Benomyl-induced aneuploidy in mouse oocytes.

Benomyl (methyl-1-[butylcarbamoyl]-2-benzimidazole carbamate) is a plant fungicide which acts by inhibiting tubulin polymerization. It is also a carcinogen and a teratogen. A substantial number of reports consistently show that benomyl inhibits cellular division and induces numerical chromosome changes in somatic cells, while results from standard genotoxicity assays which measure mutations and structural chromosome aberrations are conflicting. To define further the genotoxic effects of benomyl and to demonstrate the utility of the in vivo mouse oocyte assay for detecting chemicals which induce numerical chromosome changes, we investigated the ability of benomyl to induce numerical and structural chromosome aberrations in mouse oocytes. Superovulated female ICR mice were administered benomyl by oral gavage and oocytes were collected 17 h later. The proportions (and percentages) of hyperploid oocytes were 1/309(0.3), 6/155(3.9), 38/229(16.6), 46/130(35.4), 60/215 (27.9), 42/143(29.4) for control, 500, 1000, 1500, 1750 and 2000 mg/kg respectively. No increase in structural aberrations was observed. These results demonstrate that benomyl specifically induces numerical chromosome changes in mouse oocytes.     

Induction and transmission of chromosome aberrations in mouse oocytes after treatment with butadiene diepoxide

A study was conducted on the genotoxicity of butadiene diepoxide (DEB) in mouse oocytes. Superovulated female mice were injected intraperitoneally with DEB and mated with untreated males. Oocyte exposure occurred approximately 1.5 days before ovulation. DEB doses ranged between 26 and 52 mg/kg. Chromosome aberrations were scored in C-banded metaphases of one-cell embryos. The percentage of mated females, the average number of zygotes harvested per female, the frequencies of unfertilized oocytes and developmentally delayed zygotes did not reveal any overt sign of chemical toxicity which hindered the propensity of animals to mate or affected the ovulation, fertilization, or cell cycle progression of treated oocytes. A dose-dependent induction of chromosome aberrations was observed which was best fitted by a linear quadratic equation. Half of all the aberrations transmitted by DEB-treated oocytes were chromatid-type breaks or exchanges. Among chromosome-type aberrations, double fragments far exceeded chromosome exchanges. This spectrum of structural aberrations differed markedly from what was previously observed in one-cell embryos conceived by DEB-treated sperm, where 97% were chromosome-type aberrations and 40% were dicentrics or translocations. This difference suggests that chromosome damage in one-cell embryos can be fixed by different mutagenic pathways influenced by the targeted gamete and its specific chromatin configuration. After exposure to the same dose, oocytes transmitted to one-cell embryos between 4 and 8 times fewer aberrations than DEB-treated sperm. While the rate of aberration induction suggests that female germ cells may be less at risk than mature sperm,especially at low-dose levels, the higher threshold for reproductive toxicity observed in female than in male mice may justify inclusion of data on female germ cell mutagenicity in the genetic risk assessment of butadiene exposure. Environ. Mol. Mutagen. 30:403–409, 1997 © 1997 Wiley-Liss, Inc.     

A detailed cytogenetic analysis of large numbers of fresh and frozen-thawed human sperm after ICSI into mouse oocytes

BACKGROUND: Since information about chromosome aberrations in micro-manipulated sperm is still inadequate, cytogenetic analysis was performed on large numbers of fresh and frozen-thawed (FT) human sperm after injection into mouse oocytes. The effects of the ICSI procedure on oocytes are also discussed based on analysis of the mouse chromosome complements. METHODS: After the injection of fresh and FT human sperm into mouse oocytes, chromosomes of the hybrid oocytes were analysed at first cleavage metaphase. RESULTS: Incidences of the hybrid oocytes at the first cleavage metaphase were significantly different between fresh (71.5%) and FT sperm groups (80.1%) (P < 0.05). The chromosome analysis of 477 fresh and 141 FT sperm showed no difference in the incidences of aneuploidy (1.6/0.7%), structural aberrations (8.8/7.8%) or diploidy (0.0/0.0%) between these categories. The cytogenetic result did not differ from our previous result using IVF between human sperm and hamster oocytes. In an additional cytogenetic study on 615 mouse chromosome complements, the incidence of diploidy (5.4%) was significantly higher than those (0.3-2.8%) in the previous mouse cytogenetic studies, and the hybrid oocytes with no mouse chromosomes (2.0%) existed. CONCLUSIONS: This result suggests that the ICSI procedure induces no sperm chromosome aberrations but increases numerical aberrations in oocyte chromosome complements.     

Carcinogenicity of chloral hydrate administered in drinking water to the male F344/N rat and male B6C3F1 mouse

Male B6C3F1 mice and male F344/N rats were exposed to chloral hydrate (chloral) in the drinking water for 2 years. Rats: Measured chloral hydrate drinking water concentrations for the study were 0.12 g/L, 0.58 g/L, and 2.51 g/L chloral hydrate that yielded time-weighted mean daily doses (MDDs) of 7.4, 37.4, and 162.6 mg/kg per day. Water consumptions, survival, body weights, and organ weights were not altered in any of the chloral hydrate treatments. Life-time exposures to chloral hydrate failed to increase the prevalence (percentage of animals with a tumor) or the multiplicity (tumors/animal) of hepatocellular neoplasia. Chloral hydrate did not increase the prevalence of neoplasia at any other organ site. Mice: Measured chloral hydrate drinking water concentrations for the study were 0.12 g/L, 0.58 g/L, and 1.28 g/L that gave MDDs of 13.5, 65.0, and 146.6 mg/kg per day. Water consumptions, survival, body and organ weights, were not altered from the control values by any of the chloral hydrate treatments. Enhanced neoplasia was observed only in the liver. Prevalence and multiplicity of hepatocellular carcinoma (HC) were increased only for the high-dose group (84.4%; 0.72 HC/animal; p < or = 0.05). Values of 54.3%; 0.72 HC/animal and 59%; 1.03 HC/animal were observed for the 13.5- and 65.0-mg/kg per day treatment groups. Prevalence and multiplicity for the control group were 54.8%; 0.74 HC/animal. Hepatoadenoma (HA) prevalence and multiplicity were significantly increased (p < or = 0.05) at all chloral hydrate concentrations: 43.5%; 0.65 HA/animal, 51.3%; 0.95 HA/animal and 50%; 0.72 HA/animal at 13.5, 65.0, and 146.6 mg/kg per day chloral hydrate compared to 21.4%; 0.21 HA/animal in the untreated group. Altered foci of cells were evident in all doses tested in the mouse, but no significant differences were observed over the control values. Hepatocellular necrosis was minimal and did not exceed that seen in untreated rats and mice. Chloral hydrate exposure did not alter serum chemistry and hepatocyte proliferation in rats and mice or increase hepatic palmitoyl CoA oxidase in mice at any of the time periods monitored. It was concluded that chloral hydrate was carcinogenic (hepatocellular neoplasia) in the male mouse, but not in the rat, following a lifetime exposure in the drinking water. Based upon the increased HA and combined tumors at all chloral hydrate doses tested, a no observed adverse effect level was not determined.     

Chloral hydrate induced spindle aberrations, metaphase I arrest and aneuploidy in mouse oocytes

In view of tissue- and sex-specific differences in chemically-inducedaneuploidy, we analysed the effects of chloral hydrate (CH)on cell-cycle progression, spindle formation and aneuploidyin in vitro maturing mouse oocytes with cytogenetic and immunofluorescentmethods. CH blocks oocyte maturation irreversibly and concentrationdepend-ently. During culture in 125 µg/ml CH, germinalvesicle breakdown is delayed, and most oocytes become arrestedin meiosis I with bivalent chromosomes. Their spindles are asymmetricor attain fusiform poles. Oocytes which progress to metaphaseII also possess astral instead of barrel-shaped, anastral spindlesas characteristic for the controls. Resolution of chiasmatawithout polar body extrusion during exposure to 50 and 125 µ/mlCH results in significant rises in ‘diploid’ metaphaseII oocytes. Hyperplo-idy rates do not increase significantlyat any concentration of CH, but hypoploidy levels are elevatedat 125 µ/m\ CH. CH induces lagging of chromosomes duringtelophase I, inhibits spindle elongation in anaphase B and causeschromosome displacement from the spindle equator in metaphaseI and II. Oocytes also become irreversibly arrested in maturationwhen exposed to CH prior to resumption of maturation, or whenCH is present during the first or second 8 h of maturation.Therefore, these data show unequivocally that CH is a potentaneugen in female germ cells, affecting spindle shape, cytokinesisand cell-cycle progression. However, a metaphase I checkpointin mammalian oogenesis sensing disturbances in the spindle appearsto prevent nondisjunction efficiently in most oocytes. In thisin vitro model the aneugenic activity of drugs, critical periodsin oocyte maturation, threshold concentrations, and targetsof drug action can be directly assessed. ¹To whom correspondence should be addressed     

Mouse genetics provides insight into folliculogenesis,fertilization and early embryonic development

After colonization of the gonad, mouse female germ cells enter into the prophase of the first meiotic division as a mid-gestational hallmark of gender. Perinatally, oocytes interact with granulosa cells to form primordial follicles which, with cyclic periodicity, enter into a 3-week growth phase that culminates in meiotic maturation and ovulation. Successful fertilization in the oviduct results in the onset of embryogenesis. Genes expressed in oocytes encode maternal factors that control many of these developmental processes. The establishment of mouse models in which specific genes have been disrupted offers robust insights into molecular mechanisms that control oogenesis, folliculogenesis, fertilization and early embryogenesis. Although relatively few developmental circuits have been characterized in genetic detail, the ongoing revolution in mouse genetics holds great promise. These model systems provide novel information into the molecular basis of the pathways required for oocyte-specific processes as well as for interactions with the temporally changing environment of female germ cells. The similarities between the mouse and human genomes provide assurance that this knowledge will rapidly translate into a better understanding of human reproduction.     

Further evidence for the aneuploidogenic properties of chelating agents: induction of micronuclei in mouse male germ cells by EDTA.

A micronucleus assay based on cytogenetic analysis of early spermatids (Tates et al.; Mutation Research 121:131-138, 1983) was applied to determine if the chelating agent ethylenedinitrilotetraacetic acid (EDTA) may induce aneuploidy in mouse meiotic cells. Previous results indicated aneuploidogenic activity of EDTA in Drosophila female germ cells (induction of chromosome loss; Zordan et al.: Environ Mol Mutagen 15:205-213, 1990). In the same study, a standard aneuploidy test based on chromosome counting in mouse secondary spermatocytes failed however to show aneuploidogenic properties of EDTA in mouse somatic and germ cells. In the present study the effects of two clastogens, adriamycin (ADM) and mitomycin C (MMC), and of the aneuploidogenic agent chloral hydrate (CH) were also evaluated. All compounds were tested at a single dose level and at two time intervals corresponding to the treatment of diakinesis/metaphase I/metaphase II spermatocytes. The clastogenic potential of the compounds under study was also evaluated, by chromosomal aberration analysis in mouse spermatogonia, in an independent set of experiments. The results obtained indicate that ADM, CH and EDTA are able to induce micronuclei at meiosis. On the contrary, only ADM and MMC induced chromosomal aberrations in mouse spermatogonia. Therefore, the most probable origin of micronuclei produced by CH and EDTA is whole chromosome lagging. These results provide further evidence for the aneuploidogenic properties of these chelating agents.     

A cytogenetic study of in-vitro matured murine oocytes after ICSI by human sperm

BACKGROUND: The purpose of this study was to investigate the chromosomal complement and developmental potential of in-vitro matured murine oocytes following ICSI by human sperm. METHODS: Heterologous ICSI fertilization between mouse oocytes and human sperm was employed in order to overcome the reduced fertilization rates observed after conventional IVF due to zona hardening during in-vitro maturation, and to assess separately maternal and paternal chromosome complements. Cytogenetic analyses were performed in four types of oocytes: (i) in-vitro matured metaphase II (MII) oocytes; (ii) in-vivo matured MII oocytes; (iii) in-vitro matured oocytes after ICSI; (iv) in-vivo matured oocytes after ICSI. RESULTS: Activation rates after ICSI of in-vitro matured oocytes was lower than that of in-vivo matured oocytes (69.9 versus 97.2%, P < 0.01), and premature chromosomal condensation was only observed in in-vitro matured oocytes. However, there were no significant differences in developmental rates after successful activation between in-vivo and in-vitro matured ICSI oocytes (69.7 versus 76.6%). The incidences of aneuploidy and structural aberrations were similar between the ICSI embryos and non-ICSI (MII) oocytes. Furthermore, the frequency of chromosomal aberrations was not associated with in-vitro or in-vivo maturation. Similar analyses of paternal chromosomes indicated that there were no significant differences in the incidence of chromosomal aberrations between the embryos derived from in-vitro and in-vivo matured oocytes. CONCLUSIONS: These results suggest that in-vitro matured oocytes following ICSI do not lead to an increase in the frequency of aneuploidy and structural aberrations when human sperm are injected into mouse oocytes.     

Genetic toxicology testing of the antimalarial drugs chloroquine and a new analog, AQ-13

AQ-13 ([N1-(7-chloro-quinolin-4yl)-3-(N3,N3-diethylamino)propylamine] dihydrochloride trihydrate) is an aminoquinoline antimalarial drug that is effective against chloroquine-resistant strains of Plasmodium falciparum. It is structurally similar to the widely used chloroquine diphosphate (CQ). We evaluated these drugs in the three assays currently recommended by the International Conference on Harmonization (ICH): bacterial mutagenesis in Salmonella typhimurium and Escherichia coli, mammalian cell mutagenesis in L5178Y mouse lymphoma cells, and micronucleus induction in rat bone marrow. A small but statistically significant increase in revertant colonies was produced by CQ with Salmonella tester strain TA98 without metabolic activation (MA) and by AQ-13 with strain TA1537 both with and without MA. In L5178Y cells, testing of CQ and AQ-13 up to cytotoxic concentrations with and without MA produced no increase in mutant colonies and no increase in the numbers of small colonies. Slight decreases in the ratio of polychromatic erythrocytes (PCE) to red blood cells (RBC) were observed in male and female rats treated with CQ and in females only treated with AQ-13; however, none of these changes was statistically significant. No increases in the frequency of micronucleated PCE were observed at any dose level of CQ or AQ-13. Although both CQ and AQ-13 showed weak bacterial mutagenicity, this mutagenic effect was not confirmed in either the mouse lymphoma mutagenesis assay or the micronucleus assay. These results indicate that CQ and AQ-13 should pose minimal risk of genotoxic damage in human populations being administered these drugs.     

Mouse spermatid nuclei can support full term development after premature chromosome condensation within mature oocytes.

The nucleus of round spermatids, the earliest haploid male germ cells, can participate in the formation of normal zygotes when incorporated into activated oocytes. In this study, we injected mouse round spermatids into homologous mature oocytes that were kept arrested at metaphase II to induce premature chromosome condensation (PCC) of the spermatid nuclei. After full condensation of the spermatid chromosomes, the oocytes were activated by Sr2+-containing medium, into which cytochalasin B was added to prevent extrusion of the segregated female and male chromosomes as polar bodies. Out of 142 oocytes examined, 104 (73%) formed two male (pseudo)pronuclei and two female pronuclei. To restore the diploid state of these zygotes, one of the female pronuclei was removed. When cultured in vitro for 72 hours, all (n = 37) of the constructed embryos developed to the morula/blastocyst stage. When 2-cell embryos and morulae/blastocysts were transferred into pseudopregnant females, 14 (13/96) and 24% (9/37), respectively, developed into term offspring. This study indicates that the spermatid chromosomes, which had undergone PCC, moved safely to opposite poles after oocyte activation. Since round spermatids contain no (in the mouse) or little (in patients with spermatogenic failure) oocyte-activating factor, this method may be used to rescue oocytes that fail to be activated at the time of spermatid injection.     

Frequent structural chromosome aberrations in immotile human sperm exposed to culture media

BACKGROUND: The influence of culture media or centrifugation on chromosomes of immotile human sperm was examined using ICSI into mouse oocytes. METHODS: In experiment 1, immotile and motile human sperm retrieved directly from ejaculates were injected into mouse oocytes. In experiment 2, immotile human sperm were exposed to seminal plasma or one of four kinds of culture media (HEPES-BWW, modified-BWW, modified-human tubal fluid (HTF) and Dulbecco's phosphate-buffered saline) for 1.5-2.5 h at 18 degrees C in air before microinjection. In experiment 3, immotile human sperm were centrifuged along with HEPES-BWW before microinjection. In experiment 4, frozen-thawed immotile human sperm washed with seminal plasma or HEPES-BWW were injected into mouse oocytes. The hybrid oocytes were prepared for chromosome slides at first cleavage metaphase and were then examined cytogenetically. RESULTS: In experiment 1, there was no significant difference in the incidences of structural chromosome aberrations between motile and immotile sperm (4.3% versus 5.8%). In experiment 2, culture media caused more frequent structural chromosome aberrations (14.3-32.6%) in immotile sperm than did seminal plasma (5.4%). In experiment 3, structural chromosome aberrations were found in 48.1% of the centrifuged immotile sperm, and a live/dead sperm viability test intimated that the aberrant sperm were probably dead. In experiment 4, the incidence of structural chromosome aberrations in frozen-thawed immotile sperm was significantly higher in HEPES-BWW (62.2%) than in seminal plasma (17.2%). CONCLUSIONS: The results indicate that immotile sperm do not have significantly more DNA lesions than motile sperm, although DNA of immotile sperm appears to be vulnerable to damage caused by different culture media.     

Chromosome anomalies in mouse zygotes treated by growth hormone-releasing factor.

The incidence of chromosome anomalies was studied in fertilized oocytes in two groups of hybrid mice in which superovulation was induced by gonadotrophins and growth hormone-releasing factor (GRF) supplementation or gonadotrophins alone (controls). The rate of fertilization was significantly higher among GRF-treated females than among controls (74.1 versus 84.7%; P < 0.013). Cytogenetic data were obtained in 262 fertilized oocytes (89 from control females and 173 from GRF-treated females). The frequency of aneuploidy, calculated as twice the frequency of hyperhaploidy was 2.31% in GRF-treated females and 2.24% in controls (NS). The use of GRF to treat female mice did not adversely affect the maturation process of oocytes nor did it induce an increased frequency of aneuploidy.     

Nonrandom karyotypic changes in immortal and tumorigenic Syrian hamster cells induced by diethylstilbestrol

Treatment of Syrian hamster embryo cells with diethylstilbestrol (DES) resulted in the induction of immortal cell lines that progressed and formed tumors in nude mice. Four independently treated cell lines were analyzed cytogenetically at several passages during neoplastic progression. The immortal cell lines at the early passages had no structural abnormalities but did have numerical changes. For example, gain of chromosome 11 was found in all immortal cell lines, and gain of chromosome 19 was found in two of four cell lines. Tumorigenic cells showed not only a variety of numerical abnormalities but also structural abnormalities. Loss of a sex chromosome and gain of chromosome 19 were found in six of seven tumors. Gain of chromosome 11, which was found in all immortal cell lines, disappeared in five of seven tumors. Structural abnormalities involving chromosomes 2 and 3 were found in three of seven tumors. Many marker chromosomes were also found in the tumors. These results support our hypothesis that DES-induced nondisjunction is important in its ability to induce cell transformation and suggests that gain of chromosome 11 and/or 19 may play a role in DES-induced neoplastic progression. Furthermore, these results indicate that for the acquisition of tumorigenicity, additional numerical or structural changes are needed, suggesting that multiple genetic events are required in the multistep process of carcinogenesis.     

The adverse outcome pathway (AOP) for chemical binding to tubulin in oocytes leading to aneuploid offspring

The Organisation for Economic Co‐operation and Development (OECD) has launched the Adverse Outcome Pathway (AOP) Programme to advance knowledge of pathways of toxicity and improve the use of mechanistic information in risk assessment. An AOP links a molecular initiating event (MIE) to an adverse outcome (AO) through intermediate key events (KE). Here, we present the scientific evidence in support of an AOP whereby chemicals that bind to tubulin cause microtubule depolymerization resulting in spindle disorganization followed by altered chromosome alignment and segregation and the generation of aneuploidy in female germ cells, ultimately leading to aneuploidy in the offspring. Aneuploidy, an abnormal number of chromosomes that is not an exact multiple of the haploid number, is a well‐known cause of human disease and represents a major cause of infertility, pregnancy failure, and serious genetic disorders in the offspring. Among chemicals that induce aneuploidy in female germ cells, a large majority impairs microtubule dynamics and spindle function. Colchicine, a prototypical chemical that binds to tubulin and causes microtubule depolymerization, is used here to illustrate the AOP. This AOP is specific to female germ cells exposed during the periovulation period. Although the majority of the data come from rodent studies, the available evidence suggests that the MIE and KEs are conserved across species and would occur in human oocytes. The development of AOPs related to mutagenicity in germ cells is expected to aid the identification of potential hazards to germ cell genomic integrity and support regulatory efforts to protect population health. Environ. Mol. Mutagen. 57:87–113, 2016. © 2015 Her Majesty the Queen in Right of Canada.     

Andrology: Analysis of chromosome constitution of human spermatozoa with normal and aberrant head morphologies after injection into mouse oocytes

The chromosome constitution of human spermatozoa was determinedafter injecting individual spermatozoa into mouse oocytes. Ofa total 279 eggs arrested at first cleavage metaphase, 200 (71.7%)were suitable for the analysis of sperm chromosomes. Incidencesof spermatozoa with numerical and structural chromosome aberrationswere 1.3 and 6.9% respectively in spermatozoa with normal headmorphology, showing values comparable with those found in previousstudies using the hamster oocyte-human sperm fusion system.The ratio of X- to Y-bearing spermatozoa did not differ significantlyfrom the expected 1:1 ratio. The incidence of structural chromosomeaberrations was about four times higher in spermatozoa withamorphous, round and elongated heads (26.1%) than in those withmorphologically normal heads, whereas the incidence of aneuploidywas not significantly different between the two groups. No increasein chromosome aberrations was found in spermatozoa with largeheads. The same was true for spermatozoa with small heads. Althoughthe sample size used in this study is rather small, the resultsnevertheless indicate that some morphological abnormalitiesin the sperm heads are associated with their chromosome defects.     

In vitro studies with nine known or suspected spindle poisons: results in tests for chromosome malsegregation in Aspergillus nidulans

Within the framework of a coordinated collaborative study for evaluating assays for aneuploidy, nine known or suspected spindle poisons were tested in mitotic segregation assays with Aspergillus nidulans. Experiments with A. nidulans diploid strain P1 revealed a statistically significant increase of whole chromosome segregants (non-disjunctional diploids and haploids) after treatments with chloral hydrate (CH), thiabendazole (TB), thimerosal (TM) econazole (EZ) and hydroquinone (HQ). The latter two chemicals also increased the frequency of mitotic cross-overs. Colchicine (COL), diazepam (DZ), cadmium chloride (CD) and pyrimethamine (PY) were ineffective. Further experiments with CH, TB, TM and EZ in the haploid strain 35 demonstrated that CH, TB and TM induced hyperploid types, thus indicating a primary effect on chromosome segregation in A. nidulans. However, since EZ did not induce putative hyperploids in strain 35 and trisomics in diploid 31, it is suggested that EZ affects chromosome segregation by an indirect mechanism, possibly related to induced structural chromosome damage, as previously shown for HQ.     

Micronucleus test and metaphase analyses in mice exposed to known and suspected spindle poisons

Micronucleus (Mn) and metaphase chromosome analyses were performedin mouse bone marrow cells with two known and eight suspectedmitotic spindle poisons. Polychromatic (PCEs) and normochromatic(NCEs) erythrocytes were scored for presence of Mn, while structural(CAs) and numerical chromosome aberrations (NCAs), i.e. hyperploidcells, were evaluated by metaphase analysis. CAs were scoredin first, and NCAs in the second metaphases, identified by BrdUrddifferential staining. Hydroquinone induced Mn, NCAs and CAs;colchicine, vinblastine and, to a lesser extent, chloral hydrate,diazepam and econazole induced both Mn and NCAs; cadmium chlorideand thimerosal induced Mn and CAs, while pyrimethamine and thiabendazoleinduced Mn only. The proposed stepwise protocol allowed satisfactorystatistical evaluation of the effects induced with a reductionin the number of animals killed. An acceptable agreement wasfound between induction of Mn and NCAs, suggesting a possibleuse of the Mn test for revealing compounds with aneugenic properties.     

Sequential G-banding FISH on human sperm chromosomes

A method that allows the performance of double-colour chromosome painting (FISH) on previously G-banded human sperm metaphases has been developed. Sperm chromosomes were obtained by using the fusion technique between zona-free hamster oocytes and human spermatozoa. Single- and double-colour chromosome painting was performed using DNA libraries specific for chromosomes X, Y and 21 on either unstained or G-banded preparations. The hybridization efficiency was very high (98%). The sequential staining technique is very useful for analyses of structural (stable) and numerical chromosome aberrations in human sperm and thus can increase the efficiency of the human sperm—hamster oocytes fusion system to assess the risk to human germ cells as a result of endogenous and exogenous factors.This revised version was published online in November 2005 with corrections to the Cover Date.     

Gene expression in the axolotl germ line: Axdazl, Axvh, Axoct-4, and Axkit.

Primordial germ cells (PGCs) in embryos of mammals and urodele amphibians are formed by induction in the absence of germ plasm. We describe expression of four germ cell-related genes through the germ cell cycle of the axolotl. The orthologs of vasa and daz-like are up-regulated in PGCs of tail bud embryos before the gonad forms and are expressed throughout the female germ cell cycle. Mammalian Oct-4 is a marker of pluripotency in embryonic cells. Axolotl Oct-4 has higher homology to Oct-4 than that found in other vertebrates. It is expressed in the equivalent of the mouse epiblast, in the posterior mesoderm of late gastrulae that gives rise to PGCs, and in diplotene growing oocytes, but not in presumptive PGCs after gastrulation. Finally, a c-kit homolog is expressed in gonadal oogonia and growing oocytes as in mice but is also not found in PGCs. The expression pattern in urodele gonadal germ cells is similar to that of other vertebrates, although the pattern in pregonadal PGCs is distinctly different from that of mice. We conclude that PGCs are restricted to the germ line later in urodeles than in mice or lack migration and proliferation programs.     

TNF—α对小鼠卵母细胞成熟过程纺锤体组装的影响

目的探讨TNF—α对小鼠卵母细胞成熟过程纺锤体组装及染色体排列的影响。方法收集小鼠未成熟卵母细胞,在含不同浓度,TNF-α（0,5,10,100ng／m1）的培养液中进行培养3、9、12h,所有卵母细胞体外培养12h后固定观察纺锤体及染色体分布情况。结果本研究结果表明当TNF-α浓度为5或10ng／ml时,与对照组相比,纺锤体组装正常及染色体排列正常的卵母细胞比例无明显差异（P〉0．05）。当TNF—α浓度升高至100ng／ml时,与低浓度组及对照相比,卵母细胞纺锤体组装及染色体排列均呈现明显异常,差异有统计学意义（P〈0．05）。结论一定浓度的TNF—α影响卵母细胞纺锤体的组装及染色体的排列从而影响卵的质量。