Our previous studies have shown that oocytes collected from prepubertal calves lack developmental competence. The overall objective of this study was to assess causes by comparing biochemical and physiologic changes during in vitro maturation of oocytes collected from ovaries of adult cattle at slaughter and from superstimulated calves (<6 mo old) by either laporotomy or ultrasound-guided follicular aspiration. Activity and/or concentrations of maturation-promoting factor (MPF), mitogen-activated protein kinase (MAPK), and inositol 1,4,5-trisphosphate receptor (IP(3)R) were determined by measuring phosphorylation of histone H-1 kinase, phosphorylation of myelin basic protein, or Western blotting, respectively, and were compared between oocytes collected from calves and for those collected from cows. The activities of MPF and MAPK and the relative amount of IP(3)R were significantly lower in calf oocytes. The physiologic significance of these observations was determined by assessing the developmental potential of embryos derived by reciprocal transfer of metaphase II (M-II) chromosomes between cow and calf ooplasts and transfer of adult cumulus cells (G0/G1) into cow and calf ooplasts. Procedural controls consisted of transfer of M-II between adult oocytes and parthenogenic activation of adult and calf oocytes. Adult parthenogenically activated oocytes cleaved and developed to blastocysts at a higher rate than did similarly activated calf oocytes (42.1% vs. 3.4%, P < 0.05). Cleavage was also higher in reciprocal M-II transfer embryos containing adult ooplasm (46.2% vs. 12.0%, P < 0.05). Cleavage (66.7% vs. 21.9%, P < 0.05) and development to blastocyst (20.1% vs. 4.8%, P < 0.05) of nuclear transfer embryos reconstructed from adult cumulus cells was higher after transfer to adult ooplasts. Collectively, these results support the hypothesis that lack of developmental competence of calf oocytes is due to their failure or inability to complete ooplasmic maturation.

The present study investigated the global pattern of two histone modifications and methylation of DNA during in vitro maturation of bovine oocytes retrieved from follicles of two different sizes (<2 mm and 2-8 mm). The methylation status of histone H3 at position lysine K9 (H3K9 me2), the acetylation status of histone H4 at position lysine K12 (H4K12ac) and the methylation of DNA were assessed by immunocytochemistry. In parallel, the relative abundance of mRNAs coding for proteins specifically involved in reprogramming, including HLA-B associated transcript 8 (G9A), suppressor of variegation 3-9 homolog 1 (SUV39H1), the somatic isoform of DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3b (DNMT3b) and zygote arrest 1 (ZAR1) was determined by RT-PCR. The alpha-H3K9 me2 signal was present in the GV stage and remained detectable until the end of the maturation period. alpha-H4K12ac antibody gave a stronger signal in GV and GVBD oocytes and markedly decreased after GVBD. The signal showing the methylation of DNA was present during the entire maturation period. The five transcripts showed a gene-specific expression profile. Results revealed the global patterns of H3K9 me2, H4K12ac, DNA methylation and the mRNA pool profiles of genes critically involved in epigenetic modifications during bovine oocyte maturation and their possible relationship with the acquisition of oocyte developmental competence and follicular development.

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Background The segregation of the hypoblast and the emergence of the pluripotent epiblast mark the final stages of blastocyst formation in mammalian embryos. In bovine embryos the formation of the hypoblast has been partially studied, and evidence shows that MEK signalling plays a limited role in the segregation of this lineage. Here we explored the role of different signalling pathways during lineage segregation in the bovine embryo using immunofluorescence analysis of NANOG and SOX17 as readouts of epiblast and hypoblast, respectively. Results We show that SOX17 starts to be expressed in 16–32-cell stage embryos, whereas NANOG is first detected from 8-cell stage. SOX17 is first co-expressed with NANOG, but these markers become mutually exclusive by the late blastocyst stage. By assessing the expression kinetics of NANOG/SOX17 we show that inhibition of MEK signalling can eliminate SOX17 expression in bovine blastocysts, without altering NANOG expression. Modulation of WNT, PKC and LIF did not affect NANOG expression in the epiblast when used in combination with the ERK inhibitor. Conclusions This study shows that SOX17 can be used as a reliable early marker of hypoblast in the bovine, and based on its expression profile we show that the hypoblast segregates in day 7 blastocysts. Furthermore, SOX17 expression is abolished using 1 μM of PD0325901, without affecting the NANOG population in the epiblast. Modulation of WNT, PKC and LIF are not sufficient to support enhanced NANOG expression in the epiblast when combined with ERK inhibitor, indicating that additional signalling pathways should be examined to determine their potential roles in epiblast expansion. Electronic supplementary material The online version of this article (10.1186/s12861-019-0193-9) contains supplementary material, which is available to authorized users.