Cyclic AMP Affects Oocyte Maturation and Embryo Development in Prepubertal and Adult Cattle

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.

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

“…However, reports on bovine oocytes using other cAMP modulators are conflicting, probably due to the differences in protocols and compounds [43,57,58]. In the present study, we did not observe improvement in these parameters but developmental rates using the 10-mM caffeine treatment were similar to the standard control, which prompted us to use this concentration for the vitrification studies.…”

Delay in maternal transcript degradation in ovine embryos derived from low competence oocytes

“…The accumulation of transcripts during oocyte growth occurs during follicle recruitment, it is thus likely affected by the different hormonal status encountered during initial or cyclic recruitment. In accordance, abundant literature sustains the cytoplasmic immaturity of prepubertal oocytes in terms of abundance of specific transcripts (Bebbere et al, ; Bernal‐Ulloa et al, ; Leoni et al, ; Masala et al, ; Oropeza, Wrenzycki, Herrmann, Hadeler, & Niemann, ).…”

Consequences of assisted reproductive techniques on the embryonic epigenome in cattle

“…Studies in ruminants are conflicting as to whether DNA methylation is different between pre-and postpubertal animals. Using limiting dilution bisulfite sequencing, Diederich et al (2012) found that DNA methylation levels at repeat sequences were not different between groups, a finding also reported by Bernal-Ulloa et al (2016) but in contrast with findings in the sheep, in which oocytes from young females had reduced levels of immuoreactive global DNA methylation (Ptak et al 2006). More extensive work using next-generation sequencing would perhaps need to be done in order to identify whether there are specific loci that are not properly methylated in oocytes of prepubertal compared with adult females.…”