Dose-response relationship for the induction of structural chromosome aberrations in human spermatozoa after in vitro exposure to tritium beta-rays

The effects of tritium (HTO) beta-rays on human sperm chromosomes were studied using our interspecific in vitro fertilization system between human spermatozoa and zona-free hamster oocytes. Semen samples were treated with media containing 1.53-24.3 mCi/ml HTO for about 80 min. 1290 spermatozoa from the controls and 1842 spermatozoa from the irradiated groups were karyotyped. The incidence of spermatozoa with structural chromosome aberrations increased linearly with increasing dosage. Breakage-type aberrations occurred far more frequently than exchange-type. Chromosome-type aberrations appeared far more frequently than chromatid-type. All of these types of aberrations showed linear dose-dependent increases. The RBE values of HTO beta-rays relative to X-rays were calculated for the above-mentioned 5 indices, respectively. Their RBE values ranged from 1.89 to 3.00 when the absorbed dose was estimated to be the minimum, whereas the values ranged between 1.04 and 1.65 when the absorbed dose was estimated to be the maximum.     

Effect of lifetime intake of organically bound tritium and tritiated water on the oocytes of rats

Summary Rats were continuously exposed to constant activity of tritium in drinking water (HTO group) or to tritium organically bound in food (T-food group) in the period from conception ofF ₁ generation through maturity. Female offspring were killed at the age of 21 and 71 days and the oocytes in their ovaries were counted. Mean dose rates absorbed in the ovaries were for the HTO groups 7.25 ± 0.37 and 14.73 ± 0.79 mGy/day and for the T-food group 4.84 ± 0.25 mGy/day.Reduction in the oocyte number in the ovaries of females exposed to tritiated food was bigger than in the ovaries of females exposed to tritiated water. The dependence of the survival of small oocytes on the dose rate and the corresponding total accumulated dose had an exponential character. The damaging effect of tritium was for the period from conception to 21 days of age bigger than from 21 to 71 days of age. Of all stages of oocyte development, the highest sensitivity to tritium irradiation was observed in small oocytes and oocytes with one complete layer of follicle cells. As a result, relative number of the growing and large oocytes increased.     

Preventive effect of Ninjin-to (Ren-Shen-Tang), a Kampo (Japanese traditional) formulation, on spontaneous autoimmune diabetes in non-obese diabetic (NOD) mice

We previously found that ingestion of an extract of Ninjin-to (NJT; Ren-Shen-Tang) suppressed the development of autoimmune diabetes in C57BL/KsJ mice induced by multiple low doses of streptozotocin. To verify this effects on spontaneous autoimmune diabetes, the effects of NJT on NOD mice were investigated in the present study. NJT, provided in drinking water (0.25%, 450 mg/kg/day) from 6 weeks of age, significantly prevented the incidence of spontaneous diabetes in female NOD mice at 30 weeks of age (2/10) compared with that of the controls (7/10), with no effects on body growth or food intake. Even in non-diabetic mice, the blood glucose levels of the NOD controls gradually increased with age, while such increase in NJT-treated mice was significantly suppressed by preventing any deficiency of glucose tolerance. NJT also significantly suppressed the progression of insulitis, which causes insulin deficiency and diabetes. It is well known that NOD mice develop insulitis and diabetes because of their Th1-dominant autoimmune response. IFN-gamma production from splenic T lymphocytes stimulated with anti-CD3 monoclonal antibodies was increased, whereas IL-4 production was decreased in NOD controls compared to age- and sex-matched normal ICR mice. NJT-treatment reduced these deviations of cytokine production in NOD mice. These data all suggest that NJT can prevent spontaneous insulitis and diabetes by the modification of deviated cytokine production in NOD mice.     

Chromosomal aberrations induced by (12)C6+ ions and 60Co gamma-rays in mouse immature oocytes

The ovaries of Kun-Ming strain mice (3 weeks) were irradiated with different doses of (12)C6+ ion or (60)Co gamma-ray. Chromosomal aberrations were analyzed in metaphase II oocytes at 7 weeks after irradiation. The relative biological effectiveness (RBE) of (12)C6+ ion was calculated with respect to 60Co gamma-ray for the induction of chromosomal aberrations. The (12)C6+ ion and 60Co gamma-ray dose-response relationships for chromosomal aberrations were plotted by linear quadratic models. The data showed that there was a dose-related increase in frequency of chromosomal aberrations in all the treated groups compared to controls. The RBE values for (12)C6+ ions relative to 60Co gamma-rays were 2.49, 2.29, 1.57, 1.42 or 1.32 for the doses of 0.5, 1.0, 2.0, 4.0 or 6.0 Gy, respectively. Moreover, a different distribution of the various types of aberrations has been found for (12)C6+ ion and 60Co gamma-ray irradiations. The dose-response relationships for (12)C6+ ion and 60Co gamma-ray exhibited positive correlations. The results from the present study may be helpful for assessing genetic damage following exposure of immature oocytes to ionizing radiation.     

Chromosome aberrations induced by tritiated water or 60Co gamma-rays at early pronuclear stage in mouse eggs

The induction of chromosome aberrations in mouse eggs by exposure to HTO beta-particles and 60Co gamma-rays at the early pronuclear stage was examined at the first-cleavage metaphase by using an in vitro fertilization technique. Eggs at the pronuclear stage were exposed to beta-particles in a chemically defined medium containing tritiated water (HTO) for 2 h at 3-5 h after insemination. Other eggs at the same stage were exposed to gamma-rays from 60Co during the same period. The dose-response relationships for frequencies of chromosome aberrations per egg were fitted to a linear-quadratic model for HTO beta-particles, and to a linear model for 60Co gamma-rays. The chromosome aberrations were mainly chromosome-type, and the majority of all aberrations were fragments. RBE values of HTO beta-particles relative to 60Co gamma-rays and acute X-rays, which were estimated from the ratio of the linear regression coefficients over 0.05-Gy range, were 2.0 and 1.6, respectively.     

Types of structural chromosome aberrations and their incidences in human spermatozoa X-irradiated in vitro

We studied the effects of in vitro X-irradiation on human sperm chromosomes, using our interspecific in vitro fertilization system between human spermatozoa and zona-free hamster oocytes. 28 semen samples from 5 healthy men were exposed to 0.23, 0.45, 0.91 and 1.82 Gy of X-rays. Totals of 2098 and 2862 spermatozoa were karyotyped in the control and the irradiated groups, respectively. The incidence of spermatozoa with X-ray-induced structural chromosome aberrations (Y) increased linearly with increasing dosage (D), being best expressed by the equation, Y = 0.08 + 34.52 D. The incidence of breakage-type aberrations was more than 9 times higher than that of exchange-type aberrations. Both of them showed linear dose-dependent increases, which were expressed by the regression lines, Y = -0.014 + 0.478 D and Y = -0.010 + 0.057 D, respectively. The incidence of chromosome-type aberrations was about 6 times higher than that of chromatid-type aberrations. Their dose-dependent increases were expressed by the regression lines, Y = -0.015 + 0.462 D and Y = -0.006 + 0.079 D, respectively. These results are discussed in relation to the previous data obtained with gamma-rays. The repair mechanism of X-ray-induced sperm DNA lesions is also discussed.     

Frequent occurrence of polyovular follicles in ovaries of mice exposed neonatally to diethylstilbestrol

The occurrence of polyovular follicles (PF) was examined at 10-34 days of age in the ovaries of BALB/cCrgl female mice given five daily injections of 0.1 microgram diethylstilbestrol (DES), 2 micrograms DES, 100 micrograms progesterone (P), 137 micrograms 17 alpha-hydroxyprogesterone caproate (HPC), 20 micrograms testosterone (T), 20 micrograms 5 alpha-dihydrotestosterone (5 alpha-DHT), or oil vehicle alone starting on the day of birth, and of C57BL/Tw females given five neonatal injections of 1 microgram DES, 20 micrograms 17 beta-estradiol (E2), 50 micrograms 5 alpha-DHT, 50 micrograms 5 beta-DHT, or the vehicle alone. Ovaries of 30-day-old C57BL mice given five daily injections of 1 microgram DES starting at 3-25 days of age were also examined. PF incidence (% of PF per ovary) and PF frequency (% of mice with PF) were significantly greater in BALB/c mice receiving injections of DES, P, HPC, and T than in the controls. In DES-treated mice at 34 days, PF incidence (2-13 oocytes/follicle) was 120-340 times higher than in the controls. BALB/c mice treated with T, P, and HPC showed PF incidence (two to four oocytes/follicle) three- to six-fold higher than in the controls. In 30-day-old C57BL mice treated with T, E2, and DES, PF incidence also increased by two- to 50-fold. 5 alpha-DHT and 5 beta-DHT failed to increase PF incidence. PF incidence was significantly increased only when neonatal DES treatment was begun on days 0 to 3, but was reduced when started at days 10-25.(ABSTRACT TRUNCATED AT 250 WORDS)     

关节镜手术联合胫骨高位截骨治疗内侧膝关节骨关节炎的临床研究

目的:观察关节镜手术联合胫骨高位截骨(HTO)治疗内侧膝关节骨关节炎(KOA)的临床疗效。方法:本研究为回顾性研究,将2018年2月~2020年9月间在我院接受治疗的内侧KOA患者63根据手术方式的不同分为A组和B组,分别为30例和33例。A组进行HTO手术,B组进行关节镜手术联合HTO治疗。术前、术后6周、术后12周采用美国纽约特种外科医院(HSS)评分、视觉模拟量表(VAS)评分评价两组患者膝关节功能、疼痛情况。采用36项简明健康状况调查表(SF-36)评价两组患者术前与术后12周的生活质量变化情况。记录两组术后并发症发生情况。术前、术后12周采用MB-Ruler软件测量两组患者机械胫骨近端内侧角(mMPTA)、解剖股胫角(aFTA)。结果:术后6周、术后12周,B组HSS评分高于A组,VAS评分低于A组(P<0.05)。术后12周,B组SF-36量表各维度评分高于A组(P<0.05)。术后3周,B组mMPTA、aFTA小于A组(P<0.05)。两组术后并发症发生率组间对比无差异(P>0.05)。结论:相对于单纯的HTO手术,关节镜手术联合HTO治疗内侧KOA患者,可有效促进膝关节功能改善,减轻疼痛症状,调整下肢力线,近期疗效肯定。     

Effect of hypophysectomy on mouse oocyte maturation in vitro.

There was no difference in frequency of maturation of oocytes obtained from mice hypophysectomized for 2 weeks compared to those from sham-operated or untreated (control) animals of the same age. By 7 weeks, and also at 12 and 17 weeks, the incidence of polar body formation in vitro was significantly reduced. The number of oocytes which remained meiotically inactive in culture was increased at 7, 12 and 17 weeks after hypophysectomy. This decrease in spontaneous oocyte maturation in vitro could be partly overcome by administering exogenous PMSG, oestradiol-17beta or PMSG + oestradiol-17beta, but not progesteron or hCG, to hypophysectomized mice.     

The influence of maternal age on radiation-induced chromosome aberrations in mouse oocytes

The relative sensitivities of dictyate oocytes from young and old female mice to radiation-induced chromosome damage were examined in 2 separate experiments. Firstly, females were given either 2 or 4 Gy of X-rays and metaphase I stage oocytes collected 16.5 days later. Analysis of these cells showed dose-related increases in chromosome aberrations in both age groups. The response was significantly greater in oocytes of older females. In the second experiment, females were given 4 Gy of X-rays and metaphase I stage oocytes collected 3.5 days later. Again, a significantly larger frequency of aberrations was present in cells from older animals. Overall, these 2 experiments provide unambiguous evidence that the radiosensitivity of mouse dictyate oocytes increases with advancing maternal age.     

Maternal ageing and nondisjunction: A comparative study of two chromosomal techniques on the formation of univalents in first meiotic metaphase oocytes of the mouse

The incidence of univalents was compared between slides prepared according to two clearly different chromosomal methods, i.e. Tarkowski's method and ours, in order to examine whether a univalent pair could be formed artifactually at the first meiotic metaphase (MI). The oocytes used were obtained from young (2–3 months) and old (12–15 months) age groups of both C57BL/6 and dd mice. In Tarkowski's method only a single fixative was used, while in our method three different fixatives were used successively in order to fix oocytes without their being ruptured. Despiralized, fuzzy and loosely associated chromatids were seen frequently in the slides prepared by Tarkowski's method, while such features were seen less frequently in the slides prepared by our method. The incidence of oocytes with univalents in the slides made by Tarkowski's method was much higher than in those made by ours in both age and strain groups (P<0.05–0.001). Thus, it was confirmed that the so-called univalents could be produced artifactually. The results did not support the production line hypothesis of Henderson and Edwards (1968) which was based on their observation of an increased incidence of univalents in MI oocytes from aged female mice.     

Relative biological effectiveness and dose rate effect of tritiated water on chromosomes in human lymphocytes and bone marrow cells

Tritriated water (HTO) is a major toxic effluent from the nuclear power industry, that is released into the environment in large quantities. The low dose radiation effect and dose rate effect of HTO on human lymphocytes and bone marrow cells have not been well studied. The present study was therefore undertaken to investigate the HTO dose-response relationship for chromosomal aberrations in human lymphocytes and bone marrow cells at low in vitro radiation doses ranging from 0.1 to 1 Gy. Lymphocytes (G₀ stage) and bone marrow cells were incubated for 10–150 min with HTO at a dose rate of 2cGy/min (555 MBq/ml). The relative biological effectiveness (RBE) of HTO was calculated with respect to ₆₀Co γ-rays for the induction of dicentric and centric ring chromosomes at low radiation doses. The RBE value for HTO β-rays relative to ⁶⁰Co γ-rays was 2.7 for lymphocytes and 3.1 for chromatid aberrations in bone marrow cells. Lymphocytes were also chronically exposed to HTO for 6.7–80 h at dose rates of 0.5 cGy/min (138.5 MBq/ml) and 0.02 cGy/min (5.6 MBq/ml). There was a 71.5% decrease in the yield of dicentrics and centric rings at the dose rate of 0.02 cGy/min, indicating a clear dose rate effect of HTO. The RBE value for HTO relative to ¹³⁷Cs γ-rays was 2.0 at a dose rate of 0.02 cGy/min, suggesting that low HTO dose rates produce no increase of the RBE values and that the values may be constant between 2 and 3 within these dose rates. These results should prove useful in assessment of the health risk for humans exposed to low levels of HTO.     

Aneuploidy determination in C-banded mouse metaphase II oocytes following cyclophosphamide treatment in vivo

The ability of cyclophosphamide (CYC) to induce aneuploidy in metaphase II oocytes of mice was evaluated. Various dosages (50, 100, 150, 250 and 350 mg/kg body weight) of CYC were injected intraperitoneally prior to induced ovulation. Chromosomes were C-banded to allow a more accurate counting of chromosomes and chromatids, and analysis of structural aberrations. Structural aberrations were not observed. Although the incidence of aneuploid oocytes in the treated groups was greater than that in the control group, the differences were not statistically significant. This result was unlike those of other relevant reports and several plausible explanations for such a disparity are discussed.     

Cell death (apoptosis) in mouse intestine after continuous irradiation with gamma rays and with beta rays from tritiated water

Apoptosis is a pattern of cell death involving nuclear pycnosis, cytoplasmic condensation, and karyorrhexis. Apoptosis induced by continuous irradiation with gamma rays (externally given by a 137Cs source) or with beta rays (from tritiated water injected ip) was quantified in the crypts of two portions of mouse bowel, the small intestine and descending colon. The time-course change in the incidence of apoptosis after each type of radiation could be explained on the basis of the innate circadian rhythm of the cells susceptible to apoptotic death and of the excretion of tritiated water (HTO) from the body. For 6-h continuous gamma irradiation at various dose rates (0.6-480 mGy/h) and for 6 h after injection of HTO of various radioactivities (0.15-150 GBq per kg body wt), the relationships between dose and incidence of apoptosis were obtained. Survival curves were then constructed from the curves for dose vs incidence of apoptosis. For the calculation of the absorbed dose from HTO, the water content both of the mouse body and of the cells was assumed to be 70%. One megabecquerel of HTO per mouse (i.e., 40 MBq/kg body wt) gave a dose rate of 0.131 mGy/h. The mean lethal doses (D0) were calculated for gamma rays and HTO, and relative biological effectiveness values of HTO relative to gamma rays were obtained. The D0 values for continuous irradiation with gamma rays were 210 mGy for small intestine and 380 mGy for descending colon, and the respective values for HTO were 130 and 280 mGy, indicating the high radiosensitivity of target cells for apoptotic death. The relative biological effectiveness of HTO relative to 137Cs gamma rays for cell killing in both the small intestine and the descending colon in the mouse was 1.4-2.1.     

The CBA mouse as a model for age-related aneuploidy in man: studies of oocyte maturation,spindle formation and chromosome alignment during meiosis

To elucidate the possible mechanism of disturbances in chromosome segregation leading to the increase in aneuploidy in oocytes of aged females we examined the meiotic spindles of CBA/Ca mice. Employing immunofluorescence with an anti-tubulin antibody, and human scleroderma serum, as well as 4-6-diamidino-2-phenylindole (DAPI) staining of chromosomes the microtubular cytoskeleton could be visualized, and the behaviour of chromosomes and centromeres of oocytes spontaneously maturing in vitro could be studied. The morphology of spindles during the first meiotic division was not conspiciously different in oocytes from young and aged mice as far as the cytoskeletal elements were concerned. Neither multipolar spindles nor pronounced cytoplasmic asters appeared in oocytes of mice approaching the end of their reproductive life (9 months and older). Oocytes of aged females also did not exhibit any sign of premature separation of parental chromosomes at prophase, obvious malorientations of bivalents, or significant lagging of chromosomes during ana and telophase. Metaphase I with all bivalents aligned at the spindle equator appeared to be a relatively brief stage in oocyte development compared with pro-and prometaphase. Therefore, already slight disturbances occuring in the timing of the developmental programme which leads to a premature anaphase transition may be responsible for the high incidence of chromosomally unbalanced gametes in aged females, rather than non-separation and lagging of chromosomes during late ana-and telophase. In a second set of experiments we compared the metaphase II spindles of spontaneously ovulated oocytes obtained from animals at different ages. Previous studies have shown that spindle length and chromosome alignment may be altered in cells predisposed to aneuploidy. To distinguish between the significance of the chronological age of the female and the physiological age of the ovaries (as indicated by the total number of oocytes remaining) we examined the spindle apparatus in young (3–4 months old) and aged (9 months and older) mice as well as CBA females which had been unilaterally ovariectomized (uni-ovx) early in adult life and were approaching the end of their reproductive life at 6–7 months of age. Measurements of the pole-to-pole distance implied that spindle length may be related to maternal age. In oocytes of aged (9 month), uni-ovx (6 month) as well as 6-month-old sham-operated controls the metaphase II spindle was significantly shorter than in oocytes of young mice. By contrast, chromosome disorder and displacement was most pronounced in the aged and uni-ovx mice whilst most oocytes from young mice and moderately aged shamtreated controls exhibited a more regular alignment of chromosomes. These results, which are consistent with recent findings in CBA mice of an increased rate of aneuploidy in females approaching the end of their reproductive life, are discussed with respect to the hypothesis that the aetiology of aneuploidy rests on the critical timing of different events in oocyte development.     

Disturbances of nuclear maturation in BCB positive oocytes collected from peri-pubertal gilts

The developmental competence (quality) of oocytes is affected by several factors linked to their intrinsic properties and also to growth and maturation environment. Donor puberty and chromosomal complement are one of the main factors influencing oocyte quality. A high rate of porcine oocytes matured in vitro is chromosomally imbalanced. Moreover, there is no published data on chromosomal aberrations in oocytes selected by the brilliant cresyl blue (BCB) test. Therefore, the aim of this study was to analyze whether BCB positive (BCB+) oocytes derived from ovaries of peripubertal gilts (prepubertal NCL and cyclic CL) differ with respect to the incidence of numerical chromosome aberrations. COCs collected from NCL and CL ovaries were selected by the BCB test. Only BCB+ oocytes were matured in vitro and subjected to FISH analysis using molecular probes for chromosome pairs 1 and 10. The rate of BCB+ oocytes was similar for both groups of ovaries (NCL 80%, CL 92%). Altogether 554 oocytes were fixed and 471 oocytes at the MII stage were analyzed cytogenetically. Diploid (2MII) and aneuploid oocytes were detected. The contribution of MII oocytes was similar for NCL (85%) and CL (90%) group. Chromosomally aberrant BCB+ oocytes accounted for 18.0% and ranged from 13.7% for CL and 22.2% for NCL ovaries. Diploidy was a predominant anomaly observed (11.2%) with a significantly higher frequency in BCB+ oocytes of pre-pubertal (16.7%) than cyclic gilts (5.6%, P < 0.05). Aneuploid oocytes occurred with similar rate in NCL (6.7%) and CL (8.5%) females. The majority of aneuploid spreads (72.2%; P < 0.01) concerned the chromosome pair 10. The overall rate of disomy (56%) and nullisomy (44.4%) was similar. We have shown that donor puberty affects the incidence of chromosomal abnormalities in porcine oocytes matured in vitro. Significantly more diploid oocytes was derived from prepubertal ovaries, whereas the frequency of aneuploidy was similar in NCL and CL gilts.     

Cytogenetic studies in mouse oocytes irradiated in vitro at different stages of maturation, by use of an early preantral follicle culture system

In vivo studies on X-irradiated mice have shown that structural chromosome aberrations can be induced in female germ cells and that the radiation-induced chromosomal damage strongly depends on the stage of maturation reached by the oocytes at the time of irradiation. In the present study, the sensitivity of oocytes to induction of chromosome damage by radiation was evaluated at two different stages, by use of a recently developed method of in vitro culture covering a crucial period of follicle/oocyte growth and maturation. A key feature of this system is that growth and development of all follicles is perfectly synchronized, due to the selection of a narrow class of follicles in the start-off culture. This allows irradiation of well-characterized and homogenous populations of follicles, in contrast to the situation prevailing in vivo. Follicles were X-irradiated with either 2 or 4 Gy, on day 0 of culture (early preantral follicles with one to two cell layers) or on day 12, 3h after hormonal stimulation of ovulation (antral Graafian follicles). Ovulated oocytes, blocked in metaphase I (MI) by colchicine, were fixed 16 h after hormonal stimulation and analyzed for chromosome aberrations. The results confirm the high radiosensitivity of oocytes at 2 weeks prior to ovulation and the even higher radiosensitivity of those irradiated a few hours before ovulation, underlining the suitability of the in vitro system for further studies on the genetic effects of ionising radiation in female mammals.     

The influence of mating status and age on the induction of chromosome aberrations and dominant lethals in irradiated female mice

Young and old hybrid female mice were given 0.5 Gy or 2 Gy acute x-irradiation, followed by (i) in utero examination for dominant lethal mutations, or (ii) examination of metaphase I oocytes for chromosome aberrations 2-3 weeks after the irradiation. Some of the old females had been mated when young to males of a specific locus stock. Others were left unmated until after the irradiation when they, and the young females, were mated to the same specific locus stock and allowed to have 1 (if given 2 Gy) or 2 (if given 0.5 Gy) litters before the dominant lethal test. In both the 0.5-Gy and 2-Gy series, mean sizes of first litters in the old late-mated group were markedly lower than in the old early-mated or young groups, the differences being significant at the 2-Gy level. The intrauterine examinations showed that this difference was largely the result of a reduced ovulation rate in the old late-mated females. Preimplantation loss tended to be higher in all the old females than in the young ones, but differences between the groups in postimplantation lethality were less pronounced. In the chromosome studies, only about half as many oocytes were recovered from the ovaries of old females than from young ones. At both the 0.5-Gy and 2-Gy dose levels interchange frequencies were non-significantly higher in old than in young females (with no clear-cut effect of mating status), while the overall frequency of aberrations (interchanges + fragments) was significantly higher in oocytes of old than young females after 2 Gy X-rays (35.5% against 12.5%). No specific locus mutations were found in 5616 offspring of unirradiated females.     

Evaluation of genotoxicity of clofazimine, an antileprosy drug, in mice in vivo. I. Chromosome analysis in bone marrow and spermatocytes

Clofazimine, an antileprosy drug, was tested for its cytogenetic effect in mouse bone marrow and testis. Bone marrow metaphase analysis in adults treated directly for different periods (1, 2 and 4 weeks, 40 mg/kg/day) and with different doses (4, 20 and 40 mg/kg/day for 7 days) as well as in young animals exposed through lactation for different periods (2, 3, and 4 weeks) revealed significant increases in chromosomal aberrations over the controls. Analysis of diakinesis-metaphase I stages also exhibited a significantly elevated incidence of chromosome aberrations over controls after treatment for different periods. On the basis of the present result the drug may be considered a potential clastogen in mice.