from the cleaved embryos: pre-IVM = 45.16% ± 1.73% vs. control = 32.88% ± 1.73%). Considering that oocytes are highly vulnerable to the accumulation of DNA damage because of exposure to in vitro culture conditions, our results suggest that the modulation of intra-oocyte cAMP levels with forskolin and IBMX before IVM might afford oocytes a more effective DNA repair mechanism to overcome damage obstacles and ultimately improve developmental competence. This previously unappreciated action of cAMP modulators could help to develop improved methods for assisted reproduction technologies in animal and clinical research.

Keywords: DNA damage; IBMX; cAMP; embryo; forskolin; in vitro maturation; oocyte; sheep.

Figure 1

Representative images of (a) germinal vesicle (GV) stage oocyte and (b) MII oocyte. Scale bar = 20 µm.

Figure 2

Representative images of (a) a viable sheep oocyte, (b) early apoptotic, and (c) dead. Scale bar = 20 µm.

Figure 3

Representative images of intracellular (a) ROS and (b) GSH levels in mature oocytes. Scale bar = 50 µm.

Figure 4

Representative images of mitochondrial distribution patterns at MII stage in IVM sheep oocytes. (a) Fine, (b) small, and (c) large granulations, and (d) clustered distribution. Scale bar = 20 µm.

Figure 5

Representative images of cortical granules’ distribution patterns at the MII stage in IVM sheep oocytes. (a) Heterogeneous aggregated or clustered distribution, and (b) homogeneous fine distribution. Scale bar = 20 µm.

Figure 6

Representative images of a TUNEL-positive MII oocyte stained with (a) Hoechst 33342 and (b) fluorescein. Scale bar = 20 µm.

Figure 7

Effect of forskolin and IBMX during pre-IVM on intra-oocyte cAMP levels (pmol/mL). Results are expressed as mean ± SEM. ^a,b Different letters indicate differences between treatments.

Figure 8

The effect of pre-IVM on the DNA fragmentation of matured sheep oocytes. Results are expressed as mean ± SEM. ^a,b Different letters indicate differences between treatments.

Figure 9

The effect of pre-IVM on the number of viable, early apoptotic, and dead sheep oocytes after IVM. Results are expressed as mean ± SEM.

Figure 10

The effect of pre-IVM on the intracellular levels of ROS and GSH in matured sheep oocytes. Results are expressed as mean ± SEM.

Figure 11

Relative mRNA transcript abundance pattern of genes of interest in sheep IVM oocytes subjected to pre-IVM. Results are expressed as mean ± SEM.

Figure 12

The effect of pre-IVM on the mitochondrial distribution patterns of matured sheep oocytes. Results are expressed as mean ± SEM.

Figure 13

The effect of pre-IVM on the cortical granule distribution patterns of matured sheep oocytes. Results are expressed as mean ± SEM.