Combined use of dbcAMP and IBMX minimizes the damage induced by a long‐term artificial meiotic arrest in mouse germinal vesicle oocytes

Abstract

Phosphodiesterase (PDE)‐mediated reduction of cyclic adenosine monophosphate (cAMP) activity can initiate germinal vesicle (GV) breakdown in mammalian oocytes. It is crucial to maintain oocytes at the GV stage for a long period to analyze meiotic resumption in vitro. Meiotic resumption can be reversibly inhibited in isolated oocytes by cAMP modulator forskolin, cAMP analog dibutyryl cAMP (dbcAMP), or PDE inhibitors, milrinone (Mil), Cilostazol (CLZ), and 3‐isobutyl‐1‐methylxanthine (IBMX). However, these chemicals negatively affect oocyte development and maturation when used independently. Here, we used ICR mice to develop a model that could maintain GV‐stage arrest with minimal toxic effects on subsequent oocyte and embryonic development. We identified optimal concentrations of forskolin, dbcAMP, Mil, CLZ, IBMX, and their combinations for inhibiting oocyte meiotic resumption. Adverse effects were assessed according to subsequent development potential, including meiotic resumption after washout, first polar body extrusion, early apoptosis, double‐strand DNA breaks, mitochondrial distribution, adenosine triphosphate levels, and embryonic development. Incubation with a combination of 50.0 μM dbcAMP and 10.0 μM IBMX efficiently inhibited meiotic resumption in GV‐stage oocytes, with low toxicity on subsequent oocyte maturation and embryonic development. This work proposes a novel method with reduced toxicity to effectively arrest and maintain mouse oocytes at the GV stage.

The role of forskolin as a lipolytic stimulator during in vitro oocyte maturation and the in vitro embryo production of livestock

“…Furthermore, it has been reported that forskolin supplemented to the pre‐maturation medium in bovine and pigs can increase oocyte cAMP levels (Park, Lee, et al., 2016), decrease intra‐oocyte H 2 O 2 and increase intra‐oocyte glutathione and maintain meiotic arrest (Leal et al., 2019). A high level of forskolin in the culture medium produced a superior apoptosis rate, leading to lower quality of oocytes (Wu et al., 2020). These data concur with a prior work screening that a high dose of forskolin triggered cellular apoptosis and DNA damage (Paschoal et al., 2016).…”

“…These data concur with a prior work screening that a high dose of forskolin triggered cellular apoptosis and DNA damage (Paschoal et al., 2016). An increase of cAMP synthesis in the cytoplasm may lead to a consistent with boosting the mitochondrial function in oocyte (Wu et al., 2020). Previously, it was evidenced that the appropriate level of forskolin could maintain the stability of DNA integrity in the cell and reduced the DNA damage in oocytes (Paschoal et al., 2016).…”