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1.
C Rodríguez-Burford D K Oelschlager L I Talley M N Barnes E E Partridge 《Biotechnic & histochemistry》2013,88(1):17-21
We describe here a computational morphology-based approach to the investigation of possible causes of chromatin alterations in sperm. A comprehensive set of state-of-the-art and geometric measures are computationally extracted from toluidine blue stained images and analyzed to infer the possible processes leading to normal and abnormal chromatin formation while seeking a possible taxonomy of chromatin alterations and their influence on sperm head morphology. Using this methodology, we have identified higher chromatin fragility at some specific points of the sperm head. Despite the lack of correlation between morphologies of sperm head and chromatin structure, four main morphological types of chromatin alterations in bull spermatozoa have been identified and their possible causes discussed. 相似文献
2.
Sperm chromatin alteration is an important feature that can affect fertility of the male rabbit. This study compared toluidine blue staining with Feulgen reaction (as methods for evaluating chromatin alteration) and investigated the relationship between sperm morphology and chromatin alteration. Seven hundred rabbit ejaculates of animals with unknown fertility were used. Primary and secondary morphological sperm abnormalities were evaluated in semen smears with phase-contrast microscopy. Chromatin alterations were evaluated in semen smears stained with toluidine blue (pH 4.0 and 5.0) and with the Feulgen reaction. While the three methods were equally efficacious for identification of chromatin alterations, toluidine blue staining was more appropriate to characterize the intensity of chromatin alterations. The correlation between primary sperm defects and chromatin alteration was high and positive, suggesting that sperm chromatin structure affected sperm head morphology. The correlation between secondary sperm defects and chromatin alteration was also positive, but lower. The final chromatin compaction occurs in the epididymus, where secondary sperm defects originate. Therefore, the causes of secondary sperm defects could also intervene with final chromatin compaction. In summary, the toluidine blue stain was an effective means of evaluating the sperm chromatin alteration in rabbit spermatozoa. 相似文献
3.
Flow cytometric analysis of mouse spermatogenic function following exposure to ethylnitrosourea 总被引:7,自引:0,他引:7
The effects of the mutagenic agent ethylnitrosourea (ENU) on spermatogenic function and sperm chromatin structure were studied by flow cytometry and the results compared with sperm head morphology measurements. Groups of mice received daily exposures ranging from 0 to 75 mg/kg body weight X 5 days and were sacrificed 28 days later. Fresh testicular cell suspensions and epididymal sperm were stained with acridine orange (AO) and measured by flow cytometry. Sperm nuclei were isolated, fixed, rehydrated, and then either subjected to thermal stress or not prior to staining with AO. Body weights were unaffected by the chemical exposure while the testicular weights were reduced by about 50%. Two-parameter (DNA, RNA) flow cytometry measurements showed a dose-response relationship in the loss of certain cell types, particularly the elongated spermatids, from the testes of treated animals. Flow cytometric analysis of both heat-stressed and non-heat-stressed nuclei showed a relationship between dosage and the coefficient of variation of alpha t [red/(red + green fluorescence)] measurements of AO stained nuclei, thereby demonstrating that alterations of chromatin structure occurred in response to ENU. Enzymatic digestions with RNAse, DNAse, and nuclease S1 suggest that the increase in red fluorescence is due to an increase of single-stranded DNA induced by heat or acid treatment of chemically altered chromatin structure. The lowest daily dosage used (5 mg/kg) caused no significant changes in ratios of testicular cell types, a questionable increase in abnormal sperm head morphology and a detectable change in chromatin structure expressed as alpha t. This report shows that our technique for assaying sperm nuclear chromatin structure appears to have the same level of sensitivity to ENU induced nuclear alterations as the sperm head morphology test. 相似文献
4.
Elisson Terêncio Souza Cláudio Vieira Silva Bruno Augusto Nassif Travençolo Benner Geraldo Alves Marcelo Emílio Beletti 《Reproductive biology》2018,18(2):177-181
Alterations in sperm chromatin have been related with subfertility in several mammals. In this study, chromatin alteration types (Base, Basal half, Central axis, Dispersed, and Whole) were assessed by toluidine blue (TB) staining, 6-diamidino-2-fenilindole (DAPI) and anti-protamine 1 antibody (anti-PR1) labeling in sperm samples of fertile and subfertile bulls. Semen samples were obtained from bulls kept in Artificial Insemination Center (fertile bulls) or from bulls subjected to scrotal insulation (subfertile bulls). The percentage of chromatin alterations identified by TB was similar (P?>?0.05) in semen samples of fertile and subfertile bulls. In contrast, a greater (P?<?0.01) chromatin decondensation and heterogeneity were recorded in semen samples of subfertile bulls. In DAPI and anti-PR1 methods, the subfertile bulls samples had a higher (P?<?0.05) percentage of alteration in the base as well as overall chromatin alterations (P?<?0.05). Moreover, the chromatin alterations recorded with TB, DAPI, and anti-PR1 were compared in semen samples of fertile and subfertile bulls. In fertile bulls, the overall chromatin alterations were similar (P?>?0.05) among the methods In contrast, semen samples of subfertile bulls had a higher (P?<?0.05) percentage of overall chromatin alterations when labeled with DAPI. In conclusion, our findings shown that all dye tested had specific sperm stainability and can be feasible to monitor subfertility condition in bulls. Also, different chromatin alteration types in sperm samples of fertile and suberftile bulls were recorded. 相似文献
5.
Nongnuj Tanphaichitr Prasert Sobhon Panpaka Chalermisarachai Chutima Chutatape 《Molecular reproduction and development》1982,6(3):235-255
Preparation of human sperm heads containing mainly nucleoprotamines as their chromatin constituent was described. Acrosome-depleted human sperm heads were treated with micrococcal nuclease followed with 2 M NaCl. The treatment specifically removed nucleohistone as small chromatin fragments from the human sperm heads. The nuclease-NaCl-treated sperm head pellets primarily contained nuclease-resistant nucleoprotamine as their chromatin. Transmission electron microscopic study showed that there were two populations of the nuclease-NaCl-treated human sperm head pellets. One population had thick nucleoprotamine cords of ?650–1,200 Å in diameter, and the other had thin nucleoprotamine cords of ?330–420 Å. 相似文献
6.
AN ELECTRON MICROSCOPE STUDY OF SPERMATID DIFFERENTIATION IN THE TOAD, BUFO ARENARUM HENSEL 总被引:26,自引:22,他引:4
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The differentiation of the spermatids of Bufo arenarum has been described from a study of electron micrographs of thin sections of testis. The development of the acrosome from the Golgi complex takes place in much the same manner as in mammalian spermatogenesis but no acrosome granule is formed. A perforatorium is described for the first time in this species. It is formed by a convergence of dense filaments that arise between the nuclear membrane and the head cap. During maturation of the spermatid the chromatin undergoes striking physicochemical alterations. Fine chromatin granules uniformly dispersed in the karyoplasm are replaced by larger and larger aggregates and these ultimately coalesce to form a very dense sperm head. Two centrioles of cylindrical form are situated very near the base of the sperm head. The longitudinal fibrils of the tail flagellum take origin from one, and the dense fibrous substance of the undulating membrane is closely related to the other. Phase contrast cinematographic observations on the swimming movements of living toad sperm, when considered in relation to the fine structural components of the tail, suggest that there is a contractile component in the undulating membrane as well as in the axial fibrils. The differences in the structure of mammalian and amphibian sperm tails are discussed in relation to differences in the character of their movements. 相似文献
7.
The ultrastructure of spermatophores was studied in seven species of Oribatei. Each spermatophore is composed of a head and a stalk which is attached to the substratum. The spherical head consists of two distinct portions: (1) the sperm package; and (2) the head matrix. Position and structure of the sperm package are described in the different species, as well as the various structures in the head matrix. The sperm package contains sperm embedded in secretory products. The most characteristic feature of the sperm is an electron-dense chromatin body. Mitochondria lie adjacent to it or are partly or completely incorporated into the chromatin body. The cytoplasmic surface of the plasma membrane regularly bears an electron-dense layer; the plasma membrane is covered by a secretory sheath. The spermatophores of the Oribatei are structurally complicated and not uniform. The possible role of the spermatophore's structural elements is discussed and an attempt is made to evaluate characteristic features taxonomically. 相似文献
8.
When "denuded spermatozoa" (spermatozoa stripped of the greater part of their acrosomes and resembling in may respects spermatozoa after acrosomal reaction) of the bull are incubated with 0.1 M 2-mercaptoethanol (pH 8), sperm chromatin is degraded extensively by a protease in the sperm head. The morphological pattern of sperm nuclear dispersion upon in vitro incubation is similar to that observed in the newly fertilized egg. Following disintegration of the outer layers of the sperm nucleus, chromatin dispersion commences from the periphery of the posterior half and proceeds to the anterior end and to the core of the head. Less basic N- and C-terminal portions of bull sperm histone molecules are digested quickly. The central, very arginine-rich portions of the molecules degrade gradually, yielding an heterogeneous series of arginine-rich peptides (molecular weight, 400-1500). Evidence suggests that the protease which is responsible for the degradation of sperm chromatin is a small fraction of acrosin. This fraction of acrosin appears to be arranged along the nuclear surface and to become associated with sperm chromatin during structural changes of the nuclear surface. A similar proteolysis of rabbit, hamster and guinea pig sperm chromatin has also been observed. The resulting pattern of dissolution of the sperm nucleus is proposed as a model of some of the steps involved in male pronucleus formation from the sperm head after fertilization. Histones H2a, H2b, H3, and H4 associated with DNA are relatively resistant to acrosin. 相似文献
9.
The dark staining of human sperm heads by Diff Quik is significantly correlated with abnormal sperm head morphology (r=0.51, p<0.0001), but is not associated with changes in sperm chromatin detected by a sperm chromatin structure assay (SCSA; r=0.18, p>0.09). Whilst valuable in the assessment of head morphology, it is concluded that Diff Quik staining is not a useful substitute for the SCSA to assess human sperm chromatin. 相似文献
10.
The structure of rabbit, fowl, and Xenopus laevis sperm chromatin was explored by study of the reaction of their decondensed nuclei with DNase 1 and micrococcal nuclease. Those of rabbit and fowl were readily digested by DNase 1, and the polyacrylamide gel electrophoresis profiles of DNAs extracted from the digests were similar, each being polydisperse with a single discrete band of DNA smaller than 72 base pairs. There were differences, however, between the sperm chromatins in the course of their digestion by micrococcal nuclease. A limit digest at about 45% acid solubility was obtained with Xenopus sperm chromatin, while 90% of fowl sperm DNA was rendered acidsoluble by the enzyme. The gel profiles of the limit digests were polydisperse, but only those of rabbit and fowl sperm chromatins possessed a discrete band of DNA smaller than 72 base pairs. Bleomycin did not react with DNA of rabbit, fowl, or Xenopus spermatozoa. Since bleomycin reacts with somatic cell chromatin, and the course of DNase 1 or micrococcal nuclease digestion of sperm chromatin was different from that found for somatic cell chromatin, it would appear that sperm chromatin does not have the repeating nucleosometype structure of somatic cell chromatin. The nuclease digestion studies further suggest that the organization of rabbit and fowl sperm chromatins is similar, and is different from that of Xenopus sperm chromatin. The dependence of the structure of sperm chromatin on the composition of its basic proteins, and a possible structure for a protamine-type sperm chromatin, are discussed. 相似文献
11.
M Brito I I Concha C A Pessot S Mezzano L O Burzio 《Comparative biochemistry and physiology. B, Comparative biochemistry》1992,103(1):197-200
1. Immunoblot analysis of rat sperm head proteins revealed the presence of polypeptides recognized by anti-Sm serum obtained from patients with systemic lupus erythematosus. 2. Two of these polypeptides have molecular weights of 26,000 and 15,000 and they were identified as small nuclear ribonucleoprotein components present in other rat tissues. 3. When the autoimmune serum was used in the immuno-gold procedure for electron microscopy, gold particles were found only on the sperm nucleus. 4. The results indicate that some polypeptides of the small nuclear ribonucleoprotein complex are components of the rat sperm chromatin. 相似文献
12.
Sperm head cytometry provides a useful assay for the detection of radiation-induced damage in mouse germ cells. Exposure of the gonads to radiation is known to lead to an increase of diploid and higher polyploid sperm and of sperm with head shape abnormalities. In the pilot studies reported here quantitative analysis of the total DNA content, the morphology, and the chromatin distribution of mouse sperm was performed. The goal was to evaluate the discriminative power of features derived by high resolution image cytometry in distinguishing sperm of control and irradiated mice. Our results suggest that besides the induction of the above mentioned variations in DNA content and shape of sperm head, changes of the nonhomogeneous chromatin distribution within the sperm may also be used to quantify the radiation effect on sperm cells. Whereas the chromatin distribution features show larger variations for sperm 21 days after exposure (dpr), the shape parameters seem to be more important to discriminate sperm 35 dpr. This may be explained by differentiation processes, which take place in different stages during mouse spermatogenesis. 相似文献
13.
Intracytoplasmic sperm injection (ICSI) has heralded an era of tremendous improvements in treating male infertility leading to the births of thousands of babies. However, recent concerns over possible long-term effects of ICSI on offspring has prompted the development of a preclinical, nonhuman primate model to assess the safety of ICSI. Fluorescent imaging of rhesus macaque IVF zygotes revealed that this species shares many similarities with humans in terms of cytoskeletal and chromatin dynamics during fertilization. However, rhesus monkey zygotes fertilized by ICSI resulted in abnormal nuclear remodeling leading to asynchronous chromatin decondensation in the apical region of the sperm head, delaying the onset of DNA synthesis. The persistence of the acrosome and perinuclear theca on the apex of sperm introduced into the oocyte by ICSI may constrict the DNA in this region. Despite these differences, normal rhesus monkey ICSI embryos have been produced and have lead to several births after transfer. The irregularities described in this paper raise concerns that the ICSI procedure may result in chromatin damage during DNA decondensation and further highlight the need for devising improved pre-clinical assessment prior to global acceptance of this, and other, novel methods of assisted reproduction. 相似文献
14.
Failure of differentiation of the nuclear-perinuclear skeletal complex in the round-headed human spermatozoa 总被引:6,自引:0,他引:6
D Escalier 《The International journal of developmental biology》1990,34(2):287-297
Acrosomeless round-headed spermatozoa from three men were studied under electron microscopy and indirect immunofluorescene microscopy using the anti-calicin antibody that recognizes a basic protein of the sperm perinuclear theca (Longo et al., 1987). Electron microscopy revealed the existence of anomalies of the nuclear envelope, the nuclear matrix underlying the nuclear envelope, and the perinuclear layer. The absence of sperm labeling with the anti-calicin antibody confirmed that the formation of the perinuclear theca was impaired. Data obtained from both mature spermatozoa and ejaculated spermatids suggest that i) round-headed sperm head anomalies result from a failure of differentiation of the sperm-specific skeletal complex related to the nucleus, and ii) the acrosome spreading over the nucleus, the nuclear elongation and the post-acrosomal sheath formation are dependent on such nuclear-perinuclear differentiations. In contrast, chromatin condensation, cytokinesis and some events of the acrosomal shaping appear not to depend on those nuclear-related differentiations. The possible processes allowing the maintenance of the sperm head structures and their subsequent morphogenesis are discussed. 相似文献
15.
The perinuclear theca (PT) is a cytoskeletal structure that surrounds the mammal sperm nucleus which must be disrupted once the sperm has penetrated the oocyte to permit normal chromatin decondensation and formation of male pronucleus. F-actin is a thermo sensitive protein found in the equatorial segment which is involved in the stability of PT. It has been reported that cryopreservation induces alterations in nuclear decondensation of spermatozoa, which have been interpreted as an over condensation. The aims of the present study were identified the presence of changes in sperm sPT integrity of frozen–thawed boar spermatozoa and its effect in sperm nuclei decondensation; and whether changes in the actin cytoskeleton are involved using an in vitro model to test probably differences in a chemical decondensation (DTT/heparin) between fresh (FS) and frozen–thawed (TS) spermatozoa. Results showed an increase on sPT damage in TS (P < 0.001), and significant changes in sperm chromatin nuclear decondensation (P < 0.05). In same way differences on the swelling degree was found assessed by measures in equatorial region of head sperm (P < 0.05). Evaluation with rodamine-labeled actin (0.2 μM) showed two different patterns with differences in percentages before and after cryopreservation (P < 0.001). F-actin stabilization constrained the equatorial segment of FS while this was not observed in TS. The data showed that the presence of early changes in sPT integrity and changes in the F-actin localization on TS may suggest the participation in F-actin in decondensation process and probably that the disruption of actin-PT interaction during freezing–thawing process could have far-reaching consequences for the subsequent fertility of spermatozoa. 相似文献
16.
1. In the heads of maturing sperm of a locust the chromatin becomes arranged in a highly regular manner so as to produce many parallel lines about 60 A thick in longitudinal section and contiguous polygons in transverse section. 2. This configuration appears after fixation in osmium tetroxide, formaldehyde, or acetic acid; intermediate stages in its development are illustrated. 3. These electron micrographs are interpreted to mean that, during sperm maturation, the chromatin becomes formed into sheets and then into tubes running parallel with the long axis. 4. In the mature sperm head we have been unable so far to detect this structure. This may be because the chromatin becomes so compact during the shrinkage of the nucleus which occurs during formation of the mature sperm. 相似文献
17.
Spartaco Santi Silvia Rubbini Caterina Cinti Stefano Squarzoni Alessandro Matteucci Elisabetta Caramelli Lia Guidotti Nadir M. Maraldi 《Biology of the cell / under the auspices of the European Cell Biology Organization》1994,81(1):47-57
Summary— In the sperm nuclei the DNA is packaged into a highly condensed form and is not organized into nucleosome and solenoid but is bound and stabilized mainly by the protamines that arrange the DNA in an almost crystalline state. As demonstrated for somatic cells, the sperm DNA has been reported to be organized in loop domains attached to the nuclear matrix structures. However, the possible role of the sperm head matrix in maintaining the loop organization in absence of a typical nucleosomal structures has not been fully elucidated. By using in situ nick translation at confocal and electron microscope level, we analyzed the organization of the DNAprotamine complex and its association with the sperm nuclear matrix. The data obtained indicate that the chromatin organization in sperm nuclei is maintained during the sperm condensation by means of interactions with the nuclear matrix at fixed sites. The fine stucture of sperm nucleus and of sperm nuclear matrix, investigated on sections and replicas of freeze-fractured specimens, suggests that the lamellar array, observed by freeze-fracturing in the sperm nuclei, could depend on the inner matrix which presents a regular organization of globular structures possibly involved in the maintenance of chromatin domains in highly condensed sperm nuclei also. 相似文献
18.
M G Manfredi Romanini M Biggiogera D Formenti A Fraschini S Garagna C Pellicciari C A Redi 《Histochemistry》1986,84(4-6):484-491
Cytochemical techniques were used to study chromatin during spermiogenesis and sperm maturation in the mouse, starting from the stages at which the substitution of somatic histones by testis-specific proteins occurs. It was possible to distinguish and analyze the different temporal incidence of two processes involved in sperm maturation, i.e. chromatin condensation (a tridimensional highly compacted arrangement) and chromatin stabilization (a tough structure, which protects the genome DNA). The first process, involving a reduction in the nuclear size and a decrease in the amount of sperm DNA accessible to specific cytochemical reactions and stainings, was found to reach its maximum in caput-epididymidis spermatozoa, in which electron microscopy revealed that the sheared chromatin was mainly organized into 120-A-thick knobby fibers. No further changes were found in sperm up to their appearance in the fallopian tubes. On the contrary, chromatin stabilization, the onset of which occurs in the testis (at the late spermatid stage) via the formation of -S-S- cross-links, is completed in the vas deferens, where chromatin has a superstructure consisting of thicker fibers, with diameters of 210 and 350 A. The reductive cleavage of disulfides in vas-deferens spermatozoa does not completely destroy the superstructure of sperm chromatin, which could indicate 'coiling' of the basic knobby fiber. In fact, when the ion concentration was increased, the chromatin of vas-deferens spermatozoa appeared to be organized into fibers with diameters similar to those of the caput epididymidis. This unique organization of mature sperm chromatin should have an essential role in the fast swelling of spermatozoa during fertilization. 相似文献
19.
20.
Cadmium concentrations in the testes, sperm, and spermatids of mice subjected to long-term cadmium chloride exposure. 总被引:4,自引:0,他引:4
BACKGROUND: Exposures to cadmium have been reported to reduce male fertility and there are several hypotheses that suggest how reduced male fertility may result from incorporation of cadmium into sperm chromatin. The purpose of this study was to determine whether mice subjected to long-term intraperitoneal cadmium exposure incorporated cadmium into their sperm chromatin. METHODS: Male mice were exposed to 0.1 mg/kg body weight cadmium in the form of CdCl2 via intraperitoneal injection once per week for 4, 10, 26, and 52 weeks and then sacrificed. The cadmium contents of the liver, testes, pooled sperm, and pooled spermatids from dosed and control animals were determined by atomic absorption spectroscopy. Cadmium and zinc contents in individual sperm and spermatid heads were determined by particle-induced x-ray emission. RESULTS: Atomic absorption spectroscopy revealed that although cadmium accumulated in the liver and testes, cadmium was not detected in pooled sperm or spermatid samples down to minimum detectable limits of 0.02 microg/g dry weight. Particle-induced x-ray emission analyses did not show the presence of cadmium in any sperm or spermatid head down to minimum detectable limits of 15 microg/g dry weight. Particle-induced x-ray emission analyses also demonstrated that phosphorus, sulfur, and zinc concentrations in individual sperm and spermatid heads were not altered by exposure to CdCl2. CONCLUSIONS: Because cadmium was not incorporated into sperm chromatin at levels above 0.02 microg/g dry weight, the data cast doubt on hypotheses that suggest that reduced male fertility may result from incorporation of cadmium into sperm chromatin. 相似文献