Increased incidence of abnormal foetuses in the offspring of cyclophosphamide-treated male mice

The incidence of morphologically abnormal foetuses in the litters of cyclophosphamide (CP)-treated male mice was investigated and compared with control values. In two experiments (100 mg/kg) CP was shown to increase the incidence of grossly abnormal foetuses over that seen in the controls, although neither was statistically significant in isolation. When the probabilities from the two tests of significance were combined using the method of Fisher the result was significant (P = 0.02). These results suggest that an acute exposure to a mutagen in male mice can cause genetic damage that results in an increased incidence of phenotypically abnormal offspring. However, the large numbers of animals required and the variable control level of abnormalities, indicate that this dosing regimen is an inefficient method of studying the genetic mechanisms responsible for the effects seen.     

Pituitary-testicular axis abnormalities in immature male hypothyroid rats

The pituitary-testicular disturbances which follow the onset of hypothyroidism were studied in immature male Wistar rats rendered hypothyroid by treatment with methimazole (MMI) given in drinking water, starting at 40 days of age. Half of the animals continued on MMI (MMI group) up to 140 days of age; the remaining rats were withdrawn MMI at 100 days and injected thereafter s.c. with 3 micrograms of T3 daily, during the last 40 days (MMI + T3 group). Ten rats were used as controls (C group). Hypothyroidism induced in immature animals significantly decreased serum T4, T3, LH, PRL, and testosterone levels, and also impaired the normal growth of body and sex accessory glands. T3 replacement therapy helped to normalize serum hormonal levels, but the body and sex accessory gland weights were not fully corrected. Hypothyroidism also reduced the [125I]LH/hCG binding sites of testicular homogenates. T3 replacement was not able to improve the binding; nonetheless, the hormone-receptor affinity constant remained unaltered among the groups. Leydig cell responsiveness to hCG stimulation in vitro (0-82 nM) showed impaired testosterone production in the MMI group (25% of that found in the C group) and also in the MMI + T3 group (80% of that found in the C group). These data demonstrate that induction of hypothyroidism in the immature male rat leads to alterations in serum LH, PRL and testosterone levels, and suggest that thyroid hormones have a modulating action on the testis as far as LH-mediated testosterone secretion is concerned.     

An examination of respiratory distress and chromosomal abnormalities in the offspring of male mice treated with ethylnitrosourea

A functional defect (respiratory distress), in addition to morphological defects, was induced in the offspring of male ICR mice treated with ethylnitrosourea (ENU) before mating. ENU (100 and 50 micrograms/g) was injected intraperitoneally into adult male ICR mice that were then mated with untreated females. After the cesarian operation on the 18th day of gestation, fetuses were resuscitated. In the apneic fetuses showing respiratory distress, the lung was collapsed and the ductus arteriosus was not closed. The incidence of fetuses showing respiratory distress was significantly increased with the high dose (100 micrograms/g) of ENU, and it was higher after spermatogonial exposure than after postmeiotic exposure. There was no linearity in the dose-response relationship at the lower dose (50 micrograms/g), as was the case with the specific-locus mutation. The frequency per microgram ENU of fetuses showing respiratory distress was 3.7 X 10(-4) for spermatogonial treatment (calculated at a dose of 100 micrograms/g), the value being about 10-20 times higher than that of ordinary mutations in mice. About half of the fetuses showing respiratory distress often had specific anomalies (dwarfism and gigantic thymus), but the remainder showed no morphological changes. Spermatogonial treatment produced a zero or very low incidence of translocations in the meiotic configurations of primary spermatocytes. G-band analysis of the affected F1 fetuses also revealed no visible chromosomal abnormalities (there could be small deletions or inversions) except that trisomy 19 was found in a dwarf fetus.     

Histological changes in the liver of fetuses of alcohol-treated pregnant rats

In this study, immunolocalization of tenascin (TN), type IV collagen and histopathological changes in the liver of fetuses of pregnant Wistar albino rats treated with two doses of alcohol (1 and 5 g kg(-1) day(-1)) were determined. The samples of liver obtained from fetuses of rats on day 21 of gestation were evaluated morphologically and fixed for histology and immunohistochemistry. The sections stained with H&E and peroxidase-antiperoxidase (PAP) method for TN and type IV collagen were evaluated and immunohistochemical staining results were examined by the intensity of labelling. Statistical evaluation of data showed that there were no differences in liver weights between the control and calorie control groups; but some differences in relative liver weight between the control and alcohol treatment groups were determined. The changes in the liver histology such as degeneration of the cytoplasm of hepatocytes and increases in the number of megakaryocytes were seen in the fetuses of groups receiving alcohol during pregnancy. Increases in the immunolocalization of TN and type IV collagen in the liver of fetuses of alcohol-receiving rats were also found.     

Induction of external abnormalities in offspring of male mice irradiated with 252Cf neutron.

To assess the genetic effects of fission neutron, the induction of external malformations was studied in F1 fetuses after F0 male mice were irradiated. Male mice of the ICR:MCH strain were irradiated with 252Cf neutron at doses of 0.238, 0.475, 0.95 and 1.9 Gy. They were mated with non-irradiated female mice at 71-120 days after the irradiation. Pregnant females were autopsied on day 18 of gestation and their fetuses were examined for deaths and external abnormalities. No increases of pre- and post-implantation losses were noted at any dose. External abnormalities were observed at rates of 1.40% in the 0.238 Gy, 2.23% in the 0.475 Gy, 3.36% in the 0.95 Gy and 3.26% in the 1.9 Gy groups; the rate in the control group was 1.65%. The dose-response curve was linear up to 0.95 Gy, and then flattened out; the induction rate of external abnormalities was 2.7 x 10(-4)/gamete/cGy based on the linear regression. These results indicated that fission neutron effectively induces external abnormalities in F1 fetuses after spermatogonial irradiation.     

Hyperthermia of male Drosophila melanogaster meiocytes induces abnormalities in both paternal and maternal sex chromosome sets of the offspring

Nondisjunction and loss of sex chromosomes caused by exposure of male Drosophila melanogaster to heat shock (HS) (37 degrees C for 1 h) has been studied to determine the role of mutation l(1)ts403 (sbr10) in the control of chromosome segregation during cell division. Hyperthermia of males at the pupal stage has been demonstrated to increase the number of offspring with abnormalities of not only paternal, but also maternal sex chromosome sets. According to the criterion used, there is a temperature-sensitive period of spermatogenesis, which presumably coincides with meiosis. Phenotypes of some individuals correspond to the presence of two sex chromosomes of obtained from the same parent. The frequency of abnormal chromosome sets in the offspring of male carriers of the sbr10 mutation is about two times higher than in the offspring of males without this mutation.     

Post-meiotic cell mediation of behavior in progeny of male rats treated with cyclophosphamide

Transmission of the effects of paternal pre-fertilization exposure to a genotoxic drug being manifested in the F₁ progeny is well established using the traditional rodent dominant lethal protocol [7]. More recently, the induction of genotoxic effects in the F₁ progeny following chronic paternal exposure prior to fertilization has been described in rats using behavioral testing as an endpoint [1]. In our initial study, male F344 adult rats were treated chronically with 10 mg/kg cyclophosphamide (CP) or saline for 5 weeks (5 days of daily treatment and 2 days of rest). The present study describes the behavior of progeny whose fathers were treated with a single intraperitoneal injection of 10 mg/kg CP and mated at different times post-injection. This design showed that post-meiotic germ cells are the most sensitive to the effects of CP as seen by behavioral testing of the F₁ progeny.     

Impaired glucose homeostasis and mitochondrial abnormalities in offspring of rats fed a fat-rich diet in pregnancy

This study examined the role of heating on oxidative stress and muscle mass in immobilized limbs. Rats were divided into three groups (n = 9/group): a control group (Con), an immobilized group (Im), and an immobilized and heated group (ImH). Rats were immobilized in the plantarflexed position for 8 days. The core temperature of the ImH group was elevated to 41-41.5 degrees C on alternating days and maintained for 30 min before cooling. On day 8, both heat shock protein 25 (HSP25) and HSP72 were markedly elevated in the ImH compared with the Im group, whereas results in the Im group were not different from Con. Most notably, the ImH group had significantly larger solei compared with the Im group, which were less than those shown in the Con group. Furthermore, immobilization alone caused a significant increase in oxidative damage, and the addition of heating to immobilization significantly reduced oxidative damage. In an effort to further identify the cause of this protective effect, antioxidant enzyme activities were assessed. CuZnSOD was sharply elevated in Im compared (P < 0.025) with that in the Con and reduced in the ImH group compared with that in the Im group (P < 0.025). Catalase was elevated 8% (P < 0.025) in the Im group compared with the Con group and was similar to the ImH group. Glutathione peroxidase, glutathione reductase, and MnSOD did not differ between groups. These data indicate that heating provides protection against oxidative stress and preserves muscle mass during disuse atrophy. These data also suggest that antioxidant protection is not conferred via antioxidant enzymes, and HSPs may play an important role.     

Malformed offspring,sibling matings,and selection against inbreeding in the sand lizard (Lacerta agilis)

We demonstrated that sand lizards (Lacerta agilis) are more likely to have malformed offspring when they mate with siblings. Offspring with malformations, such as deformed limbs and heads, have zero survival in a natural population. Normal-looking siblings of malformed hatchlings also had a reduced survival in the wild, compared to offspring from clutches in which all siblings appeared normal. The proportion of malformed hatchlings in the natural population was ca. 10%, in spite of differences in juvenile dispersal between males and females. Male juveniles disperse significantly further from their natal sites than do female juveniles.     

Protein malnutrition during fetal programming induces fatty liver in adult male offspring rats

We evaluated the effects of protein malnutrition on liver morphology and physiology in rats subjected to different malnutrition schemes. Pregnant rats were fed with a control diet or a low protein diet (LPD). Male offspring rats received a LPD during gestation, lactation, and until they were 60 days old (MM group), a late LPD that began after weaning (CM), or a LPD administrated only during the gestation-lactation period followed by a control diet (MC). On day 60, blood was collected and the liver was dissected out. We found a decrease in MM rats’ total body (p < 0.001) and liver (p < 0.05) weight. These and CM rats showed obvious liver dysfunction reflected by the increase in serum glutamic pyruvic transaminase (SGOT) (MM p < 0.001) and serum glutamic pyruvic transaminase (SGPT) (MM and CM p < 0.001) enzymes, and liver content of cholesterol (MM and CM p < 0.001) and triglycerides (MM p < 0.01; CM p < 0.001), in addition to what we saw by histology. Liver dysfunction was also shown by the increase in gamma glutamyl transferase (GGT) (MM, MC, and CM p < 0.001) and GST-pi1 (MM and CM p < 0.001, MC p < 0.05) expression levels. MC rats showed the lowest increment in GST-pi1 expression (MC vs. MM; p < 0.001, MC vs. CM; p < 0.01). ROS production (MM, CM, and MC: p < 0.001), lipid peroxidation (MM, CM, and MC p < 0.001), content of carbonyl groups in liver proteins (MM and CM p < 0.001, MC p < 0.01), and total antioxidant capacity (MM, CM, and MC p < 0.001) were increased in the liver of all groups of malnourished animals. However, MM rats showed the highest increment. We found higher TNF-α (MM and CM p < 0.001), and IL-6 (MM and CM p < 0.001) serum levels and TGF-β liver content (MM p < 0.01; CM p < 0.05), in MM and CM groups, while MC rats reverted the values to normal levels. Pro-survival signaling pathways mediated by tyrosine or serine/threonine kinases (pAKT) (MM and CM p < 0.001; MC p < 0.01) and extrasellular signal-regulated kinase (pERKs) (MM p < 0.01; CM p < 0.05) appeared to be activated in the liver of all groups of malnourished rats, suggesting the presence of cells resistant to apoptosis which would become cancerous. In conclusion, a LPD induced liver damage whose magnitude was related to the developmental stage at which malnutrition occurs and to its length.     

孕期应激下调成年雄性子代大鼠海马HDAC2表达

目的明确母代大鼠孕期应激对雄性子代海马中组蛋白去乙酰化酶2(histone deacetylase 2, HDAC2)表达变化的影响。方法将16只SD孕鼠随机分为应激组和对照组,每组8只,应激组在孕16-21天进行限制性应激,对照组不做任何处理。ELISA检测成年子代外周血血浆糖皮质激素,用旷场、高架十字迷宫、新物体识别及巴恩斯迷宫四种行为学评估成年子代认知功能,免疫组织化学、Western blot、RT-PCR 检测成年雄性子代海马HDAC2表达变化。结果母代孕期应激成年子代糖皮质激素升高,旷场与高架十字迷宫中表现出焦虑样行为,新物体识别与Barnes迷宫表现出学习记忆与空间记忆能力下降,海马HDAC2 mRNA和蛋白水平均下调。结论孕期应激对子代成年的认知功能损害可能与子代海马HDAC2的表达下调有关。     

Chromosome abnormalities in spermatozoa of patients with azoospermia and normal somatic karyotype

Since epididymal and testicular spermatozoa of azoospermic patients are frequently used for intracytoplasmic sperm injection (ICSI), many studies have been carried out to evaluate their karyotype. This article will review all published data on this topic. In most of the studies, spermatozoa have been retrieved from the testis or the epididymis of patients with nonobstructive (NOA) or obstructive (OA) azoospermia, respectively. Sperm aneuploidy has been evaluated by fluorescence in situ hybridization using probes for sex chromosomes and an array of autosomes. A significantly higher sperm aneuploidy rate has been reported in patients with NOA and OA compared to ejaculated spermatozoa, mainly for sex chromosomes. The magnitude of the increase varies between studies, probably because of the heterogeneity of case selection as well as of the methodology employed. The majority of the studies reported that patients with NOA have a greater sperm aneuploidy rate compared to OA. The greater frequency of sperm aneuploidy in azoospermic patients increases the risk of transmitting a karyotype abnormality to the offspring generated by ICSI.     

Correlation of glutamate binding activity with glutamate-binding protein immunoreactivity in the brain of control and alcohol-treated rats

Specific antibodies raised against a glutamate binding protein purified from bovine brain were used to trace the immunoreactivity of this protein in rat brain subcellular fractions. In the subcellular fractions obtained from whole brain homogenates, the synaptic membranes had the highest immunochemical reactivity towards the anti-glutamate-binding protein antibodies. The combination of measurements of glutamate binding activity and glutamate-binding protein immunoreactivity indicated that in brain synaptic membranes from control animals the highest activity in these two measures was associated with a synaptic plasma membrane subfraction that was enriched with synaptic junctions. In animals treated with ethanol for 14 days, there was a significant increase in the density of synaptic membrane glutamate binding sites. This increase in glutamate binding capacity was correlated with a greater than two-fold increase in the glutamate binding activity and binding protein immunoreactivity of the light synaptic membrane subfraction, a subfraction which does not contain many recognizable synaptic junctions. Acute administration of ethanol to rats produced a moderate but non-significant decrease in glutamate binding capacity of synaptic membranes. The increase in the number of glutamate binding protein subunits in brain plasma membranes may be an adaptive response of central nervous system neurons to the acute effects of ethanol on glutamate synaptic transmission.     

生命早期PM2.5暴露对子代雄性大鼠前额皮层的影响*

目的: 探究生命早期不同阶段PM_2.5暴露对子代大鼠前额皮层的影响。方法: 将12只受孕后的SD孕鼠按体重随机分为对照组(CG)、母亲孕期暴露组(MG)、出生早期暴露组(EP)和全围产期暴露组(PP),每组3只。进行孕鼠与子鼠的清洁空气或8倍浓缩PM_2.5的暴露,其中CG组全程不暴露,MG组从妊娠第1日(GD1)暴露到GD21,EP组从出生第1日(PND1)暴露到PND21,PP组从GD1一直暴露到PND21。暴露完成后,取各组6只子代大鼠的前额皮层,采用HE染色进行病理学检测;酶联免疫吸附实验(ELISA)进行神经炎性因子检测;高效液相色谱-质谱分析进行神经递质检测;免疫印迹实验(Western blot)进行星形胶质细胞标志物检测;比色法进行脑组织氧化应激检测。结果: 与MG组和CG组子鼠比较,PP组和EP组子鼠前额皮层的病理学变化更加明显。与MG组和CG组子鼠比较,PP组和EP组大鼠的神经炎性因子IL-1、IL-6和TNF-α均显著增加(P＜0.01),且MT水平显著减少(P＜0.05),OT水平呈现下降趋势;神经递质Ach水平也显著增加(P＜0.01)。与MG组和CG组子鼠比较,PP组和EP组子鼠的GFAP水平呈升高趋势。与MG组和CG组子鼠比较,PP组和EP组子鼠的氧化应激指标SOD水平显著减少(P＜0.01),ROS水平显著增加(P＜0.01)。与CG组子鼠比较PP组子鼠的CAT水平显著减少(P＜ 0.01),与MG组子鼠比较PP组子鼠的CAT水平显著减少(P＜0.05);与CG组子鼠比较EP组子鼠的CAT水平显著减少(P＜0.05)。尚未发现PP组子鼠与EP组子鼠之间、MG组子鼠与CG组子鼠之间在IL-1、IL-6、TNF-α、MT、OT、Ach、GFAP、SOD、ROS和CAT水平存在差异。结论: 生命早期PM_2.5暴露可对子代雄性大鼠前额皮层产生不良影响,出生早期暴露可能更为敏感。     

Increased cardiovascular reactivity to acute stress and salt-loading in adult male offspring of fat fed non-obese rats

Diet-induced obesity in rat pregnancy has been shown previously to be associated with consistently raised blood pressure in the offspring, attributed to sympathetic over-activation, but the relative contributions to this phenotype of maternal obesity versus raised dietary fat is unknown. Sprague-Dawley female rats were fed either a control (4.3% fat, n = 11) or lard-enriched (23.6% fat, n = 16) chow 10 days prior to mating, throughout pregnancy and lactation. In conscious adult (9-month-old) offspring cardiovascular parameters were measured (radiotelemetry). The short period of fat-feeding did not increase maternal weight versus controls and the baseline blood pressure was similar in offspring of fat fed dams (OF) and controls (OC). However, adult male OF showed heightened cardiovascular reactivity to acute restraint stress (p<0.01; Δ systolic blood pressure (SBP) and Δheart rate (HR)) with a prolonged recovery time compared to male OC. α1/β-adrenergic receptor blockade normalised the response. Also, after dietary salt-loading (8%-NaCl ad libitum for 1 week) male OF demonstrated higher SBP (p<0.05) in the awake phase (night-time) and increased low/high frequency ratio of power spectral density of HR variability versus OC. Baroreflex gain and basal power spectral density components of the heart rate or blood pressure were similar in male OF and OC. Minor abnormalities were evident in female OF. Fat feeding in the absence of maternal obesity in pregnant rats leads to altered sympathetic control of cardiovascular function in adult male offspring, and hypertension in response to stressor stimuli.     

Postparturitional testosterone surge in male offspring of rats stressed and/or fed ethanol during late pregnancy

Male offspring of rats exposed to restraint stress and/or alcohol during late pregnancy show aberrant patterns of sexual behavior masculinization and defeminization that vary as a function of treatment. The impact of these treatments on the postparturitional testosterone (T) surge that contributes to sexual behavior differentiation was investigated. Plasma T was measured using radioimmunoassay in individual males sampled on day 21 of gestation within 10 min of cesarean delivery or 1, 2, or 4 h thereafter. Neonatal T in the group exposed only to stress did not differ from that in the control group. T was lower than control levels at birth in both alcohol groups. The magnitude of the T surge that occurred during the first hour of birth in the control group was diminished by 50% in both alcohol groups, whose T pattern was very similar. There was no common alteration in postparturitional T associated with the increased lordotic behavior potential that males in all three treatment groups typically share, nor were there idiosyncratic endocrine abnormalities linked to the very different male copulatory pattern each exhibits. Exposure to an abnormal T milieu during fetal as well as neonatal ontogeny may underlie the etiology of the different sexual behavior patterns exhibited by males exposed to stress and/or alcohol. Possible unique effects each treatment exerts on perinatal plasma T and it's aromatization to estradiol in hypothalamic targets are discussed.