Abstract
High cAMP levels during in vitro maturation (IVM) have been related to improved blastocyst yields. Here, we employed the cAMP/cGMP modulators, forskolin, IBMX, and cilostamide, during IVM to unravel the role of high cAMP in early embryonic development produced from prepubertal and adult bovine oocytes. Oocytes were collected via transvaginal aspiration and randomly assigned to three experimental groups: TCM24 (24h IVM/control), cAMP30 (2h pre-IVM (forskolin-IBMX), 30h IVM-cilostamide), and DMSO30 (Dimethyl Sulfoxide/vehicle control). After IVM, oocytes were fertilized in vitro and zygotes were cultured in vitro to blastocysts. Meiotic progression, cAMP levels, mRNA abundance of selected genes and DNA methylation were evaluated in oocytes. Blastocysts were used for gene expression or DNA methylation analyses. Blastocysts from the cAMP30 groups were transferred to recipients. The cAMP elevation delayed meiotic progression, but developmental rates were not increased. In immature oocytes, mRNA abundance of PRKACA was higher for cAMP30 protocol and no differences were found for PDE3A, SMAD2, ZAR1, PRDX1 and SLC2A8. EGR1 gene was up-regulated in prepubertal cAMP30 immature oocytes and down-regulated in blastocysts from all in vitro treatments. A similar gene expression profile was observed for DNMT3b, BCL2L1, PRDX1 and SLC2A8 in blastocysts. Satellite DNA methylation profiles were different between prepubertal and adult oocytes and blastocysts derived from the TCM24 and DMSO30 groups. Blastocysts obtained from prepubertal and adult oocytes in the cAMP30 treatment displayed normal methylation profiles and produced offspring. These data indicate that cAMP regulates IVM in prepubertal and adult oocytes in a similar manner, with impact on the establishment of epigenetic marks and acquisition of full developmental competency.
Highlights
Cyclic adenosine monophosphate is a second messenger involved in many cellular functions
At 9 h, Cyclic adenosine monophosphate (cAMP) regulators maintained the majority of oocytes from prepubertal and adult donors (GV: 68.1% and 55.5%, respectively) in meiotic arrest compared to standard in vitro maturation (IVM)
The present study investigated for the first time the influence of artificially induced high cAMP levels on acquisition and maintenance of developmental capacity of oocytes collected from bovine prepubertal and adult donors using the simulated physiological oocyte maturation (SPOM) system
Summary
Cyclic adenosine monophosphate (cAMP) is a second messenger involved in many cellular functions. In mammalian oocytes it maintains meiotic arrest by inactivation of maturation promoting factor (MPF) and by stimulating cAMP-dependent protein kinase A (PKA). During in vivo maturation in the mouse model, LH induces a decrease in cyclic guanosine monophosphate (cGMP) in the mural granulosa cells and the oocyte, which in turn relieves the inhibition of phosphodiesterase 3A, by hydrolyzing cAMP to AMP, inducing activation of MPF and germinal vesicle breakdown (GVBD) [1]. Since the oocyte receives cAMP from the adjacent cumulus cells via gap junctions, the lower cAMP levels associated with lower developmental capacity in prepubertal oocytes have been linked either to decreased LH/FSH ovarian receptor expression [5], altered adenylate cyclase response [4], different phosphodiesterase (PDE) activities [4], and/or defective gap junction communication [6]
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