RESUMO
Faithful DNA replication is essential for genome integrity1-4. Under-replicated DNA leads to defects in chromosome segregation, which are common during embryogenesis5-8. However, the regulation of DNA replication remains poorly understood in early mammalian embryos. Here we constructed a single-cell genome-wide DNA replication atlas of pre-implantation mouse embryos and identified an abrupt replication program switch accompanied by a transient period of genomic instability. In 1- and 2-cell embryos, we observed the complete absence of a replication timing program, and the entire genome replicated gradually and uniformly using extremely slow-moving replication forks. In 4-cell embryos, a somatic-cell-like replication timing program commenced abruptly. However, the fork speed was still slow, S phase was extended, and markers of replication stress, DNA damage and repair increased. This was followed by an increase in break-type chromosome segregation errors specifically during the 4-to-8-cell division with breakpoints enriched in late-replicating regions. These errors were rescued by nucleoside supplementation, which accelerated fork speed and reduced the replication stress. By the 8-cell stage, forks gained speed, S phase was no longer extended and chromosome aberrations decreased. Thus, a transient period of genomic instability exists during normal mouse development, preceded by an S phase lacking coordination between replisome-level regulation and megabase-scale replication timing regulation, implicating a link between their coordination and genome stability.
RESUMO
The study of the size of cells and organelles has a long history, dating back to the 1600s when cells were defined. In particular, various methods have elucidated the size of the nucleus and the mitotic spindle in several species. However, little research has been conducted on oocyte size and organelles in mammals, and many questions remain to be answered. The appropriate size is essential to cell function properly. Oocytes have a very large cytoplasm, which is more than 100 times larger than that of general somatic cells in mammals. In this review, we discuss how oocytes acquire an enormous cytoplasmic size and the adverse effects of a large cytoplasmic size on cellular functions.
Assuntos
Meiose , Oócitos , Animais , Citoplasma , Fuso Acromático/fisiologia , MamíferosRESUMO
Oocytes communicate with the surrounding somatic cells during follicular development. We examined the effects of two oocyte-derived growth factors, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), on the development of porcine oocyte-cumulus cell complexes (OCCs) in vitro. We collected OCCs from early antral follicles (1.2-1.5 mm) and prepared oocytectomized cumulus cell complexes (OXCs), which were then cultured in a growth medium supplemented with 0-100 ng/ml GDF9 and/or BMP15 for 7 days. In the medium without GDF9 or BMP15, OCCs developed during culture, and approximately 30% of them formed antrum-like structures. GDF9 promoted OCC development and structure formation in a dose-dependent manner. However, OXCs did not form antrum-like structures without growth factors. GDF9 promoted the development of OXCs, and 50 and 100 ng/ml GDF9 promoted the formation of the structures by 8% and 26%, respectively; however, BMP15 did not promote the formation of these structures. OXCs were then cultured with 100 ng/ml GDF9 and various concentrations of BMP15 to investigate their cooperative effects on the formation of antrum-like structures. BMP15 promoted the formation of antrum-like structures in a dose-dependent manner. In conclusion, GDF9 derived from oocytes is probably important for the formation of antrum-like structures in porcine OXCs, and BMP15 cooperates with GDF9 to form these structures.
Assuntos
Proteína Morfogenética Óssea 15 , Células do Cúmulo , Animais , Proteína Morfogenética Óssea 15/metabolismo , Proteína Morfogenética Óssea 15/farmacologia , Feminino , Células da Granulosa/metabolismo , Fator 9 de Diferenciação de Crescimento/metabolismo , Fator 9 de Diferenciação de Crescimento/farmacologia , Oócitos , Folículo Ovariano/metabolismo , SuínosRESUMO
Microinjection of spermatozoa or spermatids into oocytes is a major choice for infertility treatment. However, the use of premeiotic spermatocytes has never been considered because of its technical problems. Here, we show that the efficiency of spermatocyte injection in mice can be improved greatly by reducing the size of the recipient oocytes. Live imaging showed that the underlying mechanism involves reduced premature separation of the spermatocyte's meiotic chromosomes, which produced much greater (19% vs. 1%) birth rates in smaller oocytes. Application of this technique to spermatocyte arrest caused by STX2 deficiency, an azoospermia factor also found in humans, resulted in the production of live offspring. Thus, the microinjection of primary spermatocytes into oocytes may be a potential treatment for overcoming a form of nonobstructive azoospermia caused by meiotic failure.
Assuntos
Azoospermia , Espermatócitos , Animais , Humanos , Masculino , Meiose , Camundongos , Oócitos , EspermátidesRESUMO
During female germline development, oocytes become a highly specialized cell type and form a maternal cytoplasmic store of crucial factors. Oocyte growth is triggered at the transition from primordial to primary follicle and is accompanied by dynamic changes in gene expression1, but the gene regulatory network that controls oocyte growth remains unknown. Here we identify a set of transcription factors that are sufficient to trigger oocyte growth. By investigation of the changes in gene expression and functional screening using an in vitro mouse oocyte development system, we identified eight transcription factors, each of which was essential for the transition from primordial to primary follicle. Notably, enforced expression of these transcription factors swiftly converted pluripotent stem cells into oocyte-like cells that were competent for fertilization and subsequent cleavage. These transcription-factor-induced oocyte-like cells were formed without specification of primordial germ cells, epigenetic reprogramming or meiosis, and demonstrate that oocyte growth and lineage-specific de novo DNA methylation are separable from the preceding epigenetic reprogramming in primordial germ cells. This study identifies a core set of transcription factors for orchestrating oocyte growth, and provides an alternative source of ooplasm, which is a unique material for reproductive biology and medicine.
Assuntos
Oócitos/metabolismo , Oogênese/genética , Fatores de Transcrição/metabolismo , Animais , Linhagem da Célula , Epigênese Genética , Feminino , Fertilização , Meiose , Metilação , Camundongos , Oócitos/citologia , Folículo Ovariano/citologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismoRESUMO
Acentrosomal meiosis in oocytes represents a gametogenic challenge, requiring spindle bipolarization without predefined bipolar cues. While much is known about the structures that promote acentrosomal microtubule nucleation, less is known about the structures that mediate spindle bipolarization in mammalian oocytes. Here, we show that in mouse oocytes, kinetochores are required for spindle bipolarization in meiosis I. This process is promoted by oocyte-specific, microtubule-independent enrichment of the antiparallel microtubule crosslinker Prc1 at kinetochores via the Ndc80 complex. In contrast, in meiosis II, cytoplasm that contains upregulated factors including Prc1 supports kinetochore-independent pathways for spindle bipolarization. The kinetochore-dependent mode of spindle bipolarization is required for meiosis I to prevent chromosome segregation errors. Human oocytes, where spindle bipolarization is reportedly error prone, exhibit no detectable kinetochore enrichment of Prc1. This study reveals an oocyte-specific function of kinetochores in acentrosomal spindle bipolarization in mice, and provides insights into the error-prone nature of human oocytes.
Assuntos
Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Oócitos/metabolismo , Fuso Acromático/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Segregação de Cromossomos , Proteínas do Citoesqueleto/metabolismo , Feminino , Gametogênese/fisiologia , Meiose/fisiologia , Camundongos , Microtúbulos/metabolismo , Proteínas Nucleares/metabolismoRESUMO
Mammalian oocytes and zygotes have nucleoli that are transcriptionally inactive and structurally distinct from nucleoli in somatic cells. These nucleoli have been termed nucleolus precursor bodies (NPBs). Recent research has shown that NPBs are important for embryonic development, but they are only required during pronuclear formation. After fertilization, multiple small NPBs are transiently formed in male and female pronuclei and then fuse into a single large NPB in zygotes. In cloned embryos produced by somatic cell nuclear transfer (SCNT), multiple NPBs are formed and maintained in the pseudo-pronucleus, and this is considered an abnormality of the cloned embryos. Despite this difference between SCNT and normal embryos, it is unclear how the size and number of NPBs in pronuclei is determined. Here, we show that in mouse embryos, the volume of NPB materials plays a major role in the NPB scaling through a limiting component mechanism and determines whether a single or multiple NPBs will form in the pronucleus. Extra NPB- and extra MII spindle-injection experiments demonstrated that the total volume of NPBs was maintained regardless of the pronucleus number and the ratio of pronucleus/NPB is important for fusion into a single NPB. Based on these results, we examined whether extra-NPB injection rescued multiple NPB maintenance in SCNT embryos. When extra-NPBs were injected into enucleated-MII oocytes before SCNT, the number of NPBs in pseudo-pronuclei of SCNT embryos was reduced. These results indicate that multiple NPB maintenance in SCNT embryos is caused by insufficient volume of NPB.
Assuntos
Nucléolo Celular/metabolismo , Núcleo Celular/metabolismo , Oócitos/crescimento & desenvolvimento , Zigoto/crescimento & desenvolvimento , Animais , Transferência Embrionária , Desenvolvimento Embrionário , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Endogâmicos ICR , Microscopia de Fluorescência , Proteínas Nucleares/metabolismo , Técnicas de Transferência Nuclear , Oócitos/metabolismo , Gravidez , Zigoto/metabolismoRESUMO
Meiotic divisions in females occur in fully grown oocytes that have a large cytoplasmic volume. The intracellular processes that are needed to accomplish meiotic divisions, such as spindle formation, chromosome segregation, and polar body extrusion, are controlled by the concerted actions of nuclear and cytoplasmic factors, which exhibit dynamic quantitative and spatiotemporal changes during meiotic maturation. Thus, distinguishing between meiotic controls that are mediated by cytoplasmic factors and those mediated by nuclear factors helps in the understanding of the mechanisms underlying meiotic divisions. Here, we describe a method to artificially modify the number of nuclei and the volume of the cytoplasm of mouse oocytes through cytoplasmic removal, enucleation, and cell fusion. The oocytes generated by this method are viable and undergo reproducible meiotic divisions exhibiting the effects of altered amounts of cytoplasmic and nuclear factors, which can be analyzed by various assays, such as live imaging microscopy.
Assuntos
Fusão Celular/métodos , Núcleo Celular/metabolismo , Oócitos/citologia , Animais , Separação Celular , Feminino , Camundongos , Fuso Acromático/metabolismoRESUMO
It is well known that nucleoli of fully grown mammalian oocytes are indispensable for embryonic development. Therefore, the embryos originated from previously enucleolated (ENL) oocytes undergo only one or two cleavages and then their development ceases. In our study the interspecies (mouse/pig) nucleolus transferred embryos (NuTE) were produced and their embryonic development was analyzed by autoradiography, transmission electron microscopy (TEM) and immunofluorescence (C23 and upstream binding factor (UBF)). Our results show that the re-injection of isolated oocyte nucleoli, either from the pig (P + P) or mouse (P + M), into previously enucleolated and subsequently matured porcine oocytes rescues their development after parthenogenetic activation and some of these develop up to the blastocyst stage (P + P, 11.8%; P + M, 13.5%). In nucleolus re-injected 8-cell and blastocyst stage embryos the number of nucleoli labeled with C23 in P + P and P + M groups was lower than in control (non-manipulated) group. UBF was localized in small foci within the nucleoli of blastocysts in control and P + P embryos, however, in P + M embryos the labeling was evenly distributed in the nucleoplasm. The TEM and autoradiographic evaluations showed the formation of functional nucleoli and de novo rRNA synthesis at the 8-cell stage in both, control and P + P group. In the P + M group the formation of comparable nucleoli was delayed. In conclusion, our results indicate that the mouse nucleolus can rescue embryonic development of enucleolated porcine oocytes, but the localization of selected nucleolar proteins, the timing of transcription activation and the formation of the functional nucleoli in NuTE compared with control group show evident aberrations.
Assuntos
Blastocisto/citologia , Nucléolo Celular/fisiologia , Nucléolo Celular/transplante , Embrião de Mamíferos/citologia , Desenvolvimento Embrionário/fisiologia , Oócitos/citologia , Oogênese/fisiologia , Animais , Blastocisto/metabolismo , Clonagem de Organismos , Transferência Embrionária , Embrião de Mamíferos/metabolismo , Feminino , Camundongos , Oócitos/fisiologia , Gravidez , SuínosRESUMO
Chromosome segregation during meiosis in oocytes is error prone. The uniquely large cytoplasmic size of oocytes, which provides support for embryogenesis after fertilization, might be a predisposing factor for meiotic errors. However, this hypothesis remains unproven. Here, we show that cytoplasmic size affects the functionality of the acentrosomal spindle. Artificially decreasing the cytoplasmic size in mouse oocytes allows the acentrosomal spindle poles to have a better-focused distribution of microtubule-organizing centers and to biorient chromosomes more efficiently, whereas enlargement of the cytoplasmic size has the opposite effects. Moreover, we found that the cytoplasmic size-dependent dilution of nuclear factors, including anaphase inhibitors that are preformed at the nuclear membrane, limits the spindle's capacity to prevent anaphase entry with misaligned chromosomes. The present study defines a large cytoplasmic volume as a cell-intrinsic feature linked to the error-prone nature of oocytes. This may represent a trade-off between meiotic fidelity and post-fertilization developmental competence.
Assuntos
Segregação de Cromossomos/fisiologia , Meiose/fisiologia , Microtúbulos/metabolismo , Oócitos/citologia , Fuso Acromático/metabolismo , Anáfase/fisiologia , Animais , Citoplasma/metabolismo , Fertilização/fisiologia , CamundongosRESUMO
In 1999, Tesarik and Greco reported that they could predict the developmental potential of human zygotes from a single static evaluation of their pronuclei. This was based on the distribution and number of specific nuclear organelles - the nucleoli. Recent studies in mice show that nucleoli play a key role in parental genome restructuring after fertilization, and that interfering with this process may lead to developmental failure. These studies thus support the Tesarik-Greco evaluation as a potentially useful method for selecting high-quality embryos in human assisted reproductive technologies. In this opinion article we discuss recent evidence linking nucleoli to parental genome reprogramming, and ask whether nucleoli can mirror or be used as representative markers of embryonic parameters such as chromosome content or DNA fragmentation.
Assuntos
Nucléolo Celular/genética , Embrião de Mamíferos/embriologia , Oócitos/citologia , Zigoto/citologia , Animais , Nucléolo Celular/metabolismo , Fragmentação do DNA , Embrião de Mamíferos/metabolismo , Epigênese Genética , Fertilização , Genoma Humano , Humanos , Camundongos , Oócitos/metabolismo , Técnicas de Reprodução Assistida , Zigoto/metabolismoRESUMO
Nucleoli in mammalian oocytes and zygotes, sometimes referred to as nucleolus precursor bodies (NPBs), are compact and morphologically different from nucleoli in somatic cells. We applied a unique NPB analyzing method "enucleolation" technique to zygotes to remove the NPBs. It has been reported that oocyte NPBs are essential for embryonic development; in their absence, the oocytes complete maturation and can be fertilized, but no nucleoli are formed in the zygotes and embryos, leading to developmental failure. However, we found that when NPBs were removed from zygotes, the zygotes developed successfully to live-born pups. These results indicated that oocyte NPBs are essential for embryonic development, but zygote NPBs are not. In addition, the enucleolated zygotes formed somatic-type nucleoli during early embryonic development, demonstrating that somatic-type nucleoli do not originate from zygote NPBs. We summarize our recent investigation on NPBs, and provide additional comments and findings.
Assuntos
Nucléolo Celular/genética , Desenvolvimento Embrionário/genética , Corpos de Inclusão Intranuclear/genética , Oócitos/crescimento & desenvolvimento , Animais , Blastocisto , Feminino , Mamíferos , Camundongos , Gravidez , Zigoto/crescimento & desenvolvimentoRESUMO
The large, compact oocyte nucleoli, sometimes referred to as nucleolus precursor bodies (NPBs), are essential for embryonic development in mammals; in their absence, the oocytes complete maturation and can be fertilized, but no nucleoli are formed in the zygote or embryo, leading to developmental failure. It has been convincingly documented that zygotes inherit the oocyte nucleolar material and form NPBs again in pronuclei. It is commonly accepted that during early embryonic development, the original compact zygote NPBs gradually transform into reticulated nucleoli of somatic cells. Here, we show that zygote NPBs are not required for embryonic and full-term development in the mouse. When NPBs were removed from late-stage zygotes by micromanipulation, the enucleolated zygotes developed to the blastocyst stage and, after transfer to recipients, live pups were obtained. We also describe de novo formation of nucleoli in developing embryos. After removal of NPBs from zygotes, they formed new nucleoli after several divisions. These results indicate that the zygote NPBs are not used in embryonic development and that the nucleoli in developing embryos originate from de novo synthesized materials.
Assuntos
Nucléolo Celular/fisiologia , Zigoto/crescimento & desenvolvimento , Animais , Blastocisto/citologia , Divisão Celular , Células do Cúmulo/citologia , Técnicas de Cultura Embrionária , Transferência Embrionária , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Endogâmicos ICR , Micromanipulação , Células NIH 3T3 , Oócitos/citologia , GravidezRESUMO
Recent research has shown that nucleoli of oocytes at the germinal vesicle (GV) stage (GV nucleoli) are not necessary for oocyte maturation but are essential for early embryonic development. Nucleoli of 2-cell embryos (2-cell nucleoli) have morphology similar to that of nucleoli in oocytes at the GV stage. In this study, we examined the ability of 2-cell nucleoli to substitute for GV nucleoli in terms of supporting early embryonic development by nucleolus aspiration (enucleolation) and transfer into metaphase II (MII) oocytes or 2-cell embryos that were derived from enucleolated oocytes at the GV stage in the pig. When 2-cell embryos were centrifuged to move the lipid droplets to one side of the blastomere, multiple nucleoli in the nucleus fused into a single nucleolus. The nucleoli were then aspirated from the 2-cell embryos by micromanipulation. The injection of 2-cell nucleoli to GV enucleolated oocytes at the MII stage rescued the embryos from the early embryonic arrest, and the resulting oocytes developed to blastocysts. However, the injection of 2-cell and GV nucleoli to 2-cell embryos derived from GV enucleolated oocytes rarely restored the development to blastocysts. These results indicate that 2-cell nucleoli support early embryonic development as GV nucleoli and that the presence of nucleoli is essential for pig embryos before the 2-cell stage.
Assuntos
Nucléolo Celular/fisiologia , Nucléolo Celular/transplante , Embrião de Mamíferos/ultraestrutura , Oócitos/crescimento & desenvolvimento , Oócitos/ultraestrutura , Sus scrofa/embriologia , Animais , Blastocisto/fisiologia , Blastômeros/ultraestrutura , Células Cultivadas , Desenvolvimento Embrionário/fisiologia , Feminino , MetáfaseRESUMO
Compared with advanced developmental stage embryos and somatic cells, fully grown mammalian oocytes contain specific nucleolus-like structures (NPB - nucleolus precursor bodies). It is commonly accepted that they serve as a store of material(s) from which typical nucleoli are gradually formed. Whilst nucleoli from somatic cells can be collected relatively easily for further biochemical analyses, a sufficient number of oocyte nucleoli is very difficult to obtain. We have found that isolated oocytes nucleoli fuse very efficiently when contact is established between them. Thus, well visible giant nucleoli can be obtained, relatively easily handled and then used for further biochemical analyses. With the use of colloidal gold staining, we estimated that a single fully grown mouse oocyte nucleolus contains approximately 1.6 ng of protein. We do believe that this approach will accelerate further research aiming at analyzing the composition of oocyte nucleoli in more detail.
Assuntos
Nucléolo Celular/metabolismo , Regulação da Expressão Gênica , Técnicas de Transferência Nuclear , Oócitos/citologia , Animais , Técnicas de Cultura de Células , Nucléolo Celular/fisiologia , Núcleo Celular , Cromatina/metabolismo , Dano ao DNA , Técnicas de Cultura Embrionária , Feminino , Coloide de Ouro/farmacologia , CamundongosRESUMO
During the final stage of oocyte growth, the morphology of the oocyte nucleoli changes into a compact structure. The objective of this study was to determine the involvement of the proteasome, which is a large protein complex responsible for degrading intracellular proteins, in the nucleolar compaction. The mean nucleolar diameter of growing porcine oocytes (about 100 µm in diameter) was larger than that of fully grown (120 µm) oocytes (15.5 ± 0.3 vs. 13.2 ± 0.1 µm, P<0.05). When fully grown oocytes were treated with proteasome inhibitors, MG132 (10 and 20 µM) and lactacystin (100 and 200 µM), the nucleolar diameter significantly increased from 12.9 µm to 14.9-16.1 µm. In contrast, transcription inhibitors, actinomycin D (0.8-8 µM) and α-amanitin (10-100 µM) reduced the nucleolar diameter of growing oocytes to 9.4-12.4 µm. MG132 partially prevented this reduction in nucleolar diameter. These results suggest that the proteasome regulates the nucleolar size in porcine oocytes perhaps through the degradation of nucleolar proteins.
Assuntos
Nucléolo Celular/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Oócitos/ultraestrutura , Inibidores de Proteassoma , Suínos/crescimento & desenvolvimento , Acetilcisteína/análogos & derivados , Acetilcisteína/farmacologia , Alfa-Amanitina/farmacologia , Animais , Nucléolo Celular/fisiologia , Inibidores de Cisteína Proteinase/farmacologia , Dactinomicina/farmacologia , Feminino , Leupeptinas/farmacologia , Proteínas Nucleares/metabolismoRESUMO
In mammals, the nucleolus of full-grown oocyte is essential for embryonic development but not for oocyte maturation. In our study, the role of the growing oocyte nucleolus in oocyte maturation was examined by nucleolus removal and/or transfer into previously enucleolated, growing (around 100 µm in diameter) or full-grown (120 µm) pig oocytes. In the first experiment, the nucleoli were aspirated from growing oocytes whose nucleoli had been compacted by actinomycin D treatment, and the enucleolated oocytes were matured in vitro. Most of non-treated or actinomycin D-treated oocytes did not undergo germinal vesicle breakdown (GVBD; 13% and 12%, respectively). However, the GVBD rate of enucleolated, growing oocytes significantly increased to 46%. The low GVBD rate of enucleolated, growing oocytes was restored again by the re-injection of nucleoli from growing oocytes (23%), but not when nucleoli from full-grown oocytes were re-injected into enucleolated, growing oocytes (49%). When enucleolated, full-grown oocytes were injected with nucleoli from growing or full-grown oocytes, the nucleolus in the germinal vesicle was reassembled (73% and 60%, respectively). After maturation, the enucleolated, full-grown oocytes injected with nucleoli from full-grown oocytes matured to metaphase II (56%), whereas injection with growing-oocyte nucleoli reduced this maturation to 21%. These results suggest that the growing-oocyte nucleolus is involved in the oocyte's meiotic arrest, and that the full-grown oocyte nucleolus has lost the ability.
Assuntos
Nucléolo Celular/metabolismo , Desenvolvimento Embrionário/fisiologia , Oócitos/metabolismo , Oogênese/fisiologia , Animais , Nucléolo Celular/efeitos dos fármacos , Nucléolo Celular/transplante , Dactinomicina/farmacologia , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/efeitos dos fármacos , Feminino , Meiose/fisiologia , Metáfase/efeitos dos fármacos , Inibidores da Síntese de Ácido Nucleico/farmacologia , Oócitos/citologia , Gravidez , SuínosRESUMO
Recent research has shown that the maternal nucleolus is essential for embryonic development. The morphology of the nucleolus in growing oocytes differs from that in full-grown oocytes. We determined the ability of nucleoli from growing oocytes to substitute for nucleoli of full-grown oocytes in terms of supporting embryonic development in this study. Growing (around 100 microm in diameter) and full-grown porcine oocytes (120 microm) were collected from small (0.6-1.0 mm) and large antral follicles (4-5 mm), respectively. The nucleolus was aspirated from full-grown oocytes by micromanipulation, and the resulting enucleolated oocytes were matured to metaphase II; the nucleoli originating from full-grown and growing oocytes were then injected into the oocytes. The Chromatin of growing oocytes was aspirated with the nucleolus during the enucleolation process. Growing oocytes were thus treated with actinomycin D to release the chromatin from their nucleoli, and the nucleoli were collected and transferred to the enucleolated and matured full-grown oocytes. After activation by electro-stimulation, nucleoli were formed in pronuclei of sham-operated oocytes. Enucleolated oocytes that had been injected with nucleoli from either full-grown or growing, however, did not form any nucleoli in the pronuclei. No enucleolated oocytes developed to blastocysts, whereas enucleolated oocytes injected with nucleoli from full-grown oocytes (15%) or growing oocytes (18%) developed to blastocysts. These results indicate that the nucleoli from growing oocytes can substitute for nucleoli from full-grown oocytes during early embryonic development.