Patrick Aldag

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

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.

Extended in vitro maturation affects gene expression and DNA methylation in bovine oocytes

To mimic post-ovulatory ageing, we have extended the in vitro maturation (IVM) phase to 48 h and examined effects on (i) developmental potential, (ii) expression of a panel of developmentally important genes and (iii) gene-specific epigenetic marks. Results were compared with the 24 h IVM protocol (control) usually employed for bovine oocytes. Cleavage rates and blastocyst yields were significantly reduced in oocytes after extended IVM. No significant differences were observed in the methylation of entire alleles in oocytes for the genes bH19, bSNRPN, bZAR1, bOct4 and bDNMT3A. However, we found differentially methylated CpG sites in the bDNMT3Ls locus in oocytes after extended IVM and in embryos derived from them compared with controls. Moreover, embryos derived from the 48 h matured oocyte group were significantly less methylated at CpG5 and CpG7 compared with the 24 h group. CpG7 was significantly hypermethylated in embryos produced from the control oocytes, but not in oocytes matured for 48 h. Furthermore, methylation for CpG5-CpG8 of bDNMT3Ls was significantly lower in oocytes of the 24 h group compared with embryos derived therefrom, whereas no such difference was found for oocytes and embryos of the in vitro aged group. Expression of most of the selected genes was not affected by duration of IVM. However, transcript abundance for the imprinted gene bIGF2R was significantly reduced in oocytes analyzed after extended IVM compared with control oocytes. Transcript levels for bPRDX1, bDNMT3A and bBCLXL were significantly reduced in 4- to 8-cell embryos derived from in vitro aged oocytes. These results indicate that extended IVM leads to ageing-like alterations and demonstrate that epigenetic mechanisms are critically involved in ageing of bovine oocytes, which warrants further studies into epigenetic mechanisms involved in ageing of female germ cells, including humans.

Effects of long-term dietary supplementation with conjugated linoleic acid on bovine oocyte lipid profile

Nutritional and environmental conditions around conception and during early embryonic development may have significant effects on health and well-being in adult life. Here, a bovine heifer model was used to investigate the effects of rumen-protected fat supplementation on oocyte quality and embryo development. Holstein-Friesian heifers (n=84) received a dietary supplement consisting of rumen-protected conjugated linoleic acid (CLA) or stearic acid (SA), each on top of an isocaloric basic diet. Oocytes were collected via ultrasound-guided follicular aspiration and subjected to in vitro maturation followed by either desorption electrospray ionisation mass spectrometry (DESI-MS) for lipid profiling of individual oocytes or in vitro fertilisation and embryo culture. The type of supplement significantly affected lipid profiles of in vitro-matured oocytes. Palmitic acid and plasmalogen species were more abundant in the mass spectra of in vitro-matured oocytes after rumen-protected SA supplementation when compared with those collected from animals supplemented with CLA. Lipid concentrations in blood and follicular fluid were significantly affected by both supplements. Results show that rumen-protected fatty-acid supplementation affects oocyte lipid content and may pave the way for the establishment of a large-animal model for studies towards a better understanding of reproductive disorders associated with nutritional impairments.

148 EFFECTS OF CAFFEINE SUPPLEMENTATION ON BOVINE OOCYTE DEVELOPMENTAL CAPACITY

Oocyte culture in the presence of the nonspecific competitive phosphodiesterase inhibitor caffeine has been reported to increase developmental capacity of oocytes in different mammalian species. Here, we evaluated the effects of caffeine supplementation during the final phase of in vitro maturation (IVM) on developmental rates and blastocyst cell numbers. Bovine ovaries were collected from a local abattoir. A total of 1142 cumulus-oocyte-complexes were obtained by slicing. Cumulus-oocyte complexes were either in vitro matured for 24h (Standard) or matured for 20h followed by additional culture for 6h in fresh IVM medium supplemented with 10mM caffeine (Caffeine 6h). In vitro fertilization was performed for 19h using frozen-thawed sperm from 2 different bulls. After IVF, presumptive zygotes were cultured in vitro for 8 days until the blastocyst stage. Cleavage and blastocyst rates were evaluated 3 and 8 days after IVF, respectively. Expanded blastocysts from the different treatments were submitted to differential staining. SAS/STAT software (SAS Institute Inc., Cary, NC, USA) was used to evaluate cleavage and blastocyst rates using the Glimmix procedure and blastocyst cell numbers were compared using the linear model procedure. Cleavage rates were lower using caffeine for bull B and blastocyst production decreased for bull A. Caffeine treatment increased inner cell mass (ICM) number for bull B and decreased trophectoderm (TE) and total cell numbers for bull A. However, similar TE and total cells were obtained for bull B (Table 1; P<0.05). Results show that developmental competence can be affected by caffeine supplementation at the final phase of IVM probably due to oocyte-sperm interaction changes.

197 EFFECTS OF PRE-IN VITRO MATURATION WITH CAFFEINE ON BOVINE OOCYTE DEVELOPMENTAL CAPACITY

High cyclic adenosine monophosphate (cAMP) concentrations are critical for maintaining oocyte meiotic arrest in vivo. For in vitro maturation (IVM), the oocyte is released mechanically from the follicle, which induces a significant drop in intra-oocyte cAMP levels, triggering non-physiological meiotic resumption. It has been proposed that modulation of cAMP before IVM can increase bovine blastocyst rates in vitro. Caffeine is a nonspecific competitive phosphodiesterases (PDE) inhibitor and can inhibit meiotic resumption of oocytes due to maintenance of cAMP levels. It has been reported that gamete treatment with caffeine can increase developmental potential. The current study evaluated the effects of pre-in vitro maturation culture with different concentrations of caffeine on meiotic progress, developmental rates and blastocyst cell numbers. Bovine ovaries were collected from a local abattoir. A total of 6648 cumulus-oocyte complexes were obtained by slicing. Caffeine was used in 5 different concentrations (1, 5, 10, 20, and 30 mM) during slicing, searching, and 2 h pre-IVM culture. A control group, with 2 h pre-IVM without caffeine (0 mM) and a standard control were also included. Oocytes were washed either after standard or pre-IVM treatments and cultured for 24 h in vitro without caffeine. After IVM, oocytes were fertilised in vitro for 19 h, and zygotes were cultured in vitro for 8 days until the blastocyst stage. Subsets of oocytes were fixed in 2% glutaraldehyde at 9, 20, and 24 h after IVM. Hoechst staining was performed to evaluate nuclear status of matured oocytes. Cleavage and blastocyst formation rates were evaluated at Days 3 and 8 after IVF. Expanded blastocysts from all treatments were submitted to differential staining. One-way ANOVA from R software was applied to evaluate differences in cleavage and blastocysts rates and blastocyst cell numbers. Fisher’s exact test complemented by Bonferroni correction was used to determine meiotic progress. Caffeine maintained oocytes in meiotic arrest after 9 h of IVM in a concentration-dependent manner (germinal vesicle: 79.0%, 92.2%, 66.7%, 55.1%, 56.9%, 43.9%, 30.2%, respectively, for 30, 20, 10, 5, 1, 0 mM and standard; P   0.05). Developmental competence was not affected by 2 h pre-IVM culture. Caffeine supplementation before IVM delayed resumption of meiosis and affected embryo development. Table 1.In vitro developmental competence of oocytes treated with different caffeine concentrations before IVM