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 (24 h IVM/control), cAMP30 (2 h pre-IVM (forskolin-IBMX), 30 h 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.

Fig 1. Progression through meiosis of oocytes derived from adult and prepubertal donors collected via OPU and treated prior to and during IVM with or without cAMP modulators.

Oocytes were fixed after: A) 9h, B) 20h, and C) 24/30h of in vitro maturation. Bars represent the percentages calculated using the total number of oocytes per treatment per fixation time from four replicates. Columns with different superscripts differ significantly among treatments within the group with the respective meiotic status for oocytes obtained from adult (a, b, c) or prepubertal donors (x, y, z). Data were compared using absolute numbers by Fisher´s Exact Test complemented by Bonferroni correction. The percentages were calculated using the total number of samples per treatment and statistical analyses were performed with the absolute values. Therefore, no averages or SEMs are presented. The cAMP modulators delayed progression of meiosis in adult and prepubertal oocytes; DMSO used as solvent for cAMP modulators (vehicle control) accelerated meiotic resumption in oocytes from both types of donors (P < 0.016). GV, germinal vesicle stage; GVBD, germinal vesicle breakdown; MI, metaphase I; MII, metaphase II.

Fig 2. Cyclic AMP levels in prepubertal and adult oocytes treated with or without cAMP modulators prior to and during IVM.

Data are presented as mean ± SEM (n = 3). Optical density reads from ELISA test were analyzed using two-way ANOVA. Bars labeled with different superscripts represent statistical significance among treatments (a, b); P < 0.05. Oocytes were retrieved via OPU. The cAMP modulators increased intra-oocyte cAMP levels in both prepubertal and adult oocytes after pre-IVM (P < 0.05). No differences in cAMP profiles were found among treatments after IVM (P > 0.05).

Fig 3. Gene expression profiles in adult and prepubertal oocytes treated with or without cAMP modulators prior to and during IVM.

A) single immature oocytes and B) single MII oocytes. Data are presented as mean ± SEM (n = 12). Data were analyzed using two-way ANOVA followed by a Tukey's range test. Columns with different superscripts differ significantly among treatments within the respective meiotic status for oocytes obtained from adult (a, b) and prepubertal donors (x, y). The asterisk represents a statistically significant difference between immature and matured oocyte treatments for the same transcript; P < 0.05. Oocytes were obtained via OPU. In vivo matured oocytes were used for comparison. PRKACA was upregulated in adult oocytes treated with cAMP modulators and down-regulated in prepubertal oocytes under the DMSO30 treatment. EGR1 was upregulated in prepubertal immature oocytes. Matured oocytes from all treatments displayed lower transcript levels for ZAR1, PRDX1 and SLC2A8 after IVM compared to immature oocytes.

Fig 4. Gene expression profiles in expanded blastocysts produced from adult and prepubertal oocytes treated with or without cAMP modulators prior to and during IVM.

Data are shown as the mean ± SEM (n = 12). Data were analyzed using two-way ANOVA followed by a Tukey's range test. The asterisk represents statistical significance among treatments for the same transcript; P < 0.05. In vivo produced expanded blastocysts were used for comparison. The mRNA relative abundance of the EGR1 gene was lower in all in vitro derived blastocysts compared to in vivo produced counterparts. No differences among treatments were found for DNMT3b, BCL2L1, PRDX1, SLC2A8.

Fig 5. DNA satellite methylation profiles in immature oocytes, MII oocytes and expanded blastocysts derived from adult and prepubertal donors.

Oocytes were obtained via OPU and treated prior to and during IVM with cAMP modulators. A) Immature oocytes, B) Matured oocytes and C) Expanded blastocysts. The percentages were calculated using the total number of samples per treatment and statistical analyses were performed with the absolute values. Therefore, no averages or SEMs are presented. Bars represent the percentages of DNA methylation calculated using the total number of CpG analyzed in each treatment. Columns with different superscripts differ significantly for the respective satellite and developmental status (a, b, c). Data were analyzed by Chi-squared test followed by pairwise comparison of proportions method; immature and MII oocytes, n = 3; blastocysts, n = 5; P < 0.05. DNA methylation profiles were similar in immature oocytes from all treatments. Hypermethylation was observed for adult matured oocytes treated with cAMP modulators compared with in vivo matured oocytes. Lower DNA methylation levels were found for prepubertal oocytes after DMSO treatment compared to adult in vitro matured oocytes. Similar DNA methylation levels of both satellite sequences were observed for blastocysts produced from oocytes treated with cAMP modulators and in vivo produced blastocysts from both types of donors. DNA methylation levels were aberrant for blastocysts from TCM24 and DMSO30 treatments compared to in vivo counterparts. BTS, Bovine testis satellite I; BTαS, Bos taurus alpha satellite I.