In Vitro Grown Sheep Preantral Follicles Yield Oocytes with Normal Nuclear-Epigenetic Maturation

Abstract

BackgroundAssisted reproductive technologies allow to utilize a limited number of fully grown oocytes despite the presence in the ovary of a large pool of meiotically incompetent gametes potentially able to produce live births. In vitro folliculogenesis could be useful to recruit these oocytes by promoting their growth and differentiation.Methodology/Principal Findings In vitro folliculogenesis was performed starting from sheep preantral (PA) follicles to evaluate oocyte nuclear/epigenetic maturation. Chromatin configuration, quantification of global DNA methylation, and epigenetic remodelling enzymes were evaluated with immunocytochemistry, telomere elongation was assessed with the Q-FISH technique, while the DNA methylation status at the DMRs of maternally IGF2R and BEGAIN, and paternally H19 methylated imprinted genes was determined by bisulfite sequencing and COBRA. Specifically, 70% of PA underwent early antrum (EA) differentiation and supported in culture oocyte global DNA methylation, telomere elongation, TERT and Dnmt3a redistribution thus mimicking the physiological events that involve the oocyte during the transition from secondary to tertiary follicle. Dnmt1 anticipated cytoplasmic translocation in in vitro grown oocytes did not impair global and single gene DNA methylation. Indeed, the in vitro grown oocytes acquired a methylation profile of IGF2R and BEGAIN compatible with the follicle/oocyte stage reached, and maintained an unmethylated status of H19. In addition, the percentage of oocytes displaying a condensed chromatin configuration resulted lower in in vitro grown oocytes, however, their ability to undergo meiosis and early embryo development after IVF and parthenogenetic activation was similar to that recorded in EA follicle in vivo grown oocytes.Conclusions/SignificanceIn conclusion, the in vitro folliculogenesis was able to support the intracellular/nuclear mechanisms leading the oocytes to acquire a meiotic and developmental competence. Thus, the in vitro culture may increase the availability of fertilizable oocytes in sheep, and become an in vitro translational model to investigate the mechanisms governing nuclear/epigenetic oocyte maturation.

Genomic imprints as a model for the analysis of epigenetic stability during assisted reproductive technologies

“…ART-conceived AS patients often possess maternal SNRPN LOM. human, it is important to note that work in other animal models, such as sheep and cattle, support the studies described herein (for example, see Young et al (2001), Suzuki et al (2009), Hori et al (2010, Barboni et al (2011) and Heinzmann et al (2011)). Paramount to assisted reproduction is that timing of ARTs coincides with crucial epigenetic events during gametogenesis and early embryogenesis (Fig.…”

H3K9 Trimethylation Precedes DNA Methylation during Sheep Oogenesis

“…These two epigenetic marks are probably realized also thanks to the recruitment of HDAC1, SUV39H1, G9a, HP1, Dnmt1 (Russo et al 2007;Barboni et al 2011), and Dnmt3a (Fig. 6).…”

“…The level of DNA methylation then increased before stabilizing in oocytes enclosed in tertiary follicles. The global DNA methylation results already have been analyzed in sheep in vivo-and in vitro-grown oocytes (Russo et al 2007;Barboni et al 2011), but for the first time we have studied this process also in primary follicles and simultaneously with the H3K9me3 signal. Thus, in the present study, we have observed that in sheep female germ cells, H3K9me3 precedes global DNA methylation.…”

“…In particular, under the stereomicroscope, only the translucent follicles displaying the compactness of follicular layers, the absence of free particles within the antral cavity, and the presence of blood vessels were considered healthy (Cecconi et al 1999;Mattioli et al 2001). Single healthy follicle diameters were then measured using an inverted-phase microscope equipped with an ocular micrometer (×40 magnification) and classified (Russo et al 2007;Barboni et al 2011) as follows:…”

“…Then, after washing in PBS, a secondary goat anti-rabbit conjugated Cy3 antibody (1:400 PBS/1% BSA; Chemicon International), for 1 hr at room temperature, was used to reveal Dnmt3a (Barboni et al 2011).…”