Nuclear distribution of RNA polymerase II in human oocytes from antral follicles: dynamics relative to the transcriptional state and association with splicing factors.

The intranuclear distribution of two (unphosphorylated and hyperphosphorylated) forms of RNA polymerase II (Pol II) was studied in human oocytes from antral follicles using immunogold labeling/electron microscopy. The distribution of Pol II was analyzed relative to the transcriptional state of the oocyte as well as to the distribution of two splicing factors (snRNPs and SC-35) in the intranuclear entities, namely, interchromatin granule clusters (IGCs), nucleolus-like bodies (NLBs), and perichromatin fibrils (PFs). The results showed that (1) antibodies directed against two forms of Pol II have similar pattern of intranuclear distribution, (2) both Pol II and splicing factors progressively accumulate in IGCs with decrease in the transcriptional activity of the oocyte nucleus, (3) both Pol II and splicing factors localize to PFs, and (4) Pol II is present in the NLBs at all transcriptional states of the oocyte nucleus. These studies confirm earlier proposals that PFs represent a nuclear domain in which RNA transcription/processing are spatially coupled. The accumulation of Pol II and splicing factors in IGCs concomitant with a decrease in the transcriptional activity suggests a coordinated mechanism for the movement of both Pol II and splicing factors from the sites of action to the sites of storage.

Dynamics of distribution of splicing components relative to the transcriptional state of human oocytes from antral follicles.

The distribution of two splicing components (snRNP and SC-35) and coilin were studied by immunogold/electron microscopy in human oocytes from antral follicles at different levels of transcriptional activity (i.e., active, intermediate, and inactive). The results showed a decrease of snRNPs and SC-35 in the karyoplasm as the oocytes progress from a transcriptionally active to the inactive state. The main areas of accumulation of both these splicing components in all stages of oocytes appeared to be the interchromatin granule clusters (IGCs). Within the IGCs, the two splicing components seemed to be spatially segregated, with the snRNPs predominantly bound to the fibrillar region, whereas the SC-35 factors are being enriched in the granular zone. The p80 coilin was found only in the nucleolus-like body (NLB), which is present in all three stages of oocytes; no coiled bodies were evident. These data are consistent with the notion that splicing occurs in the karyoplasm and that the splicing components are mobilized from a storage site (IGCs) to the site of action.

Human antral follicles: oocyte nucleus and the karyosphere formation (electron microscopic and autoradiographic data).

The functional organization of the nucleus in the oocytes from human antral follicles was examined by morphological and autoradiographic analysis methods at the light and electron microscopic level. According to the position of the nucleus, the level of its transcriptional activity, and the pattern of distribution of structures in it, oocytes fall into two groups. In the first one, the oocytes with the nucleus in the central position are characterized by the distribution of numerous structures all over the nucleus or by a different extent of aggregation of chromatin around the nucleolus. The nuclei of these oocytes are characterized by [3H]uridine incorporation, the label being localized over purely fibrillar, agranular nucleoli and over dispersed fibrillar chromatin adjacent to either the regions of densely packed chromatin or fibrillar-granular material of the nucleolus-like bodies. The latter, the same as condensed chromatin, do not incorporate [3H]uridine. In the second group, the nuclei are displaced towards the oocyte's periphery, and chromosomes surround the nucleolus as a continuous mass closely adjacent to its surface, thus forming a karyosphere. The karyosphere formation takes place on the background of cessation of nuclear transcriptional activity. A fully formed karyosphere represents a complex of closely associated inactivated structures: Nucleolus, chromosomes, and nucleolus-like bodies. The karyosphere nucleolus bears no granules and consists of densely packed finely fibrillar material (fibrils 3 nm thick). Two zones (central and peripheral) can be distinguished in a nucleolus. Nucleolus-like bodies, consisting of granules 20 nm in diameter embedded in finely fibrillar material, are often associated with chromosomes. In this study, data obtained by observations on the loss of association between the oocyte (with karyosphere) and corona radiata cells are evaluated. The relation of the karyosphere formation to the atresia process and the duration of karyosphere existence in human antral follicles are also discussed.