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

Abstract

During the last stages of oogenesis, oocyte chromosomes condense and come close together, forming the so-called karyosphere. Karyosphere formation is accompanied by an essential decrease in transcriptional activity. In the grass frog Rana temporaria, the karyosphere is surrounded by an extrachromosomal capsule that separates the chromosomes from the rest of the nucleoplasm. The karyosphere capsule (KC) of R. temporaria has been investigated in detail at the ultrastructural level, but its protein composition remained largely unknown. We demonstrate here that nuclear actin, especially F-actin, as well as lamins A/C and B are the most abundant proteins of the KC. Key proteins of nuclear pore complexes, such as Nup93 and Nup35, are also detectable in the KC. New antibodies recognizing the telomere-binding protein TRF2 allowed us to localize TRF2 in nuclear speckles. We also found that the R. temporaria KC contains some proteins involved in chromatin remodeling, including topoisomerase II and ATRX. Thus, we believe that KC isolates the chromosomes from the rest of the nucleoplasm during the final period of oocyte growth (late diplotene) and represents a specialized oocyte nuclear compartment to store a variety of factors involved in nuclear metabolism that can be used in future early development.Abbreviations: BrUTP: 5-bromouridine 5’-triphosphate; CytD: cytochalasin D; IGCs: interchromatin granule clasters; IgG: immunoglobulin G; KC: karyosphere capsule; Mw: molecular weight; NE: nuclear envelope; PBS: phosphate buffered saline; SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis; Topo II: topoisomerase II

The Long Linker Region of Telomere-Binding Protein TRF2 Is Responsible for Interactions with Lamins

“…Lamin A/C could mediate nuclear envelope–telomere attraction. TRF2 co-localized with lamins in the nuclear envelope of the oocyte nucleus of Rana temporaria [ 13 ]. We demonstrated that udTRF2 interacted with purified lamins from the NL extract.…”

“…The two components isolated during NM preparation are the internal NM and the nuclear lamina (NL) [ 8 ]. The internal NM can be observed in vivo during oogenesis, where it supports the chromosomes in large germinal vesicles of the oocyte [ 9 , 10 , 11 , 12 , 13 , 14 , 15 ], but its presence in ordinary somatic cells is questionable [ 16 , 17 ]. NL, which lies beneath the inner nuclear membrane, is a relatively insoluble fibrous structure [ 18 ].…”

Actin depolymerization disrupts karyosphere capsule integrity but not residual transcription in late oocytes of the grass frog Rana temporaria

“…It is generally assumed that the KC represents a specialized component of the oocyte nuclear matrix, supporting the chromosomes in large oocyte germinal vesicles . The bundles of intranuclear actin were shown to represent an essential component of the KC in grass frog oocytes …”

“…1,2,4 The bundles of intranuclear actin were shown to represent an essential component of the KC in grass frog oocytes. 5 The morphology of KC formation in frog oocytes is relatively well described. KC reaches approximately 150 μm in width, and represents a complicated multilayered fibrous structure involving at least five morphological zones composed of various elements.…”

Special Nuclear Structures in the Germinal Vesicle of the Common Frog with Emphasis on the So-Called Karyosphere Capsule

“…Considering that nuclear actin is the main structural protein of the KC in other organisms-namely, in some insects [25][26][27]-F-actin is considered to be a signature component of the KC in a general sense, including in the common frog [8]. Another prediction was made, according to which nucleoplasmic lamins could participate in KC formation, and also some nucleoplasmic nucleoporins were revealed in the karyosphere area of the R. temporaria GV [28], although the precise ultrastructural localization of these NE-related proteins has not been documented yet.…”