Chromosomal aberrations in the bone marrow cells of mice induced by accelerated (12)C(6+) ions

The whole bodies of 6-week-old male Kun-Ming mice were exposed to different doses of (12)C(6+) ions or X-rays. Chromosomal aberrations of the bone marrow (gaps, terminal deletions and breaks, fragments, inter-chromosomal fusions and sister-chromatid union) were scored in metaphase 9h after exposure, corresponding to cells exposed in the G(2)-phase of the first mitosis cycle. Dose-response relationships for the frequency of chromosomal aberrations were plotted both by linear and linear-quadratic equations. The data showed that there was a dose-related increase in the frequency of chromosomal aberrations in all treated groups compared to controls. Linear-quadratic equations were a good fit for both radiation types. The compound theory of dual radiation action was applied to decipher the bigger curvature (D(2)) of the dose-response curves of X-rays compared to those of (12)C(6+) ions. Different distributions of the five types of aberrations and different degrees of homogeneity were found between (12)C(6+) ion and X-ray irradiation and the possible underlying mechanism for these phenomena were analyzed according to the differences in the spatial energy deposition of both types of radiation.     

Chromosomal aberrations induced by (12)C6+ ions and 60Co gamma-rays in mouse immature oocytes

The ovaries of Kun-Ming strain mice (3 weeks) were irradiated with different doses of (12)C6+ ion or (60)Co gamma-ray. Chromosomal aberrations were analyzed in metaphase II oocytes at 7 weeks after irradiation. The relative biological effectiveness (RBE) of (12)C6+ ion was calculated with respect to 60Co gamma-ray for the induction of chromosomal aberrations. The (12)C6+ ion and 60Co gamma-ray dose-response relationships for chromosomal aberrations were plotted by linear quadratic models. The data showed that there was a dose-related increase in frequency of chromosomal aberrations in all the treated groups compared to controls. The RBE values for (12)C6+ ions relative to 60Co gamma-rays were 2.49, 2.29, 1.57, 1.42 or 1.32 for the doses of 0.5, 1.0, 2.0, 4.0 or 6.0 Gy, respectively. Moreover, a different distribution of the various types of aberrations has been found for (12)C6+ ion and 60Co gamma-ray irradiations. The dose-response relationships for (12)C6+ ion and 60Co gamma-ray exhibited positive correlations. The results from the present study may be helpful for assessing genetic damage following exposure of immature oocytes to ionizing radiation.     

Induction of adaptive response by low-dose radiation in RIF cells transfected with Hspb1 (Hsp25) or inducible Hspa (Hsp70)

An adaptive response results in a reduced effect of a high challenging dose of a stressor after a smaller, inducing dose has been applied a few hours earlier. Radiation-induced fibrosarcoma (RIF) cells did not show an adaptive response, i.e. a reduced effect from a high challenging dose (2 Gy) of a radiation after a priming dose (1 cGy) had been applied 4 or 7 h earlier, but cells of a thermoresistant clone (TR) derived from RIF cells did. Since the expression of inducible Hspa (also known as Hsp70) and Hspb1 (also known as Hsp25) was different in these two cell lines, the role of inducible Hspa and Hspb1 in the adaptive response was examined. When RIF cells were transfected with inducible Hspa or Hspb1, both radioresistance measured by clonogenic assays and a reduction of apoptosis were detected. The adaptive response was also acquired by these two cell lines. The inducible Hspa transfectant showed a more pronounced adaptive response than the Hspb1 transfectant. Based on these results, it appears that inducible Hspa and Hspb1 are at least partly responsible for the induction of the adaptive response in these cells. Moreover, when inducible Hspa or Hspb1 was transfected into RIF cells, co-regulation of the two genes was detected. Heat-shock factor (Hsf) was found to be at least partially responsible for the induction of the adaptive response in these cells.     

Kit ligand mediates survival of type a spermatogonia and dividing spermatocytes in postnatal mouse testes

In the mouse testis, spontaneous death of spermatogonia has a large impact on the output of differentiating spermatids. The tyrosine kinase receptor c-kit is expressed in type A, intermediate, and B spermatogonia, and kit-ligand (KL) is expressed in Sertoli cells. Previous work indicated a depletion of type A spermatogonia after in vivo exposure to an antibody that blocks c-kit function. The present work was undertaken to determine whether blocking c-kit function results in apoptosis of spermatogonia or in an inability of spermatogonia to proliferate. Testes sections were stained by a method that detects apoptotic cells in situ. In testes of 8-day postnatal (P8) males, type A spermatogonia are the predominant germ cell type present. Stained sections from P8 males injected with the c-kit antagonistic antibody ACK2 showed a fivefold higher rate of cell death than uninjected controls. At least a twofold increase was observed in P12 and P30 injected males and in P30 SI^d + males as compared to uninjected controls. Determination of the stage of germ cell development that was affected in P30 males indicated that the frequency of gonial cell death was increased fourfold, but the frequency of death in spermatocytes around the time of the meiotic division was increased 15-fold. It is concluded that KL acts to prevent apoptosis in the testis in vivo, that the membrane bound form of KL may be more effective, and that survival of late meiotic and dividing spermatocytes is regulated by KL through an indirect mechanism probably mediated by Sertoli cells. Thus, KL is an important regulator of spermatid output. © 1995 wiley-Liss, Inc.     

Ultrastructure of spermatogonia, spermatocytes, and sertoli cells in the testis of the crayfish, Procambarus paeninsulanus

The testes of the crayfish, Procambarus paeninsulanus, were prepared for light and transmission electron microscopy. During early stages of spermatogenesis, when the spermatogonia are dividing, processes from a single Sertoli cell extend between numerous spermatogonia. As the cells enter meiosis, many points of contact can be observed between the Sertoli cell processes and spermatocytes. These desmosome-gap junctions are maintained between the germ and Sertoli cells until the early spermatid stage.     

Evidence against a (2)n synchronous increase of spermatogonia to produce spermatocytes in drosophila hydei

The numbers of primary spermatocytes within cysts as well as numbers of postmeiotic spermatids in bundles in Drosophila hydei were determined. Within the contents of a single testis the cysts of primary spermatocytes are found to contain 5–11 germ cells. Furthermore, the number of spermatocytes per cyst is age-dependent, in that pupae have a mean of 8.1 cells whereas fertile adult males have a mean of 7.1 cells. Counts of spermatids in section of testes add further support to the view that the primary spermatocytes, from which the spermatids originated, were not formed in a strict geometric progression.     

Ribosomal RNA synthesis in spermatogonia and Sertoli cells of the mouse testis

Simultaneously cloned monkey CV-1 cell sublines were found to differ in morphology, cloning efficiency, chromosome number, and sensitivity to SV40 virion productive infection. A fibroblast-like clone, FC7, when compared with an epithelioid clone, TC7, had a lower mean chromosome number and was resistant to SV40 virion infection. Virion adsorption and penetration were similar in both the FC7 and TC7 cells, and both cell types were equally sensitive to first cycle SV40 DNA infection. As the subdiploid mean chromosome number of the FC7 cells increased with passage time toward the TC7 subtetraploid number, the FC7 cells became more sensitive to virion infection. This host gene-dosage effect on SV40 productive infection suggests that a monkey cell function participates in the SV40 uncoating and/or viral genome activation process.     

Induction of translocations in mouse spermatogonia after fractionated exposure to 60Co gamma-rays

Male mice of the Q strain were exposed to 60Co gamma-rays at 2 Gy and 2 X 2 Gy separated by increasing time intervals (from 0 min to 4 min). The chromosome translocations induced in spermatogonia were scored at diakinesis-metaphase I. A significant decrease of the translocation frequency at time intervals higher than 2 min was observed, confirming results obtained with plant materials.     

Cytogenetic adaptive response induced by pre-exposure in human lymphocytes and marrow cells of mice

The frequencies of chromosomal aberrations bith in human lymphocytes and in mouse marrow cells exposed to low-level radiation were higher than in their unexposed controls. However, the frequencies of chromosomal aberrations in two kinds of cells pre-exposed to low-level radiation induced by a subsequent high dose of X-rays or γ-rays were lower than those of the groups only exposed to high-level radiation. This implies that adaptive responses for cytogenetic indicators might be induced by pre-exposure to low-level radiation. The results also show the existence of possible variations between individual lymphocytes.     

Correlation of meiotic events in testis sections and microspreads of mouse spermatocytes relative to the mid-pachytene checkpoint

In mouse, asynaptic meiotic mutants arrest at Testis Epithelial Stage IV. This arrest is 4.5 days after homologous chromosomes begin to synapse and approximately 2.5 days after synapsis is usually completed. To correlate cytological events with meiotic progression in testis and to determine which meiotic events are normally completed by Stage IV, we induced spermatogenic arrest by placing mice on a vitamin A deficient diet. Subsequent injection of retinoic acid and a return to a normal diet resulted in resumption of spermatogenesis with all spermatocytes proceeding through meiosis in a highly synchronous cohort. Between Days 11 and 16 post-injection we prepared one testis for immunocytological and the other for histological evaluation, then used antibodies to SCP3 and either RPA, or MLH1 to follow quantitative changes in synapsis and recombination. RPA was found at sites along the synaptonemal complex as soon as homologs synapsed, and most, but not all, RPA disappeared by Stage IV. MLH1 foci appeared between Stage II and IV and remained through Stage VII, the end point of the study. The data suggest that the earliest the mid-pachytene checkpoint can be activated is Epithelial Stage IV, but that activities monitored by the checkpoint may not be completed by this time.     

Induction of endocytic vesicles by exogenous C(6)-ceramide.

Ceramide is a newly discovered second messenger that has been shown to cause cell growth arrest and apoptosis. Here, we present evidence that exogenously added C(6)-ceramide induces enlargement of late endosomes and lysosomes. 10 microM C(6)-ceramide caused the formation of numerous vesicles of varying sizes (2-10 micrometers) in fibroblasts (3T3-L1 and 3T3-F442A), without toxic effects. Vesicle formation induced by C(6)-ceramide was time- and dose-dependent, rapid, and reversible. Numerous small vesicles appeared within 8 h of treatment with 10 microM C(6)-ceramide. They enlarged with time, with large vesicles found in the perinuclear region and small ones observed at the cell periphery. Within 24 h of treatment, approximately 30% of the cells exhibited these vesicles. Removal of ceramide from the culture medium caused disappearance of the vesicles, which reappeared upon readdition of ceramide. Confocal immunofluorescence microscopic analysis using an anti-lysosome-associated membrane protein antibody identified the enlarged vesicles as late endosomes/lysosomes. The fluorescent C(6)-NBD-ceramide, a vital stain for the Golgi apparatus, did not stain these vesicles. The effect on vesicle formation was influenced by ceramide structure; D-erythro-C(6)-ceramide was the most active ceramide analogue tested. Short chain ceramide metabolites, such as sphingosine, sphingosine 1-phosphate, N-hexanoyl-sphingosylphosphorylcholine, N-acetylpsychosine, and C(2)-ceramide G(M3), (G(M3), N-acetylneuraminosyl-alpha(2, 3)-galactosyl-beta(1,4)-glucosylceramide), were inactive in causing vesicle formation when added exogenously. Together, these studies demonstrate that exogenous C(6)-ceramide induces endocytic vesicle formation and causes enlarged late endosomes and lysosomes in mouse fibroblasts.     

Vitamin A deficiency results in meiotic failure and accumulation of undifferentiated spermatogonia in prepubertal mouse testis

Vitamin A (retinol) is required for maintenance of adult mammalian spermatogenesis. In adult rodents, vitamin A withdrawal is followed by a loss of differentiated germ cells within the seminiferous epithelium and disrupted spermatogenesis that can be restored by vitamin A replacement. However, whether vitamin A plays a role in the differentiation and meiotic initiation of germ cells during the first round of mouse spermatogenesis is unknown. In the present study, we found that vitamin A depletion markedly decreased testicular expression of the all-trans retinoic acid-responsive gene, Stra8, and caused meiotic failure in prepubertal male mice lacking lecithin:retinol acyltransferase (Lrat), encoding for the major enzyme in liver responsible for the formation of retinyl esters. Rather than undergoing normal differentiation, germ cells accumulated in the testes of Lrat(-/-) mice maintained on a vitamin A-deficient diet. These results, together with our previous observations that germ cells fail to enter meiosis and remain undifferentiated in embryonic vitamin A-deficient ovaries, support the hypothesis that vitamin A regulates the initiation of meiosis I of both oogenesis and spermatogenesis in mammals.     

Induction of minisatellite mutations in the mouse germline by low-dose chronic exposure to gamma-radiation and fission neutrons

Germline mutation induction at mouse minisatellite loci by paternal low-dose (0.125-1 Gy) exposure to chronic (1.66 x 10(-4) Gy min(-1)) low-linear energy transfer (low-LET) gamma-irradiation and high-LET fission neutrons (0.003 Gy min(-1)) was studied at pre-meiotic stages of spermatogenesis. Both types of radiation produced linear dose-response curves for mutation of the paternal allele. In contrast to previous results using higher doses, the pattern of induction of minisatellite mutation after chronic gamma-irradiation was similar to acute (0.5 Gy min(-1)) exposure to X-rays, indicating that the elevated mutation rate was independent of the ability of the cell to repair damage induced immediately or over a period of up to 100 h. Chronic exposure to fission neutrons was more effective than acute or chronic low-LET exposure (relative biological effectiveness, RBE=3.36). The data also provide strong support for the previous conclusion that increases in minisatellite mutation rate are not caused by radiation-induced DNA damage at minisatellite loci themselves, but rather from damage induced by ionising radiation elsewhere in the genome/cell.     

LY6A/E (SCA-1) expression in the mouse testis

Recently, it was found by two research groups that LY6A, known widely in the stem cell community as stem cell antigen-1 or SCA-1, is expressed on testicular side population (SP) cells. Whether these SP cells are spermatogonial stem cells is a point of disagreement and, therefore, the identity of the LY6A-positive cells as well. We studied the expression pattern of LY6A in testis by immunohistochemistry and found it to be expressed in the interstitial tissue on peritubular myoid, endothelial, and spherical-shaped peritubular mesenchymal cells. To address the question whether LY6A has a function in spermatogenesis or testis development, we studied the testis of Ly6a(-/-) mice (allele Ly6a(tm1Pmf)). We found no morphological abnormalities or differences in numbers of spermatogonia, spermatocytes, Leydig cells, or macrophages in relation to the number of Sertoli cells. Therefore, we conclude that LY6A expression does not influence testis development or spermatogenesis and that spermatogonial stem cells are LY6A negative.     

Induction of meiosis in fetal mouse testis in vitro by rete testis tissue from pubertal mice and bulls.

To test whether a meiosis-inducing substance (MIS) is responsible for the induction of meiosis in the testis at puberty, pubertal mouse rete testis was grown with (1) fetal undifferentiated mouse testis attached to the other side of a filter and (2) the used medium obtained from culture of the rete testis of a pubertal bull for 2 days. In both systems meiosis was induced in the fetal testis showing that MIS is not species specific. No meiosis-preventing effect was seen and it is concluded that meiosis in the testis is triggered at puberty as a result of the activity of the MIS concomitant with decreased activity of the meiosis-preventing substance.