Methylation dynamics during folliculogenesis and early embryo development in sheep

Abstract

Genome-wide DNA methylation reprogramming occurs during mammalian gametogenesis and early embryogenesis. Post-fertilization demethylation of paternal and maternal genomes is considered to occur by an active and passive mechanism respectively, in most mammals but sheep; in this species no loss of methylation was observed in either pronucleus. Post-fertilization reprogramming relies on methylating and demethylating enzymes and co-factors that are stored during oocyte growth, concurrently with the re-methylation of the oocyte itself. The crucial remodelling of the oocyte epigenetic baggage often overlaps with potential interfering events such as exposure to assisted reproduction technologies or environmental changes. Here, we report a temporal analysis of methylation dynamics during folliculogenesis and early embryo development in sheep. We characterized global DNA methylation and hydroxymethylation by immunofluorescence and relatively quantified the expression of the enzymes and co-factors mainly responsible for their remodelling (DNA methyltransferases (DNMTs), ten-eleven translocation (TET) proteins and methyl-CpG-binding domain (MBD) proteins). Our results illustrate for the first time the patterns of hydroxymethylation during oocyte growth. We observed different patterns of methylation and hydroxymethylation between the two parental pronuclei, suggesting that male pronucleus undergoes active demethylation also in sheep. Finally, we describe gene-specific accumulation dynamics for methylating and demethylating enzymes during oocyte growth and observe patterns of expression associated with developmental competence in a differential model of oocyte potential. Our work contributes to the understanding of the methylation dynamics during folliculogenesis and early embryo development and improves the overall picture of early rearrangements that will originate the embryo epigenome.

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, 2014;Bernal-Ulloa et al, 2016;Leoni et al, 2007;Masala et al, 2017;Oropeza, Wrenzycki, Herrmann, Hadeler, & Niemann, 2004).…”

“…The efforts in identifying the reasons for the reduced developmental competence not only improved the rates of embryo production from prepubertal donors (Earl, Irvine, Kelly, Rowe, & Armstrong, 1995;Ledda et al, 1999), but interestingly shed light into numerous aspects involved in oocyte potential. Morphological, cellular, biochemical and molecular studies confirmed the differential competence of gametes derived from adult and prepubertal donors in terms of resistance to cryopreservation (Leoni et al, 2006), metabolism and ultrastructure (O'Brien, Dwarte, Ryan, Maxwell, & Evans, 1996), transcript abundance (Bebbere et al, 2014;Ledda et al, 2012;Leoni et al, 2007;Romar et al, 2011), in vitro developmental competence and kinetics (Landry et al, 2016;Leoni et al, 2006Leoni et al, , 2015Majerus et al, 1999), mitochondrial distribution (Leoni et al, 2015) and methylation dynamics (Masala et al, 2017). Although the differential model has become a useful research tool independently on its breeding application, the limited competence of prepubertal oocytes is yet not fully understood.…”

The effects of biological aging on global DNA methylation, histone modification, and epigenetic modifiers in the mouse germinal vesicle stage oocyte

“…Oocytes were then incubated with a primary antibody against 5MeC (mouse monoclonal Calbiochem NA81, EMD Biosciences, Inc., La Jolla, CA; 1:500 dilution) for one hour at room temperature in a humidified chamber. Primary antibody specificity was previously validated by other groups (Rougier et al, 1998;Mayer et al, 2000;Barton et al, 2001;Masala et al, 2017;Salmina et al, 2017). Following washes in blocking solution, samples were stained with secondary antibody (Alexafluor 555 rabbit anti-mouse, Invitrogen, Grand Island, NY; 1:200 dilution) for 30 minutes at room temperature in a humidified chamber in the dark followed by extensive washing in blocking solution.…”

A Lexicon of DNA Modifications: Their Roles in Embryo Development and the Germline

“…DNA methylation and hydroxymethylation profiling in early embryos of domestic animals, including bovine (Dobbs et al, 2013 ), porcine (Cao et al, 2014 ; Lee K. et al, 2014 ), ovine (Jafarpour et al, 2017 ; Masala et al, 2017 ), and equine (Heras et al, 2017 ), have mainly been reported via immunochemistry, separating 5mC and 5hmC at limited resolution. Although active demethylation is not observed in the maternal pronucleus, it has been reported in the paternal pronucleus of pig (Cao et al, 2014 ; Lee K. et al, 2014 ).…”

“…Both paternal and maternal pronuclei in bovine show 5hmC signal, despite a much less pronounced 5hmC level detected in the maternal pronucleus (Wossidlo et al, 2011 ; Bakhtari and Ross, 2014 ). Controversial data have been reported for ovine embryos (Jafarpour et al, 2017 ; Masala et al, 2017 ). Jafarpour et al reported a similar active-demethylation pattern between male and female pronucleus, i.e.…”