Polo-like kinase 1 (PLK1), a member of the serine/threonine protein kinases family, is involved in multiple steps of mitotic progression. It regulates centrosome maturation, mitotic spindle formation, and cytokinesis. While studied extensively in somatic cells, little is known about PLK1 activities in the mammalian preimplantation embryo. We examined the role of PLK1 in the one-cell mouse embryo. Western blotting showed that the PLK1 protein content increased significantly during the S-phase of the one-cell stage and declined during the first mitotic division. Activation of PLK1 preceded nuclear envelope breakdown (NEBD) in both pronuclei at the entry to first embryo mitosis. Immunofluorescence revealed the presence of phosphorylated, active PLK1 (pThr(210) -PLK1) in both male and female pronuclei, and in the microtubule-organizing centers (MTOCs) shortly before NEBD. During the first mitotic metaphase, pThr(210) -PLK1 accumulated at the spindle poles and was also associated with condensed chromosomes. Inhibition of PLK1 activity with a specific PLK1 inhibitor, BI 2536, at the one-cell stage induced the formation of a bipolar spindle that displayed disordered microtubular arrangements and dislocated, condensed chromosomes. Although such embryos entered mitosis, they did not complete mitosis and arrested at metaphase. Time-lapse recording revealed progressive misalignment of condensed chromosomes during first mitotic metaphase. These data indicate that PLK1 activity is not essential for entry into first mitosis, but is required for the events leading up to metaphase-anaphase transition in the one-cell mouse embryo.

Insulin-like growth factor I (IGF-I) has been shown to promote mammalian early embryo development. Increased cell division or decreased cell death have been proposed as two main possible mechanisms in its effect. Here we examine the nature of this promoting effect in a model situation.Camptothecin (0.01 mug/ml) and actimomycin D (0.005 mug/ml) were used to induce apoptosis. Four-cell mouse embryos were cultured in vitro to blastocyst stage in the temporary (15 h) presence or absence of apoptotic inductors and in the permanent presence or absence of IGF-I (100 ng/ml). Embryos were assessed by morphological triple staining (Hoechst 33342, propidium iodide, Calcein AM) and comet assay on Day 5, 120 h after administration of hCG. The number of nuclei, the blastocyst formation, the proportion of embryos containing fragmented DNA and the percentage of apoptotic and secondary necrotic nuclei were assessed.Both inductors of apoptosis significantly increased the percentage of apoptotic and secondary necrotic cells and reduced total cell counts (camptothecin, P > 0.001; actinomycin D, P > 0.001). When IGF-I was added to the culture medium in the presence of an apoptosis inductor, apoptosis incidence was significantly decreased (P < 0.001). The addition of IGF-I into control samples also decreased the percentage of apoptotic and secondary necrotic cells. In contrast, IGF-I addition had no significant influence on embryo development (P > 0.05).Our data suggest a primary role for IGF-I as an apoptotic survival factor in mouse preimplantation embryos in specific conditions. (C) 2003 Elsevier Inc. All rights reserved.

The occurrence of apoptosis in mouse preimplantation embryos was analyzed using DNA staining (Hoechst 33342, PI) for the visualization of nuclear changes and by the comet assay, a single-cell gel electrophoresis assay, modified for the analysis of blastocysts. Mouse preimplantation embryos isolated 56 h after superovulation were cultured in vitro for 64 h. Apoptosis was induced by treatment with camptothecin and actinomycin D during the first 15 h of culture. After culture in vitro, a number of embryos were stained and analyzed using morphological criteria. The remaining embryos were examined using the comet assay for the detection of DNA fragmentation. The proportion of damaged embryos in experimental groups, in comparison to controls, was dependent on the dose of apoptosis inductor. At high doses (camptothecin, microg/ml and actinomycin D, 0.05 microg/ml) over 90% (chi-square test, P<0.001) of embryos had apoptotic comets, at medium doses (camptothecin, 0.01 microg/ml and actinomycin D, 0.005 microg/ml) comets appeared only in 30-70% of embryos (camptothecin, P<0.01 and actinomycin D, P<0.001). At low doses (camptothecin, 0.001 microg/ml and actinomycin D, 0.0005 microg/ml) the increase in damaged embryos was not statistically significant. Hoechst/PI staining showed a higher percentage of damaged blastomeres at high doses. Morphological changes correlated with the outcome of the comet assay. Our results show that comet assay is an appropriate method for studying apoptosis in preimplantation embryos, and it appears to be more sensitive than the classically used morphological analyses.

Apoptosis may occur in early embryos in which the execution of essential developmental events has failed. Thus the initiation of the apoptotic mechanism may be related to activation of the embryonic genome. In this way, developmentally incompetent cells or whole embryos are eliminated. It is likely that some link exists between failed resumption of rRNA synthesis and the incidence of apoptosis in cleaving embryos. In this context, decreased developmental potential in cleaving nucleotransferred embryos is consistent with cell loss, and very likely due to programmed cell death. The effects of apoptosis inducers on cleaving embryos have not been characterised in comparable detail to that in the case of somatic cells. Early embryos provide a very good model for study of these processes because of the specificity of rRNA transcription resumption after fertilization. In our experiments three apoptosis inducers (staurosporin 10 mM, actinomycin D 0.05 mg/ml and camptothecin 0.1 mg/ml) were used in a culture medium for 15 h at the 4-cell stage (day 2) of mouse embryos, followed by further development in a pure culture medium until fixation on days 3, 4 and 5. In staurosporin-induced embryos, light microscopy immunostaining of nucleolar proteins (fibrillarin, Nopp140, protein B23) did not reveal changes in nucleolar morphology on day 3. On days 4 and 5, more compact (roundish) nucleoli (in comparison with controls) were observed. The embryos treated with camptothecin displayed a similar staining pattern to those with staurosporin at each day. In actinomycin-D-treated embryos, marked changes in nucleolar appearance were visible as early as day 3. These changes in nucleolar morphology consisted of loss of the reticulation appearance and fragmentation of nucleoli. In addition to nucleolar changes, significantly decreased cell proliferation was observed. The induced embryos did not reach the blastocyst stage. The number of blastomeres was decreased, and staining with Hoechst 33342 revealed a significant percentage of apoptotic nuclei (condensed/fragmented nuclei) from day 4.

As it was shown earlier, resumption of rRNA transcription in early mouse embryo is localized in the peripheral region of nucleolus precursor body/NPB/during the two‐cell stage. Recently, nucleolar phosphoprotein Nopp140 was presented to shuttle between the nucleolus and cytoplasm as chaperone of snoRNPs. Nopp140 interacts with RNA polymerase I in nucleolus and also accumulates in CBs, suggesting a pathway between the two organelles. The aim of the study was to describe the changing location of Nopp140 during the first cleavage stages of mouse embryos and its re‐location after inhibition of rRNA synthesis with actinomycin D. Light microscope immunocytochemical staining showed Nopp140 in the periphery of NPBs before activation of rDNA transcription and in addition confirmed its localization in CBs. Immunolabelling with antibodies against RNA Pol I and UBF gave co‐localization of these proteins, implicating that Nopp140 may actively participate to rDNA transcription. We suggest that fundamental differences in molecular organization of rDNA synthesis and postranscriptional processes between cycling somatic and pre‐implantation embryonic cells may be in selective transport of transcription and/or processing‐complexes of proteins to the nucleolar organizer regions (NOR). Mol. Reprod. Dev. 59:277–284, 2001. © 2001 Wiley‐Liss, Inc.

Molecular Reproduction and DevelopmentVolume 53, Issue 3 p. 306-317 Embryo Development Cellular and subcellular localization of stathmin during oocyte and preimplantation embryo development J. Koppel, Corresponding Author J. Koppel koppel@saske.sk Institute of Animal Physiology, Slovak Academy of Sciences, Košice, SlovakiaInstitute of Animal Physiology, Slovak Academy of Sciences, Šoltésovej 4, 040 01 Košice, Slovakia.Search for more papers by this authorP. Rehák, P. Rehák Institute of Animal Physiology, Slovak Academy of Sciences, Košice, SlovakiaSearch for more papers by this authorV. Baran, V. Baran Institute of Animal Physiology, Slovak Academy of Sciences, Košice, SlovakiaSearch for more papers by this authorJ. Veselá, J. Veselá Institute of Animal Physiology, Slovak Academy of Sciences, Košice, SlovakiaSearch for more papers by this authorD. Hlinka, D. Hlinka Second Clinic of Gynaecology and Obstetrics, University Hospital of L. Pasteur, Košice, SlovakiaSearch for more papers by this authorV. Manceau, V. Manceau INSERM U 440, IFM, Paris, FranceSearch for more papers by this authorA. Sobel, A. Sobel INSERM U 440, IFM, Paris, FranceSearch for more papers by this author J. Koppel, Corresponding Author J. Koppel koppel@saske.sk Institute of Animal Physiology, Slovak Academy of Sciences, Košice, SlovakiaInstitute of Animal Physiology, Slovak Academy of Sciences, Šoltésovej 4, 040 01 Košice, Slovakia.Search for more papers by this authorP. Rehák, P. Rehák Institute of Animal Physiology, Slovak Academy of Sciences, Košice, SlovakiaSearch for more papers by this authorV. Baran, V. Baran Institute of Animal Physiology, Slovak Academy of Sciences, Košice, SlovakiaSearch for more papers by this authorJ. Veselá, J. Veselá Institute of Animal Physiology, Slovak Academy of Sciences, Košice, SlovakiaSearch for more papers by this authorD. Hlinka, D. Hlinka Second Clinic of Gynaecology and Obstetrics, University Hospital of L. Pasteur, Košice, SlovakiaSearch for more papers by this authorV. Manceau, V. Manceau INSERM U 440, IFM, Paris, FranceSearch for more papers by this authorA. Sobel, A. Sobel INSERM U 440, IFM, Paris, FranceSearch for more papers by this author First published: 21 May 1999 https://doi.org/10.1002/(SICI)1098-2795(199907)53:3<306::AID-MRD6>3.0.CO;2-YCitations: 9AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract Stathmin is a 19 kDa cytosolic phosphoprotein, proposed to act as a relay integrating diverse intracellular signaling pathways involved in regulation of cell proliferation, differentiation, and function. To gain further information about its significance during early development, we analyzed stathmin expression and subcellular localization in mouse oocytes and preimplantation embryos. RT-PCR analysis revealed a low expression of stathmin mRNA in unfertilized oocytes and a higher expression at the blastocyst stage. A fine cytoplasmic punctuate fluorescent immunoreactive stathmin pattern was detected in the oocyte, while it evolved toward an increasingly speckled pattern in the two-cell and later four- to eight-cell embryo, with even larger speckles at the morula stage. In blastocysts, stathmin immunoreactivity was fine and intense in inner cell mass cells, whereas it was low and variable in trophectodermal cells. Electron microscopic analysis allowed visualization with more detail of two types of stathmin immunolocalization: small clusters in the cytoplasm of oocytes and blastocyst cells, together with loosely arranged clusters around the outer membrane of cytoplasmic vesicles, corresponding to the immunofluorescent speckles in embryos until the morula stage. In conclusion, it appears from our results that maternal stathmin is accumulated in the oocyte and is relocalized within the oocyte and early preimplantation embryonic cell cytoplasm to interact with specific cytoplasmic membrane formations. Probably newly synthesized, embryonic stathmin is expressed in the blastocyst, where it is localized more uniformly in the cytoplasm mostly of inner cell mass (ICM) cells. These expression and localization patterns are probably related to the particular roles of stathmin at the successive steps of oocyte maturation and early embryonic development. They further support the proposed physiologic importance of stathmin in essential biologic regulation. Mol. Reprod. Dev. 53:306–317, 1999. © 1999 Wiley-Liss, Inc. Citing Literature Volume53, Issue3July 1999Pages 306-317 RelatedInformation

To investigate the significance of impaired insulin secretion on preimplantation embryo development, outbred ICR female mice received a single injection of streptozotocin 130 mg (low) and 160 mg (subdiabetic) kg(-1), 14-17 days before fertilization. Preimplantation embryos were collected on day 3 of pregnancy, four to eight-cell embryos were cultured in vitro 48 h (day 5) and their cell number was estimated. After spontaneous ovulation, the significantly different distribution pattern in comparison with the controls was detected only in preimplantation embryos isolated from subdiabetic (160 mg x kg(-1) streptozotocin) mice. Furthermore, the incidence of degenerated embryos was significantly increased after 48 h in vitro cultivation. The analysis of cell number distribution in embryos after cultivation in vitro indicated a significant delay in cell proliferation in both experimental groups (130 and 160 mg x kg(-1) streptozotocin) in comparison with control mice. After superovulation, the only significant difference was found in the distribution pattern of embryos isolated on day 3 of pregnancy from subdiabetic (160 mg x kg(-1) streptozotocin) mice. No significant differences were found after embryo cultivation in vitro. It could be concluded that, in outbred ICR mice, lower streptozotocin treatment (130 mg x kg(-1)) influenced only cell distribution of in vitro cultured embryos after spontaneous ovulation. In ICR mice, marked changes in preimplantation embryo development were detected only after subdiabetic (160 mg x kg(-1)) streptozotocin treatment. During in vitro cultivation delayed effects of impaired insulin secretion resulted in an increase of embryo degeneration at the time after the third mitotic cleavage. Our results indicate that the effects of impaired maternal insulin secretion on preimplantation embryo development in mice are marked and consistent after spontaneous ovulation. Superovulation apparently disguises subtle changes in preimplantation embryo development after low and subdiabetic streptozotocin treatment.

The structure-function relationships of the nucleolar substructures were studied in preimplantation rabbit embryos, where nucleologenesis is extending over the first four cell cycles and may not be synchronous in each blastomere. Immunocytochemical methods using light and electron microscopy were applied for protein and RNA localization as well as nick translation and terminal deoxynucleotidyl transferase techniques for DNA detection. DNA was gradually associated with the periphery of the compact nucleolar precursor bodies (NPBs) but was never found inside NPBs at the four-cell stage. In 16-cell embryos, some NPBs displayed a reticulated periphery forming the branching network of the dense fibrillar component (DFC) surrounding the ''residual body'' (remnant of NPB) in the process of activation. At the 32-cell stage, fully reticulated nucleoli were observed in each blastomere. DNA was then associated with the DFC of reticulated nucleoli. RNA was first detected at the 16 cell-stage in close contact with the DFC as well as inside the ''residual body'' which was not immunolabeled with the DNA antibodies used. When observed by light microscopy, fibrillarin, nucleolin, and protein B23 displayed a changing distribution pattern during nucleologenesis. At early stages (up to the 16-cell stage), small dot- and spot-like structures were distributed within the whole nuclei. In 16-cell embryos, these proteins started to accumulate in an irregular thin layer around the NPBs in the process of activation. The reorganization process described may be in relation with the redistribution of chromatin and nuclear/nucleolar matrix components during the activation of rDNA transcription localized in the NPB shell. In conclusion, nucleologenesis is only achieved at the fourth cell cycle in the cleaving rabbit embryo at the corresponding time when the first detectable nucleolus-associated RNA is detectable. Our results show a good correlation between the establishment of structure and function. (C) 1997 Wiley-Liss, Inc.

To investigate the significance of impaired insulin secretion on preimplantation embryo development, outbred ICR female mice received an injection of a single dose of streptozotocin 200 mg.kg-1 14-17 days before fertilization. Oocytes were collected 24-26 h after hCG injection. Morphological evaluation revealed a lower percentage of oocytes with second polar bodies from streptozotocin-treated females in comparison with controls. Furthermore, in this group the incidence of degenerated embryos significantly increased after 120 h in vitro cultivation. Insulin (5 U per 100 g b.w.) administered twice daily to streptozotocin-treated mice significantly improved the Embryonic development. Morphological analysis of oocyte maturation in streptozotocin-treated mice showed no significant differences in comparison with control mice. It could be concluded that marked changes in preimplantation embryo development were detected in outbred ICR mice after streptozotocin administration and this process was partly reversible by insulin treatment. Furthermore, it was shown that the process of fertilization was negatively influenced and that during in vitro cultivation the delayed effects of impaired insulin secretion resulted in an increase of embryo degeneration at the time following the third mitotic cleavage.

In vivo nucleologenesis was studied in bovine embryos by electron microscopic immunogold labelling of DNA, RNA, protein C23 and protein B23. We have used the classification of Kopecný et al. (1989b) and Kopecný (1990) dividing nucleologenesis in four steps: compact nucleolar precursor body (NPB), monovacuolated NPB, NPB containing secondary vacuoles and fully reticulated nucleolus. These different features of early bovine embryo nucleologenesis were mainly observed during the eight-cell stage. In the first step of nucleolar development, the association of compact NPB with DNA structures was observed. DNA was also labelled in some small secondary vacuoles appearing during the third developmental step. From the second step onward, the labelling of protein C23 was observed in the compact fibrillar network of the NPB. Protein B23 started to be labelled in the compact fibrillar mass at the third step. RNA labelling was also observed for the first time in NPB containing secondary vacuoles. Labelled RNA was located in the peripheral region of compact fibrillar mass as well as along the border of the vacuoles. In the reticulated nucleolus, the dense fibrillar component was found to contain both proteins and RNA.

We have studied the effect of moderately impaired maternal insulin secretion on oocyte chromosomal constitution, fertilization and zygote DNA synthesis. Female mice were injected with a single dose of streptozotocin (65 mg/kg) 14 days before fertilization/ovulation. Zygotes/oocytes were recovered from control and subdiabetic mice on day 1 of pregnancy. Compared with control animals, subdiabetic females showed a significant difference in the proportion of zygotes/oocytes. The subdiabetic mothers had a lower percentage of zygotes and a higher percentage of unfertilized and degenerated oocytes in comparison with control animals. The investigation of [3H]thymidine incorporation did not show any influence of the maternal subdiabetes on the initial zygote DNA synthesis. An analysis of the ovulated oocytes at the metaphase II stage isolated from subdiabetic mice did not reveal increased chromosomal anomalies in comparison with the controls. Control and subdiabetic mothers had a similar percentage of oocytes with a normal haploid set of chromosomes, and the incidence of aneuploidy/diploidy did not differ significantly. These observations suggest that insulin changes in subdiabetic mothers had a deleterious influence on oocyte fertilization in mice, but apparently they did not have any effect on the nuclear events.

The localization of fibrillarin and nucleolin in the nuclei of mouse two-cell, four-cell, and eight-cell embryos has been studied using immunofluorescent staining with specific antibodies. In all of these cleavage stages, both antigens were associated exclusively with the peripheral region of the nucleolus precursor bodies (NPBs). The original speckled fluorescent staining pattern in the early two-cell stage was progressively changed into a continuous fluorescent-positive layer localized in the cortex of the NPBs in the four-cell embryos. The compact central area of NPBs was never stained. Both proteins were colocalized in the same substructures of developing nucleoli. In order to analyze the interaction of chromatin with NPBs, DNA structures were specifically immunolabelled. At the time of resumption of nucleolar transcription (in the two-cell mouse embryo), DNA was detected at the periphery of, but not penetrating into, NPBs. Our results confirm the view that the cortical region of NPBs could represent a nucleolonemal area involved in the resumption of nucleolar transcription in the early mouse embryo. (C) 1995 Wiley-Liss, Inc.

Female mice were injected with a single dose of streptozotocin (65 mg kg-1) 14-17 days before fertilization to investigate the significance of impaired insulin secretion induced by subdiabetic streptozotocin treatment on preimplantation embryo development. Subdiabetic mice (streptozotocin-treated) had significantly different glucose tolerance from that of control animals, despite similar basal glycaemia. Morphological analysis of preimplantation embryos collected on day 2 of pregnancy revealed no significant changes in the number of two-cell embryos recovered from streptozotocin-treated females compared with controls. Two-cell embryos were transferred into the oviducts of healthy, synchronous pseudopregnant females and recovered 24-28 h later. Morphological evaluation revealed a significantly greater percentage of degenerated embryos from streptozotocin-treated females than from control females. Morphological analysis of preimplantation embryos collected on day 2.5 of pregnancy revealed no significant changes in the number of two- to four-cell embryos recovered from streptozotocin-treated females compared with controls, but there was a significant increase in the number of degenerated embryos in streptozotocin-treated females that did not receive insulin therapy. Insulin (1-1.5 iu per 100 g) administered twice a day to streptozotocin-treated mice significantly improved the altered development of embryos in both experiments. It is possible that the impaired insulin secretion in female mice adversely affected the growth of preimplantation embryos. Almost half of the morphologically normal two-cell embryos isolated from subdiabetic females were incapable of development to the eight-cell stage even in a non-diabetic maternal environment. the morphologically distinct degenerative changes were first detected at the time of the second mitotic cleavage.

To estimate the significance of insulin in the regulation of preimplantation embryo growth, female mice received a single subdiabetogenic dose of streptozocin (65 mg/kg intraperitoneally) 8-11 days or 14-17 days before fertilization. Mean glycaemia levels and the number of embryos per mouse did not differ significantly between the streptozocin-treated and control groups. Morphological analysis of preimplantation embryos collected on day 3 of pregnancy revealed significant changes in the distribution pattern of preimplantation embryo stages recovered from streptozocin-treated females. Continuous insulin treatment of streptozocin-treated mice improved the impaired development of preimplantation embryos only in short-lasting experiments. After a long subdiabetic period (14-17 days) the incidence of degenerated embryos was increased in both streptozocin-treated groups. It can be concluded that the subdiabetic state in female mice impairs preimplantation embryo development which could partly be prevented by insulin treatment.

The distribution of U1, U2, U4, U5 and U6 small nuclear ribonucleoproteins (snRNPs) and interchromatin granules (IGs) was studied by electron-microscopic immunocytochemistry (EMI) in early 2-cell mouse embryos at the onset of embryonal transcription. The localization of these antigen structures was evaluated with respect to nucleoplasmic ribonucleoprotein (RPN) regions consisting of interchromatin and perichromatin areas. SnRNP structures of maternal origin (labelled with anti-Sm antibody) were widely distributed throughout the nucleoplasm. Specifically labelled IGs were detected by gold particle clusters distributed in the interchromatin regions of the nucleoplasm. Both immunodetections were negative in nucleolar precursor bodies (NPBs). In addition, the labelling of condensed chromatin blocks with anti-DNA antibody showed heterochromatin topology at this developmental stage. Small condensed chromatin blocks were distributed throughout the nucleus and also appeared in association with the NPB rim. The observed status quo represents a transient state of nuclear structure rearrangement.

Distribution of embryonic DNA in mouse preimplantation embryos was studied by the immunoelectron microscopic method with colloidal gold. The embryos (one-cell, early or late two-cell, four-cell) were sampled separately, and immunogold labelling has been performed on thawed ultrathin cryosections. Condensed chromatin blocks were labelled very intensively. DNA labelling was predominantly visible over peripheral chromatin blocks and over small patches of chromatin localized throughout the nucleus of one-cell embryo. DNA distribution appeared to be more extended in the whole nucleus of early two-cell embryo. A few labelled chromatin clumps were observed to be associated with prenucleolar body (nucleolar precursor body--NPB) in this stage. Convex-shaped electron-light areas (fibrillar centres?) were found at the periphery of NPB in the late two-cell stage. These areas were labelled by scattered individual gold particles. In reticulated nucleolar regions (detected in four-cell embryos) immunolabelling appeared as individual gold particles present over the periphery of areas with loosely arranged material (fibrillar centres) surrounded by a denser component (dense fibrillar component). These consecutive changes are correlated with nucleologenesis and with the onset of transcriptional activity. We conclude that at the onset of nucleolar transcriptional activity condensed chromatin does not penetrate the NPB of mouse two-cell embryo.

Immunolocalization of cyclin/PCNA (proliferating cell nuclear antigen) was performed with monoclonal antibody using immunogold methods on ultrathin cryosections of fertilized mouse eggs. Immunolabeling in pronuclei was checked 20, 22, 24 and 26 h after HCG injection. A relation between onset of pronuclei migration (early S-phase) and appearance of colloidal particle clusters was found. Afterwards, (mid S-phase) the increase of labelling and the localization of cyclin/PCNA were found throughout the pronuclei, except in the nucleolar bodies. Lower labelling appeared at the time of close reciprocal pronuclei contact (late S-phase). It is concluded that bulk and distribution of cyclin/PCNA in pronuclei is closely related to the progression of first interphase after fertilization.