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Biomedical subjects

L Selwood

Publications and source records attributed to L Selwood.

At least 37 records · Page 2Linked to original sources
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

There is no highly conserved embryonic stage in the vertebrates: implications for current theories of evolution and development.

Embryos of different species of vertebrate share a common organisation and often look similar. Adult differences among species become more apparent through divergence at later stages. Some authors have suggested that members of most or all vertebrate clades pass through a virtually identical, conserved stage. This idea was promoted by Haeckel, and has recently been revived in the context of claims regarding the universality of developmental mechanisms. Thus embryonic resemblance at the tailbud stage has been linked with a conserved pattern of developmental gene expression - the zootype. Haeckel's drawings of the external morphology of various vertebrates remain the most comprehensive comparative data purporting to show a conserved stage. However, their accuracy has been questioned and only a narrow range of species was illustrated. In view of the current widespread interest in evolutionary developmental biology, and especially in the conservation of developmental mechanisms, re-examination of the extent of variation in vertebrate embryos is long overdue. We present here the first review of the external morphology of tailbud embryos, illustrated with original specimens from a wide range of vertebrate groups. We find that embryos at the tailbud stage - thought to correspond to a conserved stage - show variations in form due to allometry, heterochrony, and differences in body plan and somite number. These variations foreshadow important differences in adult body form. Contrary to recent claims that all vertebrate embryos pass through a stage when they are the same size, we find a greater than 10-fold variation in greatest length at the tailbud stage. Our survey seriously undermines the credibility of Haeckel's drawings, which depict not a conserved stage for vertebrates, but a stylised amniote embryo. In fact, the taxonomic level of greatest resemblance among vertebrate embryos is below the subphylum. The wide variation in morphology among vertebrate embryos is difficult to reconcile with the idea of a phyogenetically-conserved tailbud stage, and suggests that at least some developmental mechanisms are not highly constrained by the zootype. Our study also highlights the dangers of drawing general conclusions about vertebrate development from studies of gene expression in a small number of laboratory species.

Amphibians↗
PubMed1997

Early cleavage to formation of the unilaminar blastocyst in the marsupial Antechinus stuartii: ultrastructure.

The development of Antechinus stuartii from the 2-cell stage to the blastocyst stage in vivo was examined by routine transmission electron microscopy. The 2-8-cell stages had a similar organization of organelles, whereas the 16- to 32-cell stages had pluriblast cells and trophoblast cells forming an epithelium closely apposed to the zona pellucida. Specialized cell-zona plugs were formed at the 8-cell stage, and primitive cell junctions appeared in later conceptuses. The cytoplasmic organelles included mitochondria, lysosomes, aggregates of smooth endoplasmic reticulum, lipid and protein yolk bodies and fibrillar arrays, possibly contractile in function. Nuclei had uniformly-dispersed dense chromatin. Nucleoli of 2-4-cell conceptuses were dense, compact and fibrillar, and those of 8-cell conceptuses and later conceptuses were finely granular and became progressively reticulated. The embryonic genome is probably not switched on before the 8-cell stage. Sperm tails were detected in cells in several early conceptuses. The yolk mass had the same organelles as cells. Centrioles were discovered for the first time in marsupial conceptuses. These were prominently situated at a spindle pole in a 32-cell blastomere and were associated with a nucleus and sperm tail at the 4-cell stage. It is very likely that the paternal centrosome is inherited at fertilization and perpetuated in Antechinus embryos during cleavage.

Animals↗
PubMed1997

Histological differences between gravid and non-gravid uteri in the dasyurid marsupial, Sminthopsis macroura (Spencer).

Uterine samples from pregnant Sminthopsis macroura representing the first 10 days of its 11 day gestation period and samples from non-pregnant animals were compared histologically and examined for differences in the following characteristics: thickness of the endometrial stroma, luminal epithelium, myometrium and glandular epithelium, and the density of stromal glands and number of lymphocytes at the endometrial basal lamina. A highly significant difference between gravid and non-gravid uteri with respect to thickness of the endometrial epithelium was found on day 3, when lineage divergence occurs between the pluriblast and trophoblast. The endometrial stroma was significantly thicker in pregnant animals on day 8, when the epiblast differentiates into ectoderm, endoderm and mesoderm. Other differences between gravid and non-gravid uteri were detected in myometrial thickness on days 1 and 5. Taken together, these results indicate that despite similar endocrinological profiles of pregnant and non-pregnant marsupials, there are subtle, but significant, differences in uterine histology. The observed concordance between histological differences and differentiative events in embryogenesis is considered is indicative of embryo-maternal signalling.

Analysis of Variance↗
PubMed1997

Development in vitro of Marsupials: a comparative review of species and a timetable of cleavage and early blastocyst stages of development in Monodelphis domestica.

The development of marsupial oocytes and embryos in vitro is reviewed. Most stages of development have been cultured successfully, usually in a complex medium with added fetal calf serum. Simpler media without added serum have been developed for fertilization and cleavage in vitro. Culture systems have been established for oocyte maturation and fertilization in the grey short-tailed opossum and for cleavage from the zygote to the early expanding unilaminar blastocyst in a number of other marsupials. Survival in vitro of the unilaminar and early bilaminar blastocyst stages is limited in all species examined. In contrast, late bilaminar, trilaminar, embryonic and fetal stages develop at rates approximating those in vivo. More stages have been cultured successfully in Sminthopsis macroura than in any other species. It has been cultured from the late bilaminar blastocyst to within 18 h of birth. Stages of cleavage and unilaminar blastocyst formation of Monodelphis domestica timed by videotaping mating animals, proceeded at similar rates in vivo and in vitro. As in other marsupials, cleavage in this opossum is characterized by a polarized conceptus. This polarity is expressed in the distribution of organelles in the zygote and the localization of secretion of the extracellular matrix material into the cleavage cavity and of the initial cell-zona attachment. Because cell-cell adhesion follows cell-zona adhesion, a unilaminar blastocyst forms without the development of an intervening morula stage.

Animals↗
PubMed1996

A light microscopic study of oogenesis in the brushtail possum Trichosurus vulpecula.

Ovaries from young of the brushtail possum, Trichosurus vulpecula, were examined histologically and histochemically to determine stages of oogenesis. Groups of dividing oogonia were first present in a 13-day-old pouch young, with extensive oogonial proliferation after about 23 days of age. Meiosis was initiated in some oogonia by 48 days of age, and by 88 days numerous early primordial follicles were present. The first primary follicles had formed by 103 days of age. In oocytes of quiescent primordial follicles, dark granular material, which stained positively for protein, mainly occupied the perinuclear cytoplasm. Associated with the transition to primary follicles and continuing throughout oocyte growth was the production within the central cytoplasm of large clear vesicles and, to a lesser extent, lipid-like yolk bodies. The former were analogous to similar vesicles found in other species of marsupial, and have a putative role in development of the blastocyst. By contrast, yolk-like bodies are not found in all marsupial oocytes and their importance in development is unknown.

Animals↗
PubMed1996

Nutrient uptake and culture of Sminthopsis macroura (stripe-faced dunnart) embryos.

Glucose and pyruvate uptake by individual embryos were measured in a marsupial species (stripe-faced dunnart) and a eutherian species (mouse). At each stage of development, nutrient uptake by the dunnart embryo was around an order of magnitude greater than that of the mouse embryo. The pattern of glucose uptake by the dunnart embryo was not like that for any eutherian embryo, all of which have a low glucose uptake before the blastocyst stage. Rather, in the dunnart embryo there was a significant increase in glucose uptake after the third cleavage division, increasing from 13.6 pmol embryo h-1 at the 4-cell stage to 34.9 pmol embryo h-1 by the 8-cell stage. This increase in glucose uptake before blastocyst formation may be attributed to an increased energy demand associated with the movement of cells within the dunnart embryo. Using a new culture system, it was possible to culture 66% of dunnart embryos at the 2-4-cell stage and 80% of those at the 8-16-cell stage to the unilaminar blastocyst stage. Embryos cultured from the 2-cell to the 4-cell stage were retarded by around 12 h when they reached the blastocyst stage. Developmental retardation was also reflected in the pattern of nutrient uptake, which lagged behind that of embryos developed in vivo. The present study has shown that it is possible to culture the early marsupial embryo to the blastocyst stage in a serum-free culture system, while concomitantly quantifying embryonic nutrient requirements. Such an approach is essential for species where there is a paucity of material for study.

Animals↗
PubMed1996

The blastocyst epithelium is not a protoderm in dasyurid marsupials: a review of the evidence.

Evidence from studies of cleavage and blastocysts in dasyurids in reviewed to show that the unilaminar blastocyst is not a protoderm but consists of two cell types. Cleavage and blastocyst formation in marsupials has been most comprehensively studied in dasyurids, in which the secondary oocyte and zygote are polarized with respect to the position of the nucleus, cytoskeletal elements and cytoplasmic vesicles. Polarity is reinforced by fertilization. Early cleavage divisions are associated with the polarized elimination of a yolk mass and many vesicular structures into the perivitelline space. Because secretion of the vesicular structures, of which several types are found, facilitate blastomere-zona then blastomere-blastomere associations during cleavage, a unilaminar blastocyst forms without an intervening morula stage. Polarization of the cleavage cavity is related to the appearance of two cell lineages, pluriblast and trophoblast, at the 16-cell stage. In species in which the yolk mass persists, the tier of eight cells lying nearest the yolk mass forms the pluriblast (future embryonic and extra-embryonic lineages), and the other tier forms the trophoblast (future extra-embryonic ectoderm of the yolk sac). Thus, the unilaminar blastocyst epithelium is not a protoderm. Blastocyst expansion is associated with increased cell numbers mainly in the trophoblast. Pluriblast cells begin to increase just before the appearance of the hypoblast. The two cell populations can be distinguished by ultrastructural and histological features at the end of cleavage. During blastocyst expansion they differ in appearance and behaviour in vitro and in cell-doubling time. Two populations of cells have also been found in cleavage and unilaminar blastocyst stages in other marsupials, such as opossums, brushtail possums and bandicoots.

Animals↗
PubMed1996

The type and differentiation of cells in vitro from unilaminar and bilaminar blastocysts of two marsupials, Antechinus stuartii and Sminthopsis macroura.

The type and ability to differentiate in vitro of cells found in blastocysts of two marsupials were examined. Thirteen unilaminar blastocysts on Day 7 to Day 12 of gestation and 24 bilaminar blastocysts on Day 16 and Day 18 of gestation were collected from 11 brown antechinus, Antechinus stuartii. A total of of 77 unilaminar blastocysts on Day 5 and Day 6 of gestation and a total of 61 bilaminar blastocysts on Day 6 and Day 7 of gestation were collected from 40 stripe-faced dunnarts, Sminthopsis macroura. Pluriblast and trophoblast cells, confined to separate hemispheres, were found in unilaminar and bilaminar blastocysts, establishing that the blastocyst epithelium was not a protoderm. Hypoblast cells were found only in bilaminar blastocysts. Pluriblast and hypoblast cells did not differentiate or proliferate in up to eight weeks in culture. A small number of trophoblast cells transformed to a multinucleate state but the remainder did not proliferate or differentiate further. The presence of murine leukaemia inhibitory factor or medium conditioned by exposure to marsupial fibroblast feeder layer was not required for the maintenance of an undifferentiated state. Differentiation, proliferation and attachment of cells were not influenced by the presence of the yolk mass or the egg coats in culture. The time taken for attachment of dissociated cells varied significantly between cultures with no substrate and with collagen, fibronectin or laminin (P = 0.001, ANOVA) but did not vary significantly between substrates. The substrates did not influence the state of differentiation of the cells.

Animals↗
PubMed1996

Fate-map analysis of the epiblast of the dasyurid marsupial Sminthopsis macroura (Gould).

Allocation of cells in the marsupial epiblast to embryonic and extra-embryonic domains has to date been studied only histologically. An unresolved issue in marsupial embryology has been the existence of a medullary plate. We re-examined the hypotheses that the medullary plate, or neurectoderm, arises before notochord formation and that the integumentary ectoderm is segregated from the ectoderm after the formation of the medullary plate. By marking epiblast cells in 65 Day-8 embryos of the dasyurid marsupial Sminthopsis macroura, with the lipophilic cell-surface marker, DiI, we demonstrated that the so-called medullary plate contains progenitors of the integumentary ectoderm of the neck, chest, limbs and flank of the embryo. Thus, cell-allocation processes in the epiblast must have segregated the entire complement of embryonic ectoderm in one event, not separate events. It is concluded that the embryonic structure called 'medullary plate' in marsupial embryos is misnamed since, in fact, it consists of both integumentary ectoderm and neurectoderm.

Animals↗
PubMed1995

Evolution of transthyretin in marsupials.

The evolution of the expression and the structure of the gene for transthyretin, a thyroxine-binding plasma protein formerly called prealbumin, was studied in three marsupial species: the South American polyprotodont Monodelphis domestica, the Australian polyprotodont Sminthopsis macroura and the Australian diprotodont Petaurus breviceps. The transthyretin gene was found to be expressed in the choroid plexus of all three species. In liver it was expressed in P. breviceps and in M. domestica, but not in S. macroura. This, together with previous studies [Richardson, S. J., Bradley, A. J., Duan, W., Wettenhall, R. E. H., Harms, P. J., Babon, J. J., Southwell, B. R., Nicol, S., Donnellan, S. C. & Schreiber, G. (1994) Am. J. Physiol. 266, R1359-R1370], suggests the independent evolution of transthyretin synthesis in the liver of the American Polyprotodonta and the Australian Diprotodonta. The results obtained from cloning and sequencing of the cDNA for transthyretin from the three species suggested that, in the evolution of the structure of transthyretin in vertebrates, marsupial transthyretin structures are intermediate between bird/reptile and eutherian transthyretin structures. In marsupials, as in birds and reptiles, a hydrophobic tripeptide beginning with valine and ending with histidine was found in transthyretin at a position which has been identified in eutherians as the border between exon 1 and intron 1. In humans, rats and mice, the nine nucleotides, coding for this tripeptide in marsupials/reptiles/birds, are found at the 5' end of intron 1. They are no longer present in mature transthyretin mRNA. This results in a change in character of the N-termini of the subunits of transthyretin from hydrophobic to hydrophilic. This change might affect the accessibility of the thyroxine-binding site in the central channel of transthyretin, since, at least in humans, the N-termini of the subunits of transthyretin are located in the vicinity of the channel entrance [Hamilton, J. A., Steinrauf, L. K., Braden, B. C., Liepnieks, J., Benson, M. D., Holmgren, G., Sandgren, O. & Steen, L. (1993) J. Biol. Chem. 268, 2416-2424].

Amino Acid Sequence↗
PubMed1995

Cytoskeletal organization in the oocyte, zygote, and early cleaving embryo of the stripe-faced dunnart (Sminthopsis macroura).

Ovulation occurs in Sminthopsis macroura approximately 160 hr after administration of 1.3 IU PMSG, and yields significantly more oocytes than does spontaneous ovulation (P = 0.001). Germinal vesicle (GV)-stage oocytes have a thin cortical rim of microfilaments, which is disrupted by exposure to cytochalasin D. After GV breakdown, the first meiotic spindle forms subcortically and parallel to the oolemma. It rotates during anaphase and telophase to extrude the first polar body. This rotation is associated with a local cortical concentration of microfilaments, which is extruded in the first polar body. The second meiotic spindle is orthogonal to the surface, and extrusion of the second polar body is not associated with obvious local changes in cortical actin, resulting in a polar body containing little polymerized actin. The sites of second polar body emission and sperm entry are always in the half of the oocyte opposite the concentrating yolk mass, and are within 60 degrees of each other in most oocytes. During the concentration and eccentric movement of the yolk, microfilaments condense around it. During yolk expulsion, these microfilaments become continuous with those located subcortically. During early cleavage, the cytocortex of the zygote, but not of the extruded yolk mass, stains heavily for polymerised actin. Multiple sites of pericentriolar material are detectable in the cytoplasm of some secondary unfertilized oocytes which, in the presence of taxol, generate large cytasters and pseudospindle structures. After fertilization, a large aster is formed in association with the sperm entry point and serves as the center of an extensive cytoplasmic network of microtubules which surrounds but does not enter the yolk mass. Taxol treatment generates small cytasters within this meshwork and promotes selective stabilization of some periyolk microtubules opposite to the sperm aster.

Actin Cytoskeleton↗
PubMed1994

Development of early cell lineages in marsupial embryos: an overview.

All major embryonic and extra-embryonic cell lineages are established before implantation in marsupials. In reptiles, birds, monotremes and most marsupials, the zygote is polarized, sometimes markedly so, and the cleavage pattern in relation to the polarized state provides the mechanism for the generation of positional signals. These ensure that the embryonic cell lineages develop in the centre of the developing blastoderm or blastocyst epithelium and the extra-embryonic lineages at the periphery. The evolution of the vertebrate yolky egg was accompanied by a decreasing dependence on maternal determinants and increasing dependence on positional signals to determine cell fate. It is proposed that when a less yolky egg evolved, the mechanisms for determination of cell fate in a developing epithelium were retained. It is proposed that in marsupials, positional signals are involved in the determination of cell fate of embryonic and trophectoderm cells but do so in a two-dimensional epithelium not a three-dimensional morula. The next lineage formed is the primary endoderm which separates off from the primitive ectoderm in the embryoblast and eventually lines the blastocyst cavity. Positional signals are responsible for the determination of primary endoderm in eutherian mammals, birds and probably also marsupials. Order of cell division during cleavage provides a mechanism whereby some cells in the embryoblast of marsupials have earlier and greater contact with their neighbouring cells. The mechanism for determination of primary endoderm cells in the blastocyst epithelium is examined in the Virginia opossum and the stripe-faced dunnart.

Animals↗
PubMed1994

Structural development in the newborn marsupial, the stripe-faced dunnart, Sminthopsis macroura.

The structure of various sense organs and endocrine glands was examined in the stripe-faced dunnart, Sminthopsis macroura. This marsupial has the shortest gestation known for all mammals, 10.5-11 days. The morphology of the anterior pituitary, adrenal gland, olfactory epithelium, Merkel cells around the mouth and the utricle of the vestibular system of S. macroura was similar to that observed in other newborn marsupials. These structures are thought to be required by the newborn to transfer from the uterus to the pouch. The stage of development of the urinary system, the semicircular canals of the vestibular system, eyes and lungs was slightly different to that of other newborn marsupials. These structures are thought to have secondary importance in allowing the newborn to reach the teat. Although marsupials display variation in gestation length and produce newborn of differing body weights, there is little difference in morphology between various newborn marsupial species.

Animals↗
PubMed1993

The expression of the transthyretin gene in liver evolved during the radiation of diprotodont marsupials in Australia.

Thyroid hormone-binding proteins in blood plasma were identified in 28 different marsupial species by their capacity to bind radioactive thyroxine. All species contained albumin. Transthyretin was not found in the blood from any of 12 polyprotodont marsupial species, but was abundant in the blood from all of 16 diprotodont marsupial species investigated. Transthyretin mRNA was absent from the liver of the stripe-faced dunnart, a polyprotodont marsupial, but abundant in the liver of the diprotodont grey kangaroo. Diprotodont marsupials probably evolved in Australia from polyprotodont marsupials after their transantarctic migration from South America, about 40 million years ago. It is suggested that hepatic transthyretin expression evolved in marsupials during the radiation of herbivorous, diprotodont species in Australia. The earlier appearance of transthyretin gene expression in the choroid plexus of the stem reptiles, about 300 million years ago, contrasts with hepatic transthyretin synthesis, a relatively late evolutionary event, occurring independently in at least three lineages.

Animals↗
PubMed1993

Embryonic development in culture of the marsupials Antechinus stuartii (Macleay) and Sminthopsis macroura (Spencer) during preimplantation stages.

Forty-nine blastocysts from 11 brown antechinus, Antechinus stuartii, and 96 blastocysts from 17 stripe-faced dunnarts, Sminthopsis macroura, were used to develop a culture system for embryos during preimplantation stages. Blastocysts of brown antechinus were collected on Days 6-9 for unilaminar stages, Days 16-21 for bilaminar stages and Days 20 and 21 for trilaminar stages. Blastocysts of stripe-faced dunnarts were collected on Day 6 for unilaminar stages, Days 6-8 for bilaminar stages and Day 8 for trilaminar stages. Culture media were Dulbecco's modified Eagle's medium (DMEM) with 4.5% glucose and Whittingham's T6 medium both of which were supplemented with 5, 10, 12.5 and 20% fetal calf serum (FCS). Antechinus serum (5%) and bovine serum albumin (0.1%, 0.2%) were also added to some media. Human amniotic fluid (HAF) and Monomed media were also tested. Blastocysts were cultured at 35 degrees C in 5% CO2 in air. DMEM + 10% FCS and HAF supported normal development for the longest periods and over the greatest range of stages. Developmental failure of blastocysts in vitro during expansion of the unilaminar blastocyst and formation of the bilaminar blastocyst suggests that these stages may be dependent on uterine signals. When cultured in DMEM + 10% FCS, the rate of development of bilaminar and trilaminar blastocysts into organogenesis was 4 h slower than in vivo in the stripe-faced dunnart and about 6 h slower than in vivo in the brown antechinus. Embryos of stripe-faced dunnarts were cultured to within 18 h of birth.

Animals↗
PubMed1993

Uterine histology of the dasyurid marsupial, Antechinus stuartii: relationship with differentiation of the embryo.

Uterine samples from Antechinus stuartii on days 1, 4, 6, 8, 11, 13, 15, 18, 21 and 23 after ovulation were examined histologically. Animals were pregnant, nonpregnant and unmated, or nonpregnant and mated but found to have only unfertilized eggs on autopsy. The histological parameters used were thickness of the myometrium, endometrial stroma, and endometrial epithelium, and density of uterine stromal glands and of lymphocytes at the endometrial basal lamina. Overall, the fluctuation patterns of these parameters were superficially similar between pregnant and nonpregnant animals (mated or unmated). However, statistically significant differences were detected between pregnant and unmated nonpregnant animals in every parameter examined at nearly every time point except day 13. Comparison of these results with known data on embryonic stages, corpus luteum development and plasma progesterone concentrations revealed that the gravid uterus underwent histological changes co-incident with changes in both progesterone concentration and developmental delay or embryonic arrest. It was concluded that the uterus mediates progesterone-driven changes in embryonic developmental rate. Although determination of number of lymphocytes provided inconclusive evidence of cellular immunity against embryos, the possibility that embryonic signalling to the uterus occurs is discussed.

Animals↗