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. Anat Rec 254:307–321, 1999. © 1999 Wiley‐Liss, Inc.     

Molecular confirmation of an adenovirus in brushtail possums (Trichosurus vulpecula)

Partial genome characterisation of a non-cultivable marsupial adenovirus is described. Adenovirus-like particles were found by electron microscopy (EM) in the intestinal contents of brushtail possums (Trichosurus vulpecula) in New Zealand. Using degenerate PCR primers complementary to the most conserved genome regions of adenoviruses, the complete nucleotide sequence of the penton base gene, and partial nucleotide sequences of the DNA polymerase, hexon, and pVII genes were obtained. Phylogenetic analysis of the penton base gene strongly suggested that the brushtail possum adenovirus (candidate PoAdV-1) belongs to the recently proposed genus Atadenovirus. Sequence analysis of the PCR products amplified from the intestinal contents of brushtail possums originating from different geographical regions of New Zealand identified a single genotype. This is the first report of molecular confirmation of an adenovirus in a marsupial.     

Determination of auditory thresholds in the brushtail possum (Trichosurus vulpecula)

The auditory abilities of the brushtail possum (Trichosurus vulpecula) have been measured by cochlear potential readings, but no behavioural determinations of their auditory abilities have been published. Six experimentally naive possums were trained to perform a two-response conditional discrimination between the presence and absence of an 880-Hz tone (at 80 dB). All six possums readily learned this task. The behavioural threshold for this tone was determined using a modified tracking procedure and found to be similar to that reported using cochlear microphonic potentials. One concern with the current method was the communal nature of the experimental environment so a further threshold determination in a sound-attenuating chamber was conducted. No substantial difference was noted between the results obtained in the two threshold determinations. The success of the current method means that a full, behavioural audiogram for the brushtail possum, which would complement the existing cochlear potential data, is now possible.     

The lymphoid and immunohaematopoietic tissues of the embryonic brushtail possum ( Trichosurus vulpecula)

The lymphoid and immunohaematopoietic tissues of the embryonic and full-term brushtail possums was investigated histologically and immunohistochemically using antibodies to the T- and B-cell markers, CD3, CD5, CD79a and CD79b. No clearly defined thymus, bone marrow, spleen, lymph nodes, gut-associated lymphoid tissues or bronchus-associated lymphoid tissues were observed histologically. The liver was haematopoietic and contained erythrocytic and granulocytic precursors. No mature lymphocytes were observed histologically or detected using antibodies to T- and B-cell markers in any of the tissues. These results are consistent with other studies of the early postnatal tissues of other marsupials and support the proposition that neonatal marsupials are substantially reliant on maternal immunological protection at the time of birth and for a significant period of pouch life.     

A new papillomavirus of possums (Trichosurus vulpecula) associated with typical wart-like papillomas

Summary.  A previously unknown, cutaneous papillomavirus (Papovaviridae) in a brushtail possum (Trichosurus vulpecula) was demonstrated. This represents one of the first viruses reported in this species. Possum papillomas were identified by typical wart-like appearance and histology. Papillomavirus particles were detected by electron microscopy in tissue homogenates following purification and negative staining. The polymerase chain reaction amplified a conserved portion of the L1 gene which was purified and sequenced. Comparison of the DNA and deduced amino acid sequence from the possum papillomavirus with other papillomavirus sequences, together with phylogenetic analysis, indicated that this was a new papillomavirus. Received September 6, 1999 Accepted December 8, 1999     

Thyroid gland development in the brushtail possum,Trichosurus vulpecula

Background: The onset of thyriod function occurs at different times with respect to birth in different mammals. In most marsupials studied thus far, thyroid function begins during the latter half of pouch life. However, in marsupials, thyroxine can be transported from the mother to the young via milk. In this study the plasma concentration of the thyroid hormones, thyroxine (T₄) and triiodothyronine (T₃), in the young was correlated with the ultrastructural appearance of the thyroid of the young. Methods: Thyroid glands at 40, 65, 70, 96, 113, 115, and 129 days postpartum and from an adult were examined with the light and electron microscope. Plasma concentrations of both T₄ and T₃ in young possums were determined by radioimmunoassay. Results: Plasma concentrations of T₄ in the young were less than 10 ng/ml prior to day 60 and 12 ng/ml on day 60; they rose to a peak of 45 ng/ml on day 120 postpartum and then diminished to below 10 ng/ml by day 160 postpartum. Plasma T₃ concentrations were similar in profile, the peak concentration being 0.25 ng/ml on day 120 postpartum, and, as with T₄, T₃ concentrations decreased in the latter part of the lactation period. The morphology of the follicular cells of the thyroid gland would suggest that the developing brushtail possum is capable of producing thyroxine at about day 65 postpartum. Densely staining bodies, presumably lysosomes that are required for the conversion of thyroglobulin to thyroxine, were first seen on day 65, and greater quantities were noted in day 96, 113, 115, and 129 thyroid tissue. Conclusions: The plasma concentrations of T₄ and T₃ of the young are greater than that of the adult by day 60 postpartum, with peak concentrations of thyroid hormones on day 120 postpartum. Ultrastructural examination would suggest that the possum thyroid gland is capable of producing thyroid hormones at about day 65 postpartum. The activation of the thyroid gland precedes the reported increase in growth rate of the young possum on day 96 (Gemmell and Hendrikz, 1993. Aust. J. Zool., 41:141 – 149 ). © 1995 Wiley-Liss, Inc.     

Development of the lung of the brushtail possum, Trichosurus vulpecula

The developing lung of the brushtail possum, Trichosurus vulpecula, was studied by light microscopy, and transmission electron microscopy was used to study the morphology of the conducting airways in the adult. Bronchi did not extend beyond the hilus of each of the six lobes of the lung, and lobules were supplied by major bronchioles. By 105 days post partum, bronchi and bronchioles were fully formed, coinciding with the emergence of mucosal associated lymphoid tissue (MALT), which preceded alveolar maturation by approximately 20 days. In the adult lung, goblet cells were rarely observed in the mucosal epithelium of bronchi, whereas Clara cells were present in the mucosa of all airways, increasing proportionately as the conducting and respiratory portions narrowed distally. Although the airways of the possum lung have a poorly developed mucociliary blanket, this may be compensated for by the large numbers of Clara cells and adequate supply of MALT.     

Retinal epithelial fine structure in the brush-tailed possum (Trichosurus vulpecula)

As part of a comparative morphological study, the fine structure of the retinal pigment epithelium (RPE), the choriocapillaris and Bruch's membrane (complexus basalis) has been studied by electron microscopy in the brush-tailed possum (Trichosurus vulpecula), an Australian marsupial. In this species, the RPE consists of a single layer of squamous cells which show numerous shallow basal (scleral) infoldings as well as extensive apical (vitreal) processes enclosing rod outer segments. The epithelial cells are joined laterally by prominent, apically-located tight junctions. Internally smooth endoplasmic reticulum is the most abundant cell organelle with only small amounts of rough endoplasmic reticulum present. Basally-located mitochondria and apically-located melanosomes are abundant while polysomes and the occasional Golgi zone are also noted. The epithelial cell nucleus is large and vesicular. Bruch's membrane shows the typical pentalaminate structure noted for mammalian species. The choriocapillaris is a single layer of anastomosing capillaries which are heavily fenestrated facing the retinal epithelium.     

Ontogeny of the immune system of the brushtail possum, Trichosurus vulpecula

The numbers and distribution of T and B cells in the thoracic thymus, spleen and intestinal tissue and the proliferation of T lymphocytes were examined during pouch life and in the adult to determine when the developing brushtail possum reaches immunological maturity. CD3-positive cells were observed in the thoracic thymus at day 2 post-partum indicating that the thymus produces T lymphocytes at or soon after birth. By day 25 the thymus was fully populated with CD3-positive T lymphocytes and they were observed in distinct regions of the cortex and medulla. By day 48 post-partum, B and T lymphocytes were identified in the follicles and parafollicular areas of the spleen. Although the numbers of T and B cells in the spleen increased significantly from day 25 to day 100 post-partum (P < 0.005), fewer cells were present at day 150 post-partum than in the adult (P < 0.05). Peyer's patches were not observed in the intestines up to day 73 post-partum. However, both T and B cells were observed in the intestinal lymph nodes. Although the T lymphocytes at weaning showed a proliferative response, the response was not as great as that observed in the adult possum. Thus, the immune system of the possum is not fully developed at weaning but continues its development after pouch life.     

Cloning of the MHC class II DRB cDNA from the brushtail possum (Trichosurus vulpecula)

The cell-surface glycoproteins encoded by the major histocompatibility complex (MHC) bind to processed foreign antigens and present them to T lymphocytes. Two classes of MHC molecules and their corresponding gene sequences have been extensively studied in eutherian mammals and birds, but data on the marsupial MHC are limited. Marsupials split from eutherian mammals about 125 million years ago and represent a distinct branch in mammalian evolution. Here the cDNA cloning of MHC class II genes of the brushtail possums (Trichosurus vulpecula) is reported. The sequences obtained were found to be relatively conserved when compared to the red-necked wallaby (Macropus rufogriseus) and an South American marsupial, Monodelphis domestica. The T. vulpecula sequence shared an average overall sequence identity of 75.4% at the deduced amino acid level with M. rufogriseus and M. domestica, respectively.     

Uterine remodelling during pregnancy and pseudopregnancy in the brushtail possum (Trichosurus vulpecula; Phalangeridae)

The formation of a placenta is critical for successful mammalian pregnancy and requires remodelling of the uterine epithelium. In eutherian mammals, remodelling involves specific morphological changes that often correlate with the mode of embryonic attachment. Given the differences between marsupial and eutherian placentae, formation of a marsupial placenta may involve patterns of uterine remodelling that are different from those in eutherians. Here we present a detailed morphological study of the uterus of the brushtail possum (Trichosurus vulpecula; Phalangeridae) throughout pregnancy, using both scanning and transmission electron microscopy, to identify whether uterine changes in marsupials correlate with mode of embryonic attachment as they do in eutherian mammals. The uterine remodelling of T. vulpecula is similar to that of eutherian mammals with the same mode of embryonic attachment (non‐invasive, epitheliochorial placentation). The morphological similarities include development of large apical projections, and a decrease in the diffusion distance for haemotrophes around the period of embryonic attachment. Importantly, remodelling of the uterus in T. vulpecula during pregnancy differs from that of a marsupial species with non‐invasive attachment (Macropus eugenii; Macropodidae) but is similar to that of a marsupial with invasive attachment (Monodelphis domestica; Didelphidae). We conclude that modes of embryonic attachment may not be typified by a particular suite of uterine changes in marsupials, as is the case for eutherian mammals, and that uterine remodelling may instead reflect phylogenetic relationships between marsupial lineages.     

Molecular cloning of the brushtail possum (Trichosurus vulpecula) immunglobulin E heavy chain constant region

The immunobiology of marsupial IgE is poorly understood. As a first step towards the development of immunological reagents for marsupials and to obtain a further understanding of immunoglobulin evolution, a brushtail possum (Trichosurus vulpecula) mesenteric lymph node cDNA library was screened for the heavy chain constant region of IgE (Cε), using a partial Cε probe from the American marsupial, Monodelphis domestica. The cDNA sequence for T. vulpecula Cε was determined and found to be most similar to the M. domestica Cε sequence [(76%) at the amino acid level]. T. vulpecula Cε has amino acid sequence similarities ranging from 43–52% with various eutherian Cε sequences. The secondary structure of T. vulpecula Cε, based on loops formed by internal disulfide bonds, more closely resembles rodent Cε than the American marsupial sequence.     

Expression of the Fc receptor in the mammary gland during lactation in the marsupial Trichosurus vulpecula (brushtail possum)

One of several functions described for the Fc receptor is regulation of IgG isotype transport into milk. The first marsupial homologues of the Fc receptor heavy and light chains, FcRn and beta-2 microglobulin, from the brushtail possum have been cloned and characterised. The level of FcRn mRNA in the possum mammary gland was highest at the start of lactation, and decreased slowly thereafter. Expression of FcRn mRNA did not increase during the switch phase when the concentration of IgG in milk is highest. In contrast, the level of beta-2 microglobulin mRNA in the mammary gland increased during the switch phase when milk IgG concentration also increases. This correlation between beta-2 microglobulin mRNA expression in the mammary gland with the time of active IgG-transfer into milk was also observed in the bovine and murine mammary gland. This suggests that expression of the Fc receptor in the mammary gland is controlled by the expression of beta-2 microglobulin and that its expression is upregulated during the period of highest IgG-transfer into milk.     

Histology of the pouch epithelium and the mammary glands during chemically induced oestrus in the brushtail possum (Trichosurus vulpecula)

Changes in the epithelium of the maternal pouch and the mammary gland of brushtail possums (Trichosurus vulpecula) were examined after animals were treated to induce ovulation with follicle-stimulating hormone (FSH), luteinizing hormone (LH), pregnant mares' serum gonadotrophin (PMSG) and oestradiol. The mammary glands were similar in appearance to those described in eutherian mammals and in previous studies on other marsupials. Exposure of possums to these compounds, particularly PMSG, appeared to result in changes in the mammary glands that could be associated with milk/secretion production. In contrast, the pouch epithelium had a similar histological appearance to that of epithelium from other parts of the body regardless of whether the animal was exposed to stimulants. These preliminary observations are discussed in the context of the purported role of the pouch epithelium and the mammary gland in production of secretions at oestrus and provision of immunological protection to the neonatal marsupial.     

Differentiation of the gonads and initiation of mammary gland and scrotum development in the brushtail possum Trichosurus vulpecula (Marsupialia)

The first appearance of the mammary and scrotal primordia and the sexual differentiation of the gonads of the brushtail possum, Trichosurus vulpecula, are described. Primordial germ cells were first observed, in fetuses of 7.5 mm crown-rump length, in the gonadal ridges and migrating up the dorsal mesentery. Mammary primordia were first observed in fetuses of 11 mm, and scrotal primordia in those of 12 mm crown-rump length. These structures were diagnostic of female and male brushtail possums respectively. Processus vaginales and gubernacula showed sexual dimorphism, being better developed and appearing earlier in males than in females. Sexual differentiation of the gonads occurred after the appearance of mammary and scrotal primordia, the testes being first recognisable in a 14.5-mm fetus and the ovaries postnatally. Birth occurred between the stages of 14 and 15 mm crown-rump length. These observations appear to indicate that the development of mammary and scrotal primordia are not under gonadal hormonal control, but under direct genetic control, as suggested for the tammar wallaby by previous authors.     

Molecular identification of interleukin-2 in the lymphoid tissues of the common brushtail possum, Trichosurus vulpecula

The common brushtail possum (Trichosurus vulpecula) is an Australian marsupial. Here we describe the identification of possum interleukin-2 in mitogen-stimulated lymph node cells. We used a strategy of Rapid amplification of cDNA ends using probes designed from recently-sequenced marsupial genomes to identify the IL2 gene and then confirmed that IL-2 expression in possum immune tissue occurs in a similar manner to that in their eutherian counterparts. The predictive possum IL-2 peptide showed 28% and 35% amino acid sequence homology with the mouse and human IL-2 molecules, respectively, consistent with the divergence found within this cytokine family. Despite this low sequence identity, possum IL-2 still possessed the characteristic hallmarks of mammalian IL-2, such as a predicted signal peptide and conserved family motifs.     

Molecular cloning of four lambda light chain cDNAs from the Australian brushtail possum Trichosurus vulpecula

A brushtail possum mesenteric lymph node cDNA library was screened with a grey short‐tail opossum Cλ probe and four immunoglobulin lambda cDNAs were isolated. Two of the isolated clones (L5 and L10) contained identical framework 4 regions and constant regions (but different variable regions), suggesting that the possum lambda locus is organized as multiple J‐C pairs — a feature seen in the opossum and placental mammals. The cloning of the lambda light chain cDNAs signifies the completion of the basic molecular characterization of the brushtail possum immunoglobulin repertoire. The availability of this sequence data will allow extensive analysis of the immune response of the brushtail possum at the molecular level, as well as the development of specific immunological reagents for detection of immunoglobulin molecules at the protein level.