Early development in canine species follows a very specific pattern. Oocytes are ovulated at the germinal vesicle stage and meiotic resumption occurs in the oviduct. However, because of difficulties in the accurate determination of ovulation time and in the observation of oocyte nuclear stage by light microscopy, these early events have not been fully described. Moreover, the oocyte stage at which sperm penetration occurs is still uncertain since fertilization of immature oocytes has been reported in vivo and in vitro. The aim of this study was to establish the exact timing of in vivo meiotic resumption, fertilization and early embryo development in the bitch with reference to ovulation. Ovulation was first determined by ultrasonography, artificial inseminations were performed daily and oocytes/embryos were collected between 17 and 138 h after ovulation. After fixation and DNA/tubulin staining, the nuclear stage was observed by confocal microscopy. Of the 195 oocytes/embryos collected from 50 bitches, the germinal vesicle stage was the only one present until 44 h post-ovulation, and the first metaphase II stage was observed for the first time at 54 h. Sperm penetration of immature oocytes appeared to be exceptional (three out of 112 immature oocytes). In most cases, fertilization occurred from 90 h post-ovulation in metaphase II oocytes. Embryonic development was observed up to the eight-cell stage. No significant influence of bitch breed and age on ovulation rate, maturation and developmental kinetics was observed. However, some heterogeneity in the maturation/development process was observed within the cohort of oocytes/embryos collected from one bitch. In conclusion, the most peculiar aspect of the canine species remains oocyte meiotic maturation whereas fertilization follows the same pattern as in other mammals. Reproduction (2005) 130 193-201

After fertilization, ribosomal RNA synthesis is silenced during a period which depends on the species. Data concerning the reassembly of a functional nucleolus remain scarce. We have examined by immunocytochemistry, Western blots, and BrUTP microinjection the dynamics of major nucleolar proteins during the first cycles of mouse embryogenesis, in relation to rDNA transcription sites and coilin, a marker of Cajal bodies. We show that: (1) the reinitiation of rDNA transcription occurs at the two-cell stage, 44-45 h after hCG injection (hphCG), at the surface of the nucleolar precursor bodies (NPBs), where the RNA polymerase I (pol I) transcription complex is recruited 4-5 h before; (2) the NPBs are not equal in their ability to support recruitment of pol I and rDNA transcription; (3) maternally inherited fibrillarin undergoes a dynamic redistribution during the second cell stage, together with coilin, leading to the assembly of the Cajal body around 40 hphCG; and (4) the pol I complex is first recruited to the Cajal body before reaching its rDNA template. We also find that fibrillarin and B23 are both directly assembled around NPBs prior to ongoing pre-rRNA synthesis. Altogether, our results reveal a role of the Cajal bodies in the building of a functional nucleolus.

Mouse embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs) represent naive and primed pluripotency states, respectively, and are maintained in vitro by specific signalling pathways. Furthermore, ESCs cultured in serum-free medium with two kinase inhibitors (2i-ESCs) are thought to be the ground naïve pluripotent state. Here, we present a comparative study of the epigenetic and transcriptional states of pericentromeric heterochromatin satellite sequences found in these pluripotent states. We show that 2i-ESCs are distinguished from other pluripotent cells by a prominent enrichment in H3K27me3 and low levels of DNA methylation at pericentromeric heterochromatin. In contrast, serum-containing ESCs exhibit higher levels of major satellite repeat transcription, which is lower in 2i-ESCs and even more repressed in primed EpiSCs. Removal of either DNA methylation or H3K9me3 at PCH in 2i-ESCs leads to enhanced deposition of H3K27me3 with few changes in satellite transcript levels. In contrast, their removal in EpiSCs does not lead to deposition of H3K27me3 but rather removes transcriptional repression. Altogether, our data show that the epigenetic state of PCH is modified during transition from naive to primed pluripotency states towards a more repressive state, which tightly represses the transcription of satellite repeats.

During the final step of oogenesis, the oocyte nucleus is subject to large-scale modifications that correlate with transcriptional silencing. While oocytes with dense chromatin around the nucleolus are silent (SN, surrounded nucleolus), oocytes with uncondensed chromatin (NSN, non-surrounded nucleolus) are transcriptionally active. It is believed that epigenetic mechanisms that participate in gene expression regulation could play a role in this event. In this context, we examined the behaviour of heterochromatin and related histone modifications during the NSN to SN transition by immunostaining. Using fluorescent in situ hybridization on three dimensionalpreserved nuclei (3D-FISH), we also studied the distribution of centromeric, pericentromeric and ribosomal (rDNA) sequences in relation to the nucleolus (also called the nucleolus-like body, NLB). We observed that in NSN-type oocytes, pericentromeric heterochromatin is aggregated within chromocenters. In SN-type oocytes, pericentromeric heterochromatin and centromeres form a discontinuous ring around the NLB. rDNA sequences, which initially present a pearl necklace structure, gather together in seven highly condensed foci at the NLB periphery. H3K9me3 and H4K20me3 heterochromatin marks clearly label chromocenters, whereas H3K4me3 and H4K5ac are totally excluded from heterochromatin regions, even in the very compact SN-nuclei. Remarkably, H3K27me3 displays an intermediate behavior. It appears that GV oocyte nuclei exhibit a specific epigenetic landscape. Histone modifications, related to both active and repressive chromatin structures, seem to follow the large-scale chromatin movements that occur during the NSN to SN transition. We also demonstrate that, while heterochromatin regions re-localize around the NLB, rDNA sequences adopt a highly compact structure in SN-type oocytes.

The biology of the canine oocyte is unusual compared with that of other mammalian females. The present paper reviews both in vivo and in vitro specificities of canine oocytes. Final follicular growth in the bitch is characterised by an early appearance of LH binding sites in the granulosa, a high proportion of polyovular follicles and a preovulatory luteinisation, starting at the time of the LH surge. Through follicular fluid, preovulatory oocytes are thus exposed to high levels of progesterone, as high as 1000-fold plasma concentrations. The composition of the follicular fluid is affected by the size of the female. The more specific aspect of oocyte biology in the bitch is ovulation: oocytes are expelled immature, at the Prophase I stage. Ovulatory follicles are 6-8 mm in diameter, releasing oocytes from 110 µm, with dark cytoplasm. Resumption of meiosis occurs from 48 h postovulation, MII stages appearing 48-54 h after ovulation. The mechanisms controlling such a late meiotic resumption are still unknown. Granulosa cells seem to play a central role as in other mammalian species, but not with cAMP as the principal mediator. The importance of a transient reactivation of oocyte transcription a few hours before meiotic resumption is to be explored. These specific features may contribute to the low efficiency of IVM. Only 10-20% oocytes reach the metaphase stage and suffer from a poor cytoplasmic maturation. Moreover, in vitro culture of canine oocytes is associated with a high proportion of degeneration. To date, IVM of the oocytes is the main limiting factor for the development of assisted reproductive techniques in the canine. A better knowledge of the basic physiology of folliculogenesis and the molecular mechanisms controlling oocyte meiosis resumption in this species may allow us to overcome this obstacle.