Induction Of Oocytes Research Articles

Trichlorfon effects on mouse oocytes following in vivo exposure

Trichlorfon (TCF) is a widely used pesticide, which according to some epidemiological and experimental data, is suspected of being aneugenic in human and mouse cells. In particular, in vitro studies in mouse oocytes showed the induction of aneuploidy and polyploidy at the first meiotic division and of severe morphological alterations of the second meiotic spindle. We have tested the hypothesis that an acute treatment of mice with TCF might similarly affect chromosome segregation in maturing oocytes. Superovulated MF-1 mice were intraperitoneally injected with 400 mg/kg TCF or orally administered with 600 mg/kg TCF either at the time of or 4 h after human chorionic gonadotrophin (HCG) injection. Oocytes were harvested 17 h after HCG and metaphase II chromosomes were cytogenetically analyzed. No significant increase of aneuploid or polyploid cells was detected at any treatment condition. A significant ( p < 0.001) decrease of metaphases showing premature chromatid separation or premature anaphase II in all TCF-treated groups with respect to controls suggested that TCF treatment may have delayed the first meiotic division. To evaluate possible effects of the pesticide upon the second meiotic division, a group of females orally treated with 600 mg/kg TCF at resumption of meiosis was mated with untreated males and zygotes were collected for cytogenetic analysis. No evidence of aneuploidy induction was obtained, but the frequency of polyploid zygotes was increased fivefold over the control level ( p < 0.01). Such polyploid embryos might have arisen from fertilization of oocytes that were either meiotically delayed and still in metaphase I at fertilization or progressed through anaphase II without cytokinesis. These findings show that in vivo studies on aneuploidy induction in oocytes may yield results different from those obtained by in vitro experiments and that both kinds of data may be necessary for risk assessment of environmentally relevant exposures.

RBAT, related to L-cysteine transport, is localized to the microvilli of proximal straight tubules, and its expression is regulated in kidney by development.

We have recently isolated a renal cDNA clone (rBAT) that induces amino acid transport in oocytes either as a component or as a specific activator of a system bo,(+)-like transporter. (Bertran, J., Werner, A., Moore, M. L., Stange, G., Markovich, D., Biber, J., Testar, X., Zorzano, A., Palacín, M., and Murer, H. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 5601-5605. In order to obtain additional information on the rBAT protein, we have investigated the cellular localization of rBAT and its expression during development in rat kidney. A polyclonal antibody raised against rBAT recognizes a specific protein band of approximately 90 kDa, which is highly enriched in rat renal brush border membranes. Immunofluorescence and immunoelectron microscopy studies demonstrated that rBAT is expressed in the microvilli domain of S3 epithelial cells (i.e. straight tubules). The onset of rBAT mRNA expression in kidney was detected during late fetal life. In keeping with this, induction in oocytes of L-cystine uptake due to fetal rBAT-related mRNA was approximately 10% of the induction obtained with rBAT-related mRNA from adult kidneys. rBAT mRNA levels were low in early postnatal life, and only at the end of lactation did they increase steeply, attaining approximately 50% of adult values after weaning. rBAT protein was undetectable in total membrane preparations of kidneys from fetuses and early neonates, weakly detected during lactation, and represented < 15% of adult values after weaning. The postnatal expression of rBAT and its specific location in the microvilli of epithelial cells from the S3 segment of the proximal tubule coincide with postnatal maturation of cystine resorption and with the site of high affinity resorption of cysteine in kidney. This is consistent with the involvement of rBAT in a b(o,+)-like high-affinity resorption system for cysteine in the proximal straight tubule of the nephron.

Effects of the v-mos oncogene on Xenopus development: meiotic induction in oocytes and mitotic arrest in cleaving embryos.

Previous work has demonstrated that the Xenopus protooncogene mosxe can induce the maturation of prophase-arrested Xenopus oocytes. Recently, we showed that mosxe can transform murine NIH3T3 fibroblasts, although it exhibited only 1-2% of the transforming activity of the v-mos oncogene. In this study we have investigated the ability of the v-mos protein to substitute for the mosxe protein in stimulating Xenopus oocytes to complete meiosis. Microinjection of in vitro synthesized RNAs encoding either the mosxe or v-mos proteins stimulates resting oocytes to undergo germinal vesicle breakdown. Microinjection of an antisense oligonucleotide spanning the initiation codon of the mosxe gene blocked progesterone-induced oocyte maturation. When oocytes were microinjected first with the mosxe antisense oligonucleotide, and subsequently with in vitro synthesized v-mos RNA, meiotic maturation was rescued as evidenced by germinal vesicle breakdown. The v-mos protein exhibited in vitro kinase activity when recovered by immunoprecipitation from either microinjected Xenopus oocytes or transfected monkey COS-1 cells; however, in parallel experiments, we were unable to detect in vitro kinase activity associated with the mosxe protein. Microinjection of in vitro synthesized v-mos RNA into cleaving Xenopus embryos resulted in mitotic arrest, demonstrating that the v-mos protein can function like the mosxe protein as a component of cytostatic factor. These results exemplify the apparently conflicting effects of the v-mos protein, namely, its ability to induce maturation of oocytes, its ability to arrest mitotic cleavage of Xenopus embryo, and its ability to transform mammalian fibroblasts.

MUTAGENESIS IN OOCYTES OF DROSOPHILA MELANOGASTER. I. SCHEDULED SYNTHESIS OF NUCLEAR AND MITOCHONDRIAL DNA AND UNSCHEDULED DNA SYNTHESIS

As a model system for studying mutagenesis, the oocyte of Drosophila melanogaster has exhibited considerable complexity. Very few experiments have been conducted on the effect of exposing oocytes to chemical mutagens, presumably due to their lower mutational response relative to sperm and spermatids. This lower response may be due either to a change in probability of mutation induction per adduct due to a change in the type of DNA repair or to a lower dose of the mutagen to the female germ line. To study molecular dosimetry and DNA repair in the oocyte, the large number of intracellular constituents (mtDNA, RNA, nucleic acid precursors and large quantities of proteins and lipids) must be separated from nuclear DNA. In this paper we present results showing reliable separation of such molecules enabling us to detect scheduled nuclear and mitochondrial DNA synthesis. We also, by understanding the precise timing of such events, can detect unscheduled DNA synthesis (UDS) as a measure of DNA repair. Furthermore, by comparing the UDS results in a repair competent (Ore-R) vs. a repair deficient (mei-9(L1 )) strain, we have shown the oocyte capable of DNA repair after treatment with ethyl methanesulfonate (EMS). We conclude that the important determinant of mutation induction in oocytes after treatment with EMS is the time interval between DNA alkylation and DNA synthesis after fertilization, i.e., the interruption of continuous DNA repair.

Cytogenetic effects of X-rays and fission neutrons in female mice

The induction by X-rays of chromosomal damage in oocytes was studied, while the genetic consequences of X- and neutron-induced damage in female mice were looked for by testing offspring for dominant lethality and semi-sterility. None out of 386 sons of hybrid females given 300 rad X-rays showed evidence of semi-sterility or translocation heterozygosity, but 9 out of 294 daughters were diagnosed as semi-sterile. At least 3 and probably 4 of these (1.4%) carried reciprocal translocations, 2 of which caused male sterility. Complete or partial loss of the X-chromosome may have been responsible for some of the other sermi-steriles. Examination of oocytes at metaphase-I during the first and third weeks after X-irradiation with 100 or 400 rad revealed both multivalents (some of the ring quadrivalent type) and fragments (mainly double). These were thought to arise mainly from chromatid intercchanges (both symmetrical and asymmetrical) and isochromatid intrachanges respectively. Since neither the proportion of asymmetrical interchanges nor the amount of hidden damage was known it was not thought possible to predict the magnitude of F 1 effects from metaphase-I findings. The aberration frequency in oocytes rose with dose and (at the 400 rad level only) with time after irradiation, reaching a maximum of 10% multivalents and 22% fragments in the third week after 400 rad. The frequency of univalents showed no consistent trend, but chiasma counts decreased in the first week after 400 rad. The increase in levels of chromosomal damage with dose and time after irradiation was reflected in dominant lethal frequencies after the same radiation-conception intervals and doses of 0–400 rad. Induced post-implantation lethality was over twice as high in the third week after 200–400 rad than in the first. Pre-implantation loss also greatly increased in the third week after 300 or 400 rad; this was associated with increased non-fertilization of ova. No evidence for the induction of translocations in oogonia or resting oocytes by fast neutron irradiation was obtained, although there was evidence for X-chromosomal loss after 200 rad to oocytes. The relative biological effectiveness (RBE) for fission neutrons vs. X-rays with respect to dominant lethal induction in oocytes was found to vary with dose, but seamed to be around 1 at lower levels.