Analysis for pheromone-induced cytogenetic disturbances depending on major urinary proteins of male laboratory mice

Young male CBA/LacStoRap mice for 2 h were exposed to pheromones that are transferred by major urinary proteins of sexually mature males of the same line. The treatment was conducted using either a complete set of the major urinary proteins typical of the CBA mice or incomplete set of protein fractions detected in some animals. The effect of pheromones was estimated 24 h after treatment by cytogenetic analysis for disturbances in dividing bone marrow cells at anaphase--telophase and in germ cells at metaphase I. The frequency of both mitotic and meiotic disturbances was significantly increased after exposure to pheromones associated with the complete spectrum of the major urinary proteins. Conversely, no cytogenetic effect was observed in the absence of particular protein fractions. Possible consequences of the pheromonal effect on the genomes of somatic and germ cells are discussed as well as the relationships between pheromones and the major urinary proteins.     

Induction of meiotic disturbances in spermatocytes I by pheromones as an inhibiting mechanism of male reproductive function in house mice

The influence of pheromons on reproduction and other important physiological characteristics has been reported for many mammalian species. However, mechanisms of this action at the level of target cells still remain unclear. A study was made of the influence of non-identified pheromones from adult males and a female pheromone 2,5-dimethylpyrazine on germ cells of CBA inbred strain mice. Cytogenetic analysis shows a significant increase in such meiotic disturbances as multivalent associations and autsomal univalents 24 h after exposure to pheromonal cues. Results of in situ hybridization show that the level of c-fos and c-jun expression is significantly higher 3.5 h after exposure to pheromones of adult males. It is likely that destabilization of chromosomal apparatus in dividing meiotic cells forms the basis of some reproductive effects of murine pheromones. Possible mechanisms of pheromone influence on reproduction are discussed.     

The pattern of major urinary proteins (MUPS) expression during postnatal ontogenesis of the laboratory mouse depends on genotype and sex

We investigated the specific pattern of major urinary proteins (MUPs) expression in 3-, 4-, and 12-week old mice of CBA/LacY and C57BL/6JY inbred strains using polyacrylamide gel electrophoresis. Quantitative evaluation of 8 protein fractions A-H with regard to sex, age, and genotype of the animals is presented for the first time. Actual problems of genetic control and neuroendocrine regulation of MUPs expression during ontogenesis are discussed. In the light of current views on MUPs as a key component in intrapopulation information exchange via pheromones, we put forward the idea that the genetically determined structure of the olfactory code of the definitive type is formed at an early ontogenetic stage on the basis of the MUPs combinatorial pattern.     

The balance hypothesis of the effect of socially important volatile chemosignals on reactivity of chromosome machinery of bone marrow dividing cells in the house mouse <Emphasis Type="Italic">Mus musculus</Emphasis>

Volatile chemosignals released by female CBA mice are shown to affect the chromosome machinery of bone marrow cells in mature syngenic males in different ways depending on the experimental conditions. Chemosignals excreted by solitary adult females decrease the frequency of mitotic disturbances in bone marrow dividing cells of male recipients as compared with the spontaneous level in control animals. At the same time, 2,5-dimethylpyrazine, a pheromone released only by females caged at high densities, increases the frequency of mitotic disturbances. A preliminary 24-h treatment of males with chemosignals excreted by solitary females reduces the effect of a subsequent exposure to 2,5-dimethylpyrazine, however, the frequency of disturbances is still higher than that in the control. The simultaneous exposure to both chemosignals results in complete neutralization of the 2,5-dimethylpyrazine effect, and the frequency of mitotic disturbances does not differ from that observed after the exposure to solitary female chemosignals. It is hypothesized that the cytogenetic effects found in male recipients depend on the social housing conditions under which female chemosignal donors were kept.     

The Female Pheromone 2,5-Dimethylpyrazine Induces Sperm-Head Abnormalities in Male CBA Mice

The effect of the house mouse female pheromone 2,5-dimethylpyrazine (2,5-DMP) on sperm differentiation in male CBA mice has been studied. For this purpose, mature males were treated with a 0.01% aqueous solution of the pheromone for six days. Control mice were similarly treated with physiologic saline. The mice were sacrificed 23 days after the treatment, and material for the analysis of sperm-head abnormalities was sampled from the caudal portion of the epididymis. Analysis of the frequency of abnormal sperms has demonstrated that the pheromonal treatment significantly increases the frequencies of various sperm-head abnormalities. Apparently, this results from disturbances in germ cell differentiation caused by the induction of genetic damage at stages immediately preceding meiosis, as well as during the first and second meiotic divisions. The relationship between the effect of 2,5-DMP and the decrease in the fertility of male CBA mice that was earlier observed after a similar treatment is discussed.     

Myelotoxicity of epirubicin]

Epirubicin (pharmorubicin, India), an antitumor antibiotic of the anthracycline group, was studied in regard to its effect on peripheral blood, bone marrow and lymphoid organs (the thymus and spleen) of CBA mice after its intraperitoneal administration in a single dose equal to the MIC (7.8 mg/kg) and in a course dose (1/5 of the MIC 5 times a day). The cytogenetic impairments induced by the cytostatic were estimated on metaphase plates with the bone marrow specimens and by counting the peripheral blood erythrocytes with micronuclei (the micronuclear test). It was shown that epirubicin induced cytogenetic disturbances in the hemopoietic cells within the first 72 hours. The antibiotic had a marked reversible effect on the erythroid population and lymphoid tissues and a moderate toxic action on the granulocyte population. The antibiotic did not affect thrombocytopoiesis. The single administrations had a more pronounced and prolonged myelotoxic and lymphotropic effect.     

Effect of the estrous cycle stage on sensitivity to pheromone 2,5-dimethylpyrazine in the house mouse Mus musculus

Frequency of cytogenetic disturbances was estimated in mitotically dividing bone marrow cells of CBA strain female mice after the 24-h long action of pheromone 2,5-dimethylpyrazine (2,5-DMP). The stage of the estrous cycle of each animal was taken into account at the moment of the end of the pheromone action. The analysis was performed using the anatelophase method that allows evaluating frequencies of various types of disturbances--bridges, fragments, delayed chromosomes. The spontaneous level of the mitotic disturbances revealed by the anatelophase method in animals of the control group amounts to 5.4 %. Action of pheromone 2,5-dimethylpyrasine induced the mitosis disturbances detected in the dividing bone marrow cells at the anaphase-telophase stage in the females at the di- + postestrus stage. The corresponding frequency of disturbances after the pheromone action was equal to 9.2%. In the female in estrus, the mitotic disturbance level amounted 6.7%, which did not differ statistically significantly from control. It is suggested that differences in the female mouse hormonal state at different estrous cycle stages affect sensitivity to olfactory signals. Mechanisms of the revealed effect and significance of the differences in sensitivity to pheromone for reproductive processes are discussed.     

Effect of the estrus cycle stage on sensitivity to pheromone 2,5-dimethylpyrazine in the house mouse <Emphasis Type="Italic">Mus musculus</Emphasis>

Frequency of cytogenetic disturbances was estimated in mitotically dividing bone marrow cells of CBA strain female mice after 24-h long action of pheromone 2,5-dimethylpyrazine (2,5-DMP). The stage of estrus cycle of each animal was taken into acount at the moment of the end of the pheromone action. The analysis was performed using the anatelophase method that allows evaluating frequencies of various types of disturbances—bridges, fragments, delayed chromosomes. The spontaneous level of the mitotic disturbances revealed by the anatelophase method in animals of the control group amounts to 5.4%. Action of pheromone 2,5-dimethylpyrazine induced the mitosis disturbances detected in the dividing bone marrow cells at the anaphase-telophase stage in the females at the di-+ postestrus stage. The corresponding frequency of disturbances after the pheromone action was equal to 9.2%. In the female in estrus, the mitotic disturbance level amounted 6.7%, which not differed statistically significantly from control. It is suggested that differences in female mouse hormonal state at different estrus cycle stages affect sensitivity to olfactory signals. Mechanisms of the revealed effect and significance of the differences in sensitivity to pheromone for reproductive processes are discussed.     

Balance hypothesis of action of socially significant volatile chemosignals on reactivity of chromosome machinery of the bone marrow dividing cells in the house mouse Mus domesticus L

Volatile chemosignals released by female CBA laboratory mice have been shown to produce action of different direction, depending on conditions of performance of experiment, on chromosome machinery of bone marrow cells in syngenic adult males. Thus, chemosignals secreted into environment by isolated adult females decrease frequency of mitotic disturbances in bone marrow dividing cells in male recipients as compared with spontaneous level in control animals. At the same time, 2,5-dimethylpyrazine - pheronome released only by high density caged females - increases frequency of mitotic disturbances. Preliminary 24-h-long action of chemosignals of isolated females decreases effect of the subsequent action of 2,5-dimethylpyrazine, although the level of disturbances exceeds that in control animals. The simultaneous action of used chemosignals neutralizes completely the 2,5-dimethylpyrazine action, the frequency of mitotic disturbances being not different from that after chemosignals of isolated females. The hypothesis is put forward about dependence of the revealed cytogenetic effects in male recipients on zoosocial conditions of maintenance of female donors of chemocommunication signals.     

The female pheromone 2,5-dimethylpyrazine induces sperm head abnormalities in male CBA mice

The effect of the house mouse female pheromone 2,5-dimethylpyrazine (2,5-DMP) on sperm differentiation in male CBA mice has been studied. For this purpose, mature males were treated with a 0.01% aqueous solution of the pheromone for six days. Control mice were similarly treated with physiologic saline. The mice were sacrificed 23 days after the treatment, and material for the analysis of sperm-head abnormalities was sampled from the caudal portion of the epididymis. Analysis of the frequency of abnormal sperms has demonstrated that the pheromonal treatment significantly increases the frequencies of various sperm-head abnormalities. Apparently, this results from disturbances in sex-cell differentiation germline cells caused by the induction of genetic damage at stages immediately preceding meiosis, as well as during the first and second meiotic divisions. The relationship between the effect of 2,5-DMP and the decrease in the fertility of male CBA mice that was earlier observed after a similar treatment is discussed.     

Vomeronasal/accessory olfactory system and pheromonal recognition

Pregnancy block in mice requires exposure of recently mated females to urinary pheromones of a strange male, and when working with inbred strains this invariably requires urine from an outbred line. The pheromones which induce oestrus and early puberty in mice have been identified as the brevicomins and dihydrothiazoles. Since the same vomeronasal, neural and neuroendocrine pathways are also activated in pregnancy block, these compounds are likely candidates for pregnancy blocking pheromones. However, these relatively simple chemicals lack the capacity to code for differing mouse strains. Since large quantities of the polymorphic major urinary proteins from the lipocalin family found in urine serve as transporters for the dihydrothiazoles and brevicomins, and differ across strains, then these proteins must participate in pheromone recognition in the context of pregnancy block.      

Immunomodulating effect of protein fractions isolated from bifidobacteria

The screening of the immunomodulating activity (IMA) of different protein fractions isolated from bifidobacteria was carried out and the capacity of these fractions for changing the proliferative activity of immunocompetent cells was evaluated. Soluble proteins were extracted from lyophilized and sonicated bacterial mass of B. bifidum strain 1 in Na2HPO4 (pH 8) in a water bath at 65 degrees C for 30 minutes. After the formation and removal of nucleic acid sediment the resulting supernatant fluid was dialyzed, its adsorption spectra were analyzed and the fluid was fractionated in a specially proposed device for preparative electrophoresis. Protein fractions were tested for IMA on spleen cells of CBA mice in the reaction of lymphocyte blast-transformation by the level of the inclusion 3H thymidine. The analysis of IMA of protein fractions revealed that their high-molecular components produced a pronounced dose-dependent effect on the proliferative activity of spleen cells. The fractions containing low-molecular components were either inactive (fraction 4) or active only in the maximum dose (fraction 5).     

Altered production and renewal of natural killer cells in B-lymphocyte-deficient CBA/N mice

The present study was designed to measure by quantitative and kinetic methods the production and renewal of natural killer (NK) cells in congenitally B-lymphocyte-deficient (CBA/N) mice. The total NK activity (percent specific lysis corrected for changes in whole organ cellularity) of the bone marrow and spleen of immunologically normal (CBA/CaJ) and CBA/N mice was assayed prior to and immediately after 48 h treatment (2 X/day, i.p.) with the cell cycle poison hydroxyurea (HU) and at various intervals throughout the subsequent post-HU recovery period. The total NK activity (TNKA) of untreated CBA/N bone marrow was 154% of that of CBA/CaJ bone marrow while the TNKA of CBA/N spleen was not significantly different (112%) from that of CBA/CaJ spleen. At the conclusion of 48 h HU, bone marrow TNKA of CBA/N and CBA/CaJ mice fell to 60 and 49%, respectively, of their saline-injected (2 X/day, i.p.) control levels, while spleen TNKA fell to 42 and 61%, respectively, of their saline-injected control levels. In the bone marrow, NK cell depletion in response to HU was more rapid in CBA/N mice (day 0.5 after HU) than in CBA/CaJ mice (day 2 after HU). TNKA of the spleen also decreased more rapidly in CBA/N mice (day 2 after HU) than in CBA/CaJ mice (day 3 after HU). The data indicate an enhanced production and turnover of NK cells in CBA/N mice relative to CBA/CaJ mice. Moreover, increased production and renewal of NK cells in CBA/N mice together with virtually unchanged levels of NK activity (112% of CBA/CaJ mice) in CBA/N mouse spleens indicate that mature lytic NK cells in CBA/N spleen but not bone marrow have a significantly shorter post-mitotic life span than do NK cells in the spleens of immunologically normal (CBA/CaJ) mice.     

Effect of the synthetic preparation B 58 on the activity of natural killer and cytostatic cells of the mouse spleen

The effect of the in vivo treatment with synthetic interferon inducer B-58 on natural killer (NK) and cytostatic cell activity was studied in CBA and A/Sn mice. A marked increase in NK cell activity against target cells YAC-1 was observed in the spleen of CBA mice within 4 days after treatment. On the other hand, NK activity in A/Sn mice was not affected by B-58. However, B-58 was shown to enhance cytostatic cell activity both in CBA and A/Sn mice, when tested against target cells P 815.     

Effect of low-level radiation on the death of male germ cells

Hormetic and adaptive responses induced by low-level radiation in hematopoietic and immune systems have been observed, as shown by stimulatory effects on cell growth and resistance to subsequent radiation-induced cytogenetic damage. However, in terms of cell death by apoptosis, the effects of low-level radiation are controversial: Some studies showed decreased apoptosis in response to low-level radiation while others showed increased apoptosis. This controversy may be related to the radiation doses or dose rates and also, more importantly, to the cell types. Testes are one of the most radiosensitive organs. The loss of male germ cells after exposure to ionizing radiation has been attributed to apoptosis. In the present study, the effects of low-level radiation at doses up to 200 mGy on mouse male germ cells in terms of apoptosis and the expression of apoptosis-related proteins were examined at different times after whole-body exposure of mice to low-level radiation. In addition, the effect of pre-exposure to low-level radiation on subsequent cell death induced by high doses of radiation was examined to explore the possibility of low-level radiation-induced adaptive response. The results showed that low-level radiation in the dose range of 25-200 mGy induced significant increases in apoptosis in both spermatogonia and spermatocytes, with the maximal effect at 75 mGy. The increased apoptosis is most likely associated with Trp53 protein expression. Furthermore, 75 mGy low-level radiation given pre-irradiation led to an adaptive response of seminiferous germ cells to subsequent high-level radiation-induced apoptosis. These results suggest that low-level radiation induces increased apoptosis in male germ cells but also induces a significant adaptive response that decreases cell death after a subsequent high-dose irradiation.     

The second messenger pathway for germ cell-mediated stimulation of Sertoli cells.

Treatment of cultured rat Sertoli cells with FSH or dibutyryl cAMP for 30 min resulted in phosphorylation of the same Sertoli cell proteins. Different Sertoli cell proteins were phosphorylated after calcium ionophore A23187 and 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. A23187 stimulated the phosphorylation of hsp27, while TPA alone had no effect. TPA plus A23187 resulted in phosphorylation of a 14 kDa protein, in addition to hsp27. The effect of TPA plus A23187 was identical to that of germ cells on Sertoli cell protein phosphorylation. FSH-stimulated cAMP production by Sertoli cells was reduced by prior exposure of Sertoli cells to germ cells. The results indicate that germ cells stimulate Sertoli cells by the inositol trisphosphate/diacylglycerol mediated second messenger pathway. The results also suggest that the germ cell-activated pathway interacts within Sertoli cells to modulate Sertoli cell response to FSH.     

Antimutagenic effect of chemosignals from isolated female house mouse on male germ cells (Mus musculus L.)

The influence of chemosignals from isolated mature females of the CBA strain on level of spontaneous and radiation-induced meiotic disturbances in spermatocytes I of males of the same strain was studied. Using an ana-telophase method, 24-hour exposure of males to soiled bedding containing isolated females’ chemosignals was shown to lead to a significantly lower frequency of chromosomal aberrations and other meiotic disturbances in spermatocytes I as compared to males kept on clean bedding. The same effect of female chemosignals was found in the germ cells of irradiated males (4 Gr). The mechanisms and importance of the revealed antimutagenic effect of mouse female chemosignals on the male reproductive cells in the reproduction process are discussed.     

Cytogenetic studies in human cells exposed in vitro to GSM-900 MHz radiofrequency radiation using R-banded karyotyping

It is important to determine the possible effects of exposure to radiofrequency (RF) radiation on the genetic material of cells since damage to the DNA of somatic cells may be linked to cancer development or cell death and damage to germ cells may lead to genetic damage in next and subsequent generations. The objective of this study was to investigate whether exposure to radiofrequency radiation similar to that emitted by mobile phones of second-generation standard Global System for Mobile Communication (GSM) induces genotoxic effects in cultured human cells. The cytogenetic effects of GSM-900 MHz (GSM-900) RF radiation were investigated using R-banded karyotyping after in vitro exposure of human cells (amniotic cells) for 24 h. The average specific absorption rate (SAR) was 0.25 W/kg. The exposures were carried out in wire-patch cells (WPCs) under strictly controlled conditions of temperature. The genotoxic effect was assessed immediately or 24 h after exposure using four different samples. One hundred metaphase cells were analyzed per assay. Positive controls were provided by using bleomycin. We found no direct cytogenetic effects of GSM-900 either 0 h or 24 h after exposure. To the best of our knowledge, our work is the first to study genotoxicity using complete R-banded karyotyping, which allows visualizing all the chromosomal rearrangements, either numerical or structural.     

Radiation- and chemically-induced chromosome aberrations in mouse oocytes: a comparison with effects in males.

Data from studies on radiation- and chemically-induced chromosome aberrations in mouse oocytes have been summarized. An attempt has been made to assess the relative sensitivity to mutagenic agents of female and male germ cells through comparison of observations from mutation studies of female and male mice. No unequivocal evidence of a mutagenic effect limited to a single sex could be found in the cytogenetic data, although differences in relative germ cell sensitivity could be inferred for ionizing radiation and some chemicals. However, the pattern of inter-sex variations was not consistent: for example, irradiation of dictyate oocytes yielded a lower rate of heritable chromosome translocations than the same dose to spermatogonia; in contrast, some chemicals, such as mitomycin C, yielded a larger incidence of chromosome anomalies after treatment of dictyate oocytes than spermatogonia. Overall, the limitations in quality and quantity of cytogenetic data, and the uncertainties associated with comparing information obtained in disparate assays, place severe constraints on the use of observations on induced chromosome aberrations to assess the relative sensitivities of female and male germ cells to environmental mutagens.     

Induction of Dominant Lethals in Progeny of CBA Male Mice after Pheromonal Action

The inhibiting effect of pheromone 2,5-dimethylpyrazine of house mouse females on the reproductive function of the CBA male mice was studied. The mutagenic effect of six-day pheromonal treatment was assessed by dominant lethal test. Analysis for the frequency of dominant lethals showed that the pheromonal effect results in an increased death rate of the progeny of the treated males. This is probably explained by implantation failure and is expressed in a reduced average number of the implants and low live embryos per female. The proportion of females with live embryos decreased significantly. The implication of the effect of female mouse pheromone 2,5- dimethylpyrazine on the genetic processes in germ cells of male mice is discussed.