Biomedical subjects

W G Breed

Publications and source records attributed to W G Breed.

At least 37 records · Page 2Linked to original sources

PubMed1997

Isolation of histones and related chromatin structures from spermatozoa nuclei of a dasyurid marsupial, Sminthopsis crassicaudata.

The spermatozoa of a dasyurid marsupial, Sminthopsis crassicaudata, have two distinct nuclear regions: uniformly electron-dense chromatin (C1) in the interior and fissured chromatin (C2) at the periphery. To investigate whether the differences in morphology are due to incorporation of different packaging proteins, spermatozoa nuclear proteins were characterised by acetic acid-urea polyacrylamide gel electrophoresis (PAGE) and fractionated by reverse-phase high-pressure liquid chromatography (HPLC). The main protein component was protamine I, but a complete histone complement (H1, H2A, H2B, H3, and H4) was also detected. Immunocytochemistry showed localisation of H4, H2B, and H2A histones to the periphery of the nuclei, a region that corresponded to the C2 chromatin. The fissures in the chromatin of this region disappeared following incubation with fish protamines, indicating that the nucleohistone C2 region may be incompletely condensed relative to nucleoprotamines. This observation is consistent with the view that 60% of phosphodiester charges remain negative in nucleohistone DNA, whereas all DNA charges are neutralised in highly compact nucleoprotamines. Treatment of spermatozoa with micrococcal nuclease showed that the C1 chromatin was resistant to digestion, whereas the C2 region was cleaved into 30- to 38-nm agglomerates and 11-nm nucleosomal-size structures. Thus, this study demonstrates that spermatozoa nuclei of this marsupial species contain peripherally localised histones, and the nucleohistone chromatin accounts for the different morphology of the C2 region compared with the rest of the nucleus.

Amino Acid Sequence↗

PubMed1997

Antiserum to the egg coats of the fat-tailed dunnart (Marsupialia, Dasyuridae) cross-reacts with egg coats of other marsupial and eutherian species.

We are examining the extracellular coats of the brush-tailed possum as a possible target for an immunocontraceptive vaccine for biocontrol of this pest species in New Zealand. In this study we have compared the composition of the extracellular coats of the fat-tailed and stripe-faced dunnarts, brush-tailed possum, domestic rabbit, and laboratory mouse using histochemistry, immunocytochemistry, and immunofluorescence. The histochemistry of the luminal epithelium of the oviduct and mucoid coats of the marsupials and rabbit indicated that they contain acidic glycoproteins. Immunofluorescence showed that polyclonal antiserum raised against the extracellular coats of the oocyte and early embryo of the fat-tailed dunnart, cross-reacted with the extracellular coats of the oocytes of all five species. These results suggest that there are common epitopes on the extracellular coats of oocytes and early embryos of distinctly related therian species. Further work to characterise these proteins is required to determine whether there is close homology between the oviductal glycoproteins of these species.

Animals↗

PubMed1997

Atomic force microscopy and cytochemistry of chromatin from marsupial spermatozoa with special reference to Sminthopsis crassicaudata.

Atomic force microscopy (AFM) of the nuclear topology of spermatozoa from two marsupial species, Smithopsis crassicaudata and Trichosurus vulpecula was investigated to determine the structural organisation of the chromatin subunits. That of the former species is of special interest as it has a peripheral nucleohistone region (C2) as well as a nuclease-resistant, nucleoprotamine core region (C1). Atomic force microscopy showed that the C2 region contained clusters of 120-160 nm nodules, whereas the C1 region exhibited smaller 50-80 nm nodules. The spermatozoa nuclei of Trichosurus, which has mainly nucleoprotamines, contained higher order chromatin structures of similar size to those from the C1 region of Sminthopsis. This study shows that nucleohistones and nucleoprotamines of marsupial sperm form distinct higher order conformations. For the second part of this work, the chromatin density and affinity for cationic stains of Sminthopsis spermatozoa were determined. Spermatozoa were observed with the transmission electron microscopy (TEM) either unstained or stained with metal salts. In the unstained specimens, the C2 nucleohistone region appeared more electron-lucent than the C1 region. When large cations such as uranyl were used, the reverse situation was observed. Therefore, the electron-dense appearance of the C2 chromatin in conventionally stained material may be due to the presence of net negative DNA charges that attract the cations used for EM staining, whereas the C1 chromatin may lack excess DNA negative charges that attract these stains and thus appears less electron-dense.

Animals↗

PubMed1997

Unusual chromatin structural organization in the sperm head of a murid rodent from southern Africa: the red veld rat, Aethomys chrysophilus type B.

The structural organization of the chromatin of cauda epididymal spermatozoa of the red veld rat Aethomys chrysophilus type B was investigated by fluorescence microscopy after staining with DNA specific dyes and by transmission electron microscopy after incubation with Triton X100, dithiothreitol, and SDS. Staining with DNA dyes showed variation in intensity of fluorescence of the sperm chromatin, with an anterior spherical region staining far more intensely than the surrounding chromatin. Transmission electron microscopy of these spermatozoa indicated that this region was composed of cords and fibres. This chromatin region dispersed more readily than the surrounding chromatin when spermatozoa were incubated with the detergents, and it is suggested that, unlike the rest of the sperm chromatin, it may be a histone-rich region, with protamine(s) being either scarce or absent.

Animals↗

PubMed1996

Successful embryo transfer in a small dasyurid marsupial, Sminthopsis crassicaudata.

A technique for the transfer of early embryos from the uterus of donors to recipient females of a small dasyurid marsupial, Sminthopsis crassicaudata , is described. When the recipients were killed 3 to 5 d later, a few expanded blastocysts were found to be present in the uteri of most of the females provided that corpora lutea were present in the ovaries. This study demonstrates successful embryo transfer in a small marsupial species and supports the view that secretion of the endometrium due to luteal activity may be necessary for development of the expanded unilaminar blastocyst stage.

Journal Article↗

PubMed1996

Ultrastructure of nuclear condensation and localization of DNA and proteins in spermatozoa of a dasyurid marsupial, Sminthopsis crassicaudata.

In the dasyurid marsupial, Sminthopsis crassicaudata, the mature spermatozoon has an inner homogeneous (C1) and a peripheral indented (C2) region. Using DNase-gold conjugates, and biotinylated genomic DNA probes, DNA was found to occur in both C1 and C2 regions. The morphogenesis of the spermatozoon nucleus was investigated using ultrastructural and cytochemical studies. Spermiogenesis was divided into 15 steps. By step 10, condensation of the C1 region was complete, and at the caudal extremity of the spermatid nucleus, the nuclear envelope enclosed an electron-lucent space. This space and the surrounding nuclear envelope became very enlarged at step 11. At this stage, a plate of approximately 70 nm in thickness was present along the caudal segment of the C1 region; this "nuclear mantle" did not bind DNase-gold conjugates but stained for lysine-rich proteins using alcoholic phosphotungstic acid. Chromatin condensation resumed at step 12 with the appearance of spherical chromatin structures peripheral to the C1 chromatin. These structures then partially coalesced and the indentations of the C2 region were observed. The expanded nuclear envelope at the caudal extremity persisted in caput epididymal spermatozoa. Spherical inclusions within it did not bind to DNase-gold conjugates but stained for lysine-rich proteins. As the sperm traveled down the epididymis, these inclusions amassed near the nuclear pores and were then removed from the nucleus. In addition, the nuclear mantle was found to have disappeared by the time the spermatozoa reached the corpus epididymidis.

Animals↗

PubMed1996

The marsupial shell membrane: an ultrastructural and immunogold localization study.

In the dasyurid marsupial, Sminthopsis crassicaudata, as the oocytes/embryos travel down the female reproductive tract two extracellular coats, the mucoid and shell membrane, come to surround them. Embryos recovered from the oviduct have a mucoid coat but no shell membrane which is only found surrounding uterine embryos. Initially, the shell membrane has a compact granular consistency but it later thins and becomes fibrous in texture with fibres oriented mainly in the plane of the membrane. Immunogold labelling with polyclonal antibodies raised against the extracellular coats was employed to determine the location and ultrastructural appearance of the secretory granules which contain mucoid and shell membrane precursors. Secretory granules in the luminal epithelium of the ampulla of the oviduct are of irregular electron density, while those in the isthmus are electron-dense and homogeneous. Both types give rise to the mucoid coat. Secretory granules in the epithelia of the utero-tubal junction and some endometrial glands are electron-lucent and contain some flocculent material which, after exocytosis, gives rise to the shell membrane.

Animals↗

PubMed1996

Egg maturation and fertilization in marsupials.

This brief review summarizes our knowledge of the morphological events that are associated with oocyte maturation and fertilization in marsupials in which it has been suggested that there are marked differences from eutherians in both the developmental timetable of oocyte maturation and in some of the processes associated with sperm-egg interaction. Most of the data have been obtained from studies on four species: Monodelphis domestica, Sminthopsis crassicaudata, Sminthopsis macroura, and Trichosurus vulpecula. Differences between the species have been described for: (1) the arrangement of 'yolk' in the oocyte cytoplasm; (2) the time of formation of cortical granules: (3) the mode of sperm penetration through the zone pellucida: (4) the sperm membrane involved in sperm-egg fusion: (5) the fate of inner acrosomal and sperm plasma membranes: and (6) the rapidity of sperm chromatin decondensation in the ooplasm. Such differences suggest considerable variation in these processes between different marsupial species although some of the variation described may be due to technical differences in the obtaining of the data. Thus, whether there are fundamental differences between the two major extant infraclasses of mammals, marsupials and eutherians, in some of the processes associated with fertilization is conjectural at the present time. The interspecific variation in the results obtained cautions one in extrapolating from observations on one or two 'model' species to the infraclass as a whole: a conclusion that might not, on reflection, be too surprising bearing in mind the long and separate evolutionary history of the major extant marsupial lineages.

Animals↗

PubMed1996

Variation in ultrastructure of mucoid coat and shell membrane secretion of a dasyurid marsupial.

In the dasyurid marsupial Sminthopsis crassicaudata, the shell membrane of cleaving embryos has a compact granular structure but becomes fibrous around blastocysts. Polyclonal antibodies were raised against the extracellular coats, mucoid and shell membrane, of oocytes and early embryos. Immunogold cytochemistry resulted in labelling of secretory granules in the epithelia of both the ampulla and isthmus of the oviduct, although the secretory granules of these two regions differed in their ultrastructural appearance. Those in the ampulla were heterogeneous with areas of varying electron density, whereas those in the isthmus were electron dense and homogeneous. Shell membrane precursors were found in secretory granules in the epithelia of the uterotubal junction and endometrial glands and were electron lucent.

Animals↗

PubMed1996

Effect of cooling and cryopreservation on sperm motility and morphology of several species of marsupial.

The effects of long-term cooling and freezing on sperm motility are described for six marsupial species: the fat-tailed dunnart, koala, brushtail possum, long-footed potoroo, northern brown bandicoot and ring-tailed possum. The effects of up to eight days of cooling at 4 degrees C on the motility of dunnart spermatozoa and the effect of cryopreservation on spermatozoa of the other species were determined. The cryoprotectant used was a Tris-citrate-fructose-egg yolk-glycerol diluent. The percentage and rating of sperm motility, and sperm structure, as determined by light microscopy, were investigated. Sperm motility in the fat-tailed dunnart was retained for up to six days when cooled to 4 degrees C, suggesting that sperm from this species have some degree of tolerance to cold shock. After this time, however, the percentage of motile spermatozoa and their motility rating declined. In all species except the fat-tailed dunnart, reinitiation of motility following cryopreservation occurred across a range of glycerol concentrations (4-17%). Cryoprotectant containing 6% and/or 8% glycerol resulted in little change of motility rating or of the percentage of live sperm after thawing, although there was some decline in the percentage of motile sperm. The unusual structural and motility characteristics of dunnart spermatozoa may account for the lack of success of sperm cryopreservation in this species.

Animals↗

PubMed1996

Changes in structure of the trophectoderm of a marsupial in Mid-pregnancy up to the time of implantation.

Pre- and peri-implantation embryos of the dasyurid marsupial Sminthopsis crassicaudata were examined for morphological differentiation of the trophectoderm. The cells of unilaminar blastocysts were all squamous and stained intensely with toluidine blue. In bilaminar blastocysts and embryos at the early embryonic-disc stage, the trophectoderm was similar in appearance to, but stained more lightly than, the underlying endoderm. Trophoblast differentiation did not appear to occur until the mesoderm had begun to migrate between the trophoblast and endoderm beyond the embryonic disc. At this stage, trophoblasts had three distinct morphologies: (1) vacuolated, tall and columnar cells in the trilaminar region; (2) large cuboidal cells in the adjacent bilaminar region; and (3) squamous cells in the abembryonic pole of the bilaminar region. These variations in cell structure correlate with differences in subsequent functional activity in these three regions of the yolk sac placenta.

Animals↗

PubMed1996

Changes in distribution of labile zinc in mouse spermatozoa during maturation in the epididymis assessed by the fluorophore Zinquin.

The Zn(II)-specific fluorophore Zinquin was used to determine the regional distribution of free or loosely-bound Zn(II) in mouse spermatozoa. Spermatozoa from the testes exhibited bright fluorescence over the entire head; those from the caput epididymides generally fluoresced more brightly in the post-acrosomal region; and spermatozoa from the caudae epididymides fluoresced less brightly, with foci of fluorescence over the sperm head which were lost after extraction with Triton X-100 and hence appeared to be membrane-associated. Treatment of cauda sperm with sodium dodecyl sulfate resulted in a bright uniform Zinquin fluorescence in the heads, similar to that observed in caput sperm, indicating that the two types of sperm have similar amounts of head Zn(II) but that the availability of Zn(II) for binding Zinquin is different. By contrast, the intensity of tail fluorescence was similar in spermatozoa from different regions of the male reproductive tract and was largely unaffected by Triton X-100 extraction, consistent with an intracellular location. Similar differences were observed between caput sperm and cauda sperm in the rat. It is concluded that visualization and measurement of free or loosely-bound Zn(II) in subcellular compartments of spermatozoa should facilitate investigation of the role of this metal in the development and function of spermatozoa and abnormalities that might accompany infertility and Zn(II) deficiency.

Animals↗

PubMed1995

Ultrastructure and motility of spermatozoa in macropodid and potoroidid marsupials.

In order to gain some understanding of the significance of the morphological features of spermatozoa within the Macropodoidea, the motility of spermatozoa from two macropodids (Petrogale xanthopus and Dendrolagus matschiei) and the motility, number and distribution of spermatozoa from three potoroidids (Aepyprymnus rufescens, Bettongia penicillata and Potorous tridactylus) were examined. Sperm were collected by electro-ejaculation or from the cauda epididymides. Epididymides from the potoroidids were divided into 12 regions. One epididymidis per animal was fixed for light and transmission electron microscopy and, on the contralateral side, the number of sperm, their distribution and motility were determined. In general, spermatozoa of all five species differed markedly from one another in head and flagella dimensions. Spermatozoa from B. penicillata and P. tridactylus were significantly longer and broader and had a smaller acrosome relative to head length, and there was a radial displacement of dense fibres. They also progressed more rapidly in standard culture media. Spermatozoa from at least three species were able to alter their motility pattern in vitro as media viscosity increased. Sperm movement in all species appeared to be restricted to one plane and showed no evidence of rotation, whereas lateral head displacement was often pronounced; there was no evidence of a sinusoidal mode of progressive motility. Testicular and epididymal sperm numbers in A. rufescens and P. tridactylus were relatively high (approximately 17.5-50 x 10(6)). In A. rufescens, approximately 69% of all epididymal sperm were located in the cauda epididymidis compared with approximately 40% in P. tridactylus. This study demonstrated that marked radial displacement of the dense fibres is probably closely associated with the ability to develop a sinusoidal mode of progressive movement, and that this feature of the sperm tail structure is not just linked with sperm size. Sperm size, however, is associated with sperm velocity.

Animals↗

PubMed1995

Ultrastructure and motility of spermatozoa in the male reproductive tract of perameloid marsupials.

The number, distribution, maturation, motility and ultrastructure of spermatozoa from both northern (Isoodon macrourus) and southern (Isoodon obesulus) brown bandicoots were examined. One epididymidis per animal was fixed for light microscopy and transmission electron microscopy, and the contralateral side was used for the determination of sperm number, distribution and motility. Sperm form was similar between the two species. Approximately 56 x 10(6) testicular sperm and 100 x 10(6) epididymal sperm per side were present in I. macrourus, about 60% of which were in the caudal region. Initiation of sperm nuclear rotation and loss of the cytoplasmic droplet was first observed in distal caput or proximal corpus segments along with slow progressive motility. In these sperm, dislocation and anterior movement of the sperm neck from the implantation fossa and the modification of the distal margins of the sperm acrosome were evident. Motility of cauda epididymidal spermatozoa was rapid and coordinated, movement was restricted to one plane, and lateral head displacement was marked. As media viscosity increased, sperm velocity decreased, as did the amplitude of the tail beat, its frequency, and lateral head displacement but, in viscous mineral oil and mixtures of media and prostatic exudate, extremely rapid sinusoidal motility occurred. This study has detailed unusual morphological changes in bandicoot sperm during epididymal maturation and has shown that, although bandicoot sperm differ morphologically from those of the dasyurids, particularly in relation to head-tail orientation and tail ultrastructure, they exhibit similar motility.

Acrosome↗

PubMed1995

The fluorescent dye 3, 3' dihexyloxacarbocyanine iodide selectively stains the midpiece and apical region of the heads of murid rodent spermatozoa.

Fluorescence microscopy of caudal epididymal spermatozoa stained with 3, 3' dihexyloxacarbocyanine iodide (DiOC6(3)) showed intense fluorescence along the concave surface of the apical hook of spermatozoa of Rattus species and along the upper concave margin of the sperm head in Mus musculus. In the spermatozoa of Hydromys chrysogaster, Melomys cervinipes, and Pseudomys australis, the two ventral processes also fluoresced brightly. In P. australis, fluorescence in the apical hook of sperm heads was largely localized to its upper and lower surfaces. The sperm of N. alexis did not show consistent positive fluorescence. The localization of fluorescence in these spermatozoa after staining with DiOC6(3) was mainly restricted to regions where a large accumulation of perinuclear theca material lies beneath the plasmalemma. The reason for this remains to be determined, but DiOC6(3) may be useful for quickly demonstrating areas of abundant perinuclear thecal material in sperm heads of eutherian mammals by light microscopy.

Animals↗

PubMed1994

Origin of the oocyte shell membrane of a dasyurid marsupial: an immunohistochemical study.

Polyclonal antibodies were raised against the extracellular coats (mucoid and shell membrane) of unfertilized oocytes and early embryos of a dasyurid marsupial, the fat-tailed dunnart (Sminthopsis crassicaudata). Indirect immunofluorescence was used to test the specificity of the antibodies to the shell membrane. Streptavidin/biotin immunoperoxidase cytochemistry revealed that precursors of the tertiary egg membranes are secreted by the luminal epithelium of the ampulla, isthmus (including the crypts), utero-tubal junction, and adjacent endometrial glands. Histochemistry distinguished between the sites of mucoid and shell membrane precursor secretion. The mucoid coat stained positively with Alcian blue at pH 1.0 and 2.5, with Alcian blue at pH 0.2 after performic acid oxidation, and with PAS which was amylase resistant. Some of the luminal epithelial cells of the ampulla and isthmus, as well as its crypts, also stained positively by these histochemical methods but the luminal epithelium of the utero-tubal junction and endometrial glands were negative for all methods. The shell membrane did not stain with any of the above methods nor with dihydroxy-dinapthyl-disulphide (DDD) or ferric ferricyanide but it was eosinophilic and stained positively with the red cytoplasmic stain of Masson's trichrome. Therefore, it is concluded that shell membrane precursors are secreted by the luminal epithelium of the utero-tubal junction, adjacent glands, and by scattered glands in the anterior region of the uterus but not by any cell population of the oviduct.

Animals↗

PubMed1994

Embryonic-maternal cell interactions at implantation in the fat-tailed dunnart, a dasyurid marsupial.

BACKGROUND: In marsupials implantation occurs about two-thirds the way through the short gestation before which time the embryo is surrounded by the permeable shell membrane which prevents physical contact between the trophoblast and uterine epithelium. Although the trophoblast has been shown to be invasive to varying degrees in several species of marsupials, the ultrastructure of the embryonic-uterine cell interactions at the time of implantation has not been described in this group. METHODS: Thick plastic sections and transmission electron microscopy were employed to investigate the cellular interactions at implantation in the fat-tailed dunnart (Sminthopsis crassicaudata), a dasyurid Australian marsupial. RESULTS: Our results show that epithelial penetration begins when the embryo is at the late presomite/early somite stage. In the trilaminar region of the yolk sac (TYS), trophoblast cells adjacent to the embryo form desmosomes with uterine epithelial cells and also appear to fuse with them to form hybrid cells, the cytoplasm of which resembles that of trophoblast. Later in the TYS, as the placenta develops, trophoblast microvilli and larger cell processes invaginate, and interdigitate with, the highly folded maternal epithelium but do not invade it. At this time in the bilaminar, or avascular, yolk sac (BYS), multinucleate trophoblast giant cells (TGCs) from an annular region adjacent to the sinus terminalis intrude between, and possibly fuse with, the maternal epithelium. The invading TGCs spread laterally above the residual basal lamina before migrating into the stroma. CONCLUSIONS: In this species of marsupial at least, the cell interactions at the time of implantation are similar to those seen in some eutherian species despite the fact that the fetal chorion is of yolk sac rather than allantoic origin.

Animals↗

PubMed1994

Unusual nuclear structure of the spermatozoon in a marsupial, Sminthopsis crassicaudata.

The organization of sperm chromatin in the dasyurid marsupial, Sminthopsis crassicaudata, was investigated using various morphological techniques. Transmission electron microscopy indicates two quite distinct chromatin regions became evident late in spermiogenesis with an outer globular region containing blocks of very electron-dense chromatin. Fluorescent light microscopical studies after staining with DNA dyes and 7-amino actinomycin D of testicular, caput, and cauda epididymal spermatozoa showed that this region fluoresced less brightly than the rest of the nucleus, indicating the presence of fewer DNA binding sites. Freeze fracture showed that the chromatin in most of the nucleus had randomly arranged particles of various sizes, but that of the outer region was composed entirely of small particles. This outer region was more resistant to low concentrations of the ionic detergent, SDS, whereas both guanidine hydrochloride and urea together with sodium chloride generally dispersed all the chromatin except that in the outer globular region and in a localized area of the nucleus beneath the acrosome. This study has thus revealed that the outer globular chromatin of these spermatozoa responds differently to ionic detergents and protein denaturing agents and has a different chromatin organization than most of the rest of the nucleus. The significance of these differences remains, however, to be determined.

Animals↗