Premature separation of centromere and aneuploidy: an indicator of high risk in unaffected individuals from familial breast cancer families?

OBJECTIVES: Injury is the leading cause of death in the male working population of Brazil. An important fraction of these deaths are work related. Very few cohort studies of steel workers, and none from developing countries, have reported on mortality from injuries. This paper analyses mortality from work and non-work related injuries among Brazilian steel workers. METHODS: Deaths during employment from 1 January 1977 to 30 November 1992 were analysed in a cohort of 21,816 male steel workers. Mortality rates specific for age and calendar year among the workers were compared with those of the male population of the state where the plant is located. Work related injuries were analysed by comparing the mortality rates for different subgroups of the cohort. RESULTS: The number of deaths (391) was less than half that expected based on death rates of the general population. Over 60% (242) of deaths were due to injuries. Mortality from most causes was substantially below that in the general population, but that from unintentional injury, was 50% above that of the general population. Standardised mortality ratios (SMRs) were highest for the youngest and the oldest employees and for labourers and clerical workers. Mortality from motor vehicle injury was twice that expected from population rates (SMR = 209, 95% confidence interval (95% CI) 176-244). There was a 67% fall in the age adjusted mortality from occupational injuries in the study period. CONCLUSION: The healthy worker effect in this cohort was greater than that commonly found in studies of occupational groups in developed countries, probably because of a greater socioeconomic gap between employed and unemployed populations in Brazil, and unequal distribution of health care resources. Mortality was especially high for motor vehicle injuries. The fall in mortality from occupational injuries during the study period was probably due to improvement in safety standards, increased automation, and better medical care. There is a need to investigate risk factors for unintentional injuries among steel workers, especially those due to motor vehicle injuries. Prevention of occupational and nonoccupational injuries should be a main priority in Brazil.     

Diazepam induces meiotic delay, aneuploidy and predivision of homologues and chromatids in mammalian oocytes

The tranquilizer and anti-convulsant diazepam (DZ) is a suspected aneugen. In order to assess its aneugenic potential in mammalian oogenesis we exposed in vitro maturing mouse oocytes to the drug. Spindle formation and cell cycle progression, the behaviour of chromosomes and the distribution of mitochondria were characterized with respect to induction of numerical chromosomal aberrations. A concentration of 25 microg/ml DZ induced a pronounced delay in maturation and blocked a high percentage of oocytes in meiosis I. This arrest was partly reversible. Hyperploidy was slightly increased in oocytes matured in the presence of 5 microg/ml DZ and became significantly elevated in oocytes matured with 25 microg/ml DZ, relative to controls. Concomitantly, DZ induced spindle aberrations and displacement of chromosomes from the equator, but unlike in mitosis and in male meiosis most oocytes still possessed bipolar spindles. A significant fraction of meiotically delayed, metaphase I-blocked oocytes exposed to 25 microg/ml DZ contained univalents. Some DZ-treated oocytes progressing to meiosis II exhibited one or multiple single chromatids. Precocious chiasma resolution and equational segregation of chromatids from functional univalents in first anaphase (predivision) may be responsible for this condition, a mechanism also discussed in the aetiology of maternal age-related aneuploidy. DZ disturbed the spatio-temporal distribution of mitochondria during oocyte maturation, possibly by binding to peripheral-type benzodiazepine receptors on mitochondria, thus affecting the availability of ATP and calcium homeostasis. Blocks in maturation may also relate to binding of DZ to calmodulin. Data suggest that DZ exposes mammalian oocytes to predivision and aneuploidy. Thresholds, long lasting effects of DZ in vivo and sex-specific sensitivities in chemically induced aneuploidy of mammalian germ cells are critically evaluated.     

RNA synthesis in preovulatory mouse oocytes

RNA synthesis, previously shown to take place during oocyte growth, has been demonstrated throughout the growth-quiescent period preceding ovulation of the mouse oocyte. In the final 7-day preovulatory period, the level of incorporation of (5,6-3H)uridine into ovulated oocytes decreased as the interval between exposure to precursor and ovulation decreased; significant incorporation was detectable within 2 days before ovulation. Analysis of the frequency and density of label in ovarian oocytes at successive stages of meiosis in relation to the interval between adminstration of labeled precursor and collection of oocytes revealed that RNA synthesis continues up to within 2 h before GVBD.     

Meiotic and developmental competence of mouse antral oocytes

Mouse antral oocytes show two different patterns of chromatin organization, defining oocytes with or without chromatin surrounding the nucleolus (SN: surrounded nucleus; NSN: not surrounded nucleus). We have previously shown that upon injection of eCG, NSN antral oocytes shift towards the SN kind of chromatin organization. We hypothesized that these newly formed SN oocytes were those that would have been ovulated after an ovulatory stimulus. The main objective of this study was to investigate the meiotic and developmental competence of these two types of oocytes. SN and NSN antral oocytes were isolated after i.p. administration of eCG + hCG or eCG-only, in vitro-cultured until completion of metaphase II, and inseminated with capacitated spermatozoa; and their development to the 4-cell stage was examined. This study demonstrates 1) that SN and NSN oocytes isolated after injection of eCG + hCG are capable of embryonic development, but not beyond the 2-cell stage; and 2) that SN and NSN oocytes isolated after injection of eCG-only are capable of developing to the 2-cell stage, but a significantly higher number (11.9%) of SN oocytes than NSN oocytes (1.5%) reach the 4-cell stage. SN- and NSN-like oocytes have also been described in the antral compartment of human, rat, monkey, pig, and bovine ovaries. The findings reported in this paper may contribute to improved procedures for in vitro fertilization of humans and farm animals.     

Aging and diethylstilbestrol-induced aneuploidy in male germ cells: a transgenic mouse model

The incidence of aneuploidy in male germ cells was evaluated by analyzing extra marker chromosome(s) signal(s) in round and/or hook spermatids of transgenic mice. Two types of transgenic mice were used as models. The inserted foreign DNA (lambda-gt10LacZ shuttle vector and/or pSVc-myc plasmid) was located at the middle of the long arms of chromosome 2 (lambda DNA) and/or chromosome 8 (c-myc). The number of marker chromosomes present could easily be detected after fluorescence in situ hybridization (FISH) in testicular cells. The frequency of spontaneous aneuploidy of chromosome 2 was similar in round spermatids of lambda and lambda-myc mice. Differential involvement of chromosomes 2 and 8 was observed in both round and hook spermatids. The frequency of spontaneous aneuploidy in round spermatids was higher than that in hook spermatids. The frequency of aneuploidy of marker chromosomes was significantly higher in older mice (2 years old) than in younger ones. Diethylstilbestrol (DES)-induced aneuploidy was dose dependent, and was not influenced by the stage at which germ cells were treated with DES. These results demonstrate the usefulness of a transgenic mouse model for the study of aneuploidy in germ cells.     

Protein neosynthesis by porcine oocytes matured in vivo and in vitro

The protein pattern of individual porcine oocytes matured as intact cumulus oocyte complexes either in vivo or in vitro with or without FSH and LH for 43 h were investigated. The synthesis of a 53 kDa polypeptide ceased 21 h after hCG administration whereas a 44 kDa polypeptide were consistently absent in the protein patterns of nearly all of the in vivo maturing oocytes. Further on, a polypeptide with a relative molecular weight of 46000 persisted throughout maturation. A precipitous increase in the synthesis of two other proteins with relative molecular weights of 38000 and 28000, respectively, was observed at 9 and 21 h after hCG injection. In in vitro matured oocytes with or without FSH and LH the synthesis of the 53 kDa band decreased after a culture period of 9h. Further on, the production of the 44 kDa polypeptide ceased only in oocytes incubated in FSH and LH supplemented media after 21 h of culture. On the other hand, the two proteins of Mr 38000 and 28000 appeared only in most of the protein profiles of oocytes cultured with FSH and LH for 43 h. The production of the 46 kDa polypeptide during a 21 h culture period was significantly affected by the presence of additional granulosa cells in connection with the cumulus oocyte complex. Neither the appearance nor the disappearance of the 5 investigated bands was influenced by the presence or absence of the germinal vesicle after 21 h of culture. It is concluded that at least the addition of FSH and LH to the culture medium is necessary for cumulus oocytes complexes to synthesize a protein pattern closely corresponding to that produced by in vivo matured oocytes.     

Cytochalasin B-induced triploidy in mouse oocytes fertilized in vitro

Mouse eggs and spermatozoa were treated in various ways with 5 or 10 mug cytochalasin B/ml. The fertilization rate in vitro was reduced by treatment with the drug but 80-90% of the eggs fertilized were triploid. Many of the experimental eggs were penetrated by one or more spermatozoa but remained unfertilized (75% compared with 9% in control eggs). It is suggested that cytochalasin B weakens the zona reaction and interferes with fusion of gametes but does not prevent the block to polyspermy.     

Aging-related changes in calcium oscillations in fertilized mouse oocytes

In vivo studies of lymphocyte biology have used intravenous (i.v.) injection as the primary mode of cell transfer, a protocol consistent with the anatomic distribution of most lymphocytes. However, for study of peritoneal cavity B cells, i.v. injection does not correlate with anatomical localization. This report describes the restoration of B-cell function in B lymphocyte-defective X-chromosome-linked immune-defective (XID) mice after intraperitoneal transfer of immunoglobulin heavy chain (Igh)-disparate peritoneal cavity (PerC) cells. In contrast to i.v. transfer, intraperitoneal (i.p.) transfer restored B-cell function in young, but not adult (> 8 weeks), XID mice. When host and donor Igh allotype matched, PerC B-cell engraftment was noted in older recipients; this reconstitution however, was also age-dependent. Migration from the peritoneum to systemic circulation was necessary for serum IgM production as shown by the presence of donor antibody-secreting cells in the host spleen. Host lymphocytes also influenced the success of i.p. transplantation as severe combined immune-deficient mice, regardless of age, exhibited donor serum IgM production. Recipient age, Igh allotype, and immune-deficiency were found to have an impact on the ability of i.p.-transferred PerC B cells to restore B-cell function in XID mice.     

Embryonic lethality and tumorigenesis caused by segmental aneuploidy on mouse chromosome 11

Chromosome engineering in mice enables the construction of models of human chromosomal diseases and provides key reagents for genetic studies. To begin to define functional information for a small portion of chromosome 11, deficiencies, duplications, and inversions were constructed in embryonic stem cells with sizes ranging from 1 Mb to 22 cM. Two deficiencies and three duplications were established in the mouse germline. Mice with a 1-Mb duplication developed corneal hyperplasia and thymic tumors, while two different 3- to 4-cM deficiencies were embryonically lethal in heterozygous mice. A duplication corresponding to one of these two deficiencies was able to rescue its haplolethality.     

Timing of in vivo maturation of equine preovulatory oocytes and competence for in vitro maturation of immature oocytes collected simultaneously

The objects of this study were to monitor the development of the cumulus complex and nuclear maturation in oocytes recovered from preovulatory follicles following treatment to induce ovulation and to investigate the in vitro maturation competence of oocytes recovered from smaller nonpreovulatory follicles of varying size. All follicles > or =5 mm in pony mares were individually punctured at 0, 6, 12, 24 and 35 h after an injection of LH to induce ovulation. The recovery rates of oocytes were 64% from 55 preovulatory follicles, 22% from 32 subordinate follicles and 52% from 227 small follicles. Cumulus expansion of the preovulatory oocytes occurred at 12 h post LH treatment while the metaphase I and II components of nuclear maturation were not completed until 24 and 35 h post LH respectively. For nonpreovulatory follicles, the frequency of atresia and oocyte competence for in vitro nuclear maturation both increased with increasing follicular size.     

Fragmentation of centromeric DNA and prevention of homologous chromosome separation in male mouse meiosis in vivo by the topoisomerase II inhibitor etoposide

The mechanism of action of the topoisomerase II inhibitor etoposide (VP-16) was investigated in male mouse meiosis using the spermatid micronucleus (MN) test and two molecular cytogenetic approaches: (i) fluorescence in situ hybridization (FISH) with a mouse centromere specific minor satellite DNA probe; and (ii) immunolabelling of kinetochore proteins with CREST autoimmune serum. VP-16 caused significant increases in the frequencies of MN at all meiotic stages studied. VP-16 induced MN showed significantly elevated frequencies of centromeric hybridization signals compared to the controls. Similarly, after CREST immunostaining the majority of MN induced by the drug showed kinetochore signals when meiotic S phase and diplotene-diakinesis were treated. This would suggest that most induced MN were due to lagging of whole chromosomes. However, more than 80% of the small MN observed were signal-positive and a large pool of minute MN almost exclusively (92%) contained a kinetochore or centromere-DNA signal. This indicates that VP-16 causes chromosome fragmentation at centromeres. In addition, arrested first division (MI) anaphase figures with stretched bivalent(s) at the spindle equator were observed when diplotene-diakinesis and MI were targeted. Moreover, many small and medium size MN had two centromere or kinetochore signals at opposite sides, suggesting that inhibition of topo II at MI causes lagging of whole bivalents. Together, these results indicate that VP-16 acts by several genotoxic mechanisms at male meiosis: (i) fragmentation of centromeres possibly as a result of inhibition of the DNA strand religation reaction in a topoisomerase II mediated decatenation process of sister centromeres; and (ii) the induction of aneuploidy as a result of failures in separation of homologous chromosome arms possibly due to disturbances of chiasma resolution and decatenation processes during MI. Our results indirectly suggest that topoisomerase II plays an important role in male meiosis and its activity is needed at the metaphase-anaphase transition of both meiotic divisions for proper chromosome disjunction.     

Cyclin synthesis controls the progression of meiotic maturation in mouse oocytes

To study the mechanisms involved in the progression of meiotic maturation in the mouse, we used oocytes from two strains of mice, CBA/Kw and KE, which differ greatly in the rate at which they undergo meiotic maturation. CBA/Kw oocytes extrude the first polar body about 7 hours after breakdown of the germinal vesicle (GVBD), whilst the oocytes from KE mice take approximately 3-4 hours longer. In both strains, the kinetics of spindle formation are comparable. While the kinetics of MAP kinase activity are very similar in both strains (although slightly faster in CBA/Kw), the rise of cdc2 kinase activity is very rapid in CBA/Kw oocytes and slow and diphasic in KE oocytes. When protein synthesis is inhibited, the activity of the cdc2 kinase starts to rise but arrests shortly after GVBD with a slightly higher level in CBA/Kw oocytes, which may correspond to the presence of a larger pool of cyclin B1 in prophase CBA/Kw oocytes. After GVBD, the rate of cyclin B1 synthesis is higher in CBA/Kw than in KE oocytes, whilst the overall level of protein synthesis and the amount of messenger RNA coding for cyclin B1 are identical in oocytes from both strains. The injection of cyclin B1 messenger RNA in KE oocytes increased the H1 kinase activity and sped up first polar body extrusion. Finally, analysis of the rate of maturation in hybrids obtained after fusion of nuclear and cytoplasmic fragments of oocytes from both strains suggests that both the germinal vesicle and the cytoplasm contain factor(s) influencing the length of the first meiotic M phase. These results demonstrate that the rate of cyclin B1 synthesis controls the length of the first meiotic M phase and that a nuclear factor able to speed up cyclin B synthesis is present in CBA/Kw oocytes.     

Exaggerated precocious centromere separation in cells of a human breast cancer line treated with a green tea extract

In a breast cancer cell line, MDA-MB-468, established in our laboratory, an average of 3% of the mitotic cells exhibited a phenomenon known as centromere splaying, which is a characteristic feature of cells of patients with Roberts syndrome. However, centromere splaying in cells of Roberts syndrome patients is limited to i) the centromere region and ii) chromosomes with large amounts of heterochromatin. When the breast cancer cells were treated with an extract of green tea GTE-TP91, up to 45% of the metaphases were observed to exhibit this behavior; and the precocious centromere separation was highly exaggerated, affecting all chromosomes in such metaphases. Apparently, as the sister centromeres continued to pull apart, they carried the chromatids with them, except for the telomere regions, giving a ring-like configuration. Eventually, the sister chromatids became completely separated. Whether this bizarre phenomenon was induced by the polyphenols contained in this green tea extract GTE-TP91 is not known, but this phenomenon, upon further investigation, may throw some light on chromosomal proteins, centromere behavior, telomere behavior and related questions.     

The response of mouse oocytes injected with sea urchin spermatozoa

Entrainment to the 24-hour light-dark cycle is of adaptive significance to mammals. Human infants are no exception, but some postnatal care habits prevalent in developed countries can interfere with the physiological mechanisms underlying circadian synchronization. We describe the physiological mechanisms of entrainment to the light-dark cycle in fetuses and newborns, and some common parental care behaviors which subject the developing circadian system of the newborn to conflicting temporal cues. Improvements in parental care are proposed which may improve the circadian synchronization of newborns, and their parents or caregivers.     

Quantitative evaluation of chronological ageing and photoageing in vivo: studies on skin echogenicity and thickness

Skin ageing is divided into chronological ageing and photoageing due to the cumulative effects of solar ultraviolet radiation. It is, however, difficult to measure the degree of photoageing and chronological ageing in humans in vivo. Here, we have evaluated the usefulness of ultrasonography for measurement of chronological ageing and photoageing in vivo. Twenty megahertz ultrasonography was performed in 90 individuals (29 men, 61 women, age 18-94) to describe age-related changes in sun-exposed regions with different levels of sun exposure (dorsal and ventral forearm, forehead, ankle) and non-exposed buttock skin. Skin thickness and skin echogenicity in different layers of the dermis were measured in ultrasound images. Additionally, cutaneous photodamage was scored clinically. Age-related changes were dependent on body site as well as layer of the dermis. A progressive, age-related decrease in echogenicity of the upper dermis was found in sun-exposed regions (dorsal forearm, forehead), but not in moderately exposed regions (ventral forearm, ankle). In the buttock an increase in echogenicity was observed. The echogenicity of the lower dermis increased in all examined sites. Skin thickness increased with age in the forehead and buttock, but decreased in the extremity skin. Our findings show that photoageing causes a decrease in echogenicity in the upper dermis. In contrast, chronological ageing is associated with an increase in echogenicity in the lower dermis. Although both increases and decreases in skin thickness were observed in different anatomical regions, there was no general relationship between skin thickness and age. Dermal echogenicity was deemed valuable for in vivo study of chronological ageing and photoageing.     

In vivo determination of patello-femoral separation and linear impulse forces

Problems associated with the patellofemoral joint account for nearly half of all total knee arthroplasty (TKA) revisions. Under in vivo conditions, we previously determined that TKA subjects experience patellofemoral separation while performing dynamic, weight-bearing activities. This study investigates the impulse loading conditions that may exist at the time the patella impacts the femur during knee flexion. Fifty-seven subjects (68 knees) performed three successive deep knee bends under fluoroscopic surveillance. Eleven subjects (14 knees) had a posterior cruciate retaining (PCR) TKA, 19 subjects (25 knees) had a posterior cruciate substituting (PS) TKA, 15 subjects (17 knees) had a normal knee, and 12 subjects (12 knees) had an anterior cruciate ligament deficient (ACLD) knee. Velocities of each subjects' patella relative to a fixed point on the tibia were used as input to a mathematical model incorporating the impulse-momentum equation. At full extension, 12 of 14 PCR knees, 11 of 25 PS knees, 1 of 12 ACLD knees, and none of the 17 normal knees exhibited patellofemoral joint separation. The maximum separation, detected in a PCR knee, was 12 mm. The relative force determined upon patellofemoral impact was minimal (1.0 N). Simulated walking conditions for each subject were then entered into the mathematical model at a rate of 100 Hz and the calculated patellofemoral impact forces ranged from 78 N to 213 N. Since impulse loading conditions occur over a very small period of time, it was concluded that capturing fluoroscopy images at a rate of 30 Hz was too slow. Under simulated walking conditions, the impact forces due to impulse loading could contribute to polyethylene failure if these conditions induce fatigue of the polyethylene.     

Human sperm motility-enhancing agents have detrimental effects on mouse oocytes and embryos

OBJECTIVES: To test the artificial activating properties of the human sperm motility-enhancing agents pentoxifylline, caffeine, 2-deoxyadenosine, and cyclic adenosine 3':5' monophosphate (cAMP) on mouse oocytes and determine if the agents exhibit an inhibitory effect on in vitro development of mouse embryos. DESIGN: CD-1 mouse oocytes were exposed to 1, 2.5, 5, or 10 mM pentoxifylline, caffeine, 2-deoxyadenosine, or cAMP for 10, 30, or 60 minutes and their activation and development was scored over 96 hours of culture. A 10% ethanol solution and aging unstimulated oocytes served as controls. Pronuclear embryos from CD-1, CF-1, and B6C3 F1 hybrid mice were cultured in 0.16, 0.33, 0.66, 1.25, 2.5, 5.0, or 10 mM of pentoxifylline, caffeine, 2-deoxyadenosine, or cAMP and development was scored over 96 hours of culture. RESULTS: Exposure to pentoxifylline, caffeine, and 2-deoxyadenosine, but not cAMP, artificially activated mouse oocytes in a concentration- and exposure time-dependent manner. The level of activation was significantly greater than that associated with oocyte aging but less than ethanol-induced activation. Agent-activated oocytes had limited developmental capacity compared with the ethanol-activated oocytes. Pentoxifylline and 2-deoxyadenosine were more toxic than caffeine, especially at the higher concentrations and after prolonged exposure. All of the agents affected embryo development in a dose-dependent manner with developmental inhibition and embryotoxicity that was often not evident until after one to three cell cycles. CONCLUSIONS: Pentoxifylline, caffeine, 2-deoxyadenosine, and cAMP have adverse effects on mouse oocytes or embryos at concentrations commonly used to activate sperm in human IVF. Therefore, care should be taken to minimize the exposure of human oocytes and embryos to these agents until their direct effects have been investigated more fully.