Sperm dimorphism in terms of nuclear shape and microtubule accumulation in Cyrtanthus mackenii

Pollen tubes of Cyrtanthus mackenii, a species with bicellular pollen, were cultured in vitro to investigate nuclear phase changes during generative cell division and male germ unit (MGU) formation, using flow cytometric analysis. Results revealed that sperm cells were formed after 12 h of culture. During sperm maturation, the nuclei of sperm cells were not associated with the vegetative nucleus (unassociated sperm cells; Sua) and became longer than those of sperm cells associated with the vegetative nucleus (Svn). These findings indicate that the pair of sperm cells in the C. mackenii MGU is dimorphic in terms of nuclear shape. Dimorphism coincides with anti-α-tubulin antibody immunofluorescence, which was higher in the Sua than in Svn. Following treatment with oryzalin, triggering microtubule depolymerization, differences between nuclear shapes in the two sperm nuclei disappeared, suggesting that microtubule accumulation between sperm cells in the MGU correlates with differences in the nuclear shape.     

Determinants of sperm nuclear shaping in the genus Xenopus

The morphogenesis of sperm nuclei was investigated in six different species or subspecies of the genus Xenopus (Pipidae, Anura). The sequence of nuclear morphogenesis was similar in each species used in this study. Electrophoretic comparison of the basic chromatin proteins from late spermatids and sperm of each species demonstrated that the complements of histones and spermatid-sperm-specific basic proteins were extremely diverse suggesting that shape was not determined by specific basic proteins or mechanisms of histone removal. This conclusion was reinforced by the observation that Xenopus sperm DNA decondensed by 2.0 M NaCl remained contrained in residual structures which resembled intact sperm nuclei. These observations suggested that morphogenesis of sperm nuclei is directed by proteins or RNA molecules which are not directly responsible for chromatin condensation.     

Nuclear assembly of demembranated Xenopus sperm in plant cell-free extracts from Nicotiana ovules

The cell-free preparation derived from Nicotiana tabaccum ovules induced chromatin decondensation and pronuclear formation from demembranated Xenopus laevis sperm nuclei. Fluorescent microscope and phase-contrast microscope visualization of assembly intermediates indicated that 95.6% of X. leavis sperm changed their tadpole-like shape to circular shape or elliptical shape after over 1.5 h of incubation. Transmission electron microscope visualization showed that nuclear membrane was assembled around the periphery of the dispersed chromatin. Nuclear envelope of most reassembled nuclei was composed of a double membrane inlaid with a little single membrane. Nucleosome assembly was verified by means of micrococcal nuclease digestion. After 2 to 5 h of incubation, digestion of the product of nuclear assembly with micrococcal nuclease produced at least six nucleosome fragments of about 250 bp each.     

Physical characteristics of mouse sperm nuclei.

The nuclei of epididymal sperm, isolated from C57BL/6J and CBA/J inbred mice by their resistance to trypsin digestion, retain the shape differences of the intact sperm head. Various physical characteristics of these nuclei were measured and compared. The measurement of the projected dimensions of nuclei showed that the CBA nuclei are 13.5% longer than C57BL/6 nuclei (8.64 +/- 0.02 mum compared with 7.61 +/- 0.02 mum), 0.8% narrower (3.51 +/- 0.01 vs. 3.54 +/-0.01 mum) with 6.8% more area (22.34 +/- 0.10 vs. 20.91 +/- 0.09 mum2). However, the volumes of the nuclei as based on reconstructing calibrated electronmicrographs of serial sections of the nuclei indicated that CBA are about 7% smaller than C57BL/6 nuclei (3.72 +/- 0.08 vs. 4.01 +/- 0.03 mum3). The buoyant density of the CBA nuclei is 1.435 +/- 0.002 g/cm3 compared with 1.433 +/- 0.002 g/cm3 for the C57BL/6 nuclei as determined on linear CsCl and Renografin-76 density gradients and confirmed by a technique utilizing physiological tonicities. Therefore, the average mass of the CBA nuclei is less than that of the C57BL/6 nuclei (5.34 +/- 0.12 vs. 5.75 +/- 0.05 pg). The sedimentation velocities at unit gravity of nuclei from 11 inbred strains differ over a range of more than 6% with CBA nuclei sedimenting about 2.0% more slowly than C57BL/6 nuclei. We show that for these nuclei the sedimentation velocity can be related to their buoyant density, volume and a sedimentation shape factor. Within the errors of our measurements of these various characteristics, it was found that C57BL/6 and CBA nuclei have similar sedimentation shape factors. Therefore, the difference in sedimentation velocity between these nuclei appears to be primarily a result of differences in volume. The possible applications of these techniques to the physical separation of sperm are evaluated in the discussion.     

Intérêt de l’analyse de la morphologie fine et de la qualité nucléaire des spermatozoïdes dans le cadre des techniques d’AMP

Routine semen examination does not identify minor malformations of the sperm nucleus and chromatin architectural defects, which may be associated with ART outcome and cannot be detected by the embryologist even at 1000x magnification. Recent publications have demonstrated the advantages, compared to routine analysis, of a new method of real-time detailed morphological evaluation of motile spermatozoa: motile sperm organellar morphology examination (MSOME). MSOME is performed with an inverted light microscope equipped with high-power differential interference contrast optics enhanced by digital imaging to achieve a magnification of 10000x. To be considered morphologically normal, a sperm nucleus must have both a normal shape and a normal chromatin content. The aim of the present study was to combine MSOME and sperm DNA fragmentation characteristics to assess reproductive outcome. The study population consisted of the male partners of 52 couples referred for conventional IVF or split cycles (half IVF-half ICSI cycles) and exhibiting normal routine sperm parameters. Spermatozoa were analysed by examining the fine nuclear morphology and DNA integrity using the sperm chromatin dispersion test (SCD test), based on the principle that the deproteinized nuclei of spermatozoa with nonfragmented DNA show extended halos of DNA dispersion that are either absent or only minimally present in sperm nuclei with fragmented DNA. Fertilization rates were significantly lower in the group showing less than 8% of normal spermatozoa according to MSOME criteria, but early embryo development was not affected. Fine sperm morphology correlated with DNA fragmentation rate. These results demonstrate that the assessment of sperm nuclear normality by MSOME analysis and SCD test improves characterization of the semen sample and should be evaluated as a tool for allocating patients to specific assisted reproduction treatments.     

Flowering plant sperm cells: Isolation from pollen of Gerbera jamesonii (asteraceae)

Sperm cells were isolated from pollen of Gerbera jamesonii by gentle grinding in a medium containing buffer, osmoticum, protectants and ions. Sperm nuclei were identified by means of the DNA fluorochrome DAPI. The sperm cells were observed by Nomarski optics and by scanning electron microscopy. Isolated sperm were osmotically sensitive and excluded Evans Blue. The structure of the isolated sperm cells was similar in shape and size to that of sperm cells extruded from fresh pollen and in fixed pollen.     

Sperm decondensation in Xenopus egg cytoplasm is mediated by nucleoplasmin

At fertilization, sperm chromatin decondenses in two stages, which can be mimicked in extracts of Xenopus eggs. Rapid, limited decondensation is followed by slower, membrane-dependent decondensation and swelling. Nucleoplasmin, an acidic nuclear protein, occurs at high concentration in Xenopus eggs and has a histone-binding role in nucleosome assembly. Immunodepleting nucleoplasmin from egg extracts inhibits the initial rapid stage of sperm decondensation, and also the decondensation of myeloma nuclei, relative to controls of mock depletion and TFIIIA depletion. Readdition of purified nucleoplasmin recues depleted extracts. A physiological concentration of purified nucleoplasmin alone decondenses both sperm and myeloma nuclei. We conclude that nucleoplasmin is both necessary and sufficient for the first stage of sperm decondensation in Xenopus eggs.     

Evidence for nuclear internalization of exogenous DNA into mammalian sperm cells

Mature sperm cells have the spontaneous capacity to take up exogenous DNA. Such DNA specifically interacts with the subacrosomal segment of the sperm head corresponding to the nuclear area. Part of the sperm-bound foreign DNA is further internalized into nuclei. Using end-labelled plasmid DNA we have found that 15–22% of the total sperm bound DNA is associated with nuclei as determined on isolated nuclei. On the basis of autoradiographic analysis, nuclear permeability to exogenous DNA seems to be a wide phenomenon involving the majority of the sperm nuclei. In fact, the foreign DNA, incubated with sperm cells for different lengths of time, is found in 45% (10 min) to 65% (2 hr) of the sperm nuclei. Ultrastructural autoradiography on thin sections of mammalian spermatozoa, preincubated with end-labelled plasmid DNA, shows that the exogenous DNA is internalized into the nucleus. This conclusion is further supported by ultrastructural autoradiographic analysis on thin sections of nuclei isolated from spermatozoa preincubated with end-labelled DNA. © 1993 Wiley-Liss, Inc.     

Flow cytometry of mammalian sperm: progress in DNA and morphology measurement.

Variability in DNA content and head shape of mammalian sperm are potentially useful markers for flow cytometric monitoring of genetic damage in spermatogenic cells. The high refractive index and extreme flatness of the sperm heads produce an optical effect which interferes with DNA measurements in flow cytometers which have dye excitation and fluorescence light collection normal to the axis of flow. Orientation of sperm in flow controls this effect and results in coefficients of variation of 2.5% and 4.2%, respectively, for DNA measurements of mouse and human sperm. Alternatively, the optical effect can be used to generate shape-related information. Measurements on randomly oriented sperm from three mammalian species using a pair of fluorescence detectors indicate that large shape differences are detectable. Acriflavine-Feulgen stained sperm nuclei are significantly bleached during flow cytometric measurements at power levels routinely used in many flow cytometers. Dual beam studies of this phenomenon indicate it may be useful in detecting abnormally shaped sperm.     

Fluorescent in situ hybridization of the telomere repeat sequence in hamster sperm nuclear structures

The flat, hooked-shaped architecture of the hamster sperm nucleus makes this an excellent model for in situ hybridization studies of the three dimensional structure of the genome. We have examined the structure of the telomere repeat sequence (TTAGGG)_n with respect to the various nuclear structures present in hamster spermatozoa, using fluorescent in situ hybridization. In fully condensed, mature sperm nuclei, the telomere sequences appeared as discrete spots of various sizes interspersed throughout the volume of the nuclei. While the pattern of these signals was non-random, it varied significantly in different nuclei. These discrete telomere foci were seen to gradually lengthen into linear, beaded signals as sperm nuclei were decondensed, in vitro, and were not associated with the nuclear annulus. We also examined the relationship of telomeres to the sperm nuclear matrix, a residual nuclear structure that retains the original size and shape of the nucleus. In these structures the DNA extends beyond the perimeter of the nucleus to form a halo around it, representing the arrangement of the chromosomal DNA into loop domains attached at their bases to the nuclear matrix. Telomere signals in these structures were also linear and equal in length to those of the decondensed nuclei, and each signal represented part of a single DNA loop domain. The telomeres were attached at one end to the nuclear matrix and extended into the halo. Sperm nuclear matrices treated with Eco RI retained the telomere signals. These data support sperm DNA packaging models in which DNA is coiled into discrete foci, rather than spread out linearly along the length of the sperm nucleus.     

Effect of pH value of freeze-drying solution on the chromosome integrity and developmental ability of mouse spermatozoa

The nuclei of freeze-dried mouse spermatozoa are able to retain their chromosome integrity and developmental potential. To optimize the conditions of freeze-drying, we examined whether pH values of the freeze-drying solution affect the chromosome integrity and developmental potential of sperm nuclei. The sperm freeze-drying solution we used contained a high concentration (50 mM) of calcium-chelating EGTA. Sperm chromosomes were examined at the metaphase of the first mitosis after injection of freeze-dried spermatozoa into matured oocytes. The developmental potential of sperm nuclei was assessed by examining the development of fetuses in midgestation. The results showed that both sperm chromosomes and sperm developmental potential are maintained better when the freeze-drying solution was slightly alkaline (pH 8.0) rather than near neutral or acidic (pH 7.4-6.0). The data indicated that the chromosome integrity and developmental ability of mouse spermatozoa are affected by the pH value of freeze-drying solution.     

Light and electron microscopical observations on sperm cells of Guizotia abyssinica (Asteraceae)

The sperm cells of Guizotia abyssinica were studied during pollen development by light microscopy and at anther dehiscence by transmission electron microscopy. During development, the nuclei change shape from spherical to elongate, thread-like and banded. They are straight or folded, and rarely spiral-shaped when present in the pollen tube. Electron microscopy disclosed that the elongated sperm nuclei are apparently lobate. Intermittently, they are constricted and attenuated or convoluted. The major part of the sperm chromatin is condensed and peripheral, while a minor part is dispersed and central. The scanty sperm cytoplasm contains mitochondria and starch granules. The cytoplasm is mainly restricted to spaces adjoining constricted, lobed and convoluting nuclear sites. Some cytoplasmic patches become embayed in the nucleus at these sites. The periplasm bordering the sperm cells may originate from lucid dilations of the lumen between the plasma membranes of the sperm and vegetative cells. The periplasm is sometimes partially or entirely surrounded by double-membraned endoplasmic reticulum. Folded sperm cells with less coherent periplasm possibly represent a late stage preceding discharge into the pollen tube. The sperm cells always precede the vegetative nucleus into the pollen tube.     

Continuous live-dead discrimination of ram sperm during freezing

Use of the dye amaranth (Color Index 16185) as a supravital stain for ram sperm is described. At a concentration of 0.4% in diluted semen, the dye was completely excluded by motile sperm and had no effect on sperm motility. The nuclei of immotile sperm were stained pink by amaranth. The decrease in sperm motility during 24-h storage at 5°C was accompanied by a corresponding increase in stained sperm nuclei. The presence of the dye during freezing had no effect on sperm cryosurvival but tended to reduce sperm motility during post-thaw incubation. Insemination of ewes with fresh semen containing amaranth or with semen frozen in the presence of amaranth resulted in pregnancies in 7/10 ewes in each group, compared to 6/9 in the case of ewes inseminated with fresh semen without dye.     

In vitro decondensation of the sperm chromatin in Holothuria tubulosa (sea cucumber) not affecting proteolysis of basic nuclear proteins

Sea urchin and sea star oocyte extracts contain proteolytic activities that are active against sperm basic nuclear proteins (SNBP). This SNBP degradation has been related to the decondensation of sperm chromatin as a possible model to male pronuclei formation. We have studied the presence of this proteolytic activity in Holothuria tubulosa (sea cucumber) and its possible relationship with sperm nuclei decondensation. The mature oocyte extracts from H. tubulosa contain a proteolytic activity to SNBP located in the macromolecular fraction of the egg‐jelly layer. SNBP degradation occurred both on sperm nuclei and on purified SNBP, histones being more easily degraded than protein Ø_o (sperm‐specific protein). SNBP degradation was found to be dependent on concentration, incubation time, presence of Ca²⁺, pH, and this activity could be a serine‐proteinase. Thermal denaturalization of the oocyte extracts (80°C, 10–15 min) inactivates its proteolytic activity on SNBP but does not affect sperm nuclei decondensation. These results would suggest that sperm nuclei decondensation occurs by a mechanism different from SNBP degradation. Thus, the sperm nuclei decondensation occurs by a thermostable factor(s) and the removal of linker SNBP (H1 and protein Ø_o) will be a first condition in the process of sperm chromatin remodeling.     

Interspecies differences in the stability of mammalian sperm nuclei assessed in vivo by sperm microinjection and in vitro by flow cytometry

To assess the structural stability of mammalian sperm nuclei and make interspecies comparisons, we microinjected sperm nuclei from six different species into hamster oocytes and monitored the occurrence of sperm nuclear decondensation and male pronucleus formation. The time course of sperm decondensation varied considerably by species: human and mouse sperm nuclei decondensed within 15 to 30 min of injection, and chinchilla and hamster sperm nuclei did so within 45 to 60 min, but bull and rat sperm nuclei remained intact over this same period of time. Male pronuclei formed in oocytes injected with human, mouse, chinchilla, and hamster sperm nuclei, but rarely in oocytes injected with bull or rat sperm nuclei. However, when bull sperm nuclei were pretreated with dithiothreitol (DTT) in vitro to reduce protamine disulfide bonds prior to microinjection, they subsequently decondensed and formed pronuclei in the hamster ooplasm. Condensed rat spermatid nuclei, which lack disulfide bonds, behaved similarly. The same six species of sperm nuclei were induced to undergo decondensation in vitro by treatment with DTT and detergent, and the resulting changes in nuclear size were monitored by phase-contrast microscopy and flow cytometry. As occurred in the oocyte, human sperm nuclei decondensed the fastest in vitro, followed shortly by chinchilla, mouse, and hamster and, after a lag, by rat and bull sperm nuclei. Thus species differences in sperm nuclear stability exist and appear to be related to the extent and/or efficiency of disulfide bonding in the sperm nuclei, a feature that may, in turn, be determined by the type(s) of sperm nuclear protamine(s) present.     

Sea urchin sperm chromatin structure as probed by pancreatic DNase I: Evidence for a novel cutting periodicity

Chromatin from mature sea urchin spermatozoa is highly compacted and composed almost entirely of DNA and the five histones, although sperm H1, H2A, and H2b histones differ from those found in embryo or somatic cell nuclei. Release of acid-soluble DNA during pancreatic DNase I digestion is 20-fold slower from sperm nuclei than from embryonic nuclei. Following DNase I digestion, most sperm nuclear DNA remains at high molecular weight, although there appears to be some release of 10 base oligomer fragments. Size analysis of the higher molecular weight DNA reveals a series of fragments that indicate a cutting periodicity of approximately 500 base pairs. This pattern remains when electrophoretic separation is carried out under denaturing conditions. The 500 base pair cleavage pattern was not detected in digestions of embryonic nuclei. Nucleosomes reconstituted with fractionated core histones from sperm gave, upon digestion, a characteristic 10 base “ladder,” with no resistant high molecular weight DNA. Micrococcal nuclease and DNase II digested sperm nuclei to produce DNA fragments with a calculated repeat length of 248 ± 3 and 246 ± 6 base pairs, respectively. The structural basis for the 500 base pair cutting periodicity in sperm nuclei may reside in the unique sperm H1 histone.     

利用非洲爪蟾精子染色质和卵提取物在体外重建细胞核

应用非洲爪蟾去膜精子染色质和卵提取物成功地进行了细胞核本外重建。当精子染色质加入卵提取物后,首先发生染色质去浓缩作用,染色质整体结构膨胀;膜泡在膨胀的染色质外周聚集并逐渐彼此融合成双层膜;核孔复合体以某种未知方式组装入双层膜而形成核膜结构,并逐渐完全覆盖膨大的染色质,最终形成典型的间期核结构。     

Nuclear and manchette development in spermatids of normal and azh/azh mutant mice

Germinal cells or nuclei with attached cytoskeletal elements were prepared from the testes and epididymides of normal mice and mice homozygous for the recessive azh mutation, which results in abnormal sperm heads. To make observations, we utilized phase-contrast microscopy, immunofluorescence microscopy with antitubulin antibodies, and a direct-view stereo electron microscope system developed by A. Cole. Sperm nuclei, tails, manchettes, and other cytoskeletal structures were studied at various stages of development. The tail architectures were similar in the normal and mutant forms, but the shape of the heads at the attachment regions were markedly different. Normal sperm nuclei were very flat, whereas the posterior regions of mutant nuclei were tapered cylinders. The manchette, an organized microtubular structure that girdles the posterior region of the spermatid nucleus, differed in size and configuration between normal and mutant forms. In normal midstage spermatids, the manchette microtubules extended outward at a 45 degree angle from the long axis of the flattened head, whereas in mutant spermatids, the microtubules formed tapered cylinders around the long axis of the caudal part of the nucleus. Radical differences in head shapes between normal and mutant sperm could be related, in part, to the manner in which manchettes formed and matured on the spermatids.     

Protamine dissociation before decondensation of sperm nuclei during in vitro fertilization of pig oocytes

The correlation between morphological changes and the dynamics of protamine in boar sperm chromatin during in vitro fertilization of pig oocytes matured in vitro was assessed. For this purpose, protamine was purified from boar sperm nuclei and an antiserum against protamine was developed. After affinity purification, the antiserum reacted exclusively with boar protamine during western blotting, showing no crossreactivity with core histones. Immunohistochemical evaluation revealed that only fully developed spermatid nuclei in boar testes stained strongly with the antiserum. When pig oocytes matured in vitro were fertilized in vitro, sperm penetration was observed in 37% of oocytes at 2 h after insemination and the penetration rate increased to 99% by 5 h after insemination, accompanied by an increase in polyspermic penetration. Paraffin wax sections of the inseminated oocytes were examined by immunohistochemical analysis with the antiserum. The proportion of condensed sperm nuclei that reacted with the antiserum was 87% of the sperm nuclei that penetrated by 2 h after insemination, and this decreased to 20 and 13% at 3 and 5 h after insemination, respectively. However, none of the decondensing sperm nuclei or male pronuclei reacted with the antiserum during the entire insemination period. These results indicate that a specific antiserum against boar protamine can be raised and, using this serum, it has been demonstrated that protamine is dissociated from boar sperm nuclei before decondensation during in vitro fertilization.