Evaluation of developmental competence of Sus scrofa domesticus (L.) oocyte-cumulus complexes after intra- and extraovarian vitrification

The aim of the present study was to identify the influence of extra- (EOV) and intraovarian vitrification (IOV) on mitochondrial activity (MA) and chromatin state in porcine oocytes during maturation in vitro. During EOV porcine oocytes were exposed in cryoprotective solutions (CPS): CPS-1 – 0.7 M dimethyl sulfoxide (DMSO)+0.9 M ethylene glycol (EG); CPS-2 – 1.4 M DMSO+1.8 M EG; CPS-3 – 2.8 M DMSO+3.6 M EG+0.65 M trehalose. At IOV the ovarian fragments were exposed in CPS-1 – 7.5 % EG+7.5 % DMSO, then in CPS-2 – 15 % EG, 15 % DMSO and 0.5 M sucrose. Straws with oocytes and ovarian fragments were plunged into LN2 and stored. For devitrification, the EOV oocytes were washed in solutions of 0.25, 0.19 and 0.125 M of trehalose, the IOV – in 0.5 and 0.25 М trehalose. Oocytes were cultured in NCSU-23 medium with 10 % fluid of follicles, follicular walls, hormones. 0.001 % of highly dispersed silica nanoparticles (ICP named after A.A. Chuyko of the NAS of Ukraine) were added to all media. The methods of fertilization and embryo culture are presented in the guidelines developed by us. MA and chromatin state were measured by MitoTracker Orange CMTMRos and the cytogenetic method. Significant differences in the level of oocytes with high-expanded cumulus between control and experimental vitrified groups (81 % versus 59 % and 52 %, respectively, p ≤ 0.001) were observed. The percentage of pyknotic cells in native oocytes was 19 %, EOV or IOV oocytes were 39 % and 49 %, respectively. After culture, the level of matured native oocytes was 86 %, 48 % EOV and 33 % IOV cells finished the maturation (p ≤ 0.001). Differences were also observed in the level of MA between groups treated by EOV and IOV (89.4±7.5 µA and 149.2±11.3 µA, respectively, p ≤ 0.05). For the first time, pre-implantation embryos were obtained from oocytes treated by IOV.

LOCALIZATION OF MITOCHONDRIA IN DONOR OOCYTES OBTAINED FROM COW OVARIES POST MORTEM

Проведен сравнительный морфофункциональный мониторинг популяции донорских ооцитов, выделенных из яичников коров post mortem на разных стадиях овариального цикла, с учетом статуса хроматина и интрацитоплазматической локализации митохондрий ооцит-кумулюсных комплексов. Анализу подвергались ооциты, полученные из антральных фолликулов (Ø 2—6 мм), окруженные не менее, чем 5—6 слоями клеток кумулюса. Показано, что 45% и 50% ооцитов, выделенных из яичников на стадии фолликулярного роста и развитого жёлтого тела, соответственно, имеют периферическую локализацию митохондрий в ооплазме. Доля созревших ооцитов с периферической и равномерной локализацией митохондрий на момент аспирации их из яичников составила 88 и 85% соответственно, а уровень созревших ооцитов в группе с кластерным типом локализации достиг лишь 66%. В группе ооцитов с кластерной локализацией митохондрий отмечен высокий уровень ооцитов с дегенерированным хроматином после 24 ч культивирования (90%) по сравнению с группами с периферической и равномерной локализацией митохондрий (2 и 41% соответственно, P < 0,001). Ооциты с периферической локализацией митохондрий характеризовались гомогенной ооплазмой (58%). Гаметы с кластерным распределением митохондрий в основном имели гетерогенную ооплазму (52%), а клетки с равномерным распределением — гомо- и гетерогенную ооплазму (29 и 36% соответственно). A comparative monitoring of the population of cow donor oocytes isolated from ovaries at different stages of the ovarian cycle was carried out, the chromatin status and the intracytoplasmic localization of mitochondria of oocyte-cumulus complexes were evaluated. Oocytes obtained from antral follicles (Ø 2-6 mm) were analyzed. Gametes were cultured at a temperature of 38.5°C, in an atmosphere with 5% CO2, 90% humidity for 24 hours in T-199 medium supplemented with 10% fetal bovine serum, 106 granulosa cells/ml, 50 ng/ml bovine prolactin. Mitochondria were visualized with using of vital dye rhodamine 123, the chromatin status was assessed by Tarkowski`s cytogenetic method. It was demonstrated that 45% and 50% of oocytes isolated from the ovaries in the stage of follicular growth and developed corpus luteum had a peripheral localization of mitochondria in ooplasm. The proportion of mature oocytes with the peripheral and spread localizations of mitochondria were 88% and 85%, respectively, while the maturation rate of oocytes with the mitochondrial clusters reached only 66%. Among oocytes with cluster distribution of mitochondria a high percent of cells with signs of chromatin degeneration after 24 h culture was noted (90%) compared with groups of oocutes with peripheral and spread distribution (2% and 41%, respectively, P<0,001). Oocytes with the peripheral localization of mitochondria were characterised by homogeneous ooplasm (58%). Gametes with the clusters mostly had a heterogeneous ooplasm (52%), and cells with the spread distribution – homo- and heterogeneous ooplasm (29% and 36%, respectively).

Polyploidy in the Ginger Family from Thailand

Polyploidy is common in the ginger family Zingiberaceae. The aims of the present paper are (1) to provide a general introduction on species diversity with emphasis on conservation; (2) to highlight the human-use significance of this family, focusing on the two major genera, Zingiber (ginger) and Curcuma (turmeric); (3) to present chromosome number data from 45 natural and cultivated Curcuma taxa from Thailand, of which polyploids are predominant; and (4) to describe our own work on cytotaxonomy of selected Thai Curcuma species. We obtained somatic chromosome numbers from root tips and analysed meiotic chromosome behaviour from flowers. We also used the molecular cytogenetic method of ribosomal gene mapping on chromosomes to infer mechanism of polyploidization and reveal genomic relationships among closely related species. The main results of our cytogenetic studies include the following. The most sought-after medicinal Curcuma cultivars growing on a large-scale basis are secondary triploids, so as taxa in natural habitats that are harvested for local utilisation. These triploids are sexually deficient, due to meiotic pairing abnormalities, but they are propagated asexually via rhizomes. The ribosomal mapping results indicate natural triploidization process via hybridisation, either within populations or across the species boundaries.

FUNCTIONAL STATE OF NEUTROPHIC GRANULOCYTES’ GENOME OF THE PERIPHERAL BLOOD IN PATIENTS WITH CHRONIC HEPATITIS C WITH CONCOMITANT DIABETES MELLITUS TYPE II

The aim: To determine changes of FSG of neutrophilic granulocytes of peripheral blood (NGPB) of patients with CHC with concomitant DM-2. Materials and methods: We’ve examined 180 patients with CHC: 160 with concomitant diabetes mellitus and 20 ones without it. The NGPB genome was studied using cytogenetic method. There were analyzed 100 interphase NGPB nuclei in the preparation, structural characteristics were evaluated according to indices: chromatization (IC), nucleolar (IN), pathologically altered nuclei (PAN), micronuclei (MNI). Results: Violations of FSG OF NGPBwere found according to all indices in patients with CHC, they were more pronounced in patients with concomitant DM-2. Conclusions: FSG NGPB is more disordered in CHC with concomitant DM-2. The reduction of IC in CHC with concomitant DM-2 is more pronounced in men. Reduction of IN in patients with CHC with and without DM-2 is a marker of violations of the second stage of realization of hereditary information. The tendency to change the components of the cytogenetic status of all examined patients due to the frequency of MNI was determined.

A Comparison of Ice Cold Water Pretreatment andα-Bromonaphthalene Cytogenetic Method for Identification ofPapaverSpecies

The plants belonging to many species in genus Papaver are very similar and have very small chromosomes that make identification very difficult. The study aimed to compare the effects ofα-bromonaphtalene and ice cold water pretreatment to identify chromosomes of Papaver species collected from different areas of Iranian West Azerbaijan and Turkish Van, Agri and, Hakkari provinces. The seeds were germinated in Jacobson trays at 24°C under continuous light. Thereafter, roots from 1.5 cm long plantlets were pretreated withαbromonaphtalene for 15, 30, and 45 min or at 0°C in ice cold water for 24 h before fixing, hydrolyzation, and feulgen staining. The ice cold water pretreatment was more appropriate and easy to determine chromosomes. Seeds from seven samples did not germinate. Sixty samples out of the rest of 62 samples were identified asP. pseudo orientale, one sample was identified asP. bracteatum, and another asP. orientale. This is the first study that used ice cold water to determine the chromosomes in papaver species. It is hoped that it will also facilitate to determine chromosome number and identify other papver species.

Approach to estimate mutagenic effect of polluted water by cytogenetic method on bioindicator species Asellus aquaticus (Isopoda)

Elevated frequency of chromosomal aberrations revealed by ana-telophase method in ponds and lakes corresponds to higher degree of anthropogenic pressure. Data obtained are compared with the influence of low-dose of ionizing radiation. Validity of the model for estimation of pollution degree and its mutagenic influence risk for human being is discussed.