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Characterization of Tribolium castaneum oocyte nuclear structures using microinjection of a fusion nuclear protein mRNA

Nuclear architecture comprises a number of extrachromosomal structures, nuclear compartments, or domains. We use the oocytes of the red flour beetle, Tribolium castaneum, as an experimental model. This species was introduced as a novel laboratory insect to complement studies performed in Drosophila for genetic (the T. castaneum genome is fully sequenced and aligned), developmental, andcell-biology questions.Many researchers consider T. castaneum ‘‘less derived’’ and ‘‘more ancestral’’ than fruit flies. In many organisms, oocyte chromosomes join together in a limited volume of the oocyte nucleus, forming a karyosphere (karyosome) at the diplotene stage of meiotic prophase (GruzovaandParfenov, 1993). Chromosomes in the karyosphere may be further separated from the nucleoplasm by a complex, extrachromosomal karyosphere capsule (KC), whose biological significance is still unclear. We employed a gene-engineering approach to obtain a dynamic picture of the distribution of a newly synthesized protein in the T. castaneum oocyte nucleus. We microinjected an in vitro-synthesized, 5’-capped mRNA containing the coding sequence of the exon junction complex protein Y14, fused to a myc tag (Fig. S1, D) into oocytes. This approach allowed us to trace the nuclear dynamics of newly synthesized protein. This RNA was successfully translated within 3 h (Goodman et al., 2012) of microinjection, and the Y14 fusion protein is imported into the oocyte nucleus. A significant level of fluorescence was registered in the perichromatin region of the karyosphere,where loose DNA loops are located. Y14 was differentially localized at each oocyte stage. In the previtellogenesis stage (Fig. 1A), when the KC begins to form, Y14 resides in SC35 domains. By the vitellogenic stage, however, Y14 is completely redistributed to the well-formed KC rather than SC35 domains (Fig. 1B). Based on these dynamics, it could be supposed that, like in somatic cells, oocyte SC35-containing nuclear bodies represent transient domains where some proteins involved in mRNA transport and export can reside. Somatic extrachromosomal nuclear domains contain resident, slow-mobile proteins, including the proteins of the nuclear matrix. In contrast, pre-mRNAsplicing factorsarehighly dynamicand, despite their apparent residence in nuclear speckles (5 10 times higher concentration than elsewhere in the nucleus), donotdwell in thesespeckles longer than inotherpartsof the nucleoplasm. It is therefore possible that oocyte nuclear bodies contain other proteins that will contribute to theKCat the end of oocyte growth, of which the Y14-myc fusion protein is the first to be confirmed. Thus, the KC may be a site of residual transcription within the vitellogenic oocyte. Chromatin rearrangement, which is a characteristic feature of oocyte growth, is therefore clearly accompanied by a redistribution of nuclear proteins between different oocyte nuclear structures. This change in localization could affect how nuclear maturation proceeds.