First Report on Karyotypic, Morphometric and Meiotic Analysis of a Predatory Bombardier Beetle Pherosophus catoirai (Coleoptera: Carabidae) from Jammu region of Outer Himalayas, India

Chromosomal studies and manual karyotyping are the aged techniques for determining the identity of a species on evolutionary scale; however, these techniques are simple, reliable and inexpensive to authenticate the existence of a particular species. In the present work, the chromosome complement and meiotic processes of a predatory bombardier beetle Pherosophus catoirai were investigated. This species presented 2n=35 as diploid chromosome number and the chromosomal formula was found to be 12m+8sm+12st+X0. Sex mechanism was X0 type with metacentric X chromosome. Y chromosome was absent in this species. Karyotype revealed small chromosomes except X chromosome which is found to be largest in the spermatogonial metaphase stage. Meiotic stages were pachytene, diplotene, diakinesis and metaphase-I. Present study may find importance to analyse evolution of chromosomes in order Coleoptera particularly in family Carabidae.

Micronuclei in germ cells of hybrid frogs from Pelophylax esculentus complex contain gradually eliminated chromosomes

In most organisms, cells typically maintain genome integrity, as radical genome reorganization leads to dramatic consequences. However, certain organisms, ranging from unicellular ciliates to vertebrates, are able to selectively eliminate specific parts of their genome during certain stages of development. Moreover, partial or complete elimination of one of the parental genomes occurs in interspecies hybrids reproducing asexually. Although several examples of this phenomenon are known, the molecular and cellular processes involved in selective elimination of genetic material remain largely undescribed for the majority of such organisms. Here, we elucidate the process of selective genome elimination in water frog hybrids from the Pelophylax esculentus complex reproducing through hybridogenesis. Specifically, in the gonads of diploid and triploid hybrids, but not those of the parental species, we revealed micronuclei in the cytoplasm of germ cells. In each micronucleus, only one centromere was detected with antibodies against kinetochore proteins, suggesting that each micronucleus comprises a single chromosome. Using 3D-FISH with species-specific centromeric probe, we determined the role of micronuclei in selective genome elimination. We found that in triploid LLR hybrids, micronuclei preferentially contain P. ridibundus chromosomes, while in diploid hybrids, micronuclei preferentially contain P. lessonae chromosomes. The number of centromere signals in the nuclei suggested that germ cells were aneuploid until they eliminate the whole chromosomal set of one of the parental species. Furthermore, in diploid hybrids, misaligned P. lessonae chromosomes were observed during the metaphase stage of germ cells division, suggesting their possible elimination due to the inability to attach to the spindle and segregate properly. Additionally, we described gonocytes with an increased number of P. ridibundus centromeres, indicating duplication of the genetic material. We conclude that selective genome elimination from germ cells of diploid and triploid hybrids occurs via the gradual elimination of individual chromosomes of one of the parental genomes, which are enclosed within micronuclei.

Cytogenetic impact of sodium chloride stress on root cells of Vigna radiata L. seedlings

ObjectivesThe high salinization stress to seedling is the substantial ecological problem in the ongoing era. It negatively influences the growth that retard mitotic division by enhancing aberrations in nuclear chromatin. In the light of these views, the current work was designed to investigate the response of Vigna seedlings root tip cells to the presence of NaCl ions.Materials and methodsNM-92 and NM19-19 seeds were imbibed separately in distilled water for 24 h and allowed to grow into 0, 50, 150, 250, and 350 mM NaCl solution for 24 h. Excised root tips were stained, and slides were scored at 100× objective for the mitotic index (MI) and chromosomal aberrations.ResultsOur data demonstrated that as NaCl molarity increased, the MI was declined along with various chromatin abnormalities. The 150 mM of NaCl showed more lagging (69%) of chromosomes during anaphase in NM19-19. The highest stickiness at metaphase stage (68%) was found in 250 mM NaCl in variety NM19-19. However, both varieties were differed non-significantly for c-mitosis that was recorded 99% at 350 mM NaCl concentration.ConclusionsThe NaCl ions toxicity induced various cytological anomalies in seedling roots that adversely affect the growth of Vigna seedlings.

A Cytotaxonomic study on Minuartia L. (Caryophyllaceae) in Iraq

Chromosome count, karyotypic character analysis, meiotic studies, monoploid karyograms and ideograms were performed in six taxa of Minuartia growing in Iraq (M. hamata, M. hybrida subsp. hybrida, M. intermedia, M. meyeri, M. picta and M. hybrida subsp. turcica). Species of M. hamata and M. meyeri showed 2n=2x=30 chromosome number, while M. hybrida subsp. hybrida and M. intermedia were diploid (26). The chromosome number (n=x) of six species was studied, and was found to be n=15 in M. hamata and M. meyeri, 13 in M. hybrida and M. intermedia, while in M. picta we recorded values of n= 11 and 14. Karyotype analysis of this species was first carried out in our study. Analysis of metaphases showed that the karyotype formula was mainly metacentric, submetacentric, and sub acrocentric. The sizes of the chromosomes were mainly small and very small. The course of meiosis varied from normal to abnormal. Abnormal microsporogenesis formation of two bridge chromosomes was detected in M. hamata and one bridge chromosome in M. intermedia and M. meyeri. Formation of laggard’s chromosomes was detected in M. hamata, M. meyeri and M. intermedia. As well as ring chromosome was showed in M. hybrida subsp. hybrida, also, some cells contain triad cell in metaphase stage instead four cells, as well as founded cell, contains two nuclei in same species which led to reduced pollen fertility and differences in pollen grain size.

Centromeric CENP-A loading requires accurate mitotic timing, which is linked to checkpoint proteins

A defining feature of centromeres is the presence of the histone H3 variant CENP-A that replaces H3 in a subset of centromeric nucleosomes. In Drosophila cultured cells CENP-A deposition at centromeres takes place during the metaphase stage of the cell cycle and strictly depends on the presence of its specific chaperone CAL1. How CENP-A loading is restricted to mitosis is unknown. We found that overexpression of CAL1 is associated with increased CENP-A levels at centromeres and completely uncouples CENP-A loading from mitosis. Moreover, CENP-A levels inversely correlate with mitosis duration. We found that CAL1 interacts with the spindle assembly checkpoint protein and RZZ complex component Zw10 and thus constitutes the anchor for the recruitment of RZZ. Therefore, CAL1 controls CENP-A incorporation at centromeres both quantitatively and temporally, connecting it to the spindle assembly checkpoint to ensure mitotic fidelity.

Effect of antioxidant ascorbic acid on in vitro maturation of Caprine oocytes under normal and elevated temperatures

The aim of the present study was to assess the effect of ascorbic acid on in vitro maturation of caprine oocytes under normal and elevated temperatures. Goat ovaries were collected at slaughter and both A and B grade cumulus-oocyte-complexes (COCs) were aspirated out and were matured in vitro under normal (38.5°C) and elevated temperatures (41°C). On the basis of cumulus expansion and nuclear maturation, the maturation competencewere compared with and without ascorbic acid supplementation (100 µM). Heat stress significantly (P£ 0.01) reduced cumulus expansion, maturation rate and lowered metaphase stage II of nuclear maturation. Ascorbic acid improved developmental competence of oocytes during heat stress (41 °C) and ascorbic acid supplemented COCs demonstrated significantly (P£ 0.05) higher maturation rates when compared to non-supplemented groups.

Meiosis, embryonic, and larval development of the Black Sea scallop Flexopecten glaber ponticus (Bucquoy, Dautzenberg & Dollfus, 1889) (Bivalvia, Pectinidae)

The bivalve Flexopecten glaber ponticus is an endemic and extinguishing species of the Black Sea listed in Red book of Crimea. The sequence of meiosis stages following fertilization; embryonic and larval development of F. glaber ponticus were studied. The bivalve reproduces in June and July; these mollusks are simultaneous hermaphrodites. When spawning stimulated, fertilization of mature ova 54.7 ± 2.11 mkm in diameter take place at the first metaphase stage of meiosis. There were 12 bivalents with the sizes from 1.59 to 4.54 mkm on the metaphase plate. The duration of meiosis stage, embryonic and larvae development were determined. The structure of veliger provinculum was described. Right and left lock-valves consist of 4 teeth: two of them are on the front and back side on each valve. Measurements of larvae are presented. The description of shell and lock morphology will enable identification of F. glaber ponticus larvae at planktonic veliger stage. The data on duration of meiosis stage, embryonic and larvae development can be used in developing biotechnics of bivalve reproduction.

FahamecV1:A Low Cost Automated Metaphase Detection System

In this study, FahamecV1 is introduced and investigated as a low cost and high accuracy solution for metaphase detection. Chromosome analysis is performed at the metaphase stage and high accuracy and automated detection of the metaphase stage plays an active role in decreasing analysis time. FahamecV1 includes an optic microscope, a motorized microscope stage, an electronic control unit, a camera, a computer and a software application. Printing components of the motorized microscope stage (using a 3D printer) is of the main reasons for cost reduction. Operations such as stepper motor calibration, are detection, focusing, scanning, metaphase detection and saving of coordinates into a database are automatically performed. To detect metaphases, a filter named Metafilter is developed and applied. Average scanning time per preparate is 77 sec/cm2. True positive rate is calculated as 95.1%, true negative rate is calculated as 99.0% and accuracy is calculated as 98.8%.

THE QUANTITY OF CELLS NECESSARY FOR THE ANALYSIS OF CHROMOSOMAL ABERRATIONS AT RESIDENTS OF KUZBASS PATIENTS WITH CANCER OF LUNG

The pilot research executed in 2014 in selection of patients with lung cancer (LC) and healthy residents of Kemerovo region (338 Caucasians: 159 having LC and 179 healthy) has shown statistically significant distinction between groups of level and range of chromosome aberrations in blood lymphocytes. Patients of cancer detection centre (before treatment) had increased chromosome aberration values, both of chromatid and chromosomal types. 250 metaphases were analyzed for each individual. For accuracy test of the received quantitative characteristics of mutational process and definition required and enough number of cells necessary for the analysis, at essential increase in further selection volume, 50 LC patients (selected randomly), comparison of cytogenetic data has been carried out in the analysis of 200 cells, then 400, 600, 800, 1000 and over 1000 metaphases. Totally, in this subgroup 50 000 cells which were at a mitosis metaphase stage have been studied. Statistical data processing was carried out with the use of a software package for Windows Statistica 8.0. Using Mann-Whitney U-criterion it is confirmed that frequency of aberrant metaphases doesn't change significantly for statistics in case of increase in analyzed cells quantity; the analysis of 200 cells gives no less reliable information about the individual CA level, than the analysis of 1000 and more cells. In this regard it is possible to make the conclusion that 200 cells is necessary and enough number of analysed metaphases at the assessment of individual level and chromosomal aberrations' range for Kemerovo region residents who have lung cancer.

182 EFFECTS OF IGF-1 OR IGF-1-LongR3 ON CELLULAR AND MOLECULAR ASPECTS OF CUMULUS-OOCYTE COMPLEXES DURING IN VITRO OOCYTE MATURATION IN CATTLE

The insulin-like growth factor-1 recombinant -3 (IGF-1-LongR3), a synthetic analogue of IGF-1 with increased bioavailability has not yet been used in vitro maturation (IVM) medium of bovine oocytes. Therefore, the aim of this study was to evaluate and compare the addition effects of IGF-1-LongR3 or IGF-1 in IVM bovine oocytes on meiotic progression, apoptosis, and profile of oocytes genes (GDF9, BMP15, BAX, BCL2, OOSP1, IGFBP2, IGFBP4 and IGFBP5) and genes in cumulus cells (AREG, EGFR, FSHR, COX2, BAX, BCL2, IGFBP2, IGFBP4 and IGFBP5). Bovine ovaries were collected in slaughterhouses, and 739 oocytes with grades 1 or 2 were selected after aspiration of 2- to 8-mm follicles. IVM was carried out in TCM199 with FSH, LH, and antibiotics (BM) supplemented with 100 ng mL–1 IGF-1 or 100 ng mL–1 LongR3-IGF-1. Control oocytes were matured in BM supplemented with 0.1% polyvinyl alcohol (PVA) or 10% FCS. For all groups, maturation was performed during 22–24 h in an incubator at 38.5°C and 5% CO2 in air. Subsequently oocytes were denuded and analysed for apoptosis, nuclear maturation, and gene expression by TUNEL assay, staining Hoechst 33342, and RT-qPCR, respectively. Statistical analysis was performed using a linear mixed effects model, which correlated the change in metaphase stage 1 to 2 and the absence of apoptosis among the experimental groups. ANOVA and Tukey tests were used to analyse the results obtained by RT-qPCR. After 10 replicates of IVM, 339 oocytes were evaluated for meiotic progression and apoptosis and 400 oocytes for gene expression. There was no statistical difference between the experimental groups with respect to meiotic progression and apoptosis. BCL2 and IGFBP4 gene were less expressed in oocytes matured with IGF-1 and LongR3-IGF-1 compared with control groups. GFBP4 was also less expressed in cumulus cell of oocytes from the experimental groups. Moreover COX2 expression was statistically elevated in cumulus cells matured in the presence of IGF-1 and LongR3-IGF-1 It was possible to perform IVM of bovine oocytes in the presence of LongR3-IGF-1, allowing its use in replacement of IGF-1 and FCS. The results of this study will provide more information on the interaction of IGF with the IGFBP and its importance for oocyte maturation.