[Morphofunctional characterization and DNA stability of spermatozoa in primary examination of men fertility].

AIM: To assess the morphological and functional characteristics of spermatozoa and DNA stability in patients with normal seed parameters and with an ITZ exceeding the threshold value. PATIENTS AND METHODS: 85 males were studied for semen quality parameters. The morphological anomalies of head, middle piece, and tail of spermatozoa were estimated and presented in illustrations. Due to results elaborated the index of teratozoospermia was calculated. Then the Comet assay was performed. RESULTS: It has been found that spermatozoa from patients with high teratozoospermia index possessed decreased motility as compared to ones from patients with low index of teratozoospermia. It was not found any difference in concentration of spermatozoa between groups of males which possess low and high index of teratozoospermia. The spontaneous frequency of DNA damage estimated by means of single cell gel electrophoresis followed by silver staining in spermatozoa from healthy donors did not exceed 3,1+/-0,6. The frequency of DNA damages in sperm of patients with abnormal index of teratozoospermia exceeded 39,6+/-7,4% (p<0,01). CONCLUSIONS: Our results suggest that minor morphological changes in spermatozoa do not affect their motility, while significant morphological abnormalities with ITZ exceeding threshold values are closely associated with impaired DNA stability and dramatically reduce the quality of patients seed. SUMMARY: In the initial assessment of patient fertility the attention should be focused on a comprehensive study of sperm DNA morphology and stability.

Oxytocin-Induced Changes in Monoamine Level in Symmetric Brain Structures of Isolated Aggressive C57Bl/6 Mice.

Changes in activity of monoaminergic systems of the left and right brain hemispheres after administration of saline and oxytocin were studied in male C57Bl/6 mice subjected to social isolation. The concentrations of dopamine, norepinephrine, serotonin, and their metabolites dihydroxyphenylacetic, homovanillic, and 5-hydroxyindoleacetic acids were measured in the cerebral cortex, hippocampus, olfactory tubercle, and striatum of the left and right brain hemispheres by HPLC. In isolated aggressive males treated intranasally with saline, the content of serotonin and 5-hydroxyindoleacetic acid was significantly higher in the right hippocampus. Oxytocin reduces aggression caused by long-term social isolation, but has no absolute ability to suppress this type of behavior. Oxytocin reduced dopamine content in the left cortex and serotonin content in the right hippocampus and left striatum. Furthermore, oxytocin evened the revealed asymmetry in serotonin and 5-hydroxyindoleacetic acid concentrations in the hippocampus. At the same time, asymmetry in dopamine concentration appeared in the cortex with predominance of this transmitter in the right hemisphere. The data are discussed in the context of lateralization of neurotransmitter systems responsible for intraspecific aggression caused by long-term social isolation.

[The time course of experimentally altered thyroid status manifestations in female C3H-A mice].

The female C3H-A mice with agouty fur color were used to model hyper- and hypothyroidism in the long lasting experiment. The study was carried out for 44 weeks. Hyperthyroidism was induced by the administration of the L-thyroxine injections on alternate days during the whole period of the investigation. Hypothyroidism was achieved by adding propylthiouracil to the drinking water. The change of thyroid state was characterized by biphasic change in body weight. At the beginning of the experiment the hypothyroid animals were retarding by their weight. Otherwise the hyperthyroid animals were advancing by their weight. But since the 18th-21st week the initial trends changed, i. e. the hypothyroid mice body weight started ahead the hyperthyroid one. In the open field test both hypo- and hyperthyroid animals demonstrated the higher level of the investigating activity in comparison with the euthyroid mice. In the hyperthyroid mice the frequency of side-activity acts (grooming) increased significantly. Thus, the hyperthyroid animals appeared to be more anxious. To the 18th week of the experiment the animals of study groups started to demonstrate the apparent visual difference in their fur color. The hyperthyroid mice fur color became darker than one of the hypothyroid and the euthyroid mice. It is worthy of note that the hyperthyroid mice fur color was getting lighter than one of the euthyroid animals. The results are discussed in the context of hypothalamic-pituitary-thyroid axis functioning. The possible mechanisms of hormonal regulation of the fur color in mice are considered to include the hypothalamic-pituitary-thyroid axis hormones activities.

The incidence of breast tumor during experimental hyperthyroidism.

The incidence of breast tumor in inbred C3H-A mice (virgin female) from hyperthyroid, hypothyroid, and euthyroid control groups at week 44 of the experiment was 86.67, 30, and 62.5%, respectively. The percentage of metastatic lymph nodes in these groups was 65.9±16.7, 35.9±5.9, and 53.9±7.2%, respectively (p<0.005). It was shown that hyperthyroidism was associated with increased incidence and aggressiveness of breast tumor, while hypothyroidism was associated with lower incidence of breast tumor in comparison with the control.

[Clinical and immunological efficiency of sodium nucleospermate in treating chronic endometritis and infertility complicated by HPV infection].

Clinical efficacy of sodium nucleospermate (SNS) has shown in the treatment of 26 female patients with chronic endometritis, unsuccessful attempts at in vitro fertilization, and persistent viral infection. Cells from endometrium were processed by liquid cytology and studied by immunocytochemistry techniques. It has been clearly established that, after SNS treatment, the frequency of CD20- and CD56-positive cells was significantly decreased (p < 0.05) and the frequency of TLR4 and TLR9 expressing cells in endometrium has been significantly improved (p < 0.05). In addition, the human papilloma virus (HPV) load was reduced as manifested by the real-time PCR test. The obtained results show good prospects for successful use SNS as a means of pathogenetic therapy in patients with this diagnosis.

Sexual function in adult male rats after prenatal modulation of the cholinergic system.

Prenatal administration of the n-cholinolytic ganglerone to pregnant female rats at different periods of gestation was found to lead to long-term changes in sexual behavior in pubescent offspring: there was a reduced dynamic of acquiring sexual experience and a very low level of sexual activity, with significant impairment to the motivational and ejaculatory components of sexual behavior. The number of males with reduced sexual activity in the experimental groups was significantly greater than that in control offspring. The results obtained here provide evidence that impairments of sexual function in adult offspring induced by prenatal administration of the n-cholinolytic ganglerone at 9-11 and 12-14 days of gestation and, to a lesser extent, the m-cholinolytic metamyzil at 9-11 days of gestation, were due to impairment to the central mechanisms regulating sexual function due to stable changes in neurotransmitter activity in the hippocampus and hypothalamus, along with a significant reduction in the blood testosterone level.

Cells of rhabdomyosarcoma PA-23 tumor clones with high and low metastatic potential differ by activity of lysosomal sialidase.

We studied activity of lysosomal sialidase in cells of rhabdomyosarcoma PA-23 tumor clones with high and low metastatic potential. Low activity of lysosomal sialidase was found in clones characterized by high metastatic potential.

[The sexual function of mature rat males after prenatal modulation of cholinergic system].

The data obtained have shown that prenatal exposure of pregnant rat females of 9-19-day pregnancy to N-cholinolytics as compared to M-cholinolytics produce long-term behavioural changes in pubescent rat progeny. Pubescent rat progeny had low dynamics of gaining sexual experience and decreased sexual activity with equal disturbance of motivation and coitus. The number of males with absence of sexual activity was above that of the control group. We suggest that sexual dysfunction of offspring adulthood was provoked by introduction of ganglerone (N-cholinolytic) which had been injected on 9-11 and 12-14 days of gestation, and metamyzil (M-cholinolytic) injected on 9-11 days of gestation. Apparently, regulation of neuronal mechanisms for sexual function is disturbed as a consequence of lasting change in neurotransmitter activity. It is suggested that dopaminergic activity in brain limbic structures was affected the most. The significant decrease in blood testosterone values has also been elucidated.

[Micronuclei in mucose cells and colonization of human stomach epithelium with coccoid forms of Helicobacter pylori].

International Agency for Research on Cancer recognized as sufficient the evidence of Helicobacter pylori (HP) infection carcinogenicity and placed it into the 1 st group of carcinogens. Micronucleus level in gastric epithelial cells of antral stomach region of patients with chronic non-atrophy gastritis (n = 62) was studied. 40 patients of 62 had HP-associated gastritis. The HP-bacterium exists in a spiral and coccoid form. Both morphological forms were examined using immunocytochemistry. Significantly increased micronucleus number was observed in the cells of HP-infected patients compared with non-infected person (P < 0.05). The frequency of stomach epithelium cells with micronuclei was enhanced considerably in the patients infected with the coccoid HP form. Therefore the patients with HP-associated chronic gastritis caused by the coccoid form with high degree of colonization must be considered as a group of enhanced risk of gastric carcinogenesis.

[The study of DNA integrity in somatic cells and spermatozoa by single cell gel electrophoresis with silver staining].

In present work we studied DNA damage in human and bovine lymphocytes and spermatozoa by means of single cell gel electrophoresis followed by silver staining. The spontaneous frequency of DNA damage estimated manually in spermatozoa from healthy donors did not exceed 9% (on average -- 4.8 +/- 1.2%). The frequency of DNA damages in bull sperm after short (less than a year) and long period (more than 20 years) of cryopreservation was assessed as 3.1 +/- 0.9 and 4.3 +/- 0.5%, correspondingly. The comparative estimation of DNA damages in lymphocytes followed by silver staining is a valuable tool to estimate DNA damage in populations of somatic and reproductive cells.

[Structural changes of the nucleolar organizing regions in rhabdomyosarcoma RA-23 tumor cells induced by okadaic acid]. / Strukturnye preobrazovaniia interfaznykh IaOR v opukholevykh kletkakh rabdomiosarkomy PA-23, indutsirovannye okalaikovoi kislotoi.

It has been recently shown that okadaic acid (OA), an inhibitor of dephosphorilation, is capable of inducing changes in the nucleolar organizer regions (AgNORs) of some mammalian cells. Our work was focused on studying the structural changes in AgNORs of tumour cells of rat rhabdomyosarcoma RA-23 by their exposure to 100 nM OA. A standard silver staining procedure of interphase AgNORs in tumour cells was used. We measured no less than 100 tumour cells in each clone. In the examined tumour cell populations, the index of interphase AgNORs varied from 1.54 to 4.35. A clear structure and form of AgNORs was not observed in 30% okadaic acid-treated tumour cells, as opposed to 10% of the control ones. AgNORs in these cells looked like a mixture of thin threads encompassing some dark dots lying, mostly, separately. Additional procedures of tumour cell staining with Giemsa and fluorescent dye Hoechst 33,258, respectively, revealed that such structures were not chromosomes. Meanwhile, the frequency of cells at the stage of prophase exceeded 3%, as opposed to the control, where the frequency of cells at this stage was less than 0.5%. Thus, we can conclude that we have detected specific changes in AgNORs and chromatin structure of okadaic acid-treated tumour cells.

[Effect of cycloferon on dissemination of Lewis lung carcinoma in mice and rhabdomyosarcoma ra-23 in rats]. / Vliianie tsikloferona na metastazirovanie kartsinomy l]egkikh Lewis u myshei i rabdomiosarkomy ra-23 u kryc.

Tumor growth and proliferative activity of tumor cells were suppressed and the number of pulmonary metastases in C57B16 mice decreased 3.3-fold following seven injections of cycloferon (100 mg/kg body) to induce interferon production. Injections were carried out 1-16 days after subcutaneous transplantation of Lewis lung carcinoma. After mice were immunized with ovine red blood cells, cycloferon administration raised thymus-dependent humoral immune response. After eight injections of cycloferon (50 mg/kg body) into rats, from day of intravenous transplantation of rhabdomyosarcoma RA-23 until day 20, no significant effect on metastasizing into the lung was recorded. However, single injection of cyclophosphamide 50 mg/kg inhibited metastasis formation. The highest suppressor effect was registered with combination cycloferon-cyclophophamide treatment: mean weight of metastasis decreased by half, as compared with treatment with cyclophosphamide alone. Both drugs caused karyotypical abnormalities to occur in metastatic cells. Tumor growth and spreading suppression after cycloferon should be attributed to cytotoxic antitumor action, cell proliferation inhibition and immunomodulating effect.

[Chromosome bridges and tailed nuclei in malignant cell populations]. / Khromosomnye mosty i "kvostatye" iadra v populiatsiikh zlokachestvennykh kletok.

Cell clones of the finite organospecific rat rhabdomyosarcoma RA-23 were selected in vivo for high and low frequency of interphase cells with chromosome bridges. After selection for high frequency of cells with bridges, the frequency of cells with anomalies of nuclear form sharply increased in the cell populations. These anomalies were manifested by long nuclear protrusions into the cytoplasm. This type of anomaly was termed tailed nuclei. In the studies populations of RA-23, the frequency of cells with "tailed" nuclei positively correlates with the frequency of interphase cells with chromosome bridges and the frequency of ana- and telophases with chromosome bridges. These parameters might be genetically associated: dicentric chromosomes form chromosome bridges in ana- and telophases of mitosis. Then, in some cases, the bridges are maintained, which result in the appearance of interphase cells with bridges, and, in other cases, the bridges are ruptured, which result in the appearance of cells with tailed nuclei.

AgNORs in cardiomyocytes from surgical patients with coronary heart disease.

AIM: To evaluate the interphase ribosomal RNA cistron activity of cardiomyocytes in surgical patients with chronic ischaemic heart disease by means of the nucleolar organiser region silver staining (AgNOR) technique. METHODS: Nucleoli were investigated in myocardial samples obtained from 46 patients with chronic ischaemic heart disease before, during, and soon after cardioplegia ischaemia. Cryostat sections of 10 microns thickness were air dried, fixed in methanol/glacial acetic acid (3:1) for 15 minutes, rinsed carefully with distilled water, incubated in 2 N formic acid for 10 minutes, and impregnated with silver colloid solution for 2.5-3 minutes at 68-70 degrees C. The lightly counterstained sections were examined under oil immersion at x1000 magnification. For the estimation of AgNOR numbers at least 100 silver stained cardiomyocyte and fibroblast nuclei were counted in each section. On the basis of these data, the mean number of AgNORs in each nucleus was determined. The Student's t test was used to compare the groups tested. RESULTS: The initial mean numbers of AgNORs varied greatly, demonstrating a difference between groups of patients with or without antecedent myocardial infarction (9.5 v 11.0; p < 0.05). During myocardial arrest, the numbers of AgNORs in cardiomyocytes were decreased in all but seven patients, while those in fibroblasts tended to increase. At the stage of reperfusion and myocardial warming, in all but three patients the numbers of AgNORs in cardiomyocytes either normalised or were even higher than the initial value. CONCLUSIONS: The AgNOR count in cardiomyocytes is a very sensitive test for the measurement of cardiac function in surgical patients with chronic ischaemic heart disease and could be useful for monitoring myocardial status during the course of surgery, including cardioplegia. The high risk group for surgery included patients with antecedent myocardial infarction and severe heart failure. It is thought that a reversible nucleolin/fibrillarin/pre-rRNA/small nucleolar RNA modification might account for this fast decline then rise in the AgNOR count in cardiomyocytes at the stages of cardioplegia and reperfusion, respectively.

[Selection in vivo in cell populations of rat RA-23 rhabdomyosarcoma at the trait "frequency of cells with bridges"]. / Selektsiia in vivo v kletochnykh populiatsiiakh rabdomiosarkomy RA-23 krys po priznaku "chastota kletok s mostami".

Selection in vivo of cell clones of rat transplantable organospecific rhabdomyosarcoma RA-23 for increased and decreased frequencies of cells with chromosomal bridges (FCB) was performed. The initial average FCB in clones was 0.8% at a range of variation of from 0 to 3.0%. Selection for an increase, as well as for a decrease, in the FCB in RA-23 clones was effective. After one step of selection for an increase in the FCB, the average FCB increased to 3.0%. Thereafter, selection for an increase in the FCB was impracticable due to loss of transplantability in cell populations in which the FCB exceeded 5.0%. Over five steps of selection for a decrease in the FCB, the average FCB significantly decreased to 0.3% (P < 0.001). The heritability coefficient h2 of the trait FCB upon selection ranged from 0.25 to 0.30. The high and low FCBs attained by selection correlated with the index "frequency of cells with micronuclei." The population of RA-23 cells after selection for a decrease in the FCB differed from the population of cells of the original RA-23 strain by significantly lower karyotypic heterogeneity. The values of h2 obtained upon selection for the FCB and the effectiveness of selection for an increase, as well as for a decrease, in the FCB show that, with selection at the trait "frequency of cells with bridges," which characterizes the stability of the karyotype, existing cell strains can be subjected to karyotypic stabilization or destabilization.