The nucleolus

Many of the maternally derived products needed to prepare the biological machinery, such as re-assembly of the nucleolus, as factory for ribosomes necessary for protein synthesis are stored in the oocytes during its growth (DIELEMAN et al. 2002). The nucleolus contains large loops of DNA emanating from several chromosomes, each of which contain a cluster of rRNA genes, which are transcribed by RNA polymerase I [Pol I] (ALBERTS et al. 1994e). Each such gene cluster is known as nucleolar organizer region (NOR), where pre-ribosomal RNA is synthesized, processed and assembled with specific proteins into pre-ribosomal particles (KING et al. 1988). The transcription of these genes is considered a prerequisite for the formation of the nucleolus that develops around the NORs (HYTTEL et al. 2000b; HYTTEL et al.

2001). The number of NORs, like the chromosome number, is characteristic of the species. The diploid bovine genome comprises ten NORs localized to the telomere regions of chromosome 2, 3, 4, 11 and 29 (DI BERARDINO et al. 1979).

The nucleolus is the most prominent structure within the eukaryotic cell nucleus and is the site where ribosomal RNAs (5.8S, 18S, and 28S) are transcribed by specific

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nucleolar transcription factors and enzymes. These transcripts become associated with specific ribosomal proteins to form ribosomal subunits (SCHWARZACHER and WACHTLER 1993; ALBERTS et al. 1994e). Subsequently the subunits leave the nucleus through the pores of the nuclear envelope and associate to form ribosomes in conjunction with translation of mRNAs (HYTTEL et al. 2000b). Growing cells require continuous ribosome synthesis to ensure that subsequent generations contain the ribosomes necessary to support protein synthesis. The actively ribosome-synthesizing nucleolus consists of three main components. The fibrillar centres (FCs), the dense fibrillar component (DFC) and the granular component (GC). The FCs are frequently situated in the central region of the nucleolus. The DFC forms a network of strands surrounding the FCs, but may sometimes stretch out towards the periphery of the nucleolus. The GC is usually situated in the peripheral regions of the nucleolus (SCHWARZACHER and WACHTLER 1993). These components of the so-called fibrillo-granular nucleolus reflect the steps in the biosynthesis of ribosomes according to the following model: The fibrillar centres house the enzymatic apparatus for the transcriptional process, the dense fibrillar component carries the primary unprocessed transcripts, whereas the granular component represent the processed transcripts associated with proteins in the form of preribosomal-particles (FAIR et al.

1996; HOZAK et al. 1994; HYTTEL et al. 2000b). It is believed that rRNA transcription occurs at the interphase between the fibrillar centre and the dense fibrillar component, and that the latter is formed by the newly synthesized rRNA (HYTTEL et al. 1997). In cells with a low level of ribosomal biosynthesis the nucleoli are small, usually with a single FC and little surrounding DFC and GC ("ring-shaped nucleolus"). In active cells the DFC forms a large network enclosing several, sometimes up to hundreds of FCs, and the GC covers a large area in the periphery ("compact nucleoli"). In cells at the onset of a new stimulation, the DFC is prominent whereas FCs are few and small, and the GC is not very extensive ("reticulate nucleoli") (SCHWARZACHER and WACHTLER 1993). Studies on involving embryos produced in vitro (IVP) have revealed that transcription of ribosomal RNA (rRNA) genes during embryo development occurs at a species-specific stage of preimplantation embryonic development (THOMPSON 1996). Nucleolar function is

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gradually acquired at follicle activation and the acquisition of fibrillar centres in oocyte nucleoli is accompanied by the formation of the antral cavity in early tertiary follicles (FAIR et al. 1997a). When the oocyte reaches approximately 110 µm in diameter, corresponding to a follicle of about 3 mm in diameter, transcription decreases and the nucleolus is inactivated, forming a dense spherical remnant [Fig. 1] (FAIR et al.

1996). During the final phase of follicular dominance this remnant becomes vacuolated and, in conjunction with resumption of meiosis, disperses. There are no nucleoli during or after germinal vesicle breakdown (HYTTEL et al. 2001). The formation of a fibrillo-granular nucleolus in the bovine embryo is a sequential process, which does not reach complete functional activity until late in the fourth cell cycle when the nucleolar proteins necessary for rRNA transcription and processing are assembled (LAURINCIK et al. 2000; VIUFF et al. 1998). In the pig, fibrillo-granular nucleoli are observed at the 4-cell stage in in vivo developed embryos (HYTTEL et al. 2000a) while in in vitro produced embryos it appears for first time at the 16-cell stage, displaying a substantial delay in nucleolar activation (BJERREGAARD et al. 2003). Bovine embryos produced in vitro and embryos developed in vivo display allocation of nucleolar proteins to fibrillar and granular compartments of the developing nucleoli during the fourth cell cycle (8-cell stage).

However, among in vivo developed embryos, the formation of tentative fibrillar centres might be seen as early as during the 2^nd cell cycle showing some differences with respect to the chronology of the nucleolar development. On the other hand, parthenogenetically activated bovine embryos display a delayed nucleolar activation, which occurs during the fifth cell cycle (HAY-SCHMIDT et al. 2001; LAURINCIK et al.

2003).

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Oocyte and follicle growth

primordial, primary, secondary and tertiary follicle

Fibrillar centres

Oocyte capacitation and maturation Resumption of meiosis LH

surge

<100 100 -110 >110 µm Oocyte size

Nucleolus

Dense nucleolar remnant Oocyte and follicle growth

primordial, primary, secondary and tertiary follicle

Fibrillar centres

Oocyte capacitation and maturation Resumption of meiosis LH

surge

<100 100 -110 >110 µm Oocyte size

Nucleolus

Dense nucleolar remnant

Fig: 1. Nucleolus ultrastructure during growth, capacitation and maturation of the bovine oocyte.

Fibrillar centres invade the granular nucleoli in the secondary follicle and are marginalized towards the end of oocyte growth with the formation of the dense nucleolar remnant as the result. The remnant is vacuolized towards the end of oocyte capacitation and is dispersed in conjunction with resumption of meiosis. There are no signs of the nucleolus during or after germinal vesicle breakdown. Adapted from HYTTEL et al. 2001.

2.1.2 Developmental differences of oocytes derived from prepubertal calves