Evaluation of spermatogenic response of mice to the induction of mutations by combined treatment with X rays and antineoplastic drugs

Combined treatment with low doses of X-rays plus cyclophosphamide (0.25 Gy+25 mg/kg body weight) or X-rays plus mitomycin C (0.25 Gy+1.75 mg/kg body weight) did not induce significant dominant lethal effects in any stage of spermatogenesis when a parameter representing pre- and postimplantation loss, such as the decrease of live implants per female, was applied. After combined exposure to high doses of X-rays plus cyclophosphamide (1.00 Gy+100 mg/kg body weight) an increase of dominant lethal mutations (DLMs) was observed in differentiating spermatogonia, spermatids, and spermatozoa with the same parameter. Combined treatment with high doses of X-rays plus mitomycin C (1.00 Gy+5.25 mg/kg body weight) produced DLMs in differentiating spermatogonia and late spermatocytes. A calculation of enhanced risk was applied to the data of DLMs from the combined treatment regimen and was based on the proportion of dead implants (postimplantation loss only). Enhanced risk could be shown not only after high but also after low combined exposure to X-rays plus cyclophosphamide and X-rays plus mitomycin C. With low doses this enhanced risk was observed in spermatids for X-rays plus cyclophosphamide and in differentiating spermatogonia to early spermatocytes for X-rays plus mitomycin C.     

Induction of specific-locus and dominant lethal mutations in male mice by ifosfamide (Holoxan)

Ifosfamide induced dominant lethal mutations in spermatozoa of mice at doses of 200 and 300 mg/kg and in spermatids and spermatocytes at 600 mg/kg. The highest dose also induced specific-locus mutations in post-spermatogonial germ-cell stages of mice but not in spermatogonial stem cells. The nature of the induced mutations suggests they are intergenic. The spermatogenic specificity of ifosfamide in mouse germ cells is similar to that of the structurally related cytostatic drugs cyclophosphamide and trofosfamide. Due to the post-spermatogonial germ cell specificity of ifosfamide, the genetic risk is limited to a few weeks after exposure.     

Dominant lethal mutations induced by MMS and mitomycin C in the fish Oryzias latipes

Males of the fish Oryzias latipes were treated with various chemicals and then mated with normal females. The fertility and hatchability of the eggs laid by the parents were examined, and the dominant lethal effects were estimated. Mitomycin C induced dominant lethals in the fish spermatids and spermatocytes after the males had been treated with concentrations of 2.5 and 25 micrograms/ml. Methyl methanesulfonate (MMS) induced dominant lethals in spermatozoa and spermatozoa and spermatids after the injection of 200 and 400 mg/kg. These results are in good agreement with the results obtained with mice. However, the effects of ethyl methanesulfonate (EMS) were not clear on spermatogenic cells at any stage. We could not recognize any significant induction of dominant lethals by urethanes, bleomycin, caffeine, and two kinds of food-color additives, at least under the present experimental conditions.     

Comparison of radiation-and chemically-induced dominant lethal mutations in male mice

Ionizing radiation-induced dominant lethal mutations in all spermatogenic stages. After irradiation of male mice with 200 R the yield of induced mutations in early spermatids was twice the yield in spermatozoa, late spermatids, and spermatocytes. After irradiation with 400 R or 800 R the spermatocytes were the most sensitive stage for the induction of dominant lethal mutations. The frequency of radiation-induced dominant lethal mutations in postspermatogonial stages was dose-dependent. The yield of dominant lethal mutations in spermatogonia was independent of the dose.     

Male and female reproductive toxicity induced by sub-chronic ethanol exposure in CF-1 mice

Since genetic damage induced by ethanol exposure is controversial and incomplete and because germ and somatic cells constitute bioindicators for monitoring reproductive toxicity and genotoxic actions of ethanol consumption, the purpose of the present investigation was to evaluate morphological sperm, oocyte alterations and parental genotoxic effects after sub-chronic ethanol intake in the CF-1 outbred mouse strain. Ethanol 10% was administered to CF-1 adult male (treated males, TM) and female (treated females, TF) mice for 27 days, whereas water was given to controls from both sexes too (CM and CF). Post-treatment micronucleus frequency (MN-PCE/1,000/mouse) and gamete morphology were evaluated. To test whether change of female reproductive status results in maternal genotoxicity, CF-1 females received ethanol 10% (exposed group, periconceptionally treated females (PTF)) or water (control group, pregnant control females (PCF)) in drinking water for 17 days previous and up to 10 days of gestation. TM had a high percentage of abnormal spermatozoa vs CM (p < 0.001) and elevated parthenogenetic activated oocyte frequency appeared in TF vs CF (p < 0.001). Sub-chronic ethanol ingestion induced increased MN frequency in TM and TF (p < 0.01). In PTF, where blood alcohol concentrations were between 19–28 mg/dl, very significantly increased MN frequency was found vs PCF (p < 0.01), whereas MN values were similar to TF. These results show that sub-chronic alcohol ingestion in CF-1 mice produces sperm head dysmorphogenesis and oocyte nuclear anomalies, suggesting that morphological abnormalities in germ cells are probably related to parental genotoxicity after ethanol consumption.     

Relationship between age of the male and spontaneous or radiation induced prenatal mortality in mice]

Control and X-irradiated male mice from the BALB/c strain, three or twelve months old, have been mated with 3 months old virgin females from the same strain. Dissection of the females on the seventeenth day after the beginning of the mating has shown that the preimplantation is increased when the males are twelve months old whereas exposure to ionizing radiations enhances only the postimplantation loss. It is possible that the increase in preimplantation loss results from some changes in the vitality or fertilizing capacity of the spermatozoa.     

Testicular Volume and Reproductive Status of Wild <Emphasis Type="Italic">Callithrix jacchus</Emphasis>

The social relationship dynamic among callitrichid females is well known. Breeding exclusivity by dominant females involves female-female competition, usually resulting in the inhibition of subordinate reproduction. However, the strategies to maintain the male breeding position are still unclear. Researchers have observed no overt aggression between males, and differences in testosterone levels between dominant and subordinate individuals do not correlate with differences in reproductive success. In Callithrix monogamy is the predominant mating system, and testicular size is compatible with the absence of sperm competition. We analyzed testicular volume during development in 95 individuals at different ages (infant n = 12, juvenile n = 9, subadult n = 15, and adult n = 59). We also investigated if the ratio between testicular volume and body mass correlates with breeding position in the social group. The ratio was significantly higher in breeding males and a positive correlation between body mass and testicular volume is significant only for nonbreeding males. The findings suggest that testicular size varies with male reproductive status in the social group and that the enlargement of testicular volume in breeding common marmoset males seems to be a result of proximate causes and to depend on social and reproductive contexts acting together or separately.     

Germ line transposition of the copia retrotransposon in Drosophila melanogaster is restricted to males by tissue-specific control of copia RNA levels

Germ line transposition rates of the retrotransposon copia were directly measured in males and females of an inbred Drosophila melanogaster line, 2b3, which is highly polymorphic for copia insertion sites. The elevated germ line transposition rate of copia in this line (3–8 × 10⁻³ per generation per element) is confined to males, with transposition in females being undetectable under the conditions of the experiment but at most 50-fold lower than the rate for males. To determine the molecular basis of this effect, copia RNA levels were measured in whole bodies and germ lines of male and female flies of both the unstable 2b3 line and a stable line, Oregon RC-iso, which shows normal rates of copia transposition. Both male and female 2b3 flies contain much more copia RNA than flies of the stable line. However, 2b3 male germinal tissues contain much higher levels of copia RNA than the equivalent female tissues. The highest copia expression is detected in maturing primary spermatocytes. Our data show that high rates of germ line copia transposition are restricted to males by tissue-specific control of RNA levels and suggest that transposition of copia only occurs in fly tissues containing more than a relatively high threshold level of copia RNA. Received: 8 October 1996 / Accepted: 7 January 1997     

Sensitivity of Drosophila melanogaster to low concentrations of gaseous mutagens. II. Chronic exposures.

Sex-linked recessive lethal mutations were induced in Drosophila melanogaster males by gaseous 1,2-dibromoethane at concentrations ranging from 0.2 to 2 parts per million. Significant numbers of mutations could be induced at all these concentrations. Pronounced germ-cell sensitivity differences were observed. For low exposures, spermatids and spermatocytes were about 10--20 times more sensitive than spermatozoa. The dose-effect relation was linear below 60 ppm . h for the 3 cell types. At higher exposures, sterility prevented mutation detection in spermatocytes and in spermatogonia. The lowest effective exposure for spermatozoa was 18 ppm . h (0.25 ppm for 72 h). In spermatids, the lowest exposure tested, 2.3 ppm . h (0.2 ppm for 11 h) induced 4 times the spontaneous mutation rate. Therefore, using prolonged exposure periods one may be able to detect concentrations in the range of parts per billion. Thus, Drosophila appears suitable as a system for detecting very low concentrations of gaseous mutagens in industrial, agricultural and environmental atmospheres.     

Completion of meiosis in male zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) despite lack of DNA mismatch repair gene <Emphasis Type="Italic">mlh1</Emphasis>

Mlh1 is a member of DNA mismatch repair (MMR) machinery and is also essential for the stabilization of crossovers during the first meiotic division. Recently, we have shown that zebrafish mlh1 mutant males are completely infertile because of a block in metaphase I, whereas females are fertile but have aneuploid progeny. When studying fertility in males in a two-fold more inbred background, we have however observed low numbers of fertilized eggs (approximately 0.4%). Histological examination of the testis has revealed that all spermatogenic stages prior to spermatids (spermatogonia, primary spermatocytes, and secondary spermatocytes) are significantly increased in the mutant, whereas the total weight of spermatids and spermatozoa is highly decreased (1.8 mg in wild-type vs. 0.1 mg in mutants), a result clearly different from our previous study in which outbred males lack secondary spermatocytes or postmeiotic cells. Thus, a delay of both meiotic divisions occurs rather than complete arrest during meiosis I in these males. Eggs fertilized with mutant sperm develop as malformed embryos and are aneuploid making this male phenotype much more similar to that previously described in the mutant females. Therefore, crossovers are still essential for proper meiosis, but meiotic cell divisions can progress without it, suggesting that this mutant is a suitable model for studying the cellular mechanisms of completing meiosis without crossover stabilization. Marcelo C. Leal and Harma Feitsma contributed equally to this work. This work was supported by the Brazilian Foundation CAPES, the Cancer Genomics Center (Nationaal Regie Orgaan Genomics), the European Union-funded FP6 Integrated Project ZF-MODELS, and Utrecht University.     

Foraging for mates in the hyperparasitic wasp, Dendrocerus carpenteri: impact of unfavourable weather conditions and parasitoid age

Males of the aphid hyperparasitoid Dendrocerus carpenteri (Curtis) were attracted by a sex pheromone released by conspecific females. The intensity of this cue, and thus female attractiveness, depended both on the female's mating status and her age. Only virgin females younger than 2 h were consistently recognized as mates by foraging males. Male age did not influence foraging and mating success. Empty mummies, from which females had emerged within the previous 10 min were attractive to males and examined intensively. Rain reduced the searching success of males, although the host plant Vicia faba provided sheltered places. Wind did not reduce mating success but prevented both sexes from leaving the host plant. Since the time of female attractiveness seems to be very limited, wind may have an enormous effect on the mating success of D. carpenteri in the field and thus on the population dynamics of this species. Received: 5 October 1998 / Accepted: 16 December 1998     

Induction of specific-locus and dominant lethal mutations in male mice by busulfan

The chemotherapeutic agent busulfan was tested for the induction of dominant lethal and specific-locus mutations in male mice. A dose of 5 mg/kg b.w. of busulfan induces dominant lethal mutations in spermatozoa. A dose of 20 mg/kg b.w. induces dominant lethal mutations in spermatozoa and spermatids. A total of 83,196 offspring were scored in the specific-locus experiments. Busulfan-induced specific-locus mutations were recovered in spermatozoa and spermatids, but not in spermatogonia. The sensitivity patterns for the induction of dominant lethal and specific-locus mutations by busulfan in germ cells of male mice are similar but not identical.     

Sperm allocation pattern during a reproductive season in the copulating marine cottoid species, <Emphasis Type="Italic">Alcichthys alcicornis</Emphasis>

The sperm allocation pattern of a copulating marine cottid fish, Alcichthys alcicornis, was investigated. A total of 86 mating events using six males were conducted in aquarium tanks over 10 days, and in 36 of them, spermatozoa were collected using a false copulation method. Males released 3–8 × 10⁸ spermatozoa in early events, with the number decreasing gradually during subsequent mating events. This sperm allocation was represented as an “early investment and tapering” pattern. It was discussed why males have significantly higher sperm release in early spawning events. The reproductive behavior consists of spawning and subsequent copulation. Spermatozoa have the ability to fertilize eggs from multiple clutches, and in earlier produced clutches the level of sperm competition should be relatively low. In addition, if early spawn happens to be the first spawn with a female, spermatozoa that are released into the water column after spawning are responsible for fertilizing the female’s first clutch. The probability of this occurring should decrease dramatically as the season progresses, due to the highly synchronous seasonal spawning of females. All of these factors should select for high sperm numbers in early ejaculates. Based on such reproductive ecology of A. alcicornis we hypothesize that this sperm allocation pattern is an adaptive reproductive strategy in response to egg availability and sperm competition occurring within the ovarian cavity.     

Aspects of body size and gonadal histology in the Antarctic toothfish, Dissostichus mawsoni, from McMurdo Sound, Antarctica

Twenty-eight specimens of the large notothenioid Dissostichus mawsoni were dissected after capture on a set line near the southern limit of its range in McMurdo Sound, Antarctica. Total length (L _T) averaged 127.3 cm (range 90–162 cm) and weight (W ) was 26.7 kg (range 10.4–60.3 kg). The length-weight relationship was W=3.44×10⁻⁵ L _T ^2.85 (n=28, r ²=0.96). Subcutaneous lipid thickness averaged 2.6 mm and showed no difference due to sex, but a significant weak relationship to W and L _T. Hepatosomatic index (I _H) was 1.6% for females and 1.7% for males; gonadosomatic index (I _G) was 0.9% for females and 0.2% for males. Although specimens were large and sexually mature, the histology indicated that the gonads of this November sample were in a resting stage. Testes lacked spermatids and spermatozoa. Oocytes were in the previtellogenic stage (91.2%) or in the first stage of vitellogenesis (8.8%). A few atretic oocytes and empty follicles indicated that some females in this sample had spawned previously. A summary of the life-cycle is also presented. Accepted: 27 September 1999     

Sexual Attraction,Male Courtship and Female Remating in the Tomato Fruit Fly, <Emphasis Type="Italic">Neoceratitis cyanescens</Emphasis>

Sexual attraction in the tomato fruit fly, Neoceratitis cyanescens (Bezzi) (Diptera: Tephritidae) was tested in an olfactometer while major behavioral traits during courtship at a short range were described by video recording. In addition, 30 pairs of flies were monitored for mating activity in individual cages, during a 38-day study following adult emergence. Virgin females of N. cyanescens showed a significant attraction when males were placed in the upwind section of the olfactometer. Males adopted a typical calling behavior characterized by immobility, wings perpendicular to the body axis, swollen abdominal pleura and presence of a small transparent droplet at the tip of the abdomen. In addition, a specific odor was clearly perceptible by the observer. In individual cages, the first mating of N. cyanescens females took place on the second day after emergence. Females showed a high mating propensity, with 73 ± 18% females mated daily. Sexual conflicts due to mating system and applications to pest management strategies are discussed.     

Characterization and immunolocalization of beta-D-glucuronidase in mouse testicular germ cells and spermatozoa

Spermatozoa released from the seminiferous tubules are terminally differentiated cells with no known synthetic activity. Their components are synthesized in the spermatogenic cells during spermatogenesis. In this study, we report the characterization and immunolocalization of beta-glucuronidase in mouse testicular germ cells and spermatozoa. The enzyme is an exoglycohydrolase with dual localization, being present in lysosomes and endoplasmic reticulum of several mouse and rat tissues. The purified germ cell preparations (spermatocytes, round spermatids, and condensed/elongated spermatids) when assayed for beta-glucuronidase activity showed that the spermatocytes contained five times more enzyme activity per cell than the spermatids. Polyacrylamide gel electrophoresis, carried out under native and denaturing conditions, demonstrated that the germ cells express only the lysosomal form of the enzyme (pI 5.5-6.0) with a subunit molecular mass of 74 kDa. Immunocytochemical studies revealed a positive reaction in the Golgi membranes, Golgi-associated vesicles, and lysosomes of late spermatocytes (pachytene spermatocytes) and a stage-specific localization during spermiogenesis. The forming or formed acrosome of the elongated spermatids (stages 9-16) and epididymal spermatozoa was highly immunopositive. Comparison of immunoprecipitation curves and kinetic properties of the enzyme present in spermatocytes and spermatozoa revealed no major differences. Taken together, our results demonstrate that beta-glucuronidase activities present in the lysosomes of spermatocytes and the sperm acrosome are kinetically and immunologically similar.     

Studies on mutagen-sensitive strains of Drosophila melanogaster. IV. Modification of genetic damage induced by X-irradiation of spermatozoa and spermatids in N2 or O2 by mei-9a, mei-41D5 and mus(1)101D1

The influence of defects in DNA repair processes on X-ray-induced genetic damage in post-meiotic male germ cell stages of Drosophila melanogaster was studied using the 'maternal effects approach'. Basc males were irradiated in N2, air or O2 either as 48-h-old pupae (to sample spermatids) or as 3-4-day-old adults (to sample mature spermatozoa) and mated to females of 3 repair-deficient strains (mei-9a: excision-repair-deficient; mei-41D5: post-replication-repair-deficient; mus(1)101D1: post-replication-repair-deficient and impaired in DNA synthesis). Simultaneous controls involving mating of males to repair-proficient females (mei+) were run. The frequencies of sex-linked recessive lethals and of autosomal translocations were determined following standard genetic procedures. The responses elicited in the different crosses with repair-deficient females were compared with those in mei+ crosses. The main findings are the following: with mei-9 females, the frequencies of recessive lethals are higher after irradiation of spermatids in N2, but not after irradiation in air of O2 (relative to those in the mei+ crosses); this result is different from that obtained in earlier work with spermatozoa, in which cell stage, higher yields of recessive lethals were obtained after irradiation of males in either N2 or air; in the mei-9 crosses, there are no significant differences in response (relative to mei+) after irradiation of either spermatozoa or spermatids in O2; the translocation frequencies in the mei-9 crosses are similar to those in the mei+ crosses, irrespective of the treated germ cell stage or the irradiation atmosphere; irradiation of either spermatozoa or spermatids in N2, air or O2 does not result in any differential recovery of recessive lethals in the mei-41 relative to mei+ crosses; irradiation of spermatids in N2 and of spermatozoa in air leads to a higher recovery of translocations in the mei-41 crosses; and after irradiation of spermatids or spermatozoa in any of the gaseous atmospheres, the frequencies of recessive lethals and of translocations are lower in the mus-101 crosses. The differences in responses (between cell stages, in different gaseous atmospheres and with different repair-deficient females) are explained on the basis of both qualitative and quantitative differences in the composition of the initial lesions and the extent to which their repair may be affected by the defects present in the different repair-deficient females. Several discrepancies between expectations based on biochemical results and the genetic results are pointed out.(ABSTRACT TRUNCATED AT 400 WORDS)     

The effects of chloramphenicol, streptomycin and penicillin on the induction of mutations by X-rays inDrosophila melanogaster

Summary Injection of chloramphenicol, streptomycin or penicillin intoDrosophila males just before exposure to X-irradiation causes a reduction in the yield of sex linked recessive lethal mutations. The effect appears to be primarily on spermatids and possibly spermatocytes.With I Figure in the Text     

Restriction of PGK-B to spermatogenic cells: Evidence from experimentally cryptorchid mice

The hypothesis that PGK-B, like LDH-C₄, is restricted to spermatogenic cells was explored by examining isozyme patterns in testes from mice depleted of germinal cells by surgical cryptorchism. In experimentally cryptorchized C57BL/10Sn males, decline in PGK-B activity paralleled decline in LDH-C₄ activity and was correlated with degeneration of spermatocytes, spermatids, and spermatozoa. Trace amounts of these sperm isozymes found in cryptorchid testes after the depletion of maturing germ cells probably came from degenerated spermatids and spermatocytes and not from somatic testicular cells.     

Differential expression of vasa homologue gene in the germ cells during oogenesis and spermatogenesis in a teleost fish, tilapia, Oreochromis niloticus

Vas (a Drosophila vasa homologue) gene expression pattern in germ cells during oogenesis and spermatogenesis was examined using all genetic females and males of a teleost fish, tilapia. Primordial germ cells (PGC) reach the gonadal anlagen 3 days after hatching (7 days after fertilization), the time when the gonadal anlagen was first formed. Prior to meiosis, no differences in vas RNA are observed in male and female germ cells. In the ovary, vas is expressed strongly in oogonia to diplotene oocytes and becomes localized as patches in auxocytes and then strong signals are uniformly distributed in the cytoplasm of previtellogenic oocytes, followed by a decrease from vitellogenic to postvitellogenic oocytes. In the testis, vas signals are strong in spermatogonia and decrease in early primary spermatocytes. No vas RNA expression is evident in either diplotene primary spermatocytes, secondary spermatocytes, spermatids or spermatozoa. The observed differences in vas RNA expression suggest a differential function of vas in the regulation of meiotic progression of female and male germ cells.