Human Chromosomes: I. Preparation,Analysis and Diagnostic Implications of Abnormalities

This presentation is designed to help persons unfamiliar with cytogenetic terms and techniques to understand how chromosomes are prepared and analyzed to determine if abnormalities exist. A diagrammatic flow-sheet has been used to show how peripheral blood is treated to stimulate growth of cells, and how chromosomes are dispersed and made readily visible. Technical details have been replaced by simple statements explaining the various steps. By careful attention to differences (over-all length, position of centromere, length of arms, presence or absence of satellites), chromosomes may be arranged in order of length and into definite groups to form a typical karyotype of a person''s cells. Familiarity with chromosomes makes it possible to determine (a) if an extra chromosome is present, (b) if a chromosome is missing, or (c) if an abnormal chromosome exists.     

Roles of FliK and FlhB in determination of flagellar hook length in Salmonella typhimurium.

The length of flagellar hooks isolated from wild-type and mutant cells with various hook lengths were measured on electron micrographs. The length of the wild-type hook showed a narrow distribution with a peak (+/- standard deviation) at 55.0 +/- 5.9 nm, whereas fliK mutants (so-called polyhook mutants) showed a broad distribution of hook lengths ranging from 40 to 900 nm, strongly indicating that FliK is involved in hook length determination. Among pseudorevertants isolated from such polyhook mutants, fliK intragenic suppressors gave rise to polyhook filaments. However, intergenic suppressors mapping to flhB also gave rise to hooks of abnormal length, albeit they were much shorter than polyhooks. Furthermore, double mutations of flhB and flgK (the structural gene for hook-associated protein 1; HAP1) resulted in polyhooks, suggesting another way in which hook length can be affected. The roles of FliK, FlhB, and HAP1 in hook length determination are discussed.     

Primary cilium alterations and expression changes of Patched1 proteins in niemann‐pick type C disease

Niemann‐Pick type C disease (NPC) is a disorder characterized by abnormal intracellular accumulation of unesterified cholesterol and glycolipids. Two distinct disease‐causing genes have been isolated, NPC1 and NPC2. The NPC1 protein is involved in the sorting and recycling of cholesterol and glycosphingolipids in the late endosomal/lysosomal system. It has extensive homology with the Patched1 (Ptc1) receptor, a transmembrane protein localized in the primary cilium, and involved in the Hedgehog signaling (Shh) pathway. We assessed the presence of NPC1 and Ptc1 proteins and evaluated the relative distribution and morphology of primary cilia in fibroblasts from five NPC1 patients and controls, and in normal fibroblasts treated with 3‐ß‐[2‐(diethylamino)ethoxy]androst‐5‐en‐17‐one (U18666A), a cholesterol transport‐inhibiting drug that is widely used to mimic NPC. Immunofluorescence and western blot analyses showed a significant decrease in expression of NPC1 and Ptc1 in NPC1 fibroblasts, while they were normally expressed in U18666A‐treated fibroblasts. Moreover, fibroblasts from NPC1 patients and U18666A‐treated cells showed a lower percentage distribution of primary cilia and a significant reduction in median cilia length with respect to controls. These are the first results demonstrating altered cytoplasmic expression of Ptc1 and reduced number and length of primary cilia, where Ptc1 is located, in fibroblasts from NPC1 patients. We suggest that the alterations in Ptc1 expression in cells from NPC1 patients are closely related to NPC1 expression deficit, while the primary cilia alterations observed in NPC1 and U18666A‐treated fibroblasts may represent a secondary event derived from a defective metabolic pathway.     

An anomalous specimen of Rhamdia guatemalensis (Günther, 1864)

In March of 1984 an immature Rhamida guatemalensis (Günther, 1864) lacking the right pectoral fin (Fig. 1) was captured in the Sangregado sector, Arenal Dam, Guanacaste, Costa Rica (10°28' N, 84°46'W) The body colouration and morphometric characteristics were as described for this species (Bussing 1966; Zúñga, 1980). X-ray examination revealed the absence of the radial bones and the fin rays of the right peoctoral fin. External examination of the tegument of this region showed no scars or evidence of accidental loss of the appendage, indicating that the fin had never developed.
Morphometric data from this abnormal specimen are: total length, 14.25 cm, standard length, 11.98cm; maximum height of body, 22.35 cm; maximum width of body, 2.40 cm, caudal peduncle width, 1.27 cm; head length, 3.00cm.     

秋水仙素浓度和处理时间对茉莉腋芽生长发育的影响

为了解秋水仙素处理对茉莉﹝Jasminum sambac (Linn.) Aiton〕腋芽生长发育的影响,以长度为0(未萌发)、3~5和8~10 mm腋芽为实验材料,对500、1000和2000 mg·L-1秋水仙素溶液分别处理24和48 h后腋芽的生长状况以及萌发枝条和叶片的生长和发育状况进行分析,同时,对各处理组花粉母细胞的减数分裂行为进行观察;在此基础上,对秋水仙素处理后茉莉腋芽及萌发枝条的各生物学效应指标进行相关性分析。结果显示：经秋水仙素处理后,各处理组腋芽的生长率和处理指数,以及萌发枝条的长度、最大节间长、最大叶长、最大叶宽、现蕾数、现蕾率、最大蕾长、最大蕾宽和开花率总体上显著(P<0.05)低于对照组(用清水处理未萌发腋芽48 h),而腋芽的抑制率和死亡率显著高于对照组。总体上看,随秋水仙素质量浓度提高,腋芽的生长率和处理指数降低,而抑制率和死亡率升高,萌发枝条的长度和最大节间长缩短,开花率升高,其他指标呈波动变化;随处理时间延长,腋芽的生长率、死亡率和处理指数降低,但抑制率升高,萌发枝条仅现蕾数和开花率降低,其他指标均逐渐提高;随腋芽长度增加,腋芽的抑制率和处理指数降低,但死亡率升高,萌发枝条的现蕾率、现蕾数、最大蕾长和开花率均呈波动变化,其他指标均降低。经秋水仙素处理后,茉莉花粉母细胞在减数分裂过程中存在落后染色体、染色体桥和微核等异常现象,且随秋水仙素质量浓度和腋芽长度增加及处理时间延长,减数分裂后期的细胞异常率逐渐升高。相关性分析结果显示：细胞异常率与腋芽生长率呈显著负相关,与腋芽死亡率呈极显著正相关;腋芽生长率与腋芽抑制率和开花率分别呈显著和极显著(P<0.01)负相关;腋芽抑制率与现蕾率呈显著正相关;处理指数与细胞异常率呈极显著负相关。研究结果表明：秋水仙素浓度和处理时间对茉莉腋芽的生长发育有一定影响;综合考虑各生物学效应指标,茉莉腋芽适宜的诱导条件为用500 mg·L-1秋水仙素溶液处理3~5 mm腋芽24 h。此外,建议将处理指数作为秋水仙素对茉莉腋芽细胞减数分裂影响效应的评价指标之一。     

Enhancement of germination, growth, and photosynthesis in soybean by pre-treatment of seeds with magnetic field

Experiments were conducted to study the effect of static magnetic fields on the seeds of soybean (Glycine max (L.) Merr. var: JS-335) by exposing the seeds to different magnetic field strengths from 0 to 300 mT in steps of 50 mT for 30, 60, and 90 min. Treatment with magnetic fields improved germination-related parameters like water uptake, speed of germination, seedling length, fresh weight, dry weight and vigor indices of soybean seeds under laboratory conditions. Improvement over untreated control was 5-42% for speed of germination, 4-73% for seedling length, 9-53% for fresh weight, 5-16% for dry weight, and 3-88% and 4-27% for vigor indices I and II, respectively. Treatment of 200 mT (60 min) and 150 mT (60 min), which were more effective than others in increasing most of the seedling parameters, were further explored for their effect on plant growth, leaf photosynthetic efficiency, and leaf protein content under field conditions. Among different growth parameters, leaf area, and leaf fresh weight showed maximum enhancement (more than twofold) in 1-month-old plants. Polyphasic chlorophyll a fluorescence (OJIP) transients from magnetically treated plants gave a higher fluorescence yield at the J-I-P phase. The total soluble protein map (SDS-polyacrylamide gel) of leaves showed increased intensities of the bands corresponding to a larger subunit (53 KDa) and smaller subunit (14 KDa) of Rubisco in the treated plants. We report here the beneficial effect of pre-sowing magnetic treatment for improving germination parameters and biomass accumulation in soybean.     

Evidence of a role for endogenous electrical fields in chick embryo development.

We have tested directly the hypothesis that the endogenous electrical field in the chick embryo plays a causal role in development. Conductive implants, which shunt currents out of the embryo and thus alter the internal field, were placed under the dorsal skin at the mid-trunk level of stage 11-15 embryos. Currents leaving the posterior intestinal portal (p.i.p.) of these embryos were reduced by an average of 30%. Control embryos receiving non-conductive implants showed no change in p.i.p. currents. In the group receiving current shunts, 92% of the embryos exhibited some developmental abnormality. Only 11% of the control group displayed defects. The most common defect in the experimental group (81%) was in tail development. Tail defects ranged from complete absence to the formation of a normal length, but morphologically abnormal tail. Internally, tail structures (neural tube, notochord and somites) were frequently absent or aberrantly formed. In 33% of the experimental embryos, the notochord continued lengthening in the absence of any other tail development. This led to the formation of ourenteric outgrowths from the hindgut. Defects in limb bud and head development were also found in experimentally treated embryos, but at a much lower frequency than tail defects. The abnormalities observed in experimental embryos were very similar to those produced naturally in rumpless mutant chicks. A vibrating probe analysis of these mutants (from both dominant and recessive strains) showed that currents leaving the p.i.p. were significantly lower in phenotypically abnormal mutants than in wild-type and phenotypically normal mutant embryos from both strains. There was no apparent correlation between the average transepithelial potential (TEP) of these mutants and the development of tail abnormalities. The possible role of endogenous electrical fields in chick tail development is discussed.     

Effects of an intrauterine copper device on serum copper, endometrial histology, and ovarian, hepatic, and renal functions in the Japanese monkey (Macaca fuscata fuscata).

A copper intrauterine device (Cu-IUD) was inserted transabdominally into the uterine cavity of eight Japanese monkeys (Macaca fuscata fuscata) for 4 to 6 months, and effects on various organ functions were examined. Results showed no significant effects on the menstrual cycle length, serum levels of LH, estradiol-17 beta, progesterone or clinical biochemical data such as serum copper, glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, lactic dehydrogenase, and blood urea nitrogen. Histology revealed edema and infiltration of eosinophilic leukocytes in the endometrium treated with a Cu-IUD.     

Influence of cadmium and nickel on growth,net photosynthesis and carbohydrate distribution in rice plants

Six day old rice seedlings (Oryza sativa L. cv. Bahía) were grown for 5 or 10 days in a nutrient solution with either Cd (0.01, 0.1 mmol/l) or Ni (0.1, 0.5 mmol/l). Both Cd and Ni reduced the length of shoots and roots depending on the concentration and type of ion tested. On the other hand, the dry weight to fresh weight ratio was increased by heavy metal treatments, especially in the aerial part of 0.5 mmol/l Ni treated plants. The application of 0.1 mmol/l Cd and 0.5 mmol/l Ni to the seedlings produced an inhibition of the transport of carbohydrate reserves from the seeds from which plants were developing, to the rest of the plant. Net photosynthesis was also inhibited in treated plants. However, the total carbohydrate content in the shoots of these plants was higher than in controls. Thus, the starch, soluble sugars and sucrose content in the shoots of 0.5 mmol/l Ni treated plants was respectively up to 2.6, 2.8 and 4 times greater compared to controls. The distribution of assimilates between organs was also affected by the treatment: the carbohydrate content increased in the stem and second leaf but it was not affected or decreased in the root and third leaf. Although less evident, the effect of Cd on carbohydrate distribution and content was similar to that of Ni. The possible mechanisms involved in the abnormal carbohydrate accumulation and distribution are discussed.Abbreviations 0 DT plants at day zero of treatment - 5 DT five days treated plants - 10 DT ten days treated plants - DW dry weight - FW fresh weight     

Genital fibroblasts from normal individuals and patients with androgen insensitivity: demonstration of reduced or absent levels of a specific protein (Mr approximately 41K, pI approximately 6) in receptor negative cells

Genital fibroblasts were obtained from normal individuals and from patients with a variety of syndromes of defective androgenization (complete androgen insensitivity, partial androgen insensitivity, microgenitalia, hypospadias, infertility). Cells were labelled with [35S]methionine, and patterns of protein synthesis compared by two-dimensional gel electrophoresis, with isoelectrofocusing electrophoresis gels or non-equilibrated pH gradient electrophoresis (NEPHGE) gels as the first dimension. A protein (mol. wt approximately 41K, pI approximately 6) was found on NEPHGE gels to be reduced or absent in fibroblasts in which androgen receptor levels were abnormal. The protein was unaltered by prior incubation with 1-100 nM dihydrotestosterone for 48 h, and was present in cells both from normal controls, and from patients with abnormal sexual differentiation showing normal androgen receptor levels. The coincidence of low or absent 41K with low or absent androgen receptors suggested the possibility that it may constitute a steroid-binding moiety of the androgen receptor. To test this possibility cytosols from normal foreskins or normal cultured fibroblasts were adsorbed with testosterone-sepharose affinity resin to remove androgen receptors. Cytosols so treated showed levels of 41K on NEPHGE indistinguishable from those in untreated cytosols, or in cytosols treated with underivatized sepharose. We therefore conclude that the 41K protein, while an accurate marker of the presence or absence of androgen receptors over a range of clinical disorders, is neither an androgen-induced protein nor an androgen-binding protein.     

Methylmercury: some effects on embryogenesis in the Japanese Medaka, Oryzias latipes

Japanese Medaka (Oryzias latipes) eggs were treated with 40, 60, and 80 parts per billion (ppb) methylmercuric chloride (MMC) for a period of 10 to 25 days in order to observe gross teratogenic and lethal effects caused by methylmercury during embryogenesis. Over the period tested, 40 ppb-treated embryos were less affected than other treated animals. Embryos treated with 60 ppb MMC showed greater effects in that there was a higher death rate and those which survived ranged considerably in size, degree of gross teratogenic defects, and behavior if development was normal enough for hatching to occur. Most did not hatch during the 25-day test period. Embryos treated with 80 ppb MMC were very abnormal in development, being stunted, poorly pigmented, and they showed various eye defects. None hatched in 25 days. Many did not incorporate the heart into the body. Methylmercuric chloride in trace amounts caused various defects and presents a potential hazard to development of embryos of aquatic vertebrates.     

Effects of exogenous progesterone on gestation length, foetal survival and colostrum yield in ewes

Twin bearing mature ewes (n=40) were treated with exogenous progesterone (100mg daily in oil) or vehicle (oil control) from Day 143 of gestation until lambing to investigate the effects on gestation length, foetal survival and colostrum yield and composition. Compared to control ewes, progesterone treated ewes had increased (P<0.05) serum progesterone concentrations (by 4.3 ng/ml) before lambing and in the first day post-partum (by 10 ng/ml). Progesterone treatment increased gestation length (150.4+/-0.6 days versus 147.8+/-0.6 days, P<0.05) and colostrum yield at 1h after lambing (P<0.05) but the colostrum had a lower concentration of IgG (P=0.02). In the first 24h after lambing, total colostrum and IgG yields were not different between groups. Four (20%) of the progesterone treated ewes produced either one or two dead lambs, while one ewe died on day 155 without initiating the birth process. We conclude that the daily administration of 100mg progesterone resulted in extended gestation length and reduced lamb survival but did not lower colostrum yield.     

Genetic nature of abnormal larval development in the progeny of l(1)ts403(sbr10) females of Drosophila melanogaster

In Drosophila melanogaster the small bristles (sbr) gene is vital and evolutionary conservative and controls nuclear export of mRNA. Sbr mutant alleles had a broad pleiotropic effect. High frequency of abnormal larva dying (up to 18 %) at the first instar stage in progeny of heat shock (37 degrees C, 1 h) treated mutant females is one of the most interesting l(l)ts403(sbr10) allele effects. Abnormal larvae display characteristic phenotype that involves the Malpighian tubules defect. Using interphase FISH method (fluorescence in situ hybridization), we showed that abnormal larvae had monosomy on chromosomes 2 and 3. DNA content in neuroblast interphase nuclei of abnormal larvae is 2.1 times less than in normal larvae. We suggest that abnormal larvae could be full or mosaic haploids that appeared as a result of maternal genome loss during fertilization or the mitotic division. Larvae with the same abnormalities appear in a progeny of females with different genotypes mating with males carrying compound chromosomes 2 or 3. FISH analysis showed that such larvae had monosomy only on a chromosome that is compound in paternal strain. Thus, monosomy on large autosomes may cause aspecial phenotype of abnormal larvae in D. melanogaster.     

Partitioning, Movement, and Positioning of Nucleoids in Mycoplasma capricolum

The nucleoids in Mycoplasma capricolum cells were visualized by phase-combined fluorescence microscopy of DAPI (4', 6-diamidino-2-phenylindole)-stained cells. Most growing cells in a rich medium had one or two nucleoids in a cell, and no anucleate cells were found. The nucleoids were positioned in the center in mononucleoid cells and at one-quarter and three-quarters of the cell length in binucleoid cells. These formations may have the purpose of ensuring delivery of replicated DNA to daughter cells. Internucleoid distances in binucleoid cells correlated with the cell lengths, and the relationship of DNA content to cell length showed that cell length depended on DNA content in binucleoid cells but not in mononucleoid cells. These observations suggest that cell elongation takes place in combination with nucleoid movement. Lipid synthesis was inhibited by transfer of cells to a medium lacking supplementation for lipid synthesis. The transferred cells immediately stopped dividing and elongated while regular spaces were maintained between the nucleoids for 1 h. After 1 h, the cells changed their shapes from rod-like to round, but the proportion of multinucleoid cells increased. Inhibition of protein synthesis by chloramphenicol induced nucleoid condensation and abnormal positioning, although partitioning was not inhibited. These results suggest that nucleoid partitioning does not require lipid or protein synthesis, while regular positioning requires both. When DNA replication was inhibited, the cells formed branches, and the nucleoids were positioned at the branching points. A model for the reproduction process of M. capricolum, including nucleoid migration and cell division, is discussed.     

镉对小鼠精子和生精细胞超微结构及生精细胞bcl-2、bax基因表达的影响

研究镉暴露对小鼠附睾精子和睾丸生精细胞超微结构的变化以及镉对生精细胞凋亡相关基因bcl-2、bax表达水平的影响。采用24只雄性ICR小鼠随机分为4组,每组6只,分别以0.183、0.915、1.83mg/kg氯化镉腹腔注射,每天1次,连续5次,设阴性对照生理盐水组。于第6天透射电镜观察附睾精子超微结构、睾丸生精细胞核和线粒体超微结构的变化,免疫组化方法检测生精细胞Bcl-2、Bax表达水平。透射电镜观察显示,0.183mg/kg组精子超微结构无显著性变化,0.915mg/kg组精子头部两侧膜与头部胞质间隙轻微扩大,线粒体嵴间腔扩大且轻度空泡化,但与对照组相比无统计学意义(P>0.05)。1.83mg/kg组头部两侧膜与胞质间隙扩大,与对照组相比有显著性差异(P<0.05),尾部线粒体嵴间腔扩大且轻度空泡化,与对照组相比有显著性差异(P<0.05)。3种剂量处理组睾丸生精细胞核超微结构异常发生率显著高于对照组(P<0.05),且随着处理浓度的升高异常发生率升高;1.83mg/kg组线粒体肿胀空泡化发生率显著高于对照组(P<0.05)。3种剂量实验组生精细胞Bcl-2表达水平(吸光度)显著低于对照组(P<0.01),0.915mg/kg组Bax表达水平显著高于对照组和0.183、1.83mg/kg组(P<0.01)。3种剂量实验组Bcl-2/Bax吸光度比值显著低于对照组(P<0.01);0.915mg/kg组Bcl-2/Bax比值显著低于1.83mg/kg组(P<0.01)。上述结果提示:高浓度镉诱导附睾精子超微结构改变,高中低浓度镉致睾丸生精细胞超微结构的改变,生精细胞超微结构发生凋亡现象。镉对Bcl-2、Bax表达水平的改变可能是生精细胞凋亡的分子机制之一。     

Turnover of cell wall components during internode growth in <Emphasis Type="Italic">Merremia emarginata</Emphasis>

The internodes of Merremia emarginata plant showed exceptionally high stretchability throughout the development period. Therefore, it provides excellent material to study the changes undergoing during cell elongation. In this study, the influence of the hormone treatments (GA₃, PAA and NAA) on the wall component synthesis was analyzed in relation to elongation growth during internode development. A clear increasing trend of wall components was observed with increase in internode length. The non-esterified pectic substances were markedly correlated with internode length while esterified pectic substances showed correlation only in hormone treated internodes. Low molecular weight xyloglucans content showed correlation only in GA₃ and NAA treated internodes, while high molecular weight xyloglucans were significantly correlated with length of internodes treated with PAA and NAA.     

Mitigation of growth retardation effect of plant defense activator,acibenzolar-<Emphasis Type="Italic">S</Emphasis>-methyl,in amaranthus plants by plant growth-promoting rhizobacteria

Four rhizobacterial strains and acibenzolar-S-methyl (ASM), a chemical activator, which suppressed foliar blight of amaranthus (Amaranthus tricolor L.) caused by Rhizoctonia solani Kühn were evaluated for their effect on plant growth. The experiments were performed both under sterile and non-sterile soil conditions, in the presence or absence of the pathogen. In all cases, plants treated with ASM showed significant reduction in growth, as determined by shoot length, and shoot and root dry weight when compared to other treatments. The growth retardation effect of ASM was more profound with respect to shoot length. Reduction in shoot length was least when plants were treated with a combination of the chemical activator and Pseudomonas putida 89B61 under non-sterile soil conditions in the absence of the pathogen. Both under sterile and non-sterile soil conditions, in the presence of the pathogen, reduction in shoot length due to application of ASM was diminished significantly when plants were treated with rhizobacterial strain Pseudomonas fluorescens PN026R. Combined use of plant growth-promoting rhizobacteria (PGPR) and ASM was found to be beneficial as the growth retardation effect of the plant defense activator was reduced by the growth-promoting ability of the rhizobacteria.     

Observation of Androgenesis in Cultured Wheat Anthers at Meiosis,Tetrad, Early Uninucleate and Trinucleate Stage

The obtaining of calluses and plantlets from cultured wheat anthersat the stages from pollen mother cell to trinucleate microspore has been reported previously. Haploids as well as diploids existed among the regenerated plantlets derivedfrom anthers at these stages. Present paper reports the study on androgenesis patter-ns of cultured anthers at meiosis, tetrad, early mid- and late uninucleate and trinucleate stage. Cytological evidence of pollen-origin of calluses produced by anthers atthese stages was given. Observation showed that meiosis of wheat anthers was able tocomplete under culture conditions, resulting in releasing microspores, from which multinucleate and multicellular pollen grains formed. In meiosis anthers, abnormal cells,including syncytium and two kinds of binueleate calls were sometimes observed. Theymight be products of abnormal meiosis and abnormal development of tapetum cells. Itwas noted that failure and/or uncomplction of forming callus wall and/or pollen wallin in vitro anthers at meiosis, tetrad and early uninucleate stage occured often. Itmight lead to the low frequency of callus induction. Mature wheat anthers (trinucleate stage) contained both normal and abnormal pollen grains (pollen dimorphism); onlythe abnormal pollen grains developed into embryoids while all the normai trinucleatepollen grains degenerated rapidly. However, the date of the frequency of equal divisionof microspores suggested that abnormal pollen (N pollen, small pollen) could not be theonly source of androgenic pollens in cultured anthers at late uninucleate and other earlier stages.     

Morphology of spermatozoa bound to the zona pellucida of human oocytes that failed to fertilize in vitro.

The morphology of spermatozoa bound to the zona pellucida (ZP) of 264 oocytes that had failed to fertilize in 58 in vitro fertilization procedures was studied by light microscopy. The percentage of spermatozoa with normal morphology bound to the ZP (mean 77, range 8-100) was significantly higher than in the insemination medium (mean 42, range 2-80). The abnormal spermatozoa bound to the ZP had small oval (47%), pyriform (46%), amorphous (5%) and tapering (2%) heads. Other abnormalities of morphology were not observed in ZP-bound spermatozoa. The mean rates of binding to the ZP of spermatozoa with normal morphology (44, 95% confidence limits 42-46, number bound/ZP/10(5)/ml inseminated) were much higher than for abnormal spermatozoa with small oval 6.5 (5.5-7.6), pyriform 5.9 (4.8-7.3), amorphous 1.0 (0.5-2.0) and tapering 2.5 (1.2-5.3) heads. The morphologically abnormal forms not found on the ZP were infrequent in the insemination medium (tail defects, large oval, round, pin and duplicate heads and cytoplasmic droplets) but upper 95% confidence limits for standardized binding ratios of less than 60% indicated that these were unlikely to bind to the zona with rates approaching those of normal spermatozoa (standardized binding ratio 180%). A large number of uniformly normal spermatozoa bound to the ZP when the percentage normal morphology in the insemination medium was greater than 40%. The proportions of abnormal spermatozoa in the insemination medium. Spermatozoon head dimensions were measured with a micrometer in samples from 14 patients. While there were no consistent changes in all samples, the means for head width and area were significantly larger and the ratio of the length to width was smaller for spermatozoa on the ZP than for those in the insemination medium. The head length and ratio of length to width of spermatozoa in samples with good morphology were significantly less than in samples with poor morphology. The percentage of spermatozoa with normal morphology, motility and the ratio of head length to width in the insemination medium were positively correlated with the percentage of spermatozoa with normal morphology bound to the ZP. Logistic regression analysis showed that the diagnosis of tubal infertility and the rate of binding of spermatozoa with normal morphology to the ZP were positively related to fertilization rates in vitro while the rate of binding of spermatozoa with pyriform heads was negatively related. In conclusion. human ZP are highly selective for spermatozoa with normal morphology. The frequency of binding of abnormal spermatozoa to the ZP was mainly dependent on semen quality.(ABSTRACT TRUNCATED AT 400 WORDS)     

Decomposition of N-nitrosamines,and concomitant release of nitric oxide by Fenton reagent under physiological conditions

Our previous work has shown that prooxidant treatment has the propensity to induce male-mediated dominant lethal (DL) type mutations in mice. The present investigation is aimed to understand the effect of oxidative stress (OS) on DNA damage in testis, epididymal sperms and its propensity to induce sperm head abnormalities as well as its implications on male fertility in mice. Initially, employing two organic hydroperoxides, (t-butyl hydroperoxide, t-bHP and cumene hydroperoxide, cHP) as model prooxidants, induction of oxidative stress was ascertained following single/multiple sublethal doses. Further, the multiple exposure model was utilized to characterize effects on testicular weights, histoarchitecture, caudal sperm counts, lipid peroxidation, DNA damage and frequency of abnormal sperms. Single sublethal doses (1/20, 1/10 and 1/5 LD(50)) of t-bHP and cHP administered (i.p.) to adult mice resulted in only a marginal increase (20% at the highest dosage) in testicular MDA levels. However, multiple doses (1/10 and 1/5 LD(50) per day for 5 days) induced marked OS in testis and epididymal sperms as evidenced by a marked increase in lipid peroxidation at 24h after the last dose. This was associated with significant increase in the DNA damage (FADU assay) in the testicular tissue. While caudal sperm counts determined at all sampling weeks showed no treatment related alterations, analysis for head abnormalities revealed nearly 2-3-fold increase in the percent abnormal sperms among the hydroperoxide treated mice during the first 3 weeks. Furthermore, mating of prooxidant treated males sequentially for a period of 5 weeks with untreated females resulted in a significant reduction in average pup number per litter during the first 3 weeks. These results suggest that oxidative stress in testicular milieu is associated with DNA damage and produces higher frequency of abnormal sperms with significant effect on male fertility.