Chromosomes In Metaphase Plate Research Articles

168 EFFECT OF WORTMANNIN IN IN VITRO MATURATION OF BOVINE OOCYTES

It has been shown that phosphatidylinositol 3 kinase (PI3K) participates in oocyte maturation by regulating the activity of important reactions related to the resumption of meiosis and energy metabolism. Changes in the enzyme activity caused by in vitro conditions may impair important events of oocyte maturation and consequently the production of blastocysts. The study aimed to verify the effect of the addition of wortmannin (a specific inhibitor of PI3K) to the in vitro maturation medium on nuclear and cytoplasmic maturation of bovine oocyte, as well as on the production of blastocysts. Cumulus-oocyte complexes (COCs) were matured in vitro in medium supplemented with FCS and 0 (control) or 20 nM of wortmannin and then fertilized and cultured in vitro in the absence of inhibitor. Twenty-two hours after in vitro maturation the determination of PI3K activity of oocytes by Western blot was performed using anti-PI3K subunit P85 antibody/peroxidase. The activity quantification was performed by densitometry of the bands using the Gel Perfect software (Bozzo and Retamal 1991 Arch. Biol. Med. Exp. 63, 510). To check the effect of treatment on energy metabolism, glucose and glycogen concentration of oocytes was quantified by Glucox 500 test (Doles Reagentes e Equipamentos Para Laboratórios Ltd., Goiânia, Brazil). The oocyte viability determination was performed by double labelling with propidium iodide and calcein AM. To determine the nuclear maturation, the oocytes were stained with 2% acetic orcein, being considered matured those with chromosomes in metaphase plate. To assess the meiotic spindle organisation, the oocytes were labelled with anti-α tubulin-FITC and propidium iodide. The distribution of actin filaments and mitochondria was determined by rhodamine-phalloidin labelling. The distribution of cortical granules was observed by labelling the oocytes with Lens culinaris–fluorescein isothiocyanate (FITC). The cleavage and blastocyst rates were determined at 48 and 168 h post-fertilization, respectively, both calculated on the number of oocytes placed to mature. The treatment means were compared by t-test (SAS Institute Inc., Cary, NC, USA, 2003). Wortmannin produced a reduction around 40% of PI3K activity; however, the levels of glucose and glycogen were not altered. No changes were found in the viability of in vitro matured oocytes or in nuclear maturation rate (P ≥ 0.05). Treatment did not promote any change in the organisation of meiotic spindle, distribution of actin filaments, and positioning of mitochondria. However, oocytes treated with wortmannin showed a significant increase (P ≤ 0.05) in the migration of cortical granules compared with controls (87.4% ± 11.4 and 72.8 ± 11.8%, respectively). The cleavage rate was not influenced by treatment, but the blastocyst rate was higher when oocytes were matured in presence of wortmannin (34.2 ± 6.4% v. 20.0 ± 5.0%, respectively; P ≤ 0.05). The results indicate that partial inhibition of PI3K activity in bovine oocytes treated with wortmannin during in vitro maturation did not affect nuclear maturation, but improved the migration of cortical granules, which seems to have contributed to the increased the blastocyst rate.

Karyotypes of Garra rufa and G. ghorensis (Pisces, Cyprinidae) inhabiting the inland water systems of the Jordan basin

The karyotypes of two cyprinid species Garra rufa and G. ghorensis (Cyprinidae), belonging to the inland ichthyofauna of Israel and Jordan, were studied. For both species, diploid numbers (2n) were represented by 50 chromosomes. The number of chromosome arms (NF) was relatively similar in both species and varied from 86 (76+10m) to 92 (80+12m), depending on the degree of condensation of chromosomes in metaphase plates. In both species, karyological examinations indicated some morphological differences between female and male karyotypes.

Chromosome stickiness during meiotic behavior analysis of Passiflora serrato-digitata L. (PassifloraCEAE)

Almost 90% of species of the genus Passiflora are native to the American continent, with high commercial value due to the fact that some species are used for human food while others have ornamental and medical qualities. Passiflora serrato-digitata is one of the species that integrates the Paraná Agronomic Institute germoplasm bank at its experimental base in Londrina, PR, Brazil. Collected flower buds were fixed in ethanol/acetic acid (3:1 v/v) for 24h, transferred to 70% alcohol and stored under refrigeration. Slides were prepared by the squashing technique and stained with 1.0% propionic carmine; they were analyzed under an optic microscope. Irregularities in the chromosome segregation process of P. serrato-digitata have been verified by meiotic behavior analysis. These comprised precocious migration to poles in metaphase I and II, non-oriented chromosomes in metaphase plate in metaphase I and II, laggard chromosomes in anaphase I and II towards the formation of micronucleus in telophase I and II, and microspores in tetrads. Chromosome stickiness was another irregularity reported in the Passiflora genus for the first time. These irregularities which also contributed to the formation of monads, dyads and triads, resulted in normal imbalanced 2n and 4n microspores. According to the observed Meiotic Index of 71.83%, this species is not meiotically stable.

Induction of meiotic gynogenesis in paddlefish (Polyodon spathula) and its confirmation using microsatellite markers

A protocol for gynogenesis of P. spathula was developed in order to produce inbred lines and to develop a technique to preserve the endangered Chinese paddlefish (Psephurus gladius). Diploid gynogenesis was induced in P. spathula using ultraviolet (UV)-irradiated Amur sturgeon (Acipenser schrenckii) sperm for 4.5-min, 5-min or 5.5-min and subsequent heat shock treatment. UV irradiation of sperm for 5 min at a UV intensity (254 nm) of 863 μw cm−2 was the optimum dose to achieve diploid gynogenesis on the basis of observations on hatching rate of eggs. Three microsatellite loci were used to monitor exclusive maternal inheritance of gynogenetic progenies. The results showed all maternal genome among offspring with no paternal genome. The cytogenetic analysis showed that meiotic gynogenetic diploids possessed 120 chromosomes in metaphase plates, while haploid control groups N1–3 possessed 60 chromosomes.

Localization of AT-clusters in normal mouse chromosomes by netropsin prefixation treatment in vitro

Fetal fibroblasts of the NMRI mouse strain were treated in vitro with the oligopeptide antibiotic netropsin for different time periods before harvest. Netropsin led to centromeric elongation in all chromosomes of the murine karyotype except chromosome no. 1 and the Y chromosome. Only the centromeric C-bands are affected. Since netropsin is a specific probe for AT-clusters in duplex DNA and because netropsin linkage changes DNA conformation, the results show that AT-rich DNA is preferentially localized in the C-bands of the mouse karyotype. This confirms the data on hybridization of RNA complementary to light satellite DNA in this species. Differences in the extent of centromeric elongation by netropsin suggest that the quantity of AT-clusters varies between different chromosome pairs. Furthermore, netropsin pretreatment influences the orientation of chromosomes in metaphase plates.

Computer graphics of SEM images facilitate recognition of chromosome position in isolated human metaphase plates.

There is general agreement that at the time of mitosis chromosomes occupy precise positions and that these positions likely affect subsequent nuclear function in interphase. However, before such ideas can be investigated in human cells, it is necessary to determine first the precise position of each chromosome with regard to its neighbors. It has occurred to us that stereo images, produced by scanning electron microscopy, of isolated metaphase plates could form the basis whereby these positions could be ascertained. In this paper we describe a computer graphic technique that permits us to keep track of individual chromosomes in a metaphase plate and to compare chromosome positions in different metaphase plates. Moreover, the computer graphics provide permanent, easily manipulated, rapid recall of stored chromosome profiles. These advantages are demonstrated by a comparison of the relative position of group A-specific and groups D- and G-specific chromosomes to the full complement of chromosomes in metaphase plates isolated from a nearly triploid human-derived cell (HeLa S3) to a hypo-diploid human fetal lung cell.

Distribution of chromosomes in metaphase plates of Mesocricetus newtoni.

SUMMARYFifty-one metaphase figures from male Mesocricetus newtoni bone-marrow cells were analysed with respect to the distribution of chromosomes. The peripheral location of the sex chromosomes (XY) and that of the chromosome pairs 3 and 9, as well as the non-peripheral location of the chromosome pairs, 16, 17, 18 was revealed. The significance of the peripheral location of the X chromosome and a possible explanation of the characteristic distribution of the chromosomes are discussed.