Chromosomal control of early embryonic development in mice I. Experiments on embryos with autosomal monosomy

The effects of autosomal monosomy on early embryonic development were studied in mice with Robertsonian and reciprocal (non-Robertsonian) translocations. It was found that monosomy for autosomes 1, 2, 3, 5, 6, 16, 17 and 19 had substantially different effects on preimplantation development and survival of mouse embryos. Monosomy for autosomes 1, 3, 6, 16 and 19 does not affect cleavage, compaction and blastulation and in some cases is compatible with implantation. Most of these embryos, however, die as early blastocysts (Ms 3, 6 or 19) and some of them are eliminated at early postimplantation stages (Ms 1 or 16). The embryos with monosomy for autosomes 2, 5 or 17 can be identified during cleavage owing to the reduced blastomere number and pathological changes in the nuclei. Most of these monosomies do not survive beyond the morula stage. The results indicate that differential genetic activity of autosomes in mice becomes already evident in very early embryonic development. A hypothetical mechanism for homologous autosome activation at the onset of embryonic development in mice is suggested.     

6-Mercaptopurine,an inducer of cytogenetic and dominant-lethal effects in premeiotic and early meiotic germ cells of male mice

Dominant-lethal effects of 6-mercaptopurine on male mice were studied using eight doses, ranging from 150 to 482 mg/kg. Effects of the 150-mg/kg dose were studied over the entire spermatogenic cycle, and those of the higher doses for matings made between days 28.5 and 41.5 after treatment. It was found that, with low doses, there was only one period in which clearcut increases in induced dominant-lethal mutations were detected, namely in matings that occurred 32.5 to 35.5 days after treatment. With higher doses, effects could be detected beyond that period through day 39.5. Spermatozoa utilized for matings during the period of greatest response were presumably derived from germ cells that were in late differentiating spermatogonial and early meiotic spermatocyte stages at the time of treatment. These results are similar to those of Ray and Hyneck. To date, 6-mercaptopurine is unique in inducing dominant lethality only at these particular stages.A study of chromatid aberration induction in the treated males themselves was carried out for 150 and 250 mg/kg doses of 6-mercaptopurine over the period of 9 to 16 days after treatment. A considerable increase in isochromatid and chromatid deletions was observed in diakinesis-metaphase-I spermatocytes on days 14 and 15 after treatment. For reasons discussed, the cells sampled at this time may be assumed to have been in early meiosis (preleptotene), with some in late differentiating spermatogonial stages, at the time of treatment. The rough agreement in sensitive cell type for dominant lethality and chromatid aberration induction suggests that chromatid deletions are the cause of dominant lethality in this study. Conservative estimates of the frequency of dominant lethality expected from the chromatid aberration frequencies tend to substantiate this suggestion.     

Specific Features of Phenotypic Expression of Autosomal Monosomies during Pathological Postimplantation Development of Man

Autosomal monosomies represent a severe form of genomic disbalance which determines elimination of human embryos already at the preimplantation stages. As a rule, they occur very rarely in the materials of spontaneously aborted embryos and fetuses. Molecular-cytogenetic studies were carried out on the karyotype of cells of 60 spontaneous abortuses of I trimester of pregnancy with cell degeneration or absence of cell proliferation in the cultures, as a result of which the cells could not be studied using the standard metaphase analysis. The embryos were characterized by an unexpectedly high frequency of mosaic variants of monosomies for chromosomes 7, 15, 21, and 22, which amounted to 19% of all chromosome aberrations. Lethal forms of monosomies for human chromosomes 7 and 15 were described for the first time, since they are not found in spontaneous abortuses by standard cytogenetic methods. A hypothesis was proposed which accounts for the possibility of early postimplantation lethality of the embryos with mosaic forms of autosomal monosomies. The differences were found between the cells with monosomies for different autosomes in the mechanisms of origin, intertissue localization, and phenotypic effects. It was shown that monosomies for chromosomes 7, 15, 21, and 22 in a mosaic state with the normal cell line can be compatible with the early stages of postimplantation differentiation of the cytotrophoblast. Predominant compartmentalization of the cells with monosomies for chromosomes 21 and 22 in the extraembryonic mesoderm, a derivative of epiblast, can be a critical factor, which makes the normal morphogenesis of embryonic structures impossible.     

The in vitro survival of human monosomies and trisomies as embryonic stem cells

Chromosomal aneuploidies are responsible for severe human genetic diseases. Aiming at creating models for such disorders, we have generated human embryonic stem cell (hESC) lines from pre-implantation genetic screened (PGS) embryos. The overall analysis of more than 400 aneuploid PGS embryos showed a similar risk of occurrence of monosomy or trisomy for any specific chromosome. However, the generation of hESCs from these embryos revealed a clear bias against monosomies in autosomes. Moreover, only specific trisomies showed a high chance of survival as hESC lines, enabling us to present another categorization of human aneuploidies. Our data suggest that chromosomal haploinsufficiency leads to lethality at very early stages of human development.     

Characteristics of phenotypic expression of autosomal monosomies during pathological postimplantation human development

Autosomal monosomies represent a severe form of genomic disbalance which determines elimination of human embryos already at the preimplantation stages. As a rule, they occur very rarely in the materials of spontaneously aborted embryos and fetuses. Molecular-cytogenetic studies were carried out on the karyotype of cells of 60 spontaneous abortuses of I trimester of pregnancy with cell degeneration or absence of cell proliferation in the cultures, as a result of which the cells could not be studied using the standard metaphase analysis. The embryos were characterized by an unexpectedly high frequency of mosaic variants of monosomies for chromosomes 7, 15, 21, and 22, which amounted to 19% of all chromosome aberrations. Lethal forms of monosomies for human chromosomes 7 and 15 were described for the first time, since they are not found in spontaneous abortuses by standard cytogenetic methods. A hypothesis was proposed which accounts for the possibility of early postimplantation lethality of the embryos with mosaic forms of autosomal monosomies. The differences were found between the cells with monosomies for different autosomes in the mechanisms of origin, intertissue localization, and phenotypic effects. It was shown that monosomies for chromosomes 7, 15, 21, and 22 in a mosaic state with the normal cell line can be compatible with the early stages of postimplantation differentiation of the cytotrophoblast. Predominant compartmentalization of the cells with monosomies for chromosomes 21 and 22 in the extraembryonic mesoderm, a derivative of epiblast, can be a critical factor, which makes it impossible the normal morphogenesis of embryonic structures.     

Embryonic and post-embryonic lethals induced by diethyl sulfate in mature sperm of Drosophila melanogaster.

It has recently been reported that, in Drosophila melanogaster, when sperm treated with diethyl sulfate was stored in the females, II–III translocations were detected as from the 6th day after the treatment, though none was recovered without storage. Chromosome breaks being currently considered the main cause of dominant lethality and the embryonic period lasting about one day at 25°C, it was thought of interest to study the ability of DES to induce this type of damage with and without storage. It was found that the treatment increased embryonic lethality (measured as frequency of unhatched eggs) and post-embryonic lethality (measured as frequency of larval and pupal death) over the control values. The frequency of embryonic lethals after storage in the females for 6 days was similar to that shown by the unstored samples. In contrast with this, the yield of post-embryonic lethality was markedly raised by that storage time. It is suggested that: (1) lesions are induced as “pre-breaks”, and storage and cell divisions are instrumental in their opening; (2) potential breaks can undergo DNA replication and cell division as such and become open in different cell cycles, impairing embryonic and post-embryonic development; (3) chromosome breaks induced by DES seem to behave in a way similar to those induced by other mono- and poly-functional alkylating agents; and (4) when the potential ability of chemical compounds to induce chromosome breaks is assessed, post-embryonic lethality can be used as a simple one-generation preliminary test, to establish delayed effects.     

Day of cycle affects changes in equine intrauterine pressure in response to teasing

Oxytocin is released in response to teasing during both estrus and diestrus in mares, and at least during estrus, teasing results in an increase in electromyographic activity in the uterus. Exogenous oxytocin causes an increase in intrauterine pressure and prior studies have shown that this response is correlated to the day of the estrous cycle. To determine if teasing causes an increase in intrauterine pressure and if this response varies by day of the cycle, intrauterine pressure was measured while mares were teased with a stallion 2 days before ovulation, on the day ovulation was detected and 2 days after ovulation. A significant increase in intrauterine pressure was observed in response to teasing both 2 days before ovulation and on the day of ovulation, when plasma concentrations of progesterone were low. No significant increase in intrauterine pressure was observed in response to teasing 2 days after ovulation when progesterone concentrations were elevated. Management practices that include teasing or stallion exposure may be beneficial in stimulating uterine clearance mechanisms in mares during the preovulatory period.     

Onset of the hormone-sensitive perinatal period for sexual differentiation of the sexually dimorphic nucleus of the preoptic area in female rats

The volume of the sexually dimorphic nucleus of the preoptic area (SDN-POA) of the rat brain is severalfold larger in males than in females. The volume of the SDN-POA can be influenced significantly by the hormonal milieu during the perinatal "critical period" of sexual differentiation of the brain. The purpose of the present study was to determine the onset of this period of sexual differentiation of the SDN-POA. Pregnant rats received no treatment or were injected subcutaneously with oil on day 17, 18, or 20, or testosterone (T;5 mg) on days 16-22 of gestation. On postnatal day 15, unilateral SDN-POA volumes from female offspring prenatally exposed to testosterone on day 16 or 17 were not different from values of control (untreated or oil-injected) offspring. Female offspring from mothers treated with testosterone on day 18, 19, or 20 of gestation showed a significant and similar increase in SDN-POA volume over values from control animals. SDN-POA volumes from female offspring exposed to testosterone on day 21 or 22, although larger than those of controls, were not different statistically. We conclude that with the specific paradigm used in this study SDN-POA development is insensitive prior to day 18 of gestation, the day on which the onset of the hormone-sensitive period occurs.     

Influence of altered afferent input on the number of trochlear motor neurons during development

A loss of about half of the trochlear motor neurons occurs during the course of normal development. The present investigation was undertaken to examine the role of afferent input in regulating the number of surviving or dying trochlear motor neurons. A majority of the afferent input to the trochlear nucleus comes from the vestibular nuclei of the hindbrain via the medial longitudinal fasciculus. Portions of the hindbrain were lesioned in duck embryos on embryonic day 3, considerably prior to the time motor neurons send their axons out and cell death begins. The effectiveness of hindbrain lesion was verified by electron microscopical examination of synapses. There was a significant decrease in the number of synapses on trochlear motor neurons following hindbrain lesion. Cell counts made after the period of cell death indicated a significant decrease in the final number of surviving trochlear motor neurons. Cell counts made prior to the onset of cell death indicated that there was a drastic reduction in the initial number of trochlear motor neurons produced in hindbrain lesion embryos. In spite of a significant reduction in the initial number of neurons, the percentage loss of neurons was about the same as during normal development. Since trochlear motor neurons are generated prior to the formation of afferent synapses on them, it is unlikely that the reduction in the number of motor neurons initially produced is due to reduced afferent synaptic input. Since the percentage of cell loss in hindbrain lesion and normal embryos is about the same, it seems that the magnitude of cell death is genetically programmed. These observations suggest that the afferent synaptic input to the trochlear motor neurons may not be as important as previously thought in regulating cell number during development. They also suggest that some afferent input, of nonsynaptic type, available at very early stages of development may specify the initial number of trochlear motor neurons produced and that a fixed percentage of that number is programmed to die during development. It is suggested that this early influence may be provided by the embryonic medial longitudinal fasciculus.     

The reproductive biology of Theromyzon tessulatum (Glossiphoniidae: Hirudinoidea), with comments on Theromyzon rude

The reproductive biology and behaviour of Theromyzon tessalatum is described from the commencement of copulation to the release of the young and the death of the parent. Copulation occurs several times over a10–20 day period with either the same or different partners. Eggs were laid6–14 days after copulation in usually four cocoons each containing approximately 100 eggs. The cocoons were brooded by the parents and the young, on development of the embryonic attachment organ and later by the posterior sucker, attached to the ventral surface of the parent's abdomen. The young detached from the parent either on contact with a suitable host for a blood meal or after approximately five months just prior to the parent's death.     

Comparison of the gastrointestinal syndrome after total-body or total-abdominal irradiation

In pathogen-free mice, but not standard conventionally housed laboratory rodents, two distinctly different modes of early radiation lethality can be identified by modifying the irradiation technique (total-body versus abdominal irradiation) or by therapeutic intervention such as rescue of total-body-irradiated mice with syngeneic bone marrow or spleen. While damage to the gastrointestinal tract is usually designated as the predominant cause of death occurring within 10 days of radiation exposure, it was demonstrated that damage to the hematopoietic/lymphopoietic system can result in animal lethality over the same period as the gastrointestinal syndrome and that this target cell population is more radiation-sensitive than the gastrointestinal epithelium.     

Heat potentiation of radiation damage versus radiation potentiation of heat damage

The enhanced lethality of mammalian cells after combined treatment with hyperthermia and radiation is usually attributed to heat potentiation of radiation damage. However, it has been suggested that the situation may be reversed and that radiation may act as a modifier for heat damage. To test this hypothesis, BP-8 murine sarcoma cells were subjected to sequential radiation and heat treatments and the kinetics and extent of cell death were evaluated with the [125I]-iododeoxyuridine prelabeling assay. Cell death after heating was rapid and essentially complete within 2 days after heat exposure, whereas radiation death was slow and became apparent only after a delay period of 3 days. Combined exposure of cells to radiation and heat caused a pronounced increase in the delayed component of cell death, that is, the radiation component of death. Irradiation of cells before heating did not change the early heat component of cell death even in cells that were exposed to massive radiation doses of up to 300 Gy prior to heating. These results indicate that the increased cell death observed in hyperthermia/radiation-treated cells results from heat potentiation of radiation damage, not radiation potentiation of heat damage.     

Enhanced susceptibility of mouse embryos heterozygous for oligosyndactyly (Os/+) to mitomycin C-induced skeletal abnormalities

The mouse mutation, oligosyndactyly (Os), results in syndactyly, muscle anomalies, and deficiency of nephrons in heterozygous animals and early embryonic lethality in homozygotes. Since the homozygous lethality results from mitotic arrest with intact spindles at the time of implantation, we have hypothesized that the heterozygous manifestations may result from impairment of cell proliferation in regions with high proliferative rates. To test this hypothesis, Os/+ and +/+ mouse embryos at 6.5 days of gestation were exposed to mitomycin C (MMC), an agent that causes a high degree of embryonic cell death which is "compensated" for by a period of rapid cell proliferation. 17.9% of MMC-treated +/+ fetuses had fused vertebrae, a significant increase over untreated fetuses, and this frequency was further increased to 33.6% in MMC-treated Os/+ fetuses. Saline treated Os/+ and +/+ fetuses showed the same low rate (0-3%) of vertebral fusion. These results indicate that Os/+ embryos have an increased sensitivity to the vertebral fusion-inducing effect of MMC at 6.5 days of gestation, a finding compatible with the hypothesis that rapid cell proliferation may be impaired in Os/+ embryos and fetuses.     

Spontaneous embryonic death on Days 20 to 40 in heifers

Transrectal ultrasound examinations were used in nulliparous Holstein heifers to study the association between time of spontaneous embryonic death (cessation of heartbeat) and luteal regression, and to determine the fate of the conceptus after embryonic death. There was no significant difference between nonbred heifers (n = 135) and bred, nonpregnant heifers (embryonic heartbeat never detected, n = 40) for day of onset of luteal regression (means, 17.6 and 17.9, respectively) or for length of interovulatory interval (means, 20.6 and 20.9 days, respectively). Pregnancy was confirmed by detection of an embryonic heartbeat on Day 24 (ovulation = Day 0) or later, or on two consecutive days prior to Day 24; on average, an embryonic heartbeat was detected on Day 22.0 (n = 104). Pregnancy rate on Day 24 was higher (P<0.02) in heifers bred on Day -1 (116/149, 77.8%) than in heifers bred on Day -2 (51/79, 64.6%), and was higher (P<0.05) in heifers with an embryo transferred ipsilateral to the corpus luteum than in heifers with an embryo transferred contralateral to the corpus luteum (3/4 vs 0/5). Embryonic death (lack of embryonic heartbeat following confirmation of pregnancy) and presumptive embryonic death (embryonic heartbeat detected on one day only, prior to Day 24) were detected prior to Day 25 in one and two bred heifers, respectively, and in one and two heifers with an embryo transferred contralateral to the corpus luteum, respectively. In these six heifers, luteal regression preceded, and apparently caused, embryonic death. In seven of eight heifers in which embryonic death was detected between Days 25 and 40, the onset of luteal regression was detected at least 3 d (range, 3 to 42 d) after detection of embryonic death. The incidence of embryonic death on Days 29 to 32 was lower (P<0.02) in heifers bred on Day -1 than in heifers bred on Day -2 (0 of 96 vs 3 of 40, respectively). In heifers in which luteal regression preceded embryonic death, the conceptus was lost rapidly, with minimal evidence of degeneration. In heifers in which embryonic death preceded luteal regression, there was ultrasonic evidence of conceptus degeneration, but conceptus fluid and tissue appeared to be maintained. In all heifers with embryonic death, the conceptus and its breakdown products apparently were eliminated by expulsion through the cervix rather than by resorption.     

Thyroid hormone-deficient period prior to the onset of hearing is associated with reduced levels of beta-tectorin protein in the tectorial membrane: implication for hearing loss

The genes for alpha- and beta-tectorin encode the major non-collagenous proteins of the tectorial membrane. Recently, a targeted deletion of the mouse alpha-tectorin gene was found to cause loss of cochlear sensitivity (). Here we describe that mRNA levels for beta-tectorin, but not alpha-tectorin, are significantly reduced in the cochlear epithelium under constant hypothyroid conditions and that levels of beta-tectorin protein in the tectorial membrane are lower. A delay in the onset of thyroid hormone supply prior to onset of hearing, recently described to result in permanent hearing defects and loss of active cochlear mechanics (), can also lead to permanently reduced beta-tectorin protein levels in the tectorial membrane. beta-Tectorin protein levels remain low in the tectorial membrane up to one year after the onset of thyroid hormone supply has been delayed until postnatal day 8 or later and are associated with an abnormally structured tectorial membrane and the loss of active cochlear function. These data indicate that a simple delay in thyroid hormone supply during a critical period of development can lead to low beta-tectorin levels in the tectorial membrane and suggest for the first time that beta-tectorin may be required for development of normal hearing.     

Long-term alcohol exposure prior to conception results in lower fetal body weights

BACKGROUND: It is well known that alcohol consumption during pregnancy can result in lower birth weight babies but many women stop consuming alcohol prior to conception as a part of pregnancy planning. The purpose of this study was to determine whether alcohol consumption prior to conception may also have an effect on fetal development. METHODS: Male and female C57BL/6J mice at 4, 6, or 8 weeks of age received either a single administration of alcohol (3.0 g/kg) via intragastric gavage (IG) each day for at least 60 days, or an isovolumetric IG administration of sterile water. After 60 treatment days, males and females within each age and treatment group were mated overnight. Females continued to receive daily alcohol treatments until conception. Males continued to receive treatments until all females were successfully mated. At conception, females were isolated and left undisturbed. On embryonic day 14, fetus number, size, and weight was determined. RESULTS: Maternal food consumption, body weight at conception, and delay to conception onset did not differ between the two treatment groups or among the three age groups. Fetal body weights did not differ among the three age groups. Fetuses from females treated with alcohol had lower body weights compared to those treated with water. Male treatments did not seem to affect fetal body weight. CONCLUSIONS: Fetal growth and development can be affected by alcohol consumption prior to the time of conception. Alcohol consumption prior to conception is a potential risk factor to fetal outcome and an important consideration for those females planning to have children.     

Characterization of the early events of potato tuber development

The early events of potato ( Solanum tuberosum L. cv. Superior) tuberization were examined by using a model system of axillary bud tuber development from petiole-leaf single-node cuttings. Both fresh weight and starch accumulation were monitored to establish a developmental framework for morphological changes. Fresh weight and starch content began to increase in axillary buds after 2 days. Visible changes in bud morphology could be detected 4 days after the start of incubation. Substantial increases in both total protein and total RNA were observed at the onset of tuber morphology. Immunoblot analysis showed that the major tuber protein, patatin, could be initially detected in day 4 buds and that a 22-kDa proteinase inhibitor could be initially detected at day 8. Northern blot analysis corroborated this pattern of accumulation at the RNA level for both protein types. Substantial accumulation of the two proteinase inhibitor mRNAs occurred later than patatin mRNA accumulation. The results of this study showed that there is considerable accumulation of both protein and mRNA occurring during the early stages of tuber development prior to the substantial accumulation of the major tuber storage proteins.     

Increase in endogenous spleen colonies without recovery of blood cell counts in radioadaptive survival response in C57BL/6 mice

The radioadaptive survival response induced by a conditioning exposure to 0.45 Gy and measured as an increase in 30-day survival after mid-lethal X irradiation was studied in C57BL/6N mice. The acquired radioresistance appeared on day 9 after the conditioning exposure, reached a maximum on days 12-14, and disappeared on day 21. The conditioning exposure 14 days prior to the challenge exposure increased the number of endogenous spleen colonies (CFU-S) on days 12-13 after the exposure to 5 Gy. On day 12 after irradiation, the conditioning exposure also increased the number of endogenous CFU-S to about five times that seen in animals exposed to 4.25-6.75 Gy without preirradiation. The effect of the interval between the preirradiation and the challenge irradiation on the increase in endogenous CFU-S was also examined. A significant increase in endogenous CFU-S was observed when the interval was 14 days, but not 9 days. This result corresponded to the increase in survival observed on day 14 after the challenge irradiation. Radiation-inducted resistance to radiation-induced lethality in mice appears to be closely related to the marked recovery of endogenous CFU-S in the surviving hematopoietic stem cells that acquired radioresistance by preirradiation. Preirradiation enhanced the recovery of the numbers of erythrocytes, leukocytes and thrombocytes very slightly in mice exposed to a sublethal dose of 5 Gy, a dose that does not cause bone marrow death. There appears to be no correlation between the marked increase in endogenous CFU-S and the slight increase or no increase in peripheral blood cells induced by the radioadaptive response. The possible contribution by some factor, such as Il4 or Il11, that has been reported to protect irradiated animals without stimulating hematopoiesis is discussed.     

Siberian hamsters that fail to reentrain to the photocycle have suppressed melatonin levels

Siberian hamsters readily reentrain to a 3-h phase delay of the photocycle (16 h light/day) but fail to reentrain to a 5-h phase delay. This study tested whether melatonin production was suppressed in animals that failed to reentrain. Melatonin was measured on the day before, day of, or several days after each phase shift. Melatonin levels measured 4 h after dark onset were approximately 83 microg/ml on the day before each phase delay and undetectable (<6 microg/ml) during the light phase on the day of the phase shift. Activity onsets regained their prior phase relationship to the photocycle 4 (3 h) or 5 (5 h) days after the phase shift; on that day, melatonin levels were measured 4 h after dark onset. Melatonin levels were unaffected by the 3-h phase delay (>57.6 microg/ml) but were undetectable after a 5-h phase delay (<8 microg/ml). Thus melatonin remained suppressed only after the phase delay to which hamsters also fail to reentrain. This relationship suggests that the propensity for reentrainment may be influenced by changes in melatonin production following a phase shift of the photocycle.     

Analysis of the frequency of spontaneous aneuploidy in human somatic cells using interphase cytogenetic technology

The frequency of spontaneous aneuploidy of the four autosomes and sex chromosomes in the interphase nuclei of cultivated and uncultured lymphocytes from clinically healthy men was examined by use of two-color fluorescent in situ hybridization (FISH). It was shown that in uncultured cells from the individuals examined autosomal nullosomies were practically not detected (the frequency 0 to 0.01%). At the same time, the frequency of such cells with either Y, or X nullosomy was at least an order of magnitude higher (about 0.15%). This frequency was comparable with the level of Y- or X-disomic cells, and also with autosomal monosomies, precluding from considering the X-nullosomic cells as hybridization artifacts. During lymphocyte cultivation, a statistically significant increase in the total frequency of Y- or X-nullosomic cells was observed already after the first cell division cycle. Thus, interphase FISH analysis is a sufficiently sensitive method enabling detection of higher, compared to the autosomes, loss of sex chromosomes in the process of cell division, a phenomenon observed during replicative cell aging, as well as during natural aging of the organism. Male cells with the de novo lost singular X chromosome, probably, switch to apoptosis and do not survive during further life of a cell population. The frequency of total aneuploidy in human somatic cells with the correction for the resolution capacity of the interphase FISH analysis constituted 5.62 and 6.90% for uncultured and cultivated cells, respectively. This aneuploidy level is close to that in spermatozoa. The data obtained can serve as the basis for the examination of the aneugenic (aneuploidy-inducing) genotoxic effects and for the analysis of interindividual genetic instability.