Polo-like kinase 1 is essential for the first mitotic division in the mouse embryo.

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.

Subdiabetogenic streptozocin treatment impairs preimplantation development of mouse embryos.

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.

Effects of impaired maternal insulin secretion on preimplantation embryo development in ICR mice.

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.

Preimplantation embryo development in ICR mice after streptozotocin treatment.

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.

Immunolocalization of DNA in preimplantation mouse embryos.

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.

Corona radiata density as a non-invasive marker of bovine cumulus-corona-oocyte complexes selected for in vitro embryo production.

The density of the corona radiata as a marker for the quality of cumulus-corona-oocyte complexes (CCOC's) for in vitro embryo production was tested. The CCOC's in which the corona radiata appeared as a dark rim surrounding the zona pellucida (Group 1) and CCOC's in which the corona had the same density as the rest of the cumulus investment (Group 2) were assessed with respect to nuclear ultrastructure, corona-cumulus expansion and capacity for sustaining embryonic development in vitro. An intermediate Group 3 with characteristics between Groups 1 and 2 was also assessed for in vitro development capacity. The CCOC's in Group 1 were typically meiotically unactivated and presented a nonundulating nuclear envelope. More than half of the CCOC's in Group 2 showed some degree of meiotic activation, and those that were nonactivated displayed "holes" in the nuclear envelope or dilatations of the perinuclear cisterna. The CCOC's in Group 2 were characterized by partial corona-cumulus expansion already at collection. The CCOC's from Groups 1 and 3 sustained embryonic development in vitro at a significantly higher rate than CCOC's from Group 2. It is concluded that CCOC's in which the corona radiata has the same density as the rest of the cumulus investment are less competent candidates for in vitro embryo production.

Transposition of the sacrotuberous ligament for the treatment of coxofemoral luxation in dogs.

A surgical technique is described for transposition of the sacrotuberous ligament to replace the teres ligament in the treatment of coxofemoral luxation in dogs. Ten dogs with coxofemoral luxation were treated using this technique and all animals regained full limb function within two months of surgery. It is suggested that the technique could be employed in dogs suffering from all types of hip luxations.

[An intramammary implant in the prevention of infectious mastitis in cows]. / Intramammálne teliesko v boji proti infekcným mastitídam kráv.

A sterile polyethylene device was introduced in the milk cistern of two mammary quarters of forty dairy cows. The cows were divided into three groups according to the length of exposure of the device in the cistern (3 days, 14 days, 365 days). The somatic cell counts were studied for a year in the first 10 ml fraction of milk and an increase in the somatic cell counts was found in the quarters having the intramammary device, as compared with the control quarters (having no devices). This difference was marked during lactation and prior to the onset of drying off. For the reduction in the frequency of occurrence of new natural intramammary infections, the activity of the device against S. aureus was limited and no activity against S. agalactiae was proved. The proportions of polymorphonuclears, round-cell elements and macrophages were histologically studied and compared for the udder quarters with and without the intramammary body in correlation with the time of exposure of the device to the milk cistern milieu. The most marked differences in favour of the udder quarters with the intramammary device were recorded in the alveoli containing more cells with a significant proportion of polymorphonuclear leucocytes. Small differences were found in the interstitial and subepithelial zone of the milk cistern. The activity of acid and alkaline phosphatase and adenosine triphosphatase was histochemically determined in the tissue structures of the udder and was found not to change under the influence of the device. Leucocytes and macrophages adhering to the surface of the body were observed under scanning electron microscope.

[Dynamics of ultrastructural morphology of the nucleolar apparatus in bovine preimplantation embryos collected in an area of chronic irradiation]. / Dynamická ultramikromorfológia nukleolárneho aparátu preimplantacných embryí kráv z oblasti chronického rádioaktívneho oziarenia.

Ultrastructural morphology and immunoelectron microscopy of the nucleus and nucleologenesis in early preimplantation cow embryos were applied in an attempt to demonstrate a possible radiation injury to that early stage of development due to chronical irradiation of the animals in the Tchernobyl area. Mostly eight cell embryos as well as morulae were collected from superovulated cows which were previously constantly kept in zones of different levels of radioactive irradiation. In addition to the normometric status of reproductive organs in no case was it possible to detect an apparent deviation in the nuclear morphology or in the process of nucleologenesis as compared to the physiological situation (Kopecný et al., 1989b, 1991, 1996). This observation was supported by an immunoelectron microscope study of DNA association and penetration in the differentiated nucleolus in the late 8-cell stage. These observations show that the otherwise demonstrated radiation injury localized in the genome does not probably influence markedly the early events of the developing embryo and that the aberrant cytoplasmic command of the nuclear events known in other types of oocyte/early cow embryo impairment (review Kopecný and Nicmann, 1993; Kanka et al. 1991; Pavlok et al., 1993) is not seen in early embryos collected from chronically irradiated animals.

[Electron microscopy immunolocalization of ribosomal ribonucleoproteins using autoantibodies from patients with systemic lupus erythematosus]. / Elektrónovomikroskopická imunolokalizácia ribozomálnych ribonukleoproteínov autoprotilátkami pacientov so systémovým lupus erythematosus.

Serum of patients against rRNP was tested by Western blot method. Four ribonucleoproteins (15, 16, 36 and 38 kDa) specifically reacted with the antiserum used. The autoantibodies were used for immunolocalization of antigenic structures by immunogold method on ultrathin cryosections of hepatocytes, cumulus cells of antral follicle and spermatocytes. Mice and hamsters were used. The cytoplasm appeared to be labelled most intensively. Gold particles were loosely arranged in the cytoplasm (cumulus cells) or they were associated with endoplasmatic reticulum (hepatocytes). Nucleoli were labelled by a lower incidence of the marker. Gold particles were found over the dense fibrillar component bordering on the granular component. In the cytoplasm individual gold particles were observed. (Fig. 7, Ref. 31.).

Fast ternary and quaternary breakup of the 197Au + 197Au system in collisions at 15 MeV/nucleon.

A new reaction mechanism of violent reseparation of a heavy nucleus-nucleus system, 197Au + 197Au, into three or four massive fragments in collisions at 15 MeV/nucleon has been observed. After reseparation, the fragments are almost exactly aligned, thus showing a very short time scale of the reseparation process, of about 70-80 fm/c.

Nucleolus in apoptosis-induced mouse preimplantation embryos.

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.

Nucleologenesis in the cleaving bovine embryo: immunocytochemical aspects.

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.

Effects of healthy pseudopregnant milieu on development of two-cell subdiabetic mouse embryos.

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.

Nuclear fragmentation: sampling the instabilities of binary systems.

We derive stability conditions of asymmetric nuclear matter (ANM) and discuss the relation to mechanical and chemical instabilities of general two-component systems. We show that the chemical instability may appear as an instability of the system against isoscalarlike rather than isovectorlike fluctuations if the interaction between the two constituent species has an attractive character as in the case of ANM. This leads to a new kind of liquid-gas phase transition, of interest for fragmentation experiments with radioactive beams.

Immunoelectron microscopic localization of small nuclear ribonucleoproteins and interchromatin granules in the 2-cell mouse embryo.

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.