Differential effect of recipient cytoplasm for microtubule organization and preimplantation development in rat reconstituted embryos with two-cell embryonic cell nuclear transfer

In the present study, we examined the developmental ability of enucleated zygotes, MII oocytes, and parthenogenetically activated oocytes at pronuclear stages (parthenogenetic PNs) as recipient cytoplasm for rat embryonic cell nuclear transfer. Enucleated zygotes as recipient cytoplasm receiving two-cell nuclei allowed development to blastocysts, whereas the development of embryos reconstituted with MII oocytes and parthenogenetic PNs was arrested at the two-cell stage. Previous observations in rat two-cell embryos suggested that the distribution of microtubules is involved in two-cell arrest. Therefore, we also examined the distribution of microtubules using immunofluorescence. At the two-cell stage after nuclear transfer into enucleated zygotes, microtubules were distributed homogeneously in the cytoplasm during interphase, and normal mitotic spindles were observed in cleaving embryos from the two- to four-cell stage. In contrast, embryos reconstituted with MII oocytes and parthenogenetic PNs showed aberrant microtubule organization. In enucleated zygotes, fibrous microtubules were distributed homogeneously in the cytoplasm. In contrast, dense microtubules were localized at the subcortical area in the cytoplasm and strong immunofluorescence intensity was observed at the plasma membrane, while very weak intensity was detected in the central part of enucleated MII oocytes. In enucleated parthenogenetic PNs, high-density and fibrous microtubules were distributed in the subcortical and central areas, respectively. Pre-enucleated parthenogenetic PNs also showed lower intensity of microtubule immunofluorescence in the central cytoplasm than zygotes. In conclusion, the results of the present study showed that zygote cytoplasm is better as recipient than MII oocyte and parthenogenetic PNs for rat two-cell embryonic cell nuclear transfer to develop beyond four-cell stage. Furthermore, microtubule organization is involved in the development of reconstituted embryos to overcome the two-cell arrest.     

Nuclear and microtubule dynamics of G2/M somatic nuclei during haploidization in germinal vesicle-stage mouse oocytes

During the haploidization process, it is expected that diploid chromosomes of somatic cells will be reduced to haploid for the generation of artificial gametes. In the present study, we aimed to use enucleated mouse oocytes at the germinal vesicle-stage (G2/M) as recipients for somatic cells that are also synchronized to the G2/M stage for haploidization. The reconstructed oocytes were then induced to undergo meiosis in vitro and observed for their nuclear morphology and microtubule network formation at various expected stages of the meiotic division. Following in vitro maturation, more than half (62/119, 52.1%) of the reconstructed oocytes completed the first round of meiosis-like division, as evidenced by the extrusion of pseudopolar bodies (PBs). However, accelerated PB extrusion, approximately 3-4 h earlier than that by control oocytes occurred. Furthermore, abnormally large pseudo-PBs, as large as four times the normal PB sizes, were observed. During the process of in vitro maturation at both the expected stages of metaphase I (MI) and metaphase II (MII), condensed chromosomes were observed in 38.7% and 55.2% of oocytes, respectively. However, two other types of nuclear configurations were also observed: 1) uneven distribution of chromatin and 2) an interphase-like nucleus, indicating deficiencies in chromosome condensation. Following oocyte activation, more than half (21/33, 63.6%) of the reconstructed oocytes with pseudo-PBs formed separated pseudopronuclei (PN), suggesting formation of functional spindles. The formation of bipolar spindle-like microtubule network at both the expected MI and MII stages during in vitro maturation was confirmed by immunohistochemistry. In summary, this study demonstrated that a high proportion of G2/M somatic nuclei appear to undergo meiosis-like division, in two successive steps, forming a pseudo-PB and two separate pseudo-PN upon in vitro maturation and activation treatment. Moreover, the enucleated geminal vesicle cytoplast retained its capacity for meiotic division following the introduction of a somatic G2/M nucleus.     

Spindle Dynamics during Meiosis in Drosophila Oocytes

Mature oocytes of Drosophila are arrested in metaphase of meiosis I. Upon activation by ovulation or fertilization, oocytes undergo a series of rapid changes that have not been directly visualized previously. We report here the use of the Nonclaret disjunctional (Ncd) microtubule motor protein fused to the green fluorescent protein (GFP) to monitor changes in the meiotic spindle of live oocytes after activation in vitro. Meiotic spindles of metaphase-arrested oocytes are relatively stable, however, meiotic spindles of in vitro–activated oocytes are highly dynamic: the spindles elongate, rotate around their long axis, and undergo an acute pivoting movement to reorient perpendicular to the oocyte surface. Many oocytes spontaneously complete the meiotic divisions, permitting visualization of progression from meiosis I to II. The movements of the spindle after oocyte activation provide new information about the dynamic changes in the spindle that occur upon re-entry into meiosis and completion of the meiotic divisions. Spindles in live oocytes mutant for a lossof-function ncd allele fused to gfp were also imaged. The genesis of spindle defects in the live mutant oocytes provides new insights into the mechanism of Ncd function in the spindle during the meiotic divisions.     

Changes in global histone acetylation pattern in somatic cell nuclei after their transfer into oocytes at different stages of maturation

In our study, we have examined the pattern of global histone modification changes in somatic cell nuclei after their transfer into mouse oocytes at different stages of maturation or after their parthenogenetic activation. While germinal vesicle (GV) staged immature oocytes are strongly labeled with anti-acetylated histone H3 and H4 antibodies, the signal is absent in both metaphase I and metaphase II oocytes (MI, MII). In contrast, the oocytes of all maturation stages show a presence of trimethylated H3/K4 in their chromatin. When somatic cells were fused to intact or enucleated GV oocytes, both the GV and the somatic cell nucleus showed a very strong signal for all the antibodies used. On the other hand, when somatic cells nuclei that are AcH3 and AcH4 positive before fusion are introduced into either intact or enucleated MI or MII oocytes, their acetylation signal decreased rapidly and was totally absent after a prolonged culture. This was not the case when anti-trimethyl H3/K4 antibody was used. The somatic cell chromatin showed only a slight decrease in the intensity of labeling after its transfer into MI or MII oocytes. This decrease was, however, evident only after a prolonged culture. These results suggest not only a relatively higher stability of the methylation modification but also some difference between the oocyte and somatic chromatin. The ability to deacetylate the chromatin of transferred somatic nuclei disappears rapidly after the oocyte activation. Our results indicate that at least some reprogramming activity appears in the oocyte cytoplasm almost immediately after GV breakdown (GVBD), and that this activity rapidly disappears after the oocyte activation.     

Nek9 regulates spindle organization and cell cycle progression during mouse oocyte meiosis and its location in early embryo mitosis

Nek9 (also known as Nercc1), a member of the NIMA (never in mitosis A) family of protein kinases, regulates spindle formation, chromosome alignment and segregation in mitosis. Here, we showed that Nek9 protein was expressed from germinal vesicle (GV) to metaphase II (MII) stages in mouse oocytes with no detectable changes. Confocal microscopy identified that Nek9 was localized to the spindle poles at the metaphase stages and associated with the midbody at anaphase or telophase stage in both meiotic oocytes and the first mitotic embyros. Depletion of Nek9 by specific morpholino injection resulted in severely defective spindles and misaligned chromosomes with significant pro-MI/MI arrest and failure of first polar body (PB1) extrusion. Knockdown of Nek9 also impaired the spindle-pole localization of γ-tubulin and resulted in retention of the spindle assembly checkpoint protein Bub3 at the kinetochores even after 10 h of culture. Live-cell imaging analysis also confirmed that knockdown of Nek9 resulted in oocyte arrest at the pro-MI/MI stage with abnormal spindles, misaligned chromosomes and failed polar body emission. Taken together, our results suggest that Nek9 may act as a MTOC-associated protein regulating microtubule nucleation, spindle organization and, thus, cell cycle progression during mouse oocyte meiotic maturation, fertilization and early embryo cleavage.     

Septin 7 is required for orderly meiosis in mouse oocytes

Septin 7 is a conserved GTP-binding protein. In this study, we examined the localization and functions of Septin 7 during mouse oocyte meiotic maturation. Immunofluorescent analysis showed that intrinsic Septin 7 localized to the spindles from the pro-MI stage to the MII stage. Knockdown of Septin 7 by siRNA microinjection caused abnormal spindles and affected extrusion of the first polar body. Septin 7 mRNA tagged with myc was injected into GV stage oocytes to overexpress Septin 7. Overexpressed Myc-Septin 7 localized to the spindle and beneath the plasma membrane displaying long filaments. Fluorescence intensity of spindle α-tubulin in myc-Septin 7-injected oocytes was weaker than that of the control group, demonstrating that Septin 7 may influence recruitment of α-tubulin to spindles. MII oocytes injected with myc-Septin 7 exhibited abnormal chromosome alignment, and parthenogenetic activation failed to allow extrusion of the second polar body, suggesting that overexpression of Septin 7 may affect extrusion of the polar body by disturbing the alignment of chromosomes and regulating α-tubulin recruitment to spindles. In summary, Septin 7 may regulate meiotic cell cycle progression by affecting microtubule cytoskeletal dynamics in mouse oocytes.     

The Microtubule-Associated Protein ASPM Regulates Spindle Assembly and Meiotic Progression in Mouse Oocytes

The microtubule-associated protein ASPM (abnormal spindle-like microcephaly-associated) plays an important role in spindle organization and cell division in mitosis and meiosis in lower animals, but its function in mouse oocyte meiosis has not been investigated. In this study, we characterized the localization and expression dynamics of ASPM during mouse oocyte meiotic maturation and analyzed the effects of the downregulation of ASPM expression on meiotic spindle assembly and meiotic progression. Immunofluorescence analysis showed that ASPM localized to the entire spindle at metaphase I (MI) and metaphase II (MII), colocalizing with the spindle microtubule protein acetylated tubulin (Ac-tubulin). In taxol-treated oocytes, ASPM colocalized with Ac-tubulin on the excessively polymerized microtubule fibers of enlarged spindles and the numerous asters in the cytoplasm. Nocodazole treatment induced the gradual disassembly of microtubule fibers, during which ASPM remained colocalized with the dynamic Ac-tubulin. The downregulation of ASPM expression by a gene-specific morpholino resulted in an abnormal meiotic spindle and inhibited meiotic progression; most of the treated oocytes were blocked in the MI stage with elongated meiotic spindles. Furthermore, coimmunoprecipitation combined with mass spectrometry and western blot analysis revealed that ASPM interacted with calmodulin in MI oocytes and that these proteins colocalized at the spindle. Our results provide strong evidence that ASPM plays a critical role in meiotic spindle assembly and meiotic progression in mouse oocytes.     

Septin 7 is required for orderly meiosis in mouse oocytes

Septin 7 is a conserved GTP-binding protein. In this study, we examined the localization and functions of Septin 7 during mouse oocyte meiotic maturation. Immunofluorescent analysis showed that intrinsic Septin 7 localized to the spindles from the pro-MI stage to the MII stage. Knockdown of Septin 7 by siRNA microinjection caused abnormal spindles and affected extrusion of the first polar body. Septin 7 mRNA tagged with myc was injected into GV stage oocytes to overexpress Septin 7. Overexpressed Myc-Septin 7 localized to the spindle and beneath the plasma membrane displaying long filaments. Fluorescence intensity of spindle α-tubulin in myc-Septin 7-injected oocytes was weaker than that of the control group, demonstrating that Septin 7 may influence recruitment of α-tubulin to spindles. MII oocytes injected with myc-Septin 7 exhibited abnormal chromosome alignment, and parthenogenetic activation failed to allow extrusion of the second polar body, suggesting that overexpression of Septin 7 may affect extrusion of the polar body by disturbing the alignment of chromosomes and regulating α-tubulin recruitment to spindles. In summary, Septin 7 may regulate meiotic cell cycle progression by affecting microtubule cytoskeletal dynamics in mouse oocytes.     

Effects of MEK inhibitor U0126 on meiotic progression in mouse oocytes: microtuble organization, asymmetric division and metaphase II arrest

In this study we used U0126, a potent and specific inhibitor of MEK, to study the roles of MEK/ERK/p90rsk signaling pathway in the meiotic cell cycle of mouse oocytes. The phosphorylation of MAP kinase and p90rsk in the oocytes treated with 1.5 microM U0126 was the same as that in oocytes cultured in drug-free medium. With 1.5 microM U0126 treatment, the spindles appeared normal as they formed in oocytes, but failed to maintain its structure. Instead, the spindle lost one pole or elongated extraordinarily. After further culture, some oocytes extruded gigantic polar bodies (>30 microm) that later divided into two small ones. Some oocytes underwent symmetric division and produced two equal-size daughter cells in which normal spindles formed. In oocytes with different division patterns, MAP kinase was normally phosphorylated. When the concentration of U0126 was increased to 15 mM, the phosphorylation of both MAPK and p90rsk were inhibited, while symmetric division was decreased. When incubating in medium containing 15 microM U0126 for 14 h, oocytes were activated, but part of them failed to emit polar bodies. MII oocytes were also activated by 15 microM U0126, at the same time the dephosphorylation of MAP kinase and p90rsk was observed. Our results indicate that 1) MEK plays important but not indispensable roles in microtubule organization; 2) MEK keeps normal meiotic spindle morphology, targets peripheral spindle positioning and regulates asymmetric division by activating some unknown substrates other than MAP kinase /p90rsk; and 3) activation of MEK/ERK/p90rsk cascade maintains MII arrest in mouse oocytes.     

Nuf2 is required for chromosome segregation during mouse oocyte meiotic maturation

Nuf2 plays an important role in kinetochore-microtubule attachment and thus is involved in regulation of the spindle assembly checkpoint in mitosis. In this study, we examined the localization and function of Nuf2 during mouse oocyte meiotic maturation. Myc₆-Nuf2 mRNA injection and immunofluorescent staining showed that Nuf2 localized to kinetochores from germinal vesicle breakdown to metaphase I stages, while it disappeared from the kinetochores at the anaphase I stage, but relocated to kinetochores at the MII stage. Overexpression of Nuf2 caused defective spindles, misaligned chromosomes, and activated spindle assembly checkpoint, and thus inhibited chromosome segregation and metaphase-anaphase transition in oocyte meiosis. Conversely, precocious polar body extrusion was observed in the presence of misaligned chromosomes and abnormal spindle formation in Nuf2 knock-down oocytes, causing aneuploidy. Our data suggest that Nuf2 is a critical regulator of meiotic cell cycle progression in mammalian oocytes.     

Nek9 regulates spindle organization and cell cycle progression during mouse oocyte meiosis and its location in early embryo mitosis

Nek9 (also known as Nercc1), a member of the NIMA (never in mitosis A) family of protein kinases, regulates spindle formation, chromosome alignment and segregation in mitosis. Here, we showed that Nek9 protein was expressed from germinal vesicle (GV) to metaphase II (MII) stages in mouse oocytes with no detectable changes. Confocal microscopy identified that Nek9 was localized to the spindle poles at the metaphase stages and associated with the midbody at anaphase or telophase stage in both meiotic oocytes and the first mitotic embyros. Depletion of Nek9 by specific morpholino injection resulted in severely defective spindles and misaligned chromosomes with significant pro-MI/MI arrest and failure of first polar body (PB1) extrusion. Knockdown of Nek9 also impaired the spindle-pole localization of γ-tubulin and resulted in retention of the spindle assembly checkpoint protein Bub3 at the kinetochores even after 10 h of culture. Live-cell imaging analysis also confirmed that knockdown of Nek9 resulted in oocyte arrest at the pro-MI/MI stage with abnormal spindles, misaligned chromosomes and failed polar body emission. Taken together, our results suggest that Nek9 may act as a MTOC-associated protein regulating microtubule nucleation, spindle organization and, thus, cell cycle progression during mouse oocyte meiotic maturation, fertilization and early embryo cleavage.     

Polo-like kinase-1 is a pivotal regulator of microtubule assembly during mouse oocyte meiotic maturation,fertilization, and early embryonic mitosis

Polo-like kinases (Plks) are a family of serine/threonine protein kinases that have been activated through phosphorylation. The activity of these kinases has been shown to be required for regulating multiple stages of mitotic progression in somatic cells. In this experiment, the changes in Plk1 expression were detected in mouse oocytes through Western blotting. The subcellular localization of Plk1 during oocyte meiotic maturation, fertilization, and early cleavage as well as after antibody microinjection or microtubule assembly disturbance was studied by confocal microscopy. The quantity of Plk1 protein remained stable during meiotic maturation and decreased gradually after fertilization. Plk1 was localized to the spindle poles of both meiotic and mitotic spindles at the early M phase and then translocated to the middle region. At anaphase and telophase, Plk1 was concentrated at the midbody of cytoplasmic cleavages. Plk1 was concentrated between the male and female pronuclei after fertilization. Plk1 disappeared at the spindle region when microtubule formation was inhibited by colchicine or staurosporine, while it was concentrated as several dots in the cytoplasm after taxol treatment. Plk1 antibody injection decreased the germinal vesicle breakdown rate and distorted MI spindle organization. Our results indicate that Plk1 is a pivotal regulator of microtubule organization during mouse oocyte meiosis, fertilization, and cleavage and that its functions may be regulated by other kinases, such as staurosporine-sensitive kinases.     

Three-dimensional ultrastructure of Saccharomyces cerevisiae meiotic spindles

Meiotic chromosome segregation leads to the production of haploid germ cells. During meiosis I (MI), the paired homologous chromosomes are separated. Meiosis II (MII) segregation leads to the separation of paired sister chromatids. In the budding yeast Saccharomyces cerevisiae, both of these divisions take place in a single nucleus, giving rise to the four-spored ascus. We have modeled the microtubules in 20 MI and 15 MII spindles by using reconstruction from electron micrographs of serially sectioned meiotic cells. Meiotic spindles contain more microtubules than their mitotic counterparts, with the highest number in MI spindles. It is possible to differentiate between MI versus MII spindles based on microtubule numbers and organization. Similar to mitotic spindles, kinetochores in either MI or MII are attached by a single microtubule. The models indicate that the kinetochores of paired homologous chromosomes in MI or sister chromatids in MII are separated at metaphase, similar to mitotic cells. Examination of both MI and MII spindles reveals that anaphase A likely occurs in addition to anaphase B and that these movements are concurrent. This analysis offers a structural basis for considering meiotic segregation in yeast and for the analysis of mutants defective in this process.     

Aurora-A is a critical regulator of microtubule assembly and nuclear activity in mouse oocytes, fertilized eggs, and early embryos

Aurora-A is a serine/threonine protein kinase that plays a role in cell-cycle regulation. The activity of this kinase has been shown to be required for regulating multiple stages of mitotic progression in somatic cells. In this study, the changes in aurora-;A expression were revealed in mouse oocytes using Western blotting. The subcellular localization of aurora-A during oocyte meiotic maturation, fertilization, and early cleavages as well as after antibody microinjection or microtubule assembly perturbance was studied with confocal microscopy. The quantity of aurora-A protein was high in the germinal vesicle (GV) and metaphase II (MII) oocytes and remained stable during other meiotic maturation stages. Aurora-A concentrated in the GV before meiosis resumption, in the pronuclei of fertilized eggs, and in the nuclei of early embryo blastomeres. Aurora-A was localized to the spindle poles of the meiotic spindle from the metaphase I (MI) stage to metaphase II stage. During early embryo development, aurora-A was found in association with the mitotic spindle poles. Aurora-A was not found in the spindle region when colchicine or staurosporine was used to inhibit microtubule organization, while it accumulated as several dots in the cytoplasm after taxol treatment. Aurora-A antibody microinjection decreased the rate of germinal vesicle breakdown (GVBD) and distorted MI spindle organization. Our results indicate that aurora-A is a critical regulator of cell-cycle progression and microtubule organization during mouse oocyte meiotic maturation, fertilization, and early embryo cleavage.     

Cytoplasmic asters are required for progression past the first cell cycle in cloned mouse embryos

Unlike the oocytes of most other animal species, unfertilized murine oocytes contain cytoplasmic asters, which act as microtubule-organizing centers following fertilization. This study examined the role of asters during the first cell cycle of mouse nuclear transfer (NT) embryos. NT was performed by intracytoplasmic injection of cumulus cells. Cytoplasmic asters were localized by staining with an anti-alpha-tubulin antibody. Enucleation of MII oocytes caused no significant change in the number of cytoplasmic asters. The number of asters decreased after transfer of the donor nuclei into these enucleated oocytes, probably because some of the asters participated in the formation of the spindle that anchors the donor chromosomes. The cytoplasmic asters became undetectable within 2 h of oocyte activation, irrespective of the presence or absence of the donor chromosomes. After the standard NT protocol, a spindle-like structure persisted between the pseudopronuclei of these oocytes throughout the pronuclear stage. The asters reappeared shortly before the first mitosis and formed the mitotic spindle. When the donor nucleus was transferred into preactivated oocytes (delayed NT) that were devoid of free asters, the microtubules and microfilaments were distributed irregularly in the ooplasm and formed dense bundles within the cytoplasm. Thereafter, all of the delayed NT oocytes underwent fragmentation and arrested development. Treatment of these delayed NT oocytes with Taxol, which is a microtubule-assembling agent, resulted in the formation of several aster-like structures and reduced fragmentation. Some Taxol-treated oocytes completed the first cell cycle and developed further. This study demonstrates that cytoplasmic asters play a crucial role during the first cell cycle of murine NT embryos. Therefore, in mouse NT, the use of MII oocytes as recipients is essential, not only for chromatin reprogramming as previously reported, but also for normal cytoskeletal organization in reconstructed oocytes.     

Cytoplasmic reorganization during the resumption of meiosis in cultured preovulatory rat oocytes

Changes in organelle topography and microtubule configuration have been studied during the resumption and progression of meiosis in cultured preovulatory rat oocytes. Germinal vesicle breakdown (GVBD) was reversibly inhibited by dibutyryl cAMP (DcAMP) or nocodazole, a microtubule-disrupting agent. The microtubule stabilizing agent taxol did not inhibit GVBD, but did impair further maturation. The migration of acidic organelles and chromatin in living oocytes was analyzed using the vital stains acridine orange and Hoechst 33258, respectively. Germinal vesicle stage oocytes undergo perinuclear aggregation of acidic organelles during GVBD and these organelles subsequently disperse into the cell cortex as the first meiotic spindle migrates to the oocyte periphery. DcAMP and nocodazole block the perinuclear aggregation of acidic organelles, whereas, in taxol-treated oocytes, organelle aggregation and GVBD occur but the dispersion of acidic organelles was arrested. Dose-response studies on the effects of nocodazole showed that GVBD was generally retarded and that a 50% inhibition of GVBD was achieved at concentrations in excess of 1.0 microM. Concentrations of taxol at 10 microM or above effectively inhibited both chromatin condensation and meiotic spindle formation. Indirect immunofluorescence microscopy with anti-tubulin antibodies revealed dissolution of microtubules with 1.0 microM nocodazole. Taxol had little effect on microtubule organization in germinal vesicle or chromatin condensation stage oocytes; however, when oocytes that had formed first meiotic spindles were treated with taxol, numerous microtubule asters appeared which were preferentially associated with the oocyte cortex. The changes in organelle topography, microtubule configuration, and drug sensitivity are discussed with respect to the regulation of cytoplasmic reorganization during the meiotic maturation of rat preovulatory oocytes.     

RNA- binding protein Stau2 is important for spindle integrity and meiosis progression in mouse oocytes

Staufen2 (Stau2) is a double-stranded RNA-binding protein involved in cell fate decision by regulating mRNA transport, mRNA stability, translation, and ribonucleoprotein assembly. Little is known about Stau2 expression and function in mammalian oocytes during meiosis. Herein we report the sub-cellular distribution and function of Stau2 in mouse oocyte meiosis. Western blot analysis revealed high and stable expression of Stau2 in oocytes from germinal vesicle (GV) to metaphase II (MII). Immunofluorescence showed that Stau2 was evenly distributed in oocytes at GV stage, and assembled as filaments after germinal vesicle breakdown (GVBD), particularly, colocalized with spindle at MI and MII. Stau2 was disassembled when microtubules were disrupted with nocodazole, on the other hand, when MTs were stabilized with taxol, Stau2 was not colocalized with the stabilized microtubules, but aggregated around the chromosomes array, indicating Stau2 assembly and colocalization with microtubules require both microtubule integrity and its normal dynamics. During interphase and mitosis of BHK and MEF cells, Stau2 was not distributed on microtubules, but colocalized with cis-Golgi marker GM130, implying its association with Golgi complex but not the spindle in fully differentiated somatic cells. Specific morpholino oligo-mediated Stau2 knockdown disrupted spindle formation, chromosome alignment and microtubule-kinetochore attachment in oocytes. The majority oocytes were arrested at MI stage, with bright MAD1 at kinetochores, indicating activation of spindle assembly checkpoint (SAC). Some oocytes were stranded at telophase I (TI), implying suppressed first polar body extrution. Together these data demonstrate that Stau2 is required for spindle formation and timely meiotic progression in mouse oocytes.     

DNA fragmentation in canine oocytes after in vitro maturation in TCM-199 medium supplemented with different proteins

In the present study, the effect of different protein supplementation on meiotic nuclear configuration, DNA fragmentation (TUNEL assay) and metabolic parameters of dog oocytes cultured in vitro for 72 h was investigated. TCM-199 medium was supplemented either with 0.3% bovine serum albumin (BSA) or with 10% bitch heat inactivated plasma (OBP) collected before the LH peak or with OBP collected between the LH peak and ovulation or OBP collected after ovulation. After culture, more than 70% of the cumulus-oocyte complexes cultured in plasma groups presented extensive cell expansion, while none of those cultured in BSA showed extensive expansion of the cumulus (P < 0.05). Glucose consumption and lactate production was lower (P < 0.05) in the BSA-supplemented medium than in plasma-supplemented groups. In all groups, high amounts of alanine were produced. A higher number of oocytes with DNA fragmentation were observed in the BSA group, while in the plasma-supplemented groups more oocytes presented undistinguishable nuclear material. Only a small percentage of the oocytes (7.4-12.7%) had intact DNA after culture and within these, no differences were observed between groups in number of oocytes at each chromatin configuration stage. No differences in the percentage of oocytes reaching metaphase II (MII) were observed between experimental groups. Still, only 2% of cultured oocytes reached MII, but 85.7% of these had intact DNA. Conversely, all other chromatin configurations presented a high proportion of fragmented DNA (germinal vesicle 79.8%; meiosis resumption 73.3%; unclassified 95.2%). In conclusion, a high percentage of canine oocytes that do not complete meiotic maturation to MII are degenerated, whereas a high proportion of MII oocytes have intact DNA, independently of the protein supplement used.     

In vivo aging of oocytes influences the behavior of nuclei transferred to enucleated rabbit oocytes

The present study examined nuclear remodeling in rabbit nuclear transfer (NT) embryos formed from metaphase II (MII) oocytes aged in vivo until 19 hr postcoitum (hpc), enucleated, and fused at 22–26 hpc with 32-cell morula blastomeres by means of electric fields, which also induced recipient oocyte activation. Post-activation events observed during the first hour following the fusion/activation pulse were studied in terms of chromatin, lamins, and micro-tubules, and revealed that transferred nuclei underwent premature chromosomes condensation (PCC) in only one-third of NT embryos and remained in interphase in others. Recipient oocytes were mostly not activated by manipulations performed before the fusion/activation pulse. The persistance of transferred nuclei in interphase resulted from the rapid progression of recipient oocytes to interphase after activation, suggesting that the cytoplasmic state of MII oocytes aged in vivo was poised for the approach to interphase. Studying micro-tubular organization in MII oocytes before nuclear transfer manipulations, we found that 19 hpc MII oocytes aged in vivo differed from 14 hpc MII oocytes (freshly ovulated) and from 19-hpc MII oocytes aged in vitro (collected at 14 hpc and cultured for 5 hr), notably by the presence of microtubule asters and tubulin foci or only tubulin foci dispersed throughout the cytoplasm. When PCC was avoided, remodeling of the transferred nucleus was well advanced 1 hr after nuclear transfer, and NT embryos developed better to the blastocyst stage. Mol. Reprod. Dev. 46:325–336, 1997. © 1997 Wiley-Liss, Inc.