Chemical induction of presumed dominant-lethal mutations in postcopulation germ cells and zygotes of mice. II. Sensitivity of different postcopulation-precleavage stages to three alkylating chemicals.

The relative sensitivities of various postcopulation-precleabage and pronuclear stages to dominant-lethal effects of isopropyl methanesulfonate (IMS), ethyl methanesulfonate (EMS), and triethylenemelamine (TEM) were investigated. The pattern of sensitivity differed with the chemical. IMS was most effective when pronuclear formation was already completed and the majority of the zygotes were presumably undergoing DNA synthesis. EMS, on the other hand, induced its most pronounced effects when eggs in the course of second meiotic division and zygotes in early pronuclear stages were treated. The greatest effect of TEM was observed when zygotes were treated at the early pronuclear stage. EMS and TEM, in contrast to IMS, are similar to radiations in that zygotes undergoing DNA synthesis are more resistant to them than are the early pronuclear stages. In the case of IMS, effects induced in the most sensitive postcopulation-precleavage stage were 6 to 9 times greater than in the most sensitive precopulatory dictyate oocytes or male germ cells. On the other hand, in the case of EMS and TEM, the most sensitive precopulatory male germ cells, but not the dictyate oocytes, were more sensitive than the most sensitive postcopulation stages.     

Fetal anomalies produced subsequent to treatment of zygotes with ethylene oxide or ethyl methanesulfonate are not likely due to the usual genetic causes

Earlier studies in this laboratory revealed that ethylene oxide (EtO) or ethyl methanesulfonate (EMS) induced high frequencies of midgestation and late fetal deaths,and of malformations among some of the surviving fetuses, when female mice were exposed at the time of fertilization of their eggs or during the early pronuclear stage of the zygote. Effects of the two mutagens are virtually identical. Thus, in investigating the mechanisms responsible for the dramatic effects in the early pronuclear zygotes, the two compounds were used interchangeably in the experiments. First, a reciprocal zygote-transfer study was conducted in order to determine whether the effect is directly on the zygotes or indirectly through maternal toxicity. And second, cytogenetic analyses of pronuclear metaphases, early cleavage embryos, and midgestation fetuses were carried out.

The zygote transplantation experiment rules out maternal toxicity as a factor in the fetal maldevelopment. Together with the strict stage specificity observed in the earlier studies, this result points to a genetic cause for the abnormalities. However, the cytogenetic studies failed to show structural or numerical chromosome aberrations. Since intragenic base changes and deletions may also be ruled out, it appears that the lesions in question induced in zygotes by the two mutagens are different from conventional ones and, therefore, could be a novel one in experimental mammalian mutagenesis. Alternatively, the mechanism could invlve a non-mutational ‘imprinting’ process that caused changes in gene expression.     

Exposure of female mice to ethylene oxide within hours after mating leads to fetal malformation and death

When previously mated female mice were exposed to inhaled ethylene oxide at the time of fertilization of their eggs or during early pronuclear stage of the zygote (before DNA synthesis), a high incidence of mortality among conceptuses and of congenital abnormalities among both the dead and the surviving fetuses was observed. The developmental stage at which death occurred ranged from near the time of implantation to day 17 of gestation when examination of the uterine contents was performed. In comparison, midgestation and late fetal deaths were absent or minimal when the females were exposed either before mating or when conceptuses were in later zygotic stages (pronuclear DNA synthesis) or had reached the early two-cell stage. The random types of congenital abnormality observed and the remarkable stage-dependent sensitivity suggest a genetic basis for the response. The effects differ, both from genetic damages induced in premating germ cells, which lead only to death near the time of implantation, and from teratogenic damage, which leads to malformations only when exposure of embryos occurs during the period of major organogenesis.     

Chromosome malsegregation and embryonic lethality induced by treatment of normally ovulated mouse oocytes with nocodazole

The mouse egg is ovulated with its nucleus arrested at the metaphase-II stage of meiosis. Sperm entry triggers the completion of the second meiotic division. It has been speculated that damage to the meiotic spindle of normally ovulated eggs at around the time of sperm entry could result in chromosome malsegregation and the death of conceptuses with numerical chromosome anomalies. This hypothesis was tested using nocodazole, a microtubule inhibitor. Nocodazole was administered either to maturing preovulatory oocytes or to normally ovulated eggs at one of the following stages: (1) the time of sperm entry, (2) early pronuclear stage, (3) pronuclear DNA synthesis, (4) prior to first cleavage division, (5) early 2-cell stage, or (6) prior to the second cleavage division. Little or no effect was observed for treatment times other than the time of sperm entry, when the egg is being activated to complete the second meiotic division. Remarkably high frequencies of embryonic lethality, expressed at around the time of implantation, were induced at this stage. Cytogenetic analysis of first cleavage metaphases of zygotes treated at the time of sperm entry revealed a high incidence of varied numerical chromosome anomalies, with changes in ploidy being predominant.     

Optimization of embryo transfer protocols for mice

The efficiency of ova transfer and subsequent survivability were explored in this study. The goals of the experiment were to 1) determine the minimum number of ova necessary for pregnancy maintenance, 2) ascertain if the number of zygotes used in ova transfer approaches or exceeds uterine capacity, and 3) establish if location of deposition of ova influences embryo survival. A total of 1647 pronuclear zygotes were transferred in groups of 1, 2, 4, 6, 15 or 25 on Day 1 of gestation either via the oviducal ampulla or ostium to 156 nulliparous ICR pseudopregnant female mice. Pregnancy status was determined on Day 12 or Day 19 of gestation. Results indicated that pregnancy rates were not significantly increased by transferring larger numbers of zygotes (P < 0.1504) and that beyond transfer of 15 zygotes, the progressive increase in fetal numbers per litter declined. However, on Day 19 of gestation, no definitive evidence of limitation of uterine capacity was obtained with the numbers of zygotes transferred (P < 0.0531), and the estimates of numbers of viable and resorbed fetuses differed when determinations were made on Day 12 versus Day 19 of gestation. Mean numbers of developed fetuses per recipient declined (P < 0.0001), whereas the number of resorptions (partially resorbed fetuses or resorption sites) increased (P < 0.0001) over this period, reflecting fetal loss in mid- to late-gestation and possibly the transient nature of resorptions prior to Day 12. Additionally, there was no difference in pregnancy outcome when transferring ova into the oviducal ostium or isthmus (P < 0.5256). Finally, these results illustrated that when large numbers of zygotes were transferred into the oviducal ampulla, equivalent numbers of ova eventually implanted in the uterus; however, proportionally more of them began resorption.     

Genotoxic potency in mouse spermatogonial stem cells of triethylenemelamine, mitomycin C, ethylnitrosourea, procarbazine, and propyl methanesulfonate as measured by F1 congenital defects

Male ICR mice were intraperitoneally injected with TEM, MMC, ENU, PCZ, or PMS and mated to untreated females of the same strain on days 64-80 after the treatment. Copulations during this period involve sperm that were spermatogonial stem cells at the time of the treatment. The fetuses were examined on day 18 of pregnancy for external and skeletal abnormalities. The 5 mutagens tested all caused significant increases in the incidence of abnormal fetuses over the control level. The genotoxically effective dose, in mmole/kg, for producing fetal abnormalities with a frequency of 2% was estimated to be 0.007 for TEM and MMC, 0.6 for ENU, 1.8 for PCZ, and 3.0 for PMS. These values correlate well with the mutagenic potency estimated from the data reported for inducing specific-locus mutations in spermatogonial stem cells. Irrespective of the kind of mutagen used, external abnormalities represented by cleft palate and dwarfism occurred more frequently than skeletal abnormalities represented by rib malformations. It is concluded from these data that F1 fetal abnormalities can serve as sensitive indicators for quantitatively assessing the genotoxicity of a chemical agent in spermatogonial stem cells.     

Developmental response of zygotes exposed to similar mutagens

Exposure of mouse zygotes to ethylene oxide (EtO) or ethyl methanesulfonate (EMS) led to high incidences of fetal death and of certain classes of fetal malformations (Generoso et al., 1987, 1988; Rutledge and Generoso, 1989). These effects were not associated with induced chromosomal aberrations (Katoh et al., 1989) nor are they likely to be caused by gene mutations (Generoso et al., 1990). Nevertheless, the anomalies observed in these studies resemble the large class of stillbirths and sporadic defects in humans that are of unknown etiology, such as cleft palate, omphalocoel, clubfoot, hydrops and stillbirths (Czeizel, 1985; Oakley, 1986). Therefore, we continue to study the possible mechanisms relating to induction of these types of zygote-derived anomalies in mice. Effects of zygote exposure to the compounds methyl methanesulfonate (MMS), dimethyl sulfate (DMS), and diethyl sulfate (DES), which have similar DNA-binding properties as EtO and EMS, were studied. DMS and DES, but not MMS, induced effects that are similar to those induced by EtO and EMS. Thus, no site-specific alkylation product was identifiable as the critical target for these zygote-derived anomalies. We speculate that the developmental anomalies arose as a result of altered programming of gene expression during embryogenesis.     

The paternal genome in mouse zygotes is less sensitive to ENU mutagenesis than the maternal genome

The two parental genomes lie separate within the zygote and may be differentially affected by environmental influences. We have shown earlier (Russell et al., 1988) that the maternal genome within the mouse zygote is exquisitely sensitive to the induction of point mutations by N-ethyl-N-nitrosourea (ENU), and that the initial lesion probably occurs in one strand of the DNA. The present experiment measured specific-locus mutation induction in the paternal genome. Zygotes containing a multiple-recessive maternal genome (a; b; p cch; d se; s) and the corresponding wild-type alleles in the paternal one were exposed to 50 mg ENU/kg in vivo at one of two stages: the presumed times of sperm entry and early pronuclear stage. At weaning age, the resulting mice were examined for mutations at the marked loci as well as for other mutations producing externally visible phenotypes. At the marked loci, one possible mosaic (for b) was observed among 2113 classified offspring that had been treated with ENU as zygotes; this animal failed to transmit a mutation. By contrast, in the reciprocal cross (which tests the maternal genome) we had observed 8 specific-locus mutations (6 of them mosaics) among 1555 offspring that had received the same dose of ENU during sperm entry (and completion of oocyte meiosis II). In the present experiment, we also found one mutation at other loci (two at other loci in the reciprocal cross). The frequency of offspring with small white belly spots was significantly greater in the treated groups (3.5 and 1.9% at the earlier and later stage, respectively) than in the control (1.0%), the excess being almost entirely due to daughters. Genetic tests of a large number of such offspring failed to find a genetic cause. Instead, it appears that this phenotype may be influenced by factors in the intrauterine environment. It is concluded that shortly after sperm entry, the paternal genome of the zygote is less sensitive than the maternal one to the induction of mutations by ENU.     

Derivation of the membrane comprising the male pronuclear envelope in inseminated sea urchin eggs

Investigations were conducted in an effort to determine the origin of the membrane comprising the male pronuclear envelope of inseminated sea urchin eggs. The events of fertilization in zygotes treated with 200 μg/ml of puromycin are not impaired even though incorporation of [³H]leucine is inhibited up to 80% when compared to control specimens. Developing male pronuclei in zygotes treated with puromycin form nuclear envelopes structurally similar to and within the same period as controls. In puromycin-treated and untreated zygotes morphologically recognizable portions of the sperm nuclear envelope are incorporated into the structure of the male pronuclear envelope. Pronuclear development was also examined in inseminated ova where most of the endoplasmic reticulum (ER) was confined to a specific area of the zygote. Eggs were centrifuged in order to stratify their organelles into specific layers (stratified eggs); with further centrifugation stratified eggs are bisected to form nucleate (rich in ER) and nonnucleate halves (containing little ER). Observations of inseminated stratified eggs and nucleate and nonnucleate halves demonstrate an inverse relation between the amount of ER present in the vicinity of a reorganizing sperm nucleus and the time it takes to form the male pronuclear envelope. Computation of the maximum quantity of membrane in the male pronucleus that may be derived from the sperm nuclear envelope is approximately 15%. These investigations suggest that a major portion of the male pronuclear envelope is derived from endoplasmic reticulum within the egg and only a small portion (up to 15%) originates from the sperm nuclear envelope.     

Dithiothreitol induces sperm nuclear decondensation and protects against chromosome damage during male pronuclear formation in hybrid zygotes between Chinese hamster spermatozoa and Syrian hamster oocytes

The present study was undertaken to investigate whether a time lag in sperm nuclear decondensation and male pronuclear formation in the course of development of eggs is associated with any occurrence of structural chromosome aberrations in male genomes of hybrid zygotes between Chinese hamster spermatozoa and zona-free Syrian hamster oocytes. Shortly after insemination, hybrid zygotes were treated with dithiothreitol (DTT) at different concentrations (0.1-10.0 mM) for 30 min to reduce protamine disulphide (S-S) bonds and thereby accelerate sperm nuclear decondensation and male pronuclear formation. The incidence of sperm nuclear decondensation and male pronuclear formation increased with increasing DTT concentrations, indicating that a reduction in S-S bonds effectively induces these cytological events. Chromosomes of male genomes in hybrid zygotes generated by treatment with 1.0 mM, 2.5 mM and 10.0 mM DTT were analysed at the first cleavage metaphase. Incidence of structural chromosome aberrations in each treatment was 34.5%, 27.1% and 24.7%, respectively. There was a significant difference between the incidences with 1.0 mM and 10.0 mM DTT treatment. As the time lag in nuclear decondensation and male pronuclear formation was greatest in the 1.0 mM treatment condition, followed in order by 2.5 mM and 10.0 mM, it is suggested that the lag in sperm nuclear development behind egg development is responsible for structural chromosome aberrations in male genomes of hybrid zygotes.     

Studies on chromosome aberrations in the eggs of mice fertilized in vitro after irradiation. II. Chromosome aberrations induced in mature oocytes and fertilized eggs at the pronuclear stage following X-irradiation

Cytological analysis of the first-cleavage metaphase of eggs exposed to X-rays at the mature oocyte stage or the pronuclear stage 4 h after fertilization was performed using the in vitro fertilization technique. The frequency of chromosome aberrations in irradiated mature oocytes increased exponentially with dose, the dose-response relationship being best fitted to the linear-quadratic model. On the other hand, in eggs irradiated at the early pronuclear stage, the frequency increased linearly with dose and the dose-response relationship was best fitted to the linear model. The aberrations were mainly chromosome-type (mature oocytes: 86.0% and pronuclear stage: 88.5%) and the majority were fragments in both cases. Eggs in the early pronuclear stage were markedly more radiation-sensitive than mature oocytes. A comparison of the present results with the previous ones (Matsuda et al., 1985b) showed that the sensitivities to induction of chromosome aberrations were in the order: egg at early pronuclear stage (highest) greater than mature oocyte greater than mature sperm.     

Fetal development of mouse oocytes and zygotes cryopreserved in a nonconventional freezing medium

This study (1) analyzed fetal development of mouse embryos after oocyte cryopreservation in CJ2, a choline-based medium, (2) examined the effect of culture duration in vitro on subsequent fetal development, and (3) compared survival and fetal development of zygotes frozen in embryo transfer freeze medium (ETFM; sodium-based medium) or CJ2. Unfertilized oocytes and zygotes were cryopreserved using a slow-cooling protocol. After thawing, oocytes were inseminated after drilling a hole in their zona, cultured in vitro either to the two-cell or blastocyst stage, and transferred to the oviducts or uterine horns of recipient mice. In parallel experiments, frozen-thawed zygotes were similarly cultured and transferred. Implantation rates for transferred embryos were high (range 66-88%), regardless of whether they had been frozen as oocytes or zygotes and whether they had been transferred to the oviduct or uterus. However, fetal development was significantly higher when two-cell embryos were transferred. With blastocyst transfer, control embryos implanted and produced a greater proportion of fetuses than did oocytes frozen in CJ2, whereas transfer at the two-cell stage resulted in similar proportions of implantation sites and fetuses. Blastocyst transfer of zygotes cryopreserved in ETFM or CJ2 produced similar fetal development rates (23.6% vs 20.0%), but when frozen-thawed zygotes were transferred at the two-cell stage the fetal development rates were higher in the ETFM group (53.3%) than in the CJ2 group (32.0%). A high proportion (46.7%) of oocytes frozen in CJ2 in a nonprogrammable freezer and plunged at -20 degrees C developed into live offspring. This study shows that in the mouse (1) oocytes frozen in CJ2 can develop into viable fetuses, (2) prolonging culture in vitro has a detrimental effect on embryo transfer outcome, and (3) CJ2 offers no advantage for zygote cryopreservation.     

Preimplantation development of rabbit embryos after transfer of embryonic nuclei into different cytoplasmic environment

The development of nuclear-transfer oocytes and zygotes was tested in the rabbit. Metaphase II oocytes and zygotes in the early pronuclear stage were treated with a cytoskeletal inhibitor (cytochalasin D), enucleated, and subsequently fused either with single blastomeres from eight- and 16-cell stages (oocytes and zygotes) or with pronuclei-containing karyoplasts (zygotes only). Also, nonenucleated zygotes were fused with 1/8 blastomeres. Fusion was performed by means of an electric field. Development of reconstituted embryos was monitored mainly in vitro, but a certain number of embryos developed from oocytes and zygotes receiving nuclei from eight-cell stages were also transferred into pseudopregnant does. Development of nuclear-transfer oocytes was distinctly better than that of nuclear-transfer zygotes, since 16.9% and 9.5% oocytes vs. 8.1% and 3.7% zygotes carrying eight- and 16-cell nuclei, respectively, developed to the blastocyst stage. Two advanced but already dead fetuses were found after transfer of 27 four-cell embryos obtained after fusion of oocytes with 1/8 blastomeres. No implantations were observed after transfer of 25 four-cell embryos developed from enucleated zygotes receiving eight-cell nuclei. These findings indicate that, in the rabbit, some nuclei from 16-cell embryos are still capable of promoting at least preimplantation development. Comparison between the developmental abilities of oocyte- and zygote-derived nuclear-transfer embryos also suggests that the cytoplasmic environment of recipient cell is more crucial for the development of reconstituted embryos than the stage of introduced nuclei (at least up to the 16-cell stage). The majority of pronuclear exchange embryos (69.9%) and 40% of nonenucleated zygotes receiving eight-cell nuclei were able to develop to the blastocyst stage. This latter observation indicates, similarly as with mouse, a supporting role of residual pronuclei for participation of an eight-cell nucleus in the development of reconstituted zygotes.     

Characterization of Revertants of Unc-93(e1500) in Caenorhabditis Elegans Induced by N-Ethyl-N-Nitrosourea

Phenotypic reversion of the rubber-band, muscle-defective phenotype conferred by unc-93(e1500) was used to determine the utility of N-ethyl-N-nitrosourea (ENU) as a mutagen for genetic research in Caenorhabditis elegans. In this system, ENU produces revertants at a frequency of 3 X 10(-4), equivalent to that of the commonly used mutagen, EMS. The gene identity of 154 ENU-induced revertants shows that the distribution of alleles between three possible suppressor genes differs from that induced by EMS. A higher percentage of revertants are alleles of unc-93 and many fewer are alleles of sup-9 and sup-10. Three revertants complement the three known suppressor genes; they may therefore identify a new gene product(s) involved in this system of excitation-contraction coupling in C. elegans. Molecular characterization of putative unc-93 null alleles reveals that the base changes induced by ENU are quite different from those induced by EMS; specifically we see an increased frequency of A/T -> G/C transitions. The frequency of ENU-induced intragenic deletions is found to be 13%. We suggest that ENU, at concentrations below 5 mM, will be a superior mutagen for studies of protein function in C. elegans.     

The novel surveillance mechanism of the Trp53-dependent s-phase checkpoint ensures chromosome damage repair and preimplantation-stage development of mouse embryos fertilized with x-irradiated sperm

Cell cycle checkpoints and apoptosis function as surveillance mechanisms in somatic tissues. However, some of these mechanisms are lacking or are restricted during the preimplantation stage. Previously, we reported the presence of a novel Trp53-dependent S-phase checkpoint that suppresses pronuclear DNA synthesis in mouse zygotes fertilized with X-irradiated sperm (sperm-irradiated zygotes) (Shimura et al., Mol. Cell. Biol. 22, 2220-2228, 2002). Here we studied the role of the Trp53-dependent S-phase checkpoint in the early stage of development of sperm-irradiated zygotes. In the Trp53(+/+) genetic background, all of the sperm-irradiated zygotes cleaved successfully to the two-cell stage despite the fact that half of them carried a sub-2N amount of DNA. These zygotes progressed normally to the eight-cell stage and then implanted, but the subsequent fetal development was suppressed in a dose-dependent manner. In contrast, sperm-irradiated Trp53(-/-) embryos lacking an S-phase checkpoint exhibited an abnormal segregation of chromosomes at the first cleavage, even though they carried an apparently normal 2N amount of DNA. They were morphologically abnormal with numerous micronuclei, and they degenerated before reaching the eight-cell stage. As a consequence, no implants were observed for sperm-irradiated Trp53(-/-) embryos. These results suggest that the Trp53-dependent S-phase checkpoint is a surveillance mechanism involved in the repair of chromosome damage and ensures the preimplantation-stage development of sperm-irradiated embryos.     

Relative Rates at Which Dominant-Lethal Mutations and Heritable Translocations Are Induced by Alkylating Chemicals in Postmeiotic Male Germ Cells of Mice

There is a close relationship between the rates at which dominant lethal mutations and heritable translocations are induced by ethyl methanesulfonate (EMS) or triethylenemelamine (TEM) in male postmeiotic germ cells. This relationship does not hold for isopropyl methanesulfonate (IMS), which induced only negligible frequencies of heritable translocations at doses that induced high levels of dominant lethal mutations. Nor does IMS behave like EMS and TEM in the degree to which eggs of different stocks of females repair premutational lesions that are carried in the sperm-large differences between stocks for IMS treatment and small differences for EMS or TEM treatment. These dissimilarities between IMS and the other two alkylating chemicals are postulated to be attributable to differences in the types of lesions present at the time of repair activity and to whether or not chromosomal aberrations are already fixed prior to postfertilization pronuclear DNA synthesis.     

Subcellular distribution of mitochondrial ribosomal RNA in the mouse oocyte and zygote

Mitochondrial ribosomal RNAs (mtrRNAs) have been reported to translocate extra-mitochondrially and localize to the germ cell determinant of oocytes and zygotes in some metazoa except mammals. To address whether the mtrRNAs also localize in the mammals, expression and distribution of mitochondrion-encoded RNAs in the mouse oocytes and zygotes was examined by whole-mount in situ hybridization (ISH). Both 12S and 16S rRNAs were predominantly distributed in the animal hemisphere of the mature oocyte. This distribution pattern was rearranged toward the second polar body in zygotes after fertilization. The amount of mtrRNAs decreased around first cleavage, remained low during second cleavage and increased after third cleavage. Staining intensity of the 12S rRNA was weaker than that of the 16S rRNA throughout the examined stages. Similar distribution dynamics of the 16S rRNA was observed in strontium-activated haploid parthenotes, suggesting the distribution rearrangement does not require a component from sperm. The distribution of 16S rRNAs did not coincide with that of mitochondrion-specific heat shock protein 70, suggesting that the mtrRNA is translocated from mitochondria. The ISH-scanning electron microscopy confirms the extra-mitochondrial mtrRNA in the mouse oocyte. Chloramphenicol (CP) treatment of late pronuclear stage zygotes perturbed first cleavage as judged by the greater than normal disparity in size of blastomeres of 2-cell conceptuses. Two-third of the CP-treated zygotes arrested at either 2-cell or 3-cell stage even after the CP was washed out. These findings indicate that the extra-mitochondrial mtrRNAs are localized in the mouse oocyte and implicated in correct cytoplasmic segregation into blastomeres through cleavages of the zygote.     

Cell cycle-coupled [Ca(2+)](i) oscillations in mouse zygotes and function of the inositol 1,4,5-trisphosphate receptor-1

Sperm entry in mammalian eggs initiates oscillations in the concentration of free calcium ([Ca(2+)](i)). In mouse eggs, oscillations start at metaphase II (MII) and conclude as the zygotes progress into interphase and commence pronuclear (PN) formation. The inositol 1,4,5-trisphosphate receptor (IP(3)R-1), which underlies the oscillations, undergoes degradation during this transition, suggesting that one or more of the eggs' Ca(2+)-releasing machinery components may be regulated in a cell cycle-dependent manner, thereby coordinating [Ca(2+)](i) responses with the cell cycle. To ascertain the site(s) of interaction, we initiated oscillations at different stages of the cell cycle in zygotes with different IP(3)R-1 mass. In addition to sperm, we used two other agonists: porcine sperm factor (pSF), which stimulates production of IP(3), and adenophostin A, a non-hydrolyzable analogue of IP(3). None of the agonists tested induced oscillations at interphase, suggesting that neither decreased IP(3)R-1 mass nor lack of production or excessive IP(3) degradation can account for the insensitivity to IP(3) at this stage. Moreover, the releasable Ca(2+) content of the stores did not change by interphase, but it did decrease by first mitosis. More importantly, experiments revealed that IP(3)R-1 sensitivity and possibly IP(3) binding were altered at interphase, and our data demonstrate stage-specific IP(3)R-1 phosphorylation by M-phase kinases. Accordingly, increasing the activity of M-phase kinases restored the oscillatory-permissive state in zygotes. We therefore propose that the restriction of oscillations in mouse zygotes to the metaphase stage may be coordinated at the level of IP(3)R-1 and that this involves cell cycle stage-specific receptor phosphorylation.     

Teratologic studies on rat perinates and offspring from dams treated with ethylnitrosourea (ENU).

Ethylnitrosourea (ENU), a well known DNA alkylating agent, induces anomalies in the central nervous system (CNS), craniofacial tissues, limbs and male reproductive organs. Recently we clarified that excess cell death caused by apoptosis occurred in these organs and tissues of rat fetuses from dams treated with ENU at day 13 of gestation (GD13). In this study, we examined fetuses at GD21 and offspring at 10 weeks of age after ENU administration to pregnant rats at GD13 in order to clarify the relationship between ENU-induced apoptosis in the fetal tissues and teratogenicity of ENU. Severe intrauterine growth retardation was observed in the ENU group, and the body weight of the offspring in the ENU group was significantly lower than that of the control group throughout the experiment. In addition, a high incidence of microencephaly, ectrodactyly and curved caudal vertebrae was observed in the offspring from dams treated with ENU at GD13. Judging from the results of our previous and present studies, it was strongly suggested that ENU-induced apoptosis in rat fetal tissues may play an important role in the induction of anomalies in the corresponding tissues.     

Assessment of pronuclear formation following in vitro fertilization with bovine spermatozoa obtained after thermal insulation of the testis

This study was conducted to follow the chronology of pronuclear formation in bovine zygotes after in vitro insemination with a population of spermatozoa having abnormal morphology. Semen samples were obtained and cryopreserved from four Holstein bulls before and after a scrotal insulation period of 48 h (Day 0). A pre-insult (Day 5) and a Day 20 post-insult semen sample were evaluated for morphology and used for IVF after standard swim-up sperm separation protocols. Pronuclear formation was scored on subpopulations of presumptive zygotes after they were fixed and stained at 3-h time intervals from 6 to 18 h post in vitro insemination (hpi). Post-thaw morphological evaluation of semen samples revealed a decrease in the percentages of normal spermatozoa in the post-insult samples compared with the pre-insult samples for Bull I (74-22%) and Bull III (68-1%). The sperm penetration rate decreased (P<0.05) between the pre- and post-insult samples for Bulls I (90-76%) and III (92-70%), but was not different for Bulls II (92-90%) and IV (78-85%). The pronuclear formation rates for post-insult zygotes for Bulls II and IV had comparable increases in development over time, whereas there was no increase in the pronuclear development for the zygotes from the post-insult samples for Bulls I and III, and generally a condensed sperm head was observed in the oolemma. At 18 hpi the fertilization rate between the pre- and post-insult samples for Bulls I (51-4%), II (88-75%) and III (94-2%) decreased (P<0.01), but there was no change for Bull IV (66%). In conclusion, we inferred that the failure in normal pronuclear formation was associated with an absence of normal decondensation of the penetrating spermatozoon; this suggested that the effect of morphologically abnormal spermatozoa occurred prior to cleavage, thus limiting early development.