Competent mouse oocytes isolated from antral follicles exhibit different chromatin organization and follow different maturation dynamics

Abstract

After labelling DNA with the specific vital fluorophore Hoechst 33342, oocytes, isolated by puncture from antral follicles in adult mice, have two essentially different configurations of their nuclear fluorescence images. These have been called SN (where the nucleolus is surrounded by chromatin) and NSN (where the nucleolus is not surrounded by chromatin). Intermediate configurations are also found, although with a lower frequency. The proportion of each class is on the average equal and depends neither on the presence of cumulus cells nor on the age of the mouse. Electron microscopy confirms several ultrastructural differences between these two nuclear configurations, namely, the structure of the nucleolus, which is vacuolated in NSN-type and compact in SN-type oocytes. Using video-enhanced fluorescence microscopy at low level of excitation light, we could follow directly in vitro the meiotic maturation of both classes, without impairing their viability. We show that in germinal vessicle (GV) state, the chromatin does not change from one configuration into the other and that both classes are able to mature to metaphase II, although the maturation has slightly different characteristics.

Prediction of Developmentally Competent Chromatin Conformation in Mouse Antral Oocytes

“…The size heterogeneity itself is not striking and some groups noticed previously that on average SN oocytes are slightly larger than NSN oocytes (DEBEY et al 1993;OLA et al 2007). Surprisingly, there was no attempt until now to use the size differences for selection of oocytes with better developmental potential.…”

“…The growing oocytes possess so-called NSN type chromatin relatively evenly distributed throughout the nucleus. At the end of the growth phase, however, the NSN conformation changes to SN which is characterized by more condensed chromatin with a significant portion surrounding the nucleolus (WICKRA-MASINGHE et al 1991;DEBEY et al 1993; reviewed by TAN et al 2009). Accordingly, among the oo-cytes isolated from antral follicles, often referred to as "fully grown oocytes", there are still oocytes with NSN chromatin as well as oocytes having more developmentally advanced SN chromatin.…”

Fully-mature antral mouse oocytes are transcriptionally silent but their heterochromatin maintains a transcriptional permissive histone acetylation profile

“…1e) as the first step in the transition from the NSN to the SN antral oocyte. At this stage of maturation, NSN oocytes possess a high transcriptional activity [1,4,5,11,14,17,18], their chromatin is positive to the antibodies for the acetylated form of H3, H2B and H4, whilst CHCs are seldom positive to the immunostaining. CHCs become positive during the NSN-to-SN transition.…”

“…Depending on their chromatin organisation, mouse fullymatured antral oocytes may be classified into two groups: one group comprises oocytes that have a ring of heterochromatin surrounding the nucleolus (named SN oocytes) and the other consists of oocytes that lack a ring of heterochromatin surrounding the nucleolus (NSN oocytes) ( [5,16,24]; for a review see [26]). Oocytes possessing an NSN or SN chromatin organization have also been described in other mammals, including rat, monkey, pig, bovine and human (for a review see [23]).…”

“…Antral NSN oocytes are an immature form that in the ovary will acquire the SN-type of chromatin organisation before ovulation [15,24]. These morphological differences underline biological relevance as they have been correlated with changes in transcription: NSN oocytes are transcriptionally active and synthesise all classes of RNA, whereas SN oocytes become transcriptionally inactive [1,4,5,11,14,17,18]. Furthermore, whilst in vitro both types of oocytes are capable of meiosis resumption and reach the metaphase II (MII) stage, MII SN oocytes develop to term, whereas MII NSN oocytes consistently cease development at the 2-cell stage ( [9,25]; for a review see [29]).…”