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PubMed1996

Features of the structural development of the inferior colliculus in relation to the onset of hearing in a marsupial: the northern quoll, Dasyurus hallucatus.

The time course of synaptogenesis and the arrival and myelination of afferent connections were studied in the developing inferior colliculus (IC) of a marsupial, the Northern Quoll, and related to the onset of hearing and patency of peripheral auditory structures in that species. The quoll is born after 3 weeks of intrauterine growth and completes its development in a pouch for a further 80 days before weaning. Synaptic terminals in the IC at 9 days after arrival in the pouch were extremely rare and were associated with very low vesicle numbers. The number of synapses increased smoothly during pouch life, whereas the number of neurons with nucleoli fell over the same time period. The ratio of synapses to cells steadily increased from day 9 to day 63, then rapidly accelerated to day 73; a similar high ratio was observed in adults. Retrograde labeling from the IC of fibers projected from the medullary auditory nuclei, first observed on day 36, became progressively denser during pouch life. Myelination of lateral lemniscal fibers was absent on day 45, extremely sparse on day 54, and clear on day 63. Myelin sheaths were not observed within the IC electron microscopically until day 73. Examination of the peripheral auditory system revealed that until about day 40 the middle ear was fluid-filled, and middle ear structures were spongy. Between days 51 and 63 the middle ear cleared, the eardrum became shiny, and the ear canal became patent. The structural development of the IC is therefore very mature at the time hearing begins (67 days), and the last major anatomical change preceding hearing appears to be the opening of the external ear canal.

Animals↗
PubMed1998

First evidence of diversity in eutherian chiasmatic architecture: tree shrews, like marsupials, have spatially segregated crossed and uncrossed chiasmatic pathways.

In the optic chiasm of mammals, axons either cross the midline to the opposite side of the brain or remain uncrossed. In the eutherian species studied to date, uncrossed axons in the caudal nerve are found in all regions. In the chiasm, they are dispersed through the hemichiasm, with many axons approaching the midline and then turning back to enter the same side of the brain as the originating eye. In marsupials, by contrast, uncrossed axons never approach the midline; instead, they remain grouped in the lateral nerve and chiasm. The impression gained from these data is that there is a major difference in chiasmatic architecture between eutherian and marsupial mammals. Therefore, the mechanisms by which axons choose their route through the chiasm was also thought to differ between the two major groups of mammals. However, the present study shows that the chiasm of a highly visual eutherian mammal, the tree shrew, is similar to that found in marsupials, with uncrossed axons confined to lateral regions and not approaching the midline. However, unlike marsupials, in the tree shrew, optic fascicles in the chiasm are often separated by thick collagen bundles. It is probable that the chiasmatic structure described to date for eutherian mammals is not ubiquitous, as was previously thought, and theories explaining the mechanisms by which axons chose their route through the chiasm during development will have to be expanded.

Animals↗
PubMed1998

Anatomical comparison of the macaque and marsupial visual cortex: common features that may reflect retention of essential cortical elements.

This study identifies fundamental anatomical features of primary visual cortex, area V1 of macaque monkey cerebral cortex, i.e., features that are present in area V1 of phylogenetically distant mammals of quite different lifestyle and features that are common to other regions of cortex. We compared anatomical constituents of macaque V1 with V1 of members of the two principal marsupial lines, the dunnart and the quokka, that diverged from the eutherian mammalian line over 135 million years ago. Features of V1 common to both macaque and marsupials were then compared with anatomical features we have previously described for macaque prefrontal cortex. Despite large differences in overall area and thickness of V1 cortex between these animals, the absolute size of pyramidal neurons is remarkably similar, as are their specific dendritic branch patterns and patterns of distribution of intrinsic axons. Pyramidal neuron patchy connections exist in the supragranular V1 in both the marsupial quokka and macaque as well as in macaque prefrontal cortex. Several specific types of aspinous interneurons are common to area V1 in both marsupial and macaque and are also present in macaque prefrontal cortex. Spiny stellate cells are a common feature of the thalamic-recipient, mid-depth lamina 4 of V1 in all three species. Because these similarities exist despite the very different lifestyles and evolutionary histories of the animals compared, this finding argues for a highly conserved framework of cellular detail in macaque primary visual cortex rather than convergent evolution of these features.

Anatomy, Artistic↗
PubMed1999

Organization of somatosensory cortex in three species of marsupials, Dasyurus hallucatus, Dactylopsila trivirgata, and Monodelphis domestica: neural correlates of morphological specializations.

The organization of somatosensory neocortex was investigated in three species of marsupials, the northern quoll (Dasyurus hallucatus), the striped possum (Dactylopsila trivirgata), and the short-tailed opossum (Monodelphis domestica). In these species, multiunit microelectrode mapping techniques were used to determine the detailed organization of the primary somatosensory area (SI). In the striped possum and quoll, the topography of somatosensory regions rostral (R), and caudal (C) to SI were described as well. Lateral to SI, two fields were identified in the striped possum, the second somatosensory area (SII) and the parietal ventral area (PV); in the quoll, there appeared to be only one additional lateral field which we term SII/PV. Visual and auditory cortices adjacent to somatosensory cortex were also explored, but the details of organization of these regions were not ascertained. In these animals, electrophysiological recording results were related to cortical myeloarchitecture and/or cytochrome oxidase staining. In one additional species, the fat-tailed dunnart (Sminthopsis crassicaudata), an architectonic analysis alone was carried out, and compared with the cortical architecture and electrophysiological recording results in the other three species. We discuss our results on the internal organization of SI in relation to the morphological specializations that each animal possesses. In addition, we discuss the differences in the organization of SI, and how evolutionary processes and developmental and adult neocortical plasticity may contribute to the observed variations in SI.

Animals↗
PubMed1996

Development of hearing and vocalization in a marsupial, the Northern Quoll, Dasyurus hallucatus.

The development of hearing was studied in the Northern Quoll, a nocturnal marsupial carnivore whose young are born into a pouch in an immature state after about 21 days in utero. Startle responses to noise bursts of 105 dB sound pressure level first appeared at 60 days after arrival in the pouch, but only to occasional stimuli; forelimb rather than whole body twitches were evoked. The latter were elicited regularly at 67 days onward. Auditory brainstem response (ABR) audiograms were measured during the period when startle responses first appeared. At 68 days responses were elicited between 1 and 16 kHz with thresholds in excess of 55 dB. At 81-88 days responses occurred over the adult range at lower thresholds than observed in the adult. The waveform of the ABR increased in complexity and decreased in latency during development. Pouch-young emitted acoustically well-defined isolation cells when separated from their mothers. Fourier analysis of these calls revealed peak energy at 8-11 kHz irrespective of the sex of the pouch-young. The isolation call first became evident at about 35 days, when the young were deliberately removed from the nipple, and ceased being emitted beyond about 80 days, some 5 or so days after the eyes open, when the young leave the pouch for substantial amounts of time. The spectrum of the call was similar at all ages within this 45 day period, and its peak frequency correlated closely with the best frequency in the adult quoll's ABR audiogram. At approximately 80 days of age the calls developed major low frequency components, similar to those measured in adult vocalizations, and the contributions of frequencies between 8-11 kHz waned in importance. The results indicate that the onset of hearing in quolls occurs at a relatively early time in the course of their development, but prior to this time the pouch-young emit characteristic isolation calls easily detectable by the mother.

Acoustic Stimulation↗
PubMed1997

Cytochalasin J treatment significantly alters mitotic spindle microtubule organization and kinetochore structure in PtK1 cells.

It has previously been demonstrated that treatment of mitotic PtK1 cells with 10-20 microg/ml cytochalasin J (CJ) blocks or slows chromosome motion and has a significant effect on spindle architecture [Snyder and Cohen, 1995: Cell Motil. Cytoskeleton 32:245-257]. Spindle microtubules (MTs) were shown to reorganize within the spindle domain, with kinetochore MTs (kMTs) reduced in number and non-kinetochore MTs (nkMTs) shown to splay outside the original spindle domain. In some cases, bundles of MTs were shown to be refocused away from the original spindle poles, creating the appearance of a multi-polar spindle. In this paper we use serial section electron microscopy, coupled with computer-assisted reconstruction techniques, to determine the rearrangement of spindle MTs and chromosome position following brief treatments of mitotic cells with 10-20 microg/ml CJ at various stages of mitosis. CJ treatment of prometaphase cells reduces the number of kMTs and the size and organization of the kinetochore lamina. Instead of kinetochore bundles of MTs aligned parallel to one another and running from kinetochore to pole, this class of MTs is highly fragmented. Non-kinetochore MTs are also highly fragmented, usually less than 2 microm long, and remain relatively straight over short distances, with some MTs arranged at an oblique angle to the longitudinal spindle axis. In approximately 30% of cells treated with CJ, the failure of a small number of chromosomes to attach to spindle fibers can be documented. These chromosomes show a significant change in the organization of the kinetochore laminae. Light microscopic analysis of cells treated with CJ reveals loss of chromosome congression, with chromsomes usually located at the periphery of the spindle and some completely detached from the spindle. Cells treated with 10 microg/ml CJ for 10 min and released into tissue culture medium show a resumption of chromosome motion within a few minutes, both during congression and anaphase. Where kMTs are inserted into kinetochores, chromosome motion is seen; where chromosomes fail to attach to the spindle, no chromosome motion is observed. Cells treated in metaphase show a delayed entry into anaphase and a reduced rate of anaphase A, with the arms of some chromosomes remaining in the interzone region. Our results suggest that CJ-sensitive molecules play a role in the organization of spindle MTs, as well as their functional association to kinetochores.

Animals↗
PubMed1998

Unusual cleavage furrows in vertebrate tissue culture cells: insights into the mechanisms of cytokinesis.

In cultures of the epithelial cell lines, PtK2 and LLC-PK, some cells assume unusually large flattened morphologies and, during cell division, produce unusual cleavage furrows. We have microinjected some of these large cells with fluorescent actin and myosin probes to determine how the cell's shape and the position of its mitotic spindle affect the deposition of actin and myosin in the forming cleavage furrow. In cells with two spindles, contractile proteins were recruited not only to the cortex bordering the former metaphase plates but also to the cortex midway between each pair of adjacent nondaughter poles or centrosomes. The furrowing between adjacent poles seen in these cultured epithelial cells conformed to the furrows seen when echinoderm eggs were manipulated into a torus shape so that the poles of two mitotic spindles were adjacent to one another [Rappaport, 1961]. The recruitment of contractile proteins and the formation of furrows between adjacent centrosomes was a function of the distances between them. When adjacent centrosomes were positioned too close together neither contractile protein recruitment nor furrow formation occurred. If a normal-sized spindle was present in a very large cell, fibers of contractile protein assembled in the cortex above the former metaphase plate but they did not extend to the cell periphery, resulting in an inhibition of cytokinesis. In all injected cells, the recruitment of actin and myosin to the cell surfaces could first be detected at mid-anaphase before there was any visible sign of furrowing. Our results suggest that vertebrate cells share common mechanisms for the establishment of the cleavage furrow with echinoderm cells. The results are consistent with a model in which (1) the positions of the centrosomes and their linearly connected microtubules determine the position for the assembly of the cleavage furrow, and (2) the signal arrives at the surface within a few minutes after the initiation of anaphase. We speculate that an interaction of the ends of microtubules from adjacent centrosomes with the cell surface promotes a clustering of integral membrane protein(s) that interact with and target contractile proteins to a position midway between centrosomes where furrowing occurs.

Animals↗
PubMed1999

Cyclical changes in epithelial cells of the vaginal cul-de-sac of brushtail possums (Trichosurus vulpecula).

The aim of this study was to describe and quantify the changes that occur in cul-de-sac tissue, in particular to epithelial cells and their constituents, at specific stages of the estrous cycle in the brushtail possum. Stereological techniques were used to quantify changes in cul-de-sac epithelial cells collected at four stages of the estrous cycle; the time of removal of pouch young (RPY; n = 5), of initial follicle development (n = 5), of preovulatory follicle formation (n = 5), of midluteal stage (n = 4), and again at RPY (n = 5) after completion of the experiment to examine for any effects due to season or time. Tissue was weighed and processed for light microscopy, transmission electron microscopy, and stereological analysis. Cul-de-sac epithelial cell volume increased approximately 17-fold at the time of preovulatory follicle formation compared with that at the time of RPY, before declining (approximately four-fold greater than at RPY) during the midluteal phase. Epithelial cell volume enlargement was correlated strongly with the size of the preovulatory follicle present, and maximum size was coincidental with the formation of extracellular spaces and projection of cell processes between lateral cell membranes. Maximum cell volume was associated with an approximate 25-fold and six-fold increase in cytoplasmic and nuclear volume, respectively. Enlargement of the epithelial cells coincided with an increase in cytoplasmic organelle numbers, microvilli prominence, and accumulation of secretory vesicles. In the possum, the cul-de-sac epithelial cell undergoes phenomenal remodelling during the estrous cycle to accommodate an approximate 17-fold increase in volume. This increase in cell volume is coincident with morphological changes characteristic of secretory activity and appears to be under estrogen regulation.

Animals↗
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↗
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↗
PubMed1998

Characterisation of fibrous sheath and midpiece fibre network polypeptides of marsupial spermatozoa with a monoclonal antibody.

In this study cytoskeletal antigens common to brushtail possum and tammar wallaby spermatozoa were characterised using a monoclonal antibody (PSA-10). Using indirect immunofluorescence, the PSA-10 antibody detected antigens predominantly associated with the midpiece and principal piece of mature, permeabilised marsupial spermatozoa. The principal piece determinant, shared by a variety of other species, was found to arise in the marsupial testis. Midpiece localisation of the PSA-10 epitope was detected only in marsupial spermatozoa and shown to arise in the epididymis. Immunogold labelling demonstrated that the PSA-10 antigens were predominantly associated with the fibrous sheath and midpiece fibre network of both possum and wallaby spermatozoa. Western blotting suggested that two major possum and wallaby sperm polypeptides of 158 and 182 kDa were associated with the midpiece fibre network, a cytoskeletal structure unique to marsupial spermatozoa. A 32 kDa polypeptide was associated with the principal piece fibre network and/or fibrous sheath. The finding that these marsupial sperm cytoskeletal proteins share a common linear epitope suggests that they share some sequence similarity. The midpiece fibre network of marsupial sperm, like the fibrous sheath, has been proposed to have a structural role in providing passive stiffening for the flagellum (Harding et al., 1975, 1979; Olsen, 1975). The PSA-10 monoclonal antibody may provide a tool for comparative studies of mammalian sperm cytoskeletal proteins, particularly the marsupial midpiece fibre network. It may also allow the formation of this unique marsupial cytoskeletal structure, and its fate during the fertilisation process, to be followed by immunological means.

Animals↗
PubMed1999

Isolation and characterisation of a cDNA encoding a zona pellucida protein (ZPB) from the marsupial Trichosurus vulpecula (brushtail possum).

We have cloned a cDNA containing the entire coding sequence of a marsupial (the brushtail possum, Trichosurus vulpecula) zona pellucida protein (ZPB). The open reading frame of 1,581 nt is predicted to encode a ZPB polypeptide of 527 amino acids which contains 20 cysteine residues, 7 potential N-linked glycosylation sites, a potential N-terminal signal peptide and a potential C-terminal trans-membrane domain, preceded by a furin proteolytic processing signal. Sequence comparisons between possum ZPB and orthologous polypeptides from 7 eutherian species and from Xenopus laevis, reveal the existence of a high degree of sequence similarity, particularly in the central portion of the molecule. Cysteine residues are highly conserved, and all nine species possess potential N-terminal signal peptide sequences and C-terminal trans-membrane domains of approximately the same length. In situ hybridisation revealed that expression of ZPB was restricted to oocytes of primordial and primary follicles of adult possums; no expression was detected in the surrounding granulosa cells. The broad conservation of ZPB sequence, structure and expression over a wide range of mammalian species, revealed by our studies, makes it unlikely that these features account for the different properties of the marsupial and eutherian zona pellucidae.

Amino Acid Sequence↗