Nucleolus-like body of mouse oocytes contains lamin A and B and TRF2 but not actin and topo II

Abstract

BackgroundDuring the final stages of oocyte development, all chromosomes join in a limited nuclear volume for the final formation of a single complex chromatin structure – the karyosphere. In the majority of mammalian species, the chromosomes surround a round protein/fibrillar body known as the central body, or nucleolus-like body (NLB). Nothing seems to unite the inner portion of the karyosphere with the nucleolus except position at its remnants. Nevertheless, in this study we will use term NLB as the conventional one for karyosphere with the central body. At the morphological level, NLBs consist of tightly-packed fibres of 6–10 nm. The biochemical structure of this dense, compact NLB fibre centre remains uncertain.ResultsThe aim of this study was to determine which proteins represent the NLB components at final stages of karyosphere formation in mouse oogenesis. To determine this, three antibodies (ABs) have been examined against different actin epitopes. Examination of both ABs against the actin N-end provided similar results: spots inside the nucleus. Double staining with AB against SC35 and actin revealed the colocalization of these proteins in IGCs (interchromatin granule clusters/nuclear speckles/SC35 domains). In contrast, examination of polyclonal AB against peptide at the C-end reveals a different result: actin is localized exclusively in connection with the chromatin. Surprisingly, no forms of actin or topoisomerase II are present as components of the NLB. It was discovered that: (1) lamin B is an NLB component from the beginning of NLB formation, and a major portion of it resides in the NLB at the end of oocyte development; (2) lamin A undergoes rapid movement into the NLB, and a majority of it remains in the NLB; (3) the telomere-binding protein TRF2 resides in the IGCs/nuclear speckles until the end of oocyte development, when significant part of it transfers to the NLB.ConclusionsNLBs do not contain actin or topo II. Lamin B is involved from the beginning of NLB formation. Both Lamin A and TRF2 exhibit rapid movement to the NLB at the end of oogenesis. This dynamic distribution of proteins may reflect the NLB’s role in future chromatin organization post-fertilisation.

The karyosphere capsule inRana temporariaoocytes contains structural and DNA-binding proteins

“…The membrane telomere-binding protein TRF2 was initially isolated from R. temporaria late oocytes NE [19], but in mouse oocytes, TRF2 has been found in SC35 domains [30]. In immunoblots, the commercially available antibody recognized some minor zones with an apparent molecular mass higher than TRF2 itself, apparently an unprocessed form of the protein.…”

“…At the same time, the KC is the storage place for the DNA-related proteins, such as topo II and ATRX, which are probably required for chromatin organization during further meiotic divisions. We found that nuclear speckles (IGCs, SC35 domains) represent a storage place for TRF2 in frog oocytes (with a KC) as well as in mouse oocytes (with a central body of the karyosphere) [30]. The KC may contain many other multifarious proteins and RNAs, but a complete list of these as well as the roles of these molecules in further meiotic events and/or early embryogenesis are yet to be established.…”

“…It can relax supercoiled DNA in an ATP- and Mg 2+ -dependent manner and represents one of the first proteins identified in the scaffold of mitotic chromosomes [51,52], demonstrating highly dynamic behavior during mitosis [53]. In meiotic cells, in particular, in diplotene oocytes of mice, topo II is abundant in the nucleoplasm, but absent in the central body of the mouse karyosphere [30]. In birds, topo II is concentrated in protein bodies associated with the centromere regions of lampbrush chromosomes and, in the late diplotene, in the central body of the bird karyosphere [54].…”

“…In the nucleus of mouse oocytes derived from antral follicles, a significant portion of lamin A is stored in a nucleolus-like body, i.e. the central body of the karyosphere [30]. It is likely that the material stored in different extrachromosomal structures associated with the karyosphere, including the KC in the frog and the central body in the mouse, may be used to organize chromatin before fertilization.…”

“…Anti-udTRF2 immunostaining was much more intensive in SC35-containing nuclear bodies (splicing speckles) rather than the KC, suggesting that SC35 domains are the sites for TRF2 accumulation in the nucleus of R. temporaria late oocytes. Similarly, TRF2 also dominates in the nuclear speckles of mouse oocytes until the end of diplotene, when a portion of TRF2 begins to accumulate in the central body of the karyosphere [30]. Thus, the same extrachromosomal structures function as storage compartments for TRF2 independently of the karyosphere type.…”