Expression of p53 in normal and gamma-irradiated rat testis suggests a role for p53 in meiotic recombination and repair.

In testis, the expression of tumor suppressor protein p53 is stronger than in other tissues suggesting a role for it in spermatogenesis. We have studied the expression of p53 in both unirradiated and gamma-irradiated rat testis using the stage-specific model of rat seminiferous epithelium. Our results show that p53 is expressed during meiosis in normal rat spermatogenesis and its expression is localized to the preleptotene-early pachytene spermatocytes. The most prominent expression is in zygotene - early pachytene spermatocytes (stages XIII-I of seminiferous epithelium). After irradiation p53 levels increased in a time and a dose-dependent manner being highest with the doses of 6.0 and 12.0 Gy and 4 h after irradiation. This increase occurs in the same cells that normally express elevated levels of p53. These results support the view that p53 is involved in meiosis of the male rat and we suggest that p53 has a role in recombinational processes and/or formation of the synaptonemal complex. We also demonstrate that p53 takes part in the response of primary spermatocytes to irradiation gamma-induced DNA damage.

Stage-specific expression and phosphorylation of retinoblastoma protein (pRb) in the rat seminiferous epithelium.

To assess the potential role of retinoblastoma protein (pRb) in the regulation of cell cycle during spermatogenesis, the expression of retinoblastoma (Rb) mRNA and protein, as well as the phosphorylation states of pRb, in the rat seminiferous epithelial cycle, were studied. Two transcripts, 5.4 kb and 3.4 kb long, were detected in total RNA from the adult rat testis and only the 5.4 kb transcript was detected in poly (A)+-RNA from 8, 14 and 23-day old rat testes by Northern hybridization. Polysome analysis revealed that only a small portion of both Rb transcripts could be efficiently translated. By in situ hybridization, Rb mRNA was localized to germ cells from stage V pachytene spermatocytes to step 13 spermatids along the epithelial cycle. pRb immunoreactivity was detected in Sertoli cells and spermatogonia at all stages, as well as in the elongated steps 14-19 spermatids by immunohistochemistry. The amount of pRb and the phosphorylation status varied in a stage-specific manner in Western blots. These results show that pRb is expressed in the rat seminiferous epithelium in a cyclic fashion and suggest that it is involved in the regulation of proliferation of spermatogonia and maintenance of the differentiation status of Sertoli cells and spermatids.

Detection of aneuploidy in human spermatozoa of normal semen donors by fluorescence in situ hybridization.

We have studied human spermatozoa from 24 normal, healthy unexposed men, 18 of whom were semen donors at the Sperm Bank in Turku, using multicolor fluorescence in situ hybridization with two chromosome-specific probes. The possible age-related increase in aneuploidy frequencies was assessed. Ten thousand spermatozoa were scored per individual for the presence of hyperploid, i.e., disomic and diploid, cells. The overall hybridization efficiency was 98.8%. The frequency of spermatozoa with two chromosome 1 signals was 11.5 +/- 5.2/10,000. The frequency of spermatozoa with two chromosome 7 signals was 6.4 +/- 3.9/10,000. Diploidy was present in 15.0 +/- 8.9/10,000 spermatozoa. Interindividual variation was quite large. No statistically significant correlation between age of the donors (range = 20-46 years) and the frequency of hyperploid spermatozoa was observed. The results give background information on the incidence of hyperploid spermatozoa in unexposed men and encourage the use of this novel technique of future studies on genetic effects in men exposed to potentially aneuploidogenic agents.

Incidence of aneuploid spermatozoa among infertile men studied by multicolor fluorescence in situ hybridization.

We studied by fluorescence in situ hybridization the frequency of aneuploidy in spermatozoa of 12 infertile men: 8 with normal or nearly normal semen analysis values and 4 with oligo-astheno-teratozoospermia. The control group consisted of 18 normal healthy fertile men. Probes for chromosome 1 and 7 were used and 10,000 spermatozoa per individual were scored. The hybridization efficiency was good (higher than 98%). In the group with nearly normal semen analysis values the frequencies of spermatozoa disomic for chromosome 1 or chromosome 7 were 0.08% and 0.07%, respectively, and not elevated compared to controls (0.10% and 0.06%, respectively). The frequency of diploid spermatozoa was 0.17%, not significantly different from the control group (0.15%) either. In the group of oligoastheno-teratozoospermic men both the frequencies of disomic cells for chromosome 1 (0.22%) and for chromosome 7 (0.13%) and of diploid spermatozoa (0.56%) were significantly higher compared to controls, although this was mainly due to one patient with high frequencies of hyperploid sperm. The results indicate that infertility may be a risk factor for chromosomal aneuploidy in spermatozoa.

Effects of the DNA topoisomerase II inhibitor merbarone in male mouse meiotic divisions in vivo: cell cycle arrest and induction of aneuploidy.

In order to clarify possible risks of aneuploidy induction in germ cells by cancer chemotherapy we studied effects of a non complex-stabilizing DNA topoisomerase II (topo II) inhibitor merbarone in male mouse meiotic divisions in vivo. Two cytogenetic approaches were used: (1) C-banding on meiotic chromosome preparations and (2) analysis of spermatid micronuclei (MN) combined with immunocytochemical staining of kinetochore proteins using CREST serum. For comparison, another topo II inhibitor, VP-16, known to form cleavable complexes, was studied. The microdissection technique of mouse seminiferous tubules enabled us to carefully examine effects at specific phases of meiosis. Merbarone injections increased percentages of polyploid and hypoploid metaphase II spermatocytes at time intervals corresponding to the treatment of the first meiotic division and diplotene-diakinesis. The highest level of MN induction (5.8 MN/1000 spermatids, P < 0.001) was observed in animals injected 48 hours before the harvest, corresponding to the treatment of diplotene-diakinesis spermatocytes. Most of the induced MN (80%) contained kinetochore signals, indicating that they resulted from detachment of a whole bivalent or chromosome from the meiotic spindle. The high frequency of MN with two kinetochore signals at opposite sides (33%) most likely denotes lagging of whole bivalents during MI. Inhibition of cell proliferation was determined by scoring cells arrested at different phases of MI and MII. All groups of treated animals showed a clear increase in the frequency of arrested divisions compared to controls (P < 0.001). Thus, merbarone was shown to severely damage normal meiotic processes.

Germ cell mutagenicity of three metabolites of 1,3-butadiene in the rat: induction of spermatid micronuclei by butadiene mono-, di-, and diolepoxides in vivo.

Three metabolites of the industrial chemical 1,3-butadiene (BD), namely butadiene monoepoxide (BMO, 3,4-epoxy-1-butene), diepoxide (DEB, 1,2;3,4-diepoxybutane), and diolepoxide (DE, 3,4- epoxybutane-1,2-diol) were studied for germ cell mutagenicity using the rat spermatid micronucleus (MN) test. All three epoxides increased slightly, but significantly, the frequency of spermatid MN. The most sensitive stage to the action of BMO and DEB was preleptotene (meiotic S phase) harvested at 18-day time intervals after treatment. The dose-response for BMO followed a second order curve at this time interval, with maximum MN induction at the dose of 186 mumol/kg and lower induction of higher doses. Late stages of the meiotic prophase (late pachytene-diplotene-diakinesis) also showed some sensitivity to the three epoxides. Stem cell spermatogonia were affected by DEB as observed by a slight induction of spermatid micronuclei 50 days after treatment. No clear cytotoxic effects were observed by measuring testicular weight or cell numbers of seminiferous epithelial stage 1 18 days after the treatments. DEB at the dose 387 mumol/kg caused a slight inhibition of spermatogonial DNA synthesis in stage I and a delay of meiotic DNA replication observed in stage XII 72 hr after treatment. Since BMO is able to induce spermatid MN in the rat, the present results, together with previous data, indicate that rat bone marrow MN results that are negative for both BD and BMO cannot directly predict mutagenicity in male germ cells. The results also emphasize that tissue; species, and strain-specific differences in metabolism have to be taken into account when the genetic risks of human butadiene exposure are evaluated. The results support the conclusion that 1,3-butadiene is a germ cell mutagen-possibly also in humans.

Apoptotic response of spermatogenic cells to the germ cell mutagens etoposide, adriamycin, and diepoxybutane.

In testis, apoptosis is a way to eliminate damaged germ cells during their development. In this study, we evaluated the ability of three germ cell mutagens to induce apoptosis (or programmed cell death) at specific stages of rat seminiferous epithelial cycle. These chemicals include the cancer chemotherapy drugs etoposide and adriamycin and the butadiene metabolite diepoxybutane. According to our results, etoposide is a very potent inducer of apoptosis in male rat germ cells and the cell types most sensitive to it include all types of spermatogonia, zygotene, and early pachytene spermatocytes and meiotically dividing spermatocytes. Also, adriamycin causes an increase in apoptosis at specific stages of seminiferous epithelial cycle and the most sensitive cell types are type A3-4 spermatogonia, preleptotene, zygotene, and early pachytene spermatocytes. Diepoxybutane does not cause any significant increase in the frequency of apoptosis in rat testis. In addition, we studied whether p53 is taking part in the apoptotic response of spermatogenic cells by studying the levels of p53 protein in testis before and after chemical treatment. No accumulation of p53 in testis was seen after treatment with these three chemicals. The expression of two p53-regulated genes, p21WAF1 and mdm2, was also studied but no increase in the levels of mRNA of these genes was observed after treatment. The results indicate that apoptosis should be taken into consideration when the genotoxic effects of chemicals are evaluated in germ cells.

Effects of vinblastine and colchicine on male rat meiosis in vivo: disturbances in spindle dynamics causing micronuclei and metaphase arrest.

We have studied the effects of vinblastine sulfate (VBL) and colchicine (COL) on male rat in vivo and in vitro meiosis. A novel methodology based on isolating a segment of seminiferous tubules containing meiotically dividing spermatocytes was applied. During meiotic divisions at stage XIV of rat spermatogenesis, both chemicals induced only low frequencies of micronuclei (MN), 0.8-3.2 MN/1,000 spermatids. Fluorescence in situ hybridization experiments in mice with the mouse centromere-specific gamma-satellite DNA probe showed that 50.7% of VBL-induced MN and 56.6% of COL-induced MN were centromere positive, indicating that the MN induced by both chemicals contained detached chromosomes. The inhibition of cell proliferation was determined by counting the number of cells arrested at metaphase during the first meiotic (MI) or the second meiotic (MII) division. VBL was found to be a potent inducer of cell death while COL was not. The direct effects of VBL and COL on the meiotic spindles were evaluated using immunohistochemistry with anti-alpha-tubulin and confocal microscopy. In the control animals a significant difference was observed between the mean length of metaphase spindles of MI and MII. Both were dramatically decreased 6 hr after treatment with 2.0 mg/kg of VBL and 0.8 mg/kg of COL, respectively. At 18 hr after COL injection the spindles had about the same length as in the controls. However, the VBL-induced shortening was even more evident at 18 hr for both MI and MII. The possible reasons for observed differences between the two chemicals and between meiosis and mitosis are discussed.

Etoposide (VP-16) is a potent inducer of micronuclei in male rat meiosis: spermatid micronucleus test and DNA flow cytometry after etoposide treatment.

The genotoxic and cytotoxic effects of etoposide (VP-16), a topoisomerase II inhibitor, on male rat spermatogenic cells were studied by analysing induction of micronuclei during meiosis. Micronuclei (MN) were scored in early spermatids after different time intervals corresponding to exposure of different stages of meiotic prophase. Etoposide had a strong effect on diplotene-diakinesis I cells harvested 1 day after exposure, and a significant effect also on late pachytene cells harvested 3 days after exposure. The effect at 18 days corresponding to exposure of preleptotene stage of meiosis (S-phase) was weaker but also statistically significant. Adriamycin was used as a positive control in this study. The results indicate a different mechanism of action of etoposide compared with adriamycin and other chemicals studied previously with the spermatid micronucleus test. DNA flow cytometry was carried out to assess cytotoxic damage at the same time intervals (1, 3, and 18 days after treatment) at stages I and VII of the seminiferous epithelial cycle allowing a study of cytotoxicity to different spermatogenic cell stages. Damage of differentiating spermatogonia was observed by a decrease in the cell numbers of the 2C peak 1 and 3 days after treatment and by a reduction of the number of 4C cells (primary spermatocytes) 18 d after etoposide treatment. Adriamycin also killed differentiating spermatogonia. Since the cell population which showed a high induction of MN by etoposide was not reduced in number, the genotoxic effect is remarkable. We conclude that etoposide is a potent inducer of genotoxicity and patients treated with this agent during cancer chemotherapy are at a risk of genetic damage.

Aneuploidy in sperm and exposure to fungicides and lifestyle factors. ASCLEPIOS. A European Concerted Action on Occupational Hazards to Male Reproductive Capability.

Fungicides include chemicals that are known aneugens. The purpose of the present study was to investigate whether occupational exposure to these and other agricultural pesticides induces aneuploidy in human sperm. The contribution of lifestyle factors (smoking and alcohol consumption) to the frequency of aneuploid sperm was evaluated as well. The effects of age and sperm concentration were analyzed as confounders. Spermatozoa from 30 healthy farmers were studied before and after exposure to fungicides, using fluorescence in situ hybridization (FISH). Ten thousand spermatozoa were scored per semen sample to determine the disomy and diploidy frequencies for chromosomes 1 and 7. Exposure to fungicides was not associated with sperm aneuploidy. Smoking was significantly associated with sperm carrying an extra chromosome 1 and with diploid sperm as well as with the aggregate frequency of aneuploid sperm. Alcohol consumption, sperm concentration, and age showed inconsistent results before and after the season of exposure to fungicides. For low-level exposures, such as occupational exposures, the sensitivity of the sperm-FISH method may not be sufficient. The present study supports earlier ones showing that smoking can increase aneuploidy in human sperm.

p21WAF1 expression during spermatogenesis of the normal and X-irradiated rat.

The cyclin dependent kinase inhibitor p21WAF1 has been shown to be upregulated during differentiation and after DNA damage in somatic cells. We examined the expression of p21WAF1 mRNA during the differentiation of germ cells in normal and X-irradiated rat testis by in situ hybridization and Northern blotting. p21WAF1 was normally expressed in primary spermatocytes of the pachytene phase, but could also be detected in round spermatids. In preparations of defined segments of the seminiferous tubules, the strongest hybridization signals were detected in the segments containing stages VII VIII and IX XII of the seminiferous epithelium. Ionizing radiation (1-12 Gy) induced the expression of p21WAF1 in a dose-dependent manner and the lowest dose that showed a clear increase in mRNA levels was 3 Gy. The p21WAF1 mRNA levels peaked after 3-4 hours, but remained high compared with the control levels during the 24-h follow-up. No change in the in situ hybridization pattern was seen when comparing unirradiated and irradiated tissue. Thus, it appears that X-irradiation induces p21WAF1 in the pachytene spermatocytes. Since p21WAF1 mRNA was found in pachytene spermatocytes and in round spermatids in normal testis, the protein may take part in the regulation of meiosis and in the 'terminal' differentiation of the male germ cells.

Stage-specific expression of Gadd45 induced by X-irradiation in rat spermatogenesis.

PURPOSE: Gadd45 is involved in the response to DNA damage in somatic cells. The effect of X-irradiation and chemical treatments on expression of Gadd45 and two other 53-regulated genes, p21 and cyclin-G, was studied in rat testis. MATERIALS AND METHODS: The reverse-transcriptase polymerase chain reaction (RT-PCR) was performed on testis extracts of control, X-irradiated (6Gy), etoposide (10 mg kg(-1)) and adriamycin (5mg kg(-1))-treated rats. For stage-specific analysis, seminiferous tubules were isolated and segments representing the 14 epithelial stages were obtained. RESULTS: In whole testis extracts, increases in Gadd45, p21 and cyclin-G expression were detectable after irradiation, but not after etoposide or adriamycin treatments. Analysis of fractions consisting of defined epithelial stages showed a high expression of Gadd45 in stages VII-XII and of p21 in stages VII-VIII. Irradiation significantly increased the level of Gadd45 mRNA in stages VI-VIII and of p21 mRNA in stages VI-I. Although no overall increase could be observed in whole testis samples of the etoposide-treated rat, stage-specific analysis revealed an induction of p21 expression in stages XIII-I. Gadd45 and cyclin-G mRNA were localized to spermatocytes and round spermatids known to express p21. CONCLUSIONS: Although X-irradiation, etoposide and adriamycinare known spermatogenic mutagens and activators of apoptosis, only X-rays induce slightly Gadd45 expression in testis. This small induction was very stage specific.

Haemoglobin adducts of epoxybutanediol from exposure to 1,3-butadiene or butadiene epoxides.

Epoxybutanediol is one of the reactive metabolites of butadiene. It is formed via hydrolysis followed by oxidation of the primary metabolite of butadiene, epoxybutene, or via hydrolysis of diepoxybutane, a secondary metabolite of butadiene. Groups of male Sprague Dawley rats were treated by intraperitoneal injection of epoxybutene, epoxybutanediol or diepoxybutane. N-(2,3,4-Trihydroxybutyl)valine adducts in haemoglobin, formed from epoxybutanediol in its reaction with N-terminal valine, were measured using the N-alkyl Edman method followed by acetylation of the Edman derivatives and analysis by gas chromatography mass spectrometry. The same adducts were also measured in male Wistar rats exposed to butadiene by inhalation and in a few workers with occupational exposure to butadiene. Haemoglobin binding indexes, HBI, (pmol adduct/g per mumol of alkylating agent, or, for butadiene, per ppm x h), were calculated. The HBI for epoxybutanediol (about 10) is comparable to that of ethylene oxide in the rat demonstrating a similar capacity of the two compounds to alkylate nucleophilic sites in vivo. The HBI of diepoxybutane (about 8) for epoxybutanediol adduct formation is approximately the same as that of epoxybutanediol itself. Epoxybutanediol adduct formation was nonlinearly related to exposure in butadiene exposed rats. The epoxybutanediol-haemoglobin adduct levels were substantially higher than those of epoxybutene in both butadiene-exposed rats and humans suggesting an important role of epoxybutanediol in the toxicity of butadiene. Adducts of epoxybutanediol are probably useful for biomonitoring of human exposure to butadiene.

Effects of vinyl acetate and acetaldehyde on sperm morphology and meiotic micronuclei in mice.

The testicular genotoxic effects of vinylacetate (VA) and its hydrolysis product, acetaldehyde (AA), were studied in mice by analyzing the induction of morphologically abnormal sperm and meiotic micronuclei. VA significantly increased the frequency of sperm abnormalities at 500 mg/kg/day while lower doses were ineffective. AA did not induce abnormal sperm. Neither of the compounds influenced the frequency of meiotic micronuclei. VA, but not AA, caused a dose-dependent decrease in sperm production and a reduction of testicular weight at 500 and 125 mg/kg/day.

Induction and survival of micronuclei in rat spermatids. Comparison of two meiotic micronucleus techniques using cyclophosphamide.

The induction and survival of micronuclei (MN) in rat spermatids was studied by two different methods, the dissection method (DM) and the suspension method (SM). It was observed that MN are induced by cyclophosphamide in the S phase of meiosis, in preleptotene spermatocytes, and in an earlier cell stage, the type B spermatogonia. Both techniques showed that MN survive in spermatids at least 5 days. Advantages of the DM include the use of a DNA-specific fluorochrome for staining of MN, higher MN frequencies observed, and the possibility to gain detailed information of the kinetics of induction of MN. In the SM, slide preparation is simpler than in the DM, and several samples can be prepared simultaneously but the scoring of slides is time consuming. Improvements of the sampling system of the DM are suggested. For evaluation of clastogenic action of chemicals on male germ cells both techniques provide a simple and rapid approach.

Micronucleated spermatids in the seminal fluid of smokers and nonsmokers.

This study presents a novel approach to the analysis of chromosome damage in human male germ cells. Round spermatids are present at a low frequency in seminal fluid, and meiotic chromosome breakage may be observed in these cells by analyzing micronuclei (MN). Semen samples from 68 men, including 62 subfertile men and 6 fertile donors, were analyzed. Preparations were made of the round cell types in the semen and after PAS-hematoxylin staining, the number of MN in 100 Golgi-phase or cap-phase spermatids was scored per man. The frequencies of MN were 1.15 +/- 1.42% in the smoking subfertile men and 0.82 +/- 1.30% in the nonsmoking subfertile men. The difference was not statistically significant. When the smokers were divided into groups according to the number of cigarettes smoked per day, no significant differences were observed compared to the nonsmokers. Neither was an effect of smoking observed when the time smoked in years was taken into account. The frequency of MN in ejaculated spermatids in human males was observed to be considerably higher than that reported for testicular spermatids of unexposed rodents.

No effect of maternal smoking in early pregnancy observed on chromosome aberrations in chorionic villus samples.

The effect of maternal smoking on first trimester chorionic villus samples (CVS) was studied by analysing the frequency of chromosome aberrations (CAs) among 20 non-smoking and 20 smoking mothers. The aberrations were classified as chromosome- and chromatid-type breaks and gaps. No statistically significant differences were found in the frequencies of CAs between non-smoking mothers (5.4% or 2.0% gaps excluded) and smoking mothers (3.5% or 1.0% gaps excluded).

Meiotic micronuclei induced by X-rays in early spermatids of the rat.

In mutagenicity studies a rapid detection of chromosomal damage in mammalian germ cells would be very valuable. Encouraged by the usefulness of the bone-marrow micronucleus test, we applied analogous method to the assay of micronuclei induced during meiotic reduction divisions in the adult male rat by X-irradiation. The micronuclei were observed in early post-meiotic cells which were enriched using a transillumination phas-contrast microscopic method. The frequency of micronuclei was scored at various dose levels and at various time intervals. The results indicate a linear increase in the frequency of micronuclei 24 h after X-irradiation with doses of 0, 10, 50, 150, 300 and 600 rad. The highest frequency of micronuclei was observed after 900 rad whereas lower frequencies were found after 1200 rad. The lowest dose giving a statistically significant increase above the control level was 50 rad. The stages of meiosis showed different sensitivities to the chromosome-breaking action of X-rays. The maximal incidence of micronuclei was found 18 h after irradiation which was considered to reflect the great radiosensitivity of diakinesis-metaphase I. The anesthetized group of control animals showed a slightly higher frequency of micronuclei than the non-anesthetized controls. Potentials of the new method for mutagen testing are discussed.

Interaction of Mesna (2-mercaptoethane sulfonate) with the mutagenicity of cyclophosphamide in vitro and in vivo.

The effects of sodium 2-mercaptoethane sulfonate (Mesna) on the mutagenicity of cyclophosphamide (CP) were assessed in vitro by the Ames test and in vivo in rats by analyzing micronuclei in bone marrow and mutagenic activity in urine. Mesna alone was negative in all test systems, while CP gave a positive response in all of them. In a combined treatment there was no significant reduction of the CP-induced mutagenicity in Salmonella. In rats the frequency of bone marrow micronuclei was not diminished when Mesna was given together with CP. May-Grunwald-Giemsa staining and Hoechst-Pyronin fluorescent staining techniques for micronuclei yielded similar results. The urine of rats treated with CP was mutagenic to Salmonella and no significant difference was observed when the rats had received both Mesna and CP. The results give support to the theory that Mesna acts primarily by reducing the toxicity of metabolites of CP, particularly acrolein, in the urinary tract and not by suppressing the mutagenicity of the active metabolites of CP.

Effects of etoposide on stage-specific DNA synthesis during rat spermatogenesis.

The stage-specific effect of etoposide on spermatogenic DNA synthesis was measured 1, 3 and 18 days after a single intraperitoneal injection of etoposide. Etoposide inhibited premitotic DNA synthesis most effectively at stages II-III and IV-V of the seminiferous epithelial cycle in which DNA synthesis of late spermatogonia takes place. Compared with control levels, DNA synthesis at stages II-III was maximally inhibited 43% and 57% at doses of 5 and 10 mg/kg, respectively, and at stages IV-V the maximal inhibition was 67% and 62%, at doses of 5 and 10 mg/kg respectively. Premeiotic DNA synthesis was not as vulnerable to the etoposide action as premitotic DNA synthesis, the maximal inhibition of premeiotic DNA synthesis was 39% and 41% compared with control at doses of 5 and 10 mg/kg, respectively. Induction of most probably repair-type DNA synthesis was demonstrated in stages I-III, VIIa-b and XII of the cycle. All the effects of etoposide were most apparent 1 and 3 days after treatment but had not totally disappeared 18 days after the treatment.