Effect of different levels of intracellular cAMP on the in vitro maturation of cattle oocytes and their subsequent development following in vitro fertilization

Abstract

Serum, gonadotrophins, growth factors, and steroid hormones stimulate the in vitro maturation (IVM) of competent oocytes, acting, directly or indirectly, upon the adenylate cyclase pathway to produce the intracellular messenger, cAMP. The intracellular levels of cAMP in cattle cumulus-oocyte complexes (COC) were manipulated by adding to the collection and maturation media invasive adenylate cyclase (iAC), a toxin produced by the bacterium, Bordetella pertussis. High concentrations of iAC (1 or 5 microgram/ml) in the maturation medium inhibited the resumption of meiosis, while low concentrations (0.1 or 0.01 microgram/ml) resulted in high rates of maturation to the MII stage (92.6 +/- 2.5 and 98.5 +/- 1.4% respectively). The same low concentrations of iAC in the maturation medium resulted in rates of development to the blastocyst stage 8 days post insemination (30.1 +/- 4.2 and 45.1 +/- 3.9%, respectively), which were either not different, or significantly better, than those obtained after IVM in medium supplemented only with serum and gonadotrophins (36.1 +/- 2.9%). Finally, the addition of 0.1 microgram/ml iAC and 0.5 mM 3-isobutyl 1-methylxanthine (IBMX) in the collection medium significantly improved the blastocyst rate when IVM was performed in control medium or medium supplemented with 0.01 microgram/ml iAC (31.9 +/- 5.5 vs. 12.1 +/- 1.6 and 45.5 +/- 2.9 vs. 19.1 +/- 2.3% respectively). It is concluded that the maintenance of an optimal intracellular concentration of cAMP before and during IVM ensures a high developmental competence of bovine oocytes matured in medium without serum and hormones. Mol. Reprod. Dev. 54:86-91,1999.

Structural and functional changes linked to, and factors promoting, cytoplasmic maturation in mammalian oocytes

“…The treatment with cAMP-elevating agents, such as dibutyryl cAMP, Hx, and FSH, of CEOs during IVM improves developmental competence after in vitro fertilization (IVF) [66][67][68][69][70][71][72]. How this positive effect on cytoplasmic maturation is exerted remains unclear.…”

Developmental competence of gametes reconstructed by germinal vesicle transplantation from fresh and cryopreserved bovine oocytes

“…The stage of nuclear maturation was assessed by fluorescence microscopy following fixation in 60% methanol in phosphate-buffered saline (PBS) and staining with 0.5 mg/ mL propidium iodide (30).…”

“…Cleavage and blastocyst rates were assessed after staining with 0.5 mg/ mL propidium iodide, as previously described (30), and cell nuclei were counted under fluorescence microscopy.…”

Oocyte pre-IVM with caffeine improves bovine embryo survival after vitrification

“…It is possible that intracellular cAMP and cGMP levels were not increased after pre-IVM, instead were maintained in physiological levels during the first hours of IVM as result of a prolonged cGMP and cAMP half-life via inhibition of degradation. It has also been proposed that low levels of intraoocyte cAMP are beneficial for oocyte maturation and embryo development rather than high levels [20,52]. Therefore, it is possible that the caffeine exposure over a relatively short period of time is sufficient to modulate oocyte maturation without reaching nonphysiological cAMP levels.…”

“…This spontaneous maturation has been related to lower oocyte competence because higher blastocyst yields have been reported using in vivo matured oocytes [10]. Different strategies have been developed to avoid or delay the nonphysiological resumption of meiosis, including follicular fluid supplementation and coculture with follicular cells [14][15][16], inhibition of protein synthesis and MPF [17], and supplementation with cAMP increasing agents, including cAMP analogs such as dibutyryl cAMP [18], adenylate cyclase activators such as forskolin and invasive adenylate cyclase [19,20], and specific and nonspecific phosphodiesterase (PDE) inhibitors such as cilostamide, rolipram, milrinone, and caffeine [19,21,22]. These cAMP regulators are used in biphasic IVM systems, in which initially the agents are added to culture media designed for oocyte maturation to modulate meiotic arrest.…”