ProNGF\NGF imbalance triggers learning and memory deficits, neurodegeneration and spontaneous epileptic-like discharges in transgenic mice.

ProNGF, the precursor of mature nerve growth factor (NGF), is the most abundant form of NGF in the brain. ProNGF and mature NGF differ significantly in their receptor interaction properties and in their bioactivity. ProNGF increases markedly in the cortex of Alzheimer's disease (AD) brains and proNGF\NGF imbalance has been postulated to play a role in neurodegeneration. However, a direct proof for a causal link between increased proNGF and AD neurodegeneration is lacking. In order to evaluate the consequences of increased levels of proNGF in the postnatal brain, transgenic mice expressing a furin cleavage-resistant form of proNGF, under the control of the neuron-specific mouse Thy1.2 promoter, were derived and characterized. Different transgenic lines displayed a phenotypic gradient of neurodegenerative severity features. We focused the analysis on the two lines TgproNGF#3 and TgproNGF#72, which shared learning and memory impairments in behavioral tests, cholinergic deficit and increased Aß-peptide immunoreactivity. In addition, TgproNGF#3 mice developed Aß oligomer immunoreactivity, as well as late diffuse astrocytosis. Both TgproNGF lines also display electrophysiological alterations related to spontaneous epileptic-like events. The results provide direct evidence that alterations in the proNGF/NGF balance in the adult brain can be an upstream driver of neurodegeneration, contributing to a circular loop linking alterations of proNGF/NGF equilibrium to excitatory/inhibitory synaptic imbalance and amyloid precursor protein (APP) dysmetabolism.

Transgenic mice with dominant negative PKC-theta in skeletal muscle: a new model of insulin resistance and obesity.

Protein kinase C theta (PKC-theta) is the PKC isoform predominantly expressed in skeletal muscle, and it is supposed to mediate many signals necessary for muscle histogenesis and homeostasis, such as TGFbeta, nerve-dependent signals and insulin. To study the role of PKC-theta in these mechanisms we generated transgenic mice expressing a "kinase dead" mutant form of PKC-theta (PKC-thetaK/R), working as "dominant negative," specifically in skeletal muscle. These mice are viable and fertile, however, by the 6-7 months of age, they gain weight, mainly due to visceral fat deposition. Before the onset of obesity (4 months of age), they already show increased fasting and fed insulin levels and reduced insulin-sensitivity, as measured by ipITT, but normal glucose tolerance, as measured by ipGTT. After the 6-7 months of age, transgenic mice develop hyperinsulinemia in the fasting and fed state. The ipGTT revealed in the transgenic mice both hyperglycemia and hyperinsulinemia. At the molecular level, impaired activation of the IR/IRS/PI3K pathway and a significant decrease both in the levels and in insulin-stimulated activation of the serine/threonine kinase Akt were observed. Taken together these data demonstrate that over-expression of dominant negative PKC-theta in skeletal muscle causes obesity associated to insulin resistance, as demonstrated by defective receptor and post-receptorial activation of signaling cascade.

Engineering of a mouse for the in vivo profiling of estrogen receptor activity.

In addition to their well known control of reproductive functions, estrogens modulate important physiological processes. The identification of compounds with tissue-selective activity will lead to new drugs mimicking the beneficial effects of estrogen on the prevention of osteoporosis and cardiovascular or neurodegenerative diseases, while avoiding its detrimental proliferative effects. As an innovative model for the in vivo identification of new selective estrogen receptor modulators (SERMs), we engineered a mouse genome to express a luciferase reporter gene ubiquitously. The constructs for transgenesis consist of the reporter gene driven by a dimerized estrogen-responsive element (ERE) and a minimal promoter. Insulator sequences, either matrix attachment region (MAR) or beta-globin hypersensitive site 4 (HS4), flank the construct to achieve a generalized, hormoneresponsive luciferase expression. In the mouse we generated, the reporter expression is detectable in all 26 tissues examined, but is induced by 17beta-estradiol (E2) only in 15 of them, all expressing estrogen receptors (ERs). Immunohistochemical studies show that in the mouse uterus, luciferase and ERs colocalize. In primary cultures of bone marrow cells explanted from the transgenic mice and in vivo, luciferase activity accumulates with increasing E(2) concentration. E2 activity is blocked by the ER full antagonist ICI 182,780. Tamoxifen shows partial agonist activity in liver and bone when administered to the animals. In the mouse system here illustrated, by biochemical, immunohistochemical, and pharmacological criteria, luciferase content reflects ER transcriptional activity and thus represents a novel system for the study of ER dynamics during physiological fluctuations of estrogen and for the identification of SERMs or endocrine disruptors.

Cardiac and smooth muscle cell contribution to the formation of the murine pulmonary veins.

Previous studies have demonstrated that the primordial pulmonary veins originate as an outgrowth of the atrial cells and anastomosis with the pulmonary venous plexus. As a consequence of this embryologic origin the tunica media of these vessels is composed of cardiac cells that express atrial specific markers (Lyons et al. [1990] J Cell Biol 111:2427-2436; Jones et al. [1994] Dev Dyn 200:117-128). We used transgenic mice for the cardiac troponin I (cTNI) gene and smooth muscle (SM) myosin heavy chain as differentiation markers, to analyze how cardiac and SM cells contribute to the formation and structural remodeling of the pulmonary veins during development. We show here that the tunica media of the adult mouse pulmonary veins contains an outer layer of cardiac cells and an intermediate SM cell compartment lining down on the inner endothelium. This structural organization is well expressed in the intrapulmonary veins from the beginning of vasculogenesis, with cardiac cells accumulating over preexisting roots of endothelial and SM cells and extending to the third bifurcation of the pulmonary branches without reaching the more distal tips of the vessels. On the other hand, SM cells, which are widely distributed in the intrapulmonary veins from the embryonic stage E16, accumulate also in the extrapulmonary branches and reach the posterior wall of the left atrium, including the orifices of the pulmonary veins. This event takes place around birth when the pulmonary blood flow starts to function properly. A model for the development of the pulmonary veins is presented, based upon our analysis.

Constitutive activation of NF-kappaB and T-cell leukemia/lymphoma in Notch3 transgenic mice.

The multiplicity of Notch receptors raises the question of the contribution of specific isoforms to T-cell development. Notch3 is expressed in CD4(-)8(-) thymocytes and is down-regulated across the CD4(-)8(-) to CD4(+)8(+) transition, controlled by pre-T-cell receptor signaling. To determine the effects of Notch3 on thymocyte development, transgenic mice were generated, expressing lck promoter-driven intracellular Notch3. Thymuses of young transgenics showed an increased number of thymocytes, particularly late CD4(-)8(-) cells, a failure to down-regulate CD25 in post-CD4(-)8(-) subsets and sustained activity of NF-kappaB. Subsequently, aggressive multicentric T-cell lymphomas developed with high penetrance. Tumors sustained characteristics of immature thymocytes, including expression of CD25, pTalpha and activated NF-kappaB via IKKalpha-dependent degradation of IkappaBalpha and enhancement of NF-kappaB-dependent anti-apoptotic and proliferative pathways. Together, these data identify activated Notch3 as a link between signals leading to NF-kappaB activation and T-cell tumorigenesis. The phenotypes of pre-malignant thymocytes and of lymphomas indicate a novel and particular role for Notch3 in co-ordinating growth and differentiation of thymocytes, across the pre-T/T cell transition, consistent with the normal expression pattern of Notch3.

Sox2 regulatory sequences direct expression of a (beta)-geo transgene to telencephalic neural stem cells and precursors of the mouse embryo, revealing regionalization of gene expression in CNS stem cells.

Sox2 is one of the earliest known transcription factors expressed in the developing neural tube. Although it is expressed throughout the early neuroepithelium, we show that its later expression must depend on the activity of more than one regionally restricted enhancer element. Thus, by using transgenic assays and by homologous recombination-mediated deletion, we identify a region upstream of Sox2 (-5.7 to -3.3 kb) which can not only drive expression of a (beta)-geo transgene to the developing dorsal telencephalon, but which is required to do so in the context of the endogenous gene. The critical enhancer can be further delimited to an 800 bp fragment of DNA surrounding a nuclease hypersensitive site within this region, as this is sufficient to confer telencephalic expression to a 3.3 kb fragment including the Sox2 promoter, which is otherwise inactive in the CNS. Expression of the 5.7 kb Sox2(beta)-geo transgene localizes to the neural plate and later to the telencephalic ventricular zone. We show, by in vitro clonogenic assays, that transgene-expressing (and thus G418-resistant) ventricular zone cells include cells displaying functional properties of stem cells, i.e. self-renewal and multipotentiality. We further show that the majority of telencephalic stem cells express the transgene, and this expression is largely maintained over two months in culture (more than 40 cell divisions) in the absence of G418 selective pressure. In contrast, stem cells grown in parallel from the spinal cord never express the transgene, and die in G418. Expression of endogenous telencephalic genes was similarly observed in long-term cultures derived from the dorsal telencephalon, but not in spinal cord-derived cultures. Thus, neural stem cells of the midgestation embryo are endowed with region-specific gene expression (at least with respect to some networks of transcription factors, such as that driving telencephalic expression of the Sox2 transgene), which can be inherited through multiple divisions outside the embryonic environment.

Reproductive toxicity of 1,3-butadiene in the mouse: cytogenetic analysis of chromosome aberrations in first-cleavage embryos and flow cytometric evaluation of spermatogonial cell killing.

Reproductive effects of 1,3 butadiene inhalation have been evaluated in male mice by reduction of post-meiotic germ cells, alteration of sperm chromatin structure and transmission of chromosome aberrations to one-cell embryos. Animals were exposed for 5 consecutive days for 6 h per day to butadiene concentrations of 130, 500 or 1300 ppm. The testicular fraction of post-meiotic germ cells was measured by flow cytometric analysis on the basis of their DNA content. Round spermatids were discriminated from mature, elongated spermatids by their different degree of chromatin condensation. Butadiene-induced cytotoxic effects on differentiating spermatogonia were shown by a concentration-dependent decrease of round spermatids occurring 21 days after chemical exposure, confirmed by a similar decrease of elongated spermatids measured in testes sampled 7 days later. Statistically significant effects were seen already at 130 ppm. An incomplete repopulation of the elongated spermatid compartment observed 35 days after exposure to 1300 ppm suggested that, at the highest concentration tested, butadiene toxicity extended to stem cells. Alterations of sperm chromatin were revealed by its increased sensitivity to acidic denaturation in situ. The percentage of abnormal sperm was significantly increased after butadiene exposure of differentiating spermatogonia and spermatocytes. This suggested the induction of persistent effects interfering with chromatin remodelling during spermiogenesis. Chromosome-type structural aberrations were significantly elevated in first-cleavage embryos conceived by males mated during the first and second week after the end of exposure. The lowest effective tested concentration was 500 ppm, the same reported for dominant lethal induction under identical exposure conditions. As in the dominant lethal assay, the effect of this dose was confined to exposed sperm, while both sperm and late spermatids were affected by the inhalation of 1300 ppm. A quantitative comparison between the effects induced by intraperitoneal injections of diepoxybutane or butadiene inhalations suggested that other reactive intermediates, in addition to diepoxybutane, might contribute to mediate butadiene-induced reproductive toxicity.

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 for 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.

Flow cytometric assessment of trophosphamide toxicity on mouse spermatogenesis.

The effects of trophosphamide on mouse reproductive cells have been investigated by flow cytometric analysis of testicular cell populations and alterations of sperm chromatin structure. Mice were treated with single intraperitoneal injections of TP, the doses ranging between 50 and 150 mg/kg, and were killed after 7, 14, 21, 28, 35, or 49 days. Dose-dependent reductions of tetraploid cells, round spermatids, and elongated spermatids were detected at 7, 21, and 28 days, respectively, reflecting cytotoxic damage to the differentiating spermatogonia compartment. The dose necessary to reduce the number of differentiating spermatogonia to half the control value was approximately 70 mg/kg. Stem cells were not affected by this treatment, and the normal spermatogenic process was restored after 7 weeks. In addition, cauda epididymal sperm were analyzed by the sperm chromatin structure assay, a flow cytometric measurement of the susceptibility of the sperm nuclear DNA to in situ acid denaturation; a statistically significant increase of sperm with altered chromatin structure was detected after a TP treatment of 150 mg/kg. Together with previous findings published in the literature, where the same doses induced heritable genetic damage, this study demonstrates a marked adverse cytotoxic effect of TP on the male reproductive integrity. All this information should be taken into consideration when TP is used in chemotherapeutic regimens.

Genotoxicity of trophosphamide in mouse germ cells: assessment of micronuclei in spermatids and chromosome aberrations in one-cell zygotes.

The genotoxicity of trophosphamide (TP) in mouse germ cells was assessed by the cytogenetic analysis of micronuclei in spermatids and chromosome aberrations in one-cell zygotes and compared with the genotoxicity in somatic cells evaluated by the micronucleus reticulocyte assay. Single acute doses of 50, 75, 100 and 150 mg/kg were studied after i.p. injection. TP was only weakly mutagenic for preleptotene spermatocytes-differentiating spermatogonia, but clear-cut cytotoxic effects were demonstrated after treatment of these cells by a dose-dependent reduction of the ratio between Golgi and cap phase spermatids. Effects induced in post-meiotic stages were estimated, after mating the treated males with untreated superovulated females, by the frequencies of zygotes with chromosome aberrations: a peak of genetic damage was detected in late spermatids, with as many as 55% zygotes with aberrations, but spermatozoa and early spermatids were also clearly affected. When compared with matched solvent-injected controls, the lowest effective dose in spermatozoa and late spermatids was 100 mg/kg, although the 3- to 4-fold increases detected at 50 mg/kg were also statistically significant when compared with a pool of laboratory controls. In peripheral blood reticulocytes, the micronucleus frequencies were increased by 3-20 times the respective baseline values in the individual animals. A marked cytotoxic effect on bone marrow cells was revealed by the reduction of the proportion of early reticulocyte stages, which dropped to 20% of the control value at 150 mg/kg. Both genotoxic and cytotoxic effects were higher in bone marrow than in germ cells of the same animals, pointing to a generalized higher susceptibility of somatic cells to TP, possibly related to chemical distribution and target organ accessibility. The accurate description of stage- and dose-effect relationships in germ cells of experimental models is crucial for genetic risk assessment after chemical exposure. The approaches applied in this study may contribute to this goal.

Clastogenicity of diepoxybutane in bone marrow cells and male germ cells of mice.

The bifunctional metabolite of 1,3-butadiene, 1,2:3,4-diepoxybutane (DEB), was tested in the mouse bone marrow micronucleus assay and in male mouse germ cell tests, namely the analysis of first cleavage divisions and the dominant lethal assay. All experiments were performed with single intraperitoneal treatment of the animals. In the micronucleus test, DEB doses of 4.5, 9.0, 18.0 and 36.0 mg/kg body weight were tested at a sampling interval of 24 h for bone marrow. The dose response for the induction of micronuclei in polychromatic erythrocytes was linear with the lowest effective dose of 9.0 mg/kg body weight. No sensitivity difference was observed between male and female mice. the cytogenetic analysis of first cleavage division chromosomes was performed after treatment of male mice with 17, 26, 34, 43 and 52 mg/kg body weight of DEB and mating the males to hormonally stimulated females on days 7, 14, 21 and 28 after treatment. The two higher doses caused general toxicity evidenced by the poor mating behavior of the males. Only 13 and 20% of the mated females were fertilized on day 7 after treatment of the males with 43 and 52 mg/kg body weight of DEB, respectively. An increased number of unfertilized oocytes was obtained from fertilized females on day 7 after treatment of the males with 34 mg/kg body weight of DEB. With a dose of 26 mg/kg body weight, it was demonstrated that chromosomal aberrations were only induced in spermatozoa (mating on day 7 after treatment) while spermatids (mating on days 14 and 21) and spermatocytes (mating on day 28) were not susceptible to the clastogenic effect of DEB. The response in spermatozoa in the dose range 17-34 mg/kg body weight was linear up to 26 mg/kg body weight and reached a plateau thereafter. The results of the dominant lethal experiments performed in the dose range 18-54 mg/kg body weight gave results similar to the cytogenetic study. With the highest dose tested, the toxicity and cytotoxicity during the first 8 mating days after treatment dramatically reduced the number of pregnant females and, consequently, the total implantations, so that no significant dominant lethal effect could be assessed. During mating days 9-12 (treated late spermatids), a significant dominant lethal effect was observed. With the two lower doses (18 and 36 mg/kg body weight), the dominant lethal effect was restricted to spermatozoa. The good correlation of the chromosomal aberrations with dominant lethal mutations confirms the chromosomal origin of dominant lethal effects. The clastogenic effect of DEB in somatic cells and in germ cells of mice was of the same order of magnitude.

Susceptibility to vinblastine-induced aneuploidy and preferential chromosome segregation during meiosis I in Robertsonian heterozygous mice.

Chromosome segregation at meiosis I was studied in oocytes and spermatocytes of four different Robertsonian (Rb) heterozygous mouse stocks by cytogenetic analysis of meiotic products. Two Rb heterozygotes spontaneously yielded high frequencies of unbalanced oocytes. In one case, Rb(2.18)Rma, the excess hyperploidy was mainly accounted for by nondisjunction of normal bivalents, suggesting a generalized impairment of meiotic segregation. In each stock, frequencies of hyperploid spermatocytes were either not significantly different or significantly lower than the corresponding frequencies in the oocytes. This confirmed the greater risk of segregational errors in female than in male carriers of the same Rb metacentric. The hypothesis that an error prone system of meiotic segregation, such as the trivalent configuration of single Rb heterozygous oocytes, could be hypersensitive to chemically induced malsegregation was tested by injecting Rb heterozygous females with low doses of vinblastine (VBL). An intraperitoneal injection of 0.06 or 0.09 mg/kg VBL before the first meiotic division significantly increased the spontaneous frequency of hyperploid oocytes, inducing segregational errors of both the trivalent and normal bivalents. The comparison of these data with VBL effects in B6C3F1 mice showed that single Rb heterozygous oocytes are more sensitive to VBL-induced meiotic aneuploidy than oocytes with a standard karyotype. Although segregation distortion has been repeatedly shown in the progeny of Rb heterozygous mice with a significant excess of all telocentric balanced offspring, it has never been demonstrated whether this is a primary event occurring during meiotic segregation or a consequence of selective postconceptional death. In this study, we showed that preferential segregation occurred during female meiosis in all the Rb stocks tested. When segregation distortion was analyzed separately in balanced and unbalanced oocytes, the latter did not show preferential segregation, suggesting that, when the two telocentrics segregated from each other, then the metacentric was randomly directed to the ovum or the polar body.

Acrylamide-induced chromosomal damage in male mouse germ cells detected by cytogenetic analysis of one-cell zygotes.

Within a project coordinated by the Commission of the European Communities for the detection of germ cell mutagens, the cytogenetic analysis of first-cleavage metaphases was carried out to detect chromosomal damage induced by acrylamide (AA) in meiotic and postmeiotic stages of mouse spermatogenesis. Male mice were intraperitoneally injected with single acute doses of 75 or 125 mg/kg or treated with five daily injections of 50 mg/kg and mated either 7 or 28 days after the end of treatment. Chromosomal aberrations were scored in C-banded metaphases prepared from one-cell zygotes by a mass harvest technique. AA treatment of late spermatids-spermatozoa resulted in significant increases of structural aberrations at all doses tested. The data could be fitted to a curvilinear regression and a doubling dose of 23 mg/kg was calculated. The large majority of observed aberrations were of the chromosome type, including dicentrics, rings and translocations, in agreement with a mechanism of chromosomal damage mediated through the alkylation of DNA-associated protamines. Even though the frequency of aberrations 28 days after treatment was not significantly higher than the control value, the presence of multiple rearrangements in two cells suggested that AA might also have a minor effect on spermatocytes. The results of the cytogenetic analysis of first cleavage metaphases agreed well both qualitatively and quantitatively with the outcome of dominant lethal and heritable translocation assays. AA-induced cytotoxicity was monitored by flow cytometric DNA content analysis of testicular cells. By this method, a dose-dependent depletion of mature spermatids after treatment of spermatogonia and a toxic effect upon primary spermatocytes were detected.

Noscapine does not show aneugenic activity in mouse oocytes.

To clarify if noscapine has the ability to induce polyploidy in rodent germ cells in vivo, a cytogenetic study of mouse metaphase II oocytes was conducted after oral treatment with noscapine at the doses of 20, 120 and 400 mg/kg. Plasma concentrations of noscapine were measured by reversed-phase liquid chromatography and UV detection in three satellite groups of mice up to 8 h after administration of these doses. Thus, the relationship of the maximum plasma concentration and the area under the curve (AUC) with that of meiotic progression could be established. Although noscapine was tested at the maximum tolerated dose, no delay of meiotic progression or induction of chromosome malsegregation could be shown as no increase in the frequency of metaphase I-arrested, polyploid or hyperploid oocytes were found. At the highest dose only, noscapine affected the physiology of superovulation as shown by a significant decrease in the mean number of oocytes harvested per female. In view of the large span covered by the doses tested, corresponding to concentrations far above those detected in humans, and the similarity between the pharmacokinetics of noscapine in mouse and humans, it is unlikely that noscapine represents a genetic risk for humans at therapeutic dosages.

Griseofulvin-induced aneuploidy and meiotic delay in female mouse germ cells. I. Cytogenetic analysis of metaphase II oocytes.

Griseofulvin (GF) was tested in female mouse germ cells for the induction of aneuploidy and meiotic arrest. Superovulated mice were orally treated with 200, 666, 1332 or 2000 mg/kg in olive oil at the time of human chorionic gonadotrophin (HCG) injection and were sacrificed 18 h later. A dose-dependent increase in the frequency of metaphase I (M I) arrested oocytes was observed (maximum of 70%). Aneuploidy was not significantly induced. Also, the kinetics of meiotic progression up to the metaphase II (M II) stage was studied in untreated mice in order to correlate the time of treatment with the time of the first meiotic division. The results demonstrate that the majority of cells was treated with GF approximately 8 h before the M I stage. A second series of experiments were performed to test GF effects at a different treatment time. Doses of 200, 666 or 2000 mg/kg were administered 2 h post HCG. As in the first series of experiments, the animals were sacrificed 18 h post HCG. The results, compared with those obtained in the first experimental series, showed an inverse trend for meiotic arrest and aneuploidy induction. The frequency of M I arrested oocytes dropped from a maximum of 70% to a maximum of 20%, while, at the latest treatment time, a dose-dependent increase in the frequency of hyperploid oocytes was observed up to 56% aberrant cells at 2000 mg/kg. Altogether the results suggest that the arrest of meiotic division and the induction of aneuploidy by GF are caused by interaction with different targets or different developmental stages of the same target. In conclusion, GF has been shown to induce aneuploidy during the first meiotic division in a dose-related manner, together with other effects such as polyploidy, developmental delay and meiotic arrest. Also, these findings demonstrate that the sensitivity of the oocyte target(s) may be restricted to a specific time period and that a correct experimental protocol is critical for assessing the aneugenic activity of a chemical.

Griseofulvin-induced aneuploidy and meiotic delay in female mouse germ cells. II. Cytogenetic analysis of one-cell zygotes.

The effects of griseofulvin (GF) upon the first meiotic division of female mouse germ cells were evaluated by cytogenetic analysis of first-cleavage (1-Cl) zygotes. The present study is an extension of an investigation that began with the cytogenetic analysis of metaphase II (M II) oocytes. Different doses (200, 666, 1332, 2000 mg/kg) were tested by oral administration of GF to superovulated animals either at the time of human chorionic gonadotrophin (HCG) injection or 2 h post HCG. When GF was given at the time of HCG, significant dose-dependent increases of different types of cytogenetically abnormal cells were found. These included zygotes containing ostensibly female-derived M I or M II arrested chromosomes and polyploid zygotes. The total yields of these aberrations were 2.9, 4.3, 26.2, 60.6, and 64.1% for control, 200, 666, 1332, and 2000 mg/kg, respectively. The origin of these zygotes was attributed to the fertilization of oocytes that had been previously arrested at M I. No significant induction of hyperploidy was detected. Developmentally abnormal zygotes were still observed when GF was administered 2 h post HCG, although their frequencies were significantly lower than in the first series of experiments. The yields of developmentally abnormal zygotes were 49, 10.2, and 23.6% at 200, 666, and 2000 mg/kg. Additionally, a dose-dependent increase in the frequency of hyperploid zygotes was detected up to a maximum of 36.5% at 2000 mg/kg. These results confirm the cytogenetic observations from M II oocytes after GF treatment under the same experimental conditions; namely, a dramatic change in the oocyte target susceptibility to GF occurred within a short time period. Also, the present study demonstrated that most of GF-induced aneuploid oocytes were fertilized and reached first-cleavage metaphase.